Artigos I

ARTIGOS
Advances in Experimental Medicine and Biology 817

Microbial Endocrinology
Mark Lyte
John F. Cryan
Editors
Microbial
Endocrinology:
The Microbiota-
Gut-Brain Axis in
Health and Disease
Advances in Experimental Medicine and Biology
Mark Lyte, Texas Tech University Health Sciences Center,
Abilene, TX, USA
Volume 817
Editorial Board:
IRUN R. COHEN, The Weizmann Institute of Science, Rehovot, Israel

ABEL LAJTHA, N.S. Kline Institute for Psychiatric Research, Orangeburg, NY,
USA
JOHN D. LAMBRIS, University of Pennsylvania, Philadelphia, PA, USA
RODOLFO PAOLETTI, University of Milan, Milan, Italy
For further volumes:

http://www.springer.com/series/5584
Mark Lyte • John F. Cryan
Editors
Microbial Endocrinology:
The Microbiota-Gut-Brain
Axis in Health and Disease
Editors
Mark Lyte John F. Cryan
Department of Immunotherapeutics Department of Anatomy and Neuroscience
and Biotechnology University College
Texas Tech University Health Sciences Cork, Ireland
Center
Abilene, TX, USA
ISSN 0065-2598 ISSN 2214-8019 (electronic)

ISBN 978-1-4939-0896-7 ISBN 978-1-4939-0897-4 (eBook)
DOI 10.1007/978-1-4939-0897-4
Springer New York Heidelberg Dordrecht London
Library of Congress Control Number: 2014942372
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Prof. Mark Lyte: “To my loving wife and my
two remarkable sons who are my pillars of
strength”
Prof. John F. Cryan: “To Colleen, Ois´ın &
Alannah: For constant support and patience”
Foreword
This book is the second volume of a continuing series. The first volume published
by Springer in 2010, “Microbial Endocrinology: Interkingdom Signaling in Infec-
tious Disease and Health”, contained little in regard to brain and behavior, but
instead focused almost exclusively on aspects of infectious disease. Health conse-
quences as such were mainly concerned with the role that stress could play in
altering the interface between host and microbiota. The present volume is therefore
a testament to the great strides during the intervening years which have illuminated
the myriad ways in which microbiota interfaces with the host. It is anticipated that
future volumes in this series will reflect the ever increasing acceleration of research
into the microbiota–gut–brain axis.
Abilene, TX, USA Mark Lyte

January 2014 Series Editor
vii
If one was to ask whether a book dealing with the ability of the microbiota to
influence the brain, and ultimately cognition and behavior, would have been
possible just a few short years ago, the answer would most likely be no. A simple
search of PubMed using the index words “microbiota AND gut AND brain” reveals
only 134 publications as of 16th January 2014. However, this would not be an
accurate reflection of the work that has been ongoing for many decades, but yet
remained on the outer fringes of the disciplines that constitute the study of the
mechanisms by which the microbiota and the brain communicate with each other. A
comprehensive series of articles by Bested and colleagues [1] catalog the numerous
studies going back over a century which amply demonstrate that the investigation of
the role of the microbiota in brain function, and by extension mental health, has a
long and varied (some may say checkered) scientific history. During this time it
remained, for large measure, outside mainstream scientific inquiry following an
initial burst of enthusiasm both in the scientific and public arenas at the turn of the
twentieth century. That such scientific skepticism remained, and in many cases
became entrenched, in the very scientific disciplines that form the basis of the
microbiota–gut–brain axis is owed to a number of factors. One of these is surely the
increasing specialization that occurred within each discipline over the years and the
inherent lack of interdisciplinary thought that accompanied such specialization.
With the advent of the concerted research into the microbiota and the microbiome,
as best evidenced by the tremendous strides that the Human Microbiome Project
has made over the last decade in cataloging the incredible diversity in the
microbiota in health and disease, the realization that the microbiota has a role to
play in the development and function of the nervous system and hence behavior and
cognition, has once again entered into mainstream scientific and medical thought.
However, old beliefs die hard. The recent experience of one of us (ML) as described
in the prologue to Chap. 1 is but one example of the resistance that is still being
encountered today for a role of the microbiota in the functioning of the brain. In
many conservative Learned Societies the concept that the gut and indeed the gut
microbiota can have such an influence on brain & behavior is still looked upon with
incredulity. However, this is changing.
ix
x Preface
This book represents the realization that any attempt to understand the ability of
Preface
the microbiota to interface with the brain (and by association any part of the host’s
neurophysiology) must attempt to address multiple disciplines, such as microbiol-
ogy, anatomic neuropathology, and endocrinology to name but a few, that while on
the first examination appear to be rather disparate from each other but on further
examination are in fact highly interconnected as evidenced, for example, by the
development of the field of microbial endocrinology itself. As described in Chap. 1,
as well as detailed in a chapter in the first book of this series [2], the field of
microbial endocrinology developed out of need to understand the paradox in which
stress resulted in increased death from a bacterial challenge at the same time greatly
increasing the phagocytic activity of the immune system. In considering the
microbiota as an interactive player in the host that can both respond to signals
from the host and influence the host through the provision of the very same host
signaling molecules (i.e., neurochemicals) that are more commonly associated only
with vertebrates, but in fact have a long evolutionary history involving the pro-
karyotes, the potential role of the microbiota in brain functioning and its potential
for treatment of mental disorders becomes apparent.
As such, the book is organized along three thematic lines which will provide the
reader not only a fuller understanding of the capabilities of the microbiota to
interface with the brain and form the microbiota–gut–brain axis, but will also
provide detailed examination of the consequences of the microbiota-driven gut-
to-brain communication for both health and disease. The first four chapters cover
the “Basic Concepts Underlying the Microbiota–Gut–Brain Axis”; the next eight
chapters examine the “Mechanistic Factors Influencing the Microbiota–Gut–Brain
Axis” and the concluding seven chapters address the “Microbiota–Gut–Brain Axis
in Health and Disease”.
We have assembled a group of contributors who are recognized to be at the front
of their respective fields to review the state of the art of this growing field. As the
chapters in this book amply demonstrate, the field of microbiota–gut–brain axis is
still in its infancy although its origins are now over a century old. With the advent of
modern techniques ranging from deep pyrosequencing of the microbiota to brain
imaging, the tools are in place to address those questions which were raised many
decades ago. Given our evolving understanding of the complexity of the microbiota
which when one couples that to the complexity of the brain and nervous system, this
book represents only one more chapter in what promises to be a long and challeng-
ing story.
Abilene, TX, USA Mark Lyte

Cork, Ireland John F. Cryan
January 2014
Preface xi
References
1. Bested AC, Logan AC, Selhub EM (2013) Intestinal microbiota, probiotics and mental health:
from Metchnikoff to modern advances: Part I – autointoxication revisited. Gut Pathog 5(1):5
2. Lyte M (2010) Microbial endocrinology: a personal journey. In: Lyte M, Freestone PPE (eds)
Microbial endocrinology: interkingdom signaling in infectious disease and health. Springer,
New York, pp 1–16
xiv Contents
Part I Basic Concepts Underlying the Microbiota-Gut-Brain Axis
1 Microbial Endocrinology and the Microbiota-Gut-Brain Axis .............. 3

Mark Lyte
2 Utilizing “Omics” Tools to Study the Complex Gut Ecosystem ............. 25
Anthony Fodor
3 The Enteric Nervous System and Gastrointestinal Innervation:
Integrated Local and Central Control .................................................... 39
John B. Furness, Brid P. Callaghan, Leni R. Rivera, and Hyun-Jung Cho
4 Intestinal Barrier Function and the Brain-Gut Axis .............................. 73
Carmen Alonso, Mar´ıa Vicario, Marc Pigrau, Beatriz Lobo,
and Javier Santos
5 Vagal Pathways for Microbiome-Brain-Gut Axis Communication ......... 115
Paul Forsythe, John Bienenstock, and Wolfgang A. Kunze
6 The Brain-Gut Axis in Health and Disease ......................................... 135
Yasser Al Omran and Qasim Aziz
Part II Mechanistic Factors Influencing the Microbiota-Gut-Brain Axis
7 Gastrointestinal Hormones and Their Targets ..................................... 157

Jens F. Rehfeld
8 Microbiome, HPA Axis and Production of Endocrine Hormones
in the Gut ............................................................................................... 177
Nobuyuki Sudo
9 Neuropeptides and the Microbiota-Gut-Brain Axis.............................. 195
Peter Holzer and Aitak Farzi
xiii
10Bacterial Neuroactive Compounds Produced by Psychobiotics ........... 221
Contents
Rebecca Wall, John F. Cryan, R. Paul Ross, Gerald F. Fitzgerald,
Timothy G. Dinan, and Catherine Stanton
11 Multidirectional Chemical Signalling Between Mammalian
Hosts, Resident Microbiota, and Invasive Pathogens:
Neuroendocrine Hormone-Induced Changes in Bacterial
Gene Expression..................................................................................... 241
Michail H. Karavolos and C.M. Anjam Khan
12 Influence of Stressor-Induced Nervous System Activation
on the Intestinal Microbiota and the Importance for
Immunomodulation .....................................................................................255
Michael T. Bailey
Part III The Microbiota-Gut-Brain Axis in Health and Disease
13 The Effects of Inflammation, Infection and Antibiotics on the

Microbiota-Gut-Brain Axis ................................................................ 279
Premysl Bercik and Stephen M. Collins
14 Microbiota, Inflammation and Obesity ................................................. 291
Yolanda Sanz and Angela Moya-Pérez
15 Microbiota, Immunoregulatory Old Friends and Psychiatric
Disorders ................................................................................................ 319
Graham A.W. Rook, Charles L. Raison, and Christopher A. Lowry
16 Microbiota-Gut-Brain Axis and Cognitive Function ........................... 357
Mélanie G. Gareau
17 The Impact of Microbiota on Brain and Behavior: Mechanisms
& Therapeutic Potential ........................................................................ 373
Yuliya E. Borre, Rachel D. Moloney, Gerard Clarke, Timothy G. Dinan,
and John F. Cryan
18 Neuroimaging the Microbiome-Gut–Brain Axis ................................... 405
Kirsten Tillisch and Jennifer S. Labus
19 The Future of Probiotics for Disorders of the Brain-Gut Axis ............ 417
Eamonn M.M. Quigley and Fergus Shanahan
Index .............................................................................................................. 433
xvi List of Contributors
Yasser Al Omran, BSc (Hons) Centre for Digestive Diseases, Wingate Institute of
Neurogastroenterology, Blizard Institute, Barts and The London School of Medi-
cine and Dentistry, Queen Mary, University of London, London, UK
Carmen Alonso, MD, PhD Neuro-Immuno-Gastroenterology Group, Digestive

Diseases Research Unit, Gastroenterology Department, Hospital Universitari Vall
d’Hebron, Vall d’Hebron Research Institute, Barcelona, Spain
Department of Medicine, Universitat Autònoma de Barcelona, Centro de

Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas
(Ciberehd), Barcelona, Spain
Qasim Aziz, MRCP, PhD, FRCP Centre for Gastroenterology, The Wingate
Institute of Neurogastroenterology, Bart’s and The London NHS Trust,
London, UK
Michael T. Bailey, PhD Institute for Behavioral Medicine Research, College of

Medicine, The Ohio State University, Columbus, OH, USA
Premysl Bercik, MD Medicine, McMaster University, Hamilton, ON, Canada
John Bienenstock, MBBS, FCRP, FCRP(C) Pathology and Molecular Medicine,

Brain-Body Institute, McMaster University, St. Joseph’s Healthcare, Hamilton,
ON, Canada
Yuliya E. Borre, PhD Neurogastroenterology Lab, Alimentary Pharmabiotic

Center, University College Cork, Cork, Ireland
Brid P. Callaghan Anatomy and Neuroscience, University of Melbourne,

Parkville, VIC, Australia
xv
Gerard Clarke, PhD Department of Psychiatry and Laboratory of NeuroGastroen-
List of Contributors
terology Alimentary Pharmabiotic Centre, University College Cork, Cork, Ireland
Hyun-Jung Cho, PhD Anatomy and Neuroscience, University of Melbourne,

Stephen M. Collins, MD Medicine, McMaster University, Hamilton, ON, Canada
John F. Cryan, PhD Department of Anatomy and Neuroscience, University

College Cork, Cork, Ireland
Timothy G. Dinan, MD, PhD Psychiatry, University College Cork, Cork, Ireland
Aitak Farzi, MD Institute of Experimental and Clinical Pharmacology, Medical

University of Graz, Graz, Austria
Gerald F. Fitzgerald, PhD, DSc Microbiology and Alimentary Pharmabiotic

Centre, University College Cork, Cork, Ireland
Anthony Fodor, PhD Bioinformatics and Genomics, UNC Charlotte, Charlotte,

NC, USA
Paul Forsythe, PhD Medicine, McMaster University, Hamilton, ON, Canada
John B. Furness Anatomy and Neuroscience, University of Melbourne, Parkville,

VIC, Australia
Mélanie G. Gareau, PhD Department of Medicine, University of California San

Diego, La Jolla, CA, USA
Peter Holzer, MSc, PhD Experimental Neurogastroenterology, Institute of Exper-

imental and Clinical Pharmacology, Medical University of Graz, Graz, Austria
Michail H. Karavolos, PhD Centre for Bacterial Cell Biology, Institute for Cell
and Molecular Biosciences, Newcastle University, Newcastle upon Tyne, UK
C.M. Anjam Khan, PhD Centre for Bacterial Cell Biology, Institute for Cell and
Molecular Biosciences, Newcastle University, Newcastle upon Tyne, UK
Wolfgang A. Kunze, PhD Psychiatry and Behavioural Neuroscience, St. Joseph

Healthcare, Hamilton, ON, Canada
xvi xvii
Jennifer S. Labus, PhD Division of Digestive Diseases, Department of Medicine,

Center for Neurobiology of Stress, David Geffen School of Medicine at UCLA, Los
Angeles, CA, USA
Beatriz Lobo, MD, PhD Neuro-Immuno-Gastroenterology Group, Digestive Dis-

eases Research Unit, Gastroenterology Department, Hospital Universitari Vall

Christopher A. Lowry, PhD Department of Integrative Physiology, Center for

Neuroscience, University of Colorado Boulder, Boulder, CO, USA
Mark Lyte, PhD, MS, MT(ASCP) Department of Immunotherapeutics and

Biotechnology, Texas Tech University Health Sciences Center, Abilene, TX, USA
Rachel D. Moloney, PhD Laboratory of NeuroGastroenterology, Alimentary

Pharmabiotic Centre, University College Cork, Cork, Ireland
Department of Psychiatry, University College Cork, Cork, Ireland
Angela Moya-Pérez, BSc Microbial Ecology, Nutrition & Health, National

Research Council (IATA-CSIC), Paterna-Valencia, Spain
Marc Pigrau, MD, PhD Neuro-Immuno-Gastroenterology Group, Digestive


Eamonn M.M. Quigley, MD, FRCP, FACP, FACG, FRCPI Division of Gastro-
enterology and Hepatology, Medicine Department, Houston Methodist Hospital,
Houston, TX, USA
Charles L. Raison, MD Department of Psychiatry, College of Medicine and

Norton School of Family and Consumer Sciences, College of Agriculture and
Life Sciences, University of Arizona, Tucson, AZ, USA
Jens F. Rehfeld, MD, DMSc, DSc Department of Clinical Biochemistry,

Rigshospitalet, University of Copenhagen, Copenhagen, Denmark
Leni R. Rivera, PhD Anatomy and Neuroscience, University of Melbourne,
Graham A.W. Rook, BA, MB, BChir, MD Centre for Clinical Microbiology,
UCL (University College London), London, UK
R. Paul Ross, PhD, DSc Alimentary Pharmabiotic Centre, Teagasc Moorepark

Food Research Centre, Fermoy, Cork, Ireland
Javier Santos, MD, PhD Neuro-Immuno-Gastroenterology Group, Digestive


Yolanda Sanz, PhD Microbial Ecology, Nutrition & Health, National Research
Council (IATA-CSIC), Paterna-Valencia, Spain
Fergus Shanahan, MD, FRCP, FCCP, FRCPI Department of Medicine, Alimen-

tary Pharmabiotic Centre, University College Cork, Cork, Ireland
Catherine Stanton, PhD, DSc Alimentary Pharmabiotic Centre, Teagasc

Moorepark Food Research Centre, Fermoy, Cork, Ireland
Nobuyuki Sudo, MD, PhD Department of Psychosomatic Medicine, Kyushu

University, Fukuoka. Japan
Kirsten Tillisch, MD Division of Digestive Diseases, Department of Medicine,

Center for Neurobiology of Stress, David Geffen School of Medicine at UCLA,
Los Angeles, CA, USA
Mar´ıa Vicario, PhD Neuro-Immuno-Gastroenterology Group, Digestive Diseases

Research Unit, Gastroenterology Department, Hospital Universitari Vall d’Hebron,
Vall d’Hebron Research Institute, Barcelona, Spain

Rebecca Wall, PhD Alimentary Pharmabiotic Centre, Teagasc Moorepark Food

Research Centre, Fermoy, Cork, Ireland
xviii List of Contributors
Part I
Basic Concepts Underlying the
Microbiota-Gut-Brain Axis
Chapter 1
and the Microbiota-Gut-Brain Axis
Mark Lyte
Abstract Microbial endocrinology is defined as the study of the ability of micro-

organisms to both produce and recognize neurochemicals that originate either
within the microorganisms themselves or within the host they inhabit. As such,
microbial endocrinology represents the intersection of the fields of microbiology
and neurobiology. The acquisition of neurochemical-based cell-to-cell signaling
mechanisms in eukaryotic organisms is believed to have been acquired due to late
horizontal gene transfer from prokaryotic microorganisms. When considered in the
context of the microbiota’s ability to influence host behavior, microbial endocrino-
logy with its theoretical basis rooted in shared neuroendocrine signaling mecha-
nisms provides for testable experiments with which to understand the role of the
microbiota in host behavior and as importantly the ability of the host to influence
the microbiota through neuroendocrine-based mechanisms.
Abbreviations
CNS Central nervous system

ENS Enteric nervous system
GABA Gamma aminobutyric acid
Prologue
“If you are right that the bacteria in the gut can communicate with the brain and induce
cognitive behavioral changes such as anxiety, then why aren’t all the patients we give
M. Lyte (*)
Department of Immunotherapeutics and Biotechnology, Texas Tech University Health
Sciences Center, 1718 Pine Street, Abilene, TX 79601, USA
e-mail: mark.lyte@ttuhsc.edu
M. Lyte and J.F. Cryan (eds.), Microbial Endocrinology: The Microbiota-Gut-Brain 3

Axis in Health and Disease, Advances in Experimental Medicine and Biology 817,
DOI 10.1007/978-1-4939-0897-4_1, © Springer New York 2014
4 M. Lyte
antibiotics to in the hospital running around the floors crazy?” NIH Director’s Pioneer
Award Study Section Member—July, 2008
In July 2008 I found myself as a finalist for the coveted NIH Director’s Pioneer
Award being asked that very question following my PowerPoint presentation by a
study section member in front of not only the other assembled study section
members but also the representatives of all the NIH Institutes and the Director’s
office. Earlier that year I had submitted an application for the Pioneer Award
entitled “The Microbial Organ in the Gut” where I proposed that bacteria in the
gut were not only able to communicate with the brain and influence behavior, but
also that the brain could likewise communicate with the gut bacteria to achieve
regulation of microbial populations that would benefit the host. The mechanism by
which this bi-directional communication was governed was proposed to be that of
microbial endocrinology—the ability of bacteria to respond to as well as produce
the same neurohormones found in the host. The study section member’s question of
why people weren’t “running around crazy” was the first one asked following a
short presentation to all present. I had anticipated that questions during the 15 min
following my presentation would be probing given that from hundreds of applicants
for the first round, only 25, including myself, had been selected for a live presen-
tation to a completely new panel of experts at the Lawton Chiles International
House on the NIH campus. I also knew that the presentation would meet with some
skepticism but hadn’t been prepared for the very same study section member
spitting water in a veritable geyser after taking a drink and hearing me say, not
2 min into my talk, that bacteria can communicate with the brain and change
behavior (the incident was witnessed by all in the room for which I did receive a
telephone apology for the member’s behavior weeks later from the Director’s
office). So, the sarcastic, condescending nature of the question came as no surprise.
And it was no surprise that my answer (which in many ways forms the basis of this
chapter) satisfied neither the member nor the rest of the panel and I did not receive
one of the Pioneer Awards that year. But, as they say, times change and science
marches on.
Microbial Endocrinology: Conceptual Framework
Microbial endocrinology represents the intersection of two seemingly disparate

fields, microbiology and neurobiology. The field of microbial endocrinology was
founded in 1993 when the term was first coined by Lyte [1, 2] based on experi-
mental data obtained the prior year [3, 4]. As will be seen in this chapter, although
the concept of microbial endocrinology was founded just two decades ago [1, 3–5],
there has been published evidence by numerous investigators over the preceding six
decades going back to 1930 [6] that demonstrate the validity of uniting the fields of
microbiology and neurobiology as a conceptual framework with which to under-
stand interactions between the microbiota and the host in homeostasis and disease.
1 Microbial Endocrinology and the Microbiota-Gut-Brain Axis 5
That these two fields should intersect and play a role in not only infectious disease,
but also microbiota-gut-brain communication can be best understood when one
considers how the two fields are similar to one another. The presence of neuroen-
docrine hormones that are exactly the same in structure, as well as share the same
biosynthetic pathways, to that found in mammalian systems has been recognized
for decades (for review see [7]). Prominent examples include members of the
catecholamine family that have been found not only in bacteria [8], but in fish
[9], plants [10] and insects [11]. The complete biosynthetic pathway including co-
factors for catecholamines, from tyrosine through epinephrine, is found in
Escherichia coli as well as other bacterial species [12]. Acetylcholine [13], hista-
mine [14], serotonin [15, 16], and even more newly described neurotransmitters
such as agmatine [17–19] have all been shown to be produced by microorganisms.
The spectrum of neuroactive compounds produced by bacteria that can potentially
interact with the host also includes a number of neuropeptides [20]. That many of
the described neurohormones produced by bacteria also function in mammals as
part of the neurophysiological system suggests, as will be discussed in the
succeeding sections, that their production within the mammalian host can impact
the neurophysiological aspects of the host including cognition.
The ubiquitous presence of neuroendocrine hormones in non-mammalian sys-
tems means that the presence of the very same neuroendocrine hormones in
mammalian systems has a long evolutionary shared history. Iyer et al. [12] pro-
posed that acquisition of cell-to-cell signaling systems, such as those that charac-
terize neuroendocrine pathways in mammalian systems, are due to late horizontal
gene transfer from bacteria. The theory that neurochemical signaling in mammalian
cell systems is due to bacterial gene transfer has been bolstered by recent results
from the human microbiome project. Riley et al. [21] have shown that such
bacterial-mammalian cell lateral gene transfer of bacterial DNA into the human
somatic genome occurs via integration of a RNA intermediate and is more common
than previously recognized.
In non-mammalian systems the presence of neuroendocrine hormones often
serves in a similar capacity to that seen in mammals. For example, tomato plants
exposed to various stressors such as cold temperatures can produce large amounts
of stress-related catecholamines. As in mammals [22], stress and the production of
stress-related hormones such as norepinephrine and epinephrine in tomato plants
are also associated with increased susceptibility to infectious agents such as the
plant fungal and bacterial pathogens [23, 24]. Interestingly, in response to an
infectious insult during periods of stress and increased production of catechol-
amines tomato plants produce antimicrobial compounds that use as their backbone
the complete structure of catecholamines such as norepinephrine and dopamine [23,
24]. Whether evolution has afforded other non-plant-based systems a similar way to
deal with stress-induced susceptibility to infectious challenge by constructing
antimicrobial compounds based on neurochemical structures has not yet been
fully examined.
What is still incompletely understood for the majority of bacteria from which
neuroendocrine hormones have been isolated is the simple question of “why”. Why
6 M. Lyte
do bacteria produce neuroendocrine hormones? In large part, most reports of

neurochemical production by bacteria are mainly descriptive and the “why” aspect
is too often left unanswered. However, for some bacterial species which are known
to produce certain neurochemicals via the same mechanism found in animals, such
as gamma aminobutyric acid (GABA) which utilizes α-decarboxylation of
L-glutamic acid catalyzed by glutamate decarboxylase, a reason for its production
has been reported. For example, production of GABA can confer resistance to
acidic pH for a number of Lactobacilli species such as Lactobacillus reuteri [25] as
well as have a role in the germination of bacterial spores [26]. As an acid-protective
mechanism, the GAD system employed by Lactobacilli may offer a sound expla-
nation concerning survival of the bacterium following ingestion and subsequent
transfer through the acidic conditions within the stomach and into the intestine, but
falls short to explain from an evolutionary perspective why Lactobacilli that
normally reside in the gut should possess the biosynthetic pathway to produce
GABA. Nor for the reports that other commensal microbiota such as those belong-
ing to the Clostridia also possess the ability to decarboxylate glutamic acid and
produce GABA [27]. Can it instead be proposed that the production of GABA by
bacteria can also serve as a mechanism by which such bacteria can not only
influence the host through interaction with host cell receptors for GABA that can
be found in the intestinal tract both in neuronal cells that belong to the enteric
nervous system (ENS) [28] as well as immune cells [29], but additionally as a way
by which one bacterial species can communicate with another within the microbiota
that also possesses receptors for GABA? In fact, the presence of a high affinity
receptor for GABA in Pseudomonas spp. had formed the basis for the use of a
bacterial-based system to quantify nanomolar concentrations of GABA in clinical
fluids such as cerebrospinal fluid [30]. The isolation and characterization of the high
affinity receptor for GABA in Pseudomonas was reported a few years later [31].
The concept that the production of neuroactive chemicals by members of the
microbiota can not only serve in the capacity of interacting with the host, but also as
a means of signaling among other members of the microbiota, has been proposed
[32]. Such neurochemical-signaling mechanisms between members of the
microbiota would constitute a type of primitive nervous system and satisfy the
requirements contained with any definition of an organ—namely, that the cellular
elements which comprise the organ can be influenced, and in turn influence, the
host. From a microbial endocrinology-based standpoint the microbiota contained
within the gut can therefore be termed as a microbial organ [32].
Origins of Microbial Endocrinology: Evidence from the 1930s

to Present
Over the last decade the number of reports which have demonstrated the ability of
bacteria to respond to neuroendocrine hormones produced by the host, especially
during times of stress, have steadily increased. The first report that a stress-related
neurochemical could influence bacterial growth appeared in the early 1930s due to
an unfortunate set of occurrences. Epinephrine (adrenaline) as the first hormone
purified to homogeneity was beginning to find increasing use in the clinical arena.
One of those uses was for the treatment of urticaria. Reports began to appear almost
immediately following its use in the clinic of patients dying from fulminating sepsis
within hours after administration of epinephrine [6]. The cause was traced to the
glass syringes and metal needles that pre-dated the modern use of disposable
syringes and needles [33]. Although glass syringes and needles were cleaned with
various agents between patients, it was quickly discovered that such cleaning of a
needle and syringe set used to drain infected abscesses of patients with infections
such as the spore-forming Clostridium perfringens was inadequate. The combina-
tion of epinephrine and the very small number of spores or injured bacteria left in
the syringe and needle proved to be a dangerous combination. Since all patients
who died from epinephrine injections were traced back to syringes and needles that
had been used to drain bacterial abscesses it became standard medical practice for
decades that a syringe and needle set could not be used for epinephrine injections if
it had been recently used to drain a bacterial abscess. Although this association has
been largely lost to history, it should be noted that on occasion such associations
have proved beneficial for the evaluation of drugs to treat infectious bacteria such as
C. perfringens. Traub et al. [34] demonstrated that in order to get C. perfringens to
infect a mouse it was necessary to co-inject fresh, non-oxidized, adrenaline and that
by utilizing such a neuroendocrine hormone-based model system one could eval-
uate the efficacy of antimicrobial candidate drugs to treat gas gangrene infections.
The majority of reports that have dealt with various aspects of neuroendocrine
hormone production by bacteria or their recognition of host-produced hormones
have done so in the context of infectious disease. This is not surprising given the
fact that the first reports of hormones having a role in host health started in the
1930s with the reports of gas gangrene following injection of epinephrine. The first
report that described a direct interaction of bacteria and neuroendocrine hormones
and ascribed a role in infectious disease was the demonstration 60 years later in
1992 that the stress-related neurohormones norepinephrine and dopamine could
increase the growth of human intestinal bacterial pathogens by over six orders of
magnitude within hours [3, 4]. Importantly, intestinal pathogens which are not
commonly associated with extra-intestinal infection, such as Yersinia entero-
colitica, do not respond to the stress hormone epinephrine. This is a critical
observation as it indicates that bacteria may have developed the ability to recognize
host hormones based on evolutionary association with specific anatomical regions
of the host. Reports, such as Sperandio et al. [35], which have subsequently
appeared and suggest that epinephrine plays a key role in the pathogenesis of
bacteria within the gut critically have not recognized (or even ignored) the fact
that epinephrine does not exist in appreciable amounts within the gastrointestinal
tract. This is due to the fact that neurons contained with the enteric nervous
system (ENS) that innervates the entire length of the gut do not possess the enzyme
8 M. Lyte
phenylethanolamine-N-methyltransferase which is needed for conversion of nor-

epinephrine to epinephrine in the catecholamine biosynthetic pathway [36].
As can be expected, the more one digs into the literature to find instances of
where neurochemicals and bacteria have been examined the more one finds papers
which provided tantalizing clues that these two systems, one the neurophysiological
and the other microbial, could interact in totally unexpected ways. For example,
Campylobacter jejuni is a highly prevalent food-borne pathogen that requires a
microaerophilic environment in the laboratory for its propagation. However, the
addition of norepinephrine to the microbiological growth medium was shown by
Bowdre et al. [37] to result in tolerance to and growth of C. jejuni in an aerobic
environment. The mechanisms to account for this have not been elucidated but
further highlight the ability of neuroendocrine hormones to affect bacterial physi-
ology. Along these lines, in the succeeding years since the demonstration of
catecholamine-induced growth of bacteria and increased production of virulence-
associated factors [38, 39], numerous reports have appeared that further document
the ability of neuroendocrine hormones, chiefly the catecholamines, to influence
bacteria. For example, stress-related hormones have been shown to increase
conjugative transfer of antibiotic resistant genes between enteric bacteria thereby
contributing to the increased prevalence of antibiotic-resistant food borne bacterial
pathogens in the food supply [40]. Additionally, the ability of monoamines such as
norepinephrine and dopamine to alter gene expression has now been shown for a
number of pathogenic microorganisms including Mycoplasma hyopneumoniae
[41], Salmonella enterica serovar Typhimurium [42] and Vibrio
parahaemolyticus [43].
Evolution of Current Microbial Endocrinology-Based

Perspective of Microbiota-Gut-Brain Axis
Of specific relevance to the current study of the subject of microbiota-gut-brain axis

was the dominating scientific view of the time that sought to explain the mecha-
nisms by which stress neurohormones could influence the pathogenesis of infec-
tious disease. Miles and colleagues undertook a series of experiments starting in the
late 1940s and continuing into the 1950s in which they co-injected stress hormones
with a wide range of bacterial species into animals [33, 44, 45]. Their findings
corroborated earlier studies that showed that epinephrine had the ability to increase
the growth rate of bacteria, such as C. perfringens (referred to in this series of
papers by the former name C. welchii) and E. coli, while decreasing the dose needed
to cause infection by up to one-million fold [6, 46]. However, all attempts to
identify the involved mechanism(s) had been centered on the host side as it was
not conceived that the bacterium itself could be as active a player in the infectious
disease process as the host and most critically could utilize the host’s own neuro-
endocrine hormone production during stress to identify where it was and initiate
processes to ensure its own survival. The most prevalent reason given by the
researchers during this time to account for the ability of epinephrine to increase
bacterial numbers was that it was due to an inhibition of phagocyte migration into
the area where the bacteria were actively growing thereby allowing them to grow in
an unrestricted manner [33, 44]. However, these researchers had also observed that
epinephrine was principally effective during the early stages of infection when
bacteria were low in number and that the injection of epinephrine later in the
infective process did not appreciably inhibit the response of phagocytic cells.
This seeming contradiction was resolved decades later when it was shown that
the response of bacteria to catecholamines is highest when bacteria are in low
concentration [47, 48] and that as the bacteria increase in density their need for
catecholamines decreases at the same time a catecholamine-induced autoinducer of
growth is produced [48, 49]. The critical distinction between these two research
periods separated by nearly 40 years is the examination of the site of action of
neuroendocrine hormones in a biological system containing both prokaryotic and
eukaryotic cells, wherein during the former period researchers considered that since
neuroendocrine hormones were of mammalian origin they would naturally influ-
ence mammalian, and not prokaryotic, cells as part of the infective process. That
bacteria were known even at that time to produce neurochemicals such as acetyl-
choline [13] did not seem to enter into the infectious disease equation. That there
still is today a similar view that two systems, host and microbial, are separate and
distinct as far as behavior can be regarded is best exemplified by the skepticism
discussed in this chapter’s prologue.
As already partly discussed, there have been numerous reports since the 1930s
regarding the ability of specific bacterial species to produce and/or recognize
through specific receptors neuroendocrine hormones many of which are involved
in key aspects of neurotransmission. One of the most prominent, GABA, has been
extensively described for members of the Lactobacilli family as already discussed
in a previous section as well as for Bifidobacteria [50] and characterization of a high
affinity receptor in Pseudomonas spp. [31]. In the presence of the same substrates
and coenzymes that are found in mammalian cells involved in the production of
GABA, bacterial strains isolated from the human gastrointestinal tract have been
shown to produce over 20,000 μg ml—1 of GABA [50]. Acetylcholine [13], dopa-
mine [8, 51], norepinephrine [8, 51], histamine [14] and even precursors of benzo-
diazepine ligands [52, 53] are just a few of the examples that can be found in the
literature. Roshchina [7] has authored the most extensive review to date regarding
the capacity of bacteria to produce a wide panoply of neuroactive compounds.
Further, while the interaction of neuroendocrine hormones such as the catechol-
amines has most often been examined in bacteria, there have been reports which
demonstrate the utilization of catecholamines by other microorganisms such as the
pathogenic yeast Cryptococcus neoformans [54, 55].
10 M. Lyte
In Vivo Veritas
As noted above, the demonstration that the microbiota itself is capable of producing
neuroendocrine hormones is the crucial first step in evaluating the feasibility of
microbial endocrinology-based mechanisms in gut-to-brain interactions. Although
there have been reports which have concluded that increased neurochemicals found
in the circulation of the host, for example serotonin [56], are due to the presence of
neurochemical secreting bacteria, it has only been very recently that a comprehen-
sive study has conclusively demonstrated the production of physiological levels of
neuroendocrine hormones by bacteria within the intestinal lumen. In this study by
Asano et al. [51], levels of the catecholamines norepinephrine and dopamine were
quantitated in the gastrointestinal lumen in three microbiota-distinct types of mice:
specific pathogen-free, germ-free and gnotobiotic mice reconstituted with a mixture
of various bacterial species. Appreciable physiological amounts of both catechol-
amines were only found in specific pathogen-free mice while substantially lower
amounts were detected in luminal contents of germ-free animals. Critically,
whereas the majority of catecholamines in pathogen-free animals were structurally
determined to be free and biologically active, those found in germ-free animals
were present in a biologically inactive, conjugated form. Inoculation of germ-free
animals with the microbiota from specific pathogen-free mice resulted in the
production of free, biologically active, catecholamines within the gut lumen. As
such, this report [51] clearly established that in vivo the microbiota is capable of
producing neuroendocrine hormones that are commonly only associated with host
production. That these substances also are intimately involved in host neurophys-
iology provides solid evidence that the fields of microbiology and neurophysiology
do intersect with attendant consequences for both host and microbiota as further
discussed below.
Microbiota and Behavior: Does Microbial Endocrinology

Have a Role to Play?
The ability of microbes to influence behavior has been shown in a large number of
studies, many of which are discussed in length in other chapters in this book. What
is at question, however, is whether the ability of microorganisms to produce
neuroactive compounds provide for a mechanism(s) by which such microbial-
induced changes in behavior can be accounted for.
In many of the studies which have addressed mechanisms by which microbes
can influence behavior they have often concluded that such mechanisms involve to
some degree immune system involvement. This is not surprising given that such
studies often involve the administration of a microorganism in a manner that nearly
guarantees an immune system response. Further, microorganisms are often given in
such large doses that do not reflect actual “real-life” scenarios where infective doses
tend to be very low. Following such administration, the development of immune-

related sequelae involving the production and release of cytokines and inflamma-
tory mediators result in the interaction with well-characterized neuronal targets
both within the central nervous system (CNS) and the ENS [57]. These CNS and
ENS targets then communicate to the brain, via vagal afferents for example, and
result in altered behavioral responses.
While the sequence of pathogen infection resulting in immune activation that
then ultimately results in an alteration of behavior is well recognized, it is perhaps
somewhat surprising to learn that increasingly studies are reporting the direct, non-
immune, non-infectious, related ability of microbes to influence behavior. The first
study which demonstrated the ability of a bacterium within the gut to influence
behavior in the absence of any detectable immune response was shown in a series of
studies utilizing C. jejuni in mice [58]. Although an important human food-borne
pathogen, C. jejuni in mice, unlike in humans, does not cause diarrhea. In this series
of studies, a low per oral dose of C. jejuni was employed to introduce a novel,
replicating organism into the microbiota and examine whether this new member
could be “seen” by the brain. As reported in this series of studies, C. jejuni was able
to induce anxiety-like behavior in mice through a vagal-mediated pathway in the
absence of any immune activation [59]. Further, it was shown that within hours
following the introduction of C. jejuni into the microbiota that neuronal activation
in specific brain regions occurred as detected by expression of the neuronal
activation marker c-Fos. It is therefore evident that a mechanism exists whereby
changes in the microbiota can be “seen” by the brain and these changes can result in
modification of behavior. To date, the mechanism(s) by which this non-immune
mediated neuronal activation within the brain occurs has not been identified and
awaits to be explored.
Given that bacteria are prolific producers of neuroendocrine hormones, as well
as other neuroactive compounds [20], it would seem reasonable to conclude that
such bacterial production of neuroactive compounds within the gut lumen could
influence either host-specific neural receptors within the gut or extra-intestinal
neuronal sites following luminal uptake into the portal circulation. There are a
number of reports that provide support that neurochemical production by bacteria
within the gut can influence behavior in both humans and animal model systems
[60–62]. Most often, these reports employ probiotic bacteria, such as Lactobacillus
or Bifidobacterium, many of which species belonging to these two genera are
prolific producers of neurochemicals for which well-defined neural mechanisms
are known by which behavior may be modulated. Of particular interest, Bravo
et al. [61] observed reduced anxiety-like and depressive-like behavior in mice fed
the probiotic strain L. rhamnosus (JB-1). Following probiotic administration they
were able to demonstrate changes in the levels of GABAAα2 mRNA in those brain
regions associated with the specific behavior [61]. Although they did not quantify
the amount of GABA produced by the administered L. rhamnosus (JB-1) strain, the
demonstration of a mechanism, such as that mediated via central GABA receptor
expression, provides evidence that the ability of bacteria to influence behavior can
occur through a neurochemical-mediated route.
12 M. Lyte
And as to whether bacteria are capable of producing enough quantities of

neurochemicals to affect behavior, a recent study which employed the GABA-
producing Lactobacillus brevis FPA 3709 amply demonstrates that ability. In this
functional food study, L. brevis was used to enrich black soybean milk with GABA
which was then fed to rats subjected to a forced swim behavioral test [63]. The
forced swim test, in which animals are placed in a water-containing glass cylinder
and the duration of immobility before the animals begin to swim is measured, is a
well-recognized test of depressive-like behavior. In this study, it was shown that
GABA-enriched soybean milk significantly reduced the immobility time before rats
began to swim and was as effective as the selective serotonin reuptake inhibitor
fluoxetine as an antidepressant [63].
Experimental Challenges
While the studies described above do provide tantalizing evidence that microbial
endocrinology does indeed play a role in microbiota-gut-brain interactions that
ultimately culminate in changes in behavior, a number of experimental challenges
have yet to be addressed. To date, substantial direct cause and effect evidence to
support such a microbial endocrinology-based mechanism is still lacking. The
reasons for this are many-fold and include the only recent development of the
necessary analytical tools both on the microbiome as well as neuroimaging sides to
examine such interactions. However, the larger reason may be due to the experi-
mental rigor that must be employed to unequivocally demonstrate that it is the
actual production of a neurochemical in vivo by a specific microorganism, and not a
non-neurochemical aspect of the microorganism such as a cell wall component
interacting with immune cells in the gut, that is responsible for a specific change in
behavior. Further, receptor specific binding within the gut or extra-intestinal site
must be demonstrated for the specific neurochemical produced by the microorgan-
ism. These are only two, of a number of requirements that must be fulfilled for one
to conclude that a microbial endocrinology-based mechanism can be responsible
for a specific change in host behavior. Recently, a step-by-step experimental
approach was introduced to guide the experimental design for probiotics which
seek to examine such microbial endocrinology-based mechanisms [64]. As shown
in Table 1.1, a sequential research plan is proposed which combines in vitro and
in vivo methodologies to specifically demonstrate that a specific neurochemical
produced in vivo by the microorganism binds to a specific host receptor which
ultimately results in an alteration of behavior/cognition in the host. The use of
microorganisms that only produce one type of neurochemical is preferred as a
number of bacterial strains have been shown to produce more than one neurochem-
ical. For example, production of acetylcholine and GABA by certain Lactobacillus
has been reported [7]. Other considerations, which are more extensively covered in
hypothetical papers addressing the role of the microbiota in nutrition and appetite
[65, 66], cover aspects such as ensuring that the diet contains the neurochemical
Table 1.1 Sequential design to evaluate ability of neurochemical-producing probiotics to influ-

ence disease pathobiology
Step Comments
Identify neurochemical of interest to be pro- Physiological and/or behavioral measures
duced by probiotic based on desired physi- should be readily quantifiable. Measures that
ological and/or behavioral effect in host. are receptor-based with known antagonists
readily available are preferred as can subse-
quently be employed at in vivo steps
involving animal models.
Screen candidate probiotic in vitro for neuro- An example of a metabolomics-based screen is
chemical production using robust assay to given in [64]. More than one microbiologi-
determine if neurochemical of interest as cal growth medium should be used. Prefer-
well as other neurochemicals are produced. ably a medium that reflects the gut
environment should also be employed.
Define kinetics (i.e. time dependent achievable Identify in vitro growth conditions which result
intra- and extra-cellular concentrations) of in sustained levels of neurochemical pro-
neurochemical production. duction throughout growth period.
Obtain non-producer mutant (either through A mutant that does not produce the neuro-
in vitro screening or site-directed mutagen- chemical will provide critical control for
esis procedure). in vivo experiments.
Conduct time and dose-dependent per oral Measure levels of neurochemical of interest in
administration of neurochemical-producing intestinal luminal fluid and plasma. Deter-
probiotic to normal animals to determine mine time-dependent colonization of gut
ability of probiotic to produce neurochemi- tissue using quantitative PCR. Perform gross
cal in vivo. Employ vehicle—only animals pathology and immunohistopathology of
as control. relevant tissue and compare to control
(vehicle only) animals.
Perform per oral administration of probiotic in Animal models of specific disease pathology or
an animal model which involves a behavior are suitable candidates. Select
neurochemical-responsive element. dosage of neurochemical-secreting probiotic
from prior step that is found to result in high
and sustainable levels of neurochemical
within the gut. If known receptor antagonists
are available, give antagonist to block
neurochemical-responsive element of dis-
ease or behavioral process.
Perform control experiments utilizing per oral Quantifiable changes in animal model that are
administration of mutant (non-neuro- obtained by administration of
chemical-secreting) probiotic. neurochemical-secreting probiotic in above
step should not be present (or at lower
levels) with mutant strain.
From Lyte M. Probiotics function mechanistically as delivery vehicles for neuroactive com-
pounds: Microbial endocrinology in the design and use of probiotics. Bioessays. 2011;33
(8):574–581. Reprinted with permission from John Wiley & Sons.
precursors that are needed as substrates in the synthesis of the specific

neurochemical.
The steps outlined in Table 1.1 present a sizable research hurdle to overcome to
unequivocally demonstrate the validity of microbial endocrinology-based mecha-
nisms in the microbiota-gut-brain axis. While the research task of identifying
14 M. Lyte
microbial endocrinology-based mechanisms as regards a known neurochemical-

producing microorganism is rather straightforward, unequivocally demonstrating
such changes within the microbiota itself (i.e. identifying members that may be
responsible for such production and the neurochemicals themselves) presents
another level of difficulty. Such an approach would undoubtedly involve the use
of metabolomics to demonstrate that the microbiota is capable of producing
neuroactive compounds during various physiological conditions whether it be stress
or changes in diet that may or may not possess the necessary substrates for members
of the microbiota to synthesize the candidate neurochemicals.
Differentiation of host versus microbiota produced neurochemicals will, of
course, be an essential first step. Recently, Matsumoto et al. [67] have published
an elegant study of the mouse intestinal metabolome in which they analyzed the
metabolome from the intestine, food and host compartments thereby providing a
roadmap by which to differentiate the contribution of each compartment to the
overall metabolome within the gut. The finding of neurochemical production
exclusively within the microbiota raises the question of which member(s) are
responsible. It will be necessary, then, to employ a functional genomics approach
to sift through the genomic data obtained from the microbiota community analyzed
from the same sample that the metabolome was obtained from to identify those
members that possess the genes necessary for the biosynthetic pathways required
for the production of the neurochemical of interest. Such a functional
metagenomics-based approach has found increasing utility in applications such as
human nutrition where elucidating the roles that the human and microbial genomes
play in nutrition, starting with microbial biotransformation of food specific to the
microbiome of a particular individual and the end products that are produced that
impact human physiology for that particular individual, have been examined [68–
70]. Once a metagenomics-based approach can identify the bacterial populations
that do account for production of a particular neurochemical, it will then be
necessary to isolate that population and attempt to culture in vitro to determine if
the neurochemical in culture is in fact produced and the relative capacity for
production under varying substrate conditions. The design and use of
physiologically-relevant intestinal medium complete with the same substrates that
are present in the diet of the individual from which the bacterial population was
isolated will be crucial to the evaluation. Utilization of standard microbiological
medium without requisite concern regarding physiological relevance nor the con-
tribution of food components for substrates and co-enzymes needed for production
of a particular neurochemical will most likely lead to non-relevant results. As
shown in Fig. 1.1, food itself can contain both the substrates for neurochemicals
as well as neurochemicals themselves (such as histamine) and thus plays a critical
role in the ability of the microbiota to function in a microbial endocrinology-based
manner. Once the identification of the neurochemical-producing population has
been achieved, the remaining steps to demonstrate microbial endocrinology-based
mechanisms(s) mediating gut-to-brain changes in behavior in the normal host
microbiota would then closely follow the steps outlined in Table 1.1.
Fig. 1.1 The microbial endocrinology-based pathways by which neuroactive compounds pro-
duced by both the host and the microbiota can serve as a mechanism by which the brain and
behavior can be modulated within the microbiota-gut-brain axis. Food ingested by the host
contains both the substrates needed for neurochemical production by the host and the microbiota
as well as fully functional neuroactive components ①. The microbiota in the gut is capable of
either forming neurochemicals from the substrates present in the ingested food; or responding to
the neuroactive food components themselves; or responding to neurochemicals secreted into the
gut by components of the host enteric nervous system ②. Neurochemicals produced by the
microbiota in the gut have two pathways by which to influence the host; they can either be
taken up from the gut into the portal circulation ③ or they can directly interact with receptors
found on components of the enteric nervous system which innervates the complete length of the
gastrointestinal tract ②. Once in the portal circulation, microbiota-derived neurochemicals can
influence components of the nervous system and ultimately the brain ④. Microbiota-derived
neurochemicals can also influence components of the nervous system such as the brain through
enteric nervous system-central nervous system communication ⑤. The result of either pathway ④
or ⑤ on the brain may result in an alteration of behavior or cognition ⑥ as well as food
preferences and appetite ⑦. As described in the text, this should not be viewed as a one-way
direction of only gut-to-brain since the brain may influence the composition of the microbiota
through the specific release of neurochemicals into the gut lumen ②. From Lyte M. Microbial
endocrinology: Host-microbiota neuroendocrine interactions influencing brain and behavior. Gut
Microbes. 2014. Reprinted with permission [87]
Methodological Approach to Examining Putative

Neurochemical-Microbiota Interactions
Experimental design employed in both past and present studies involving the
examination of neurochemicals and bacteria, whether to investigate the possible
production of neuroactive compounds or test the effects of neurochemicals on
various aspects of bacterial physiology, have done so in medium that is not
reflective of the in vivo environment from which the bacteria were originally
isolated. For example, a study by Parr et al. [71] which evaluated the ability of
epinephrine to influence the growth of a number of bacterial pathogens responsible
16 M. Lyte
for nosocomial wound infections utilized the standard rich microbiological medium
such as Mueller-Hinton and not surprisingly concluded that there was no effect on
growth. When one starts from the point that the bacterium can already achieve
maximal growth in the test medium, then addition of other substances such as
neurochemicals can only affect growth in one direction, negatively. The use of a
medium more relative to the tissue environment present in wound infections with its
array of inhibiting substances such as transferrin and other specific and non-specific
immune components would have been more appropriate. Other studies which have
used more relevant medium have in fact shown that Staphylococci can dramatically
increase their rate of growth in the presence of catecholamines and catecholamine
inotropes used clinically in the maintenance of cardiac and kidney function such as
dopamine and dobutamine [72, 73]. As such, these findings are more in agreement
with the historical clinical-based literature discussed previously on the association
of catecholamines and infection.
In designing an experiment to test the ability of a bacterium to produce a putative
neuroactive substance in vivo that may then have a role in modulation of host
behavior or even immune function within the gut, the use of a medium which
incorporates in elements of the diet that the host consumes is essential. It is well
recognized (as already discussed) that the exact same biochemical pathway utilized
by eukaryotic cells to synthesis neuroactive compounds are also used by a number
of microorganisms. For the microbiota then, as well as for the host cells, the
substrates that are required are often dietary components. With that said, it is easily
recognized that the diets of most individuals can vary greatly thereby confounding
efforts to understand what neuroactive compounds may be produced in the
microbiota at any given time. Use of laboratory animals with standard diets only
offers a modicum of more control since such diets are not routinely analyzed for
substrates that may be used in the production of neurochemicals.
Impact of Diet on Determining a Role for Microbial

Endocrinology in Gut-to-Brain Communication
As discussed above, the role of diet in the ability of members of the microbiota to
produce and respond to neurochemicals is one that is often overlooked in experi-
mental design. Further complicating experimental design in animals such as rodents
is the fact that most diets are composed of plant-based materials. Since plants
themselves can produce a wide spectrum of neurochemicals as part of their own
intercellular communication and known to have neuronal function in animals [74],
it should be expected that both substrates for any putative bacterial produced
neurochemical within the microbiota may also be contained within the diet itself
being of plant origin. According to standard laboratory practice in the use of
neurochemicals, this should not be a problem since exposure to air and heating
during the preparation of diets and their formation into pellets would result in the
oxidation of most neurochemicals present in the diet. However, that would be an

incorrect assumption since neurochemicals found in high concentrations in some
plants, such as L-dopa, that are easily rendered inactive by oxidation and heating,
are hardly altered following treatment in harsh conditions such as autoclaving as
long as they are associated within a food matrix [75]. As discussed above,
Matsumoto et al. [67] have provided a technical roadmap by which the chemical
composition of the diet or diet metabolome and the metabolome of the lumen can be
simultaneously analyzed and those neurochemicals which are specific to the diet
versus from the host or microbial origin can be separated out.
As noted above, nutrition plays a significant role in shaping the microbiota [68–
70]. One of the first studies which examined the ability of diet-induced changes to
influence the microbiota and in turn influence cognition involved the feeding of a
meat-based diet to rodents [76]. In this study, mice were fed either standard rodent
chow or chow containing 50 % lean ground beef for up to 3 months. Diet-induced
changes in the microbiota revealed higher bacterial diversity in the animals which
consumed a beef-supplemented diet. Interestingly, assessment of animals for mem-
ory and learning using a hole-board open field apparatus demonstrated that
increased bacterial diversity in beef-fed animals correlated in a positive manner
with improved working and reference memory [76]. While this study provided the
first, albeit correlational, data to suggest that the composition of the microbiota may
have a role to play in memory and learning, a potential confounder for this study
[76], as well as for any other diet-based study, is the presence of nutritive or non-
nutritive elements within any diet that may also influence cognition irrespective of
any effects on the microbiota. While the two diets in the Li et al. [76] study
were balanced for a large number of these factors besides simple calories, it still
remains a potential confounder that needs to be recognized and addressed.
Location, Location, Location
In proposing that a microbial endocrinology-based mechanism is involved in the

ability of the microbiota to influence behavior the issue of the spatial juxtaposition
of the microbiota with the host neuroanatomy presents itself. The innervation by the
CNS and the ENS is extensive along the gastrointestinal tract [36, 77]. What is often
not fully appreciated it that the ENS does not uniformly innervate the intestinal
tract. Anatomical sections of the gut are differentially innervated by CNS and ENS
components with direct gut-to-brain neural-based communication dependent on the
specific anatomical region of the gut. In a similar fashion the microbiota is also not
uniform throughout the length of the gastrointestinal tract and as such it cannot be
assumed that one anatomical region of the gut possesses the same capacity to
produce neuroactive compounds as another region that even may be immediately
adjacent to one another. As such, understanding the location of the neuroactive-
producing members of the microbiota in relation to the neuronal elements that can
18 M. Lyte
communicate to the brain will be critically important if the microbiota can be

shown to have a role in determining behavior through gut-to-brain communication.
For example, could microbiota-induced neuronal activation within the brain
resulting in a quantifiable behavior be traced to a specific bacterial species that
inhabits a mucus layer immediately adjacent to a specific part of the gut from which
sensory information obtained by ENS elements travels to the CNS via extrinsic
primary afferent neurons that track along either vagal or spinal afferent routes? Can
we distinguish that from bacteria that specifically inhabit the proximal gut instead
of the distal gut where communication to the brain in that region occurs instead via
the vagus nerve? Use of multiple techniques, such as MALDI-MS image analysis of
tissue sections to demonstrate specific production of the neuroactive compounds by
the microbiota in specific anatomical regions [78], will be needed to definitively
demonstrate that microbial endocrinology-based mechanisms account for the abil-
ity of the microbiota to influence behavior.
And, it should be noted that question of location doesn’t necessarily diminish in
any way the ability of the microbiota to directly interact with extra-intestinal
neuronal elements of the CNS (effectively bypassing the ENS) and influence
behavior through the direct uptake by the host of microbially-produced neurochem-
icals within the microbiota into the systemic circulation.
Two-Way Street
The phrase “microbiota-gut-brain axis” is often mistakenly interpreted as a one-

way street—that is, communication principally in the direction of gut to brain.
While numerous reviews have emphasized the bi-directional nature of gut- to-
brain communication [79–83], the consideration of microorganisms as neuro-
chemical producers that also possess cognate high-affinity receptors, means that the
microbiota is responsive to signals from the brain to the gut and as such can alter its
function and composition in response to host-originated neurochemical signals.
One of the first demonstrations that host derived neuroendocrine hormones could
radically alter the composition of the microbiota was the observation that the
systemic wide release of catecholamines following the administration of the adren-
ergic neurotoxin 6-hydroxydopamine resulted in the shifting of the gut bacterial
populations from predominantly Gram-positive to Gram-negative with a nearly
7 log-fold increase in numbers of E. coli within 24 h following neurotoxin admin-
istration [84]. That signaling from the host to the microbiota is a determining
factor in the composition of the microbiota was further observed as the adrenergic
nerves within the gut re-healed over the ensuing 14 day post-neurotoxin adminis-
tration, the distribution of Gram-positive and Gram-negative bacteria within the gut
returned to the normal pre-neurotoxin distribution [84]. More recent work by Bailey
et al. [85] have shown that the application of social stressor altered the bacterial
population in the intestine with decreased amounts of bacteria in the genus
Bacteroides while at the same time numbers of bacteria in the genus Clostridia
increased.
The concept of a two-way street whereby elaboration of neuroendocrine hor-
mones by the host affects the community structure of the microbiota can be applied
to the clinical arena. For example, antibiotic associated diarrhea is a well-
recognized complication following the administration of wide-spectrum antimicro-
bials [86]. An unanswered question in gastroenterology is the identity of the
mechanism(s) by which the microbiota is able to reconstitute itself following the
cessation of antimicrobial treatment to the same community structure that existed
prior to the administration of antimicrobials [86]. Can release of neuroactive
compounds (hormones, peptides, etc.) by host elements within the gastrointestinal
tract, such as enterochromaffin cells which secrete serotonin or release of other
substances from neural elements that innervate the villi and crypts along the
intestinal wall, specifically stimulate those populations of microbes to grow that
are the same populations that are most beneficial to the host? And can this brain-
gut-microbiota direction essentially re-populate the gut with the pre-antimicrobial
microbial community structure? The long evolutionary symbiosis between host and
the microbial inhabitants in the gastrointestinal tract necessitate that the host’s
nervous system must have developed the means by which to not only monitor,
but also influence the composition of the microorganisms within [32]. This recog-
nition of such active monitoring by the host also implies that certain
gastrointestinal-related clinical conditions, in which the microbiota is intimately
involved such as antibiotic-associated diarrhea, can be viewed anew and hopefully
lead to new therapeutic approaches.
Concluding Thoughts: Speculation into the Unknown

of the Gut-Microbiota-Brain Axis
The unequivocal demonstration that microbial endocrinology-based mechanisms

are prime mediators of microbiota-gut-brain interactions has not yet been achieved.
While there are a number of studies which provide indirect evidence that such
mechanisms are indeed operative in the ability of the microbiota to influence
behavior, mechanisms based on the production of neuroactive compounds by the
microbiota will still need to fulfill all the steps outlined in Table 1.1. The highly
interactive (and complex) network of interactions with which the microbiota can
interface with the host as shown in Fig. 1.1 provides for a number of varied research
approaches. With that said the microbiota contains the capacity to both produce and
recognize neuroactive compounds that are recognized by most researchers to be
solely associated with a mammalian nervous system. Evolution has insured that the
microbiota possesses such neuroactive capacity and if the increasing demonstration
of the role of such microbial endocrinology-based mechanisms in the pathogenesis
20 M. Lyte
of infectious disease is any indication, a role in microbiota-gut-brain communi-

cation will also be demonstrated.
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10.4161/gmic.28682
Chapter 2
Utilizing “Omics” Tools to Study
the Complex Gut Ecosystem
Anthony Fodor
Abstract In a healthy gut, the immune system tolerates a diverse microbial

commensal community avoiding inappropriate inflammation responses and mini-
mizing the presence of pathogens. When the balance between host and microbes is
disrupted, risk for disease increases. There is mounting evidence that microbial
dysbiosis is a substantial risk factor for common gut diseases including IBS, IBD
and colorectal cancer. Understanding this dysbiosis is challenging because of the
extraordinary complexity of the gut ecosystem and the tremendous variability
between healthy individuals in the taxa that make up the human microbiome.
Advances in technology, especially sequencing technology, are beginning to
allow for a full description of this complexity. In this review, we consider how
new “omics” technology can be applied to the study of the gut ecosystem in human
and animal models with special consideration given to factors that should be
considered in the design of experiments and clinical trials.
Abbreviations
ATP Adenosine triphosphate

cDNA Complementary DNA
DNA Deoxyribonucleic acid
FDR False Discovery Rate
IBD Inflammatory Bowel Disease
IBS Irritable Bowel Syndrome
A. Fodor (*)
Bioinformatics and Genomics, UNC Charlotte, 9201 University City BLVD, Charlotte, NC
28223, USA
e-mail: afodor@uncc.edu

26 A. Fodor
OUT Operational Taxonomic Units

PCR Polymerase chain reaction
RNA Ribonucleic acid
rRNA Ribosomal ribonucleic acid
Introduction
In the healthy gut, host immune processes tolerate a diverse commensal population
avoiding excessive inflammation responses and minimizing the presence of path-
ogens. However, there is compelling evidence that microbial dysbiosis—an imbal-
ance in the microbial community—plays a formative role in many diseases of the
gut including IBD [1], IBS [2, 3] and colorectal cancer [4, 5]. All gut microbes are
acquired from the external environment and for the first 3 years of life the diversity
and complexity of the gut microbial community steadily increases [6]. By the 3rd
year of life, the microbial community is more stable but numerous studies have
repeatedly shown that there is a high degree of individual variation in the microbial
community between different people [7–13]. The factors that determine why
different people end up with such different microbial communities are poorly
understood, although twin studies suggest that host genetics does not exert sub-
stantial control over the composition of the microbial community [14].
If we are to understand how host and microbes together produce the full
spectrum of health and disease phenotypes, we will need to determine which alleles
are represented and expressed in the host, which microbes are present and where in
the gut microenvironment the microbes are found and, for both host and microbes,
how genes are expressed to produce metabolites within activated pathways. To
understand the state of the human and microbial ecosystem in the gut, therefore,
requires an accounting of an ecosystem of phenomenal complexity. There are on
the order of three billion base pairs in the human genome [15], but there are ~10
times more bacterial cells within the human body than bacterial cells [16] and
encoded within the genomes of those microbial cells is likely more than 100 times
more distinct genes than are encoded within the human genome [17]. And, of
course, only knowing the genome sequence of either host or microbes by itself
does not tell us which genes are expressed or where or when or how epigenetic
changes to genomes influence pathway structure and function. Within the last
decade, there has been explosive growth in “omics” technologies that are allowing
us to begin to approach an initial accounting of this tremendous complexity.
Development of these technologies have primarily, but not exclusively, been driven
by the stunning drop in the cost of DNA sequencing. Only 10 years ago, the cost of
sequencing a megabase of DNA was well over $1,000. Today, it is less than $0.10
and there is every reason to think that this greater than exponential drop in cost of
sequencing will continue into the future (http://www.genome.gov/sequencingcosts/).
Newly armed with ever more affordable sequencing technology, biologists have
begun to characterize in detail the complex microbial gut environment. In this
2 Utilizing “Omics” Tools to Study the Complex Gut Ecosystem 27
review, we will discuss the technologies that are making this exploration possible
together with the experimental and bioinformatics challenges inherent to performing
studies that try to link the state of the microbial community to host disease
phenotypes.
16S Sequencing Is an Economical Way to Ask “Who

Is There” for Both Common and Rare Taxa
For nearly 30 years [18], microbial ecologists have been using sequencing of the
16S rRNA gene to ask which microbes are present in complex microbial environ-
ments. The 16S rRNA gene is among the most conserved genes in bacterial
genomes. It is especially useful for phylogenetic characterization because it con-
sists of a number of “variable regions”, which tend to be different in different
bacteria, separated by “conserved regions”, which tend to be the same across a wide
phylogenetic space. The conserved regions can be used to place PCR primers that
sequence across the variable regions, yielding a surprisingly informative degree of
phylogenetic information from minimal sequencing effort. Before the advent of
next-generation sequencing, capillary-based Sanger sequencing was often
performed on clone-libraries created from the 16S gene. With a read length on
the order of 1,000 basepairs, a paired-end Sanger sequencing strategy could
sequence the entire 16S rRNA gene. This approach has been widely utilized and
successfully generated descriptions of microbial communities both associated with
the human microbiome [19, 20] and external environmental microbial communities
such as soil and ocean.
Despite these successes, the cloning approach suffers from several limitations.
Because sequences generated from clone libraries are relatively difficult and
expensive to generate, studies that characterized microbial communities via
sequencing of clone libraries generally could only achieve on the order of
100 16S sequences per sample, and only then with a great deal of expense and
effort. Next generation sequencing eliminated the need for the laborious cloning
step even as it offered nucleotide base costs that were orders of magnitude cheaper
than Sanger sequencing. Next generation sequencing platforms exploit massively
parallel chemistry in which numerous sequencing reactions are run at the same time
and the results captured with a computer camera. Because many sequencing
reactions are run in parallel, next generation sequencing platforms such as Illumina
and 454 generate sequences much more quickly than older dye-termination based
technologies. In 2005, the year in which the 454 sequencing platform was described
in a Nature paper [21], there were ~136,000 16S sequences cataloged in the
Ribosomal Database Project (http://rdp.cme.msu.edu/download/posters/
ASM2005.pdf). Today, using the Illumina HiSeq platform, we can routinely gen-
erate 100 million 16S sequences for a cost of only a few thousand dollars [4, 22, 23].
This ability to generate with next generation sequencing in a single experiment
more sequences than had been accumulated world-wide in decades of dye
28 A. Fodor
termination sequencing provides an enormous opportunity to interrogate complex

ecosystems, such as the human gut, while maintaining a sensitivity to detect even
rare taxa. But it brings with it significant bioinformatics challenges. Some of these
challenges involve finding the hard-disk space and network capacity to handle these
large volumes of sequence data. Without proper planning for these mundane
considerations, it is not uncommon for the initial analysis of metagenomics projects
to be severely impacted. There has been considerable recent interest in developing
cloud computing capacity to handle these challenges [24] and investigators consid-
ering generation of large sequence datasets may wish to explore storing and
analyzing their data in the cloud [25].
Bioinformatics challenges can also arise from the short read length inherent to
the currently popular next-generation platforms. The early 454 platforms had a read
length of only ~100 basepairs [26] and the initial 454 pyrosequencing character-
izations of ocean microbial communities therefore utilized this read-length [27,
28]. Recent Illumina platforms, while many orders of magnitude cheaper than
454 sequencing, also have a read length of only 100 basepairs, but 16S sequences
of this length can clearly distinguish the microbial community in inflamed and
non-inflamed mammalian guts [4] showing the utility of even such short reads.
Bioinformatics simulation studies have shown that the information that is available
in short reads can be reasonably close to the information available in full length
sequences [29], with the V1–V3 and V3–V5 regions of the 16S rRNA considered to
be especially appropriate targets given read-lengths of a few hundred base pairs [30,
31] such as are now achievable on 454 and Illumina platforms [26].
The ability to use 16S rRNA sequencing to characterize in-depth the microbial
community from cohorts of interest allows for the intersection of phylogeny and
traditional hypothesis testing in ways that can yield interesting insights into how the
microbial community might impact disease. As an example, a recent study used
454 sequencing of 16S rRNA amplicons to compare the microbial community in
punch biopsies taken from 33 subjects with colorectal adenomas and 38 control
subjects [12]. In total, slightly more than half a million 16S rRNA sequences with
an average length of just over 300 basepairs were generated from these 71 subjects.
In order to place the information in these sequences into a phylogenetic context, we
can build a tree that shows the relationship of the sequences to one another
(Fig. 2.1). Each node of the tree represents a cluster of sequences that have on
average 97 % identity to one another. Nodes of the tree that are close to one another
have sequences that are more similar while nodes that are further from each other.
As we would expect, most of the bacteria that we see in the human gut can be
assigned to the phyla Bacteroidetes or Firmicutes, although other phyla, notably
Proteobacteria which harbors many known pathogens, are also present. For each
taxa in the tree, we can form a null hypothesis that the relative abundance of that
taxa is not different in the case and control subjects. P-values can be generated for
each null hypothesis using a non-parametric Wilcoxon test. In setting thresholds for
significance, we must be careful to correct for testing multiple hypotheses. Rather
than using a simple-threshold of p < 0.05, we instead set a threshold based on a
10 % false discovery rate (FDR), where we expect 10 % of the taxa that we call
Fig. 2.1 Phylogenetic tree

generated from Operational
Taxonomic Units (OTUs)
representing clusters of
sequences with an average
97 % identity from a study
of colorectal adenomas in
humans [12]. Branches that
are colored red represent
taxa that are significantly
different between cases and
controls at a 10 % False
Discovery Rate (see [12] for
methodological details)
significantly different to be false positives. Each taxa that was found to be signif-
icantly different between case (adenomas) and control at this threshold is colored
red in Fig. 2.1. We see that many of the taxa that are different between case and
control are in the phyla Proteobacteria. By creating a visualization that merges
phylogeny with canonical hypothesis testing, we are therefore able to begin to
implicate specific groups of taxa in disease (see [12] for more information).
Given that early 16S sequencing experiments based on clone libraries could be
performed generating less than a hundred reads per sample, it may seem foolish to
plan 16S experiments with read depths of over a million sequences per sample. But
a simple thought experiment shows that such sequence depths are not inappropriate.
Consider E. coli, which is a possible driver of colorectal cancer in mouse and
human studies [4] but in fecal samples can represent less than 1 % of all sequences
collected. On average 100 sequences must be obtained to observe 1 sequence that
represents such a rare taxon. If one wishes to study a population with a 1,000-fold
range in such a taxon, one must utilize an additional 1,000-fold sequencing depth in
order to maintain the full dynamic, quantitative range of sensitivity across people
with different relative abundances of the taxon. Finally, in utilizing 454 and
Illumina sequences, a barcode method is used in which many samples are put
together on the same sequencing run [32, 33]. This procedure can easily introduce
a tenfold variation in how many sequences are collected per sample. Putting this
together—two orders of magnitude to detect a taxa at average 1 % abundance times
three orders of magnitude variation in that taxa between people of different
phenotypes times one order of magnitude technical variation in the number of
sequences collected per sample—we see that it is not unreasonable to produce
and analyze one million 16S sequences per sample.
30 A. Fodor
As technology continues to develop, both read length and read depth will
improve allowing for more information to be generated from each sample but
also increasing the challenges associated with managing and interpreting so much
data. Besides taxonomical considerations, there are many other challenges to the
analysis of 16S sequence data including quality assurance steps [34], choosing
appropriate clustering algorithms [35] and chimera detection [36]. The setting up of
analysis pipelines for 16S sequences has been reviewed elsewhere [37].
Individual Variation Is a Primary Challenge for Studies

in the Gut Micobiome
While there are millions of SNP variations between any two non-twin individuals,
human genomes have many essential common features that all healthy individuals
must share. Every person has to have a working copy of an actin gene, for example,
or survival will be impossible. By contrast, the structure of the microbiome does not
appear to be essential in the same way. As we will discuss below, mice can be raised
in a sterile environment with no gut microbes whatsoever, and while these mice
have a great range of phenotypic differences from control mice, they are able to
survive [38]. The mammalian gut, therefore, appears to have a certain amount of
flexibility with regards to the microbiome. This may explain why, at least at the taxa
level as measured by 16S rRNA, a high degree of variability is tolerated in the
human microbiome. Perhaps the most dramatic example of microbiome variation
was demonstrated by the Human Microbiome Project, which recruited 242 healthy
patients and characterized the microbiome by 16S sequencing at 18 distinct body
sites [8]. At all the measured body sites, there were tremendous individual differ-
ences in this healthy cohort [7, 10]. Moreover, within this cohort, associations
between individual taxa and host phenotypes were generally modest. While there
were taxa with reasonably strong associations with ethnicity and, as previously
observed [39] vaginal pH, associations with phenotypes such as BMI, gender,
temperature and blood pressure were moderate at best [10]. It is currently not
well understood to what extent the differences in the microbiome associated with
ethnicity are driven by genetic or cultural differences, but the possibility of micro-
bial variability produced by ethnicity should be explicitly considered in recruiting
cohorts for and powering clinical studies. In general, the modest correlations
between healthy human phenotypic variation and microbiome variation suggest
that many non-pathological phenotypes are not directly controlled by which taxa
are present in the microbiome.
The complexity, individual variation and weak association with phenotypes of
the healthy human microbiome represent a substantial challenge for studies that
hope to link the state of the microbiome to human phenotypes. If we each have our
own unique relationship to the microbiome that defines our own individualized
healthy or dysbiotic state, then cross-sectional studies that look across people will
have substantial difficulty in coming to any consistent conclusion. One intriguing

idea that has been proposed as a framework to deal with this complexity is
enterotypes [40, 41]; it has been argued that much of the complexity of the gut
microbiome could be summarized by two or three types of categories dominated by
distinct taxa. This hypothesis is enormously appealing as clinical studies could
dramatically reduce complexity (and hence improve power) by assigning each
participant to one of these pre-defined types before attempting to associate the
state of the microbial community to disease phenotypes. Unfortunately, subsequent
studies have demonstrated that the presence of enterotypes appears to rely on
particular methods of analyzing 16S rRNA data and does not therefore appear to
be robust and reproducible in new cohorts [10, 13, 42, 43]. The idea of distinct
microbial types likely makes sense for the low-diversity vaginal microbiome [10,
39], but for the more complex gut microenvironment, there appears to be more
evidence for a continuum of microbes rather that distinct types.
The variety of gut microbes that will be encountered, and the possibility of only
weak associations of taxa with phenotype, must be explicitly considered when
powering clinical studies of the human gut microbiome. One approach that may
help ease power concerns is to design studies around longitudinal sampling. In a
longitudinal sample, each patient in some sense can serve as their own control,
which has the potential to reduce variance and hence increase power. Ideally, a
longitudinal sampling scheme would recruit a cohort before disease developed and
then follow the cohort as some individuals developed disease and others remained
healthy. The analysis can then ask both whether the initial state of the microbial
community predicted disease and whether changes to the microbial community
differ between those who remain healthy and those who develop disease. While this
approach is often optimal from the perspective of experimental design, it can be
difficult to achieve in practice, especially if the time required to follow a cohort is
longer that the length of grant support from funding agencies interested in gut
disease.
The Fecal and Mucosal Microbiomes Are Distinct
One great challenge of surveying the gut microbiome, as opposed to more external
microbiota such as skin, is that often the microbes that we are most interested in are
not the easiest to sample. Fecal samples, obviously, are relatively easy to obtain, but
their handling and storage can provide challenging from an operations point of
view. Fortunately, it has been demonstrated that issues with how fecal samples are
handled, for example how quickly they are frozen, does not appear to have a large
effect on the measured 16S community [44]. As an alternative to fecal samples,
there has been some interest in utilizing fecal swabs [45], which are easier to collect
and store and in the future may be a standard implementation for large clinical
studies. No matter how they are collected, however, fecal samples may be inap-
propriate for studies that evaluate hypotheses regarding the mucosal microbiota.
32 A. Fodor
For example, a recent paper has suggested that microbial DNA may be more present
in cancer samples than in non-cancer [46]. Presumably, the microbial invasion that
would explain this observation is more likely to occur in the tight contact of host
and microbial cells in mucosal material than in the luminal gut. In both human and
mouse microbiomes, mucosal and fecal microbiomes generally cluster separately
[20] suggesting that there are very distinct luminal and mucosal microbial commu-
nities. Obviously, with humans, directly sampling the mucosal microbiota requires
an invasive sampling scheme and produces additional IRB requirements, although
this collection of internal gut samples can be incorporated into normal colonosco-
pies. In designing studies, thought should be given to the specific questions being
asked and sampling schemes designed accordingly in order to maximize observa-
tion of the microbial community most likely to be involved in the phenotype under
study.
Mouse Models Have Great Utility but Results Must

Be Interpreted with Great Caution
While human association studies are crucial, ultimate evaluation of mechanistic

hypotheses about how host-microbe interactions impact disease must be tested in
animal models. Because mice can be raised sterile, and then inoculated with a pre-
defined microbiome consisting of either cultured [4] or mixed microbial samples
[47, 48], gnotobiotic mice allow for testing of hypotheses about how microbes
directly cause phenotypes such as cancer [4] or obesity [48]. Despite their power, a
number of caveats must be observed when designing and performing mouse
microbiome experiments. In particular, once the gavage has been performed, a
number of factors not related to the contents of the initial gavage can substantially
alter the microbial community. These factors include the cage the mice are housed
in [49], the facilities the animals are housed in [50], the amount of time that has
elapsed since exposure to microbes [51] and (in animals not raised sterile) the line
of maternal transmission [52]. If these factors are not accounted for, they may
induce variations in the microbial community that may confound interpretation of
experimental design. In a recent study comparing animals gavaged to animals
allowed to acquire their microbial community from the environment of the animal
facility, it was found that while the initial gavage had an effect on the microbial
community, most of the composition of the microbial community was driven by the
amount of time that had elapsed since animals were removed from germ-free
conditions and the cage in which the animals were kept [53]. Clearly, experimental
designs that do not explicitly consider these factors are likely to lead to flawed
conclusions and in powering mouse studies, the number of cages, in addition to the
number of animals, must be explicitly considered.
Whole-Genome Metagenome Sequencing and RNA-Seq Can

Be Used to Interrogate Genome Function
As outlined above, small regions of the 16S rRNA sequence can be surprisingly
informative, but there are limits to how much information can be generated by
measuring a single gene. The drop in the cost of sequencing has made much more
feasible experiments which measure all the genes present in microbial genomes
(whole genome metagenome-shotgun sequencing) and experiments which measure
microbial transcripts from mixed microbial communities (metagenomic RNA-seq
experiments). As is the case for 16S sequencing, initial sequencing effort using
Sanger sequencing for whole-genome metagenome experiments required substan-
tial investments of time and expense. An early whole-genome metagenome shotgun
sequencing experiment [54] using clone libraries and Sanger sequencing produced
~78 million bases of unique sequence from fecal samples of two human subjects,
producing our first look at the genome content of the gut microbiome. Today,
through the use of Illumina HiSeq, it is not uncommon to produce ~2 gigabases of
sequences per sample, with per sample costs in the hundreds of dollars. As is the
case for 16S sequences, therefore, we can now produce in a single experiment more
sequences than were produced by multiple labs over years of experiments using
Sanger sequencing.
To be of any utility, whole-genome metagenome sequencing generally requires
many more sequences per sample than 16S sequencing. This translates both into
more expense and a more difficult analysis path. Not only does hard-disk and
network capacity need to be found for the large numbers of sequences that will
be generated by these methods, but the mapping of individual reads to reference
gene databases can require substantial computational times. Investigators wishing
to perform whole-genome or RNA-seq on microbial communities must therefore
ensure they have adequate computational resources or risk project paralysis in
attempting to sift the data once the sequences have been obtained.
Despite the increased overhead and expense of whole-genome sequencing
approaches, these experiments can yield great insights into the gut microbial
community. An intriguing result from the Human Microbiome Project found that
while across body sites and individuals there was great variability in taxonomy
(as defined by 16S sequences), if one looks at the fraction of reads assigned to gene
functions, they was much more consistency [7]. This result suggests the intriguing
hypothesis that while taxa vary substantially in the human microbiome, the gene
functions encoded in those taxa are much more constant. Of course, this interpre-
tation of these results is very dependent on the accuracy of functions that are in gene
function databases and there has been some question as to how biased these
databases may be [55]. Moreover, it is perhaps not surprising that across samples
and subjects, the fraction of genes assigned to broad categories such as “ATP
synthesis” and “central carbohydrate metabolism” is reasonably constant. It
remains an open question how much this high-level consistency is reflected in
consistency in specific metabolic pathways. It will be fascinating to watch
34 A. Fodor
resolution of the question as to the best way to biologically interpret gene function
annotations as the technologies and approaches that power the study of the human
microbiome continue to mature.
If instead of whole-genome sequencing of DNA, RNA is isolated, largely the
same informatics pipelines can be used to assign gene functions at the transcript
level. Because RNA is much less stable than DNA, these experiments are often
more difficult to perform than whole-genome shotgun sequencing, but since mes-
sage is being measured, rather than just genomic potential for message, the biolog-
ical insights generated from these experiments can be considerable. In addition to
the usual difficulties associated with any RNA preparation, RNA-seq on microbial
and metagenomic populations has its own set of challenges. These arise from the
fact that unlike eukaryotic mRNA, prokaryotic mRNA does not have a poly-A tail.
Message and ribosomal RNA therefore cannot be easily separated by the use of
poly-T primers during transcription of cDNA. Strategies that utilize beads that
preferentially bind to, or enzymes that preferentially cleave, rRNA have been
developed to separate mRNA from rRNA, although these strategies have been
found to vary substantially in effectiveness [56]. One strategy that becomes more
attractive as sequencing costs drop is to not attempt to separate rRNA from message
RNA and simply rely on sequencing depth to characterize the mRNA that may be
present in a sample. This strategy has the appeal of simplicity and will also generate
a complete rRNA profile, that can itself be useful in taxonomic assignment. Its
successful application, however, depends on sequencing being inexpensive enough
that sufficient sampling depth can be generated to characterize the small fraction of
reads that are message.
For both whole-genome metagenome sequencing and RNA-seq from mixed
microbial communities within the human microbiome, there is also the problem
of host contamination. The bulk of nucleotides in fecal samples is microbial, but in
other tissues the fraction of microbial vs. host DNA and RNA can vary substan-
tially. Again, as sequencing becomes ever cheaper, the strategy of simply applying
more sequences and computationally removing human contaminant becomes more
attractive, assuming that sufficient computational resources are available to achieve
an initial parse of sequence data.
Future Studies Will Integrate Multiple “Omics” Techniques

to Generate a Complete Picture of Host and Microbial
Pathways
In parallel to the decrease in the cost of nucleotide sequencing, metabolomic and

proteomic platforms are continuing to increase in power, robustness and accessi-
bility. In proteomics, a major challenge is identifying spectra and this challenge is
only increased in the case of mixed metagenomic communities where the genome
sequences that give rise to proteins are not necessarily known [57]. Despite this,
recent efforts have demonstrated not only that proteomics on metagenomics sam-
ples is feasible [58] but that the combination of metagenomics and metaproteomics
approaches can pinpoint particular host and microbial pathways that are associated
with disease [59]. Further integration of these techniques with metabolomics will
undoubtedly yield additional insights [60]. The principle challenge of performing
these types of studies is the integration of diverse genomics datasets, but this is an
area of active research in bioinformatics [61]. We will unquestionably see more and
more studies in the future that will combine nucleotide sequencing with proteimic
and metabolomic techniques.
While the new world of “omics” and its associated bioinformatics tools are often
thought of as the “microscope” through which we can understand the gut ecosystem
in all its complexity, the tools of traditional microbiology, having been continu-
ously refined over the last century, are powerful and should not be overlooked. It is
often stated that most gut microbes are not cultivable, but a recent study that
attempted to systematically cultivate gut microbes from fecal metagenomic sam-
ples found that a substantial proportion of microbes that were detectable with 16S
sequencing could be cultivated with high-throughput anaerobic techniques
[62]. Because these organisms can be introduced into sterile mice, creation of
these biobanks of cultivated organisms will allow for explicit testing of hypotheses
about which taxa and groups of taxa are associated with disease phenotypes.
Moreover, with newly affordable high-throughput sequencing, whole-genome
sequences can be easily obtained for these cultivated organisms, which will allow
for delineation of which genes and genome regions drive health and disease
associations in humans and produce measurable phenotypes in mice. This marriage
of classical microbiology with gnotobiotic and sequencing technology will likely
prove a powerful tool in the next decade’s attempt to understand how specific
pathways are implicated in disease phenotypes.
Conclusion
The gut ecosystem is very complex, but there has been substantial and exciting
recent progress in development of genomic and bioinformatics tools that can allow
for delineation of that complexity. The initial phase of the Human Microbiome
Project focused on utilizing sequencing to characterize variation in healthy adults.
As we move into the next phase of the study of the human microbiome, a central
focus will be on determining which microbial taxa, genes and pathways are
implicated in disease. Careful design of clinical trials and experiments in animal
models will be required to overcome the substantial background variation in the gut
microbiome and separate confounding variables that are often closely related to the
disease categories of interest. A central challenge will be the integration of different
types of “omics” data to produce mechanistic descriptions of how host and microbe
together produce phenotype.
36 A. Fodor
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Chapter 3
The Enteric Nervous System
and Gastrointestinal Innervation:
Integrated Local and Central Control
John B. Furness, Brid P. Callaghan, Leni R. Rivera, and Hyun-Jung Cho
Abstract The digestive system is innervated through its connections with the
central nervous system (CNS) and by the enteric nervous system (ENS) within
the wall of the gastrointestinal tract. The ENS works in concert with CNS reflex and
command centers and with neural pathways that pass through sympathetic ganglia
to control digestive function. There is bidirectional information flow between the
ENS and CNS and between the ENS and sympathetic prevertebral ganglia.
The ENS in human contains 200–600 million neurons, distributed in many
thousands of small ganglia, the great majority of which are found in two plexuses,
the myenteric and submucosal plexuses. The myenteric plexus forms a continuous
network that extends from the upper esophagus to the internal anal sphincter.
Submucosal ganglia and connecting fiber bundles form plexuses in the small and
large intestines, but not in the stomach and esophagus. The connections between the
ENS and CNS are carried by the vagus and pelvic nerves and sympathetic path-
ways. Neurons also project from the ENS to prevertebral ganglia, the gallbladder,
pancreas and trachea.
The relative roles of the ENS and CNS differ considerably along the digestive
tract. Movements of the striated muscle esophagus are determined by neural pattern
generators in the CNS. Likewise the CNS has a major role in monitoring the state of
the stomach and, in turn, controlling its contractile activity and acid secretion,
through vago-vagal reflexes. In contrast, the ENS in the small intestine and colon
contains full reflex circuits, including sensory neurons, interneurons and several
classes of motor neuron, through which muscle activity, transmucosal fluid fluxes,
local blood flow and other functions are controlled. The CNS has control of
defecation, via the defecation centers in the lumbosacral spinal cord. The impor-
tance of the ENS is emphasized by the life-threatening effects of some ENS
neuropathies. By contrast, removal of vagal or sympathetic connections with the
J.B. Furness (*) • B.P. Callaghan • L.R. Rivera • H.-J. Cho

Department of Anatomy and Neuroscience, University of Melbourne, Parkville, VIC 3010,
Australia
e-mail: j.furness@unimelb.edu.au

40 J.B. Furness et al.
gastrointestinal tract has minor effects on GI function. Voluntary control of defe-

cation is exerted through pelvic connections, but cutting these connections is not
life-threatening and other functions are little affected.
Abbreviations
5HT 5-Hydroxytryptamine
CA Cervical afferents
CGRP Calcitonin gene related peptide
CM Circular muscle
DRG Dorsal root ganglia
EEC cell Enteroendocrine cell
EPSP Excitatory Postsynaptic Potential
GALT Gut associated lymphoid tissue
GEP Gastroenteropancreatic
GLP-2 Glucagon-like peptide 2
ICCs Interstitial cells of Cajal
IF Intestinofugal neurons
IGLEs Intraganglionic laminar endings
IMAs Intramuscular arrays
IPANs Intrinsic Sensory Neurons (or intrinsic primary afferent neurons)
LES Lower esophageal sphincter
LM Longitudinal muscle
MMC Migrating myoelectric complexes
MP Myenteric plexus
Muc Mucosa
NHMRC National Health and Medical Research Council of Australia
NO Nitric oxide
NPY Neuropeptide Y
PVG Prevertebral ganglia
SCG Sympathetic chain ganglia
SGLT Sodium/glucose linked transporter
SMP Submucosal plexus
TRH Thyrotropin-releasing hormone
TRPV1 Transient receptor potential cation channel subfamily V member 1
VIP Vasoactive Intestinal Peptide
VMR Visceromotor Reflex
3 The Enteric Nervous System and Gastrointestinal Innervation: Integrated... 41
Introduction
The innervation of the digestive tract is involved in determining the patterns of its
movements, in the control of gastric acid secretion, in regulating movement of fluid
between the gut lumen and body fluid compartments, in changing local blood flow,
in release of gut hormones, in modifying nutrient handling and interacting with the
gut immune system.
The gastrointestinal tract differs from all other peripheral organs in that it has an
extensive intrinsic nervous system, the enteric nervous system (ENS), that can
control functions of the small and large intestines even when they are completely
separated from the central nervous system (CNS). But in reality the ENS is not
autonomous. The neuronal control of gastrointestinal function is an integrated
system in which local enteric reflexes, reflexes that pass through sympathetic
ganglia, reflexes that pass from the gut and back through the CNS and central
control systems interact (Fig. 3.1).
This review is confined to discussion of monogastric mammals, in which most
investigations have been done and which are arguably most relevant to human.
The Extrinsic Innervation of the Gastrointestinal Tract
Connections between the gut and the central nervous system can be conveniently
classified as vagal, spinal thoracolumbar and spinal lumbosacral. Each of these
includes afferent (sensory) innervation and efferent (motor innervation). The effer-
ent pathways contain pre-enteric neurons that end within enteric ganglia and control
or modify the activities of enteric neurons. Pathways from the CNS also contain
neurons that directly innervate a restricted number of gastrointestinal effectors,
such as striated muscle of the esophagus (vagal innervation), sphincters (sympa-
thetic innervation) and intrinsic blood vessels (also sympathetic innervation).
Vagal Innervation
The human abdominal vagus contains about 40,000–50,000 axons [1]. These fibers
provide a sensory innervation and efferent (motor) control pathways for the upper
gastrointestinal tract and digestive organs (Fig. 3.1). The afferents include mucosal
mechanoreceptors, chemoreceptors and tension receptors in the esophagus, stom-
ach and proximal small intestine, and sensory endings in the liver and pancreas.
There is a less prominent vagal afferent innervation of the distal small intestine and
proximal colon. Sensory information concerning luminal contents is detected by
EEC cells which release hormones that act on vagal afferent nerve endings [2]. This
indirect chemoreceptor activation is important for the detection of nutrients and
Fig. 3.1 The innervation of the gastrointestinal tract. The neural connections between the enteric
nervous system (ENS), the central nervous system (CNS) and sympathetic ganglia, and neural
connections between gastrointestinal organs are illustrated. Connections from the ENS to other
organs and the CNS are at the left, and connections from the CNS are at the right. The small and
large intestines (middle of figure) contain full ENS reflex circuits (motor neurons and interneurons
in blue, sensory neurons in purple). Pathways from the gastrointestinal tract (left) project out-
wards, via intestinofugal neurons (red), to the CNS, sympathetic ganglia, gallbladder, pancreas
and trachea. Some neurons in sympathetic prevertebral ganglia (PVG, green neurons) receive both
CNS and ENS inputs. Sensory information goes both to the ENS, via intrinsic primary afferent
(sensory) neurons (purple) and to the CNS via extrinsic primary afferent neurons (left of figure)
that follow spinal and vagal nerve connections. Cervical afferents (CA) connect the esophagus to
the cervical spinal cord. Pathways from the CNS reach the ENS and gastrointestinal effector
tissues through vagal, sympathetic and pelvic pathways (right of figure). Vagal medullary and
pelvic spinal outflows include pre-enteric neurons (ending in enteric ganglia) and most gut-
projecting sympathetic neurons with cell bodies in PVG are also pre-enteric neurons. SCG
sympathetic chain ganglia
also potentially noxious agents in the gut contents [3]. The functions that are
regulated by the vagal sensory innervation include appetite and satiety, esophageal
propulsion, gastric volume, contractile activity and acid secretion, contraction of
the gallbladder and secretion of pancreatic enzymes.
Structural and Functional Characteristics of Vagal Afferent Pathways
Three distinct types of vagal afferent ending occur in the gastrointestinal tract,
intraganglionic laminar endings (IGLEs), intramuscular arrays (IMAs) and mucosal
varicose nerve endings (Fig. 3.2) [4]. IGLEs are complex branching nerve endings
that give rise to flat (laminar) expansions within myenteric ganglia. They were
originally described in the esophagus and shown to be of vagal origin [5], and were
subsequently demonstrated throughout the gastrointestinal tract [6]. IGLEs in the
rectum, and some of those in the distal colon, arise from pelvic nerves [7]. IGLEs
that were identified by anterograde filling responded promptly to probing with a von
Frey hair [8]. Firing rates diminished within the first 2–3 s, but were maintained
above the background level for the duration of the stimulus, thus these are partially
adapting mechanoreceptors. IGLEs that responded to direct probing also responded
to stretching the stomach wall, which provides a direct proof that IGLEs are stretch
receptors [7, 8]. They almost certainly correspond to the low threshold tension
receptors that have been known for a long time, and, in the case of the stomach,
probably signal filling [9, 10].
IMAs are formed by single afferent axons that branch within the circular muscle
layer to form arrays of varicose fibers that run parallel to muscle bundles [11]. They
form synapse-like complexes with interstitial cells of Cajal (ICCs) and it has been
suggested that IMAs, ICCs and smooth muscle work cooperatively or synergisti-
cally to transduce specific stretch or muscle length information [12]. Close
approaches of IMAs to ICC include lamella structures, which have some similar-
ities to the lamellae of IGLEs [12].
Three types of vagal mucosal afferent have been identified: gastric mucosal
afferent endings, afferents supplying villi in the small intestine (villus afferents) and
afferents supplying intestinal crypts (crypt afferents) [13]. The axons of gastric
mucosal afferents branch extensively in the mucosa to provide an innervation that
lies close beneath the epithelium; there are commonly flattened structures (lamel-
lae) near the endings of these branches [13]. These are reminiscent of the
mechanosensitive lamellae of IGLEs and IMAs. Gastric mucosal receptors are
responsive to low intensity stroking of the mucosa, but not to muscle stretch or
contraction, and are also sensitive to chemical stimuli, such as acid in the lumen
[14–16]. Solid food is titurated in the stomach into smaller particles that are able to
pass through the pylorus [17]. Experiments in which the antral mucosa was
separated from the underlying muscle, a procedure that abolishes vago-vagal
reflexes, suggest that mucosal mechanoreceptors may discriminate particles by
size and regulate their passage into the duodenum [18]. Mucosal afferents may
also be involved in the control of satiety, as their mechanosensitivity is enhanced by
the satiety hormone, leptin, and reduced by the feeding hormone, ghrelin, both of
which are released from gastric enteroendocrine cells that are in close proximity to
the gastric mucosal afferent endings [19, 20]. In humans, ghrelin signalling to
hypothalamic feeding centers is via the vagus [21].
Fig. 3.2 Sensory nerve endings in the gastrointestinal tract. Different types of sensory endings in
the intestine: (a) intraganglionic laminar endings (IGLEs); (b) mucosal varicose nerve endings that
supply the villi; (c) intramuscular arrays (IMAs); (d) sensory endings around crypts in the small
intestine. IGLEs branch extensively and provide laminar endings on the surfaces of myenteric
ganglia (green). Perivascular sensory endings are not illustrated. (a) From Castelucci P, Robbins
HL, Furness JB. P2X2 purine receptor immunoreactivity of intraganglionic laminar endings in the
mouse gastrointestinal tract. Cell Tissue Res. 2003;312:167–174 [169]. Reprinted with permission
from Springer Science + Business Media. (b) From Powley TL, Spaulding RA, Haglof SA. Vagal
afferent innervation of the proximal gastrointestinal tract mucosa: Chemoreceptor and mechano-
receptor architecture. J Comp Neurol. 2011;519:644–660. Reprinted with permission from John
Wiley and Sons. (c) From Berthoud HR, Kressel M, Raybould HE, Neuhuber WL. Vagal sensors
in the rat duodenal mucosa: distribution and structure as revealed by in vivo DiI tracing. Anat
Embryol. 1995;191:203–212 [170]. Reprinted with permission from Springer-Verlag. (d) From
Berthoud HR, Powley TL. Vagal afferent innervation of the rat Fundic stomach: morphological
characterization of the gastric tension receptor. J Comp Neurol. 1992;319:261–276. Reprinted
with permission from John Wiley and Sons
Separate villus and crypt afferents innervate the mucosa of the small intestine
[13]. Villus afferents have axons that project toward the villus tip, where they
branch extensively. The branches have irregular flat expansions that tend to be close
to the internal surface of the villus epithelium. Each villus afferent fiber typically
innervates a cluster of two or more neighboring villi. The villus afferents are ideally
positioned to detect substances released from the epithelium, including local
hormones such as CCK and 5HT that are known to activate vagal nerve endings
[2, 3]. The crypt afferents form subepithelial rings of varicose processes below the
crypt-villus junction (Fig. 3.2). Assessment of single fibers filled by anterograde

transport indicates that the villus and crypt afferents are independent endings of
different vagal sensory neurons [13].
Vagal Efferent Pathways
The vagal efferent pathways arise from the dorsal motor nucleus of the vagus and
the nucleus ambiguus. Most of these neurons are pre-enteric, that is, they form
synapses with neurons in enteric ganglia, but some run directly to the striated
muscle cells of the esophagus. The major roles of the vagal innervation are to
control esophageal propulsion, to relax the lower esophageal sphincter for
swallowed food to pass, to increase gastric capacity, to facilitate antral contractions,
to relax the pylorus, to increase gastric acid secretion, to contract the gallbladder
and to promote pancreatic exocrine secretion (Fig. 3.1). Intracellular micro-
electrode recordings from individual gastric enteric neurons indicate that the
majority, at least 2/3, of gastric myenteric neurons receive direct cholinergic
excitatory synaptic inputs from pre-enteric vagal neurons [22]. These experiments
were done by stimulating a vagal branch connected to an isolated region of gastric
corpus. It is possible that not all inputs to each neuron were retained or effectively
stimulated, so the data might underestimate the numbers of neurons receiving direct
excitatory inputs from the vagus. Structural studies also indicate that the majority of
gastric neurons receive vagal input, and even suggest that the vagal inputs out-
number those that arise from intrinsic gastric neurons [23–26]. Surprisingly, only
about 10 % of myenteric ganglia in the striated muscle part of the esophagus receive
vagal efferent inputs [26].
Comparable analyses of projections of vagal pre-enteric neurons to the small
intestine do not appear to have been made. However, tracing studies indicate that
there is a sparse vagal innervation of myenteric and submucosal ganglia in the small
intestine [25]. Consistent with a minor vagal influence, structural and functional
investigations of nerve circuits in the small intestine indicate that there is a
predominance of local connections made with enteric neurons [27]. In contrast,
vagal pre-enteric neurons innervate all intrinsic neurons in the bladder [28]. The
exocrine pancreas has a strong reliance on vagal control [29], suggesting that here
also there are pre-enteric inputs to a high proportion of pancreatic neurons.
Throacolumbar Innervation
The thoracolumbar spinal cord connects with the gastrointestinal tract through
spinal afferent neurons with cell bodies in dorsal root ganglia (DRG) and through
sympathetic efferent pathways (Fig. 3.1). Thoracolumbar afferent axons are almost
all unmyelinated C-fibers. Fiber numbers have been counted in the cat. The greater
splanchnic nerve that supplies the upper abdomen contains about 3,000–4,000
afferent fibers and the lumbar splanchnic nerves contain about 4,000–5,000 afferent
axons [1]. A high proportion is immunoreactive for CGRP and tachykinins, and
they are commonly immunoreactive for the TRPV1 channel, which is associated
with pain afferents [30, 31]. Deletion of TRPV1 results in diminished afferent
responses to distension and to acid in the lumen [32]. A high proportion of the
afferent neuron endings is around arterioles in the gut wall [7]. The axons of spinal
afferent neurons also provide a sparse network of varicose axons in the myenteric
ganglia [30, 33]. Thoracolumbar afferent endings also branch within the lamina
propria of the mucosa throughout the gastrointestinal tract, although their branching
patterns have not been defined [7]. Rare thoracolumbar afferent fibers are found in
the muscle layers. As they pass through sympathetic prevertebral ganglia, the axons
of spinal afferent neurons provide collaterals that form synapses with cell bodies of
postganglionic neurons [34].
There is little evidence that pain comes from the healthy gastrointestinal tract.
In fact, it seems remarkably insensitive to stimuli, such as cutting, that would cause
pain elsewhere. Gastrointestinal pain is associated with inflammation, and post-
inflammatory disorders [35, 36]. Experimental studies indicate that inflammation
causes long term changes in the properties of spinal afferents, that causes
unresponsive neurons to become sensitive and responsive neurons to become
hypersensitive [37, 38].
The sympathetic efferent pathways have four primary targets: myenteric
ganglia, submucosal ganglia, blood vessels and sphincter muscle (Fig. 3.3). The
preganglionic sympathetic neurons have their cell bodies in the intermediolateral
columns of the spinal cord. Postganglionic neurons of vasoconstrictor pathways are
in sympathetic chain and prevertebral ganglia. Postganglionic (pre-enteric) neurons
with cell bodies in prevertebral ganglia provide a dense innervation of myenteric
and submucosal ganglia. In both cases these are inhibitory; the sympathetic inner-
vation of myenteric ganglia inhibits excitatory effects of enteric neurons on the
muscle of the stomach and intestine, thus slowing passage of the contents of the
gastrointestinal tract [39]. The innervation of submucosal ganglia inhibits
secretomotor neuron activity (see later section “Neural Control of Fluid Movement:
Secretomotor and Vasomotor Reflexes”). Sympathetic post-ganglionic neurons
contract the sphincters of the gastrointestinal tract, which, like the innervation of
myenteric ganglia, inhibits transit of contents.
Pelvic Innervation
The distal colon and rectum are provided with afferent and efferent innervation via
the pelvic nerves and sacral plexuses. The pelvic nerves are commonly regarded as
providing an innervation to the distal gut similar to that provided by the vagus to the
proximal gut. However, unlike the vagal afferent nerves, the pelvic afferents
include pain fibers [40]. Colorectal distension causes a visceromotor reflex
(VMR) contraction of abdominal muscles in rats, a response that is deduced to be
a consequence of stimulating pain pathways [41, 42]. The VMR was not affected by
Fig. 3.3 Sympathetic innervation of the gastrointestinal tract. This diagram illustrates the inner-
vation pathways for the non-sphincter regions of the stomach, small and large intestines. The
densest innervation is of the myenteric ganglia throughout these regions, the submucosal ganglia
of the small and large intestines, and intramural arteries. Few sympathetic fibers innervate the
muscle of non-sphincter regions, whereas the sphincter muscle is densely innervated. The post-
ganglionic neurons that innervate gut effectors have noradrenaline as their primary transmitter.
Intestinofugal neurons (IF) synapse with sympathetic neurons in prevertebral ganglia. Modified
from Lomax AE, Sharkey KA, Furness JB. The participation of the sympathetic innervation of the
gastrointestinal tract in disease states. Neurogastroenterol Motil. 2010;22:7–18 [171]
cutting the lumbar colonic or hypogastric nerves, but was abolished when the pelvic
(rectal) nerves were cut [40]. It is established that the pelvic nerves carry afferent
information from low threshold mechanoreceptors. These have been identified as
IGLEs, similar to those in the esophagus and stomach [43]. Action potential firing
in the preterminal axons of IGLEs was evoked by direct probing, or by stretching
the wall of the rectum. Rectal IGLEs detect stretch over a wide range, including into
the level for pain [44]. Mucosal mechanoreceptors in the large intestine are similar
to those in the stomach and proximal small intestine, in that they respond to mild
stroking of the mucosa, but not to distension or contraction of the colon [45]. There
are about 3,500 afferent axons in the pelvic nerves of the cat [1].
The efferent pathways in pelvic nerves provide innervation to enteric ganglia of

the distal colon and rectum [46]. Retrograde tracing indicates that nerve cells in the
sacral spinal cord project directly to the colon, and that there are also nerve cells
that project from the pelvic ganglia to the colon [47], suggesting that pre-enteric
neurons are in both the spinal cord and in pelvic ganglia (Fig. 3.1). For motility
control, the innervation of enteric ganglia comes from the defecation centers that
are in the lumbosacral spinal cord, between L5 and S3 (the levels being slightly
different between species) [48]. In the rat the center is located primarily at L6-S1
[49–51] and in the guinea-pig at S1–S2 [47]. Reflexes through this center can be
initiated by irritation or distension of the rectum; they persist after transection of the
more rostral spinal cord, but are eliminated by section of the sacral outflows or
the pelvic nerves [48, 52, 53]. In healthy individuals, the propulsive reflexes of the
distal colon and rectum are kept in check to maintain fecal continence by central
control centers that relay in the spinal defection center, and when defecation is
appropriate it is triggered by central commands that impinge on the defecation
center. Direct stimulation of the defecation center causes co-ordinated emptying of
the colon, via the ENS [54]. Voluntary control of defecation (both inhibition and
facilitation) is lost if cortico-spinal connections to the defecation centers are
severed by spinal injury [55]. Nevertheless, if the defecation center remains intact
after spinal injury it can be stimulated to command the ENS pathways for bowel
emptying [56]. The pelvic pathways also carry pathways that cause vasodilation in
the colorectum [57].
Cervical Spinal Afferents
Although the gut does not receive efferent inputs from the cervical spinal cord,
afferent neurons that supply the upper, striated muscle, part of the esophagus do
make connections at this level [58]. It is probable that these pathways carry
esophageal pain signals.
Essential Nature of the ENS, in Contrast to Innervation from

the CNS
In Hirschsprung’s disease, the ganglia of the ENS fail to develop in the distal bowel,
but all other tissue components are intact and functional [59]. Under these circum-
stances, no propulsive activity occurs in the aganglionic bowel, and the newborn
child will die if this region is not removed. Similar absence of enteric neurons in the
distal bowel is also lethal in other species, including horse (lethal white syndrome),
rats and mice [60]. Degeneration of colonic enteric neurons in Chagas’ disease,
precipitated by infection with the protozoan Trypanosoma cruzi, causes colorectal
propulsion to fail and megacolon to develop in the adult, similar to the problems
associated with Hirschsprung’s disease in the child [61]. Other enteric neuropathies
that have significant effects on the motor functions of the digestive tract include
esophageal achalasia, gastroparesis and hypertrophic pyloric stenosis [62]. These
diseases illustrate essential roles of the ENS.
The control of fluid movement between the intestinal lumen and body fluid
compartments (discussed below) is also subject to pathological, life threatening,
influences. The fluid movement is controlled by enteric secretomotor neurons that
are abnormally activated by certain infective agents or their products. These
pathogens, including cholera toxin and rotavirus, act directly on the secretomotor
neurons and on the mucosal epithelial cells to cause life-threatening fluid loss [63].
In contrast to the severe, even life-threatening, effects of enteric neuron loss or
dysfunction, severing connections with the CNS has relatively minor effects.
Pavlov achieved a complete vagal denervation of the abdominal organs in dogs:
these animals showed no evidence of ill-health, although responses to sham feed-
ing, which are vagally mediated, were lost [64]. In humans, total abdominal or
selective vagotomy has been used as a treatment for tens of thousands of peptic
ulcer patients, without any indication of significant morbidity due to the vagotomy
itself [65]. Minimal effects are also observed after sympathectomy. Complete
removal of the sympathetic chains in cats left the animals in good health for
many months after the surgery, although they became very sensitive to a cold
environment [66]. Likewise, in humans in which sympathetic innervation of the
gastrointestinal tract is removed for vascular disease or pain, there is no significant
morbidity [67, 68]. Denervation of the gut by destructive lesions of the pelvic
nerves or sacral plexus does not significantly disturb colorectal function, but it does
compromise voluntary control of defecation and it can cause fecal incontinence [55,
69].
Structure of the ENS and Its Constituent Neurons
The enteric nervous system is composed of thousands of small ganglia that lie
within the walls of the esophagus, stomach, small and large intestines, pancreas,
gallbladder and biliary tree, the nerve fibers that connect these ganglia, and nerve
fibers that supply the muscle of the gut wall, the mucosal epithelium, intramural
arteries and other effector tissues (Fig. 3.4). Large numbers of neurons are
contained in the enteric nervous system, about 200–600 million in human
[27]. This is more than the total numbers of neurons of all sympathetic and
parasympathetic ganglia combined and about the same number of neurons that
are in the spinal cord. The enteric nervous system originates from neural crest cells
that colonise the gut during intra-uterine life. It becomes functional in the last third
of gestation in human, and continues to develop following birth.
Figure 3.4 is representative of the ENS of the mammalian small intestine.
Enteric ganglia contain neurons and glial cells, but not connective tissue elements,
and in many respects they are similar in structure to the CNS, except that there is no
Fig. 3.4 The organisation of the ENS. This diagram illustrates the ENS of the small intestine of
human and medium sized to large mammals. It has two ganglionated plexuses, the myenteric
plexus between the longitudinal and circular layers of the external musculature and the submuco-
sal plexus (SMP), that has outer and inner components. Nerve fiber bundles connect the ganglia
and form plexuses innervating the longitudinal muscle, circular muscle, muscularis mucosae,
intrinsic arteries and the mucosa. Axons of extrinsic origin also run in these nerve fiber bundles.
There are also innervations of gastroenteropancreatic (GEP) endocrine cells and gut associated
lymphoid tissue (GALT) that are not illustrated here. From Furness JB. The enteric nervous system
and neurogastroenterology. Nat Rev Gastroenterol Hepatol. 2012;9:286–294. Reprinted with
permission from Nature Publishing Group
significant blood-enteric nervous system barrier. Two major sets of ganglia are
found, the myenteric ganglia between the external muscle layers, and the sub-
mucosal ganglia (Fig. 3.4). The myenteric plexus forms a continuous network,
around the circumference of the gut and extending from the upper esophagus to the
internal anal sphincter. A ganglionated submucosal plexus is present in the small
and large intestines. It is absent from the esophagus and almost no submucosal
ganglia occur in the stomach. These organs also lack the large fluid fluxes across the
mucosal epithelium that occur in the small and large intestines. Nerve fiber bundles
within the enteric nervous system consist of the axons of enteric neurons, axons of
extrinsic neurons that project to the gut wall, and glial cells.
There are some differences in structure and organisation between regions and
species that are reviewed elsewhere [27, 70, 71]. There is a single layer of ganglia in
the intestinal submucosa of small mammals. This is in contrast to large mammals
that have two layers of submucosal ganglia, and sometimes have an intermediate
layer, and in which there are structural and functional differences between the inner
and outer submucosal plexuses [27, 72].
The gastrointestinal tract also harbors an extensive endocrine signaling system,
and many gastrointestinal functions are under dual neuronal and endocrine control.
Enteric neurons also interact with the extensive intrinsic immune system of the
gastrointestinal tract.
Types of Enteric Neurons
Approximately 20 types of enteric neurons can be defined, the numbers differing

slightly between regions [27, 73]. Combinations of features (morphology, neuro-
chemical properties, cell physiology, projections to targets and functional roles)
help to define each type. Amongst the 20 types, three classes can be identified,
intrinsic primary afferent neurons (IPANs, also referred to as intrinsic sensory
neurons), interneurons and motor neurons (Fig. 3.5). IPANs detect the physical
state of the organs (for example, tension in the gut wall) and chemical features of
the luminal contents [74]. They react to these signals to initiate appropriate reflex
control of functions including motility, secretion and blood flow. IPANs connect
with each other, with interneurons and directly with motor neurons. Interneurons
connect with other interneurons and with motor neurons. Amongst the motor
neurons are muscle motor neurons, secretomotor neurons, secretomotor/vasodilator
neurons, motor neurons to enteroendocrine cells, and an innervation of lymphoid
follicles (Fig. 3.5).
Intrinsic Sensory Neurons (IPANs)
The intrinsic sensory neurons (or intrinsic primary afferent neurons, IPANs) were
first identified as large multi-axonal neurons (type II morphology) that respond to
changes in luminal chemistry, mechanical distortion of the mucosa, and direct
mechanical distortion of their processes in the external musculature [75–78]. It
has been more recently discovered that distortion also excites other neurons, for
example interneurons, in the enteric nerve circuits [79–81], indicating that reflexes
are not uniquely initiated or modulated through type II neurons. Cell bodies of
multi-axonal IPANs are 10–30 % of neurons in the submucosal and myenteric
ganglia of the small and large intestines. Consistent with the motor functions of the
esophagus being controlled from or via the brain stem, type II neurons are not found
in the esophagus [27]. They are rare in the stomach, where motility is primarily
controlled by vagal efferent pathways that originate in the medulla oblongata.
Motor Neurons
Muscle Motor Neurons
Excitatory and inhibitory neurons innervate the longitudinal and circular smooth
muscle and the muscularis mucosae throughout the digestive tract. These are uni-
axonal neurons that receive prominent fast excitatory synaptic potentials. The
primary transmitters of the excitatory neurons are acetylcholine and tachykinins.
Fig. 3.5 Neuron types in the ENS. The types of neurons in the small intestine, that have been
defined by their functions, cell body morphologies, chemistries, key transmitters and projections to
targets. LM longitudinal muscle, MP myenteric plexus, CM circular muscle, SM submucosal
plexus, Muc mucosa. Neuron Types: Ascending interneurons (1); Myenteric intrinsic primary
afferent neurons (IPANs) (2); Intestinofugal neurons (3); Excitatory longitudinal muscle motor
neurons (4); Inhibitory longitudinal muscle motor neurons (5); Excitatory circular muscle motor
neurons (6); Inhibitory circular muscle motor neurons (7); Descending interneurons (local reflex)
(8); Descending interneurons (secretomotor and motility reflex) (9); Descending interneurons
(migrating myoelectric complex) (10); Submucosal IPANs (11); Non-cholinergic secretomotor/
vasodilator neurons (12); Cholinergic secretomotor/vasodilator neuron (13); Cholinergic
secretomotor (non-vasodilator) neurons (14); Uni-axonal neurons projecting to the myenteric
plexus (15); motor neuron to the muscularis mucosa (16); innervation of Peyer’s patches (17).
Not illustrated, motor neurons to enteroendocrine cells. Modified from Furness JB. The Enteric
Nervous System. Oxford: Blackwell 2006
The inhibitory neurons have multiple transmitters, including nitric oxide (NO), VIP
and ATP-like transmitters [27, 82]. The primary transmitter of the neurons appears
to be NO, and deficits in transmission are observed if NO synthase is knocked
out [83].
The majority of neurons that innervate the circular muscle have their cell bodies
in the myenteric ganglia. In fact, they are almost all in myenteric ganglia in small
mammals, such as mice, rats and guinea-pigs. In larger mammals, including dog
[84, 85], pig [86], and probably human, a component of circular muscle innervation
comes from submucosal ganglia. The cell bodies of motor neurons that supply the
longitudinal muscle are in the myenteric plexus of small animals. In the pig, and
probably in other large mammals, the majority of the cell bodies are in the
myenteric plexus, but some longitudinal muscle motor neurons have cell bodies
in the outer submucosal plexus [72].
Similar to other smooth muscle of the wall of the gastrointestinal tract, the
muscularis mucosae is innervated by excitatory and inhibitory motor neurons. In
the small intestine and colon of the dog, removal of myenteric ganglia, allowing
time for axon degeneration, did not change the innervation of the muscularis
mucosae, which indicates that the innervation derives from nerve cells in submu-
cosal ganglia [85]. In the esophagus, where there are no submucosal nerve cells, and
the stomach, where there are few, the innervation must arise from nerve cells in
myenteric ganglia.
The endings of vagal motor neurons, with their cell bodies in the nucleus
ambiguus of the brain-stem, form conventional motor end-plates on the striated
muscle cells [87, 88]. However, about a third of the endplates have an additional
innervation from myenteric neurons, through which vagal excitation is modulated
(see below section “Neural Control of Gastrointestinal Muscle Activity”, “Eso-
phagus”). The distal, smooth muscle part of the esophagus and the lower eso-
phageal sphincter are innervated by enteric neurons.
Secretomotor and Secretomotor/Vasodilator Neurons Controlling Fluid

Exchange
Exocrine fluid secretion, such as that from the salivary glands, sweat glands and
pancreas, relies on supply of water and electrolytes from the blood. Because of this,
exocrine secretion is coupled to vasodilation. Coupling also occurs in the intestine,
where secretion and vasodilation are controlled together [89]. Analysis of transport
of water and electrolytes across the intestinal mucosa shows that neurally evoked
fluid transport is mediated by the active secretion of chloride ion, which is accom-
panied by sodium and water secretion [90, 91]. Pharmacological analysis of
responses to nerve stimulation indicates that there are two components of trans-
mission to the mucosa, a cholinergic component and a non-cholinergic component
[92, 93]. Consistent with this, immunohistochemical analysis of neurons projecting
to the mucosa identifies VIP-containing neurons that lack synthesizing enzymes for
acetylcholine, and other neurons that contain choline acetytransferase [94, 95].
VIP is found in neurons innervating the mucosa throughout the small and large
intestines and in the gallbladder of all mammalian species, including human. VIP
both causes fluid secretion and increases blood flow [96, 97], and there is evidence
that collaterals from the VIP containing secretomotor neurons innervate arterioles
in the submucosa [98]. In human, overproduction of VIP causes the watery diarrhea
syndrome [99]. The chemical markers of the cholinergic neurons differ between
species. In the guinea-pig there are two groups, one containing NPY and the other
immunoreactive for calretinin [94, 95]. Acetylcholine is both a stimulant of muco-
sal secretion and a vasodilator.
Motor Neuron Influence on the Glucose Transporter
There is emerging, but incomplete, evidence that enteric neurons influence the
transport of glucose across the mucosa of the small intestine. Glucose is detected by
receptors on enteroendocrine cells that release several gut hormones when stimu-
lated, including glucagon-like peptide 2 (GLP-2) [100–102]. In turn, there is an
induction and functional activation of the glucose transporter SGLT1
[100]. Although the induction of SGLT1 is mediated through GLP-2, the GLP-2
receptor is on submucosal neurons, not on the epithelium, which implies that the
increased glucose transport is a nerve-mediated effect [103]. GLP-2 excites sub-
mucosal neurons [104]. There is also evidence that vago-vagal reflexes contribute
to induction of SGLT1 in the small intestine [105]. The afferent component of the
vago-vagal reflex was blocked by capsaicin application to the abdominal vagus
[105]. The efferent pathway probably involves vagal pre-enteric neurons and
enteric final motor neurons.
Gastric Secretomotor Neurons That Stimulate Acid Output
Some secretomotor neurons govern gastric acid secretion [106]. These neurons are
cholinergic and act on the parietal cells through muscarinic receptors. Projection
studies indicate that the secretomotor neurons have cell bodies in the myenteric
plexus close to the regions of mucosa that they innervate [107].
Gastric Vasodilator Neurons
Gastric acid secretion and blood flow are enhanced when the vagus nerve is
stimulated and these effects are reduced by muscarinic antagonists. In most exper-
iments, it is not possible to determine whether vasodilation is due to a direct
vascular action of cholinergic neurons in addition to a functional hyperemia
consequent on the increased secretion [108]. However, centrally administered
thyrotropin-releasing hormone (TRH) stimulates a vagal pathway in the rat that
causes gastric vasodilation after acid secretion is blocked by omeprazole,
suggesting a direct vasodilator pathway [109]. The blood flow increase in the
absence of secretory change was antagonized by atropine. There is also evidence
for non-cholinergic gastric vasodilator neurons that use VIP as a transmitter [110],
but whether these are vasodilator alone or secretomotor/vasodilator neurons (as in
the intestine) has not been determined.
Motor Neurons to Enteric Endocrine Cells
Twelve or more classes of endocrine cells reside in the mucosa of the gastrointes-
tinal tract [3], and because the mucosa is densely innervated, these cells have nerve
fibers in close proximity, but it is not clear in all cases whether the endocrine cells
are functionally innervated. The best documented motor neurons innervating
enteric endocrine cells are those controlling release of gastrin, which is under the
influence of vagal and of intrinsic gastric pathways [27]. Transmission from the
final secretomotor neurons is mediated at least in part by gastrin-releasing peptide
[111]. Hormone release from other entero-endocrine cells is also likely to be under
neural control. Peptide YY is released from the distal small intestine by vagal
stimulation, and there is evidence of vagal reflex control of its release [112]. The
release is attenuated by the muscarinic antagonist, atropine. The basal release of
motilin is reduced by atropine and by tetrodotoxin, and stimulated by muscarinic
agonists, suggesting that motilin cells receive an excitatory cholinergic input [113].
Innervation of Lymphoid Tissue (Peyer’s Patches),

Lymphocytes and Mast Cells
Lymphoid aggregations of the gastrointestinal tract, the most prominent being

Peyer’s patches, have surrounding nerve fibers, but it is difficult to trace the fibers
into the follicles [114, 115]. However, careful examination does reveal an inner-
vation of the suprafollicular dome region, but not an innervation of the germinal
centers, in porcine jejunal lymphoid aggregations [116, 117], human ileal Peyer’s
patches [117] and follicles in the lamb small intestine [118]. Retrograde tracing
from follicles reveals that they are innervated from submucosal ganglia [118].
In addition, receptors for transmitters of enteric neurons occur on lymphocytes
that are scattered in the connective tissue (lamina propria) of the mucosa, and there
are close approaches that suggest functional innervation of isolated lymphocytes
within the connective tissue of the mucosa [119]. There are also close appositions
between axons and mast cells in the mucosa [120].
Enteric Interneurons
Studies of the projections of neurons within the gut wall have identified several
types of interneurons. However, these are more difficult to investigate physiolog-
ically than other neurons, because they can only be definitively studied by direct
recording techniques, even though elegant divided organ bath methods have pro-
vided insights into the properties of enteric interneurons [121]. Because of the
inherent difficulties in studying the neurons, knowledge of their properties, con-

nections and roles have been obtained from limited numbers of species and regions.
Within the myenteric plexus, the interneurons form chains of like neurons that
run both orally and anally [122–124]. In the guinea-pig small intestine, three classes
of descending interneurons and one class of ascending interneuron have been
identified. Detailed studies of synaptic connections indicate that the chains formed
by two of the types of descending interneuron interconnect [125]. The ascending
interneurons appear to be involved in local motility reflexes, as are two types of
descending cholinergic neurons, those which contain NOS and those containing
5HT [121]. Another type of descending interneuron, the ACh/SOM interneurons,
might be involved in the passage of the migrating myoelectric complexes (MMC)
along the intestine. The somatostatin containing neurons have numerous branching,
tapering, filamentous dendrites [123]. Recent evidence suggests that some classes
of interneurons in the colon are mechanoceptive and that reflexes can be initiated
when they are activated by stretch [126].
Neural Control of Gastrointestinal Muscle Activity
The muscle layers of the gastrointestinal tract direct propulsion, mixing of contents,
reservoir capacity (notably in the stomach) and expulsion of pathogens and noxious
chemicals. The degree to which the ENS is essential for coordinated muscle
function, and the extent to which nerve pathways that originate outside the alimen-
tary tract are necessary for adequate control vary with the region of the gastro-
intestinal tract and also with the physiological circumstance. In broad terms, the
body of the esophagus is controlled through brain stem circuits located in the
medulla oblongata and the stomach is controlled through the brain stem and vago-
vagal reflexes. Small intestine motility is primarily controlled through the ENS,
as is large bowel motility, except for the essential role of the CNS in
defecation [27].
The Esophagus
The nerve circuits for motor programs of propulsive activity in the upper, striated
muscle, part of the esophagus are in the medulla oblongata of the CNS. These
circuits relay through the nucleus ambiguous, which contains the cell bodies of the
motor neurons that innervate the striated muscle [127, 128]. Although there are
numerous ganglia that form an ENS of conventional appearance in the striated
muscle esophagus, the ENS has little influence on the pattern of propulsive activity,
and esophageal propulsion fails and never recovers its function if the vagal inner-
vation is severed [129]. Nevertheless, myenteric neurons do supply an innervation
to about a third of the end-plates and thus, unlike motor endplates elsewhere,
individual endplates in the esophagus receive dual innervation, one axon being
from a vagal motor neuron and the other originating from a cell body in the
myenteric plexus [130–133]. The endings of myenteric origin have NOS immuno-
reactivity, implying that transmission from enteric neurons is nitrergic. The
myenteric neurons exert a presynaptic inhibition of vagal excitatory transmission,
that has been demonstrated by experiments in which enteric NOS neurons were
stimulated indirectly [134]. Thus the enteric nervous system seems to have a role in
modulating peristalsis in the upper esophagus. The enteric innervation may have a
greater role in young animals, because all motor endplates receive an enteric
innervation at days 4–10 postnatal, after which there is partial withdrawal of
innervation [135].
The nerve fibers that innervate the smooth muscle of the lower esophagus have
their cell bodies in enteric ganglia. Nevertheless, peristalsis in this region is also
coordinated from the CNS. The enteric ganglia of the smooth muscle esophagus are
directly innervated by pre-enteric neurons of the dorsal motor nucleus of the vagus,
and lesion of this nucleus impairs the motility patterns of the smooth muscle
esophagus [128]. The vagus is involved in relaxing the lower esophageal sphincter
(LES), to allow passage of food, through a descending inhibitory reflex that relaxes
the sphincter when a bolus of food enters the last part of the esophageal body and its
intraluminal pressure is raised. The reflex relaxation is inhibited by cooling the
vagus nerve [136]. However, sphincter relaxation still occurs in response to disten-
sion following vagal block, indicating that a local reflex can be elicited [136].
Peak pressures during gastric mixing contractions exceed resting pressures in the
body of the esophagus and the LES has an important role in limiting reflux of the
corrosive contents of the stomach into the esophageal body. This role is apparent
when pressure in the stomach is increased and a reflex constriction of the LES is
initiated [137, 138]. This sphincter contraction is mediated by a vago-vagal reflex
pathway that passes through the brain stem. Failure of this guarding results in reflux
esophagitis and esophageal mucosal damage.
Stomach
A well-developed ganglionated myenteric plexus is found in the stomach, whose

activity is significantly controlled through the vagus (see also above section “Vagal
Efferent Pathways”).
The stomach has a reservoir function; it increases volume as it fills, and relaxes
prior to food arriving. It also has a function to mix the food with gastric juices and to
push the liquefied products of gastric digestion into the duodenum. The fundus
(proximal stomach) is primarily associated with the gastric reservoir function and
the corpus-antrum (distal stomach) is associated with gastric mixing and antral
propulsion [139]. Each antral contraction propels a small amount of liquid into the
duodenum, while solid material is retained in the stomach [17].
Gastric Reservoir Function
The pressure in the stomach does not increase as it is filled [140], implying that the
muscle of the proximal stomach relaxes to accommodate the meal. In fact, relax-
ation occurs before the food arrives, a phenomenon called receptive relaxation
[141]. The relaxation that occurs when the pharynx or esophagus is distended
occurs even when the esophagus is severed and no food reaches the stomach
[142]. The reflex is prevented if the vagus nerves are cut. Relaxation of the proximal
stomach also occurs if the gastric volume is increased, for example by distension
with an intragastric balloon. This accommodation reflex is substantially reduced
after vagotomy [143, 144]. A vagally mediated gastro-gastric reflex relaxation is
also be elicited when distension is confined to the antrum [145]. In addition, there
appears to be a small residual component of accommodation that is due to an
intrinsic reflex [146]. As the volume in the stomach reduces, the fundus contracts.
This also appears to be a vagally mediated effect [144]. Thus the stomach adjusts its
volume both by relaxation and contraction, via vago-vagal reflexes.
Gastric Peristalsis and Mixing (the Distal Stomach:

Corpus and Antrum)
Gastric peristalsis, which occurs in the body and antrum, is not prevented when the
myenteric plexus is cut through or nicotine is given in a dose that blocks peristalsis
in the intestine [147, 148]. Moreover, the frequency of peristalsis corresponds to the
frequency of gastric slow waves in the muscle, indicating that gastric peristalsis is
generated by the slow waves and, unlike peristalsis in the small intestine and colon,
it does not require activity of excitatory neurons to be observed.
The augmentation of the gastric contractions when the stomach is artificially
distended with fluid is almost entirely through vago-vagal reflexes [149]. When the
antrum, or the whole stomach, is extrinsically denervated, antral peristaltic con-
tractions are smaller and emptying times are prolonged [149–151]. Moreover, the
strengths of the antral contractions are sequentially reduced when the vagal
branches entering the antrum are successively cut, from proximal to distal [152].
Nevertheless, a number of studies indicate that there is intrinsic activity of
excitatory cholinergic neurons, even in the completely isolated stomach. Intracel-
lular microelectrodes have demonstrated the spontaneous occurrence of fast EPSPs
in some enteric neurons in the isolated stomach [153], and other investigations have
demonstrated an excitatory tone that is reduced by tetrodotoxin, or by antagonists of
muscarinic or nicotinic receptors [154–156]. The amplitudes, but not the frequen-
cies of occurrence of contractile waves are reduced when transmission from
excitatory neurons to the muscle is prevented by tetrodotoxin [156]. The effective-
ness of the excitatory neurons is enhanced when the stomach is distended [156],
presumably because their rates of firing are increased.
There is little evidence for a gastric intrinsic reflex that is organised like that in
the small intestine and which is necessary for intestinal peristalsis. After vagotomy,
gastric distension causes very much weaker phasic contractions than are seen in the
vagally innervated stomach [149]. The residual responses to distension are reduced
by hexamethonium, indicating that there is a component of the enhancement of
gastric peristaltic waves that is due to intrinsic reflexes. Furthermore, if the mus-
carinic receptor agonist, carbachol, is applied to the isolated stomach in which all
nerve-mediated events have been prevented by tetrodotoxin, gastric peristaltic
waves are restored [156]. This suggests that neuronal circuits are not required to
co-ordinate peristaltic movement, direct excitation of the muscle being sufficient.
IPANs, the types of neurons through which reflexes in the intestine are initiated are
absent or very rare in the stomach [27].
It is concluded that gastric peristalsis is a consequence of contractions that are
induced in the muscle by slow waves that are themselves generated by the pace-
maker activity of ICC [157].
The Small Intestine and Colon
These regions rely on the ENS to direct various patterns of movement. In the small
intestine, these patterns are rapid orthograde propulsion of contents (peristalsis),
mixing movements (segmentation), slow orthograde propulsion (the migrating
myoelectric complex, MMC) and retropulsion (expulsion of noxious substances
associated with vomiting). In the large intestine there are mixing and propulsive
movements, including the colonic MMC [46]. To orchestrate these movement
patterns, the state of the intestine is sensed and appropriate motor patterns are
generated through ENS circuits. The structural organisation of the circuits that
detect the state of the small intestine, integrate the information and direct the
activities of motor neurons is known (Fig. 3.6) and the colonic circuits appear to
be similar [126, 158], but the mechanisms, within the integrative circuitry, through
which one pattern of activity is converted to another are not known. Signals that
trigger changes in patterns of movement in the small intestine have been identified.
For example, fatty acids added to the luminal surface convert propulsive contractile
activity to mixing movements, through a neural mechanism [159]. Conversion from
one pattern to another can also be achieved with some drugs that target enteric
neurons [160].
Fig. 3.6 Nerve circuits for control of motility in the small intestine. This diagram is based on
studies in the guinea-pig small intestine. Similar component neurons have been identified in the
small intestine of other species, including human, and in the large intestine. This is a simplified
circuit diagram showing the major circuit features that have been identified. Networks of
interconnected intrinsic sensory neurons (IPANs, red) detect mechanical distortion and luminal
chemistry. These synapse with descending (yellow) and ascending (green) interneurons, and
connect with excitatory muscle motor neurons (blue) and inhibitory muscle motor neurons
(purple) directly and via interneurons. Based on Furness JB. The Enteric Nervous System. Oxford:
Blackwell 2006
Neural Control of Fluid Movement: Secretomotor

and Vasomotor Reflexes
It is essential that the movement of fluid between the lumen of the intestine and the
body fluid compartments is regulated. More than two blood volumes cross the
mucosal epithelial surface each day, and disruption of fluid transport regulation,
such as occurs in cholera intoxication, is life-threatening.
One reason for the large flux is that the absorption of sugars (monosaccharides)
and amino acids is through cation-coupled transporters. Thus the absorption of a
glucose molecule through the sodium/glucose linked transporter (SGLT) brings
with it a sodium ion together with counter ions, mainly chloride. It is calculated that
100 g of absorbed glucose takes with it 1.8 L of water [27, 161]. Enteric reflexes,
through activation of secretomotor neurons, return water and electrolyte to the
lumen (Fig. 3.7). This fluid is drawn from the circulation and from the absorbed
fluid. Enteric secretomotor reflexes cannot act in isolation, they must be modulated
to take into account whole body fluid balance. This control is exerted through blood
volume and blood pressure detectors that change the activity of two sympathetic
pathways, vasoconstrictor pathways and secretomotor inhibitory pathways
(Fig. 3.7) [27, 162].
The fine control of fluid balance through local (ENS) and systemic (sympathetic)
reflexes is thrown into chaos when there is an excessive luminal content of certain
pathogens or their toxins. These agents, including cholera toxin, rotavirus and
pathogenic E. coli, activate enteric secretomotor neurons. In mild cases, this
stimulates diarrhea that helps expel the pathogens and their toxic products.
Fig. 3.7 Neural control of transmucosal water and electrolyte movement in the small intestine.
The final secretomotor neuron of reflexes that play an essential role in balancing local fluid fluxes
and in whole body water and electrolyte balance is illustrated. Large volumes of fluid are absorbed
from the lumen with nutrients, such as glucose. These fluids are returned through secretomotor
reflexes. The absorption of nutrients with fluid activates enteric secretomotor reflex pathways that
impinge on the secretomotor neurons. It is important that the balance of this fluid exchange is
modulated by sympathetic vasoconstrictor and secretomotor inhibitory pathways. Activity in these
sympathetic pathways, which inhibit secretion and reduce local blood flow, is determined by
whole body fluid status, which includes sensory detection through blood volume detectors,
baroreceptors and osmoreceptors. Modified from Furness JB. The Enteric Nervous System.
Oxford: Blackwell 2006
However, when there are high levels of pathogens or toxins, the intestine is
overwhelmed and a pathological, life-threatening hypersecretion can ensue. The
hypersecretion results in copious diarrhea. Infectious diarrhea causes about 1.5
million deaths a year, primarily in underdeveloped tropical countries [163].
Entero-Enteric Reflexes
Figure 3.1 shows the intestinofugal neurons that have cell bodies in the digestive
tract and project to sympathetic ganglia, other organs and to the CNS. Only the roles
of those projecting to the sympathetic ganglia are known. These intestinofugal
neurons are in the afferent limbs of entero-enteric reflexes, that pass from distal to
proximal regions through sympathetic ganglia, where intestinofugal neurons form
synapses [27, 164, 165]. The reflex pathways bypass the CNS. Distension of
segments of intestine activates the reflex pathways, causing sympathetic inhibition
of motility in more proximal regions. In the case of the stomach, acid or hypertonic
solution in the lumen of the upper small intestine causes inhibition of gastric
motility and emptying into the duodenum through entero-enteric reflexes [166,
167]. The entero-enteric reflex that is initiated by fat in the distal intestine and
slows transit in the proximal small intestine is referred to as the ileal brake
[168]. Thus the reflexes arise in distal regions and regulate more proximal regions,
so that luminal contents that arrive at more distal regions are adequately processed
proximally.
Summary and Conclusions
Neural control of the gastrointestinal tract is exerted by integration of signals that

originate in the CNS and ENS. Gastrointestinal function is maintained in the
absence of influence from the CNS, but if ENS control of the intestine is lost,
propulsion of content in the affected region is ineffective, which is life-threatening.
Three major regions of the CNS connect with the gastrointestinal tract, the brain
stem through the vagus nerve, the thoracolumbar spinal cord through spinal afferent
and sympathetic efferent pathways, and the lumbosacral spinal cord through pelvic
nerve afferent and efferent pathways. Vagal afferents carry mechanoreceptive and
chemoceptive information from the esophagus, stomach and intestine to the CNS,
but do not signal pain. Thoracolumbar and lumbosacral afferents both signal pain of
gut origin. In addition, there is cervical afferent innervation of the upper esophagus.
The primary control centers for the smooth muscle esophagus, lower esophageal
sphincter, stomach, gallbladder and pancreas are in the CNS; they exert control
through vagal efferent pathways. The vagal neurons that control gastric motility,
acid secretion and hormone release form synapses in the ENS. The major efferent
connections of sympathetic pathways are to myenteric ganglia, through which
gastrointestinal movements are inhibited, to submucosal ganglia, through which
fluid movement into the lumen is inhibited, and to intramural arteries that are
constricted by sympathetic nerve activity. The efferent pelvic nerves convey the
outputs of the lumbosacral defecation centers.
The enteric nervous system consists of many thousands of interconnected
ganglia that extend from the upper esophagus to the internal anal sphincter. These
ganglia in human contain in total about 200–600 million neurons. Motor neurons in
the enteric ganglia supply all major effectors in the gastrointestinal tract. In the
small and large intestines, the ENS contains full reflex pathways that are essential to
direct the movements of these parts of the digestive tract. Another critical role of the
ENS, in concert with signals from the CNS, is whole body fluid balance. This is
necessary because of the very large fluid load that is contributed to by water and
electrolyte movement that is associated with nutrient digestion and absorption. The
ENS contains a type of neuron not found anywhere else in the periphery. These are
intestinofugal neurons, with cell bodies in enteric ganglia, that send their axons to
sympathetic ganglia, to other organs (the pancreas, gallbladder and trachea), and to
the CNS via the vagus and pelvic nerves. Thus the digestive tract is controlled
through integrating centers in the brainstem, spinal cord, sympathetic ganglia and
gut wall that are extensively interconnected through conventional afferent and
efferent pathways and via the intestinofugal neurons.
Acknowledgements Research in the authors’ laboratories is supported by the National Health

and Medical Research Council of Australia (NHMRC). LRR is supported by a NHMRC post-
doctoral fellowship.
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Chapter 4
Intestinal Barrier Function
and the Brain-Gut Axis
Carmen Alonso, Mar´ıa Vicario, Marc Pigrau, Beatriz Lobo,

and Javier Santos
Abstract The luminal-mucosal interface of the intestinal tract is the first relevant
location where microorganism-derived antigens and all other potentially immuno-
genic particles face the scrutiny of the powerful mammalian immune system. Upon
regular functioning conditions, the intestinal barrier is able to effectively prevent
most environmental and external antigens to interact openly with the numerous and
versatile elements that compose the mucosal-associated immune system. This
evolutionary super system is capable of processing an astonishing amount of
antigens and non-immunogenic particles, approximately 100 tons in one individual
lifetime, only considering food-derived components. Most important, to develop
oral tolerance and proper active immune responses needed to prevent disease and
inflammation, this giant immunogenic load has to be managed in a way that
physiological inflammatory balance is constantly preserved. Adequate functioning
of the intestinal barrier involves local and distant regulatory networks integrating
the so-called brain-gut axis. Along this complex axis both brain and gut structures
participate in the processing and execution of response signals to external and
internal changes coming from the digestive tract, using multidirectional pathways
to communicate. Dysfunction of brain-gut axis facilitates malfunctioning of the
intestinal barrier, and vice versa, increasing the risk of uncontrolled immunological
reactions that may trigger mucosal and brain low-grade inflammation, a putative
first step to the initiation of more permanent gut disorders. In this chapter, we
describe the structure, function and interactions of intestinal barrier, microbiota and
brain-gut axis in both healthy and pathological conditions.
C. Alonso • M. Vicario • M. Pigrau • B. Lobo • J. Santos (*)

Neuro-Immuno-Gastroenterology Group, Digestive Diseases Research Unit, Gastroenterology
Department, Hospital Universitari Vall d’Hebron, Vall d’Hebron Research Institute, Paseo
Vall d’Hebron 119-129, 08035 Barcelona, Spain
Department of Medicine, Universitat Autònoma de Barcelona, Centro de Investigación
Biomédica en Red de Enfermedades Hepáticas y Digestivas (Ciberehd), Barcelona, Spain
e-mail: javier.santos@vhir.org

74 C. Alonso et al.
Abbreviations
ACTH Corticotropin
CRF Corticotropin-releasing-factor
DSS Dextran sulphate sodium
GALT Gut-associated lymphoid tissue
GCs Goblet cells
HNPs Human neutrophil peptides
HPA Hypothalamic pituitary-adrenal axis
IBD Inflammatory bowel disease
IBS Irritable bowel syndrome
JAMs Junctional adhesion molecules
MAPKs Mitogen-activated protein kinases
MARVEL MAL and related proteins for vesicle trafficking and membrane link
MLC Myosin light chain
MLCK Myosin light chain kinase
MUC Mucins
NGF Nerve growth factor
NLRs Nod-like receptors
NOD Nucleotide-binding oligomerization domain
PAMP Pathogen-associated molecular patterns
POFUT1 Protein O-fucosyltransferase 1
PRR Pattern recognition receptors
RELM Resistin-like molecule
TJs Tight junctions
TNBS Trinitrobenzene sulphonic acid
ZO Zonula occludens
Introduction
The survival of living organisms greatly depends on the ability of species and
individuals to constantly provide a series of complex and dynamic repository
responses to counteract internal and environmental threats. This functional equi-
librium, named homeostasis, relies upon the adequate integration of every gener-
ated response to a threat. At the gastrointestinal level, the mucosal surfaces are the
first location where immunogenic particles, environmental toxins and
microorganism-derived antigens gain access to the immune system [1]. The luminal
side of the mucosa of the ileum and jejunum is coated with hundreds of tiny finger-
like structures called villi, which in turn are composed by myriads of microvilli,
rendering a final physical contact area of about 400 m2. This enormous epithelial
surface area favours nutrient absorption and water and electrolyte transport across.
However, it also designed to select which luminal antigens should face the
4 Intestinal Barrier Function and the Brain-Gut Axis 75
components of the mucosal-associated immune system. This selection process is

aimed at preventing the generation of inadequate pro-inflammatory signals
[2]. Mucosal processing of antigens and non-immunogenic molecules will at the
end, determine whether tolerogenic or non-tolerogenic immune responses are
raised to keep homeostasis [3].
The intestinal mucosal barrier consists of different consecutive layers, including
the intestinal flora and external mucus, the columnar epithelium and extracellular
matrix below, and the innermost lamina propria. Within the lamina propria we can
find blood and lymph vessels, a plethora of resident immune cells (plasma cells,
lymphocytes, macrophages, eosinophils, mast cells, dendritic cells, etc.), and a
significant number of intrinsic and extrinsic nerve terminals (Fig. 4.1). All of
these components may display effector and modulatory functions relevant to the
control of inflammation, absorption and secretion, transport of macromolecules and
metabolic processes [4]. Considerable evidence now supports the existence of
multidirectional communication between the components of this local regulatory
network [5, 6]. Communication is driven by the release of chemical mediators, such
as neuropeptides, neurohormones, neurotransmitters, cytokines, chemokines,
growth factors, and other regulatory molecules.
The regulation of gut physiology is also achieved through the activity of both the
enteric nervous system (ENS) and the central nervous system (CNS). ENS is an
extensive neural network, also known as the second brain, containing approxi-
mately 100 million neurons embedded in the gastrointestinal lining, similar number
to the spinal cord [7]. The ENS contains sensory neurons, inter-neurons, and motor
neurons, which primarily control motility, absorption and secretion, but also vis-
ceral sensitivity. In addition, the ENS is wired with multiple terminals from
ascending and descending CNS pathways that help to control gut function. To
understand gut physiology and pathology, it is of particular importance to consider
the role of the autonomic nervous system, and the hypothalamic pituitary-adrenal
axis (HPA) because both systems also establish a vast and complex array of
integrative and bidirectional interactions between the brain and the gut, the brain-
gut axis.
The Intestinal Barrier
The intestinal barrier has evolved to guarantee homeostasis through the execution
of basic weeping off functions, such as water secretion, to wash off harmful sub-
stances that may be present in the intestinal lumen, and by the development of a
programme, that includes active immunological surveillance. One of the first steps
to fight unwanted or harmful stimuli involves the release of mucus, defensins,
secretory-immunoglobulin A, and other chemical mediators to the lumen [8]. In
addition, the importance of maintaining epithelial permeability tight to prevent the
passage of noxious substances, was emphasized in the early 1990s [9], and reiter-
ated by many authors thereafter.
76 C. Alonso et al.
Fig. 4.1 Intestinal barrier function. The intestinal barrier has evolved to guarantee homeostasis
through the execution of basic weeping off functions, such as water secretion and intestinal
peristaltism, and by the development of immunological surveillance. This barrier is composed by
several levels of protection aimed at preventing and selecting toxin and antigen penetration across.
The most external laters harbours mucus, enzymes, antimicrobial peptides and the intestinal
microbiota. Just below, a single-cell layer of epithelial cells, sealed by intercellular junctions,
regulates the transcellular and paracellular passage of substances. Intermingled goblet cells secrete
mucins that dissolve in water to form mucus, a major contributor to the retention of secretions
containing antibacterial peptides and digestive enzymes, and to keep epithelial hydration. The
epithelium also displays microbial recognition receptors and is able to release immune mediators.
Lamina propria leukocytes produce proteases and cytokines to modify epithelial secretory activity
and permeability range of the epithelium. M cells are found in the follicle-associated epithelium of
the Peyer’s patches and transport antigens from the luminal side to immune cells across the
epithelial barrier. IgA is produced by plasma cells, and transported through, and secreted by, the
epithelium to the luminal side. Both, the central and the enteric nervous system, interact with
the immune system, the smooth muscle and the epithelium to regulate immune responses, absorp-
tion and secretion, motility, and also visceral sensitivity. Note: IEL intraepithelial lymphocyte
Structure and Function of Intestinal Barrier
Mucus
The entire intestinal mucosal surface is covered by a layer of mucus gel, thicker
than 100 μm secreted by goblet cells (GCs). Mucus protects the epithelial lining
from luminal sheer forces, adhesion and invasion by microorganisms, dietary,
chemical and radiation toxins, and other antigens present in the intestinal lumen
[10]. The mucus layer also contributes to the retention of mucosal secretions
containing antibacterial peptides and digestive enzymes [11, 12] and keeps epithe-
lial hydration. Mucus seems to participate in epithelial renewal, differentiation and
integrity, and relates to other biological processes [13]. More recently, mucus has
also been shown to enhance oral tolerance by imprinting dendritic cells with anti-
inflammatory properties through the assembly of a galectin-3-dectin-1-FcγRIIB
receptor complex that activated β-catenin, interfering with the expression of inflam-
matory, but not tolerogenic cytokines by dendritic cells [14].
Components of mucus include water, phospholipids, the negatively charged
mucins (MUC), which provide a chemical barrier to protect the underlying epithe-
lium, and a variety of trefoil factors and other antimicrobials such as secretory IgA
[15], cathelicidins and defensins that provide the physical and immune protection
against luminal agents [16]. Mucus secreted at the apical brush border binds the
glycocalyx to form a viscoelastic gel with hydrophobic and surfactant properties,
dependent on the presence of phospholipids at the most apical part. Hydrophobicity
helps to fight enteric bacteria and to regulate gut permeability [17].
MUC represent the most abundant component of the mucus gel. MUC are huge
glycoproteins composed of a central protein backbone rich in serine, threonine and
proline. These glycoproteins are highly glycosylated by attached oligosaccharides,
which contain blood group structures and are initiated by N-acetyl-galactosamine
that is O-linked onto serine or threonine at the protein core [18–20]. These O-linked
oligosaccharides are responsible for MUC properties. Up to 20 different MUC
genes have been identified to date (MUC1 to MUC20) [21], with site and cell-
specific expression. Several secreted mucins (MUC2, MUC5AC, MUC5B, and
MUC6) function as extracellular viscous secretions whereas others appear as
membrane-associated mucins (MUC1, MUC3 and MUC4) in the glycocalyx
[22]. MUC1–4 represent the most abundant secreted mucins in the human intestine.
The first identified human secretory mucin was MUC2 that is also the principal
secreted MUC [23], and is normally restricted to GCs [24]. In mice, it has been
shown that colonic mucus consists of two layers with similar protein composition,
being MUC2 the major structural component. The inner layer is firmly attached to
the epithelium and functions as a barrier to prevent bacterial invasion while the
outer layer is a loose matrix usually colonized by bacteria [25]. Thickness of the
inner mucus layer varies down along the intestine according to luminal concentra-
tion of bacteria, being thicker at the highly colonized colonic segment, and thinner
at the less colonized small intestine [26]. Baseline secretion of MUC is a constitu-
tive pathway where small vesicles transport MUC directly to the cell surface where
immediate and full exocytosis of their contents takes place. The release and
secretion of packaged MUC is a different pathway regulated by specific stimuli
including microbes and their products, and neuroendocrine and inflammatory/
immune mediators. Mucus production is tightly regulated by different protein
families, such as MUC and protein O-fucosyltransferase 1 (POFUT1) family
members. Dysfunction of mucus secretion can lead to the development of intestinal
inflammation as shown by the susceptibility of MUC2 KO mice to develop spon-
taneous colitis, and by a more severe intestinal response to the administration of
78 C. Alonso et al.
dextran sulphate sodium (DSS) [27]. These mice also display impaired host resis-
tance to parasitic infection [28], and over-enhanced susceptibility to Salmonella
enterica serovar typhimurium [29]. Decreased production and alteration of the O-
glycosylation profile of MUC2 has been associated with increased inflammation in
ulcerative colitis [30, 31]. Moreover, increased susceptibility to ulcerative colitis
[32] and Crohn’s disease [33] has been linked to a rare variable number of tandem
repeat alleles of the MUC3 gene. Mice defective in intestinal POFUT1 exhibit
chronic intestinal inflammation in association with an alteration of mucus-
associated flora, goblet cell hyperplasia and hypertrophy and elevated production
of mucus [34].
Resistin-like molecule (RELM)-β is a cysteine-rich protein also present in the
mucus layer and specifically produced by intestinal GCs. RELM-β upregulates
MUC2 and M1/MUC5AC gene expression in the human colonic HT29 cell line.
Pretreatment of murine colon with RELM-β significantly attenuates trinitrobenzene
sulphonic acid (TNBS)-induced colitis [35] while RELM-β deficient mice show
increased susceptibility to T-cell-dependent TNBS-induced colitis. Therefore,
available evidence suggests that RELM-β plays an important role in colonic
inflammation [36].
Trefoil factors, a group of small cysteine-rich peptides, are also essential pro-
tective components of the mucus layer and contribute to mucosal repair, particu-
larly, the trefoil factor 3 synthesized and secreted by intestinal GCs [37, 38]. Trefoil
factor 3 deficient mice are highly susceptible to DSS, chemotherapy and radiation-
induced colitis [39, 40], and display prominent hypoxia-elicited increases in intes-
tinal permeability [41].
Epithelial Lining
The intestinal epithelium is a single polarized continuous layer of columnar cells of

only 20 μm thick that covers the intestinal surface and separates the intestinal lumen
from the internal milieu. Although it functions primarily as a physical barrier, it also
regulates the absorption of dietary nutrients, water and electrolytes. The passage of
molecules from the intestinal lumen to the lamina propria takes place mainly
through two different routes: (1) The paracellular pathway, which allows small
molecules (<600 Da) diffuse through tight junctions (TJs) located between adja-
cent intestinal epithelial cells; and, (2) The transcellular pathway, which allows the
passage of larger particles through the epithelial cells via endocytosis or exocytosis
processes [42].
The intestinal epithelium contains several stem cell-derived cellular types, such
as absorptive enterocytes, GCs, Paneth cells, enteroendocrine cells, and M cells, as
shown in panel 1 of Fig. 4.2. This epithelial population renews every 3–5 days from
pluripotential stem cells located in the intestinal crypts to ensure cellular integrity
all along the intestinal epithelium. Pluripotential stem cells migrate to the tip of the
villus where final differentiation takes place [43]. Signalling cascades such as the
wnt and the Notch pathway are involved in epithelial proliferation and differenti-
ation, essential processes to regulate homeostasis in the intestinal epithelium [44].
Fig. 4.2 Ultrastructure of the intestinal mucosa. Transmission electron micrographs of the
intestinal epithelium and the lamina propria of the human jejunum. The intestinal mucosa is
responsible for nutrient absorption and water secretion, which require a selectively permeable
barrier. Panel 1—Intestinal epithelium. The epithelium functions primarily as a physical barrier
between the external environment and the internal milieu. It is composed by enterocytes, secretory
cells and immune cells, all supported on the basal side by a basement membrane underneath which
the lamina propria harbors blood and lymph vessels, resident immune cells and nerve terminals.
GC goblet cell, IEL intraepithelial lymphocyte, EC enterochromaffin cell, Ep epithelial cell. Panel
2—Intercellular junctions. The epithelial cells are polarized cells bound together through
specific junctions. The apical junctional complex delineates the apical and the basal regions of
the epithelial cells. It limits the uptake of microbial and food-derived antigens and prevents the
passage of cellular elements across. TJs are located at the most apical site of the epithelium
followed by the subjacent adherens junction and the desmosomes. TJ tight junction, AJ adherens
junction, D Desmosome. Panel 3—Lamina propria. Most of the immune elements of the
intestinal barrier are located in the lamina propria, where they develop innate and adaptive
responses in coordination with the nervous system and the epithelium. Eo eosinophil, NE nerve
endings, PC plasma cell, MC mast cell, L lymphocyte, Ep epithelial cell
Enterocytes
Enterocytes are key elements of the epithelial lining. Although, the most important
endeavour of these cells is to maintain the integrity of the intestinal physical barrier,
enterocytes reinforce barrier strength by also developing immunologic activity.
Enterocytes express innate immune receptors [45], act as non-professional antigen
presenting cells, and release several chemokines and cytokines such as fractalkine
[46] or thymic stromal lymphopoietin [47] involved in leukocyte recruitment and in
dendritic cell regulation.
Enterocytes are tightly bonded to each other through the apical junctional
complex that separates the apical membrane from the basolateral membrane. This
apical junctional complex is composed TJs, adherens junctions, and desmosomes,
80 C. Alonso et al.
as shown in panel 2 of Fig. 4.2. The junctional complex limits the uptake of
microbial and food derived antigens and prevents the passage of cellular elements
across. TJs are located at the most apical site of the epithelium and composed of
intracellular and surface-membrane proteins. Intracellular proteins are zonula
occludens (ZO)-1, ZO-2 and ZO-3, as well as cingulin. Surface-membrane or
transmembrane proteins include occludin, claudins, and junctional adhesion mole-
cules (JAMs). TJs seal the intercellular space and regulate intestinal permeability.
Adherens junctions are located below TJs and mainly composed by e-cadherin,
catenin, and actin filaments. These protein complexes provide the necessary
strength to hold the cells together.
Occludin was the first TJ transmembrane protein identified. It belongs to the
TJ-associated MAL and related proteins for vesicle trafficking and membrane link
(MARVEL) proteins, and contains a MARVEL domain. The function of occludin
remains to be elucidated. On one hand, occludin deficient mice do not show
alterations in TJ assembly and permeability [48], but, on the other hand, occludin
seems to play a role in the regulation of integrity rather than in the de novo
assembly of the TJs [21]. Furthermore, in vitro observations suggest that occludin
localization to the TJ complex is regulated by phosphorylation [49]. Regulation of
occludin phosphorylation implicates several kinases including protein kinases C,
mitogen-activated protein kinases (MAPKs), Rho kinases, and the Src-Family
kinases [50]. When occludin is highly phosphorylated on serine and threonine
residues, it is selectively located at the TJ. In contrast, occludin dephosphorylation
at those residues by protein phosphatases, results in redistribution of the protein to
the cytoplasm [24].
The claudin family of transmembrane proteins consists of 24 members with a
molecular weight ranging from 20 to 27 kDa. Each member shows a specific organ
and tissue distribution. This protein family plays a central role in the regulation of
barrier function. Some claudins make up pores that allow preferential passage of
specific ions, while others reduce the transit of specific ions. The strength, size, and
ion selectivity of TJs is determined by claudins, as reflected by massive trans-
epidermal water loss and death of mice within one day of birth affecting claudin-1
deficient mice [51]. Moreover, segmental barrier properties along the crypt-villus
axis and throughout the length of the intestine do correlate with the disposition of
claudins [52, 53]. In the human intestine, both ileal and colonic mucosa express
tightening claudins-1, -3, -4, -5 and -7 [54, 55]. However, the expression of the
permeability mediator claudin-2 is restricted to the crypt, in the colon [30, 56], yet
detected in the crypt and the villus, in the small bowel [31]. Differences in the
expression and distribution of claudins may reflect adaptation to specific physio-
logical functions carried out by the different segments down the intestinal tract.
A third group of transmembrane receptors found at TJs is the family of JAMs.
JAMs have been implicated in the construction and assembly of TJs [57], and in the
regulation of intestinal permeability and inflammation [58]. JAM-A deficient mice
display increased intestinal permeability and inflammatory cytokine production,
and marked epithelial apoptosis to DSS-induced colitis [59]. More recently,
reduced intestinal JAM-A expression has been described in irritable bowel
syndrome (IBS) patients, possibly contributing to intestinal barrier dysfunction in

these patients [60]. JAMs are also present on blood cells, such as leukocytes,
thereby contributing to the process of trans-endothelial migration [61].
The TJ transmembrane proteins, claudins, occludin, and JAMs are linked to the
actomyosin fibers of the cytoskeleton by members of the ZO family [62]. This
association to the peri-junctional actomyosin ring seems crucial for the dynamic
regulation of permeability at paracellular spaces. Interestingly, only ZO-1 and ZO-2
are relevant for claudin recruitment, TJ formation and for epithelial barrier
function [63].
Far from being static, TJs are quite mobile structures that readily adapt to
changing conditions and challenging stimuli. Regulation of intestinal permeability
involves different functional pathways. Fast changes in permeability occur usually
via myosin light chain kinase (MLCK)-mediated cytoskeleton contraction, and by
endocytosis of TJ proteins [64, 65]. In contrast, lasting permeability disturbances
involve the transcriptional modulation of TJ proteins, epithelial cell apoptosis and
ultrastructural alterations in the epithelium [66].
Phosphorylation of myosin II regulatory light chain (MLC) induces actomyosin
cytoskeleton contraction and increased TJ junction permeability. Rho GTPases
have been shown to regulate TJs through redistribution of ZO-1, and reorganization
of JAM-1 away from the TJ membrane [67]. Up-regulation of zonulin expression
increased intestinal permeability to bacterial and gliadin exposure. In fact, this
zonulin-mediated intestinal barrier defect has been advocated to play a central role
in the origin of celiac disease [68] and type 1 diabetes [69].
Secretory Cells
The intestinal epithelium also houses different types of specialized epithelial called
secretory cells that contribute to the reinforcement of the intestinal epithelial
barrier, mainly goblet cells, Paneth cells and enteroendocrine cells.
GCs are scattered through the epithelial lining. GCs that mainly secrete mucins,
but also trefoil peptides, RELM-β and Fc-γ binding protein. GC distribution varies
throughout the gastrointestinal tract, the number increasing from the duodenum to
the distal colon. The number of GCs is probably regulated by the intestinal
microbiota because germ-free mice have less and smaller GCs than regular
mice [70].
Paneth cells are located at the base of the crypts of Lieberkühn. Similar to the
other intestinal epithelial cell types, they evolve from stem cells at the bottom of the
crypt. Contrary to other cell types, Paneth cells migrate downwards, to the bottom
of the crypt, where they synthesize and secrete antimicrobial peptides and other
proteins to the intestinal lumen. Among them, lysozyme, α-defensins, TNF-α, and
secretory phospholipase A2 type IIA, contribute to maintain host-microbe homeo-
stasis and to protect stem cells from pathogens [71, 72]. Certain defects in Paneth
may be linked to the pathogenesis of Crohn’s disease [73, 74] and necrotizing
enterocolitis [75, 76].
82 C. Alonso et al.
Gut enteroendocrine cells spread all along the intestinal epithelium where they
function as highly specialized chemoreceptors sensing changes in luminal osmo-
larity, pH and nutrient composition. Although they represent less than 1 % of the
entire gut epithelial population, enteroendocrine cells constitute the largest endo-
crine organ of the human body. Products released by enteroendocrine cells include
hormones, such as ghrelin, somatostatin, cholecystokinin, gastric inhibitory poly-
peptide, glucagon-like peptides and peptide YY, and neurotransmitters such as
serotonin [77]. Enteroendocrine cells inform the brain-gut axis mostly through
the activation of neural pathways [78].
The Intestinal Immune System
Mucosa-associated lymphoid tissue is a diverse and diffuse defence system found at

most mucosal surfaces of the body, such as the respiratory system and the eye
conjunctiva. The immune response generated by this system provides generalized
immunization at all mucosal surfaces [79]. About 70 % of whole body’s immune
cells reside within the gastrointestinal tract shaping the gut-associated lymphoid
tissue (GALT), which is conformed in two different compartments: the organized
immune inductive sites, and the diffuse effector sites.
Diffuse GALT is composed of two lymphocyte populations distributed at both
sides of the basal lamina. Intraepithelial lymphocytes are found between epithelial
cells, above the basal lamina. Lamina propria lymphocytes reside in lamina propria
along with many other types of immune cell, such as eosinophils, dendritic cells,
mast cells, macrophages or plasma cells (panel 3 of Figs. 4.2 and 4.3). The majority
of intraepithelial lymphocytes are CD8+ T cells that function as surface gatekeepers
of the intestinal barrier because the constantly monitor and respond against luminal
bacteria and other antigens. Lamina propria lymphocytes constitute a much more
heterogeneous population, approximately 50 % of which correspond to plasma
cells, 30 % to T lymphocytes, and the remaining 20 % to macrophages, dendritic
cells, mast cells and eosinophils. Resident B lymphocytes complete their matura-
tion into plasma cells, mostly producing IgA, but IgM and IgG. Activated T and
B-lymphocytes express α4β7 integrin and mucosal endothelial cells of Peyer’s
patches, mesenteric lymph nodes and lamina propria of the small and large
intestine constitutively express the mucosal addressin cell adhesion molecule-1
that interacts with α4β7 integrin to recirculate lymphocytes between the blood
and the gastrointestinal tract [80].
Inductive sites of the GALT include organized lymphoid structures in the small
intestine such as Peyer’s patches, mesenteric lymph nodes, isolated lymphoid
follicles, and lymphocytes and antigen-presenting cells. Peyer’s patches are mac-
roscopic lymphoid aggregates found at the submucosal levels in the antimesenteric
border of the intestine. The follicle-associated epithelium covering Peyer’s patches
contains M cells, another special cell type that plays a role in monitoring the gut
lumen and maintaining intestinal barrier function. M cells display several unique
properties including apical microfolds instead of microvilli, no mucus layer, and a
Fig. 4.3 Resident immunocytes in the intestinal mucosa. The majority of the immune cells within
the body reside in the gastrointestinal tract (Gut-associated lymphoid tissue, GALT), and are
distributed in two different compartments: the organized inductive sites, and the diffuse effector
sites. The diffuse GALT is composed of intraepithelial lymphocytes, between the epithelial cells,
and the lamina propria lymphocytes, which reside below the basal lamina, along with other
immune cells. The figure shows intestinal micrographs ( ×400 magnification) processed for
H&E staining to identify mucosal eosinophils (1), and immunohistochemistry for T-
lymphocytes (2, CD3+), B-lymphocytes (3, CD20+), macrophages (4, CD68+), plasma cells
(5, CD138+), and mast cells (6, CD117+)
reduced glycocalyx, which facilitate the capture of luminal antigens and microor-
ganisms and their transport to contact underlying immune cells [81]. Peyer’s
patches also contain antigen-presenting cells, mainly dendritic cells, but also
macrophages. These antigen-presenting cells capture luminal antigens (taken up
by M cells in the Peyer’s patch dome), to further process and present them to
immunocompetent cells in association with the major histocompatibility complex.
Innate immunity is present in both animals and plants [82]. It serves the host
defence via immediate, but non-specific, responses to a wide variety of pathogens.
The main components of innate immune response include pattern recognition
receptors (PRRs), and antimicrobial peptides.
84 C. Alonso et al.
PPRs are a class protein that responds to small molecular sequences consistently
found on pathogens, named pathogen-associated molecular patterns (PAMP). PRRs
include Toll-like receptors (TLRs) and Nod-like receptors (NLRs).
The TLR family consists of at least 13 transmembrane receptors containing a
large leucine-rich repeats extracellular domain that recognizes different bacterial,
viral, parasite or self-derived ligands, such as lipopolysaccharide, peptidoglycan,
muramyl dipeptide, lipoteichoic acids, and bacterial DNA. After activation upon
PAMP recognition, TLRs initiate downstream signalling cascades, leading to
transcriptional responses and to the initiation of both innate immune responses
(macrophage activation and induction of antimicrobial peptides for various cell
types) and the adaptive immune response (induction of T cell responses and
maturation of dendritic cells) [83]. In many tissues, mast cells, dendritic cells,
monocytes/macrophages and B cells express TLRs [84]. Healthy intestinal epithe-
lial cells express relatively low levels of TLRs, such as TLR-4, perhaps explaining
why lipopolysaccharide does not induce a potent inflammatory response in normal
intestine [85]. By contrast, and consistent with the idea that chronic intestinal
inflammation may be the result of uncontrolled responses to components of the
intestinal bacterial flora, the intestinal epithelium of patients with inflammatory
bowel disease (IBD) shows increased expression of TLR-4 [86]. The cellular
localization of TLRs is also influenced by the polarized epithelial cell organization.
TLR5 is expressed on the basolateral surface of intestinal epithelia only, where if
becomes stimulated by luminal flagellin exposure when disruption of the epithelial
barrier. Therefore, its localization prevents inappropriate stimulation by flagellin,
but allows recognition of invasive pathogens [87]. Similarly, TLR9 activation
through apical and basolateral surface domains induces distinct transcriptional
responses. Whereas basolateral TLR9 strongly stimulates proinflammatory chemo-
kine secretion, through NF-kappaB activation, apical TLR9 stimulation invokes a
unique response in which ubiquitinated IkappaB accumulates in the cytoplasm
preventing NF-kappaB activation conferring mucosal tolerance towards microbial
exposure [88].
NLR constitutes a large family of 23 intracellular PRRs, being nucleotide-
binding oligomerization domain (NOD)1, NOD2 and NALP3 the most extensively
described. NOD1 and NOD2 recognize intracellular bacterial cell products, and
NALP3 responds to multiple stimuli to form a multi-protein complex termed the
NALP3 inflammasome, which promotes the release of the IL-1 family of cytokines.
Most NLRs share a similar structure consisting of a centrally located NOD, a C-
terminal leucine-rich repeat that detects PAMPs, and a variable N-terminal domain
that is critical for downstream signalling through the recruitment of adap-
tors or effector molecules [89]. NOD1 recognizes γ-D-glutamyl-meso-
diaminopimelic acid, which is found in the peptidoglycan structures of all gram-
negative as well as in several gram-positive bacteria [90]. In contrast, NOD2
recognizes muramyl dipeptide, which is found in nearly all gram-positive and
gram-negative organisms [91]. Upon ligand recognition, NOD1 and NOD2 induce
the activation of NF-kappaB and MAPKs pathways leading to the activation of both
innate and adaptive immune responses. In contrast, other NLRs such as Ipaf and
cryopyrin respond to microbial components through the assembly of multiprotein

complexes termed “inflammasomes” that promote caspase-1 activation to generate
the proinflammatory cytokines IL-1β and IL-18 [92]. NOD1 is expressed by
intestinal epithelial cells [93] while NOD2 expression is predominantly found in
monocytes and Paneth cells [73]. Both NOD1 and NOD2 have been shown to
modulate inflammation and mediate efficient clearance of bacteria from the muco-
sal tissue during Salmonella colitis [94]. In addition, NOD2-deficient mice display
an increased load of commensal resident bacteria, and a diminished ability to
prevent intestinal colonization by pathogenic bacteria [95]. NOD-2 mutations
have been identified in Crohn’s disease patients and could be related to an impaired
release of antimicrobial peptides from Paneth cells [96].
Antimicrobial peptides are endogenous antibiotics that are constitutively
expressed in intestinal epithelial cells, yet may be also inducible in immune cells
and Paneth cells [97]. They include compounds such as lactoferrin, hepcidin,
bactericidal/permeability increasing protein, lysozyme and overall, defensins and
cathelicidins.
Defensins are a family of small cationic peptides (29–45 amino acids) that
exhibit a wide and potent antimicrobial activity spectrum against gram-negative,
and gram-positive bacteria, fungal and yeast, parasites, viruses, and even tumor
cells [98]. Defensins have been identified in both prokaryotes and eukaryotes.
Although structurally different, most defensins display cationic and amphiphilic
properties which confer them the capacity to permeabilize the bacterial cell mem-
brane. In mammals, these peptides are expressed in mucosal epithelial cells and
phagocytes, but also are released into the intestinal lumen, several grams daily, by
Paneth cells [99]. Defensins act as effector and regulatory molecules of the innate
immune response. In addition, defensins also enhance adaptive response acting on
phagocytic cells and mast cells to induce the release of inflammatory mediators and
to regulate the complement system. Defensins also interact with dendritic cells and
T cells to increase antigen-specific immune response [100].
These peptides are classified as α and β-defensins according to their disulphide
bond pairing pattern. The human α-defensins 1–4, conventionally referred as to
neutrophil defensin (human neutrophil peptide, HNP), although defensins HNP1-3
are also expressed in epithelial cells of inflammed mucosa [101]. In contrast, human
α-defensin 5 and 6 (HD5 and HD6) are only expressed in Paneth cells of the small
intestine [102]. HD5 has been shown to induce IL-8 expression on intestinal
epithelial cells [103], and to protect mice from DSS colitis and Salmonella infection
[104]. More recently, HD6 has been shown to form fibrils and nanonets that
surround and entangle bacteria to protect the small intestine against invasion by
diverse enteric pathogens [105].
Human β-defensin-1 is constitutively expressed in the small intestine and the
colon. In contrast, Human β-defensins-2-4 expression is inducible [106] in inflam-
matory conditions such as IBD [107, 108] or infection by enteroinvasive
bacteria [109].
The other major class of antimicrobial peptides is the cathelicidin group. In
mammals, about 35 members have been identified, but only one in humans:
86 C. Alonso et al.
hCAP18/LL37 [110]. Although regarded as neutrophil specific, hCAP18/LL37is

also expressed in other leukocytes, keratinocytes and epithelial cells of the respi-
ratory, genitourinary and gastrointestinal tract [111], and in human breast
milk [112].
Expression of hCAP18/LL37 in human colonic epithelial cells has been related
to cell differentiation [113]. Infection of intestinal epithelial cells by Shigella spp.
inhibits the expression of hCAP18/LL37 [114], while bacterial components such as
sodium butyrate [115] or TLR-ligands such as bacterial DNA [116] induce its
expression.
Acquired immunity is restricted to vertebrates and constitutes a second line of
defence against pathogens. It is driven by B and T lymphocytes through specific
receptors and confers protection against re-exposure to the same antigen. Antigen
binding to these receptors results in clonal expansion of these cells and the initiation
of a directed immune response. Functionally speaking, within the adaptive immu-
nity, we can distinguish inductive and effector compartments. Antigen presentation
and naive T and B-lymphocytes activation occurs in the inductive compartment. In
the effector compartment sensitized cells against different antigens extravasate and
differentiate to carry out the destruction of pathogens. IgA secretion has been
shown to be regulated through TLR-signalling [117] but also by changes in the
composition of intestinal Microbiota [118].
Intestinal Barrier Dysfunction
Stress, Hormones and Neurotransmitters
Stress represents a threat to the internal homeostasis. In response to stress, a

coordinated response is initiated to maintain stability through the autonomic,
endocrine, and immune systems. The main systems activated during the stress
response are the sympatho-adrenomedullary, a component of the sympathetic
division of the autonomic nervous system, and the HPA axis. The autonomic
nervous system provides, through its sympathetic and parasympathetic arms, the
fastest response to stressor exposure, leading to rapid alterations in physiological
state through neural innervation of end organs. Stress activation of the HPA axis
stimulates the parvocellular neurons in the paraventricular nucleus of the hypo-
thalamus to secrete corticotropin-releasing-factor (CRF), which in turn travels to
the anterior pituitary to promote the synthesis of corticotropin (ACTH)
[119]. ACTH, when released into the systemic circulation, activates the adrenal
cortex to induce cortisol and corticosterone secretion that circulate through the
bloodstream to reach every tissue [120]. Adaptation to stress through the activation
of the sympatho-adrenomedullary system and the HPA axis to maintain homeosta-
sis is called “allostasis”. However, excessive stress exposure impairs this adaptive
response, eventually predisposing these subjects to the development of disease or to
exacerbation of previous existing ones [121], specially in stress-sensitive disorders,

like IBS.
At the experimental level, different type of stresses, acute and chronic, physical
or psychological, have been shown to influence properties of the intestinal barrier
function, including as ion and water secretion, intestinal permeability, mucus
secretion, and also intestinal flora. Ion and water secretion allows the intestine to
wash away noxious substances present in the intestinal lumen, preventing adhesion
to the mucosal surfaces and penetration to the lamina propria. The jejunum of rats
submitted to restraint stress or cold restraint stress was found to show an increase its
baseline short-circuit current, indicative of enhanced anion secretion [122]. Later, it
was observed that peripheral CRF and repetitive exposure to water avoidance stress
reproduced stress-induced rat jejunal and colonic epithelial barrier dysfunction via
cholinergic and adrenergic nerves and mast cells [123, 124]. More recently, it has
been shown that chronic psychosocial stress also activates mucosal mast cells and
increases baseline short-circuit current in both the jejunum and the colon [125]. In
humans, studies using jejunal segmental perfusion techniques reveal that acute
physical or psychological stress either reduce net water absorption or increase
secretion in healthy subjects and in patients with food allergy [126, 127] through
the parasympathetic nervous system and mast cell activation [128]. More recently,
we have extended these observations to show that in healthy female volunteers that
intestinal water secretion during cold pain stress was significantly reduced in those
with moderate background stress compared to those with low stress [129]. This
observation could indicate a loss of regulatory mechanisms in subjects suffering
from continuous life stress.
Both paracellular and transcellular permeability to small and large molecules
increased in response to acute and chronic stress in the rodent jejunum and colon
[130–133]. Several mechanisms, including mast cells, CRF [134], MLCK, and
cytokines like interferon gamma, and interleukin-4 [135] have been implicated.
In humans, it is known that surgery, trauma, and gastrointestinal infections [136]
increase intestinal permeability. CRF has been shown to enhance transcellular
uptake of macromolecules in human colonic mucosa via CRF-R1 and CRF-R2
receptors, located on subepithelial mast cells [137]. Unpublished observations from
our group indicate that intravenous CRF increased intestinal permeability in healthy
subjects and in IBS patients [138]. Acute psychological stress also increases small
intestinal permeability in humans and peripheral CRF reproduces the effect of
stress and mast cell stabilization blocks the effect of both stress and CRF,
suggesting the involvement of mast cells [139]. Cold pain stress also increased
intestinal permeability in female healthy subjects, although this response was larger
in women with moderate background stress. Increased intestinal permeability has
been found in diarrhoea prone IBS patients [140]. These findings provide new
insight into the complex interplay between the central nervous system and gastro-
intestinal function in man.
Acute stress causes mucin release in the rat colon, along with enhanced secretion
of rat mast cell protease II and prostaglandin 2. These changes were reproduced by
intravenous or intracerebral injection of CRF in non-stressed rats, and were
88 C. Alonso et al.
inhibited by the administration of a CRF antagonist or a mast cell stabilizer

[141]. In addition, stress-induced release of mucin was abolished in mast-cell
deficient mice, highlighting a key role of mast cells in stress-mediated mucin
release [142]. In contrast, rats submitted to chronic stress displayed mucus deple-
tion along with increased bacterial adhesion and penetration into enterocytes [143].
Stress can also induce microbiological changes in the intestinal flora. Maternal
separation in infant rhesus monkeys decreased faecal bacteria, especially
Lactobacilli, and increased their susceptibility to opportunistic bacterial infections
[144]. Similarly, prenatal stress reduced the overall numbers of Bifidobacteria and
Lactobacilli in the newborn infants [145]. Interestingly probiotic treatment ame-
liorates stress-induced changes in the gastrointestinal tract [146] and attenuates the
observed Lactobacilli reduction in maternally-deprived rat pups [147]. In addition
to these microbiological changes, dexamethasone administration in rats enhanced
bacterial adherence to the mucosa, decreased secretory-immunoglobulin A secre-
tion, and increased intestinal permeability [148]. More recently, Söderholm
et al. showed that chronic psychological stress in rats, leads to an increased antigen
and bacterial uptake in follicle associated epithelium from Peyer’s patches [149] as
well as in the villous ileal and colonic epithelium. Emotional stress during take-off
in cosmonauts induced changes in faecal Bifidobacteria and Lactobacillus, as well
as an increase in Escherichia coli, whereas a substantial increase in Enterobacteria
and Clostridia was found after the flight [150]. These stress-induced changes in the
faecal flora have been related to catecholamine release into the intestinal lumen
and/or into the systemic circulation, as the addition of various catecholamines to
cultures of gram negative bacteria resulted in dramatic increases in growth of
E. coli, Yersinia enterocolitica and Pseudomonas aeruginosa [151].
Mast cells are known to modulate stress-mediated responses of the epithelial
barrier function, to orchestrate the mucosal immune function and to participate in
the defence against bacteria [152, 153]. To exert these functions, enteric mast cells
are strategically located within the gastrointestinal tract, developing an optimal
sensory and effector interaction within the local regulatory neuroendocrine net-
works. Upon activation, mast cells act as effector cells, through the selective
(piecemeal degranulation) (Fig. 4.4) or massive release (anaphylactic degranula-
tion) of preformed or newly produced biological mediators. More relevant to stress-
mediated inflammation is their ability to communicate, bidirectionally, with both
the enteric, autonomic and central nervous systems. Anatomical contacts between
mast cells and enteric nerve fibres have been demonstrated in the human gastroin-
testinal mucosa and these contacts increase, when inflammation is present
[154]. An increase in the nerve-to-mast cell proximity in the colonic mucosa of
IBS patients has been positively correlated with the severity and frequency of
abdominal pain [155]. This mast cell-enteric nerve interaction provides a physical
substrate for bidirectional communication between the CNS and the gut, by which
stress might influence gastrointestinal physiology. This is reflected in vivo by the
release of mast cell products into the lumen of the human small intestine after cold
stress, which is accompanied by increased epithelial secretion [128]. Mast cell
mediators released after degranulation can sensitize mesenteric afferents and
Fig. 4.4 Intestinal mast cells. Enteric mast cells are known to modulate the epithelial barrier
function, to orchestrate the mucosal immunity and to participate in the defence against bacteria.
They are strategically located within the gastrointestinal tract, developing sensory and effector
interactions within the local regulatory neuroendocrine networks. Upon activation, mast cells act
as effector cells, through the selective (piecemeal degranulation) or massive release (anaphylactic
degranulation) of preformed or newly produced mediators. The figure shows transmission electron
micrographs of ultrastructural characteristics of mucosal mast cells: a resting mast cell in health
(H ), with granules filled (white arrows) and no signs of degranulation; piecemeal degranulation in
a mast cell from a patient with irritable bowel syndrome (IBS), identified by partial or total
emptiness of granules content (black arrows) and intact granules (white arrow); and anaphylactic
degranulation in a mast cell from a food allergy patient (FA), identified by fusion of granule
membranes devoid of content (black arrow). Barr indicates 2 μm
nociceptive receptors [156]. Among the potential mast cell mediators involved,
both histamine and serotonin induce intestinal secretion of water, electrolytes and
mucus. In addition, mast cells from IBS patients release more histamine and
tryptase than intestinal mast cells from normal subjects [157] a fact that has been
linked to the generation of visceral hypersensitivity, through the activation of
proteinase-activated receptors type 2. These receptors can modulate enteric neuro-
transmission, secretion, motility, epithelial permeability, and visceral sensitivity,
and are also known to regulate intestinal inflammation [158]. However, altered
expression of histamine H1 and H2 receptor subtypes has recently been reported in
mucosal biopsies from distal gut of IBS patients, suggesting that these receptors
could also play a role in these processes [159].
CRF and related peptides are the most important neuroendocrine factors medi-
ating the effects of stress, both at the central and peripheral level. CRF urocortin
(Ucn) 1, Ucn 2 and Ucn 3 exert their effects after binding to G protein-coupled
receptor subtypes, CRF-R1 and CRF-R2, signalling through cAMP [160]. After
physical or psychological stress, neural or immune release of CRF and urocortins
mediate autonomic, hormonal, and behavioural responses to stress and stimulate the
ENS to modulate gastrointestinal motility and secretion [161–163]. Increased CRF
and urocortin expression has been demonstrated in the colonic mucosa of IBD
patients [164, 165].
Vasoactive intestinal peptide is also involved in the regulation of chloride
secretion, mucin release, paracellular permeability and epithelial cell proliferation
90 C. Alonso et al.
[166, 167]. Psychological stress increases vasoactive intestinal peptide levels in the
small intestine of mice [168] and vasoactive intestinal peptide has been implicated
in the regulation of the intestinal barrier function, through its direct effect on tight
junction-associated protein, ZO-1, in epithelial cells [169].
Substance P participates in gut inflammation by interacting mainly with the
neurokinin-1 receptor, expressed on nerves, epithelial, endothelial and smooth
muscle cells, and immune cells, such as mast cells, macrophages, and T cells
[170]. This neuropeptide has been found to stimulate macrophage and eosinophil
secretion of pro-inflammatory cytokines, to increase NK cell activity and migration,
and to activate the release of chemokines from leukocytes. It also induces the
release of vasoactive mediators from mast cells, contributing to chloride secretion,
intestinal permeability, vascular leakiness and oedema at sites of inflammation,
modulating diarrhoea, inflammation, and motility [171]. Substance P mediates
stress-induced CRF expression in mice eosinophils, and eosinophil-derived CRF
is responsible for mast cell activation and consequently, epithelial barrier
dysfunction [172].
Nerve growth factor (NGF) has been involved in the development of stress-
induced barrier dysfunction [173] and hyperalgesia during inflammation [174,
175]. These effects seem to be mediated by CRF and mast cells [176, 177]. Maternal
deprivation has been shown to induce hyperalgesia to rectal distension and to
enhance colon permeability in association with elevated NGF expression [173]. A
subsequent study from the same group showed that CRF, acting through its receptor
CRF-R1, stimulated NGF release from mast cells, which in turn increased gut
paracellular permeability [178]. More recently, norepinephrine has been shown to
induce visceral sensitivity to colorectal distension by increasing the expression of
NGF in the rat colon wall [179]. These findings support the importance of NGF in
stress-induced visceral hypersensitivity, but also in stress-induced barrier
dysfunction.
Sex steroids also play a role in modulating intestinal barrier, although conflicting
results have been described. Estrogen can bind to two different receptors named
estrogen receptor-α and β. Estrogen receptor-α mediates estrogen signalling in the
development of secondary sex characteristics, and the regulation of the menstrual
cycle and sperm maturation [180]. In contrast, estrogen receptor-β is mainly
expressed in epithelial cells and is the most abundant estrogen receptor in the
colon [181]. Both progesterone and estradiol have been shown to reduce chloride
secretion in intestinal epithelial cells [182, 183], whereas estradiol has also been
found to reinforce epithelial permeability [184], and to up-regulate JAM-A and
occludin expression [185].
Other hormones have been involved in the regulation of intestinal barrier
function (Table 4.1).
Table 4.1 Hormones and intestinal barrier

Hormone Function References
Glucagon-like peptide 2 Decreases intestinal permeability [292, 293]
Growth hormone Decreases intestinal permeability [294, 295]
Insulin-like growth factor 1 Decreases intestinal permeability [296, 297]
Ghrelin Decreases intestinal permeability [298]
KdPT Decreases intestinal permeability [299]
KdPT a tripeptide derivative of the C-terminus of α-melanocyte-stimulating hormone
Infections
Intestinal pathogens have developed specific strategies to gain access to the lamina
propria. Strategies include direct TJ disruption, the production of toxins that induce
fluid and electrolyte secretion, and the activation of the inflammatory cascade
[186]. Vibrio cholerae can directly alter TJs through its cytotoxin hemagglutinin
protease, a metalloproteinase that disrupts occludin-ZO-1 interactions leading to TJ
and cytoskeleton anchorage destabilization [187]. In addition, other toxins have
been involved in TJ disruption by V. cholerae such as the RTX toxin, that crosslinks
actin inducing cell rounding and increased permeability [188], or the ZO toxin, that
fragments ZO-1 and occludin and disrupts the actin cytoskeleton [189, 190]. Clos-
tridium difficile infection produces two distinct exotoxins, Toxin A and B (TcdA
and TcdB), that through RhoA GTPases inactivation cause actin filament disaggre-
gation and cell rounding, resulting in increased paracellular permeability [191,
192]. Recent findings suggest that toxin A could even disrupt directly TJ proteins
[193]. Clostridium perfringens enterotoxin utilizes claudin-3 and 4 as receptors
[194] to bind the enterocyte surface where it forms small protein complexes in the
plasma membrane that interact with other proteins forming a large complex, that at
the end triggers massive permeability changes [195]. Enteropathogenic E. coli
infection directly disrupts TJ through occludin dephosphorylation and dissociation
from TJs to the cytoplasma [196] and MLC phosphorylation [197] enhancing
intestinal permeability.
Intestinal Microbiota
Intestinal microbiota has been shown to influence intestinal barrier function and the
brain-gut axis [198, 199]. Intestinal microflora displays several important functions
to maintain gut homeostasis, such as nutrient digestion, vitamin and hormone
production and most importantly, protection from microbial colonization, achieved
through competition for intestinal nutrients and for attachment sites
[200]. Probiotics are live microorganisms which, when consumed in adequate
amounts, confer a health benefit on the host. Increasing evidence suggests that
probiotics implement intestinal epithelial homeostasis and enhance barrier tightness
and integrity. In contrast with pathogens, probiotics have been shown to increase
92 C. Alonso et al.
occludin expression [201], and to enhance ZO-2 expression in parallel to its

redistribution towards the cell boundaries via silencing of PKCζ [202] thereby
leading to TJ stabilization and the restoration of the epithelial barrier. Specific
Lactobacilus salivarus strains prevent hydrogen peroxide-induced reduction in
transepithelial resistance when added to Caco-2 cell monolayers [203]. Similarly,
Lactobacillus rhamnosus GG improves intestinal barrier function in the immature
murine gut through the induction of claudin 3 expression [204], the regulation of
apoptosis and the promotion of cytoprotective responses [205]. Interestingly,
probiotics have also demonstrated beneficial effects in other tissues such as the
skin barrier [206] or the respiratory tract [207, 208].
There is a significant body of evidence indicating that probiotics can also prevent
intestinal barrier damage in conditions such as IBD or experimental stress. In rats,
DSS-induced colitis was ameliorated by Lactobacillus reuteri decreasing the bac-
terial translocation from the intestine to mesenteric lymph nodes [209]. E. coli
Nissle 1917 has been shown to confer protection against murine DSS colitis-
associated increase in mucosal permeability through up-regulation of ZO-1 expres-
sion [210]. Moreover, a probiotic mixture of Lactobacillus acidophilus,
Bifidobacterium lactis, Lactobacillus plantarum and Bifidobacterium breve helped
to maintain the integrity of colonic mucosal barrier in the DSS model by down-
regulating macrophage nitric oxide production and by enhancing mucus production
[211]. In this model, the administration of a probiotic mixture prevented not only
the decrease in TJ proteins expression, but also the increase of epithelial apoptotic
ratio induced by acute colitis [212]. Furthermore, in patients with severe pouchitis,
probiotics were able to restore the mucosal barrier, as they decreased E. coli K12
passage through the intestinal epithelium in Ussing chambers [213].
Probiotics also play a role in stress-induced intestinal damage and psychiatric
comorbidity. Lactobacillus farciminis has been shown to suppress stress-induced
hyperpermeability and endotoxemia, and to prevent HPA axis response and
neuroinflammation in rats submitted to partial restraint stress [214]. Probiotic
administration to mice submitted to food and mobility restriction increased IgA
producing cells, CD4+ cells in the lamina propria of the small intestine, and
secretory IgA in the lumen and also reduced the levels of IFN-γ
[215]. Bifidobacterium lactis CNCM I-2494 has been shown to suppress gut
hypersensitivity and colonic barrier disruption induced by partial restraint stress
in rats [216, 217]. In the last years, attention has been also pointed to the potential
role of microbiota in the pathophysiology of psychiatric disorders such as depres-
sion and anxiety [218] and neurodevelopmental disorders such as autism. Interest-
ingly, treatment with the human commensal Bacteroides fragilis restores gut
permeability, alters microbial composition, and ameliorates defects in communi-
cative, stereotypic, anxiety-like and sensorimotor behaviors in a mouse model of
the autism spectrum disorder [219]. Since psychiatric comorbidities are highly
common in functional gastrointestinal disorders, the emerging role of microbiota
and probiotics in the regulation of intestinal and brain barrier function and its
implication in behavioral changes in the host certainly will boost investigations in
this field in the years ahead.
Inflammatory Mediators
Several inflammatory mediators have been involved in intestinal barrier regulation.

In vitro experiments with epithelial cell monolayers demonstrated that interferon-γ
and TNF-α induce epithelial barrier dysfunction through MLCK up-regulation and
MLC phosphorylation [220, 221], although they can also disrupt intestinal perme-
ability through down-regulation of occludin transcription [222] and up-regulation
of the channel-forming TJ protein claudin-2 expression. In addition, TNF-α but also
IL-1β have been shown to inhibit electrogenic sodium absorption in the rat distal
colon [223], and mice injected with TNF-α present diarrhoea as a consequence of
Na+/H+ exchange inhibition [224].
Similarly, IL-13 and IL-4 increased paracellular permeability in a dose- and
time-dependent fashion and IL-4, but not IL-13, stimulated chloride secretion in
T84 cells [225] through a PI3K pathway [226]. In contrast, IL-10 has been identi-
fied as a protector cytokine in barrier function as the addition of this cytokine to T84
cells prevents interferon-γ-induced disruption of T84 monolayer barrier integrity
and limits chloride secretion [227]. Moreover, IL-10 deficient mice display
increased intestinal permeability [228] and most importantly, develop spontaneous
colitis [229], suggesting that increased permeability predisposes to intestinal
inflammation.
Although, beyond the limits of this chapter. It is to know that many other
cytokines have been involved in barrier function such as IL-17A, IL-17F, IL-22,
and IL-26, interferon-α, interferon-β, transforming growth factor-α, and -β [230,
231].
Nutritional Factors
Some dietary compounds are able to induce intestinal barrier dysfunction in

susceptible individuals such as in celiac disease and food allergy. The gliadin
fraction of wheat gluten is the environmental triggering of celiac disease. In
genetically predisposed subjects gluten exposure may lead to increased intestinal
permeability and inflammation. Recent evidence has shown that the increase in
intestinal permeability occurs through the activation of the zonulin pathway in a
MyD88-dependent fashion [232]. The protein zonulin is the target of the Zot toxin
of the V. cholerae and has been show to play a pivotal role in TJ regulation in
different autoimmune disorders such as type 1 diabetes and celiac disease
[233]. Food allergies are adverse reactions against food antigens that are IgE and
mast cell mediated. Altered intestinal permeability has also been involved in the
pathophysiology of food allergy, as these patients display an enhancement of
intestinal permeability even in the absence of food allergens [234]. Moreover,
patients under tacrolimus treatment have been shown to develop new-onset food
allergies that could be related to tacrolimus-induced increase in intestinal
permeability [235].
94 C. Alonso et al.
In contrast with these observations, several diet products such as glutamine or

butyrate have been shown to exert a protective effect on the intestinal barrier.
Butyrate, a short chain fatty acid produced by intestinal microbial fermentation of
dietary fibres, maintains intestinal barrier function through an increase in mucus
production [236] and an enhancement in TJ protein expression [237]. Glutamine
has also been shown to protect intestinal barrier function through the regulation of
TJ proteins such as claudin-1, and occludin [238].
Drugs and Toxins
Ethanol has been shown to promote separation of ZO-1 proteins in Caco-2 mono-
layers and disassembly and displacement of perijunctional actin and myosin fila-
ments from the perijunctional areas and MLCK activation [239]. Recent findings
point to one of its metabolites, acetaldehyde, as the main toxic product for intestinal
barrier because it raises tyrosine phosphorylation of ZO-1, e-cadherin, and
β-catenin [240]. Further investigations revealed that the deleterious effects of
ethanol require the presence of resident microflora, to oxidize ethanol into acetal-
dehyde in situ, and downstream mast cell activation [241], and that the ethanol-
mediated increase in intestinal permeability is modulated through iNOS-mediated
activation of RhoA [242] and IL-22 [243].
NSAIDs can increase intestinal permeability. Several factors play a role in
NSAIDs-induced intestinal barrier dysfunction. In vitro experiments with gastric
epithelial monolayers showed that barrier dysfunction was associated with
decreased expression of claudin 7 and involved phosphorylation of p38 MAPK
[244]. NSAIDs also affect intestinal barrier through inhibition of intestinal epithe-
lial restitution by decreasing calpain activity and membrane-associated expression
of calpain-2 [245], and also through the increase of intestinal NO synthase [246].
Other drugs causing intestinal barrier dysfunction appear in Table 4.2. It is of
particular interest the development of new drugs, such as larazotide, that may
decrease intestinal permeability in celiac disease by acting on TJs.
Other Disorders Associated with Barrier Dysfunction
Many other conditions such as chronic kidney disease [247], type 1 diabetes [248],
primary biliary cirrhosis and primary sclerosing cholangitis [249], liver cirrhosis
[250], alcoholic liver disease [251], autoimmune thyroiditis [252] and IgA nephrop-
athy [253] have been associated with TJ dysfunction. In addition, some life
threatening conditions have been related to intestinal barrier dysfunction and
translocation of bacteria or/and endotoxin from gastrointestinal tract. In this line,
hemorrhagic shock has been associated with increased intestinal permeability and
bacterial translocation [254] through mucus damage and the generation of free
radical species [255]. Estrogens exert a protective role against hemorrhagic shock-
induced gut and lung injury by the activation of estrogen receptor-α, β or both [256]
Table 4.2 Drugs and intestinal barrier

Drug Effect on permeability References
Ethanol Increase [239, 241]
NSAIDs Increase [244, 245]
Methotrexate Increase [300]
Corticosteroids Increase [301]
Omeprazole Increase [302]
Cyclophosphamide Increase [303]
Tacrolimus Increase [304, 305]
Vitamin D Decrease [306]
Larazotide/AT1001 Decrease [307–310]
Anti-TNF monoclonal antibodies Decrease [311]
Heparin Decrease [312]
receptors. Similarly, gut inflammation and loss of gut barrier function has been
related to splachnic ischemia-reperfusion through HIF-1 activation [257]. Multiple
injured patients also show an increased intestinal permeability that correlates with
IL-6 levels [258]. Severe burn injury also results in the loss of intestinal barrier
function involving MLCK-dependent MLC phosphorylation signalling pathway
[259] and p38 MAPK activation [260] in a TLR-4-dependent process [261].
Intestinal Barrier and Disorders of the Brain-Gut Axis
The pathophysiology of several gastrointestinal disorders involves intestinal barrier

dysfunction and dysregulation of brain-gut interactions, particularly functional
gastrointestinal disorders including IBS and functional dyspepsia. In recent years,
due to new imaging techniques, such as positron emission tomography, it has been
possible to characterize the role of the CNS in modulating gut motility and visceral
pain in patients with functional gastrointestinal diseases. There is significant over-
lap between the brain regions responsible for modulating visceral sensitivity and
regions involved in emotion processing in these patients. IBS patients display a
higher activation of the anterior cingulate cortex in response to rectal distension
[262] that correlates with the presence of psychosocial disorders when compared to
healthy subjects [263].
At the peripheral level, mucosal inflammation, increased intestinal permeability
and visceral hypersensitivity are findings associated with clinical manifestations of
IBS. Mast cells play a key role in IBS pathophysiology because they modulate
intestinal permeability, and target visceral afferents involved in abdominal pain
[155]. Stress has been associated with the development, exacerbation and perpet-
uation of IBS through the brain-gut-axis. Early life stress plays a major role in the
vulnerability of individuals to develop IBS in adult life [264–266]. Post-traumatic
stress syndrome or sexual abuse are also important risk factors in the development
of IBS and functional gastrointestinal disorders [267] and both acute psychological
96 C. Alonso et al.
and physical stress have been associated with enhancement of visceral sensitivity
[268] and small-intestine motility in IBS [269].
Functional dyspepsia is characterized by postprandial fullness and early satiation
or by epigastric pain or burning in the absence of an organic cause Functional
dyspepsia has been show to share some of the pathophysiological features of IBS.
Particularly, patients with functional dyspepsia display low-grade inflammation in
the duodenal mucosa, characterized by an increased infiltration of mucosal mast
cells and eosinophils, and increased duodenal permeability [270]. Acute gastroen-
teritis has been shown to be a risk factor for functional dyspepsia development
[271], as well as the presence of psychosocial comorbidities such as anxiety and
depression [272], and life stress [273].
Stress, acting through the brain-gut axis, also modulates intestinal inflammatory
conditions such as IBD. Social Gibbon monkeys submitted to social upheaval
develop spontaneous colon inflammation [274]. Intracolonic infusion of TNBS
induced a significantly higher inflammatory reaction in maternally deprived rats
than in control animals [275]. Collins et al. [276] found that rats recovering from
TNBS-induced colitis and submitted to mild restraint stress displayed a significant
increase in myeloperoxidase activity. Moreover, overt inflammation was induced
when animals were exposed to stress in combination with a small dose of TNBS,
suggesting an additive effect [277]. In keeping with these findings, a significant
association between stress and relapse in IBD has been reported, especially in
patients with ulcerative colitis [278, 279]. Although the mechanism underlying
the association between stress and IBD remains unclear, disturbances of brain-gut
axis, peripheral neuroendocrine-immune interactions and altered intestinal barrier
function [280–284] have been demonstrated in IBD patients [285].
Finally, a heterogeneous group of conditions associated with chronic manifes-
tations affecting the CNS and the gut may possibly reflect the existence of primary
or secondary alterations of brain-gut axis, intestinal microbiota and barrier function.
This is the case of diabetes and the metabolic syndrome [286], liver encephalopathy
[287], neuropsychiatric disorders [288], autism [289], chronic fatigue [290] or
fibromyalgia [291], although the ultimate pathophysiological mechanisms are not
well known.
Acknowledgements Supported in part by the Fondo de Investigación Sanitaria and Ciberehd,

Instituto Carlos III, Subdirección General de Investigación Sanitaria, Ministerio de Ciencia e
Innovación (PI12/00314, Alonso, C.; CM08/00229, Lobo, B; CM10/00155, Pigrau, M; CP10/
00502, PI13/00935, Vicario, M; PI11/00716 & CB06/04/0021, Santos, J.).
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Chapter 5
Vagal Pathways for Microbiome-Brain-Gut
Axis Communication
Paul Forsythe, John Bienenstock, and Wolfgang A. Kunze
Abstract There is now strong evidence from animal studies that gut microorgan-
ism can activate the vagus nerve and that such activation plays a critical role in
mediating effects on the brain and behaviour. The vagus appears to differentiate
between non-pathogenic and potentially pathogenic bacteria even in the absence of
overt inflammation and vagal pathways mediate signals that can induce both
anxiogenic and anxiolytic effects, depending on the nature of the stimulus. Certain
vagal signals from the gut can instigate an anti-inflammatory reflex with afferent
signals to the brain activating an efferent response, releasing mediators including
acetylcholine that, through an interaction with immune cells, attenuates inflamma-
tion. This immunomodulatory role of the vagus nerve may also have consequences
for modulation of brain function and mood.
What is currently lacking are relevant data on the electrophysiology of the
system. Certainly, important advances in our understanding of the gut-brain and
microbiome- gut-brain axis will come from studies of how distinct microbial and
nutritional stimuli activate the vagus and the nature of the signals transmitted to the
brain that lead to differential changes in the neurochemistry of the brain and
behaviour.
Understanding the induction and transmission of signals in the vagus nerve may
have important implications for the development of microbial-or nutrition based
therapeutic strategies for mood disorders.
P. Forsythe
Medicine, McMaster University, Hamilton, ON, Canada
J. Bienenstock (*)
Pathology and Molecular Medicine, Brain Body Institute, McMaster University, St. Joseph’s
Healthcare, Room T3304, Juravinski Tower, 50 Charlton Avenue, E, Hamilton, ON, Canada
L8N 4A6
e-mail: Bienens@mcmaster.ca
W.A. Kunze
Psychiatry and Behavioural Neuroscience, St. Joseph Healthcare, Hamilton, ON, Canada
116 P. Forsythe et al.
Abbreviations
5-HT 5-Hydroxytryptamine
CCK Cholecystokinin
CRF Corticotropin-releasing factor
DHA Docosahexaenoic acid
DRG Dorsal root ganglia
DSS Dextran sodium sulfate
EPA Eicosapentaenoic acid
FDA Food and Drug Administration
GABA Gamma-aminobutyric acid
GI Gastrointestinal
GLP-1 Glucagon-like peptide-1
HPS Hypothalamic-pituitary-adrenal
IGLE Intraganglionic laminar vagal afferent ending
IKCa Calcium dependent potassium channel
IPAN Intrinsic primary afferent neuron
LPS Lipopolysaccharide
MDD Major depressive disorder
mRNA Messenger RNA
NTS Nucleus of the solitary tract
PSA Polysaccharide
PUFA Polyunsaturated fatty acids
PYY Peptide YY
TNF Tumor necrosis factor
The Vagus Nerve
The vagus (tenth cranial nerve) innervates the pharynx, larynx and visceral organs.
While it contains both motor and sensory fibres, it is the main afferent pathway
from the abdominal cavity to the brain. Information from the heart, lungs, pancreas,
liver, stomach and intestines are delivered tonically to the brain via sensory fibres in
the vagus nerve [1]. Sensory vagal inputs arrive in the nucleus of the solitary tract
(NTS) via the nodose ganglion which is chiefly made up of sensory visceral afferent
fibres. From there fibres ramify to widespread areas of the CNS, including the
cerebral cortex and medulla oblongata.
There are 30,000–80,000 vagal afferent nerves that supply the intestine with a
9:1 ratio of afferent to efferent fibres in peripheral nerve bundles [2–4]. Vagal
primary afferents innervate the muscular and mucosal layers of the gut with the
5 Vagal Pathways for Microbiome-Brain-Gut Axis Communication 117
coeliac branch supplying the intestine from proximal duodenum to the distal part of
the descending colon [5]. Vagal innervation is densest proximally but is still
significant in the colon.
Histological and electrophysiological evidence indicates that visceral afferent
endings [3] in the intestine express a diverse array of chemical and
mechanosensitive receptors [2]. The chemosensitive receptors are the targets of
gut hormones and regulatory peptides such as ghrelin, CCK, GLP-1 and PYY(3–
36) that influence the control of food intake and regulation of energy balance
[2]. Vagal afferent fibres have been identified in the lamina propria of duodenal
and jejunal villi, and crypts of Lieberkühn, but they do not cross the basal mem-
brane to innervate the epithelial layer [5]. Thus, vagal afferents are not in a position
to sense luminal nutrients directly unless they arrive intact in the lateral intercellular
spaces, but are in close anatomical apposition to the basal membrane of
enteroendocrine cells [6].
Intraganglionic laminar vagal afferent endings (IGLEs) are located in the con-
nective tissue capsule of myenteric plexus ganglia, between the longitudinal (outer)
and circular (inner) muscle layers. These fibres respond to muscle tension generated
by both passive stretch and active contraction of the muscle layers [7]. This type of
vagal afferent ending is found in large numbers throughout the oesophagus and
gastrointestinal tract and is thought to be important for generating vagal afferent
tone which has been associated with balanced interoceptive awareness and emo-
tional well-being. Furthermore experimental data suggesting that changes in vis-
ceral sensation can affect the perception and interpretation of external inputs [8, 9]
has led to the suggestion that altered sensory vagal inputs can influence our attitude
to the outside world. The anterior insular cortex is involved in interpretation of most
if not all interoception, and therefore through these vagal and other inputs, repre-
sents most of our subjective feelings. It is suggested that pathological changes in
sensory vagal inputs may increase the risk of affective behavioural disorders.
Chronic sensory vagal inputs might then act as ‘natural’ breaks for augmentation
of stress-related behavioural responses via tonic modulation of the neuronal activity
in the locus coeruleus and in turn the forebrain [10].
The Vagus and Behaviour
Some of the earliest indication of the role of the vagus in modulating behaviour
came from studies of animals exposed to endotoxin. Sickness behaviour is a term
used to describe the motivational state responsible for re-organizing perceptions
and actions to enable ill individuals to cope better with infection [11]. The associ-
ated behaviours include lethargy, depression, anxiety, loss of appetite, sleepiness,
hyperalgesia, and reduction in grooming. These behavioural changes are mediated
by proinflammatory cytokines particularly IL-1β and TNF [11].
The role of vagal afferents in the induction of sickness behaviour following
intraperitoneal administration of the cytokine inducer lipopolysaccharide (LPS) or
IL-1β has been assessed in laboratory animals that have been submitted to
subdiaphragmatic vagotomy [12, 13] and these responses have been shown to be
entirely dose responsive [14].
A dose of IL-1β or the cytokine inducer lipopolysaccharide (LPS) that induced
consistent sickness behaviour in sham-operated animals was no longer able to
decrease social exploration in vagotomised rats and mice [15, 16]. In the same
manner, vagotomy blocked the depressing effects of LPS on food-motivated
behaviour in mice [17].
In contrast to the role of the vagus in mediating sickness and depressive type
behaviour it is also emerging that stimulating the vagus can lead to a reduction in
anxiety and depression associated behaviours. In one study, rats were exposed to
vagus nerve stimulation for 30 min per day for 4 days, and were then subjected to
the forced swim test, a well validated assessment of anti-depressant activity. Vagus
nerve stimulation significantly reduced immobility time compared to unstimulated
controls, reflective of antidepressant effects [18]. Interestingly, the vagal nerve
stimulation-induced decreases in immobility were associated with increased swim-
ming behaviour, which has been linked to a predominantly serotonergic mechanism
of action [19]. In a subsequent controlled trial, rats received desipramine or vagal
nerve stimulation for 2 h at three time points over a 24 h period, prior to undergoing
the forced swim test and both treatments resulted in reduced immobility compared
to saline control [20]. However, chronic vagal nerve stimulation for 1 month failed
to show any behavioural alterations in rats subjected to the forced swim test or the
elevated plus maze test, in contrast to treatment with a classical anti-depressant,
imipramine [21]. No careful timecourse or analysis of different dose and timing
schedules of stimulation appear to have been conducted.
Clinically, vagal stimulation has been used successfully in the treatment of
refractory epilepsy [22] and is also an FDA approved alternative treatment for
intractable depression. While this treatment for depression is controversial, largely
due to a lack of positive sham treatment controlled clinical trials, there have been
reports that vagal nerve stimulation is beneficial in at least some patients with
depression and may be particularly effective with chronic treatment [23, 24].
The Vagal Anti-Inflammatory Reflex
The vagus innervates tissues known to participate in immune functions and/or

contain important immune elements, such as thymus, lung, liver, and gastrointes-
tinal tract. Furthermore, trunks or branches of the vagus are often associated with
lymph nodes that drain regions in which immune activation occurs. The functional
relevance of vagal innervation of immune tissue has been highlighted by the
identification of a neural circuit that controls the inflammatory response in a
reflex-like manner. In this system it is suggested that the vagus nerve senses
inflammation sending afferent signals to the brain thus activating an efferent
response, releasing mediators including acetylcholine that, through an interaction

with immune cells, attenuates inflammation.
This area was first explored by Levine and colleagues [25] who suggested that
gut vagal afferents sent signals to the brain and that as a consequence, vagal
efferents could inhibit various nociceptive as well as inflammatory peripheral
events such as bradykinin induced plasma extravasation in joints. However Tracey
and colleagues were the first to highlight and delineate the anti-inflammatory role of
the vagus and its mechanism of action. They showed that direct electrical stimula-
tion of the distal end of a subdiaphragmatic sectioned vagus nerve prevented the
development of shock in rats through the inhibition of TNF synthesis by macro-
phages [26]. They considered that inhibition of macrophage function is mediated by
Ach released by the vagus acting on specific alpha7nicotinic receptors expressed by
the immune cell. Similarly, macrophages have been suggested to be the main target
of the anti-inflammatory function of the vagus nerve in a murine model of inflam-
matory bowel disease (IBD) [27]. However evidence supports the fact that this
reflex may not be monospecific for alpha7 and can also be mediated via other
nicotinic receptors such as alpha5. In addition to suppressive effects on macro-
phages the vagus nerve also acts to regulate T cell function. O’Mahony et al. [28]
demonstrated that transfer of CD4+ T cells from vagotomized donors into non-
vagotomized mice with DSS induced colitis, reduced the number of splenic
Foxp3+ regulatory T cells in recipient animals, and was associated with aggravated
disease symptoms mimicking the effects of vagotomy on colitis. Sub diaphragmatic
vagotomy leads to a dramatic increase in T cell proliferation and production of
inflammatory cytokines when compared to cells from sham-operated animals
[29]. The effect of vagotomy was not limited to the spleen as lymphocytes isolated
from the mesenteric lymph nodes also demonstrated a significant increase in
inflammatory cytokine production. Overall these data suggest that CD4+ T cells,
in addition to macrophages, are also under tonic inhibitory control from the vagus.
Further revisions of the definition of this important function of the efferent vagus
have recently been published. The source of the acetylcholine involved in this reflex
may not be coming from the vagus but norepinephrine stimulated memory T cells
[30], in keeping with the papers listed above [28, 29]. Furthermore B cells in
addition to T cells can respond to stimulation by cholecystokinin through release
of acetylcholine which controls recruitment of neutrophils but not adaptive immune
function [31]. Thus the vagus nerve is intimately involved in many immunoregu-
latory functions via a number of different cholinergic receptors and through a
number of different immune cell types.
These anti-inflammatory efferent responses may be important and play a role in
the regulation of mood in healthy conditions as well as in psychiatric disease. They
may also mediate the anti-depressive effects of vagal nerve stimulation as outlined
before. Immune system dysfunction has been linked to depression [32–34]. Approx-
imately one-third of people with depression, without co-morbid diseases, have
higher levels of inflammatory markers such as TNF and C-reactive protein, com-
pared with the normal, non-depressed population [35]. Furthermore, inflammatory
illnesses are associated with greater rates of major depression, while patients treated
with cytokines such as interferon for various illnesses, are at increased risk of
developing major depressive illness. Conversely, successful treatment with an
antidepressant decreases levels of pro-inflammatory cytokines such as IL-6 and
TNF [36–38]. While it is as yet unclear whether neurostimulation therapies for
depression affect immune function, there is evidence in vagal nerve stimulation
treated epilepsy patients that pro-inflammatory cytokine levels were reduced with
successful treatment [39, 40].
The huge population of bacteria in the gut, known collectively as the gut
microbiome, is largely responsible for the generation and control of the major
immunoregulatory pathways that exist to respond to and control external challenges
[41]. As we will discuss subsequently in this chapter, there is evidence that
commensal bacteria in the gut can directly or indirectly modulate the activity and
function of the enteric nervous system and thereby the brain and its functions,
including behaviour. Therefore, taken together, the gut microbiome and the vagus
nerve may be influencing the brain via various mechanisms. Indeed the inappro-
priate lack of regulation of inflammation in major depressive disorder (MDD) may
be put at the door of a possible imbalance or dysbiosis of the gut microbiome which
if rebalanced, might be expected to restore the proper functioning of the central
nervous system [42]. This argument is an extension of the “hygiene hypothesis” that
many autoimmune, immune and allergic diseases which have recently been shown
to be having such epidemic prevalence [43, 44] are doing so as a result of mankind’s
attention to cleanliness and the eradication of bacteria. In the main, these arguments
are based on the evidence that it is likely that evolutionary change in diet, nutrition,
environmental factors such as urbanization, concepts of cleanliness and the use of
antibiotics may all have conspired to change our previous balanced gut microbiome
to one which is not as favourable to immune regulation as it used to be.
Many chronic diseases are associated with mild or moderate inflammation, the
evidence for which is present through increase in levels of biomarkers in the blood
and also in the tissues themselves (e.g. activated macrophages in obesity). The
source(s) of the inflammatory changes noted in association with depression and
anxiety are not known, but it has been noted that stress itself is accompanied by
evidence of proinflammatory cytokine elevation both experimentally and in clinical
conditions and is at least one of the possible causes of raised inflammatory bio-
markers [45]. Increased hypothalamic-pituitary-adrenal (HPA) axis activity in
chronic stress is known to also be associated with MDD and anxiety syndromes.
In this regard it is pertinent that ingestion of beneficial bacteria has been associated
with attenuation of HPA axis hyperactivity [46] and also responses to acute stress
[47, 48].
Dietary Fatty Acids and the Vagus
Long- and short-chain fatty acids both activate rat jejunal vagal afferent nerve fibers
but do so by distinct mechanisms [49]. Short-chain fatty acids such as butyric acid
have a direct effect on vagal afferent terminals while the long-chain fatty acids
activate vagal afferents via a cholecystokinin (CCK) dependent mechanism. Inter-
action between long-chain fatty acids and the vagus results in activation of the
cholinergic anti-inflammatory pathway [50]. Luyer et al. demonstrated that admin-
istration of high fat nutrition reduced circulating levels of TNF and IL-6 in rats
subjected to hemorrhagic shock. When these experiments were repeated in
vagotomized animals, the administration of the high fat diet no longer prevented
the increase in TNF and IL-6 [50]. In addition, nicotine receptor antagonism
blocked the ability of dietary fat to suppress the cytokine increase. Similarly,
deafferentation abrogates the protective effects of lipid-rich nutrition on systemic
inflammation and loss of intestinal integrity following shock [51]. Overall these
experiments provide strong evidence of a nutritional anti-inflammatory pathway
whereby the intake of dietary fat suppresses cytokine release through activation of
peripheral afferent vagus nerves that in turn initiate the cholinergic anti-
inflammatory response. In keeping with the findings of Lal et al. [49] the mecha-
nism underlying the protective effects of long chain fatty acids include a role for
CCK. Administration of CCK receptor antagonists and specifically antagonists of
the peripheral CCK-1 impaired the fat-induced suppression of the shock
response [50].
Clinically, studies indicate that dietary n-3 PUFA levels and n-3 PUFA supple-
mentation are related to improved heart rate variability suggesting increased vagal
tone [52, 53]. The relationship between the immunomodulatory actions of n-3
PUFA and their effects upon vagal tone has yet to be established. However a
number of studies have associated control of inflammation with heart rate variabil-
ity in humans [54–57] and it is possible that diet-induced activation of the cholin-
ergic anti-inflammatory pathway contributes to the reduced mortality from sepsis
and organ damage following early enteral feeding in trauma and surgery patients
[58–60].
It has also been suggested [61] that this nutrient activated neural feedback loop
could help maintain unresponsiveness of the GI tract to luminal antigens allowing
the intestine to perform the dual roles of sensing and absorbing essential nutrients
while protecting against invasion from potentially damaging agents.
A number of studies have indicated that long chain fatty acids, and particularly
n3-PUFAs such as EPA and DHA, may have potential as therapy or adjunctive
treatment in depression and that such effects could also be related to an ability to
modulate HPA activity. In animal models, feeding DHA to rats significantly
decreased immobility time in the forced swim test, a well validated indication of
anti-depressant activity. The DHA induced behavioural change was associated with
decreased CRF levels in the hypothalamus and pituitary tissues, an indication of
changes in HPA activity [62, 63]. In human studies, Jazayeri et al. [64, 65] reported
that fluoxetine and EPA were equally effective in controlling depressive symptoms
and that a fluoxetine and EPA combination was superior to either treatment alone.
EPA and fluoxetine, alone or in combination, also decreased serum cortisol after
8 weeks of treatment in depressed patients leading to the suggestion that EPA may
exert its therapeutic effects through reduction of HPA hyperactivity [65].
DHA mediated attenuation of HPA may be explained by the demonstration that
n-3 PUFAs, can act on GABAA receptors to potentiate GABAergic inhibitory drive
on CRF-secreting hypothalamic neurons [66]. In this regard, it is interesting to note
that the decreased anxiety and HPA response to stress of mice fed with Lactoba-
cillus rhamnosus is also associated with changes in the central GABAergic
system [48].
Bacterial Communication from Gut Lumen to Enteric

Nervous System (ENS)
Luminal probiotic bacteria may alter behaviour and brain biochemistry in a variety
of ways even in the absence of changes in the inflammatory status of the host
[67]. Bacterial products could enter the circulation to pass the blood-brain barrier if
they are sufficiently small and lipophilic [68], or they might enter the brain at the
circumventricular organs where the barrier is diminished. Since prior vagotomy
abolishes behavioural and brain biochemical changes induced by certain probiotic
bacteria [48, 67], afferent vagal signalling is a necessary condition for the central
effects of these neuroactive microorganisms.
The majority of sensory fibres innervating the intestinal mucosa derive from
intrinsic primary afferent neurons (IPANs) of the enteric nervous system (ENS) [69,
70]. Therefore, IPANs are a priori likely targets for the action of neuroactive
bacteria leading to alterations in gut motility. This has been substantiated by direct
experimentation. Nine day feeding of 109 JB-1 cfu caused an increase in the
intrinsic excitability of rat colon myenteric IPANs with a decrease in the post
action potential slow afterhyperpolarisation (relative refractory period)
[71]. These results have been replicated in an acute ex vivo preparation where
beneficial bacteria (JB-1 or Bacteroides fragilis) or its capsular polysaccharide
(PSA) were applied to the epithelium while whole cell recordings were made
from nearby IPANs [72]. Application of the bacteria or PSA evoked bursts of
action potentials in the IPANs in a sensory (non-synaptic) manner as was demon-
strated by blocking all synaptic transmission. The onset latencies of the initial
sensory responses were about 8 s which then led to an increase in IPAN intrinsic
excitability via entrainment of the reciprocally connected IPAN to IPAN network.
The increase in intrinsic excitability depended on IPAN to IPAN slow synaptic
transmission via G protein coupled receptors [72]. Because the specific intermedi-
ate conductance calcium dependent potassium channel (IK Ca) blocker TRAM-34
mimics the effect of JB-1 on IPAN slow afterhyperpolarisation and intrinsic
excitability we proposed that one of the molecular mechanisms involved may have
been the inhibition of IKCa channels [72, 73]. However, a detailed examination of
the effects of JB-1 and other probiotics vs TRAM-34 on neurally mediated prop-
agated motor complexes in the mouse intestine suggests that reduction in the open
probability of IKCa channels is not sufficient to explain the entirety of the neuronal
actions of these organisms and that additional ion channel targets or regulatory
molecules must be involved [74].
Because the addition of beneficial bacteria reduces IPAN slow afterhyperpo-
larisation and increases the intrinsic excitability, the question arises whether the
absence of gut bacteria altogether might have the opposite effect. In agreement with
this notion it has been established that IPANs taken from germ free animals have
reduced intrinsic excitability while the slow afterhyperpolarisation is exaggerated
beyond that seen in normal healthy animals [75]. This finding suggests that the
microbiome itself conditions the normal functioning of IPANs and therefore gas-
trointestinal motility.
Neuroactive bacteria might alter afferent vagal signalling via two broad sensory
modalities. Beneficial luminal bacteria might act on the enteric nervous system to
alter the contractile activity of the intestine [73, 74, 76, 77] and this would be sensed
by the intramuscular arrays and intraganglionic laminar endings both of which are
vagal mechanoreceptors [78, 79]. Closer to the lumen, the vagus innervates muco-
sal villi and varied epithelial layer cells [80] with endings that are both chemo-and
mechanosensitive [80, 81]. Vagal chemoreceptors could be activated directly by
substances such as short chain fatty acids that can be transported across the
epithelial barrier to the portal circulation [82] or by paracrine mediators like 5-
HT, histamine, CCK, ATP or glucagon-like peptides released by the various
mucosal epithelial layer taste cells [83, 84]. That vagal mucosal chemoreceptors
fibres might be involved in activation of the “microbiome-gut-brain axis” [83] is
substantiated by animal studies where beneficial bacteria were applied to the
epithelium at known concentrations and vagal nerve activity recorded.
In a pioneering study, intraduodenal injection of a Lactobacillus johnsonii strain
increased gastric vagus massed multiunit firing within 15 min of application
[85]. However, the authors did not identify single unit chemoreceptors fibres, nor
were they able to rule out potentially confounding effects of the probiotic on
duodenal motility. Intraluminal JB-1 application appears to reduce single dorsal
root fibre discharge [86] as well as blocking nociceptive sensitisation of DRG
neurons innervating the rat colon [87]. This is potentially important for behaviour,
as the caudal nucleus of the solitary tract receives input from the spinosolitary tract
[88, 89]. The caudal nucleus is the part that receives projections from nodose
neurons that innervate the intestines [90]. Perez-Burgos et al. [91] recently used
an ex vivo mouse jejunum preparation to record from perivascular mesenteric
nerves in real time while known concentrations of psychoactive JB-1 bacteria
were added to the luminal perfusate. 10 9 cfu/mL JB-1 increased the constitutive
firing rate of responsive individual mesenteric nerve single units by about 70 %
above baseline rate within 10–15 min of application. This effect persisted even
when contractions were abolished by blocking L calcium channels with
nicardipine, demonstrating that the fibres were chemoreceptors. Importantly, the

bacteria did not translocate during the period of the experiment. The chemoreceptor
fibres were multimodal since they were also strongly activated by raising
intraluminal pressure to 30 hPa when the musculature was allowed to contract,
and this response was further augmented by addition of JB-1 to the lumen. When
vagal fibres were eliminated by subdiaphragmatic vagotomy the excitatory effects
of JB-1 on single units was absent demonstrating that vagal but not spinal fibres
were activated.
These results have demonstrated for the first time that multimodal chemorecep-
tor vagal afferents acutely respond to luminal application of a psychoactive probi-
otic thus delineating the peripheral sensory projection and physical basis for the
bacteria’s effects on the brain and behaviour.
The Vagus and Gut Bacteria
There is now strong evidence from animal studies that gut microorganisms can
activate the vagus nerve and that such activation plays a critical role in mediating
effects on the brain and subsequently, behaviour.
Such evidence came early from the study of animals infected with pathogens.
Subdiaphragmatic vagotomy attenuated c-fos expression in the PVN of rats inoc-
ulated with Salmonella typhimurium [92]. Although S. typhimurium infection was
accompanied by intestinal inflammation subsequent studies have indicated that
microorganisms in the gastrointestinal tract can directly activate neural pathways
even in the absence of an identified immune response [93]. The anxiogenic effect of
orally administered subclinical doses of Campylobacter jejuni, in mice was asso-
ciated with a significant increase in c-Fos expression in neurons bilaterally in the
vagal ganglia and activated visceral sensory nuclei in the brainstem. The areas of
brainstem activation, the NTS and lateral parabrachial nucleus, participate in neural
information processing that ultimately lead to autonomic neuroendocrine and
behavioural responses [93].
Non-pathogenic bacteria also activate vagal signalling from gut to brain. Tanida
et al. [85] demonstrated that intraduodenal injection of the bacterial strain
L. johnsonii La1 reduced renal sympathetic nerve activity and blood pressure
while enhancing gastric vagal nerve activity. All of these effects could be abolished
by pre-treatment with a histaminergic H3-receptor antagonist. Similarly the effects
were absent in animals that had bilateral lesions of the hypothalamic
suprachiasmatic nucleus, a major regulator of circadian rhythm. These findings
suggest that the influence of the bacteria on autonomic neurotransmission and
subsequently blood pressure, is mediated centrally, likely through histaminergic
nerves and the suprachiasmatic nucleus [85].
Consequently, subdiaphragmatic denervation of vagal nerve fibers surrounding
the oesophagus eliminated the ability of L. johnsonii La1 to reduce renal sympa-
thetic nerve activity and blood pressure indicating that at least some of the effects of
this bacteria on autonomic nerve responses were elicited by interaction with

afferent vagal nerve fibers [85].
More recently, it was demonstrated that oral administration of a L. rhamnosus
strain (JB1) could alter the normal behaviour of adult Balb/c mice [48]. Chronic
treatment with the bacteria reduced anxiety-like behaviour as assessed in an
elevated plus maze, and decreased the time spent immobile in a forced swim test.
In addition, stress-induced plasma corticosterone levels were lower in treated mice,
a similar effect to subchronic or chronic treatment with antidepressants that can
prevent forced swim stress-induced increases in plasma corticosterone in both mice
and rats. Overall, changes induced with L. rhamnousus were indicative of reduced
anxiety, and decreased depression-like behaviour. Assessment of neural correlates
to behavioural changes determined that mice receiving L. rhamnosus had alter-
ations in central GABA receptor subunit mRNA expression. L. rhamnosus admin-
istration decreased expression of GABA type B (GABAB) subunit 1 isoform b
(GABAB1b) mRNA in the amygdala and hippocampus, while increasing expres-
sion in cortical areas. Expression of GABAAα2 receptor mRNA was reduced in the
amygdala and cortical areas, whereas levels were increased in the hippocampus
[48]. It is difficult to attribute a causal relationship between behavioural effects
observed and neural correlates. However, reduced expression of GABAB1b
mRNA, in the amygdala, hippocampus, and locus ceruleus is consistent with the
antidepressant-like effect of GABAB receptor antagonists [94] and with studies of
GABAB1b-deficient animals, indicating an important role for this subunit in the
development of cognitive processes, including those relevant to fear [95, 96]. The
experimentally induced changes, especially in GABAA receptors correlates with
those seen in benzodiazepine administration, a well characterized tranquillizer
[97]. It is also interesting to note that in a recent study of transcriptomes from the
mucosa of the proximal small intestines of healthy human subjects following
intraduodenal application of different Lactobacillus species, there was a strong
correspondence between in vivo transcriptional networks altered after consumption
of one of the strains, Lactobacillus casei, and the response of human cells to the
anxiolytic GABA A receptor modulator, Tracazolate [98].
Subdiaphragmatic vagotomy blocked the anxiolytic and antidepressant effects
of chronic L. rhamnosus ingestion in normal adult Balb/c mice while also
preventing the associated alterations in GABAAα2 mRNA expression in the amyg-
dala [48]. Similarly, the ability of B. longum to attenuate DSS colitis induced
anxiety was abolished by vagotomy [67]. Ingestion of the same bacteria had similar
effects on the behaviour of normal healthy mice. However not all beneficial bacteria
seem to exert their behavioural effects via the vagus [99]. These data suggest that
gut bacteria may affect the brain through both vagal, non-vagal and other possible
systemic pathways.
Given what is known of the vagal anti-inflammatory reflex it seems plausible
that gut microbiota induced modulation of vagal mediated “periphery to brain”
signalling could translate into changes in efferent neural pathways controlling
immune responses. As yet, there is no evidence that the vagus nerve contributes
to the immunomodulatory effects of gut bacteria and at least one study suggests that
the local protective effect of Lactobacillus and Bifidobacteria strains in models of

colitis does not depend on vagal nerves [100].
Conclusions
Overall, studies indicate that vagal pathways mediate signals that can induce both
anxiogenic and anxiolytic effects depending on the nature of the stimulus and,
interestingly, the vagus appears to differentiate between non-pathogenic and poten-
tially pathogenic bacteria even in the absence of overt inflammation.
It is therefore clear that the involvement of the vagus in microbiota-gut-brain
communication is not straightforward or simply dependent on “activation”. Even
with the dubious assumption that an increase in c-fos expression always reflects an
increase in neuronal firing rates, existing anatomical data cannot answer why in
some cases vagal activation causes depression and in others, for example, electrical
stimulation of the vagus, eases depression. What is currently lacking are relevant
data on the electrophysiology of the system. Tanida et al. [85] showed that injecting
L. johnsonii into rat duodenum increased gastric vagal multiunit firing rate by about
10 % within 15 min, and this slowly grew to a 90 % increase over the baseline 1 h
after the injection was delivered. Clearly, much more work of this sort needs to be
done and should compare with vagal responses to either anxiogenic or anxiolytic
peripheral stimuli.
Electrophysiology may also be utilized to determine the nature of the peripheral
signal acting to stimulate the vagus nerve in the gut following exposure to specific
bacteria or nutrients. Single chemosensitive vagal afferent units supplying the gut
are normally silent or have a low resting discharge of 0–3 Hz [101]. They respond to
most luminal molecules by increasing their firing rate. Response latencies vary
consistently according to the chemical nature of the stimulus. The short chain fatty
acid butyrate had a response onset latency of 2–3 ms [49], the long chain fatty acid
sodium oleate had a latency of 15 ms [49], amino acids evoked responses within
about 9 ms [3]. The response to casein acid hydrolysate has a latency of 19 ms
[102], and glucose takes 20 ms [103]. S. typhimurium lipopolysaccharide evoked an
increase in the mesenteric nerve discharge within 30 min while LPS from a
commensal E. coli had no effect [104].
Certainly, important advances in our understanding of the gut-brain and
microbiome- gut-brain axis will come from studies of how distinct microbial and
nutritional stimuli activate the vagus and the nature of the signals transmitted to the
brain that lead to differential changes in the neurochemistry of the brain and
behaviour. However, while it appears that the vagus is critical to mediating gut-
brain communication by specific bacteria in some model systems, it is by no
means the only potential signalling method. Indeed, largely due to technical
difficulties, few studies have investigated the role of spinal afferents in mediating
bacteria induced changes in behaviour and brain chemistry. It is certainly possible
that the observed changes in brain chemistry behaviour induced by gut bacteria
require parallel input from both the vagal and spinal afferents. Furthermore,
behavioural changes induced through disruption of the microbiota by antibiotic
treatment have been demonstrated to be independent of vagal signalling [99] with
some additional evidence that neither sympathetic afferents nor immune modula-
tion is required. This clearly suggests that the bacteria in the gut can communicate
to the brain through multiple pathways. Nevertheless understanding the induction
and transmission of anxiolytic signals in the vagus nerve may have important
implications for the development of microbial-or nutrition based therapeutic strat-
egies for mood disorders.
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Chapter 6
The Brain-Gut Axis in Health and Disease
Yasser Al Omran and Qasim Aziz
Abstract The interaction between the brain and the gut has been recognized for
many centuries. This bidirectional interaction occurs via neural, immunological and
hormonal routes, and is important not only in normal gastrointestinal function but
also plays a significant role in shaping higher cognitive function such as our
feelings and our subconscious decision-making. Therefore, it remains unsurprising
that perturbations in normal signalling have been associated with a multitude of
disorders, including inflammatory and functional gastrointestinal disorders, and
eating disorders.
Abbreviations
5-HT 5-Hydroxytryptimaine
ACC Anterior cingulate cortex
Ach Acetylcholine
ANS Autonomic nervous system
CCK1R Cholecystokinin 1 receptor
CRH Corticotropin-releasing factor
DMN Dorsal motor nucleus of the vagus
EMS Emotional motor system
FGF19 Fibroblast growth factor 19
Y. Al Omran
Centre for Digestive Diseases, Wingate Institute of Neurogastroenterology, Blizard Institute,
Barts and The London School of Medicine and Dentistry, Queen Mary, University of London,
London, UK
Q. Aziz (*)
Bart’s and The London NHS Trust, Centre for Gastroenterology, The Wingate Institute of
Neurogastroenterology, 26 Ashfield Street, London E1 2AJ, UK
e-mail: q.aziz@qmul.ac.uk
136 Y. Al Omran and Q. Aziz
fMRI Functional magnetic resonance imaging

GALT Gut-associated lymphoid tissue
GI Gastrointestinal
GLP1 Glucagon-like peptide-1
GPR G-protein coupled receptor
HPA Hypothalamic-pituitary-adrenal
KLB Klotho beta
NF- κB Nuclear factor κB
NPY Neuropeptide Y
OFC Orbitofrontal cortex
PAG Periaqueductal grey
PFC Prefrontal cortex
TNF-α Tumor necrosis factor-α
α7nAChR α7 nicotinic acetylcholine receptor
Introduction
The interaction between the brain and the gut has been recognized for many
centuries [1]. However, it was not until the nineteenth and early twentieth centuries
that this association was critically evaluated by prominent physiologists, psychia-
trists and psychologists [2–6]. From their studies, a bidirectional relationship was
assumed, with both a brain to gut modulation of gastrointestinal (GI) function, by
stress and emotions, and a gut to brain pathway that transmits physiological
information about gut sensory motor function. More recent work demonstrates
that these interactions occur via neural, immunological and hormonal routes.
Thus the brain-gut axis plays an important role in gut regulating physiological
function and its disruption may have pathophysiological consequences. The aim of
this chapter is to discuss the basic principles, the latest research and the significance
of the brain-gut axis in both health and disease.
Brain to Gut Signalling in Health
The brain signals to the viscera, including the GI tract, through a myriad of neural,
hormonal and immunological routes. These include the sympatho-adrenal axis and
hypothalamic-pituitary-adrenal (HPA) axis, the two branches of the autonomic
nervous system (ANS), and the monoaminergic pathways, which modify dorsal
horn excitability and spinal reflexes. The hypothalamus and amygdala are two main
subcortical structures that contribute to these routes. They receive inputs from a
number of cortical areas, including the anterior cingulate cortex (ACC) and the
6 The Brain-Gut Axis in Health and Disease 137
medial prefrontal cortex (PFC) [7]. The lateral PFC and the orbitofrontal cortex
(OFC) are additionally involved in this signalling process and transmit information
relating to homeostatic body states (such as food intake, visceral pain and gut
homeostasis) to the medial PFC, which integrates this information. The output
from this complex network is integrated into clear motor patterns that are projected
to the periaqueductal grey (PAG) [8] and then relayed to the raphe nuclei, the locus
coeruleus complex and the dorsal vagal complex in the pons and medulla. This
cortico-limbic-pontine network has been labelled the emotional motor system
(EMS), with the medial component regulating spinal reflexes related to GI function
and the lateral component integrating and modulating motor autonomic, neuro-
endocrine and pain related patterns [9, 10]. Both components thus modulate gut
function. For example, the medial component has been shown to modulate pain-
related behaviours in adult rats upon the consumption of food, through activation of
the descending serotonergic pain inhibitory pathways [11]. In contrast, the lateral
component may influence distinct visceral motor patterns through activation of the
regional ANS [12, 13]. This regional ANS can be activated in a plethora of ways; by
interoceptive feedback from the gut, descending emotional, cognitive, or during
intense periods of emotion such as anger fear and sadness [14, 15].
Role of the Sympathetic Nervous System
The effects of the sympathetic nervous system on GI function have been well
established. The gut receives sympathetic innervation from subclasses of post-
ganglionic vasoconstrictor, secretion suppressing, and motility suppressing neu-
rons, with the overall effect being to slow GI transit, motility and secretion. These
inhibitory effects are mainly fulfilled by modification of cholinergic transmission
and by stimulating sphincteric contractions on smooth muscle [16–18]. Addition-
ally, sympathetic innervation may be involved in modification of mucosal immune
systems [19], and in mucosa-microflora interactions [20, 21]. The best evidence for
this sympathetic-immune interaction comes from the spleen, but this interaction has
also been shown in Peyer’s patches (lymphoid nodules in the ileum), and in
non-follicular mucosa that is in close proximity to other classes of immune cells,
and which can influence immune-related activity [21].
Role of the Parasympathetic System
The influence of the parasympathetic nervous system on GI function have also been
extensively studied [17, 18]. These wide-ranging influences are initiated from vagal
and sacral efferents, which innervate foregut and hindgut structures respectively.
For example, in addition to providing input to the stomach, small intestine, and to
the proximal portion of the colon, vagal structures may provide input to ganglia
within the ENS to facilitate the cephalic phase of gastric secretion vago-vagal
motor reflexes, and to encourage the release of peptides and 5-hydroxytryptimaine
(5-HT; a serotonin precursor) containing granules from enteroendocrine and entero-
chromaffin cells respectively [22]. Also, like the sympathetic nervous system,
parasympathetic modulation of immune cells has been reported, with vagal modifi-
cation of macrophage activation thought to be part of the vago-vagal anti-inflam-
matory reflex [23]. This reflex is driven by efferent vagal fibres, the majority of
which originate from the dorsal motor nucleus of the vagus (DMN) in the medulla,
and has been shown to attenuate circulating levels of proinflammatory cytokines.
Overall, the brain to gut interaction (Fig. 6.1), involving sympathetic and para-
sympathetic neurons, most likely facilitates emotion-related alterations in secre-
tory, motor and immune related activity in the GI tract [24]. These alterations may
even be compared to emotion-related alterations in facial expression and posture
(facilitated by the medial EMS) to reflect mood and state of mind, perhaps signi-
fying just how strong this interaction normally is.
Gut to Brain Signalling in Health
The ENS has more than 200 million neurons and thus has been colloquially referred
to as the “second brain” [25]. This comparison is not surprising when considering
that this network covers an area that is 100 times larger than the human surface area
of skin and is home to approximately three-quarters of the human body’s immune
cells [25]. This extensive network has a bidirectional interaction with the brain, and
thus may influence it. These are achieved via endocrine, neuronal and immune
afferent signalling (Fig. 6.2).
Endocrine Signalling
With over 20 different types found in the body, enteroendocrine cells form the
largest endocrine organ [26]. They are involved in the modulation of digestive
functions through the ENS network and the modulation of endocrine and paracrine
signalling to vagal afferents. Mechanisms that enteroendocrine cells use to achieve
this include the activation of Ca2+ -dependent fatty acid acyl chains [27], presum-
able through activation of the G-protein coupled receptor (GPR) 40 [28], depolar-
ization through the opening of mechanosensitive cation channels on microvilli [29],
as well as G-protein-coupled taste receptors, and vagal efferents [22, 30].
An exceptionally well-studied type of enteroendocrine cell is the 5-HT releasing
enterochromaffin cell, which is partly responsible for why 95 % of an organism’s
5-HT is present in the gut [29]. It releases 5-HT on the basolateral side, and possibly
the luminal side, in response to shearing forces in the gut [29]. Released 5-HT is
Fig. 6.1 Brain to gut signalling. In response to internal events or environmental factors, specific
regions of the brain are activated, which may cause different effects depending on the stimuli. The
hypothalamic-pituitary-adrenal axis may be activated to initiate the release of adrenal hormones
such as catecholamines and glucocorticoids. Projections from these brain regions to brainstem
nuclei, may initiate vagal (parasympathetic) output or these may project to the spinal cord and
modulate interoceptive signals such as those relating to gastrointestinal spinal reflexes or pain
sensitivity. Also, depending on which spinal cord level is activated, there may be additional
parasympathetic or sympathetic outflow. These hormonal and neural outputs have the ability to
influence gastrointestinal targets such as immune cells, enteric smooth muscle, enteric neurons and
enteroendocrine cells. See text for details. Abbreviations: ACC anterior cingulate cortex, ACTH
adrenocorticotrophic hormone, Amyg amygdala, Hypo hypothalamus, Ins insula, latPFC lateral
prefrontal cortex, medPFC medial prefrontal cortex, OFC orbitofrontal cortex. Adapted from
[104]
needed to initiate peristalsis and secretomotor reflexes, and it is yet to be deter-

mined whether it may confer signalling in the central nervous system (CNS).
Neuronal Signalling
The neurons that innervate the GI tract can be divided into primary extrinsic (vagal
and spinal afferents) or primary intrinsic afferent neurons, with the intrinsic neurons
being much more abundant [31]. Both extrinsic and intrinsic neurons respond to
mechanical and chemical noxious stimuli, however it is suggested that luminal
signals do not influence afferents nerve terminals directly, they act through multiple
reflex loops to optimize gut function [32]. Notably, there are differences in both
extrinsic and intrinsic neurons with regards to the importance of these reflex loops,
Fig. 6.2 Gut to brain signalling. Gut to brain signalling is achieved through endocrine, neuronal
and immune routes. Mechanical and chemical information relating to the luminal environment is
signaled through extrinsic (vagal and spinal) primary afferent neurons to the brain. Intrinsic
primary afferent neurons are also present and although a direct synaptic connection between
extrinsic and intrinsic neurons has yet to be found, they may communicate through intraganglionic
laminar endings. Additionally, terminals of extrinsic primary afferent neurons are in close
proximity to immune and enteroendocrine cells. Both cell types, in conjunction with enteric
microbiota produce a variety of signalling molecules that can activate a number of receptors on
extrinsic primary afferent neurons. Therefore, endocrine, neuronal and immune signals are all
integrated and are sent to specific brain regions and may alter cognition, mood and emotions.
Abbreviations: 5-HT 5-hydroxytryptamine, CCK cholecystokinin, CRH corticotropin-releasing
hormone, GLP-1 glucagon-like peptide-1, Neuropeptide Y NPY. Adapted from [105]
depending on their location. The stomach is predominantly governed by vago-vagal

reflexes, thus signals arising from extrinsic and intrinsic neurons are relatively
weak. In the intestines, intrinsic primary afferent neurons and enteric motorneurons
are important for intestinal function afferents are much stronger in the intestine,
which are reliant on these signals [17]. The intrinsic primary afferents feed and
regulate signals relating to secretion, propulsion and blood flow indirectly to the
CNS, perhaps through specific myenteric ganglia, which receive input from these
neurons as well as extrinsic afferents, and relay information to spinal, mesenteric
and supraspinal reflex loops that span much larger distances in the bowel [13]. Inter-
estingly, some of these intrinsic afferents are normally unresponsive to mechanical
stimuli, and only become responsive during periods of inflammation [33].
Certain subsets of vagal afferents terminate with great proximity to entero-

endocrine cells. These terminals contain chemosensitive receptors, which are
responsive to the peptides released by these cells [34]. These include receptors
for orexigenic (hunger inducing) or anorexigenic (satiety inducing) peptides.
Emerging evidence suggests that together with the cholecystokinin 1 receptor
(CCK1R), the expression of these receptors can be modulated by diet or nutritional
status [35]. Additionally, the receptors for anorexogenic peptides were found to be
downregulated by fasting, while those for orexigenic peptides were upregulated,
creating a greater impulse to eat. These reports reflect the phenotypic plasticity of
vagal afferents depending on the homeostatic state [36].
Immune Signalling
Approximately three-quarters of the body’s immune cells are located in the GI tract
and is often referred to as the gut-associated lymphoid tissue (GALT), suggesting
that the gut may be considered as the body’s main defence organ. A single layer of
columnar intestinal epithelial cells forms the barrier between 100 trillion micro-
organisms and the host [37]. These microorganisms are essential for normal physio-
logical functions, as the absence of intestinal bacteria has been shown to
compromise the development of the GALT and the subsequent release of antibodies
[38, 39]. With the observation that intestinal immune cells remain predominantly
hyporeactive to the commensal bacteria that live in symbiosis within us, yet are
hyperreactive against pathogens, alludes to the fact that the intestinal immune
system can identify commensal bacteria from pathogens and generate appropriate
responses in order to maintain normal well-being [40]. As the epithelial layer
samples the luminal environment, lymphoid structures (including Peyer’s patches)
located in the lamina propria in the intestine deal with immune insults [40] through
specialised immune cells that sample antigens from or on microorganisms deliver
them to antigen processing cells in Peyer’s patches, and dendritic cells in the lamina
propria, which can extend their dendritic arbours through the epithelial tight
junctions to sample the luminal environment. These cells possess a myriad of
receptors that can recognise pathogen associated molecular patterns [41]. In addi-
tion to being in close proximity and having receptors that are responsive to the
products of enteroendocrine cells, vagal afferents are close to, and possess receptors
on their terminals that are responsive to immune cells and their products, namely
cytokines, proteases, 5-HT, corticotropin-releasing factor (CRH) and histamine
[42]. Moreover, immune cells may have functional effects on enteroendocrine
cells as has been demonstrated by the increased release of cholecystokinin in an
animal model of gut inflammation [43, 44].
Integrated Gut to Brain Signalling
Overall, there is a strong integration of the endocrine, neuronal and immune signals;
and these contribute in the transmission of information from the gut to the brain.
However, only a portion of these signals are normally consciously perceived.
However, a preponderance of evidence suggests that subconscious interoceptive
inputs, in conjunction with intestinal microbiota, may effect memory, cognition and
emotions [45]. For example, primary extrinsic afferents from the GI tract may
project via the vagus nerve to the solitary tract nucleus and through spinal; afferents
to laminae I, V, VII and X of the spinal cord. The projections arriving from
laminae I, V and the solitary tract are integrated in the parabrachial complex,
which is then transmitted to forebrain regions including the hypothalamus and
amygdala [46]. The latter of which has been reported to be involved with reward
based behavior and emotions, especially fear [47].
Furthermore, collaterals from these ascending extrinsic projections may make
further contact to the raphe nuclei and the locus coeruleus complex, and thus may
alter the release of 5-HT and noradrenaline respectively, which modify cortical
arousal. Projections from Lamina I specifically, have been found to activate the
ACC and the insula via the thalamic nuclei [48]. In fact, recent studies have
identified the insula as the most likely region for allowing integration of interocep-
tive information in emotional behavior [49, 50]. This interceptive information is
projected to regions of the brain depending on the origin of the signal. For example,
both sensory and visceral stimuli are registered in the middle and posterior insula,
while gustatory or olfactory inputs are registered in the middle and anterior insula,
suggesting that different insular regions may be responsible for the perception of
different interoceptive inputs from the GI tract [49, 50]. The activation of the
anterior insula, and to a lesser extent the ACC, has additionally been reported in
individuals who inhaled pungent odors producing strong feelings of disgust, or in
individuals who viewed video clips, showing the altered emotional facial expres-
sions, of others being disgusted by the odors. This suggests that at least at the level
of the anterior insula and the ACC, homeostatic reflexes can be activated in the
paucity of interoceptive input, and can be activated by the recollection of intero-
ceptive memories [51].
There is emerging evidence that interoceptive memories may develop during
infancy, when the gut to brain interactions are beginning to be molded, and positive
and negative feeding states are being established. For example, the response to
consuming something sweet has been associated with the activation of opioids
(associated with a feeling of pleasure) in both in mice and in children [52,
53]. These neurochemical programming of feeding states develop into adulthood
and may partially explain why ingestion of food that is high in calories is accom-
panied by a feeling of pleasure. On the other hand, consumption of potentially
harmful food may initiate nausea and vomiting (via 5-HT). In fact, gut-based
neurochemicals can signal satiety [via cholecystokinin, glucagon-like peptide-1
(GLP1), and neuropeptide Y (NPY)], hunger (via ghrelin) through enteroendocrine
cells; and can signal pain (via CRH, proteases and cytokines) through intestinal
immune cells. Overall, the enteroendocrine, neuronal and immune components of
gut-brain signal intermingle with one another and under normal circumstances have
great influence in shaping normal homeostatic functions in different aspects of
physiology.
Acute Perturbations in Signalling
Substantial evidence suggests that the bidirectional brain-gut interaction can be

perturbed leading to acute physiological repercussions. For example, upon acti-
vation by possible noxious stimuli such as chemotherapeutic drugs or bacterial
toxins, enterochromaffin cells may increase their production of 5-HT, which may
activate 5-HT3 receptors on both extrinsic and intrinsic afferents. This may result
not only in hypersecretory and hypermotor reflexes, but also in the activation of
brain regions that receive input from ascending afferent pathways. Overactivation
of these pathways may be associated with nausea and vomiting in order to expel the
harmful contents out of the body. Another example includes vagal-mediated acti-
vation of the hypothalamus and limbic brain regions following the release of
proinflammatory cytokines in the liver and gut. This results in “sickness responses”
that include, fever, depression and withdrawal from usual activity [54]. Addition-
ally, a myriad of inflammatory mediators including cytokines, proteases and neuro-
peptides may be released by mucosal immune and glial cells, which may result in
sensitization of both nociceptive and innocuous ascending spinal pathways, thus
amplifying the perception of visceral pain [55, 56]. Finally, the CRH signalling
system influences brain-gut signalling. The release of CRH is a normal physio-
logical reaction to stress but may be pathologic if there it is overproduced [57]. Its
effects, being endocrine, behavioural, autonomic and visceral, may also be
reproduced if administered directly into animal brains [58]. The release of CRH
is associated with increased anxiety-like behaviours in animals; and there is also
decreased GI secretory and motor responses and acceleration of distal bowel
movements, which reduces luminal contents in the GI tract and thus GI metabolic
demand. This can result in greater distribution of blood to the skeletomotor and
gastrointestinal system for the flight and flight response. It was thought that binding
of CRH to the CRH1 receptor might cause these effects [14]. Notably, although
these perturbations are usually acute, if severe, they may contribute to chronic
diseases.
Chronic Perturbations in Signalling
Perturbations in chronic diseases affect multiple signalling pathways along the

brain-gut axis. This makes it difficult to posit specific perturbations to the specific
chronic diseases. Additionally, although many disease states may be related to
altered signalling along the brain-gut axis, convincing evidence is limited to a
few. Taken together, the following section will reflect on well-established pertur-
bations that may render chronic diseases.
Functional Gastrointestinal Disorders (e.g. Irritable Bowel

Syndrome)
Functional gastrointestinal disorders form a constellation of disorders characterized

by chronic discomfort and pain along various locations in the GI tract, and occur
without discernable physical, biological or anatomical abnormalities that would
explain their symptoms [59]. Over 40 different disorders have been postulated
including functional dyspepsia, chest pain of oesophageal origin and irritable
bowel syndrome (IBS); all of which share similar symptoms [59]. However, out
of all these disorders, IBS is the most common and most extensively studied [60].
Symptoms specific to irritable bowel syndrome are related to abnormal colonic
transit and rectal evacuation such as chronic constipation, diarrhoea and anismus
[61, 62]. A number of mechanisms have been found that may cause these symp-
toms, including luminal and mucosal irritants that alter mucosal permeability,
overactivation of immune systems, which subsequently alter motor and sensory
conditions in the GI tract.
The increased presence of undigested food in the lumen has been found to be a
trigger for the onset of IBS. These include: undigested fats [63], carbohydrates (that
lead to the production of short-chain fatty acids) [64]; bile acid malabsorption
leading to the production of more bile acid [65]; modifications of bacterial
populations in the gut, which may increase colonic secretion and have been
associated with mental disease in IBS [66]. These luminal factors and exogenous
chemicals trigger the release of several amines and peptides from enteroendocrine
cells. However, the abnormal release of 5-HT has been well characterized in IBS
[67]; increased 5-HT levels increases motor, sensory and secretory functions and is
associated the diarrhoea-predominant IBS, while the decreased 5-HT levels causes
the opposite effect and is associated with constipation-predominant IBS [68]. Acti-
vation of immune cells may also be involved; as an increased expression of T-
lymphocytes in the rectal mucosa has been associated with increased intestinal
permeability in a subset of patients with IBS [69]. Genetic predispositions to
develop IBS has also been associated with mucosal irritants. For example, a genetic
variation of KLB (Arg728Gln) leads to impaired KLB synthesis and prevents
fibroblast growth factor 19 (FGF19) bind to the KLB-FGFR4 receptor on
hepatocytes. This reduces the bile acid synthesis negative feedback, causing the
release of more bile acid, which is associated with diarrhoea-predominant IBS [70].
There is also evidence that CNS hypervigilance may be involved in IBS. One of
the first IBS related symptom reported in children is increased mucosal permeabi-
lity, which may also be triggered by stress [71]. When CRH was placed on the
serosal surface of colonic mucosal biopsy specimens in healthy individuals, there
was an increased mast cell-mediated uptake of horseradish peroxidase (used as a
marker of permeability) [72]. Increased permeability was also seen when indivi-
duals were subjected to a cold stimulus [73]. The connection between increased
intestinal permeability and IBS has been supported by observations that increased
permeability leads to inflammation and the activation of local reflex pathways,
which lead to increased visceral sensations [74]. Additionally, several non-gastro-
intestinal comorbidities have been associated with IBS, such as fibromyalgia and
temporomandibular disorder, which are known to be exacerbated by stress; and the
fact that these conditions and IBS may be treated with psychopharmacological
agents such as tricyclic antidepressants and serotonin/noradrenaline reuptake inhi-
bitors substantiates the brain’s involvement in IBS [75]. Overall, there appears to be
bidirectional interaction in the development of IBS, with both interoceptive signals
from the gut and peripheral and central sensitization mechanisms associated with
modified cognition and altered descending signals to the gut.
Inflammatory Bowel Disease
Inflammatory bowel disease (IBD) encompasses a group of inflammatory disorders

that affect the GI tract, but the two most common disorders are Crohn’s disease and
ulcerative colitis. Overwhelming evidence suggests that this disorder is caused by
exaggerated responses to enteric microorganisms in a genetically susceptible host
[76], but the brain-gut axis may be involved in modulating these responses
(Fig. 6.3).
Like in IBS, stress may play a pathological role in IBD through a plethora of
mechanisms. The products of mast cells, including numerous cytokines and chemo-
kines, may activate terminals on sympathetic spinal primary afferent neurons
[77]. However, during times of stress there is an increase in circulating level of
catecholamines that, through α- and β- adrenergic receptors, activate the pro-
inflammatory nuclear factor κB (NF- κB) signalling pathway and increase peri-
pheral and central proinflammatory cytokines production, thus potentiating
inflammatory insults [78]. Conversely, an anti-inflammatory role of vagal efferent
pathways may reduce inflammation in IBD. Acetylcholine (Ach), released at the
by vagal efferent terminals have been shown to limit the production of pro-
inflammatory cytokines such as IL-1, IL-6 and tumor necrosis factor-α (TNF-α)
through activation of the α7 nicotinic acetylcholine receptor (α7nAChR) on the
surface of macrophages [79] and thus attenuated intestinal inflammation [80].
In fact chronic vagal nerve stimulation agonists has been shown to improve IBD
Fig. 6.3 Brain-gut axis in inflammatory bowel disease (IBD). IBD is thought to be a result of
inappropriate responses of enteric immune cells against the intestinal microflora. The brain gut
axis is involved in modulating these responses. Activation of the sympatho-adrenomedullary axis
results in an increase of catecholamines, which activate receptors on immune cells and cause an
increased release of inflammatory cytokines. The terminals of vagal efferents are believed to
induce an anti-inflammatory effect via the release of acetylcholine on α7 nicotinic acetylcholine
receptor (α7nAChR), expressed on the surface of macrophages. Corticotrophin-releasing hormone
(CRH) released from the hypothalamus can be attenuated by stress, leading to an attenuated
release of plasma corticosterone; its paucity has been associated with IBD. Also depression and
anxiety have been shown to increase susceptibility to develop IBD. Alternatively, the gut may
signal to the brain via interoceptive feedback that activates descending pain inhibitory systems,
which in turn reduces pain in IBD (not shown). Abbreviations: ACTH adrenocorticotrophic-
releasing hormone, CRH corticotropin-releasing hormone
symptoms [80], and thus may be a potential alternative to anti-TNF therapy, which
is currently the gold standard treatment of moderate to severe IBD [81]. However,
stress decreases vagal outflow, and together with the increased levels of catechola-
mines, there is a greater shift towards intestinal inflammation [82]. This may be
achieved by decreased activity of the PFC, which is known to control the para-
sympathetic tone through modulation of vagal outflow [83], as well as the enhanced
activity of the amygdala which is strongly associated with stress [84]. The abnormal
activity of the PFC and the amygdala results in an imbalance between stimulation of
the HPA axis and the ANS, which favors greater proinflammatory conditions [85].
The CRH signalling system is additionally affected by stress. Chronic colitis
attenuated CRH gene activation in the parvocellular neurosecretory neurons in the
hypothalamus, cells that produce CRH and dampened the plasma corticosterone
responses to acute environmental stressors [86]. A decreased HPA axis response is
associated to a susceptibility to autoimmune and inflammatory disorders, and thus
decreased CRH production predisposes to developing IBD [87].
Early life events may influence stress and the development of IBD. Neonatal
inflammation has been shown to employ long-term effects in immune regulation
and HPA activity [88]. The modulation of stress may harbor a deleterious role of the
brain in controlling peripheral immunity. Separation of rat pups from their mothers
has been used as a model of early life stress. It produced life long abnormal
hyperactivity in the HPA and CRH signalling system, and predisposed rats to
visceral hypersensitivity, increased defecation, increased penetration of bacteria
into the lamina propria and increased levels of anxiety [89, 90], and these may be
due to modulation of immunological responses and microbiota in the
intestines [91].
A causal relationship between depression in maternal separation model and the
hypersecretion of proinflammatory cytokines and mediators has also been proposed
[92]. Mice separated from their mothers at birth exhibit a pattern of behavior
reminiscent of depression, and are more vulnerable to inflammation. This relation-
ship is further supported by the fact that treatment with tricyclic antidepressants
reversed depressive-like behavior [93]. Additionally, depression has been shown to
increase the susceptibility to inflammation under baseline conditions and during
periods of stress [94]. However, it may be that the comorbidities of depression and
anxiety are auxiliary effects of the main IBD pathology; causality has yet to be
determined [95].
Our knowledge of the brain-gut axis in IBD is not limited to preclinical research,
brain-imaging studies on patients have also been revealing. Altered sensory experi-
ences are commonly described in patients with IBD; but unlike IBS, persistent
visceral hyperalgesia and abdominal pain are variably reported [96]. Using func-
tional magnetic resonance imaging (fMRI), cortical or subcortical areas of acti-
vation or deactivation were assessed upon exposure to rectal pain in patients with
IBD or in controls. Analysis revealed that the control and IBD groups showed
distinctive profiles of response [97]. In particular, activation of both central and
peripheral pain inhibitory areas was more prominent in IBD patients which may be
a possible mechanism for the lack of visceral hyperalgeisa in IBD patients
[98]. Overall, there appears to be a strong interaction between the brain and gut
in the modulation of sensations and functions in IBD.
Eating Disorders
Eating disorders are common across society; yet it is still not understood how an
assortment of outcomes and situations, including diet, exercise and infections, may
result in syndromes such as obesity, anorexia nervosa and bulimia, which harbour
many societal problems relating to morbidity, mortality and healthcare costs
[99]. Although the causes of these disorders are likely to be multifactorial, the
brain-gut axis may have a role to play.
Obese individuals seem to eat beyond their caloric requirements, suggesting that
there is an imbalance between homeostatic and hedonic regulation of food intake.
This may be due to modulated peripheral signalling processes in the gut, which may
encourage greater food take. For example, diet-induced attenuation of gut to brain
signals relaying satiety-triggering processes have reported to be affected. These
include modulations in cholecystokinin-dependent molecular processes, and the
development of insulin and leptin resistance. These cause a switch from an anorexi-
genic to an orexigenic phenotype [100]. Hedonic causes are also present. Imaging
studies suggest that obese subjects may have compromised dopaminergic pathways
that regulate neuronal systems related to reward sensitivity, conditioning and
control. Provision of food cues (such as viewing or imagining high calorie foods)
induced an exaggerated response in the dopaminergic pathways, however actual
food intake produced an attenuated response [101]. As many gut produced peptides,
including leptin, ghrelin and insulin have the ability to activate the central dopa-
mine pathways, it seems likely that the impairments in satiety responses observed
may be also due to modulated interoceptive feedback back to the brain.
On the other hand, individuals with anorexia nervosa or bulimia have an
impaired perception of self-image, which drives an obsession with weight loss
and a preoccupation with food or food rituals [102]. Although behavioral and
brain abnormalities have been reported, potential modifications in the brain-gut
axis are not fully understood. However, it has been shown that upon oral sucrose
provision, patients with anorexia nervosa had reduced activity in the anterior insula,
striatum and ACC. This implies that interoceptive signals from the GI tract that
activate dopaminergic pathways might be dysfunctional in these disorders [103].
Conclusion
A substantial amount of progress has been made with regards to our understanding
of the brain-gut axis. This includes delineating exact functional neuroanatomical
processes, mechanisms and pathways of the ENS, and both how the brain modu-
lates gut function and how the gut modulates activity across different brain regions.
These signalling patterns are important in health and their perturbation may con-
tribute to specific disorders that are associated with chronic pain, gut inflammation,
psychosocial stressors and eating disorders. There are a multitude of unanswered
questions including what role the enteric microbiota may have in signalling.
Through close collaboration with clinical neurophysiologists, neuroradiologists,
physicists and even other specialties, gastroenterologists may be able to delve
deeper into unknown areas of physiology and pathophysiology and make further
advances in our understanding of the gut-brain axis in health and disease.
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Part II
Mechanistic Factors Influencing the
Microbiota-Gut-Brain Axis
Chapter 7
Gastrointestinal Hormones and Their
Targets
Jens F. Rehfeld
Abstract Gastrointestinal hormones are peptides released from endocrine cells

and neurons in the digestive tract. More than 30 hormone genes are currently known
to be expressed in the gastrointestinal tract, which makes the gut the largest
hormone producing organ in the body. Modern biology makes it feasible to
conceive the hormones under five headings: The structural homology groups a
majority of the hormones into nine families, each of which is assumed to originate
from one ancestral gene. The individual hormone gene often has multiple pheno-
types due to alternative splicing, tandem organization, or differentiated maturation
of the prohormone. By a combination of these mechanisms, more than 100 different
hormonally active peptides are released from the gut. Gut hormone genes are also
widely expressed in cells outside the gut, some only in extraintestinal endocrine
cells and neurons but others also in other cell types. The extraintestinal cells may
synthesize different bioactive fragments of the same prohormone due to cell-
specific processing pathways. Moreover, endocrine cells, neurons, cancer cells,
and, for instance, spermatozoa release the peptides differentially (autocrine, endo-
crine, neurocrine, paracrine, spermiocrine secretion etc.), so the same peptide may
act as a blood-borne hormone, a neurotransmitter, a local growth factor, or a
fertility factor. The molecular targets of each bioactive peptide are specific G-
protein coupled receptors expressed in the cell membranes of different target cells.
Also the target cells of gut hormones occur widespread outside the digestive tract.
J.F. Rehfeld (*)

Department of Clinical Biochemistry, Rigshospitalet, University of Copenhagen, 9
Blegdamsvej, 2100 Copenhagen, Denmark
e-mail: jens.f.rehfeld@regionh.dk
158 J.F. Rehfeld
Abbreviations
CCK Cholecystokinin
CGRP Calcitonin gene related peptide
EGF Epidermal growth factor
G-cells Gastrin-producing cells
GIP Gastric inhibitory peptide (later renamed glucose-
dependent insulinotropic polypeptide)
GLP-1 and -2 Glucagon-like peptide 1 and 2
IGF Insulin-like growth factor
L-cells GLP-producing cells
mRNA Messenger ribonucleic acid
NPY Neuropeptide Y
PP Pancreatic polypeptide
PTHrP Parathyroid Hormone-related Protein
PYY Peptide YY
TGF-α (alpha) and -β Transforming growth factor α (alpha) and -β (beta)
(beta)
TSH Thyroidea-stimulating hormone
VIP Vasoactive intestinal polypeptide
Historical Introduction
The bloodborne regulation by specific messenger molecules was discovered in

1902 in London by Bayliss and Starling [1]. Following up on the observation that
acidification in the upper small intestine, the duodenum, stimulated pancreatic
secretion, Bayliss and Starling extracted from the duodenal mucosa a substance
that released bicarbonate from the denervated pancreas when injected into blood.
They gave this substance a very broad name, secretin. In 1905, John Edkins (also
from London) suggested that extracts of the antral mucosa [2] contained an acid
stimulatory messenger (“gastric secretin”—or simply gastrin). Hence, the first two
bloodborne “chemical messengers” to be known in mammalian biology, secretin
and gastrin, were both of gastrointestinal origin. Subsequently, also in 1905,
Starling proposed the word hormone as a general designation for bloodborne
messengers [3].
In the following decades, however, other types of hormone came into focus—
steroids from the adrenals, ovaries, and testes; protein hormones from the pituitary
gland; the thyronins from the thyroid gland; and insulin from the pancreas. The
clinical implications and often life-saving effects of these discoveries made the
interest for secretin and gastrin fade in the darkness of the bowels. Subsequently,
only a small priesthood of physiologists continued to study the hormonal control of
digestion. One of them was Andrew Ivy in Chicago, who with his assistant, Eric
Oldberg, found a gallbladder emptying hormone [cholecystokinin (CCK)] in
7 Gastrointestinal Hormones and Their Targets 159
extracts of the small intestine [4]. A stimulator of pancreatic enzyme secretion

(pancreozymin) was discovered 15 years later by Harper and Raper in Newcastle
[5]. But Jorpes and Mutt showed in the 1960s in Stockholm, however, that CCK and
pancreozymin were one and the same peptide hormone [6] for which the acronym
CCK is now used.
Secretin, gastrin, and CCK constitute the classical troika of gastrointestinal
hormones, but since the early twentieth century, many more have been discovered
(Fig. 7.1). In order not to lose overview, this chapter summarizes all the gut
hormones, some of their targets, and their major biological activities in Tables 7.1,
7.2 and 7.3, but otherwise presents the general principles governing structure and
biogenesis of gastrointestinal hormones and their receptors (see also [7, 8] for
longer reviews). Readers interested in details about individual hormones, their
targets, receptors, and their effects, should consequently consult multi-author
volumes comprising the full range of gastrointestinal endocrinology [9–11]. Also,
a shorter review on the history of gastrointestinal endocrinology has recently been
published [12].
Comparative Aspects of the Development
Life in multicellular organisms began as a simple tube with only one opening. Take
coelenterates, for instance (Fig. 7.2). They live in water that runs into their lumen,
and from which nutrients are absorbed into the epithelial cell-lining. Coelenterates
have a regulatory system of singular primitive neurons spread out in the wall.
Apparently, these neurons release small regulatory peptides. Thus, multicellular
life began as an isolated ‘gut’ whose function was controlled by regulatory or
hormonal peptides. Consequently, viewing the phylo- and ontogenetical develop-
ment of life, evolutionists could say that the specific organs and tissues in vertebrate
organisms are derivatives of the primordial multicellular structure, the gut. Accord-
ingly, the regulatory or hormonal peptides of the gastrointestinal tract are from the
beginning essential caretakers of life; also human life and its disorders.
General Features of Gastrointestinal Hormones
The Structural Homology
Gastrointestinal endocrinology currently encompasses a large number of hormones,

neuropeptides and growth factors. Not only have new hormones been found in gut
extracts, but also peptides from the central nervous system and hormones first
identified in other endocrine organs have been found in endocrine cells and/or
neurons in the gastrointestinal tract. Moreover, peptides originally believed to be
160 J.F. Rehfeld
Fig. 7.1 Discovery and identification of regulatory peptides in the gastrointestinal tract 1900–
2000. Discovery is indicated by year of first report. Solid circles indicated structural identification,
and open circles indicated hormonal activities that still require structural identification. Some of
the unidentified hormonal activities are explained by later identified hormones. For instance, the
incretin activity is partly due to gastric inhibitory polypeptide (GIP) and glucagon-like peptide I
(GLP-I). Commonly used acronyms are indicated in brackets after full name, except for PACAP,
which is an acronym for pituitary adenylate cyclase-activating peptide
classical hormones but later shown to be neurotransmitters have been isolated from
gut extracts. Finally, a number of growth factors have now been found in the gut—
epidermal growth factor (EGF), originally isolated as the gut hormone, urogastrone,
from urine; insulin-like growth factors (IGF) I and II; transforming growth factors
(TGF)-α (alpha) and -β (beta); amphiregulin, and others.
The complexity is increased through individual genes for gut regulatory peptides
encoding different peptides released in a cell-specific manner. Several principles
for gene expression operate to provide such variety. Hence, alternative splicing of
the calcitonin gene transcript to express CGRP is not the only example [13]. Also,
the secretin gene is expressed in different molecular forms due to alternative
splicing [14, 15].
Additional studies indicate that there are still hormonal activities in the gut that
are not structurally identified. Perhaps some of the activities can be explained by
already identified peptides. Hence, the hormonal stimulation of insulin secretion
from the gut, originally called incretin, is today explained by at least two hormones,
GIP and truncated GLP-1—probably in combination with other gut hormones,
including gastrin and CCK peptides (for review, see [16]), whereas intestinal
Table 7.1 Peptide hormone, neuropeptide and growth factor families in the gastrointestinal tract
and the pancreas
Families and members Major regulatory activity
Secretin family
Secretin Stimulates pancreatic bicarbonate secretion
Glucagon Increases glucose production and amino acid metabolism
Glucagon-like peptide Stimulates insulin and inhibits glucagon secretion and gastric
1 (GLP-1) emptying
Glucagon-like peptide Stimulates mucosal cell growth in intestinal crypts
2 (GLP-2)
Gastric inhibitory polypeptide Enhances glucose-stimulated insulin secretion and inhibits gas-
(GIP) tric secretion
Vasoactive intestinal polypep- Inhibits gastrointestinal motility and stimulates fluid secretion
tide (VIP)
Peptide histidine isoleucine VIP-like actions
(PHI)
Growth hormone releasing Stimulates growth hormone secretion
hormone
Pituitary adenylyl cyclase- Contributes to the regulation of gastric acid secretion and gas-
activating peptide trointestinal motor function
(PACAP)
Gastrin family
Gastrin Stimulates gastric acid secretion and gastric mucosal cell growth
Cholecystokinin (CCK) Stimulates pancreatic enzyme secretion, cell growth, and gall-
bladder emptying, but inhibits gastric acid secretion
Caerulein Not expressed in Cholecystokinin-like activities
Cionin mammals
Tachykinin family
Substance P Stimulates motility
Neurokinin A Stimulates motility
Neurokinin B Stimulates motility
Ghrelin family
Ghrelin Stimulates appetite and growth hormone secretion
Obestatin Suppresses food intake (?)
Motilin Contracts gastrointestinal smooth muscles to stimulate motility
PP-fold family
Pancreatic polypeptide (PP) Involved in feeding behavior (?)
Peptide YY (PYY) Reduces gastric emptying, pancreatic exocrine secretion, and
delays intestinal transit
Neuropeptide Y (NPY) Modulates the contractility in smooth muscle cells
Somatostatin family
Somatostatin Inhibits gastric acid, gastrin secretion and other gut functions
through endocrine, paracrine, and neurocrine release
Cortistatin Somatostatin-like activities
Insulin family
Insulin Establishes energy resources in fat, liver and muscle cells
Insulin-like growth factor I Stimulates growth and differentiation in interaction with other
(IGF-I) growth factors
(continued)
162 J.F. Rehfeld
Table 7.1 (continued)

Families and members Major regulatory activity
Insulin-like growth factor II Stimulates growth and differentiation in interaction with other
(IGF-II) growth factors
Relaxin Function in the gastrointestinal tract uncertain
EGF family
Epidermal growth factor (EGF)Stimulates growth of epithelial cells and inhibition of gastric acid
secretion
Transforming growth factor α EGF-like activities
(TGFα)
Amphiregulin Growth regulation of epithelial cells
Heparin-binding EGF-like EGF-like activities
growth factor
Opioid peptide family
Enkephalins Modulates transmitter activity from nerveplexes
β-endorphins Modulates transmitter activity from nerveplexes
Dynorphins Modulates transmitter activity from nerveplexes
Table 7.2 Singular peptide hormones, neuropeptides, and growth hormones in the gastrointesti-
nal tract
Hormones and growth factors Major regulatory activity
Apelin Stimulates gastric mucosal growth and cholecysto-
kinin secretion
Bradykinin Contributes to control alkaline secretion in the
duodenal mucosa
Calcitonin gene-related peptide (CGRP) Modulates blood flow, secretion, and motility
Cocaine and amphetamine regulated tran- Increases satiety
script (CART)
Galanin Stimulates motility and luminal secretion
Gastrin-releasing peptide (GRP) Stimulates antral gastrin secretion
Neurotensin Increases the ileal brake
Orexin Stimulates gut motility (?)
Transforming growth factor β (TGFβ) Growth, differentiation, and inflammation
Thyrotropin-releasing hormone (TRH) Releases TSH from epithelial cells in the gut
inhibitory effects on stomach secretion, the gastrone effects, may be explained by

combinations of CCK, somatostatin, GIP, and EGF. However, the villikinin,
duocrinin, enterocrinin, and the more recently suggested gastrocalcin [17] still
await structural identification. At present there is, however, evidence that
gastrocalcin may be PTHrP (the parathyroidhomone-related protein) known to be
expressed as a paracrine regulator of differentiation and local intercellular signaling
[18]. The multiplicity of gut hormones may jeopardize an overview of gut endo-
crinology. Structural identifications, however, have shown striking homologies
between groups of peptides. Consequently, many of the biologically active pep-
tides, hormones, neuropeptides, and growth factors in the gastrointestinal tract can
be classified into nine families (Table 7.1). The expression of several hormone
Table 7.3 Receptors and receptor subtypes for some gastrointestinal hormones
Hormones Receptors and subtypes
Atrial Natriuretic Peptide (ANP) NPA, NPB, NPC
Brain Natriuretic Peptide (BNP) NPA, NPB, NPC
C-type Natriuretic Peptide (CNP) NPA, NPB, NPC
Calcitonin Calcitonin-R
Calcitonin Gene-Related Peptide (CGRP) CGRP1, CGRP2
Cholecystokinin (CCK) CCKA, CCKB
Gastric Inhibitory Polypeptide (GIP) GIP-R
Gastrin Gastrin/CCKB
Gastrin-Releasing Peptide (GRP) CGRP-R
Ghrelin Ghrelin-R
Glucagon-Like Peptide-1 (GLP-1) GLP-1-R
Motilin Motilin-R
Neurotensin NTR1, NTR2, NTR3
Parathyroid Hormone-related Protein (PTHrP) PTH-R
Pituitary Adenylate Cyclase Activating Peptide (PACAP) PAC1
Peptide Tyrosyl Tyrosyl (PYY) Y1, Y2, Y3, Y4, Y5
Secretin Secretin-R
Somatostatin sst1, sst2A, sst2B, sst3, sst4, sst5
Substance P NK1, NK2, NK3
Vasoactive Intestinal Polypeptide (VIP) VPAC1, VPAC2
genes both in the gut and pancreas reflects the intestinal origin of the pancreas. The
nature of the homology varies. It may be an overall similarity in the primary
structure as, for example, the PP-fold family. The similarity of the tertiary structure
in this family is due to homologous residues necessary for stabilization of the three-
dimensional structure [19].
Another type of homology is that of the gastrin family which, in addition to
mammalian gastrin and CCK, also consists of the protochordean neuropeptide
cionin [20] and frogskin peptide cerulein [21]. The decisive homology of this
family is concentrated in the primary structure around the active site, the common
C-terminal tetrapeptide amide sequence, -Trp-Met-Asp-Phe-NH2. Comparison
between propeptide and gene structures also reveals some similarity, but the family
is still defined primarily by the conserved active site sequence and by neighboring
O-sulfated tyrosyl residues.
The frequent occurrence of homology among hormones, neuropeptides, and
growth factors is not specific for bioactive peptides in the gut. It is a common
feature among all kinds of regulatory peptides, enzymes, and other proteins in the
organism [22]. Each family is assumed to reflect the phylogenetic evolution by
duplication and subsequent mutations of an ancestral gene.
The phylogenetic story shows that gastrointestinal hormones are indeed very
old, several hundred million years [23]. So far, the data also support the idea that
each hormone family has evolved from a single ancestor. An associated trait is that
gastrointestinal hormones have, to a large degree, preserved their tissue-specific
164 J.F. Rehfeld
Fig. 7.2 Scheme of the

structure of coelenterates
with endoderm (En),
ectoderm (Ek), footplate
(Fp), gaster (stomach Ga),
mouth (M ), and tentacles
(Te)
sites of expression during evolution, both in the primary and secondary sites
[24]. Accordingly, the evolution emphasizes the general significance of gut hor-
mones as intercellular messenger molecules.
At present, a few bioactive peptides in the gastrointestinal tract have no relatives
or family (Table 7.2). Time will show whether gut peptides still awaiting discovery
will show homologies with these peptides.
The Multiple Phenotypes
Three decades ago, one gene was believed to encode one hormonal polypeptide in
accordance with what we have learned about the master hormone, insulin. How-
ever, more intricate dimensions were added when it became obvious that a single
hormone gene often expresses several different bioactive peptides. Today, we know
three ways in which a gut hormone gene can express different hormonal peptides.
Alternative Splicing of Transcripts
Alternative splicing was discovered when it was shown that calcitonin gene tran-
scription generates mRNAs encoding either calcitonin peptides or calcitonin-gene
related peptides (CGRPs) [13]. CGRPs are now known also to be abundantly
expressed in intestinal neurons (see [25] for review). Moreover, a tachykinin gene
transcript [26] and the transcript encoded by the secretin gene [14, 15] are also
spliced alternatively in the gut. For many years, secretin was believed to exist only
as a carboxyamidated peptide of 27 amino acid residues [27]. However, in the
mid-1980s, two additional secretins with full bioactivity were identified in porcine
gut extracts. One was the immediate precursor of amidated secretin-27, glycine-
extended secretin-28, and the other was secretin-30 extended by a Lys-Arg
sequence. The existence of glycine- and glycyl-lysyl-arginine-extended forms of
secretin and the related VIP is not surprising. They are to be expected from what is
known about the biosynthesis of carboxyamidated peptides. The discovery of
secretin-71 [14], which contains the sequence of nonamidated secretin-27 N-termi-
nally, followed by a Gly-Lys-Arg extension and a further C-terminal extension of
41 amino acid residues did, however, come unexpectedly (Fig. 7.3). With the
exception of an arginine residue, the C-terminal sequence of secretin-71 is identical
to the C-terminal 40-amino-acid residue fragment from porcine preprosecretin.
Thus, the sequence that corresponds to secretin RNA encoding a 32-amino acid
sequence has been spliced out from the primary secretin gene transcript. For
reasons mentioned above, secretin-71 has full secretin bioactivity.
Multiple Products of Prohormones with One Active Sequence
The somatostatin and gastrin families represent peptide systems in which the gene
encodes only one prohormone that contains only one active site, but where the
prohormone is processed in a way to release peptides of different lengths with the
same active C-terminus. Although the different bioactive products of the same
precursor are bound to the same receptor, their varying clearances from plasma
affect their hormonal significance considerably. Hence, it matters whether intestinal
proCCK is processed mainly to CCK-58 or to CCK-8 (Fig. 7.3), or whether
prosomatostatin is processed to somatostatin-28 or -14. So far, the biosynthesis of
gastrin in antral G-cells has been examined particularly thoroughly. It is, therefore,
a useful illustration of the second way in which one gastrointestinal hormone gene
can encode different bioactive peptides (for reviews, see [7, 28]).
166 J.F. Rehfeld
Fig. 7.3 Multiple phenotypes of three gut hormone genes. The cholecystokinin (CCK) gene
encodes a prepropeptide which is processed to six CCK peptides varying in length from 83 to
8 amino acid residues through differentiated endoproteolytic cleavage. The six peptides have the
same C-terminal bioactive octapeptide sequence. The secretin gene encodes a prepropeptide that
through endoproteolytic cleavages and variable C-terminal trimming is processed to three bioac-
tive secretin peptides of almost similar size (secretin-27, -28, and -30). In addition, bioactive
secretion-71 is produced by splicing out RNA, encoding the midsequence of preprosecretin
(i.e. broken line of secretin-71). The glucagon gene encodes a prepropeptide that through cell-
specific endoproteolytic cleavages is processed to either genuine pancreatic glucagon
(in pancreatic α-cells) or to glucagon-like peptides I and II (GLP-I, GLP-II) [7]
Differential Processing of Prohormones Containing Two or More Active

Sequences
A third way in which one gene can express different bioactive peptides occurs when
the gene encodes a propeptide containing different but often homologous peptide
hormones or neuropeptides. Gastrointestinal hormones and neuropeptides comprise
many examples of such genes of which the opioid-peptide genes, some of the
tachykinin genes, the VIP gene, and the glucagon gene amply illustrate the phe-
nomenon. Some of the genes not only encode a peptide precursor containing
different bioactive peptides, which is then subjected to tissue-specific posttransla-
tional processing, but the primary transcripts of these gene(s) may also undergo
tissue-specific alternative splicing [26].
Proglucagon is an example of a poly-protein precursor that contains three similar
but still different peptide sequences in mammals (Fig. 7.3). In pancreatic islet α-
cells, proglucagon is processed to release the well-known pancreatic glucagon,
whereas the C-terminal part of proglucagon remains silent in that neither GLP-I nor
GLP-II is synthesized [29, 30]. The L-cells of the gut also express proglucagon but
process it in a different way to release GLP-I and GLP-II [29, 31]. Although
glucagon and, for instance, GLP-I are highly homologous peptides, and both are
glucoregulatory, they have separate activities and receptors. Proglucagon also tells
another story of interest. Deduction of its structure from cloned cDNA provided the
first evidence or suggestion of separate bioactive peptide moieties from the same
precursor due to the homologies between the sequences 33–61, 72–107, and 126–
158 [32]. Physiological studies, however, showed that the first deduced GLP-I
(proglucagon 72–107), which is situated between two dibasic sites in the precursor,
is a poorly active peptide. Instead, a truncated form of the original GLP-I, which
corresponds to the proglucagon sequence 78–107, turned out to be a highly potent
peptide [31, 33]. Thus, bioactive peptide structures cannot be predicted from cDNA
and precursor sequences. This also requires exact identification of the released
peptides accompanied by physiological studies of their activities.
Widespread Gene Expression
For gastrointestinal hormones, the expression cascade is elaborate and involves

multiple processing enzymes with cleavages and derivatizations. Each step may
control whether the initial gene transcription results in a bioactive peptide product.
Transcription can occur without translation of the transcript, and lack of parallelism
between mRNA, propeptide, and the mature bioactive peptide has been described
(for review, see [34]). Hence, gene expression in “new” sites in the body requires
specification of the sense in which the term expression is meant.
All gut hormones are widely expressed in tissues outside the gastrointestinal
tract. For some, the extraintestinal expression is confined mainly to neurons and
endocrine cells, especially neurons in the central and peripheral nervous systems.
However, several gastrointestinal hormones are also expressed in other cell types
and tissues. The literature on extraintestinal expression of gut hormones has
become overwhelming. Therefore, the phenomenon will be described for a single
hormonal system only (gastrin), which may serve as an example.
The gastrin gene is expressed in several other cell types than the antroduodenal
G-cells. Quantitatively, these other cells release only little gastrin to blood in
normal organisms since the extra-antral secretion seems to serve local purposes.
Besides that, biosynthetic processing is often so different that bioactive gastrins
may not even be synthesized. So far, extra-antral expression of progastrin and its
products has been encountered in the distal small intestinal and colorectal mucosa
[35], endocrine cells in the fetal and neonatal pancreas [36, 37], pituitary
corticotrophs and melanotrophs [38, 39], hypothalamopituitary [40] and vagal
neurons [41], and human spermatogenic cells [42].
The meaning of extraintestinal synthesis of gastrointestinal hormones is often
unknown, but some suggestions can be offered. Local growth regulation is the first
possibility. Secondly, it is possible that the low concentration of peptides is without
significant function in the adult, but is a relic of a more comprehensive fetal
168 J.F. Rehfeld
synthesis. A third possibility is that the low cellular concentration reflects consti-
tutive secretion where the peptides are not stored in secretory granules.
Cell-Specific Prohormone Processing
Gastrointestinal hormone genes and prohormone structures are often so complex

and the posttranslational processing so elaborate that the phenotypic result of gene
transcription is unpredictable. Hence, the cellular equipment with processing
enzymes and their necessary cofactors determine the structure of the particular
prohormone product. This cell-specific processing of prohormones applies to all
gastrointestinal hormones. But again, gastrin is also one of the most extensively
studied gastrointestinal hormones with regard to cell-specific prohormone
processing.
Almost every tissue in which progastrin is expressed has its own characteristic
processing pattern. Four different patterns are shown in Fig. 7.4. For members of
the gastrin family the processing varies with respect to endoproteolytic processing
and with respect to amino-acid derivatizations such as tyrosyl sulfations and
phenylalanyl amidations. In this context it is worth realizing that the different
types of processing my influence each other, presumably by changing the affinity
for the various processing intermediates as substrate for the processing enzymes.
Thus, tyrosyl sulfation, the earliest posttranslational modification for the gastrin
family of prohormones, increases endoproteolytic cleavage efficiency [43], and as
endoproteolytic cleavage efficiency increases, so does C-terminal amidation pro-
cess efficiency.
Cell-Specific Peptide Release
To understand the specific effects of the gastrointestinal peptides, it is necessary to

realize that the different types of cells that express the respective genes also release
the peptides in different ways. Secretion of gastrointestinal hormones was supposed
to be endocrine only, until 30 years ago. But today, three alternative routes of
secretion to neighboring cells and one to the secretory cell itself have been
discovered (Fig. 7.5). Firstly, the peptides synthesized in neurons are released
from synaptosomal vesicles in the nerve terminals to the receptors of adjacent
target cells as neurotransmitters. In addition, it is possible that a spill-over of gut
hormonal peptides released from peripheral neurons may be transported via blood,
analogous to other extraintestinal neuropeptides. It is also possible that some
peptidergic neurons expressing gut hormonal peptides, such as hypothalamo-
pituitary neurons, release the peptides directly to blood vessels as neurocrine
secretion. Secondly, it has been shown that there are specific paracrine cells that
release, for instance, somatostatin in the gastrointestinal mucosa [44]. These cells
Fig. 7.4 Schematic

illustration of cell-specific
processing of preprogastrin
in antral G-cells, G-cells in
fetal and neonatal pancreas,
in pituitary corticotrophic
cells, and in unidentified
cells in the colorectal
mucosa [7]
carry peptidergic granules through cytoplasmic extensions to specific target cells in

the neighborhood. Paracrine cells can be considered as hybrids of classical endo-
crine cells and neurons. It is, therefore, possible that a local spillover of peptides
from paracrine cells may also reach the circulation.
Cells stimulate their own growth through autocrine secretion. Trophic peptides
bind to specific receptors in the membranes of cells in which they are also
synthesized (Fig. 7.5). Autocrine secretion is supposed to play a decisive role in
tumor and cancer development [45–47]. There is, for instance, evidence to suggest
that the growth of certain cultured bronchial carcinoma cells [48], pancreatic tumor
cells [49] and gastric and colon cancer cells [50, 51] are stimulated by autocrine
secretion of gastrin, and that growth of certain human pancreatic cancer cell lines is
stimulated by gastrin and CCK peptides [52].
Cellular release of gastrointestinal peptides also occurs in a fifth way (Fig. 7.5).
Spermatogenic cells in mammals express the gastrin, CCK, and PACAP genes [42,
53, 54]. The gastrin and CCK peptides are fully carboxyamidated and, like PACAP
[55], concentrated in the acrosome. In accordance with the acrosomal reaction, the
peptides are released from the spermatozoon by contact with the jelly-coat of the
egg and subsequently bound to receptors in the egg membrane. Defects of the
reproductive functions have now been found in PACAP-deficient mice [55].
Acrosomal release may prove an important mechanism of secretion for gut
peptides if fertilization of the egg turns out to require such peptides. The release
of bioactive peptides from acrosomal granules could be termed spermiocrine
release (Fig. 7.5).
170 J.F. Rehfeld
Fig. 7.5 Different types of cell-specific release of regulatory gut peptides: (1) endocrine release to
capillaries from classic endocrine cells in the gastrointestinal mucosa; (2) neurotransmitter release
from central or peripheral neurons to the synaptic cleft; (3) paracrine release to neighboring cells
through short cellular processes; (4) autocrine release to receptors on membrane of same cell that
synthesizes and releases the peptides; and (5) spermiocrine release from acrosomal granule of
spermatozoa to receptors on egg cell membranes [7]
General Features of Gut Hormone Targets
Target Cells
The molecular targets of gastrointestinal hormones are specific G-protein coupled

receptors expressed on a variety of cell membranes in the body. Many of the target
cells are located in the gastrointestinal tract: Neurons (including the coordinating
myenteric and submucosal nerveplexes); other endocrine gut cells; smooth mus-
cles; secretory cells that release enzymes, amines, acid and bicarbonate etc. The
hormonal control of intestinal target cells ensure that digestion, cellular growth
turnover and motility of the gut occur in a coordinated manner in order to optimize
the utilization of food and the subsequent energy delivery to the body. However,
cells of many extraintestinal organs in the body also express receptors for gastro-
intestinal hormones, which teleologically may provide a reason for the occurrence
of hormonal peptides from the gut in general circulation. These extraintestinal
organs include for instance endocrine glands (the pituitary; thyroid C-cells; para-
thyroid glands; islets of Langerhans etc.); the liver; the gallbladder; the pancreas;
the cardiovascular system and the lungs. At low-level most other tissues in the body
also express gut hormone receptors, the significance of which is still largely
unknown. Finally, many gastrointestinal and extra-intestinal cancers express pro-
miscuously the genes of both gut hormones and their receptors whereby these
cancers are often equipped with local autocrine growth promoter mechanisms
[56]. The known target functions of each gastrointestinal hormone are outlined in
Tables 7.1 and 7.2.
Receptors
The receptors for gut hormones are as mentioned of the G-protein coupled or
rhodopsin-like type with seven transmembrane loops. The amino acid chain is
often heavily derivatized at for instance phosphorylation and glycosylation sites.
Receptors structurally identified so far are listed in Table 7.3 in relation to their
specific hormonal ligand. The relationship is often complex because a specific
ligand may be bound to several receptors.
The detection of G-protein-coupled receptors in normal tissues is difficult since
the number of receptors in normal target organs that is necessary to elicit a
functional effect is small, compared, for instance, to the large amount of hormones
synthesized in comparable sites. Therefore, the detection methods for receptors are
limited and need to be critically evaluated. Several different in vitro techniques
have been used to detect G-protein-coupled receptors: measurement of receptor
mRNA by PCR techniques is a widely used way to assess receptors in normal and
tumor tissue, with the limitations, however, that it is not the receptor protein that is
detected and that he morphological correlate is missing (except for in vitro hybrid-
ization techniques). The lack of morphology and the high sensitivity of mRNA
measurement by PCR imply that small amounts of normal cells expressing the
receptors (blood vessel cells, immune cells, endocrine cells, connective tissue, and
neurons etc.) may suggest receptor expression of the main target cells present in an
organ. Since most tissue samples are highly heterogeneous from a cellular point of
view, it is better to use a morphological method for receptor analysis. It is also
preferable to detect the receptor protein itself, and if possible, the receptor-binding
sites in these proteins, since the binding sites represent the functional molecular
basis for peptide hormones [56]. A “gold standard” example is in vitro quantitative
somatostatin receptor autoradiography on frozen tissue sections that combines
morphology, binding site detection and receptor quantification. Because of limited
cellular resolution, receptor autoradiography is optimal for the detection of recep-
tors in larger cell groups. An attractive morphological alternative is immunohisto-
chemical analysis of the receptors on formalin-fixed tissues [57–59] with the
limitations that quantification is not possible and that an epitope that may be
different from the binding site is identified. The existence of receptor subtypes
for G-protein-coupled receptors has made the evaluation of the receptor profiles
more complex.
172 J.F. Rehfeld
In principle, all the mentioned methods are capable of detecting receptor sub-
types. Unfortunately, antibodies raised against the known G-protein-coupled recep-
tors and their subtypes rarely have the necessary reliability for
immunohistochemical detection, i.e. the necessary specificity, affinity and titer.
Nevertheless, adequate antibodies against the somatostatin receptor, the sst2 and
possibly also sst5, are now available [59–61], and that is a major progress that
eventually may occur also for antibodies to the other hormone receptors [62, 63].
Perspective
Gastrointestinal endocrinology has developed from an appendix of general endo-

crinology to a biological discipline of its own over the last 40 years. Today it
comprises a multitude of more than 100 bioactive peptides expressed in a controlled
cell-specific manner all over the body. The peptides participate in intercellular
regulation from local control of growth and cell differentiation to acute systemic
effects on metabolism all over the body. Thus, in the early 1970s, a revolution
changed the fundamental concepts and opened wide perspectives for gastrointesti-
nal hormones in physiology and pathophysiology.
Gastrointestinal peptide hormones must be viewed as evolutionarily conserved
intercellular messengers of general significance. There are no obvious boundaries
between their role in food intake and digestion and their function in other bodily
regulations. Most regulatory peptides (hormones, neuropeptides, growth factors,
and cytokines) are probably expressed in the gut, at least at some stage in the
phylogenetic or ontogenetic development. Hence, the development of gastrointes-
tinal endocrinology may continue its exponential growth with a broad definition of
regulatory peptides. On the other hand, such extension almost deprives the concept
of gastrointestinal endocrinology of its meaning. And that is exactly what this is all
about: Gastrointestinal hormones should be viewed not only as local hormones of
specific interest to digestive physiologists and clinical gastroenterologists. They are
integrated chemical messengers in the coordination and regulation of many or most
bodily functions in mammals. Thus, it is not surprising that today gut hormones are
studied not only in physiology and cell biology, but also by microbiologists,
psychiatrists, zoologists, cardiologists, diabetologists, and others.
Acknowledgement The skillful and patient secretarial assistance of Connie Bundgaard is grate-
fully acknowledged. The author also wishes to thank Dr. Jean-Claude Reubi (Berne, Switzerland)
for helpful discussion and suggestions for the receptor section.
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Chapter 8
Microbiome, HPA Axis and Production
of Endocrine Hormones in the Gut
Nobuyuki Sudo
Abstract Recent accumulating evidence indicates that the gut microbiome can
affect the development and regulation of the hypothalamic-pituitary-adrenal axis
and behavior, with central integrative systems being crucial in the successful
physiological adaptation of the organism to external stressor. In contrast, host-
derived hormones increase the bacterial proliferative capacity and pathogenicity. In
the gut lumen, this type of cross-talk between microorganisms and the host is
presumed to be performed continually through various kinds of luminal molecules,
as numerous types of bacteria and host cells are in close proximity in the gastro-
intestinal tract of mammals.
We herein focus on bidirectional signaling between the gut microbiome and the
host in terms of commensal microbiota affecting the hypothalamic-pituitary-adrenal
HPA axis response and behaviors and further discuss the role of gut luminal
catecholamines and γ-aminobutyric acid, both of which are presumed to be involved
in this signaling.
Abbreviations
ACTH Adrenocorticotropin hormone

CA Catecholamines
CRH Corticotrophin-releasing hormone
DA Dopamine
E Epinephrine
EHEC Enterohemorrhagic Escherichia coli
GABA γ-Aminobutyric acid
GAD Glutamic acid decarboxylase
N. Sudo (*)
Department of Psychosomatic Medicine, Graduate School of Medical Sciences, Kyushu
University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan
e-mail: nobuyuki@med.kyushu-u.ac.jp
178 N. Sudo
GC Glucocorticoids
GUS β-Glucuronidase
NE Norepinephrine
Tir Translocated intimin receptor
Introduction
Gut microbiota have an estimated mass of 1–2 kg, numbering 100 trillion [1] and
together possessing 100 times the number of genes in the human genome [2]. These
bacteria not only play a principal role in the postnatal maturation of the mammalian
immune system [3], but also aid in the digestion and absorption of macromolecules
and act as a barrier to gut pathogens by blocking attachment to gut binding sites
[4]. Moreover, it is also rapidly becoming apparent that the gut microbiome plays a
major role in the development and regulation of the hypothalamic-pituitary-adrenal
(HPA) axis [5] and behavior [6–11].
In contrast, host hormones can signal commensal microbial cells via converging
pathways directed to bacterial signaling molecules. Lyte and colleagues first dem-
onstrated in their pioneering studies conducted in the 1990s that some species of
pathogens can recognize exogenous catecholamines (CA) in vitro and that such
recognition increases the bacterial proliferative capacity [12–15]. Sperandio and
colleagues subsequently showed that enterohemorrhagic Escherichia coli (EHEC)
virulence increases upon exposure to epinephrine (E) and norepinephrine (NE) and
that E binds and signals through the QseC receptor [16, 17]. This type of bidirec-
tional communication is called “microbial endocrinology” [15] or “interkingdom
signaling” [17, 18], which mediates the symbiotic and pathogenic relationships
between the bacteria and mammalian host. Since numerous kinds of bacteria and
host cells are in close proximity in the gastrointestinal tract of mammals,
interkingdom signaling via various kinds of luminal molecules is presumed to be
performed continually in the gut lumen [19] and to participate in the regulation of
various pathophysiological functions.
We herein focus on the bidirectional signaling between the gut microbiome and
the host in terms of commensal microbiota affecting the HPA axis response and
behavior and further discuss the possible involvement of some gut luminal mole-
cules in this signaling.
Gut Microbiota and the Stress Response of the Host
The HPA axis is considered to be a central integrative system, being crucial in the
successful physiological adaptation of the organism to stress. During stress,
corticotrophin-releasing hormone (CRH) and arginine vasopressin, the principal
8 Microbiome, HPA Axis and Production of Endocrine Hormones in the Gut 179
hypothalamic regulators of the HPA axis, are released. CRH stimulates the secre-
tion of the adrenocorticotropin hormone (ACTH) from the anterior pituitary into the
hypophyseal portal system via collateral fibers in the systemic circulation. ACTH
induces the secretion of glucocorticoids (GCs; cortisol in humans and corticoste-
rone in rodents) from the adrenal cortex, the main target of ACTH. GCs regulate
multiple bodily functions and prepare the individual to cope with the demands of
metabolic, physical and psychological stressors [20].
Critical Role of the Gut Microbiota in Determining the Set

Point of the HPA Axis
It is well known that the HPA axis is susceptible to environmental influences,

particularly early in life [21, 22]. Since indigenous microbiota constitutes a major
environmental force affecting the host physiology, we examined whether these
bacteria can alter the development of the HPA response using gnotobiotic mice.
As shown in Fig. 8.1A, the degree of plasma ACTH and corticosterone elevation
in response to a 1-h restraint stress was substantially higher in the GF mice than in
the SPF mice. When the mice were exposed to ether stimulus, no significant
differences in the plasma ACTH or corticosterone response were found in either
group of animals (Fig. 8.1B). Monoassociation with Bifidobacterium infantis, a
representative inhabitant of the neonate gut, lessened the HPA stress response to
SPF (Fig. 8.2). The hormonal stress response in the rabbit-derived EPEC-
monoassociated mice was substantially higher than that observed in the GF mice,
although no such exaggerated response was found in the mice reconstituted with an
EPEC mutant strain, ΔTir [23], which is not internalized due to defects in the
translocated intimin receptor.
Interestingly, the enhanced HPA stress response of the GF mice was partially
corrected at 3 weeks after reconstitution of SPF feces at an early stage of develop-
ment (Fig. 8.3A), while no such correction was found following reconstitution at a
later stage (Fig. 8.3B). Therefore, the microbe-induced reversal of the HPA axis set
point extended into adulthood, but only if bacterial colonization occurred before the
animals reached 6 weeks of age. Colonization of the adults was ineffective, which
suggests a critical window of susceptibility to the effects of bacteria-host
interactions.
Recently, animal studies performed by several independent groups have shown
the commensal microbiota to be a crucial factor modulating the host behavioral
profile [6–9, 11]. In our recent study performed under a strictly contamination-free
environment, EX-GF mice, gnotobiotic mice reconstituted with a normal specific
pathogen-free microbiota, were less anxious and active than the GF mice based on
open field and marble-burying tests [11]. Monoassociation with Clostridium
(Brautia) coccoides reduced the anxiety levels; however, it did not affect the
180 N. Sudo
Fig. 8.1 Increased plasma ACTH and corticosterone responses to restraint stress but not to ether
exposure in GF mice. Panel A: The mice were subjected to a 1-h period of restraint stress (GF,
n ¼6–11 per each time-point; SPF, n ¼6–11 per each time-point). The baseline data were obtained
via cardiac puncture in mice killed using cervical dislocation before stress exposure.
***P < 0.001, **P < 0.01, *P < 0.05 in a post hoc Dunnett’s test between GF and SPF. Panel
B: The GF and SPF mice failed to show any differences in the HPA response to ether exposure
(n ¼ 6 per each time point)
locomotor activity. In contrast, colonization with B. infantis decreased the locomo-

tor activity while having little effect on the anxiety level.
Therefore, the commensal gut microbiota affects the development and regula-
tion of the biobehavioral stress response of the host.
Fig. 8.2 Effects of restraint stress on the plasma ACTH and corticosterone levels in the gnoto-
biotic mice. The plasma ACTH and corticosterone levels were measured before or immediately
after the 1-h restraint test in the GF (n ¼20), SPF (n ¼ 18) and monoassociated mice (n ¼ 18–24
per group) at 9 weeks of age. ***P < 0.001, *P < 0.05 according to Dunnett’s test.
Bifidobacterium, EPEC and ΔTir indicate Bifidobacterium infantis-, enteropathogenic E. coli-
and EPEC mutant strain deficient of Tir (translocated intimin receptor)-associated mice,
respectively
Microbiota and Stress Resilience
Recently, there has been increasing interest in the individual’s response to manag-
ing adverse events and stressors [24, 25]. Such an ability to recover from adverse
changes, known as “stress resilience,” includes psychological and biological pro-
cesses that allow an individual to avoid or reduce the harmful consequences of
extreme stress. Resilient individuals encountering chronic psychosocial stress min-
imize pathophysiological outcomes, such as extended or exaggerated HPA axis
activity [26, 27], that can precipitate stress-related diseases, such as post-traumatic
stress disorder, anxiety and major depression [28, 29]. In addition to genetic factors,
a broad range of environmental factors contribute to resilience. In fact, a recent
elegant study conducted by Lehmann and Herkenham [30] showed that enriched
environmental housing (environmental enrichment) confers stress resilience
through an infralimbic cortex-dependent neuroanatomical pathway in a mouse
model of social defeat stress. Taken together, these findings lead us to the following
interesting hypothesis: newborn babies are likely to recognize colonizing bacteria
as a stressor when encountering them for the first time because the babies have little
capability to discern whether a novel stimulation from the external environment is
good or bad. This is supported by the fact that colonization of GF mice by a
nonpathogenic bacterium induces a small and transient increase in the plasma
corticosterone and IL-6 levels in addition to hypothalamic c-fos activation without
eliciting any apparent inflammation of the gut [5]. Such colonizing microbes,
however, are not harmful to the host, but rather offer beneficial stimulation for
enhancing host resistance to future severe stressors. Selye called this type of
stressor “eustress,” a positive form of stress usually related to desirable events in
a person’s life [31]. Therefore, the commensal microbiota may be a “eustress” that
182 N. Sudo
Fig. 8.3 Effects of restraint stress on the plasma ACTH and corticosterone levels in the mice
reconstituted with SPF feces. SPF flora-reconstituted mice were established by orally introducing
fresh SPF murine feces into the GF mice at either 1 or 3 weeks before being subjected to the stress
protocol. Restraint stress was applied to the reconstituted mice at 9 (panel A) and 17 (panel B)
weeks of age (n ¼ 18–24 per group). ***P < 0.001, **P < 0.01 according to Dunnett’s test
plays an important role against the development of stress-related disorders, such as

anxiety and depression, by providing the host with “stress resilience,” similar to
environmental enrichment.
Possible Luminal Molecules Mediating Gut Microbe-Host

Interactions
The exact mechanisms whereby commensal bacteria interact with the host in the
gut and what molecules are involved in this interaction remain to be elucidated.
Vagal afferent nerves have been shown to play a role in the signaling from gut
microbes to the central nervous system [32]; however, the underlying pathways and
molecules are highly complex, and it is unlikely that only one common pathway or
series of molecules is involved. However, we herein pay particular attention to CA

and γ-aminobutyric acid (GABA) present in the gut lumen.
CA as an Interkingdom Signal in the Gut Lumen
CA, such as NE and dopamine (DA), are utilized in the central and peripheral
nervous systems, which regulate various types of body functions, such as cognitive
abilities, mood and gut motility [33]. In addition to the well-established roles of CA,
recent accumulating evidence suggests CA to be important interkingdom signal
molecules in the gut.
CA Exist in a Biologically Free Form in the Lumen

of the Gastrointestinal Tract
Our recent work [34] showed that free NE and DA are present in the lumen of the
ileum, cecum and colon. The NE and DA levels in the lumen are the highest in the
colon among the three parts of the gut (Fig. 8.4). In contrast, there are no significant
differences in the tissue NE or DA levels between the ileum, cecum and colon.
Since a large proportion of peripheral CA in the blood and urine exist in a
conjugated form that is biologically inactive [35, 36], we investigated the free
and glucuronide- and sulfate-conjugated forms of CA in the lumen of the ileum,
cecum and colon.
Figure 8.5 shows that almost all of the NE and DA molecules were present in a
biologically free form in the lumen of the cecum and colon of the SPF mice,
although substantial amounts of glucuronide-conjugated NE and DA were present
in the ileum.
Crucial Role of Bacterial β —Glucuronidase in the Generation

of a Biologically Active Free Form of CA
The β-glucuronidase (GUS) from E. coli is a 290-kDa tetrameric protein that is

essentially free of sulfatase activity [37]. Its optimal pH is 6.8, while that of the
tissue-type GUS is 4.5 [38]. Since the mean pH in the intestinal lumen ranges from
6.5–7.9 (upper segment of the small intestine) to 6.8–8.0 (colon) in rodents [39–41],
we hypothesized that free CA present in the lumen of the cecum and colon are
generated via deconjugation by bacterial GUS derived from the gut microbiota. As
shown in Fig. 8.6, the luminal free NE and DA levels were lower in the GF mice
than in the SPF mice. In addition, more than 90 % of the DA in the GF mice was in
the glucuronide-conjugated form in all parts of the digestive tracts examined
(Fig. 8.7A), while approximately 40 % of the NE was in the glucuronide-conjugated
184 N. Sudo
Fig. 8.4 Luminal and

tissue CA concentrations in
the gastrointestinal tract in
the SPF mice. The luminal
11–15, panel A) and
(n ¼
tissue (n ¼ 5–8, panel B)
CA levels are shown. The
open and closed circles
indicate the NE (left vertical
axis) and DA (right vertical
axis) levels, respectively.
***P < 0.001 was
significantly higher than the
corresponding ileum value
form (Fig. 8.7B). The critical role of bacterial GUS in the production of free CA
was further verified in additional experiments using gnotobiotic mice.
Association with either a mixture of 46 Clostridia species (Clostridia) or fecal
flora from SPF mice (EX-GF) showed a drastic elevation of the free NE and DA
levels (Fig. 8.8). In another set of experiments, the changes in the luminal CA levels
were examined in the cecum after GF mice were colonized with either an E. coli
mutant strain lacking the GUS-encoded gene, uidA (JW1609: ΔGUS), or its parent
E. coli strain (BW25113). Figure 8.9 shows that 70 % of the DA remained in the
glucuronide-conjugated form even after the association with ΔGUS, although 25 %
of the total DA was in the free form. In contrast, two-thirds of the DA was converted
into the free form, while 29 % of the total DA remained conjugated after the
association with BW25113. The conjugated form of NE accounted for 29 % of
the total following inoculation with ΔGUS, while representing 15 % of the total
following inoculation with BW25113. The GUS activity in the cecal lumen of
the ΔGUS-gnotobiotic mice was only marginally detectable and almost identical
to the GF value (n ¼ 5 per each group, ΔGUS 6.7 0.4 μg PheP/h/mg protein,
Fig. 8.5 Free and

glucuronide- and sulfate-
conjugated CA in the gut
lumen in the SPF mice. The
luminal glucuronide- and
sulfate-conjugated DA
(n ¼6, panel A) and NE
(n ¼6, panel B) levels in
the ileum, cecum and colon
were analyzed with post
column HPLC using
diphenylethylenediamine as
a fluorogenic reagent. The
mean value of each form of
CA is expressed as the
percentage of the total (free
CA + conjugated CA). The
open, closed and dotted bars
indicate the free,
glucuronide-conjugated and
sulfate-conjugated CA,
respectively
GF 6.4 0.3 μg PheP/h/mg protein). On the other hand, the BW25113 E. coli-
gnotobiotic mice exhibited a small but significant increase in the GUS activity in
the cecal lumen in comparison with that observed in the ΔGUS-gnotobiotic mice
(n ¼4, 11.6 1.3** μg PheP/h/mg protein, **P < 0.01 vs. ΔGUS value). Interest-
ingly, the GUS activity in the cecal wall was significantly increased upon exposure
to ΔGUS to a comparable level found upon exposure to the BW25113 strain (n ¼5
per each group, ΔGUS 3.00 0.25** μg PheP/h/mg protein, BW25113
2.72 0.18* μg PheP/h/mg protein, GF 1.93 0.06 μg PheP/h/mg protein;
**P < 0.01 and *P < 0.05 vs. GF value).
Collectively, these results indicate that the gut microbiota plays a critical role in
the generation of luminal free CA via GUS. The bacteria-induced increase in the
tissue GUS activity may be involved in this phenomenon.
Can Commensal Bacteria Themselves Produce CA In Vivo?
Russian researchers [42, 43] have reported that some species of microorganisms
produce CA in an in vitro culture system. In fact, transcripts that have some
186 N. Sudo
Fig. 8.6 Cecal free CA

levels in the lumen and
tissue of the SPF and GF
mice. The luminal (n ¼ 6–
10, panel A) and tissue
(n ¼6–8, panel B) free CA
levels in the SPF and GF
mice are shown. The
luminal NE levels (left
vertical axis) in the SPF and
GF mice were
35 5*** ng/g and
3.8 1.3 ng/g, respectively,
and the luminal DA levels
(right vertical axis) in the
SPF and GF mice were
115 14*** ng/g and
5.0 0.5 ng/g, respectively.
***P < 0.001 was
corresponding GF value
similarity with mammalian tyrosine hydroxylase, a rate-limiting enzyme, are found

in some species of bacteria [44, 45]. In our study, no significant differences were
observed in the total DA levels (free + conjugated types) of the cecal content
between the GF and SPF mice; however, the total NE levels of the cecal and
colonic content were substantially higher in the SPF mice than in the GF mice
(n ¼ 5 per each group, cecum, GF 7.4 1.7, SPF 36.4 5.6***; colon, GF
6.4 1.3, SPF 62.6 6.7***; ***P < 0.001 vs. GF value). These results suggest that
gut microbes are a likely source of gut luminal NE. In addition, gut bacteria
enriched from murine feces actually contain substantial amounts of NE and a lesser
amount of DA (Fig. 8.10). Therefore, it is possible to speculate that gut microbes
are an important source of luminal NE. However, some species of bacteria, includ-
ing E. coli, have a functional transporter for CA, such as the bacterial neurotrans-
mitter sodium symporter family member, Leu T [46]. Therefore, there is thus far
Fig. 8.7 Free and

glucuronide- and sulfate-
conjugated CA in the gut
lumen in the GF mice. The
luminal glucuronide- and
sulfate-conjugated DA
(panel A) and NE (panel B)
levels in the ileum, cecum
and colon are shown. The
mean value of each form of
CA is expressed as the
percentage of the total (free
CA + conjugated CA). The
open, closed and dotted bars
indicate free, glucuronide-
conjugated and sulfate-
conjugated CA,
respectively
insufficient evidence to determine whether the NE and DA found in gut microbes

originate from bacterial production by tyrosine hydroxylase-like enzyme or if they
are obtained from the gut lumen via a Leu T-like transporter.
CA Receptors on Gut Epithelial Cells and Their Functions
DA 1A receptors are identified in the cells at the base of the intestinal crypts of the
rat small intestine [47]. Alpha 2-adrenergic receptors are also reported to be present
on gut epithelial cells [48]. These findings suggest the physiological importance of
luminal CA. In fact, the luminal administration of DA stimulates active ileal ion
absorption via α2-adrenergic or dopaminergic receptor activation, demonstrating
the role of luminal DA as a proabsorptive modulator of ion and water transport [49,
50]. These results were also confirmed by our recent findings using an in vivo colon
loop model in which the injection of ten micromoles of DA into the loop was found
to induce a 30 % increase in water absorption out of the gut lumen in comparison
to the injection of vehicle without DA (vehicle 55 5 μl/30 min/cm, DA
72 4* μl/30 min/cm; *P < 0.05).
188 N. Sudo
Fig. 8.8 Luminal free CA

levels and the GUS activity
in the cecum in the
gnotobiotic mice. Panel A:
The cecal luminal contents
obtained from EX-GF
(n ¼ 6) and Clostridia
(n ¼ 6)-associated mice
were processed for free NE
and DA measurement.
***P < 0.001 was
corresponding GF value.
Panel B: The cecal luminal
contents of GF, EX-GF and
Clostridia-associated mice
were subjected to
measurement of the GUS
activity. ***P < 0.001 was
corresponding GF value
It is conceivable that luminal CA are involved not only in proabsorptive func-

tions and increases in bacterial pathogenicity, but also a variety of physiological
and pathological functions, such as gut motility [51] and modulation of immune
reactions [52, 53]. Clarifying such unidentified functions will further support the
notion that CA are important molecules mediating between gut microorganisms and
the host under pathophysiological conditions.
GABA as an Interkingdom Signal in the Gut
GABA is a four carbon, nonprotein amino acid conserved from bacteria to verte-
brates. Organisms have the ability to synthesize GABA from glutamate in a
reaction catalyzed by the cytosolic enzyme L-glutamic acid decarboxylase
(GAD). Several researchers have found that some bacteria, including E. coli and
members of the Lactobacillus genus, possess a GAD activity, allowing them to
Fig. 8.9 Free and glucuronide- and sulfate-conjugated CA in the cecal lumen in the mice
colonized with the E. coli mutant strain JW1609 or its parent strain BW25113. The cecal contents
were subjected to measurement of the free and glucuronide- and sulfate-conjugated NE (panel A)
and DA (panel B) levels 4 weeks after exposure to E. coli mutant strain devoid of GUS (n ¼ 5,
JW1609: ΔGUS) or its parent strain (n¼5, BW25113). The mean value of each form of CA is
expressed as the percentage of the total (free CA + conjugated CA). The open, closed and dotted
bars indicate free, glucuronide-conjugated and sulfate-conjugated CA, respectively
Fig. 8.10 Bacteria-rich fractions enriched from the cecal contents contain NE and DA. Bacteria-
rich fractions were enriched from the cecal contents of the SPF mice according to a previously
reported method [68]. The enriched fractions were thoroughly disrupted using repeated sonication
then processed for CA measurement. A microscopic test revealed that the enriched bacteria had no
contaminants, such as epithelia or debris. The NE and DA levels (mean standard error) were
103 27 and 25 6 ng/g, respectively (n ¼ 6). *P < 0.05 indicates a significant difference
between the NE and DA values
190 N. Sudo
convert glutamic acid to GABA [54–56]. The production of GABA by bacteria

appears to naturally occur under physiological conditions, as a recent study using
metabolomics showed that the gut luminal GABA levels in Ex-GF mice are
considerably higher than those observed in GF mice [57]. It is well known that
plant-derived GABA mediates communication between organisms belonging to
different kingdoms [58]; therefore, the GABA locally produced by the resident
microbiota may play an important role as an interkingdom signal in the gut. In fact,
a recent publication by Li and colleagues [59] showed that gut epithelial cells
express several types of GABA receptors, including β2/3- and π-subunits, on their
surface. The authors also demonstrated that endogenous autocrine GABAergic
signaling in the mammalian intestinal epithelium upregulates intestinal fluid secre-
tion and becomes intensified in mice with allergic diarrhea. To date, there is no
direct evidence demonstrating that gut luminal GABA is actually involved in
signaling from the gut to the brain. However, neural and/or humoral interactions
between the intestinal GABA system and the brain GABA system comprise a
fascinating research theme, as the JB-1 strain of Lactobacillus rhamnosus reduces
stress-induced anxiety- and depression-related behavior, accompanied by an altered
GABAAα2 receptor mRNA expression in the brain [32].
Other Hormones in Microbial Cells
Hormones and hormone-binding proteins with homology to those of vertebrates are

reported to be present in fungi, yeast and bacteria [60, 61]. In particular, insulin and
insulin-like materials contained in microbes have been the most extensively studied
[62–64]. Corticotropin [65] and somatostatin [66] have also been identified in a
unicellular organism (Tetrahymena pyriformis) and Bacillus subtilis, respectively.
In this regard, Iyer and colleagues [67] proposed the interesting theory that the
evolutionary history of prokaryotic genes encoding many of the enzymes involved
in the synthetic and metabolic pathways of CA, histamine, acetylcholine and
GABA is best described by scenarios that include late horizontal gene transfer
from bacteria. This concept is substantiated by the growing body of evidence
showing that bacteria produce small molecules that are formally involved in
bacteria–bacteria communication and have now become involved in bacteria-host
communication.
While we emphasize the role of CA or GABA in this context, this is but one of
many examples of the consequences of the bacterial synthesis of neuroactive
molecules that remain to be explored.
Conclusion and Perspectives
We are living in a bacterial world. Bacterial signaling helps us maintain homeo-

stasis, keeping us healthy and happy. Given that the gut microbiome plays a crucial
role in the development of the HPA axis and behaviors, gut microbes may play a
critical role against the development of stress-related disorders, such as anxiety and
depression, by providing the host with the “stress resilience” necessary to adapt to a
changing external environment.
Clearly, further studies are called for; however, the recent findings described
herein provide strong evidence in this rapidly developing field of research. We
foresee a day when a comprehensive view regarding the interactions and pathways
involved in the “microbiota-gut-brain axis” will be unraveled.
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Chapter 9
Neuropeptides and the Microbiota-
Gut-Brain Axis
Peter Holzer and Aitak Farzi
Abstract Neuropeptides are important mediators both within the nervous system
and between neurons and other cell types. Neuropeptides such as substance P,
calcitonin gene-related peptide and neuropeptide Y (NPY), vasoactive intestinal
polypeptide, somatostatin and corticotropin-releasing factor are also likely to play a
role in the bidirectional gut-brain communication. In this capacity they may
influence the activity of the gastrointestinal microbiota and its interaction with
the gut-brain axis. Current efforts in elucidating the implication of neuropeptides in
the microbiota-gut-brain axis address four information carriers from the gut to the
brain (vagal and spinal afferent neurons; immune mediators such as cytokines; gut
hormones; gut microbiota-derived signalling molecules) and four information
carriers from the central nervous system to the gut (sympathetic efferent neurons;
parasympathetic efferent neurons; neuroendocrine factors involving the adrenal
medulla; neuroendocrine factors involving the adrenal cortex). Apart from operat-
ing as neurotransmitters, many biologically active peptides also function as gut
hormones. Given that neuropeptides and gut hormones target the same cell mem-
brane receptors (typically G protein-coupled receptors), the two messenger roles
often converge in the same or similar biological implications. This is exemplified
by NPY and peptide YY (PYY), two members of the PP-fold peptide family. While
PYY is almost exclusively expressed by enteroendocrine cells, NPY is found at all
levels of the gut-brain and brain-gut axis. The function of PYY-releasing
enteroendocrine cells is directly influenced by short chain fatty acids generated
by the intestinal microbiota from indigestible fibre, while NPY may control the
impact of the gut microbiota on inflammatory processes, pain, brain function and
behaviour. Although the impact of neuropeptides on the interaction between the gut
microbiota and brain awaits to be analysed, biologically active peptides are likely to
P. Holzer (*) • A. Farzi

Research Unit of Translational Neurogastroenterology, Institute of Experimental and Clinical
Pharmacology, Medical University of Graz, Universitätsplatz 4, 8010 Graz, Austria
e-mail: peter.holzer@medunigraz.at
196 P. Holzer and A. Farzi
emerge as neural and endocrine messengers in orchestrating the microbiota-gut-

brain axis in health and disease.
Abbreviations
AgRP Agouti-related protein
APUD Amine precursor uptake and decarboxylation
BDNF Brain-derived neurotrophic factor
CRF Corticotropin-releasing factor
MAMP Microbe-associated molecular pattern
NPY Neuropeptide Y
NR2A NMDA receptor subunit 2A
NTS Nucleus tractus solitarii
PYY Peptide YY
TLR4 Toll-like receptor 4
Neuropeptides at the Forefront of the Brain-Gut Axis
Biologically active peptides have been instrumental in the formulation of the

concept that brain and gut have much in common. When in the 1960s and 1970s
several peptides were discovered to occur both in the brain and gastrointestinal
tract, the term “gut-brain axis” was first coined, based on the prevailing concept that
the brain would be essential for controlling gut function. The way to this concept
was pathed by the so-called APUD (amine precursor uptake and decarboxylation)
hypothesis which, owing to common histochemical characteristics, held that amine-
and peptide-producing cells of the nervous system, the gut and other organs derive
from a common origin in the neural crest [1, 2]. While certain cells of the thyroid,
adrenal medulla, carotid bodies and autonomic as well as enteric ganglia originate
in fact from the neural crest, the peptide-secreting endocrine cells of the gut do not
[2]. Although the APUD hypothesis has not stood the test of time, it was an
important contribution to the current understanding of the coordinating function
of neuropeptides in many organ systems. We now know that a vast number of
neuropeptides is produced by central and peripheral neurons alongside with endo-
crine cells in the gastrointestinal tract and other endocrinologically active organs
[2–4]. Biologically active peptides, particularly neuropeptides, play many diverse
roles in the bidirectional data highway between the gut and brain and offer
unforeseen opportunities for drug development. At the same time, the multiplicity
of messengers (including neuropeptides) also represents a challenge in understand-
ing the complex interactions between gut and brain. Although their precise role in
9 Neuropeptides and the Microbiota-Gut-Brain Axis 197
the microbiota-gut-brain axis has not yet been defined, neuropeptides such as
substance P, calcitonin gene-related peptide, neuropeptide Y (NPY), vasoactive
intestinal polypeptide, somatostatin and corticotropin-releasing factor (CRF) are
candidates to play an important role in this respect.
The Gut-Brain Axis Involves Microbial, Immune, Endocrine

and Neural Signalling Pathways: Neuropeptides
May Be Involved in Each Pathway
The term “gut-brain axis” refers to the bidirectional communication between the
gut and the brain (Fig. 9.1). Apart from the autonomic regulation of digestion by the
central, parasympathetic, sympathetic and enteric nervous systems as well as by
neuroendocrine factors (derived from the adrenal medulla and cortex), there is
ongoing communication from the gut to the brain in health and disease [5,
6]. Thus, visceral information is continuously fed into subcortical regions of the
brain including the limbic system and the autonomic and neuroendocrine centres
[5]. This information is integrated with other interoceptive information from the
body and with contextual information from the environment [5]. Under patholog-
ical conditions, the interoceptive input from the periphery may reach the level of
consciousness and give rise to the sensation of nausea, discomfort and/or pain
[6]. In addition, the brain’s output to the gut via autonomic and neuroendocrine
pathways may result in gastrointestinal dysfunction. The afferent part of this gut-
brain-gut axis has recently been in the focus of investigation in order to understand
why gastrointestinal disease such as inflammatory bowel disease and irritable
bowel syndrome is associated with pain and a number of psychiatric disturbances
including anxiety, neuroticism and depression.
The gut-brain axis uses four major information carriers for the communication
between the gut and the brain (Fig. 9.1):
• neural messages carried by vagal and spinal afferent neurons,
• immune messages carried by cytokines,
• endocrine messages carried by gut hormones and
• microbial factors that may directly reach the brain via the blood stream but can
also interact with the other three transmission pathways [6–8].
These communication systems are abundantly present in the gastrointestinal
tract and, in an evolutionary perspective, are relevant for a number of vital
functions:
• The brain with its sensory systems needs to interact with the gut in finding
appropriate food and assimilating it for the sake of metabolic survival.
• The gut needs to distinguish between useful and useless as well as dangerous
(antigenic, pathogenic, toxic) ingredients of food and sort them accordingly.
Brain Appetite and metabolic homeostasis

Cognition, emotion and mood
Stress resilience and recovery
Interoception and pain
Gut-brain-gut axis Microbial factors Autonomic neurons

Gut hormones Neuroendocrine factors
Cytokines
Sensory neurons
Gut immune system
Gut mucosa
L L
Gut microbiota
Fig. 9.1 The bidirectional microbiota-gut-brain axis. Four communication pathways (microbial
factors, gut hormones, cytokines, sensory neurons) signal from the gut to the brain where they can
modify cerebral function and behaviour. Two pathways (autonomic and neuroendocrine outputs)
signal from the brain to the gut. L denotes endocrine L cells in the intestinal mucosa
• The gut needs to maintain homeostasis with the extensive community of

microbes in the intestine, which are important in supporting nutrition, educating
the immune system and communicating with other organ systems including the
brain.
Each of the communication pathways between the gastrointestinal and central
nervous system may involve neuropeptides and structurally related signalling
molecules. Ever since their gradual discovery, biologically active peptides have
been intimately related to the regulation of digestion and to the communication with
the central nervous system. Regulation of food intake (appetite), metabolic homeo-
stasis and pain have been areas that were addressed in particular detail. Neuropep-
tides comprise a class of evolutionarily well conserved molecules that, by
definition, operate as transmitters in the enteric, peripheral and central nervous
systems and share transduction mechanisms with other biologically active peptides
such as gut hormones. Apart from their origin, it is frequently difficult to distinguish
between their function as neuropeptides or gut hormones because they operate often
via the same receptors and cellular transduction systems. Thus, neurons as well as
endocrine, immune, interstitial, muscle, epithelial and microbial cells can respond
to these signalling molecules by expressing the appropriate peptide receptors. The
microbiota residing in the mucosa [9] is in immediate vicinity to the endocrine cells
of the gastrointestal mucosa which produce more than 20 different gut hormones
[10]. Apart from immune mediators, gut hormones may thus play an important role
as communicators between the gut microbiota and host functions. Gut hormone
signalling to the brain not only occurs by an endocrine route but may also involve
activation of primary afferent neurons, especially in the vagus nerve (e.g., chole-
cystokinin and ghrelin). Furthermore, it is important to realize that the four com-
munication pathways between the gut and the brain do not operate in isolation but
are closely interrelated with each other.
Direct Brain Communication Pathways Used by the Gut

Microbiota
With the emerging role of the microbiota a new gut-brain pathway has come to
light. Thus, the gut microbiota communicate not only with gastrointestinal epithe-
lial, immune and nerve cells in their immediate neighbourhood but also generate
and release molecules that can signal to distant organs. This is true for molecules
designated as pathogen-associated molecular patterns or, in a more benevolent vein,
microbe-associated molecular patterns (MAMPs) as well as for many other micro-
bial metabolites. There are experimental data to show that a significant part of the
metabolites circulating in mammalian blood are derived from the intestinal micro-
bial community [11–15]. Importantly, the presence or absence of the gut microbiota
also influences the profile of metabolites (including peptides) present in the
brain [16].
While the potential effects of the microbial metabolites on the host are still little
understood, it is obvious that they could convey messages around the whole body.
Some information in this respect can be derived from the actions of two MAMPs,
lipopolysaccharide (LPS) and peptidoglycan components such as meso-
diaminopimelic acid. These MAMPs are recognized by pattern recognition recep-
tors of the innate immune system: LPS activates toll-like receptor 4 (TLR4) while
the peptidoglycan structures stimulate nucleotide-binding oligomerization domain–
containing protein-1 (Nod1) and/or Nod2. Importantly, translocation of peptido-
glycan from the gut to the blood impacts on neutrophils in the bone marrow and
primes their capacity to defend the body against bacterial infection via stimulating
Nod1 [17].
In a similar manner, LPS translocated from the gut through a leaky mucosal
barrier carries a microbial message to distant organs including the brain. The
behavioural responses to systemic exposure of excess LPS are well characterized
in animals and humans and comprise acute sickness [18, 19] and delayed
depression-like behaviour [20–24]. LPS originating from the gut microbiota may
give rise to alterations in brain function via three different pathways. Following
translocation across the intestinal mucosa it may, on the one hand, stimulate the
intestinal immune system to produce cytokines which (1) can signal directly to the
brain or (2) sensitize/stimulate vagal and spinal afferent neurons [18, 19, 25,
26]. On the other hand, (3) the circulation may carry LPS itself to the central
nervous system where it may modify brain function.
The latter possibility need be envisaged because—apart from the innate immune
system—there is a widespread expression of TLR4 and other TLRs at several levels
of the gut-brain axis. Thus, TLRs are present on gastrointestinal epithelial cells [27,
28], neurons of the enteric nervous system [29, 30], primary afferent neurons [29]
and various cell types (neurons, microglial cells and astrocytes) in the brain [31–
33]. By stimulating TLR4 and TLRs in the brain, LPS and other bacterial factors
can stimulate the generation and release of proinflammatory cytokines and in this
way give rise to neuroinflammatory processes. These effects are not only relevant to
neurodegeneration and repair [31–33] but may also be involved in the manifestation
of psychiatric disorders. Specifically, increased levels of IgA and IgM against LPS
of commensal gut bacteria are found in the circulation of patients with depression or
chronic fatigue syndrome, and the hypothesis has been put forward that increased
translocation of LPS across a leaky gut may be a factor that contributes to these
pathologies [34, 35]. Taken all findings together, it would appear, therefore, that the
physiological roles of the symbiotic gut microbiota relate not only to the regulation
of digestion at the gastrointestinal level but also extend to systemic immunity and
brain function.
Neuroactive Factors Released by the Gut Microbiota
There is increasing evidence that the gut microbiota sheds not only ligands for
pattern recognition receptors, but also releases factors that target specific neuronal
systems involved in the gut-brain axis. Although it remains to be established
whether the microbiota can produce neuropeptide-like compounds, they are capable
of generating a number of neurotransmitters and neuromodulators [7, 14]. Members
of the genera Candida, Streptococcus, Escherichia and Enterococcus synthesize 5-
hydroxytryptamine (5-HT), members of the genera Escherichia, Bacillus and
Saccharomyces generate dopamine and/or noradrenaline, members of the genus
Lactobacillus produce acetylcholine, and members of the genera Lactobacillus and
Bifidobacterium manufacture gamma-aminobutyric acid (GABA) [7, 14, 36–
39]. The release of microbiota-derived dopamine into the lumen of the intestine
has been suggested to play a proabsorptive role in the colon [38]. Signalling via
opioid and cannabinoid receptors may also be modified by the gut microbiota, a
conclusion based on the ability of certain probiotics to alter the expression of opioid
and cannabinoid receptors in the gut [7].
Moreover, the microbiota in the intestine is able to produce metabolites with
benzodiazepine-like structures and effects [40–42]. Specifically, benzodiazepine
receptor ligands originating from the gut microbiota have been proposed to con-
tribute to the encephalopathy associated with fulminant hepatic failure [40]. Under
these conditions, benzodiazepine-like molecules are likely to reach the brain at
increased concentrations that will enhance neurotransmission via GABAA receptors
and thus contribute to the disease process [40]. The pyrrolobenzodiazepines (e.g.,
anthramycin) synthesized by a number of gut microbes display not only
benzodiazepine-like but also antibiotic and antineoplastic activities and may thus
influence the biology of the microbiota and host alike in many respects. In addition,
this circumstance indicates that the gut microbiota is a rich source of yet-to-be-
identified compounds with therapeutic potential.
Apart from producing and releasing neuroactive factors, the microbiota modifies
the levels of metabolites that are relevant to the synthesis of transmitters in the
nervous system. For instance, the concentrations of tryptophan (the precursor of 5-
HT), tyrosine (the precursor of dopamine and noradrenaline) and glutamine in the
total brain of germ-free mice are lower than in mice that have been re-colonized by
the gut microbiota [16]. In the hippocampus of germ-free mice, however, the
concentrations of 5-HT and its main metabolite 5-hydroxyindoleacetic acid are
higher than in conventionally colonized mice [43]. Colonization of the germ-free
animals restores peripheral tryptophan levels to control values but fails to reverse
the changes in hippocampal 5-HT levels [43]. The concentrations of tryptophan, 5-
HT and tyrosine in the blood plasma are likewise increased in germ-free animals
[11, 43], the elevation of tryptophan being likely due to the absence of bacterial
tryptophanase [11]. Another explanation could be that the gut microbiota re-directs
the metabolism pathways of tryptophan which lead either to the production of 5-HT
or kynurenine [7].
Interaction of the Gut Microbiota with Gut Peptides
Due to their spatial vicinity with the gastrointestinal mucosa, the gut microbiota is
in a prime position to interact with the epithelial cells and to modify their activity.
Among these cells, enteroendocrine cells are poised to govern the activity of cells in
and outside the digestive system and in this way also to convey messages from the
microbial community in the gut. The enteroendocrine L cells in the distal ileum and
colon represent a distinct example of this interactive relationship. These cells are
stimulated by particular nutrients and digestive products, which leads to the release
of PYY, glucagon-like peptide-1 (GLP-1) and GLP-2 [6, 44, 45]. L cells are also
stimulated by short chain fatty acids (e.g., acetate, butyrate, propionate), which
particular microbes generate by fermentation of otherwise indigestible carbohy-
drate fibres. Short chain fatty acids stimulate L cells via activating G protein-
coupled receptors such as Gpr41 [6, 44, 45]. The important role of this
microbiota-host interaction is underscored by the finding that colonization of the
mouse colon with a fermentative human microbial community increases the plasma
level of PYY, an effect that is blunted by knockout of Gpr41 [45]. Gpr41 deficiency
is associated with a reduced expression of PYY, an increase in intestinal transit rate
and an attenuation of energy harvest [45].
Following their release from L cells, PYY and GLP-1 not only inhibit gastric
motility and improve glucose homeostasis but also induce satiety and behavioural
changes. Thus, butyrate is able to ameliorate aging-related memory decline in rats
[46] but has inconsistent effects on anxiety and depression-like behaviour
[47]. Propionate has been shown to evoke autism spectrum disorder-related behav-
iours in rodents [48, 49].
The interaction between the gut microbiota and intestinal L cells can be modu-
lated by the use of prebiotics (fermentable carbohydrates). Prebiotic supplementa-
tion in humans increases the plasma concentrations of GLP-1 and PYY, which is
associated with satiety and a decrease of postprandial glucose levels [50]. Experi-
ments in obese mice show that prebiotic treatment causes a change in the compo-
sition of the gut microbiota alongside with a decrease of inflammatory tone and an
enforcement of mucosal barrier function [51]. The complex interactions between
gut microbiota, mucosal function and metabolic homeostasis also involve the
endocannabinoid system [52] and GLP-2 which improves intestinal function
[51]. These interrelationships suggest that prebiotic supplementation has therapeu-
tic potential as “pharmaco-nutritional” approach to reversing host metabolic alter-
ations linked to intestinal dysbiosis in obesity and diabetes [53].
Given that nutritional status, dietary factors, physical activity and age have an
important influence on the composition of the gut microbial community [54, 55] it
is not surprising that appetite-regulating hormones other than PYY, GLP-1 and
GLP-2 will also interact with the gut microbiota in shaping appetite and metabolic
status. Emerging evidence indicates that this applies to ghrelin [55, 56], cholecys-
tokinin [56] as well as leptin [56]. In addition, germ-free mice have a smaller
number of enteroendocrine cells than conventionally colonized animals [56].
Interaction of the Gut Microbiota with Brain Function

and Behaviour: Emerging Neurochemical Mediators
Accumulating evidence shows that the absence or disturbance of the gut microbiota
has a significant impact on brain function and behaviour. There is also some
information on the molecular factors that may play an important role in this
interrelationship. In a first line of research, germ-free mice have been found to
exhibit a number of neurochemical and functional alterations relative to conven-
tionally colonized animals. For instance, the expression of the NMDA receptor
subunit 2A (NR2A) in the cortex and hippocampus [57] and of the NR2B unit in the
central amygdala [58] is decreased in germ-free mice, as is the expression of the 5-
HT receptor 1A (5HT1A) in the dentate granule layer of the hippocampus [58]. In
contrast, inconsistent changes in the levels of brain-derived neurotrophic factor
(BDNF), a key neurotrophin involved in neuronal growth and survival, have been
reported: two studies hold that BDNF in the hippocampus, amygdala and cortex of
germ-free mice is decreased [57, 59], while another study purports that the level of
BDNF in the hippocampus of germ-free mice is increased [58].
At the behavioural level, germ-free animals exhibit reduced anxiety in three [43,
58, 59] but not one study [60]. This outcome is somewhat surprising, since the
hypothalamic-pituitary-adrenal axis in germ-free mice appears to be hyperactive
rather than hypoactive [57]. Germ-free mice also show increased spontaneous
motor activity, an observation that may be related to elevated dopamine, noradren-
aline and 5-HT turnover in the striatum [59]. With regard to cognition, germ-free
mice have deficits in simple non-spatial and working memory tasks [60]. It awaits
to be examined whether the cognitive deficits are related to decreased
synaptogenesis and a decrease in the expression of synaptic plasticity-related
genes [59].
The impact of the gut microbiota on brain function has been confirmed by the
impact of antibiotic-induced dysbiosis on the gut-brain axis and by the effects of
selective probiotics on behaviour and brain chemistry. Disturbance of the gastro-
intestinal microbiota with a combination of nonabsorbable antibiotics (neomycin,
bacitracin, and pimaricin) increases exploratory behaviour and enhances BDNF
expression in the hippocampus [61]. Similar observations have been made with
another combination of nonabsorbable antibiotics (neomycin, cefoperazone and
ampicillin) which has an anxiolytic-like effect and impairs learning/memory in the
object recognition test [62].
Chronic treatment of mice with the probiotic Lactobacillus rhamnosus JB-1 has
been found to cause region-dependent alterations of GABAAα2 and GABAB1b
receptor mRNA in the brain, which are associated with a decrease in the stress-
induced corticosterone response and a reduction of anxiety- and depression-related
behaviour [63]. Importantly, these neurochemical and behavioural effects of pro-
biotic treatment are prevented by bilateral subdiaphragmatic vagotomy. This role of
vagal afferent neurons in communicating between gut bacteria and brain was
confirmed by another study in which vagotomy abolished the anxiolytic effect of
the probiotic Bifidobacterium longum NCC3001 in mice with experimentally
induced colitis [64].
Interaction of the Gut Microbiota with Brain Function

and Behaviour: The Direct Involvement of Neuropeptides
Awaits Exploration
Neuropeptides such as substance P, calcitonin gene-related peptide and NPY are

expressed at all levels of the microbiota-gut-brain axis and are likely to play an
important role in the bidirectional signalling between the gut and brain. Theoreti-
cally, neuropeptide-like molecules may also be produced by certain microbes, and
the gut microbiota will respond to neuropeptides and gut hormones if it expresses
the relevant receptors. However, direct evidence that these neuropeptides contrib-
ute to the communication between the gut microbial community and the central
nervous system is sparse. It also remains to be investigated whether alterations in
the microbial community within the gut impacts on neuropeptide systems in the
central nervous system.
The information available is mostly restricted to peptide level changes associ-

ated with manipulation of the intestinal microbiota. For instance, the colonic
content of substance P is enhanced following antibiotic-induced dysbiosis of the
intestinal microbiota [65]. The expression of neuropeptides in primary afferent
neurons (e.g., substance P, calcitonin gene-related peptide) has not yet been
addressed in experimental studies, although such studies appear worthwhile in
view of two lines of research relating the intestinal microbiota to pain. On the
one hand, the establishment of inflammatory hyperalgesia is attenuated in germ-
free mice [66]. On the other hand, treatment of rodents with the probiotic Lacto-
bacillus reuteri attenuates sensory neuron excitability [67] and alleviates the pain-
related response to gastric distension [68]. Lactobacillus acidophilus also reduces
experimentally evoked visceral pain, an effect that is associated with enhanced
expression of opioid and cannabinoid receptors in the intestinal mucosa
[69]. L. paracasei has been found to attenuate antibiotic-induced visceral hyper-
sensitivity in mice [65], while L. rhamnosus GG has a beneficial effect in abdominal
pain-related functional gastrointestinal disorders in childhood [70].
Neuropeptide Autoantibodies Under the Control

of the Intestinal Microbiota
The gut microbiota is important in educating the immune system to recognize

foreign antigens and to tolerate commensal microbes [71]. In this way, the gut
microbes can modulate, tune and tame the host immune response [72]. Dysbiosis of
the microbial community can lead to the development of autoimmunity [72, 73],
and experimental findings indicate that both autoimmune encephalomyelitis [74]
and autoimmune demyelination [75] involve the gut microbiota. There is also
evidence that the formation of autoantibodies against neuropeptides is governed
by intestinal microbes [76–78].
Specifically, IgG and IgA autoantibodies against alpha-melanocyte-stimulating
hormone, NPY, PYY, agouti-related protein (AgRP), ghrelin, leptin and some other
neuropeptides/peptides involved in appetite control are present in the human blood
[76–78]. Numerous intestinal microbes including Lactobacillus, Bacteroides,
Helicobacter pylori, Escherichia coli and Candida species contain proteins that
have amino acid sequences identical to these appetite-regulating peptides [78]. The
circulating levels of autoantibodies against alpha-melanocyte-stimulating hormone,
which are increased in anorexia nervosa and bulimia nervosa, correlate with the
psychobehavioural abnormalities of these eating disorders [76]. Vice versa, germ-
free rats have decreased levels of circulating IgA autoantibodies against several
appetite-regulating peptides, while the levels of antighrelin IgG are increased
[78]. A mechanistic analysis in rats has shown that alpha-melanocyte-stimulating
hormone autoantibodies are involved in the regulation of feeding and anxiety
[78]. It thus appears conceivable that the gut microbiota control appetite and
emotional behaviour indirectly by inciting the formation of autoantibodies against

neuropeptides/peptides involved in these processes.
Interaction of Gut Microbiota with Brain Function

and Behaviour: A Potential Role for NPY
NPY is a neurotransmitter that in view of its multiple implications in brain function

may play a particular role in the microbiota-gut-brain axis. This contention is based
on this neuropeptide’s involvement in controlling inflammatory processes, pain,
emotion, mood, cognition, stress resilience, ingestion and energy homeostasis
[6]. Consisting of 36 amino acids, NPY exerts its biological actions via five NPY
receptor types, termed Y1, Y2, Y4, Y5 and y6 (a human pseudogene), which are
coupled to pertussis toxin-sensitive Gi/o protein transduction mechanisms [79]. Y
receptors occur at all levels of the gut-brain and brain-gut axis [6, 80–84], the major
systems expressing this peptide being enteric neurons, primary afferent neurons,
several neuronal pathways throughout the brain and sympathetic neurons (Fig. 9.2).
Within the brain, NPY is one of the most abundant neuropeptides. In the context
of the gut-brain axis it is particularly worth noting that NPY occurs in the nucleus of
the solitary tract and ventrolateral medulla, periaqueductal grey and locus
coeruleus, paraventricular nucleus of the thalamus, hypothalamus (arcuate nucleus,
paraventricular nucleus and other regions), septum, hippocampus, amygdala, basal
ganglia, nucleus accumbens and cerebral cortex [6, 80–84]. Several important
pathways utilizing NPY as a neurotransmitter have been identified. These include
noradrenergic neurons originating in the locus coeruleus of the brainstem and
issuing both ascending and descending projections in the central nervous system,
neurons expressing both NPY and AgRP originating in the arcuate nucleus of the
hypothalamus, and distinct pathways operating in the limbic system [80–84]. The
major receptor subtypes which NPY acts on are the Y1 and Y2 receptors, which are
widely distributed in the central nervous system, while the localization of Y4 and
Y5 receptors is restricted to particular regions of the brain [82–85].
The NPY system may impact on the microbiota-gut-brain axis at distinct levels
[6, 81–84, 86–91]. It may
• influence the vitality of certain gut bacteria,
• modify gut functions such as motility, secretion and blood flow,
• regulate the activity of the immune system,
• protect against behavioural disturbances caused by peripheral immune
challenge,
• inhibit nociceptive transmission in the spinal cord and brainstem,
• protect from the impact of stress on the brain-gut axis,
• regulate food intake and energy homeostasis, and
• play a role in the interoceptive regulation of anxiety and mood.
NPY
Brain
Y1, Y2, Y4, Y5
Food intake, energy homeostasis Interoception

Cognition, emotion, mood, stress resilience Pain
Y1, Y2, Y4, Y5 Y1, Y2, Y4
NPY NPY
Sympathetic neurons Sensory neurons
Y1, Y2
Y1, Y2 Gut motility Y1, Y2

NPY
Gut secretion Enteric neurons
Blood flow
Y1, Y2, Y4, Y5
PYY Mucosal
Mucosal L cells immune system
Gastrointestinal
microbiota
Fig. 9.2 The NPY/Y receptor system in the microbiota-gut-brain axis. The graph shows the major
sources of NPY and PYY along the gut-brain axis and the Y receptor subtypes which mediate the
effects of these peptides at the different levels of the gut-brain axis. The symbol
denotes inhibition
Effect of NPY on Gut Bacteria
There is some evidence that the NPY system has an impact on the composition and
function of the gut microbiota and its relevance to the gut-brain axis. Similarly to
substance P, calcitonin gene-related peptide and vasoactive intestinal polypeptide,
NPY has been found to exhibit a direct antimicrobial effect against various gut
bacteria including E. coli, Enterococcus faecalis, and L. acidophilus [86].
Effects of NPY on the Immune System
In the context of the microbiota-gut-brain axis it is important to mention that NPY

has a distinct impact on immune function, within and outside the gastrointestinal
tract [6, 87–89]. NPY released from the sympathetic nerve fibres acts on Y
receptors (notably of the Y1, Y2, Y4 and Y5 subtype) expressed by distinct classes
of immune cells (e.g., dendritic cells, mononuclear cells, macrophages,
granulocytes, T and B lymphocytes) to modify their activity [6, 89–91]. In addition,
NPY acts as a paracrine or autocrine immune mediator, because immune cells (e.g.,
B and T lymphocytes, macrophages) themselves can produce and release NPY [6,
88, 91]. The effects of NPY include modulation of immune cell trafficking,
activation of antigen-presenting cell function, T helper cell differentiation, negative
regulation of T cell function, cytokine secretion, phagocytosis and production of
reactive oxygen species [6, 87–91].
With this immunological activity profile, NPY regulates inflammatory processes
in the gut, given that NPY-containing nerve fibres are in close contact with immune
cells in the mouse ileum lamina propria [92]. Specifically, NPY is able to promote
colonic inflammation, an effect that is supported by several lines of evidence:
(1) NPY knockout mice are largely resistant to the induction of dextran sulfate
sodium-induced colitis [93, 94]. (2) The result of NPY deletion is reproduced by
treatment with a NPY antisense oligodeoxynucleotide [95] and by knockout or
antagonism of Y1 receptors [96]. The antiinflammatory phenotype of Y1 receptor
knockout mice results from a defect in antigen-presenting cell function, a reduction
of TNF-alpha and IL-12 production by macrophages, and a decrease in the number
of effector T cells [90]. Furthermore, experimentally induced colitis is associated
with an increase in the colonic synthesis of NPY [93, 95, 97], a reduction of colonic
Y1 receptor expression and a loss of the antisecretory action of NPY in the colon
[98]. In contrast, the colonic levels of the related gut hormone PYY are decreased in
rats with DSS-induced colitis [99]. These experimental data are in line with a
decrease of colonic PYY levels in patients with inflammatory bowel disease [100–
102], while circulating levels of PYY and NPY are enhanced [103, 104]. The
proinflammatory effect of NPY could in part be counterregulated by the
vasoconstrictor effect of the peptide [105].
Effect of NPY to Protect from Immune Challenge-Evoked

Behavioural Disturbances
Infection and inflammation are increasingly recognized to have an impact on the

pathogenesis of mood disorders [18, 19, 106, 107], and clinical evidence suggests
that activation of the intestinal immune system by constituents of the intestinal
microbiota can give rise to depression [34] and chronic fatigue syndrome
[35]. Experimentally, the impact of peripheral immune challenge on brain function
and behaviour can be modelled by systemic administration of LPS or Bacille
Calmette-Guérin [18, 19, 106, 108]. The signalling pathways whereby peripheral
immune challenge alters brain mechanisms involve proinflammatory cytokines
such as interleukin-6, tumour necrosis factor-alpha and interferon-gamma, which
reach the brain via the circulation but also excite vagal afferent neurons and lead to
the expression of cytokines by cerebral microglial cells and astrocytes [18, 19,
106–108].
The effect of peripheral immune challenge on brain function involves several

brain areas that express NPY and various Y receptors [6, 81–84]. NPY is involved
in the regulation of emotional-affective behaviour [6, 81–84], and there is indirect
evidence that NPY-expressing neurons in the arcuate and paraventricular nuclei of
the hypothalamus counteract the behavioural responses to immune stress and
infection [109–111]. This implication has been confirmed by knockout experiments
in which the NPY/Y receptor system has been found to protect against distinct
functional disturbances in response to immune challenge. For instance, deletion of
NPY as well as NPY plus PYY aggravates the Bacille Calmette-Guérin-induced
loss of body weight and markedly delays recovery from this weight loss [112]. This
finding attests to an important role of NPY and PYY in maintaining energy
homeostasis in the face of immune stimulation [112].
Analogous observations have been made when the behavioural responses to LPS
are analysed in Y2 and Y4 knockout mice [6]. Y2 receptor knockout mice are
particularly susceptible to the acute action of LPS to attenuate locomotion and
suppress social interaction [20]. In contrast, the LPS-induced rise of temperature
and circulating corticosterone is suppressed by Y2 receptor knockout [20]. The
short-term effect of LPS to enhance anxiety is enhanced in Y2 and Y4 receptor
knockout mice [20, 21]. In Y4 receptor knockout mice, the anxiogenic response to
LPS persists at least for 4 weeks post-treatment by which time it has waned in WT
mice [21]. Depression-related behaviour is enhanced 1 day post-LPS in control and
Y2 receptor knockout mice, but not in Y4 receptor knockout mice. Four weeks post-
treatment the depressogenic effect of LPS has waned in wildtype mice, but is
maintained in Y2 receptor knockout mice and first observed in Y4 receptor knock-
out mice [21]. Thus, knockout of Y2 and/or Y4 receptors unmasks the ability of
immune challenge with LPS to cause a delayed and prolonged increase in anxiety-
and/or depression-like behaviour [6]. These findings suggest that NPY acting via
Y2 and Y4 receptors prevents the development of long-term anxiety- and
depression-like behaviour caused by immune challenge [6, 21]. It awaits examina-
tion whether the behavioural disturbances associated with dysbiosis of the gut
microbiota are likewise under the control of the NPY/Y receptor system.
Effect of NPY to Inhibit Nociceptive Transmission
Spinal afferent neurons, which contain low amounts of NPY, terminate in the spinal
cord where interneurons and descending noradrenergic neurons express appreciable
amounts of NPY [6, 113, 114]. An abundant occurrence of Y1 and Y2 receptors in
the spinal cord enables NPY to play an important role in the processing of incoming
nociceptive information. Germ-line knockout of Y1 receptors or conditional knock-
down of NPY is associated with thermal, chemical and mechanical hyperalgesia [6,
113–116]. Peripheral inflammation leads to an upregulation of Y1 receptors in
spinal afferent neurons and in the dorsal horn of the spinal cord [117]. While these
studies have primarily focused on somatic pain, it remains to be investigated
whether the NPY/Y receptor system also plays a role in the impact of the gut
microbiota on visceral pain sensitivity [66–70]. Two major mechanisms whereby
NPY controls pain transmission in the spinal cord have been envisaged: inhibition
of transmitter release from the terminals of primary afferent neurons, mediated
primarily by Y2 receptors, and inhibition of postsynaptic neurons in the dorsal horn,
mediated primarily by Y1 receptors [113, 114].
Apart from spinal sensory neurons, vagal afferent neurons terminating in the
nucleus tractus solitarii (NTS) have also been established to play a role in visceral
nociception, particularly in visceral chemonociception [118]. Y2 and Y4 receptors
are the Y receptor subtypes prevailing in the NTS [6, 119], and gene deletion
experiments have revealed that endogenous NPY acting via Y2 and Y4 receptors
attenuates the chemonociceptive input from the stomach to the brainstem [119].
Effect of NPY to Protect from the Impact of Stress

on the Gut-Brain Axis
Inflammation and psychosocial stress have a marked impact on the bidirectional

communication between the gut and brain [5]. Since NPY plays a role in stress
coping [84], it may also be relevant to the impact of stress on the gut-brain axis.
NPY as well as Y1, Y2 and Y5 receptors are widely expressed in cerebral areas
critical to the regulation of stress resilience [81–84]. The expression of NPY in the
human brain is related to polymorphisms in the NPY gene, and a low NPY
expression genotype is associated with negative emotional processing, diminished
stress resilience, a risk for major depression, and a reduced antidepressant treatment
response [120–122]. If individuals with a low NPY expression genotype are
exposed to negative stimuli, there is an exaggerated activation of the amygdala,
medial prefrontal cortex and anterior cingulate cortex [120–122]. The concentration
of NPY in the cerebrospinal fluid and plasma is reduced in patients with post-
traumatic stress disorder, while trauma-exposed individuals who do not develop or
have recovered from post-traumatic stress disorder have enhanced plasma levels of
NPY [123–125]. It would appear, therefore, that the cerebrospinal and plasma
concentration of NPY is a biological correlate of resilience to or recovery from
the adverse effects of stress [124]. Animal experiments have confirmed that NPY is
involved in the emotional processing of stress [6], and the question arises whether
this role also relates to the impact of stress on the microbiota-gut-brain axis.
Since stress can alter the permeability of the gastrointestinal mucosa [126, 127],
it is very likely that stress will also alter the interaction between the gut microbiota
and the mucosal immune system. CRF is a neuropeptide and gut hormone that is
intimately related to stress, and there is considerable evidence that activation of
peripheral CRF receptors contributes to stress-related alterations of gut physiology
[126]. NPY may likewise be involved because it appears to mediate the effects of
stress on many physiological systems including the gastrointestinal and immune
systems [128, 129]. For instance, deletion of NPY alters gastrointestinal, feeding
and corticosterone responses to restraint stress, exaggerates stress-induced
defaecation and reduces food intake [128]. Trinitrobenzene sulfonic acid-induced
colitis increases the NPY concentration in brain and plasma [97], and gastrointes-
tinal inflammation enhances anxiety- and depression-related behaviour, this effect
being modified by deletion of NPY and/or PYY [94, 130]. It follows that NPY and
PYY participate in the effect of intestinal inflammation on the gut-brain axis. In
addition, the depression-like phenotype of PYY knockout animals [94] suggests
that alterations in the expression of this gut hormone modify mood and stress
coping. This contention is in line with the finding that water avoidance stress lowers
the plasma level of PYY, a change that is associated with an increase in gastroin-
testinal motility [131].
Effects of NPY and PYY to Regulate Food Intake and Energy

Homeostasis
The implications of NPY and PYY in gut-brain signalling are particularly well
exemplified by their effects on hunger, food intake, satiety and energy balance.
These roles have been extensively reviewed elsewhere [10, 132, 133] and may be of
particular relevance to the impact of the gut microbiota on metabolic regulation,
energy homeostasis and metabolic disorders. PYY is released postprandially from
intestinal L cells and acts as a satiety factor, slowing gastrointestinal transit,
inhibiting further intake of food and modifying the metabolic status of the organism
[10]. In the circulation, PYY is truncated to PYY3-36 which is a relatively selective
Y2 receptor agonist. Food intake is inhibited by PYY3-36 both via stimulation of Y2
receptors on vagal afferent neurons and an interaction with Y2 receptors in the
hypothalamus [6, 10, 134, 135]. Within the brain, PYY3-36 reduces food intake
primarily via activation of Y2 receptors in the arcuate nucleus which is an important
centre for integrating peripheral and central signals in the control of appetite and
energy homeostasis [136]. NPY, on the other hand, is one of the most potent
orexigenic peptides found in the brain [133, 136]. Specifically, it occurs in neurons
projecting from the arcuate nucleus to various areas of the hypothalamus in which
the orexigenic effect of NPY is primarily mediated by Y1 receptors, although Y5
receptors also contribute [133, 136]. Pathologies associated with a decrease in food
intake such as experimental colitis lead to increased release of NPY from the
paraventricular nucleus of the hypothalamus [137], again attesting to a role of
NPY in gut-brain signalling.
NPY, PYY and Other Gut Peptides in the Interoceptive

Regulation of Emotion and Mood
Apart from regulating ingestion and energy homeostasis, gut hormones such as
ghrelin, PYY, GLP-1 and GLP-2 have an impact on emotional-affective behaviour.
In an evolutionary point of view, co-regulation of appetite and emotional state is an
important strategy for survival, given that anxiety would be an adverse condition
when there is a need to seek food [6]. Indeed, ghrelin which is released from the
upper gastrointestinal tract under conditions of hunger reduces both anxiety-like
and depression-related behaviour [138]. Under fed conditions, behaviour is
changed to a hedonic state as observed when PYY3-36 is administered to reach
postprandial plasma concentrations of the peptide [139]. The ability of PYY to
promote hedonic behaviour is supported by the finding that knockout of PYY
increases depression-like behaviour but does not alter anxiety [94]. Physiologically,
however, emotion and mood under fed conditions will be determined by the
presence of a variety of gut hormones such as PYY, GLP-1 and GLP-2 that are
released postprandially. Thus, GLP-1 has been found to enhance anxiety-related
behaviour [140–142], while GLP-2 attenuates depression-like behaviour [143].
Gut hormones whose release from the enteroendocrine cells is likely to be
regulated by the gut microbiota thus provide a constant stream of interoceptive
input from the gut to the brain.
Conclusion: The Gut Microbiota Meets Neuropeptides
The gut microbiota has proved as a novel factor relevant to health and disease. How
the gut microbiota communicates with distant organs such as the brain is only
beginning to emerge. It is very probable that the microbiota will take use of several
information carriers from the gut to the brain including microbiota-derived signal-
ling molecules, immune mediators, gut hormones as well as vagal and spinal
afferent neurons. Biologically active gut peptides and neuropeptides play a role in
several of these communication pathways. This is true for peptides produced by
enteroendocrine cells which respond to metabolites generated with the help of the
microbiota. PYY, which is one of these peptides, acts via Y receptor types that are
also stimulated by the neuropeptide NPY. Neuropeptides are important transmitters
in afferent, central and efferent pathways of the bidirectional gut-brain communi-
cation network. It remains to be shown whether the gut microbiota itself expresses
neuropeptide receptors or releases metabolites that are ligands at neuropeptide
receptors. Although a direct link between the gut microbiota and distinct neuro-
peptide systems has not yet been revealed, NPY, CRF and tachykinins are very
likely to emerge as messengers in the microbiota-gut-brain axis.
Acknowledgements This study was supported by the Zukunftsfonds Steiermark (grant 262), the
Austrian Science Fund (FWF grants L25-B05, P23097-B18 and P25912-B23), and the Federal
Ministry of Science and Research of the Republic of Austria (grant GZ 80.104/2-BrGT/2007).
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Chapter 10
Bacterial Neuroactive Compounds Produced
by Psychobiotics
Rebecca Wall, John F. Cryan, R. Paul Ross, Gerald F. Fitzgerald,

Timothy G. Dinan, and Catherine Stanton
Abstract We recently coined the phrase ‘psychobiotics’ to describe an emerging

class of probiotics of relevance to psychiatry [Dinan et al., Biol Psychiatry 2013;74
(10):720–726]. Such “mind-altering” probiotics may act via their ability to produce
various biologically active compounds, such as peptides and mediators normally
associated with mammalian neurotransmission. Several molecules with neuroactive
functions such as gamma-aminobutyric acid (GABA), serotonin, catecholamines
and acetylcholine have been reported to be microbially-derived, many of which
have been isolated from bacteria within the human gut. Secreted neurotransmitters
from bacteria in the intestinal lumen may induce epithelial cells to release mole-
cules that in turn modulate neural signalling within the enteric nervous system and
consequently signal brain function and behaviour of the host. Consequently, neuro-
chemical containing/producing probiotic bacteria may be viewed as delivery vehi-
cles for neuroactive compounds and as such, probiotic bacteria may possibly have
the potential as a therapeutic strategy in the prevention and/or treatment of certain
neurological and neurophysiological conditions.
R. Wall • R.P. Ross • C. Stanton (*)

Alimentary Pharmabiotic Centre, Teagasc Moorepark Food Research Centre, Fermoy, Cork,
Ireland
e-mail: Catherine.stanton@teagasc.ie
J.F. Cryan (*)
Department of Anatomy and Neuroscience, University College Cork, Cork, Ireland
e-mail: j.cryan@ucc.ie
G.F. Fitzgerald
Microbiology and Alimentary Pharmabiotic Centre, University College Cork, Cork, Ireland
T.G. Dinan
University College Cork, Cork, Ireland
222 R. Wall et al.
Abbreviations
AA Arachidonic acid
ASD Autism spectrum disorders
CLA Conjugated linoleic acid
GAD Glutamate decarboxylase
GF Germ-free
GIT Gastrointestinal tract
IPA Indole-3-propionic acid
LAB Lactic acid bacteria
LC-PUFA Long-chain fatty acid
PPAR γ Peroxisome proliferator-activated receptor gamma
SCFA Short chain fatty acid
Introduction
We recently coined the phrase ‘psychobiotics’ to describe an emerging class of

probiotics of relevance to psychiatry [1]. Such “mind-altering” probiotics may act
via their ability to produce various biologically active compounds, such as peptides
and mediators normally associated with mammalian neurotransmission. Several
molecules with neuroactive functions such as gamma-aminobutyric acid (GABA),
serotonin, catecholamines and acetylcholine have been reported to be microbially-
derived, many of which have been isolated from bacteria within the human gut.
Secreted neurotransmitters from bacteria in the intestinal lumen may induce epi-
thelial cells to release molecules that in turn modulate neural signalling within the
enteric nervous system and consequently signal brain function and behaviour of the
host. Consequently, neurochemical containing/producing probiotic bacteria may be
viewed as delivery vehicles for neuroactive compounds and as such, probiotic
bacteria may possibly have the potential as a therapeutic strategy in the prevention
and/or treatment of certain neurological and neurophysiological conditions.
In recent years, interdisciplinary investigation has revealed strong evidence of
the existence of a bidirectional signalling between the intestine and the brain, the so
called “brain-gut axis”. This communication system integrates neural, hormonal
and immunological signalling between the gut and the brain and is critical to
maintain homeostasis [2]. More recently, however, this axis concept was expanded
to the “microbiota-gut-brain axis”, when it became clear that not only the intestinal
tract itself but also its 100 trillion microbial inhabitants can affect the functioning of
the central nervous system (CNS) and consequently mood and behaviour [3, 4]. The
10 Bacterial Neuroactive Compounds Produced by Psychobiotics 223
brain communicates with the enteric microbiota directly by releasing signalling

molecules into the gut lumen, and indirectly by altering gastric motility, secretion
and intestinal permeability [5]. Equally, the enteric microbiota can communicate
with the host via epithelial cells, receptor-mediated signalling, and stimulation of
cells of the lamina propria [6]. Changes in the composition of the gut microbiota
may lead to deterioration in gastrointestinal, neuroendocrine, or immune pathways
and relationships, which in turn could lead to alterations in brain-gut interactions
and consequently result in disease [7].
Recently, the microbial endocrinology-based theory was introduced which
claimed that probiotics (i.e. live microorganisms that, when ingested in adequate
amounts, exerts a health benefit on the host [8]) function as pharmacological agents
and hence act as drug delivery vehicles due to their ability to synthesize neuroactive
compounds [9]. As such, probiotics may affect the brain in a direct manner by
producing neurotransmitters and neuromodulators and may therefore have the
potential to act as a novel treatment for neuropsychiatric diseases. The delivery of
neurochemicals by probiotics may either be in the amount already contained in the
bacterium at time of ingestion or what is actively produced by the bacterium once
inside the gastrointestinal tract (GIT).
It is well recognized that some bacteria within the human GIT have the capacity
to produce many neurotransmitters and neuromodulators. For example, Lactoba-
cillus spp. and Bifidobacterium spp. have been reported to produce GABA;
Escherichia spp., and Bacillus spp. have been reported to produce norepinephrine;
Streptococcus spp., Escherichia spp. and Enterococcus spp. have been reported to
produce serotonin; Bacillus spp. have been reported to produce dopamine, and
Lactobacillus spp. have been reported to produce acetylcholine and histamine [10–
14]. It is possible that the secreted neurotransmitters from bacteria in the intestinal
lumen may induce epithelial cells to release molecules that in turn modulate neural
signalling within the enteric nervous system, or act directly on primary afferent
axons [15]. Other bacterially-produced metabolites with proven neuroactive func-
tions include short chain fatty acids (SCFAs) and long chain fatty acids such as
conjugated linoleic acid (CLA). Table 10.1 provides a list of a range of neuroactive
chemicals isolated from bacteria within the human gut (it should be noted that this
is representative of neuroactives isolated, but is not a complete and comprehensive
list). The production of these metabolites and the aforementioned neuroactives by
bacteria naturally inhabiting the human gut will be discussed in this chapter.
Bacterial Metabolites
The microbiome has a capability to produce a spectrum of neuroactive compounds,

and although we are still in an early stage of exploring its capacity, there is an
expanding volume of evidence supporting the role of our intestinal inhabitants as
being factories for neurochemicals. Studies comparing germ-free (GF) animals
(lacking gut microbiota) with conventional animals (with a normal gut microbiota)
224 R. Wall et al.
Table 10.1 Representative list of neurochemicals isolated from bacteria within the human gut
Genus Neurochemical References
Lactobacillus, Bifidobacterium GABA [10]
Streptococcus, Escherichia, Enterococcus, Lactococcus, Serotonin [17, 49]
Lactobacillus
Escherichia, Bacillus Norepinephrine [14, 49]
Escherichia, Bacillus, Lactococcus, Lactobacillus, Dopamine [14, 17, 49]
Streptococcus
Lactobacillus, Bacillus Acetylcholine [12, 13, 59, 61]
Lactobacillus, Lactococcus, Streptococcus, Enterococcus Histamine [11, 66, 67]
have demonstrated that the commensal microbiota influence monoamine levels in

specific brain regions of the host brain [3, 16]. Neurochemicals that have been
isolated from gut bacteria include GABA, noradrenaline, serotonin, dopamine and
acetylcholine [10, 17, 18], which may directly affect the brain. The use of probiotic
bacteria that can deliver neurochemicals has further been suggested as a novel
treatment for neuropsychiatric diseases [9]. Other bacterial metabolites with neuro-
active functions include SCFAs such as propionate and long chain fatty acids such
as CLA [19–22].
It is not yet clear as to why certain bacteria harbour the genes responsible for the
production of neuroactive molecules. It has been proposed that late horizontal gene
transfer can explain the existence of genes encoding many of the enzymes involved
in the synthetic and metabolic pathways of catecholamines, acetylcholine, and
GABA from bacteria. This concept is concordant with increasing evidence that
signalling molecules of quorum-sensing systems, used by bacteria to communicate
and coordinate their actions [23] can also bind to mammalian receptors and directly
influence the host [24, 25]. Neurotransmitters that are produced by the host can
furthermore influence the function of members of the microbiota. As an example,
the QseC sensor kinase, present in Escherichia coli O157:H7 is a bacterial receptor
for host-derived epinephrine/norepinephrine which triggers the transcription of
virulence genes in bacteria, a response which can be blocked by adrenergic
antagonists [26].
Gamma-Aminobutyric Acid
Gamma-aminobutyric acid (GABA) is a major inhibitory neurotransmitter in the

brain regulating many physiological and psychological processes, and dysfunctions
in GABA signalling have been linked to anxiety and depression [27]. We have
recently demonstrated that human intestinally derived strains of lactobacilli and
bifidobacteria produce GABA from monosodium glutamate (MSG) in culture [10]
and it has been suggested that microbially produced GABA may have an effect on
the brain-gut axis [28]. The production of GABA by commensal bacteria occurs via
the same biosynthetic pathway as in neuronal tissue involving conversion of

glutamate by the action of the enzyme glutamate decarboxylase (GAD) and vitamin
co-factor pyridoxal phosphate [29]. The GABA-producing capability held by some
bacterial strains is thought to protect the organism from the acidic environment
encountered in the stomach, since its synthesis involves proton exchange for the
uptake of glutamate [30]. Many studies have reported the presence of a gad gene in
lactic acid bacteria (LAB) [29, 31, 32] and given the heightened interest in the
physiological effects associated with GABA, many GABA-enriched fermented
food products, using dairy starter cultures with GABA-producing capabilities,
have been developed in the past 10 years [31, 33, 34]. The levels of GABA that
can be achieved in vitro by probiotic organisms are quite large. For example, in the
production of fermented foodstuffs, such as Japanese funa-sushi and Chinese
traditional paocai, which uses lactobacilli as starter cultures, GABA levels in the
millimolar range have been detected in the final products [35, 36].
The prevalence of GABA-producing lactobacilli and bifidobacteria in the human
GIT is not as widespread as it is among food-derived LAB. We screened 91 strains
of human-derived lactobacilli and bifidobacteria for their ability to produce GABA
from MSG, and found that five strains had the ability [10], with Lactobacillus brevis
and Bifidobacterium dentium being the most efficient GABA producers. L. brevis
DPC6108 was the most efficient of the strains tested, and it retained the capability
to produce GABA in the presence of other gut-derived bacteria (in faecal fermen-
tations). A recent study also demonstrated that GABA production in black soybean
milk by L. brevis FPA3709 and its administration to rats resulted in an antidepres-
sant effect similar to that of fluoxetine (a common antidepressant drug) but without
the side effects of lost appetite and decreased weight [37]. Interestingly, neuronal
cells have been shown to respond to nanomolar concentrations of GABA [38]. At
the level of gene expression, ingestion of the Lactobacillus strain, Lactobacillus
rhamnosus (JB-1), altered the mRNA expression of both GABAA and GABAB
receptors. These receptors are implicated in anxiety and depression, and are widely
expressed in key brain regions responsible for maintaining normal fear and mood
responses [39].
A number of further potential health benefits of GABA have been described,
including induction of hypotension, diuretic effects, and tranquilizer effects [40,
41]. Furthermore, GABA has a receptor-mediated role in a number of immuno-
logical (i.e. down-regulation of cytokine released by proinflammatory cells release)
and intestinal neurophysiological (i.e. secretion of neuropeptides by intrinsic and
extrinsic intestinal nerve fibers) processes [38, 42, 43]. Given the broad health
benefits associated with GABA, the use of a GABA-secreting bacterium, acting on
dietary glutamate, could have potential in both neuropsychiatric diseases and in
inflammatory conditions such as inflammatory bowel disease (IBD), however,
in vivo studies are required to ascertain whether the host would benefit from
microbially- produced GABA.
226 R. Wall et al.
Serotonin and 5-HT Precursors
Serotonin (5-hydroxytryptamine, 5-HT) is a metabolite of the essential amino acid

tryptophan and plays an important role in the regulation of a number of bodily
functions, including mood. Today, the vast majority of antidepressant drugs lead to
increases in the levels of serotonin in the brain. Serotonin is ubiquitously distributed
in nature and has been found in some plants (fruits and nuts) and in both vertebrates
and invertebrates animals [44]. Some studies also indicate that bacteria can syn-
thesize serotonin and/or induce its production by the host. Wikoff et al. [45] utilized
a metabolomics-based approach to study the metabolic products of the microbiome
in mice which may impact health, and unexpectedly found that serotonin plasma
levels were nearly threefold higher in conventional mice compared with GF mice,
whereas plasma concentrations of tryptophan was 40 % lower in conventional
animals than in their GF counterparts. The authors postulated that the increased
plasma serotonin levels observed could indirectly result from an as yet undefined
host microbe interaction [45]. Furthermore, increased serotonin turnover and
altered levels of related metabolites in the striatum [3] and hippocampus [46] of
GF mice have been reported. The levels of serotonin in the cortex and hippocampus
were also significantly reduced in GF mice [4], suggesting a role for the microbiota
in maintaining serotonin levels. Rats that were given Bifidobacterium infantis for
14 days had increased concentrations of the serotonin precursor tryptophan in
plasma, suggesting that commensal bacteria have the ability to influence tryptophan
metabolism [47]. This effect on tryptophan metabolism may be mediated by the
impact of the microbiota on the expression of indoleamine-2,3-dioxygenase, a key
enzyme in the physiologically dominant kynurenine pathway of tryptophan degra-
dation [48]. Moreover, Özogul [17] investigated the influences of LAB on biogenic
amine formation by foodborne pathogens using single and mix cultures. All the
LAB species used in their study, including Lactococcus lactis subsp. cremoris
(MG 1363), L. lactis subsp. lactis (IL1403), Lactobacillus plantarum (FI8595)
and Streptococcus thermophilus (NCFB2392) produced serotonin to some extent
[17]. Shishov et al. [49] also demonstrated that E. coli K-12 was capable of
producing serotonin at nanomolar concentrations in culture.
Catecholamines
Catecholamines such as dopamine and norepinephrine (also known as noradrena-

line) are the major neurotransmitters that mediate a variety of the CNS functions,
such as motor control, cognition, memory processing, emotion and endocrine
regulation [50]. Dysfunctions in catecholamine neurotransmission are implicated
in some neurological and neuropsychiatrical disorders, including Parkinson’s dis-
ease [51], Alzheimer’s disease [52] and major depressive disorders [53].
Both norepinephrine and dopamine were identified in bacteria in a study by

Tsavkelova et al. [14]. Dopamine, in concentrations from 0.45 to 2.13 mmol/L was
found in the biomass of Bacillus cereus, B. mycoides, B. subtilis, Proteus vulgaris,
Serratia marcescens, S. aureus, and E. coli, and norepinephrine was detected (0.21–
1.87 mmol/L) in B. mycoides, B. subtilis, P. vulgaris, and S. marcescens. Moreover,
it was demonstrated that bacteria, particularly B. subtilis may release norepineph-
rine and dopamine out of the cell and perhaps in this way might participate in
intercellular microbe–microbe and microbe–host communications [14]. Shishov
et al. [49] used the E. coli K-12 strain to investigate the production of catechol-
amines in vitro and demonstrated that this E. coli strain can produce dopamine and
norepinephrine and also their precursor, DOPA, in culture. The culture fluid of
E. coli contained micromolar concentrations of DOPA and nanomolar concentra-
tions of dopamine and norepinephrine [49]. Moreover, Ö zogul [17] demonstrated
the production of dopamine by some LAB species in culture, namely L. lactis
subsp. cremoris (MG 1363), L. lactis subsp. lactis (IL1403), L. plantarum (FI8595)
and S. thermophilus (NCFB2392). A recent study by Asano et al. [54] demonstrated
that bacteria which constitute the normal microbiome in mice are capable of the
in vivo production of large quantities of norepinephrine. Furthermore, adoptive
transfer of the microbiome of mice that could produce norepinephrine in vivo to GF
mice resulted in the in vivo elaboration of norepinephrine within the murine GIT
[54]. Interestingly, in many cases, the content of catecholamines found in bacteria is
higher than in human blood, for example concentrations of norepinephrine in
human blood are found to be 0.04 mmol/L [55].
Acetylcholine
Acetylcholine is a well-known neurotransmitter in the central and peripheral

nervous systems that plays a critical role in cognitive function, particularly in
memory and learning. It is synthesized by choline acetyltransferase in the CNS
and by both choline acetyltransferase and carnitine acetyltransferase in the peri-
pheral system [56, 57]. Acetylcholine has also been identified in non-neuronal
tissues, including gastrointestinal, respiratory and urogenital epithelial cells
[58]. In addition, acetylcholine has been found to be a component of bacteria and
its production was discovered in a strain of L. plantarum [13, 59, 60]. Cell free
enzyme(s) participating in acetylcholine synthesis were also found in L. plantarum
[59]. Horiuchi et al. [61] tested three different bacterial strains for acetylcholine
content and synthesis, E. coli JCM 5491, Staphylococcus aureus JCM 2151 and
Bacillus subtilis PCI 219. Among these, a substantial amount of acetylcholine was
detected in B. subtilis (55.7 pmol/1010 colony forming units), while much smaller
amounts were found in E. coli (2.22 pmol) and S. aureus (0.39 pmol). Although
acetylcholine synthesis was detected in all bacterial samples, the levels were low
and the authors suggested that an acyltransferase other than choline
228 R. Wall et al.
acetyltransferase and carnitine acetyltransferase was responsible for acetylcholine

synthesis in these bacteria [61].
Histamine
Histamine acts as a modulatory neurotransmitter in the mammalian brain and has an

important role in the maintenance of wakefulness, while dysfunction in the hista-
minergic system has been linked to narcolepsy [62]. Moreover, behavioural studies
suggest that the histaminergic system in the brain has important roles in cognitive
function [63]. Levels of histamine are decreased in the hippocampus, temporal
cortex and hypothalamus of patients with Alzheimer’s disease, suggesting that
histaminergic neurons undergo degeneration and contribute to cognitive decline
in this disorder [64].
Histamine is produced via histidine decarboxylase (HDC) of L-histidine, an
essential amino acid for humans that is present in many dietary foods [65]. Some
fermentative bacteria, including strains of Lactobacillus, Lactococcus, Strepto-
coccus, Pediococcus and Enterococcus have been reported to possess the HDC-
gene and to produce histamine at different levels [66, 67]. However, the
production of histamine by certain bacterial strains has caused alarm as a health risk
in food and as a marker of food spoilage. Ingestion of food containing high
concentrations of histamine has been linked with headaches, vomiting and hyper-
tension [68]. Nonetheless, histamine production by the probiotic strain Lacto-
bacillus reuteri (ATCC PTA 6475) was recently reported, resulting in a
suppression of human proinflammatory tumor necrosis factor (TNF) production
[11]. Moreover, histidine supplementation increased the expression of HDC-genes
and the production of histamine by L. reuteri. In addition to the role of histamine in
immunomodulation observed in the study by Thomas et al. [11], luminal production
of histamine by L. reuteri 6475 may influence signalling in the enteric nervous
system. However, in vivo studies are needed to gain a better understanding of the
role of bacterially-produced histamine in the gut and that such production does not
induce negative side-effects.
Indole-3-Propionic Acid
Indole-3-propionic acid (IPA) is a deamination product of tryptophan and is found

in plasma and cerebrospinal fluid [69, 70]. IPA has been shown to be a powerful
antioxidant [71] and has been considered as a possible treatment for Alzheimer’s
disease [72] due to its ability to protect neurons and neuroblastoma cells against
oxidative damage and death [73, 74].
Wikoff et al. [45] demonstrated that the production of IPA was completely
dependent on the presence of gut microbiota and could also be established by
colonizing germ-free mice with the bacterium Clostridium sporogenes. An earlier

study by Jellet et al. [75] also demonstrated that IPA was present in the spent
bacterial media of C. sporogenes and that addition of the precursor tryptophan to
the media greatly enhanced the formation of IPA. In addition, IPA was shown to be
produced in vitro when human large intestinal contents were incubated with
tryptophan and indolelactate [76].
Short-Chain Fatty Acids
Short chain fatty acids (SCFA) are the major products of the bacterial fermentation
of carbohydrates and proteins in the GIT [19, 77]. The main compounds are acetic,
propionic and n-butyric acids, occurring roughly in molar ratios of 60:20:20 in the
colon [78]. Through their absorption and metabolism, the host is able to salvage
energy from foodstuffs, particularly resistant starch and fibers that are not digested
in the upper part of the GIT. The main site for SCFA production and absorption is
the proximal large intestine, where the fermentation of undigested food by colonic
bacteria occurs at high rates. Bacteria that produce SCFA include, but are not
limited to, Bacteroides, Bifidobacterium, Propionibacterium, Eubacterium, Lacto-
bacillus, Clostridium, Roseburia and Prevotella [19]. SCFA have a multiplicity of
effects in the body, and affect epithelial cell transport and metabolism, epithelial
cell growth and differentiation, and hepatic control of lipid and carbohydrates,
while providing energy sources for muscles and kidneys, as well as the heart and
brain [79]. Epithelial cells in the distal colon derive 60–70 % of their energy
requirements from bacterial fermentation products [80]. SCFA also act as signal-
ling molecules. Propionate, acetate, and to a lesser extent butyrate are ligands for at
least two G protein-coupled receptors (GPCRs), Gpr41 and Gpr43, which are
broadly expressed in the distal small intestine, colon and adipocytes
[81, 82]. SCFA interaction with Gpr43 can profoundly affect inflammatory
responses. For example, mice treated with oral acetate showed a substantial
decrease in inflammation. This protection was mediated by acetate binding to
Gpr43, because acetate had no effect in Gpr43-deficient mice. Furthermore, it
was shown that Gpr43 exhibited enhanced expression in neutrophils and eosino-
phils, suggesting that SCFA-Gpr43 signalling is one of the molecular pathways
whereby commensal bacteria regulate immune and inflammatory responses
[83]. Gpr43 is also induced during adipocyte differentiation and exhibits increased
levels during high-fat feeding in rodents, suggesting that Gpr43 may also affect
adipocyte function [84]. Hong et al. [85] demonstrated that acetate and propionate
act on lipid accumulation and inhibition of lipolysis mainly through Gpr43
[85]. Gpr41 has been shown to be implicated in microbiota-dependent regulation
of host adiposity and leptin production [86].
SCFA can cross the blood-brain barrier and enter the CNS [87] and are taken up
by glia and, to a lesser extent, by neurons, where they are thought to comprise a
major energy source in cellular metabolism, particularly during early brain
230 R. Wall et al.
development [88–90]. They also play a role in cell signalling [91] and neurotrans-
mitter synthesis and release [92]. Moreover, SCFA have been shown to increase the
synthesis of dopamine and its related catecholamines through induction of tyrosine
hydroxylase, a key enzyme in the synthesis of catecholamines [93]. Propionate, in
particular has been shown to alter dopamine, serotonin, and glutamate systems in a
manner similar to that observed in autism spectrum disorders (ASD) [94, 95]. Fur-
thermore, intraventricular infusion of propionate in rats was shown to impair social
behaviour and cause brain abnormalities, similar to those detected in human autism
[96–98] and furthermore to alter brain phospholipid composition [99]. Some clin-
ical studies have also found that a subset of ASD patients have high levels of
Clostridial and Bacteriodetes species in the gut [100, 101], species which are
efficient propionate-producers [19]. This highlights that although propionate is
beneficial at appropriate levels, such as lowering lipogenesis, serum cholesterol
levels and improving insulin sensitivity [102], excessive propionate may have
negative effects on health and behaviour.
Butyrate is known to exhibit many important physiological functions in eukary-
otic cells [19]. One of the most recognised cellular mechanisms for the action of
butyrate is its effects on histone acetylation [103], where the inhibition of histone
deacetylase facilitates hyperacetylation of histone proteins to occur, thus facilitat-
ing the access of DNA repair enzymes. Interestingly, sodium butyrate has been
demonstrated to elicit an antidepressant effect in the murine brain [104]. When
injected systemically, sodium butyrate induced a short-lasting, transient acetylation
of histones in frontal cortex and hippocampus, in conjunction with dynamic
changes in expression of brain-derived neurotrophic factor (BDNF), thereby
resulting in an antidepressant-like behavioral response [104].
Long-Chain Fatty Acids
Long-chain fatty acids (LC-PUFAs) play numerous roles in the brain, including
structural (forming the physico-chemical properties in the lipid bilayer of cellular
membranes) and signalling functions. Moreover, they influence neurogenesis and
neurotransmission within the nervous tissue. Arachidonic acid (AA, C20:4n-6) and
docosahexaenoic acid (DHA, C22:6n-3) are highly concentrated in the brain and
are vital fatty acids for neurological development [105, 106]. Furthermore, there is
a growing body of evidence for their role in mental health across the lifespan
[107]. Being the major structural components of brain cells [108], AA and DHA
influence cell membrane physical properties, enzyme activity, regulation of ion
channels and neuroreceptors and their signalling (neurotransmission) [109, 110].
Recently, we have reported that administration of a Bifidobacterium breve strain,
B. breve NCIMB702258 to mice had a significant impact on the fatty acid compo-
sition of brain [111]. Mice that received this strain for 8 weeks exhibited signifi-
cantly higher concentrations of AA and DHA in the brain when compared to
unsupplemented mice. Interestingly, this effect was bacterial strain-dependent, as
it was not induced by the B. breve strain DPC6330. The mechanism by which the
B. breve strain alters the fatty acid composition is currently unknown. Possible
explanations include modulations of fat-absorption processes in the small intestine
and/or desaturase activities involved in the metabolism of fatty acids to the longer-
chain unsaturated derivatives caused either directly by the strain administered or by
alterations in the gut microbiota. Interestingly, it was previously postulated that
different members of the gut microbiota promote fatty acid absorption via distinct
mechanisms [112]. Semova et al. [112] used the zebrafish model to investigate how
microbiota and diet interact to regulate lipid absorption in the gut epithelium. By
comparing GF zebrafish with conventional zebrafish, the authors demonstrated that
the microbiota stimulate fatty acid uptake and lipid droplet formation in both the
intestinal epithelium and liver [112]. Previous studies have also demonstrated that
manipulation of the gut microbiota by probiotics resulted in altered fat composition
in the host [113–115]. Although the adult microbiome is not known to be particu-
larly enriched in genes involved in fatty acid metabolism [116], there are indica-
tions that interactions between fatty acids and components of the gut microbiota
occur which could affect the biological roles of both. However, a deeper knowledge
of such interactions and what consequences they have for the host are warranted.
Conjugated Linoleic Acid
Conjugated linoleic acid (CLA) comprises a mixture of positional and geometric

isomers of linoleic acid (cis-9, cis-12 C18:2n-6) characterised by the presence of
conjugated double bonds with cis or trans configurations. CLA is a natural com-
ponent of ruminant milk and tissue fat as a result of the action of the ruminal
microbiota on dietary linoleic acid [117]. A range of health-promoting activities
have been attributed to the consumption of CLA, most notably anticarcinogenic,
immune-modulatory, anti-obesity and anti-atherosclerotic activities [118–121].
In contrast, in certain conditions, some CLA isomers can also exert potentially
negative effects such as liver steatosis and insulin resistance [122, 123]. Regarding
the action of CLA on the CNS, it is known that CLA crosses the blood-brain barrier
and is incorporated and metabolized in the brain [124]. Dietary CLA has been
shown to reduce cerebral prostaglandin E2 in the peripheral and CNS [125], and to
exert antiangiogenic actions in the brain [126]. Furthermore, Hunt et al. [127]
showed that CLA protects cortical neurons from excitotoxicity at concentrations
likely achieved by consumption of CLA as a dietary supplement.
Evidence of the role of the gut microbiota in the endogenous production of CLA
was first reported by Chin et al. [128] who observed that increasing the amount of
linoleic acid in the diet increased the tissue content of CLA in conventional rats but
not in GF rats. Since then, commensal lactobacilli and bifidobacteria from the
human GIT, most notably B. breve strains and L. plantarum strains, have been
shown to produce CLA, predominantly the cis-9, trans-11 (c9, t11) isomer from
free linoleic acid [21–23, 129]. These bacteria convert linoleic acid to CLA in a
232 R. Wall et al.
similar manner to ruminant bacteria via the action of the enzyme linoleic acid
isomerase [130], which also has been sequenced in some Lactobacillus and
Bifidobacterium strains [131, 132]. We have reported that the CLA-producing
bacterium, B. breve NCIMB702258 converts linoleic acid to CLA in the murine
gut, resulting in significantly elevated c9, t11 CLA in the liver [133]. Moreover,
Bassaganya-Riera et al. [134] demonstrated that administration of the probiotic
mixture VSL#3 to mice with colitis resulted in high colonic concentrations of c9,
t11 CLA and that this locally produced CLA improved colitis by activating
peroxisome proliferator-activated receptor gamma (PPAR γ) in macrophages.
Two studies have also reported the in vivo production of the CLA isomer, trans-
10, cis-12 (t10, c12) CLA, by using two strains of human origin, L. rhamnosus PL60
and L. plantarum PL62. Administration of these two strains to mice resulted in
increased t10, c12 CLA concentrations in sera with subsequent reductions in
adipose tissue and body weight [135, 136]. Druart et al. [137] further demonstrated
that prebiotic supplementation increased CLA content in caecal tissue, by increas-
ing substrate availability and by modulating gut microbiota composition.
Although dietary CLA has been reported to affect the CNS, the consequences of
bacterially-produced CLA on nervous system function are yet to be discovered.
Conclusion
Although we are still at the very early stages of understanding the complex
communication systems between gut bacteria and the brain, we know that certain
bacteria within the human gut have the ability to produce molecules with neuroac-
tive functions which could affect the brain in a direct manner. However, only
cultivable bacteria have been tested for their capacity to produce neuroactive
compounds in vitro and only a limited number of bacterial strains have been tested
up to now. Moreover, in complex microbial ecosystems such as in the human gut,
interactions and competition exist between bacteria, which are not studied upon
simple culture conditions in vitro. This highlights the need for in vivo studies to
elucidate the role of metabolite-producing bacteria and what effect such bacteria,
and their components, have on nervous system function and behaviour. Such future
studies may also facilitate our understanding of the consequences of neuroactive
compound production by the microbiota and how probiotic bacteria can influence
the CNS, and could furthermore identify the potential for neurochemical
containing/producing probiotic bacteria as a therapeutic strategy in the treatment
of certain neurological and neurophysiological conditions. Given that molecular
tools have now been developed for many intestinal organisms, the possibility exists
now to overproduce neuroactive compounds and/or to regulate their production in
response to gut metabolites such as bile.
Acknowledgements This work was supported by the Science Foundation of Ireland—funded

Centre for Science, Engineering and Technology, the Alimentary Pharmabiotic Centre.
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Chapter 11
Multidirectional Chemical Signalling
Between Mammalian Hosts, Resident
Microbiota, and Invasive Pathogens:
Neuroendocrine Hormone-Induced Changes
in Bacterial Gene Expression
Michail H. Karavolos and C.M. Anjam Khan
Abstract Host-pathogen communication appears to be crucial in establishing the

outcome of bacterial infections. There is increasing evidence to suggest that this
communication can take place by bacterial pathogens sensing and subsequently
responding to host neuroendocrine (NE) stress hormones. Bacterial pathogens have
developed mechanisms allowing them to eavesdrop on these communication path-
ways within their hosts. These pathogens can use intercepted communication
signals to adjust their fitness to persist and cause disease in their hosts. Recently,
there have been numerous studies highlighting the ability of NE hormones to act as
an environmental cue for pathogens, helping to steer their responses during host
infection. Host NE hormone sensing can take place indirectly or directly via
bacterial adrenergic receptors (BARs). The resulting changes in bacterial gene
expression can be of strategic benefit to the pathogen. Furthermore, it is intriguing
that not only can bacteria sense NE stress hormones but they are also able to
produce key signalling molecules known as autoinducers. The rapid advances in
our knowledge of the human microbiome, and its impact on health and disease
highlights the potential importance of communication between the microbiota,
pathogens and the host. It is indeed likely that the microbiota input significantly
in the neuroendocrinological homeostasis of the host by catabolic, anabolic, and
signalling processes. The arrival of unwanted guests, such as bacterial pathogens,
clearly has a major impact on these delicately balanced interactions. Unravelling
the pathways involved in interkingdom communication between invading bacterial
pathogens, the resident microbiota, and hosts, may provide novel targets in our
continuous search for new antimicrobials to control disease.
M.H. Karavolos • C.M.A. Khan (*)

Centre for Bacterial Cell Biology, Institute for Cell and Molecular Biosciences,
The Medical School, Newcastle University, Newcastle upon Tyne, UK
e-mail: michail.karavolos@roslin.ed.ac.uk; Anjam.Khan@ncl.ac.uk
242 M.H. Karavolos and C.M.A. Khan
Abbreviations
AHLs N-acylhomoserine lactones

AI-2 Autoinducer-2
AI-3 Autoinducer-3
BAR Bacterial adrenergic receptor
DPD 4,5-Dihydroxy-2,3-pentanedione
GI Gastrointestinal
NE Neuroendocrine
QS Quorum sensing
Introduction
During host infection bacterial pathogens use a wide variety of molecular sensors to
monitor and adapt to changes in their environment. It is now known that specialised
mechanisms allow bacterial pathogens to listen-in on mammalian endocrine sig-
nalling molecules such as neuroendocrine (NE) stress hormones [1]. NE hormones
such as dopamine, norepinephrine, and epinephrine are synthesised from the amino
acid L-tyrosine and are assigned to the chemical family name of catecholamines.
The endocrine system represents a complex network of chemical signals or
hormones produced by endocrine glands, which relay instructions to target cells
located throughout the body. The endocrine system operates intimately with the
nervous system to coordinate activities. The responses can work in seconds such as
the “flight or fight” reflex in response to fear, to more long-term responses such as
growth and developmental processes. The ability to sense the NE landscape of the
host may aid pathogens towards their successful adaptation and survival during
infection [1–3]. This research area has been termed “microbial endocrinology” and
has largely been pioneered by Lyte and colleagues [2, 3]. On infecting the host,
bacterial pathogens encounter a variety of chemical signals including the NE stress
hormone norepinephrine found abundantly in the gut and epinephrine located
predominantly in the bloodstream [4–6]. Lipopolysaccharide (LPS) is present in
the outer membrane of Gram-negative bacteria and is potently proinflammatory.
Remarkably it has been observed that LPS may act as a signal to stimulate the
formation of epinephrine and norepinephrine by macrophages in the bloodstream
[7, 8]. This has led to the suggestion that the phagocytic system in some aspects
resembles an adrenergic organ composed of scattered cells [7, 8].
Confronted with such an extensive range of chemical signals derived from the
host, bacteria engage cooperative decision-making, coordinating their responses to
avoid host defences and prolong their survival. Successful pathogens have there-
fore, developed sophisticated communication tools involving the production and
sensing of the critical concentrations of bacterial autoinducer molecules in a
process termed quorum sensing (QS) [1, 9]. Following the establishment of a
11 Multidirectional Chemical Signalling Between Mammalian Hosts, Resident.. . 243
Fig. 11.1 Schematic representation of the role of NE hormones and bacterial autoinducers on
interactions between the host, resident microbiota, and bacterial pathogens. NE stress hormones
can crosstalk with bacterial autoinducers (AI) and other bacterially produced signalling molecules
to mediate changes in bacterial or host gene expression. The gastrointestinal (GI) tract microbiota
may modulate the host NE hormone environment as well as chemically interacting with invading
bacterial pathogens. The changing landscape in the production of biologically active host hor-
mones and bacterial signalling molecules, may be sensed by distal organs within the host to
ultimately modulate their physiology e.g. brain function. The signalling processes are likely to be
complex and multidirectional
critical autoinducer concentration, bacteria detect the autoinducer and synchronise

their gene expression and subsequent physiological responses to reflect a “multi-
cellular” response. Interestingly, the more recently described bacterial autoinducer,
AI-3, appears to have the ability to “cross-talk” with the host NE stress hormones
epinephrine and norepinephrine [10].
It is clear that the interactions of pathogens with their hosts lead to significant
changes in gene expression and are hence crucial in shaping the overall result of an
infection. Increasing evidence suggests bacteria can directly or indirectly sense host
NE stress hormones such as epinephrine and norepinephrine to modulate their gene
expression [1, 11–13]. The information in this chapter summarises the major findings
on interkingdom signalling and changes in gene regulation, revealing a variety of
interesting features and mechanisms for bacterial-host communication (Fig. 11.1).
Bacterial Autoinducers: Chemical Signals in Quorum

Sensing and Interkingdom Communication
Quorum sensing (QS) is a key facilitator of bacterial gene regulation and a major
component of crosstalk between bacterial and host produced communication sig-
nals. The majority of QS mediated communication in Gram-negative bacteria are
usually facilitated by N-acylhomoserine lactones (AHLs), 2-alkyl-4-quinolones,
and furanones such as autoinducer-2 (AI-2) [14, 15]. The ability to coordinate gene
responses via QS creates a major tactical advantage for bacteria in terms of
allowing synchronised expression of bacterial survival systems whilst simul-
taneously modulating the virulence and fitness [16–20].
Many Gram-negative bacteria, but curiously not Salmonella or Escherichia coli,
synthesise AHLs which are freely diffusible autoinducers. AHLs bind to and
activate LuxR transcriptional regulators via the LuxNUO signal transduction sys-
tem as part of QS [21, 22]. On the other hand, Gram-positive bacteria produce post-
translationally modified autoinducer peptides derived from the cleavage of larger
precursors. These autoinducer peptides are then actively secreted from the bacterial
cell, and subsequently bind to membrane receptors. These membrane receptors
usually form part of the classical two-component signal transduction system [23–
26].
AI-2 is produced by many Gram-negative and also Gram-positive bacteria and
may be a universal signal molecule. AI-2 is generated from the molecular
rearrangement of 4,5-dihydroxy-2,3-pentanedione (DPD) a by-product of the acti-
vity of the enzyme LuxS [27]. DPD can be rearranged in different ways and bacteria
may exploit different versions of these products as the AI-2 signalling molecule
[28–30]. AI-2 activity has been detected in the culture supernatants of a wide range
of bacteria [28, 31]. LuxS has been shown to modulate the regulation of genes
encoding virulence factors, antibiotic production, biofilm formation, motility, cell
division, and also carbohydrate metabolism [22, 28, 32, 33].
In E. coli [10, 34], the LuxS enzyme has a pleiotropic impact on a broad range of
metabolic pathways and has also been indirectly implicated in the production of
autoinducer-3 (AI-3) [10, 34]. AI-3 regulates the motility and virulence via the
two-component signal transduction systems QseBC and QseEF [1, 35]. Remarkably
the effects of AI-3 can be mimicked by epinephrine and norepinephrine. AI-3 was
first described almost 10 years ago, yet its structure is still unknown, and its
importance in quorum sensing remains to be fully determined. Using the existing
published methods we and others have not been able to detect AI-3 in culture
supernatants and perhaps this can be attributed to a number of reasons including
subtle differences in growth conditions [11, 36]. Thus AI-3 appears to be quite an
elusive and possibly unstable molecule, so it is now crucial the AI-3 synthase is
identified to support these concepts. This should be readily achievable by classic
bacterial genetics and AI-3 reporter screens.
NE Hormones Modulate the Fitness of Bacterial Pathogens

to Cause Disease by a Variety of Mechanisms: Sensing
and Signalling by Bacterial Adrenergic Receptors
and Growth Stimulation
A broad range of internal or external signals including stress responses can have a
major impact on the functions of the gastrointestinal tract [37]. Consequently these
signals can impact on the resident intestinal microbiota. The gastrointestinal tract
represents a highly interactive environment where bacteria-host communications
can potentially flourish [2, 3].
In mammalian tissues the responses generated by NE hormones depends on the
presence of two major types of G-protein coupled adrenergic receptors present on
the surface of cells. These are known as α(alpha)-adrenergic and β (beta)-
adrenergic receptors and have their distinct set of molecular agonists and anta-
gonists. In EHEC the effects of epinephrine and norepinephrine can be mimicked
by the bacterial autoinducer AI-3, providing opportunities for cross-talk between
the two signalling systems during infection [10]. This observation raised the
possibility of the existence of putative bacterial adrenergic receptors (BARs) [10].
Indeed, the sensor kinase QseC is autophosphorylated on binding either epi-
nephrine or norepinephrine, providing evidence for the existence of adrenergic
receptors in bacteria [38]. Furthermore, these adrenergic responses can be inhibited
by mammalian α (alpha)- and β (beta)-adrenergic antagonists like phentolamine
and propranolol. Remarkably, there is strong specificity in the antagonistic effect
with QseC only being blocked by phentolamine [39]. In E. coli O157:H7 and
Salmonella the QseBC system has been proposed as the adrenergic receptor [40–
42]. However, new emerging evidence supports the existence of alternative adre-
nergic receptors. For example, in S. Typhimurium it has been demonstrated that
QseBC is not required for norepinephrine-enhanced enteritis or intestinal coloni-
sation in calves [43].
In another example of adrenergic receptor antagonist inhibition, increased
expression of virK and mig14 in S. Typhimurium was blocked by the addition of
the β (beta)-adrenergic antagonist propranolol [12]. Some BAR-independent adre-
nergic phenotypes in bacteria (discussed below) are associated with altered iron
uptake by the siderophore enterobactin [44, 45]. A tonB mutant, defective in
siderophore uptake, showed the same differential gene regulation upon exposure
to NE stress hormones as the parent strain, implying that adrenergic regulation
operates through a TonB independent mechanism. The virK and mig14 genes are
involved in survival and persistence within the host. Genetic deletion of either gene
reduces the virulence of S. Typhimurium in a mouse infection model, and also
reduces survival in macrophages, signifying a possible role in the late stages of
infection [46, 47]. The down-regulation of mig14 by NE stress hormones may
hence reduce bacterial persistence and promote clearance of bacteria [12]. These
observations highlight the dual role of NE stress hormones in mediating host-
bacterial interactions via regulation of gene expression.
Additionally in Salmonella, the epinephrine-induced reduction in resistance to

polymyxin B was fully reversible by the addition of the β (beta)-adrenergic blocker
propranolol. This effect was dependent on the BasSR two component signal
transduction system which is the putative epinephrine sensor mediating the anti-
microbial peptide response [13]. Epinephrine may, therefore, exert its effect on the
pmr locus of S. Typhimurium through the reversible interaction of the β (beta)-
adrenergic blocker with the BasS membrane sensor in a manner similar to the
interaction of epinephrine with QseC in E. coli. The low (31 %) amino acid
sequence identity between BasS and QseC may provide the reason why we observe
β-blockage in Salmonella as opposed to α (alpha)-blockage in E. coli.
The physiological phenotypes of NE stress hormones described above are not
linked with QseBC or QseEF signalling [11–13]. This may reflect differences in
pathogenesis between S. Typhimurium, an invasive pathogen infecting macro-
phages and epithelial cells and E. coli, a mainly non-invasive pathogen which
remains in the host intestine. The significant divergence in niches occupied by
these two pathogens requires different gene expression patterns for maximum
infection efficiency; hence NE stress hormones may modulate different genetic
pathways to the advantage or disadvantage of the pathogen.
The inhibition of NE stress hormone-mediated hemolysis by the adrenergic β
(beta)-blocker propranolol in the exclusively human pathogen S. Typhi is another
example of the existence of an additional putative novel bacterial adrenergic
receptor. In S. Typhi, NE stress hormone-mediated hemolysis is clearly indepen-
dent of the known E. coli O157:H7 adrenergic receptor QseBC and is mediated via
the CpxAR two component system [11, 36].
Based on the above observations, it is evident that natural selection ensured there
is no monopoly in bacterial adrenergic signalling. Millions of years of co-existence
have culminated in a fine tuned bacterial sensing system composed of different
BARs, which, through their fastidious specificities, orchestrate the regulatory
pathways to modulate the strategic responses of pathogens within their host milieu.
NE stress hormones can modulate the growth of bacteria in iron-limited media,
which reflects the in vivo condition within the GI tract [3, 48]. NE stress hormones
can efficiently extract iron from host transferrin and lactoferrin and hence these
hormones can provide iron for bacteria to use [49, 50]. Although NE stress
hormones improve the growth of coagulase-negative Staphylococci, the hormones
do not have a significant impact on the growth of the important pathogen Staphylo-
coccus aureus [51, 52].
Independent of the growth stimulation effects, NE stress hormones modulate the
expression of the virulence-associated K99 pilus adhesin enterotoxigenic in E. coli
[53] to upregulating type 3 secretion in Vibrio parahaemolyticus [54]. In Campylo-
bacter jejuni, NE stress hormones increase invasion of epithelial cells and disrup-
tion of tight junctions [55]. Exposure to NE stress hormones modulates the
virulence of Borrelia burgdorferi [56], elevates expression of the protease arg-
gingipainB virulence factor of Porphyromonas gingivalis [57], and in the
opportunistic pathogen Pseudomonas aeruginosa increases the production of its
virulence factors pyocyanin and elastase as well as the secreted Pseudomonas
quinolone signal, PQS [70]. Collectively, these examples clearly add support to the
role for NE stress hormones in regulating the virulence of bacterial pathogens.
The S. Typhimurium epinephrine response is primarily characterised by the
upregulation of operons involved in metal homeostasis and oxidative stress
[13]. An induction of key S. Typhimurium metal transport systems and oxidative
stress responses employing manganese internalisation is typical after a 30 min
treatment with NE stress hormones [13]. The fact that the oxidative stress regulator
OxyR is important for survival in the presence of epinephrine implies that, through
affecting iron transport, epinephrine may induce oxidative stress. The sensing of
epinephrine may hence provide an environmental cue to initiate the Salmonella
oxidative stress response in anticipation of forthcoming host-based oxidative stress.
Treatment of S. Typhimurium with NE stress hormones reduces its resistance to
the human antimicrobial peptide cathelicidin LL-37 [12]. LL-37 is produced in the
gastrointestinal tract, bone marrow and macrophages. The peptide possesses anti-
microbial activity against a range of Gram-negative and Gram- positive bacteria
[58]. It could be speculated that through an increase in sensitivity to LL-37, NE
stress hormones may also act as a host defence system to combat infection. This
observation may suggest that although bacteria can sense and exploit these host
signalling molecules for their benefit, in some instances the host can use the same
signals to manipulate the bacteria [12].
Chemical Dialogues Between the Host, Microbiota,

and Bacterial Pathogens: A Multidirectional Regulatory
Liaison
Most of the studies on BAR dependent or independent signalling discussed above

have focused mainly on two enteroinvasive bacterial pathogens. Intriguingly, the
microbes that colonise humans hosts, outnumber human cells by tenfold [59]. The
collective genomes of these microbial symbionts, referred to as the microbiome,
provides a genetic repertoire with the potential to significantly affect host-
pathogen-symbiont communication [60]. Data mining of the human microbiome
suggests the presence of enzymes and pathways capable of supporting microbiota-
based production of known or novel autoinducers [59–61]. Interkingdom signalling
in the gut lumen may be crucial for maintaining the physiological balance in the gut
microbiome, thus facilitating microbiome homeostasis and preventing dysbiosis.
Even in healthy individuals, the diversity of the human microbiome is extraordi-
nary, implying the potential complexity of the interactions in these populations
[59, 60].
It is clear that some species of bacteria are able to modulate the production of NE
hormones in vitro and possibly in the gut [above; 62–64]. Genes encoding compo-
nents with high similarity to the molecular machinery needed to produce NE
hormones are indeed present in some species of bacteria [65–68]. There is
increasing evidence that gut bacteria may contribute significantly to the levels of
gut NE hormones such as norepinephrine [62]. Indeed bacteria may modulate gut
motility and/or immune functions via the production of NE hormones to steer gut
homeostasis. The complete role of bacterially-induced/produced NE hormones in
host gene regulation and microbial survival is yet to be evaluated.
The wide majority of the gut microbiome species are not culturable using
traditional laboratory techniques. The size and variation of the gut microbiome
only hints at the richness of molecules produced and potentially sensed by these
commensal populations. Microbial pathogen infection and insults like antibiotic
use, perturb such fine balance leading to dysbiosis in the gut [69]. A metabolomics
study in a murine infection model using S. Typhimurium revealed a significant
alteration in the metabolic profile of multiple pathways involved in host eicosanoid
hormone metabolism [69]. It is therefore clear that bacteria can influence their host
in a variety of ways including a possibly significant involvement into modulating
host NE homeostasis.
Microbiota Are Key Players in Modulating Levels of NE

Hormones in the Gastrointestinal Tract
The gastrointestinal microbiota represent a diverse range of balanced bacterial

communities with fundamental roles in maintaining good health and preventing
disease in humans. A recent study has revealed the involvement of gut microbiota
not only in the regulation but also the production of NE hormones in the mouse
gastrointestinal (GI) tract [62]. Asano and colleagues investigated the levels of
norepinephrine and dopamine through the GI tract by developing a robust HPLC
assay [62]. In order to examine the impact of the GI tract microbiota on catechola-
mine concentration, they determined the levels of norepinephrine and dopamine in
specific pathogen-free mice, germ-free mice, and gnotobiotic mice [62]. The lumen
of specific pathogen-free mice had much greater levels of neuroendocrine hor-
mones than those found in germ-free mice that harboured no bacteria.
Amazingly the introduction of either, specific-pathogen free mice fecal flora,
Clostridum species or E. coli into germ free mice resulted in a striking elevation in
levels of free biologically active norepinephrine and dopamine. An E. coli mutant
unable to produce β (beta)-glucuronidase was no longer able to elevate levels of
free biologically active catecholamines implying a role for this bacterial enzyme in
the process [62]. Through this comparative analysis, the investigators identified that
the resident gut microbiota and the bacterial enzyme β (beta)-glucuronidase, have
major roles in the production of biologically active norepinephrine and dopamine in
the GI tract of the host. The study provides important insights into the interactions
between the microbiota and host cells, and raises important questions. Can the
presence of the gut microbiota: be sensed by the host cells, induce host cells to
produce these catecholamine hormones, as well as play a key role in the maturation
of biologically active hormones? This study by Asano and colleagues will undoubt-
edly have a major impact in the field and stimulating further research.
Conclusions
NE hormones play an essential role in regulating the physiology of mammals

producing immediate responses within seconds such as the “fight or flight” response
to more long term developmental changes.
It is now widely acknowledged that bacteria can detect, and respond to changes
in the NE hormone landscape of their host. These hormones may provide important
environmental cues on their in vivo location and the physiological status of the host.
This sensing may take place indirectly or directly through BARs. Research from a
number of independent laboratories, including ours, vividly demonstrated that
Salmonella have evolved a number of specialised systems for sensing NE stress
hormones. Sensing of the NE hormones subsequently leads to global alterations in
bacterial gene regulation patterns. Even transient exposure to NE hormones can
have significant impact on the physiology of Salmonella modulating its ability to
cause disease. There appears to be a delicate balance, which can be shifted to favour
the pathogen or the host. The complex interactions between bacteria and their host
involving host or bacterially derived NE stress hormones is briefly depicted in
Fig. 11.1. In summary, there is increasing evidence to suggest the existence of a
complex chemical dialogue between host cells, the microbiota, and invading
pathogens. Further studies on understanding the fascinating nature of the bacterial
adrenergic receptors and signalling pathways will not only provide colourful
biological insights on pathogen-host interactions, but may also identify potential
novel targets in the treatment of disease.
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Chapter 12
Influence of Stressor-Induced Nervous
System Activation on the Intestinal
Microbiota and the Importance
for Immunomodulation
Michael T. Bailey
Abstract The body is colonized by a vast population of genetically diverse

microbes, the majority of which reside within the intestines to comprise the
intestinal microbiota. During periods of homeostasis, these microbes reside within
stable climax communities, but exposure to physical, physiological, as well as
psychological stressors can significantly impact the structure of the intestinal
microbiota. This has been demonstrated in humans and laboratory animals, with
the most consistent finding being a reduction in the abundance of bacteria in the
genus Lactobacillus. Whether stressor exposure also changes the function of the
microbiota, has not been as highly studied. The studies presented in this review
suggest that stressor-induced disruption of the intestinal microbiota leads to
increased susceptibility to enteric infection and overproduction of inflammatory
mediators that can induce behavioral abnormalities, such as anxiety-like behavior.
Studies involving germfree mice also demonstrate that the microbiota are necessary
for stressor-induced increases in innate immunity to occur. Exposing mice to a
social stressor enhances splenic macrophage microbicidal activity, but this effect
fails to occur in germfree mice. These studies suggest a paradigm in which stressor
exposure alters homeostatic interactions between the intestinal microbiota and
mucosal immune system and leads to the translocation of pathogenic, and/or
commensal, microbes from the lumen of the intestines to the interior of the body
where they trigger systemic inflammatory responses and anxiety-like behavior.
Restoring homeostasis in the intestines, either by removing the microbiota or by
administering probiotic microorganisms, can ameliorate the stressor effects.
M.T. Bailey (*)

Institute for Behavioral Medicine Research, College of Medicine, The Ohio State University,
Columbus, OH 43210, USA
e-mail: Michael.bailey@osumc.edu
256 M.T. Bailey
Abbreviations
ACTH Adrenocorticotrophic hormone

CFU Colony forming units
CRH Corticotrophin release hormone
DGGE Denaturing gradient gel electrophoresis
GABA γ-Amino butyric acid
GI Gastrointestinal
iNOS Inducible nitric oxide synthase
mRNA Messenger ribonucleic acid
NE Norepinephrine
SDR Social disruption
SNS Sympathetic nervous system
TNF-α Tumor necrosis factor alpha
Introduction
The body is heavily colonized by microorganisms collectively referred to as the

microbiota, and it is now realized that all surfaces of the body naturally harbor
unique microbial communities. While archea, protists, and viruses are known to
reside within these communities, the majority of the microbiota are bacteria that
reside within the gastrointestinal tract. Proximal sections of the gastrointestinal
(GI) tract, including the stomach and the duodenum, harbor low levels of microor-
ganisms (typically between 100 and 1,000 colony forming units (CFU) per ml of
contents), whereas distal sections of the GI tract, including the ileum and the colon,
harbor high levels of microorganisms (typically between 10 6 and 1012 CFU/ml of
contents). In the colon, the microbiota reside as a stable climax community due to
the selection of microbes that are best adapted for their given niche [1]. Although
this climax community is relatively resistant to change [2], it is well known that
factors such as diet and antibiotics can cause transient alterations in microbial
community structure [3–5]. This review will discuss the evidence that exposure
to different types of stressors can also cause transient alterations in microbial
community structure, and will discuss the evidence that even transient alterations
in the microbiota may be associated with variations in host immune and behavioral
responses.
12 Influence of Stressor-Induced Nervous System Activation on the Intestinal.. . 257
The Modern Stress Concept
Stress is an intrinsic part of life, and successfully coping with aversive stimuli is
essential for organism survival in an ever changing environment. While the concept
of stress is intuitive, there is not a single, widely accepted definition of stress. In its
simplest form, stress can be broken down into the stimulus that threatens organism
homeostasis (called the stressor) and the behavioral and physiological response to
this challenge (called the stress response). Thus, a stressor is any stimulus that
disrupts internal homeostasis, and can involve psychological, physical, or physio-
logical stimuli. This disruption to homeostasis elicits physiological responses that
are aimed at reducing the threat and re-establishing internal homeostasis. Initiation
of the stress response to physical or physiological stressors is typically subcon-
scious, but additional cognitive processing occurs in response to psychological
stressors. Psychological stressors that are perceived as exceeding available coping
strategies set into motion coordinated behavioral and physiological responses that
ultimately serve to help the organism adapt to the stressor. Interestingly, the
physiological stress responses to physical, physiological, and psychological
stressors have many similarities that can be generalized across host species.
Physiological Stress Response
There are two neuroendocrine pathways that are major contributors to the stress
response, namely the hypothalamic-pituitary-adrenal (HPA) axis and the sympa-
thetic nervous system (SNS). Activation of the HPA axis occurs through the release
of corticotrophin release hormone (CRH) from neurosecretory cells found in the
paraventricular nucleus of the hypothalamus. CRH travels a short distance from the
hypothalamus to the anterior pituitary gland where it stimulates the release of
adrenocorticotrophic hormone (ACTH). The ACTH then travels through the
blood and stimulates the release of glucorticoid hormones, namely corticosterone
in rodents and cortisol in humans, from the cortex of the adrenal glands. As the
name suggests, glucorticoids are important for increasing the bioavailability of
glucose via gluconeogenesis in the liver. The glucose is then used by the body to
cope with and adapt to the stressful stimulus.
In addition to the HPA axis, the sympathetic branch of the autonomic nervous
system becomes activated during stressor exposure. The sympathetic nervous
system (SNS) originates in the brain stem from different brain nuclei, such as the
locus coeruleus, pons, and medulla, which send projections along the spinal col-
umn. After exiting the spinal column, preganglionic SNS neurons synapse in
prevertebral ganglia using acetylcholine as the neurotransmitter. The acetylcholine
excites postganglionic neurons that innervate virtually every organ in the body
using norepinephrine (NE) as the terminal neurotransmitter.
258 M.T. Bailey
Activation of the SNS occurs very rapidly and is largely responsible for the well-
known “fight-or-flight” stress response, that is dependent upon the effects the SNS
has on the heart and lungs (i.e., increased heart rate and respiration), blood vessels
(i.e., increased vasodilation in skeletal muscle), and internal organs (e.g., increased
glycogenolysis in the liver and reduced digestive functions in the gut). Upon
stressor termination, the parasympathetic branch of the autonomic nervous system
becomes activated and releases the neurotransmitter acetylcholine to restore
homeostasis and induce the “rest-and-digest” response.
Stress Exposure and the Gut
Stressor-induced activation of the SNS and the HPA axis are well known to affect
the functioning of the gastrointestinal tract. This was first recognized when it was
observed that a patient with a gastric fistula produced significantly less gastric acid
during fearful periods [6]. Other stressors, including a cold pressure task and mental
arithmetic [7, 8] can also reduce gastric acid secretion. Studies in laboratory
animals indicate that these stressor effects are due to activation of the autonomic
nervous system. Activation of the SNS tends to suppress whereas the PNS enhances
gastric acid secretion [9].
Other components of gastrointestinal physiology such as gastrointestinal motil-
ity and mucous production are also known to be significantly changed by stressor
exposure. For example, GI motility, can be either slowed or enhanced during
stressor exposure depending upon the type of stressor and the section of the
intestine that is investigated [10, 11]. Stressor exposure has also been shown to
affect mucous secretion, depending upon the strength and duration of the stressor.
Early life or short-lasting stressors tend to increase mucous secretion throughout the
length of the gastrointestinal tract, whereas long-lasting stressors tend to deplete
mucous stores and thus decrease mucous levels in the gut [12, 13].
Gastric acid secretion, gastrointestinal motility, and mucous levels can influence
the ability of microbes to colonize within the gastrointestinal tract. For example, it
is well known that microbes must be able to survive the low acidity in the stomach
in order to colonize lower sections of the gut. Reducing acid secretion can in turn
alter gut microbiota populations [14]. Similarly, motility has long been recognized
as a primary factor controlling microbe levels the length of the GI tract [15]. Phar-
macological manipulation of gastrointestinal motility is associated with altered
microbial populations [16]. The mucous layer in the gut is also an important factor
for the development of microbial community structure, because the mucins that
comprise the mucous layer are glycosylated with O-glycans that are an important
food source for mucoadherent microbes [17]. Moreover, some microbes, such as
members in the genus Lactobacillus, contain mucous binding proteins that help
them to bind to the intestinal mucous layer [18]. Thus, changing mucous secretion
has the potential to change microbial populations.
Impact of the Stress Response on Gut Microbiota

and Colonic Inflammation
Studies in this laboratory have been influenced by the findings that gastrointestinal
physiological functions that are affected by stressor exposure can also impact gut
microbes, because they reflect a possible biological/mechanistic link between
stressor-exposure and alterations in the microbiota. However, alteration of gut
physiological functioning is not the only potential mechanism by which stress
could impact the gastrointestinal microbiota. Direct neurotransmitter/hormone-
bacterial interactions might also mediate stressor effects on the gut microbiota.
Neuroendocrine-Bacterial Interactions
The growth of many types of bacteria, including both infectious and commensal
organisms, have been shown to be significantly enhanced upon culture with cate-
cholamines, such a norepinephrine (NE), as shown in multiple chapters in this book.
While the effects of neuroendocrine hormones on microbial growth have been
amply demonstrated in both in vitro and ex vivo model systems (reviewed in [19]),
demonstrating these interactions occur in vivo has been challenging. However,
studies involving the use of a neurotoxin to lyse peripheral sympathetic neurons,
and thus causing an increase in norepinephrine levels in vivo, indicate that elevated
norepinephrine levels leads to bacterial overgrowth in the intestines [20]. The
growth of commensal Gram-negative microbes, primarily Escherichia coli, was
increased by nearly 10,000-fold after elevating NE levels through the use of the
neurotoxin [20]. The effects of norepinephrine on bacterial growth was also evident
in an ileal loop model, where growth of Salmonella enterica in the presence of NE
prior to inoculation into an ileal loop was associated with significantly elevated
pathogen levels in the ileal loop, and a more severe pathogen-induced disease
progression [21].
As these studies demonstrate, there are multiple mechanisms by which host
physiology can impact microbial populations in the intestines. And, exposure to
stressors that are physical, physiological, or psychological in nature has the capac-
ity to significantly change all of these host physiological processes. These findings
have led to testing the general hypothesis that stressor exposure can significantly
change microbial populations naturally residing within the gastrointestinal tract.
260 M.T. Bailey
Culture-Based Findings of Stressor Effects on the Structure

of the Microbiota
It has been recognized for over 30 years that changing an animal’s environment can
lead to gut microbial dysbiosis. In 1974, Tannock and Savage [22] demonstrated
that moving mice into a cage lacking bedding, food, and water reduced the number
of lactobacilli that could be cultured from the small and large intestines, with the
greatest reduction being found in the stomach. Although it was not possible to
determine whether the reduction was due to the change in environment, rather than
the lack of food and water, this was one of the earliest studies to demonstrate that
external factors could impact the microbiota. Subsequent studies confirmed and
extended the observation that environmental stimuli can impact the microbiota. For
example, chronic sleep deprivation was found to cause a significant overgrowth of
microbiota in the distal ileum and cecum [23]. This microbial overgrowth was
associated with a translocation of the microbes to the spleen, liver, and regional
lymph nodes in sleep-deprived animals [23].
It is becoming increasingly evident that physical and physiological stressor can
impact gut microbial populations, but only a few studies have focused on the impact
that psychological stressors can have on the microbiota. Data from early studies on
the composition of the microbiota in Russian cosmonauts were among the first to
suggest that psychological stimuli could impact the composition of the microbiota.
The data demonstrated that the intestinal microbiota were significantly different
during space flight as compared to training periods on Earth [24]. There are many
environmental changes associated with space flight, and it was not clear whether the
differences in the microbiota could be due to the stress associated with space flight.
However, other studies tracking microbial populations during space training found
that periods of emotional stress, such as the stress of confinement, was associated
with periods of altered microbial profiles [25], thus suggesting that emotional stress
could impact the stability of the intestinal microbiota.
The strongest evidence that stressor exposure can impact microbial populations
has come from studies involving laboratory animals. For example, studies demon-
strate that separating rhesus monkeys (Macaca mulatta) from their mothers was
sufficient to significantly change the number of bacteria that could be cultured from
the stool. Levels of total cultured bacteria tended to be significantly decreased by
3 days after separation [26], but the most consistent findings occurred when a single
type of microbe was cultured. Levels of bacteria in the genus Lactobacillus were
significantly reduced 3 days after maternal separation [26]. Of importance, this
reduction in lactobacilli was significantly correlated with the expression of stress
indicative behaviors. Those animals that displayed a larger number of stress-
indicative behaviors (such as repetitive lip smacking and cooing) tended to have
lower levels of lactobacilli. This effect lasted through 3 days post-separation.
Interestingly, as the infant monkeys formed stable social groups by 1 week post-
separation, the levels of lactobacilli returned to pre-separation values [26].
Members of the genus Lactobacillus are known to have protective effects in the
intestines, with one protective effect being the production of proteins and other
compounds that have the capacity to kill enteric pathogens. Two enteric pathogens,
namely Shigella flexneri and Campylobacter jejuni, are endemic in monkey colo-
nies. Exposure to maternal separation increased opportunistic infection with
S. flexneri and C. jejuni, and pathogen levels tended to correlate with lactobacilli
levels. In general, maternally separated infant monkeys that had high pathogen
loads also had low levels of lactobacilli [26]. This study demonstrated that a
naturally occurring stressor changed the levels of bacteria that could be cultured
from the stool and also reduced the ability of the microbiota to exclude pathogen
colonization.
The effects of stressor exposure on the microbiota also extend into the prenatal
period. Exposing monkeys to an acoustical startle stressor during gestation signif-
icantly changes the development of the intestinal microbiota in the offspring
[27]. This was manifest as a reduction in the levels of bifidobacteria and lactobacilli
that could be cultured from the stool for the first 6 weeks of life. As with previous
studies, this stressor-associated reduction in lactobacilli was associated with an
increased incidence of opportunistic infection [27].
Culture-based studies in rodents have also demonstrated that stressor exposure
reduces the number of lactobacilli cultured from the stool. Mice that were housed in
cages lacking bedding, as well as mice that were exposed to horizontal shaking, for
3 consecutive days were found to have lower lactobacilli levels shed in the feces
than did control mice [28]. This reduction in lactobacilli was consistent between the
different stressors, and led the authors to suggest that reduction in the lactobacilli
could be used as a marker for environmental stressor exposure [28]. A note of
caution is needed, however, because one study has found that inbred female mice
have low levels of Enterococcus and Lactobacillus spp., as determined using
fluorescent in situ hybridization (FISH), but exposure to water avoidance stress
during antibiotic administration causes an increase in this bacterial group, rather
than a decrease [29].
The effects of stressor exposure on lactobacilli have primarily been studied in
laboratory animals, but one study found that stressor exposure reduced the levels of
lactobacilli cultured from humans. Fecal lactobacilli levels were assessed in college
students during a low stress period (i.e., the first week of the semester) and a high
stress period (i.e., final exam week) to determine whether the stressful period was
associated with lower levels of lactobacilli. The final exam period was associated
with higher levels of perceived stress, and consistent with results from animal
studies, higher perceived stress resulted in lower levels of lactobacilli shed in the
stool [30]. It should be noted that the exam period was also associated with
significant differences in diet. Because diet can significantly impact microbial
populations [31], it is possible that the reductions in lactobacilli were dependent
upon changes in diet. However, given results demonstrating stressor-induced
reductions in fecal lactobacilli in laboratory animals consuming a standardized
laboratory diet [26, 27], it is likely that alterations in the human microbiome during
262 M.T. Bailey
the stress of the exam week were due to combined effects of the stressor on host
physiology and changes in dietary habits.
Culture-Independent Studies of Stressor Effects on Gut

Microbial Community Structure and Function
Most studies assessing the effects of stressor exposure on the gut microbiota have
relied on culture-based enumeration of only a few types of microbes within a given
sample. However, the vast majority of microbes in the gut cannot be cultured due to
undefined culture conditions [32]. As a result, there are an increasing number of
studies that have utilized culture-independent methods to demonstrate that stressor
exposure can affect more than just a few gut microbes; community-wide alterations
of the gut microbiota have been demonstrated to occur in response to multiple types
of stressors. This was first realized in rats that were separated from their mothers for
3 h per day early in life (i.e., postnatal days 3–12). This maternal separation stressor
resulted in significant community-wide alterations in the gut microbiota as assessed
using denaturing gradient gel electrophoresis (DGGE) to assess microbial
populations in the stool when the rats were 7–8 weeks of age [33]. Studies in this
laboratory have also used culture-independent methods to assess the effects of
stressor exposure on the intestinal microbiota [34, 35]. Next generation, high
throughput 454 FLX pyrosequencing was first used to characterize the microbiota
in mice exposed to a prolonged restraint stressor.
Studies Involving Prolonged Restraint
Prolonged restraint is a widely used murine stressor that has been extensively
characterized in the literature and is the most commonly used murine stressor in
biomedical and biobehavioral research [36]. This stressor involves both a physical
component (i.e., physical confinement) and a psychological component that is
thought to reflect the animal’s perception of burrow collapse and inescapability
[36]. Exposure to the prolonged restraint stressor induces a physiological stress
response that results in the elevation of endogenous corticosterone, epinephrine,
and norepinephrine [36–39]. Thus, mice were exposed to the prolonged restraint
stressor to determine the effects of the stress response on the stability of the
intestinal microbiota.
In this initial experiment, approximately 100,000 sequences from the cecal
contents of 32 mice (approximately 3,000 sequences per mouse) were analyzed to
characterize microbial diversity within the cecum. Microbial diversity encompasses
both the richness (i.e., the number of different types of bacteria in a community) and
the evenness (i.e., the distribution of the individual bacteria). In microbial ecology,
there are two primary measures of diversity, with α-diversity assessing diversity of
species within samples and β-diversity assessing diversity between samples.

Prolonged restraint affected both α- and β-diversity. Hierarchical clustering ana-
lyses indicated that the profile of the top ten most abundant bacterial types was
significantly different in the mice exposed to 3, 5, or 7 days of restraint compared to
profiles found in control animals [34]. Mice will not eat or drink while in restraining
tubes, even if food and water is provided. Because changes in diet can have a
profound impact on the microbiota [5, 40], a food and water deprived control group
was included in the study. Mice that were restrained for one night had microbial
profiles that were similar to food and water deprived control mice. However, as
mice were exposed to repeated cycles of the restraint stressor (i.e., 3, 5, or 7 repeated
nights of prolonged restraint) microbial profiles were distinct from those found in
food and water deprived mice [34]. This indicates that at least some of the effects of
the stressor on the microbiota are due to food and water deprivation, but that
repeated cycles of the stressor had additional effects on the microbiota that were
not accounted for by food and water deprivation.
In addition to changes in microbial community β-diversity, exposure to
prolonged restraint also results in changes to α-diversity. Rarefaction analysis
indicated that species diversity decreased with repeated cycles of restraint. This is
important, because it is generally believed that loss of α-diversity leads to increased
susceptibility to enteric infection [41]. Thus, it was hypothesized that mice exposed
to the prolonged restraint stressor would have an increased susceptibility to enteric
infection [34]. To test this hypothesis, mice were orally challenged with
Citrobacter rodentium, which is a natural murine colonic pathogen, with patho-
genesis and resulting colonic pathology that are nearly indistinguishable from that
produced in humans infected with enteropathogenic E. coli, and some components
of enterohemorrhagic E. coli [42–44]. As the infection progresses, the colonic
inflammatory response resembles many aspects of the colitis found in patients
with inflammatory bowel disease [44, 45].
Challenging mice with C. rodentium prior to, or during, exposing to the
prolonged restraint stressor significantly increased C. rodentium colonization in
the colon and increased pathogen-induced colitis marked by increases in inflam-
matory cytokine (e.g., TNF-α) mRNA levels and increased colonic histopathology
[34, 46]. Interestingly, exposing mice to six consecutive nights of prolonged
restraint prior to oral challenge with C. rodentium increased colonic pathogen
levels and mildly increased pathogen-induced colitis [34]. However, exposing
mice to the prolonged restraint stressor for 1 night prior to oral challenge with
C. rodentium and then exposing mice to 6 more nights of prolonged restraint (i.e.,
through day 5 post-C. rodentium challenge) resulted in significantly greater colonic
pathology [46]. Simultaneously administering the prolonged restraint stressor and
the C. rodentium challenge caused outbred CD-1 mice, which are generally con-
sidered resistant to C. rodentium infection, to develop severe colitis with lesions
containing inflammation, epithelial defects, hyperplasia, and dysplasia. In some
cases, neutrophilic inflammation extended from the mucosa to the submucosa and
was frequently associated with epithelial erosion and ulceration [46].
264 M.T. Bailey
C. rodentium lack pathogenic mechanisms to cross the intestinal epithelial

barrier, and thus are not considered an invasive pathogen. However, simply expos-
ing mice to the prolonged restraint stressor during oral challenge with C. rodentium
was sufficient to significantly increase the occurrence of C. rodentium in the spleen,
and also increased circulating levels of IL-6 and anxiety-like behavior. This sug-
gests that stressor exposure during C. rodentium challenge disrupted the tight
junctions between intestinal epithelial cells that in healthy tissue prevent the passive
transfer of non-invasive microbes, fluid, and nutrients form the lumen of the
intestines to the interior of the body. Stressor exposure is well known to affect
tight junctional protein expression and the permeability of intestinal tissue [47–
49]. Our study involving a colonic pathogen suggests that pairing stressor exposure
and colonic infection can further degrade colonic epithelial barrier integrity [46].
It is not yet known whether stressor-induced alterations in the intestinal
microbiota contribute to the enhancive effects of stressor exposure on
C. rodentium challenge. However, administering probiotic Lactobacillus reuteri
(ATCC23272) has beneficial effects on stressor-exposed mice orally challenged
with C. rodentium [46]. L. reuteri is widely recognized to limit inflammation, and in
a study involving gnotobiotic mice orally challenged with enterohemorrhagic
E. coli (which is closely related to C. rodentium), L. reuteri significantly reduced
colonic inflammation [50]. In our studies, L. reuteri administration during
prolonged restraint significantly enhanced gut barrier integrity. Providing
L. reuteri to the stressor-exposed mice prevented the ability of C. rodentium to
translocate from the lumen of the intestines to the spleen. Administering the
L. reuteri also prevented the increase in circulating IL-6 and the development of
anxiety-like behavior in mice exposed to the stressor during C. rodentium
challenge [46].
Exposure to the prolonged restraint stressor reduces both relative and absolute
levels of commensal L. reuteri that are associated with colonic tissue (Galley et al.,
under review). This was observed in a study involving 16s rRNA gene sequencing
using the 454 FLX-Titanium pyrosequencing platform followed by real-time PCR
to characterize colonic tissue-associated microbiota in mice exposed to the
prolonged restraint stressor. Considering the finding that administering probiotic
L. reuteri to stressor-exposed mice prevents some, but not all, effects of the stressor
has led to the hypothesis that stressor-induced alterations in commensal tissue-
associated microbiota result in an internal environment that is more conducive to
pathogen-induced colonic inflammation. It is further hypothesized that this internal
environment leads to increased epithelial permeability and the translocation of
pathogenic (as well as commensal) microbes from the lumen of the intestines to
the interior of the body where they stimulate increases in inflammatory cytokines
that alter the behavior of the host (Fig. 12.1). Further studies are needed to confirm
this hypothesis, and to determine whether commensal and probiotic microbes in
addition to L. reuteri are involved with, or can prevent, stressor-induced increases
in colonic inflammation.
Physical, Physiological, and/or

Psychological Stressor Exposure
Beneficial/Probiotic Microbes
Stressor-Induced e.g., Lactobacillus reuteri
Circulating cytokines
Anxiety-like behavior Physiological Response
e.g., Glucocorticoids,
Catecholamines
Disruption of Homeostatic Interactions

Between Host and Microbiota
 Susceptibility to mucosal pathogens

 Inflammation
 Epithelial barrier disruption/Bacterial
translocaiton
Fig. 12.1 Exposure to physical, physiological, or psychological stressors sets into motion a series
of physiological responses that have the capacity to disrupt homeostatic interactions between the
host and the gut microbiota. These disrupted homeostatic interactions lead to increases in suscep-
tibility to intestinal infection and inflammation, and enhances epithelial barrier permeability and
subsequent translocation from the lumen of the intestines to the interior of the body. The
disruptions in epithelial barrier integrity lead to increases in circulating cytokines that have the
capacity to change animal behavior and further stimulate the endocrine response. The hypothesis
that alterations in the intestinal microbiota are responsible for these disrupted homeostatic
interactions comes from data indicating that stressor exposure reduces beneficial microbes, such
as bacteria in the genus Lactobacillus. Feeding mice lactobacilli to prevent the stressor-induced
reduction in Lactobacillus spp. prevents the stressor-induced increase in susceptibility to colonic
infection and inflammation and prevents disruptions to epithelial barrier integrity
Studies Involving Repeated Social Defeat
The effects of stress on colonic microbiota and inflammatory responses are also
evident using a social stressor called social disruption (SDR). Social stressors often
involve aggressive interactions between dominant and subordinate animals and are
widely used to study the effects of stress on animal behavior and physiological
functioning [51–54]. Social disruption involves aggressive interactions between a
dominant intruder mouse (i.e., the aggressor) and resident subordinate mice (i.e.,
the experimental subjects). The aggressive interactions occur over a 2 h period at
the beginning of the active cycle, when the aggressor is placed into the cage of the
resident subordinate mice. The aggressor physically interacts with the residents for
short periods of time until the residents display an upright defeat posture. Because
the mice are housed together, the subordinate mice cannot escape and the aggres-
sive intruder mouse will repeatedly attack and defeat the residents. Thus, the
residents are exposed to repeated social defeat during the 2 h period.
The SDR stressor involves both physical and psychological components, and the
defeated mice develop anxiety-like behaviors [55, 56] and a physiological stress
266 M.T. Bailey
response marked by elevated corticosterone [57, 58], epinephrine, and norepineph-

rine [59]. Importantly, exposure to the SDR stressor has well defined effects on
systemic immune responses. For example, exposure to the SDR stressor is known to
increase circulating levels of cytokines, such as IL-1 and IL-6, even in the absence
of infectious challenge [60–62], which is also commonly evident in humans
exposed to different types of stressors [63–65]. In addition, exposure to the SDR
stressor reduces the sensitivity of splenic monocytes/macrophages to the suppres-
sive effects of glucocorticoid hormones and increases the ability of these splenic
monocytes/macrophages to kill target microbes [57, 58, 66–69].
Microbial populations in the cecums of mice exposed to the well-defined SDR
stressor were assessed using 454 FLX pyrosequencing. Consistent with results
obtained with prolonged restraint, exposure to the SDR stressor resulted in signif-
icant changes in both the α and β diversity of the cecal microbiota [35]. Alpha
diversity indices, including OTU, ACE, and Chao all demonstrated statistically
significant reductions in microbial diversity by 15 h after the last cycle of the SDR
stressor. In addition, the relative abundance of 8 out of the top 25 most abundant
microbes was significantly affected by exposure to the SDR stressor. These differ-
ences were evident immediately after the last cycle of stressor exposure, as well as
the morning following the last cycle of the stressor [35] indicating that the effects of
the stressor occur rapidly in response to stressor exposure and can persist for at least
15 h after termination of stressor exposure.
The conclusion that stressor exposure can enhance infectious colitis based on
studies involving outbred CD-1 mice exposed to prolonged restraint during oral
challenge with C. rodentium has been confirmed and extended in inbred mice
exposed to a social stressor during oral challenge with C. rodentium. Inbred
C57BL/6 mice were orally challenged with a low dose of C. rodentium, and in
non-stressed control mice, little pathogen colonization and pathogen-induced coli-
tis occurred with this low infectious dose. However, simply exposing the mice to
the SDR stressor again increased both pathogen colonization and associated pathol-
ogy (Galley et al., under review). Mice exposed to the social stressor during
C. rodentium challenge had higher levels of mRNA for colonic inflammatory
cytokines (e.g., TNF-α), chemokines (e.g., CCL2), and inflammatory mediators
(e.g., inducible nitric oxide synthase (iNOS)). In addition, pathogen-induced
colonic histopathology, which was mild in mice left undisturbed during oral
challenge with C. rodentium, was significantly increased in mice exposed to the
SDR stressor during challenge with the pathogen. Stressor exposed mice had
increases in colonic epithelial cell hyperplasia and dysplasia, as well as epithelial
defects, generalized edema and leukocyte infiltration. These effects were not
evident in the mice that were not exposed to the stressor during pathogen challenge
(Galley et al., under review).
Inflammatory monocytes are recruited to sites of inflammation in response to the
chemokine CCL2 and are prolific producers of tissue-damaging TNF-α and iNOS
in the colon [70]. Unpublished observations from our laboratory indicate that
L. reuteri ATCC23272 can inhibit the ability of murine colonic epithelial cells
(i.e., CMT-93 cells) to produce CCL2 (Mackos et al., unpublished observations),
while others demonstrate that L. reuteri (strain 6475) can inhibit the ability of
monocytes to produce TNF-α [71–73]. Thus, mice were administered L. reuteri to
determine whether the commensal probiotic would attenuate stressor-induced coli-
tis. Daily administration with 1×108 CFU of L. reuteri after exposure to the SDR
stressor significantly reduced the effects of the stressor on C. rodentium induced
colitis (Galley et al., under review); stressor-induced increases in TNF-α, iNOS, or
CCL2 through the peak of C. rodentium infection, which occurs on day 12 post-
challenge did not occur in probiotic-treated mice. In addition, colonic histopathol-
ogy was not evident in any of the mice fed the L. reuteri, regardless of whether they
were exposed to the stressor or not.
Much has been learned about the effects of probiotic microbes on host immune
responses over the past 10 years, and it is tempting to speculate on the mechanisms
by which L. reuteri attenuates stressor-induced colitis. L. reuteri has the capacity to
limit pathogen growth and replication, particularly in vitro [74]. However, in all of
the studies conducted in our laboratory utilizing C. rodentium [46], as well as other
laboratories using a closely related pathogen (EHEC) [50], L. reuteri did not affect
pathogen levels in vivo. Mice exposed to either the prolonged restraint stressor or
the social stressor during oral challenge with C. rodentium had similar pathogen
levels with or without being fed L. reuteri. These data demonstrate that L. reuteri
does not attenuate pathogen-induced colitis by reducing pathogen load, and sug-
gests that L. reuteri directly suppresses host inflammatory responses.
There are now multiple studies demonstrating that L. reuteri produces an
immunomodulatory factor(s) when grown to stationary phase in vitro that reduces
monocyte inflammatory cytokine production upon stimulation. Some of the effects
of L. reuteri on stimulated monocytes are thought to be dependent upon bacterial
production of histamine that when bound to H2 receptors reduces monocyte activity
[73]. However, some strains of L. reuteri, such strain as ATCC23272 used in our
studies, do not strongly reduce monocyte/macrophage activity, but rather have
strong effects on colonic epithelial cells. Administering supernatants from over-
night cultures of strain ATCC23272 reduced CCL2, TNF-α, and iNOS production
by CMT-93 colonic epithelial cells, but not RAW264.7 macrophages or CD11b
+ splenic monocytes/macrophages stimulated with C. rodentium (Mackos and
Bailey, Unpublished Observations). Thus, it is possible that some strains of
L. reuteri reduce colonic inflammation through effects directly on inflammatory
monocytes, whereas other strains might reduce colonic inflammation by reducing
the ability of colonic epithelial cells to recruit and activate inflammatory cells, such
as inflammatory monocytes and neutrophils.
It is also possible that L. reuteri has a more indirect effect on colonic inflam-
mation in stressor-exposed mice. Studies demonstrate that intestinal microbes can
impact the activation of the HPA axis and increase glucorticoid levels [75]. This
could be of particular importance, because glucocorticoids produced by activation
of the HPA axis potently suppress inflammatory responses [76], and reduced
glucocorticoid production during stressor exposure as a result of adrenal insuffi-
ciency leads to intestinal inflammation during stressor exposure [77]. It is also
possible that the production of immunomodulatory neuroendocrine mediators by
268 M.T. Bailey
L. reuteri, or by commensal microbes affected by L. reuteri, are responsible for

effects on colonic inflammation in stressor-exposed mice. It has been shown that
probiotic microbes can produce immunomodulatory neuroendocrine hormones,
such as γ-amino butyric acid (GABA), norepinephrine, dopamine, and serotonin
(reviewed in [78]), and it has been hypothesized that this hormone production can
be responsible for influencing mucosal immune responses [78]. Thus, it is conceiv-
able that L. reuteri does not directly impact host colonic inflammation, but rather
stimulates host physiological responses that are known to have suppressive effects
on the inflammatory response. Potential pathways by which stress, the microbiota,
and probiotics impact colonic inflammation are illustrated in Fig. 12.1.
Microbiota and Stressor-Induced Immunomodulation

in Systemic Compartments
Stressor exposure often results in increases in nonspecific inflammatory responses.

For example, humans under the chronic stress of caring for a spouse with
Alzheimer’s disease were found to have increases in circulating IL-6 [79], whereas
exposure to acute laboratory stressors, such as different mental tasks, causes
increases in IL-1 [80]. The mechanisms by which these stressor-induced increases
in inflammatory cytokines occur in otherwise healthy individuals are not
completely understood. But data from our laboratory, as well as others, suggest
that the intestinal microbiota are involved [35, 81–83]. Mice exposed to the SDR
stressor also show evidence of circulating cytokines, and cytokine levels directly
correlate with microbiota levels [35]. For example, the relative abundance of three
members of the microbiota (i.e., Coprococcus spp., Pseudobutyrivibrio spp., and
Dorea spp.) were inversely correlated with the stressor-induced increases in circu-
lating IL-6 [35]. This suggested that the microbiota were somehow involved in
stressor-induced increases in circulating cytokines, but it was not until mice were
given an oral cocktail of nonabsorbable antibiotics to reduce the microbiota that the
link between the microbiota and circulating cytokines began to be clarified. Expos-
ing antibiotic-treated mice to the stressor failed to increase circulating cytokines
demonstrating a direct link between the microbiota and circulating cytokines
[35]. This initial discovery led to additional studies to determine whether the
microbiota are involved in stressor-induced modulation of macrophage microbici-
dal activity.
Phagocytes from mice lacking microbiota are deficient in their ability to kill
target pathogens, including Streptococcus pneumoniae and Staphylococcus aureus
[84]. Colonizing germ free mice by transplanting fecal bacteria from conventional
mice in to the germ free mice led to effective bacterial killing by the phagocytes.
Because reconstituted germfree mice had detectable levels of bacterial peptidogly-
can in circulation, and because mice lacking the peptidoglycan receptor Nod1 were
deficient in killing target microbes [84], it is likely that peptidoglycan from the
microbiota is necessary to prime phagocytes for efficient microbicidal activity. This

led us to question whether the microbiota are also necessary for the ability of the
stress response to prime splenic macrophages for enhanced microbicidal activity.
Exposing conventional mice to the SDR stressor increases the ability of splenic
macrophages to kill target microbes, such as E. coli, through an increased produc-
tion of macrophage peroxynitrite [67, 85, 86]. This effect is dependent upon
signaling through TLR4 [67] and the IL-1 receptor type 1 [86], and fails to occur
in germfree mice that lack any microbiota [85]. Exposing germfree mice to the SDR
stressor did not increase macrophage microbicidal activity or peroxynitrite produc-
tion. However, reconstituting the germfree mice with microbiota allowed the
effects of the stressor on splenic macrophage activity to again be manifest
[85]. This demonstrates that the microbiota are necessary for stressor-induced
increases in microbicidal activity to occur. Ongoing studies are determining the
mechanisms by which the microbiota can impact splenic macrophage activity, but
as shown in Fig. 12.2, data suggests that the microbiota exert their effects through
IL-1R1 and TLR4 signaling.
Conclusions
The dense populations of microbes that naturally colonize the body are well
recognized to have beneficial effects on the host, and as microbiota research
flourishes, we are becoming increasingly aware of the function of the microbiota
in maintaining the health of the host. These functions are in part dependent upon the
structure of the microbial communities, and it is thought that structure-function
relationships have developed through the co-evolution of the host and its
microbiota, such that alterations in one are associated with alterations in the
other. This is well-illustrated in animals exposed to different types of stressors.
During periods of quiescence, homeostatic interactions occur between the host and
its microbiota to maintain beneficial microbial populations in the intestines and
limit the induction of host inflammatory responses. As outlined in this chapter,
exposing animals to experimental stressors significantly disrupts these homeostatic
interactions; stressor-induced alterations in microbiota community structure are
associated with increased host inflammatory responses.
There is now accumulating evidence that stressor-induced alterations in
microbiota community structure are not just correlated with alterations in host
inflammatory responses, but might actually be causally involved in stressor-
induced immunomodulation. Exposure to psychological and physical stressors
results in the reduction in commensal lactobacilli, with data in mice suggesting
that the abundance of colonic tissue-associated Lactobacillus spp. are strongly
reduced upon stressor exposure. The lactobacilli are known to have several bene-
ficial effects on the health of the host, thus it is somewhat counterintuitive that the
stress response, which has evolved to benefit the host in the face of environmental
270 M.T. Bailey
Physical, Physiological, and/or

Psychological Stressor Exposure
Stressor-Induced
Physiological Response
e.g., Glucocorticoids,
Catecholamines Germfree State
Disruption of Homeostatic Interactions

Between Host and Microbiota
Lack of Bacterial Translocation

Attenuated Inflammatory Cytokines
Inflammatory Cytokines Attenuated Microbicidal Activity

- e.g., IL-1
Mucosal Mast Cell

Translocation of Live Microbiota
Degranulation
or Bacterial Peptidoglycan
-stimulation of TLR4
Enhanced Microbicidal Activity
-Peroxynitrite
-Superoxide
-Nitric Oxide
Enhanced Cytokine Production
Fig. 12.2 Exposing conventional mice to a social stressor leads to the translocation of gut
microbes and their products from the lumen of the intestines to the interior of the body, through
a mast cell-dependent mechanism. It is hypothesized that this translocation is responsible for the
stressor-induced increase in circulating cytokines, and subsequent enhancement of splenic mac-
rophage microbicidal activity. The gut microbiota are hypothesized to be involved, because
germfree mice exposed to the stressor have lower levels of circulating cytokines and macrophage
microbicidal activity is not enhanced by stressor exposure
demands, would negatively impact commensal microbes. However, stressor-

induced reductions in the lactobacilli might actually be reflective of gastrointestinal
physiological responses that are meant to be protective against enteric pathogens.
Stressor exposure increases colonic secretions, including the secretion of mucous,
and colonic motility. Colonic mucous is a protective buffer that separates potential
pathogens from adhering to colonic tissue. Thus, increased colonic mucous secre-
tion, coupled with enhanced colonic motility, would help to flush potential patho-
gens from the colon. If, however, mucoadherent commensal microbes, such as
members of the genus Lactobacillus, that naturally suppress colonic inflammation
are also flushed from the intestines, it would result in a colonic environment that is
conducive to overproduction of inflammatory mediators (Fig. 12.1).
Support for this notion comes from studies demonstrating that exposure to either
prolonged restraint or the SDR stressor reduces the abundance of Lactobacillus
spp., particularly of L. reuteri, and leads to increased colonic cytokine and chemo-
kine production upon pathogen challenge. L. reuteri reduces colonic cytokine and
chemokine production, and feeding stressor exposed mice L. reuteri to prevent
stressor-induced reductions in L. reuteri prevents the exacerbating effects of the
stressor on colonic cytokine and chemokine production. These findings support a

causal role for stressor-induced alterations in lactobacilli in the stressor-induced
exacerbation of infectious colitis (Fig. 12.1).
In addition to attenuating colonic inflammation, L. reuteri helped to reinforce the
epithelial barrier. Mice exposed to either prolonged restraint or the SDR stressor
during oral challenge with C. rodentium are more likely to have C. rodentium in the
spleen than non-stressed control mice challenged with C. rodentium. This is
important, because unlike invasive enteric pathogens, such as Salmonella spp.,
C. rodentium does not have mechanisms to invade its host [44]. C. rodentium and
closely related EPEC stay within the digestive tract, attached to the apical surface of
the colonic epithelium during infection of immunocompetent hosts [44]. However,
C. rodentium can disrupt tight junctions found between colonic epithelial cells via
the injection of effector proteins through a type III secretion system. L. reuteri are
known to prevent inflammation-induced increases in colonic epithelial permeability
[87], and can prevent the translocation of C. rodentium from the colon to the spleen
in stressor-exposed mice, an effect that is associated with altered expression of
genes encoding tight junction proteins [46]. These data indicate that an important
function of the commensal microbiota is the regulation of tight junctional proteins,
and stressor-induced alterations in the microbiota can allow for the loosening of
tight junctions and the exacerbation of systemic manifestations of infectious colitis
(Fig. 12.1).
Increased epithelial barrier permeability is also evident in uninfected stressor-
exposed mice [48, 49, 85]. Several different types of stressors have been shown to
increase the permeability of the intestinal barrier through mast cell-dependent
mechanisms [47–49]. A defining characteristic of commensal microbes is their
inability to invade through an epithelial barrier. Thus, commensal microbes can
be maintained within their niche in the body by just a single layer of epithelial cells.
However, it is known that stressor exposure can increase the ability of commensal
microbes, and their products like lipopolysaccharide and peptidoglycan, to trans-
locate from the lumen of the intestines to the interior of the body [85, 88,
89]. Because stressor-exposed germfree mice do not have increases in circulating
cytokines, it is hypothesized that microbes that have translocated into circulation
during stressor exposure cause an increase in circulating cytokines, such as IL-1. It
is further hypothesized that this microbiota-dependent increase in IL-1 primes
phagocytes for enhanced microbicidal activity through TLR4 signaling, because
stressor-induced increases in microbicidal activity does not occur in TLR4-/- mice,
IL-1R1-/- mice or in germfree mice (Fig. 12.2).
These studies demonstrate that the microbiota are interactively involved in
stressor-induced immunomodulation at mucosal surfaces, as well as at systemic
sites. Intestinal epithelial cells are important in mediating interactions between the
microbiota and host immune responses. As interest in the microbiota continues to
grow, it will be of importance to understand the molecular underpinnings through
which microbiota, intestinal epithelial cells, and immune system activity are
affected by stressor exposure.
272 M.T. Bailey
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Part III
The Microbiota-Gut-Brain Axis in Health
and Disease
Chapter 13
The Effects of Inflammation, Infection
and Antibiotics on the Microbiota-Gut-
Brain Axis
Premysl Bercik and Stephen M. Collins
Abstract Animal studies have demonstrated that the early phase of enteric infec-
tion is accompanied by anxiety-like behavior, which is mediated through vagal
ascending pathways. Chronic infection alters gut function, including motility and
visceral sensitivity, as well as feeding patterns, anxiety and depression-like behav-
ior. These effects are likely immune-mediated, and involve changes in pro-
inflammatory cytokines and altered metabolism of kynurenine/tryptophan path-
ways. Clinical studies have shown that chronic gastrointestinal infections lead to
malnutrition and stunting, resulting in impaired cognitive function. Accumulating
evidence suggests that in addition to pathogens, the commensal gastrointestinal
microbiota also influences gut function and host’s behavior. Both animal and
clinical studies have demonstrated changes in behavior and brain chemistry after
induction of intestinal dysbiosis by administration of antibiotics. This concept of
microbiota-gut-brain interactions opens a new field of research aimed at developing
microbial-directed therapies to treat a broad spectrum of human conditions, includ-
ing chronic gastrointestinal and psychiatric disorders.
Abbreviations
BDNF Brain derived neurotrophic factor

CGRP Calcitonin gene-related peptide
CRP C-reactive protein
DGGE Denaturing gradient gel electrophoresis
GABA γ-Aminobutyric acid
P. Bercik (*) • S.M. Collins Department

of Medicine, McMaster University, 1200 Main Street West, HSC 2E16, Hamilton,
ON, Canada, L8S 4K1
e-mail: bercikp@mcmaster.ca
280 P. Bercik and S.M. Collins
GBA Gut brain axis

IBD Inflammatory bowel diseases
IDO Indoleamine 2,3-dioxygenase
IFN-γ Interferon-gamma
IQ Intelligence quotient
MPO Myeloperoxidase activity
NMDA N-methyl-D-aspartate
SCFA Short chain fatty acids
SP Substance P
TLR4 Toll-like receptor 4
TNF-α Tumor necrosis factor alpha
Introduction: Gut-Brain Axis
The gut brain axis (GBA) is a bi-directional neuro-humoral communication system

that links gut and brain function in health and disease. The axis utilizes neural,
endocrine and immunological signaling to integrate the two organs. The clinical
importance of this axis is examplified by its role in the Irritable Bowel Syndrome
(IBS) most common of all intestinal disorders seen in our society where it generates
a huge socio-economic burden [1, 2]. Involvement of the axis is reflected in the high
prevalence of psychiatric morbidity in IBS [3]. There is also growing awareness
that the GBA plays a role in the natural history of chronic inflammatory bowel
diseases (IBD) including Crohn’s Disease and Ulcerative Colitis [4, 5]. Patients
with depression, anxiety or under psychological stress experience more active
disease [5]. It is now evident that the brain monitors and reacts to inflammatory
activity in the gut via the vagus nerve in what is known as the inflammatory reflex
[6]. Several studies have shown that vagus-mediated central nervous system control
of the gut inflammation is tonic and that vagal integrity is critical in regulating
chronic inflammation in animal models [7, 8]. The basis of co-existence of psychi-
atric disorders with IBD is unclear in terms of cause and effect for it is known that
chronic intestinal inflammation induces altered behavior in animal models [9].
The intestinal microbiota is a vast consortium of 1014 bacteria and represents the
most densely packed ecosystem on earth. Emerging data, reviewed below, provides
evidence to support the integration of the intestinal microbiota into the GBA
[10]. This extended axis involves bidirectional signaling involving neural, hor-
monal and immunological pathways described in detail below.
13 The Effects of Inflammation, Infection and Antibiotics on the.. . 281
Inflammation, Cytokines and the Central Nervous System
There is a growing body of literature linking the immune system with psychiatric
disorders. Multiple studies have demonstrated that patients with major depression
have elevated levels of pro-inflammatory cytokines and C-reactive protein (CRP)
[11, 12]. Depression is increased in patients with chronic illnesses associated with
immune activation such as cardiovascular disease [13], rheumatoid arthritis [14],
chronic obstructive pulmonary disease [15] and type 1 diabetes [16]. Cognitive
function also appears to be decreased in patients with chronic inflammatory disor-
ders [17, 18].
It is well established that acute administration of pro-inflammatory cytokines
leads to sickness behavior, which includes depressive-like behavior and fatigue
[19]. Therapy with cytokines in cancer patients is associated with changes in CNS
function and behavior [20] and psychiatric symptoms are also a frequent side effect
of interferon treatment for hepatitis C [21, 22].
The mechanisms by which cytokines can affect central nervous system (CNS)
function are not fully elucidated. It has been shown that cytokines can directly
activate primary afferents and the vagus nerve, or access the brain via the
circumventricular organs, where the blood brain barrier is more permeable
[19]. Animal studies have demonstrated that depressive-like behavior is caused
by cytokine-induced changes in tryptophan/serotonin metabolism and production
of kynurenine, which induces anxiety-like behavior in a dose dependent fashion
[23]. Indoleamine 2,3-dioxygenase (IDO) is the extrahepatic enzyme responsible
for kynurenine production, which is present in monocytes, macrophages and brain
microglia and activated in response to pro-inflammatory cytokines, particularly
TNF-α and INF-γ [24]. Patients who receive cytokine immunotherapy and develop
major depression exhibit a prolonged decrease in circulating concentrations of
tryptophan, which is accompanied by elevated concentrations of kynurenine com-
pared to non-depressed patients [25]. By reducing tryptophan availability, IDO
activation may impact brain serotoninergic neurotransmission, as tryptophan is the
limiting factor for the synthesis of serotonin that plays a crucial role in the
regulation of mood [26]. Furthermore, neuroactive metabolites of IDO activation,
such as kynurenine and kynurenic acid, are able to directly affect CNS
function [26].
If anxiety and depression are induced by immune mediators, then immunomod-
ulatory treatment should improve mood. Two studies have demonstrated improve-
ment of comorbid depression in patients with psoriasis and rheumatoid arthritis
after treatment with etanercept [27, 28]. While infliximab had no overall benefit in a
cohort of patients with major depression, it improved depressive symptoms in
patients with higher baseline inflammatory biomarkers [29]. Interestingly, in this
subset of patients infliximab treatment benefitted a wide range of depressive
symptoms, including depressed mood, psychomotor retardation, performance of
work and other activities (anhedonia/fatigue), as well as psychic anxiety and
suicidal ideation. All together, these data suggest that immune system and its
products affect the brain function and play an important role in, at least in a subset
of, patients with psychiatric disorders.
Effects of Infections on Cognitive Function
Accumulating evidence links infectious diseases, mainly of the digestive tract, to

the function of the central nervous system. This is evident primarily in children, as
from an energy-consumption standpoint, a developing human will have difficulty
building a brain and fighting off infectious diseases at the same time, as both are
very metabolically costly tasks. A recent study has assessed the worldwide distri-
bution of cognitive ability in relationship to the load of infectious diseases. Using
three measures of average national intelligence quotient (IQ), the authors found a
robust worldwide correlation between average IQ and parasite stress. Infectious
disease remained the most powerful predictor of average national IQ even when
controlling for additional factors, such as temperature, distance from Africa, gross
domestic product per capita and several measures of education [30]. Treating
parasitic infections can positively affect brain function. A double-blind placebo-
controlled study examined the effect of treating moderate to high worm burden of
non-invasive Trichuris trichiura on the cognitive functions of Jamaican school
children. Eradication of the infection led to a significant improvement in tests of
auditory short-term memory and scanning, and retrieval of long-term memory [31].
Multiple studies have demonstrated that diarrheal diseases affect nutrition and
development, including cognitive function in children. Early childhood diarrhea
has been associated not only with impaired physical fitness and growth but also with
cognitive function 6–9 years later, hindering school performance [32, 33]. A pooled
analysis of nine studies, covering five countries and a 20-year period, investigated
the effects of the previous history of diarrhea on stunting at age 24 months. The
odds of stunting increased multiplicatively with each diarrheal episode, with the
adjusted odds of stunting increasing by 1.13 for every five episodes [34]. Stunting in
infancy was linked to cognition in a large study in Filipino children that found
children aged between 8 and 11 years stunted between birth and age of 2 years,
displayed deficits in cognitive ability compared to non-stunted children, especially
when stunting was severe [35].
Gastrointestinal Infection Affects Gut-Brain Axis
Animal models have provided insight into mechanisms involved in gut-brain

communication during gastrointestinal inflammation. A landmark study by Lyte
et al. showed that very early phase of Campylobacter jejuni infection causes
anxiety-like behavior in mice without any increase in inflammatory mediators,
likely due to activation of vagal ascending pathways [36, 37]. This suggests that the
host is able to detect a pathogen before any significant activation of immune
system. We have found that chronic infection with Helicobacter pylori induces
functional and structural changes in the enteric nervous system (ENS), including
altered release of acetylcholine upon electrical or chemical stimulation, as well as
upregulation of SP and CGRP containing nerves in the stomach and spinal cord,
which only partially normalizes 2 months post bacterial eradication [38]. This is
accompanied by changes in visceral mechanosensitivity and gastric emptying
[39]. Furthermore, the infected mice have altered feeding patterns with increased
frequency of eating bouts and decreased amount of food consumed per bout which
is associated with decreased proopiomelanocortin expression in the arcuate
nucleus, and increased TNFα in the median eminence, which remain up-regulated
even 2 months post bacterial eradication. These functional and structural abnor-
malities in the post-infectious period may be maintained by antigenic mimicry or
cross-reactivity, as experiments showed that abnormal peristalsis and visceral
hypersensitivity were maintained by feeding crude Trichinella spiralis antigen to
the previously infected mice [40]. We have also observed that mice with chronic
H. pylori infection display anxiety-like behavior and possibly impaired learning
capacity (Fig. 13.1).
Another series of experiments showed that chronic mild to moderate infection
with the non invasive colonic parasite Trichuris muris leads to anxiety-like behav-
ior, which is accompanied by decreased brain derived neurotropic factor (BDNF)
expression in the hippocampus [41], mildly elevated TNFα and INFγ, and increased
levels of kynurenine. The abnormal behavior, but not BDNF levels, was normalized
by immunomodulatory treatment with etanercept and budesonide. Interestingly,
both behavior and BDNF normalized after administration of the specific probiotic
B. longum, which likely acts through modulation of ENS and the vagal ascending
pathways [9].
Antibiotic Induced Psychosis
Psychosis (highly distorted contact with reality) is a mental disorder, which could
arise as a form of an adverse drug reaction [42]. Most classes of antibiotics
administered for the treatment of various infections have been recorded to induce
transient psychosis in patients. Some of the symptoms presented include, but are not
limited to, visual and auditory hallucinations, lost orientation to persons, space and
time, delusions, and agitation. Most common antibiotics known to have psychotic
inducing effects are penicillins [43, 44], quinolones [42, 45, 46], macrolides [47,
48], sulfonamides [6, 48, 49] and anti-tuberculosis agents [50, 51] The psychotic
events generally develop within the first days of treatment and cease after with-
drawal of the antibiotic treatment [42]. Several explanations have been put forward
to explain this adverse effect, including interaction of the antibiotics with neuro-
transmitters. Quinolones were proposed to displace GABA from its receptors,
Fig. 13.1 Mice infected with H. pylori display anxiety-like behavior. BALB/c mice infected with
H. pylori for at least 3 months appeared to display anxiety-like behavior when assessed at week
1 using the light preference test. This abnormal behavior became more evident at week 2, as
uninfected control mice spent more time in the illuminated compartment, partially due to a learned
behavior, while H. pylori infected mice did not change their behavior compared to week 1
decreasing GABAergic inhibition and leading to the stimulation of the CNS [42]. -
Antibiotic-induced psychosis may be also the result of altered NMDA-receptor
function due to the depletion of D-alanine-producing intestinal flora [52]. In our
opinion, antibiotic induced intestinal dysbiosis may lead to altered metabolism of
multiple neuroactive substances produced by bacteria, including neurotransmitters
[53, 54] and short chained fatty acids (SCFA) [55], thus affecting brain function.
Effects of Antibiotics on Gut Function and Behavior: Lesson

from Animal Models
Traditionally, insights into host-microbial interactions have used comparison

between germ free and colonized young mice. However, many host systems are
immature in germ free mice and differences seen may not be congruous with those
seen in older mice with a stable microbiota and matured host immune and physi-
ological systems including the brain. A preferred approach is to use interventions
that alter the microbiome composition in adult mice. Perturbation of the delicate
balance between the microbial composition of the gut and the host results in
changes in the gut-brain axis, at the level of the gut and the brain. Exposure of
the intestinal microbiota to antimicrobials is a well-established experimental
approach to studying the impact of gut bacteria on the host; the impact of antimi-
crobials on the microbiota is dependent on the class of antibiotics used [56]. The
combination of orally administered bacitracin, neomycin and the antifungal agent
primaricin for 10 days resulted in increase of the inflammatory state of the gut, as
reflected by a small and transient increase in myeloperoxidase (MPO) activity and
in upregulation of the sensory neurotransmitter substance P (SP) in the enteric

nervous system. These changes were accompanied by an increase in the
pseudoaffective response to colonic distension of the colon [57]. Concomitant
treatment with dexamethasone normalized these changes, indicating that the
changes in host physiology were mediated by the small increment in intestinal
inflammation secondary to the antibiotic-induced change in the microbiota profile.
Furthermore, restoration of Lactobacilli following antibiotic administration also
ameliorated intestinal function [57]. A similar approach was adopted by Anitha
et al. [58] who showed that antibiotic-induced dysbiosis resulted in altered gut
transit and that these changes were mediated by Toll-like receptor 4 (TLR4).
Antibiotic-induced changes in the intestinal microbiota also induced changes in
brain chemistry and behavior. A similar antimicrobial cocktail of bacitracin, neo-
mycin and pimaricin was gavaged to mice over 7 days and behavior was monitored
pre-, during and post administration of antibiotics. The microbiota profiles were
monitored using denaturing gradient gel electrophoresis (DGGE). While no differ-
ences in microbiota profiles were seen between test groups prior to antibiotics,
treatment resulted in increased relative abundance of Lactobacilli and
Actinobacteria and a decrease in γ-proteobacteria and Bacteroides [59]. This
change in microbiota profile was accompanied by a reduction in anxiety, like
behavior, as reflected in the step down and the light preference tests when measured
7 days after antibiotic administration. The above-described antibiotic-induced
change in microbiota reverted to normal within 2 weeks after cessation of treatment
and this was accompanied by a normalization of behavior. The anxiolytic behavior
of antibiotic-treated mice was accompanied by an increase in BDNF in the hippo-
campus, and a decrease in BDNF in the amygdala [59] and these changes normal-
ized 2 weeks post cessation of the antibiotics. Since intra-peritoneal administration
of the same antibiotic mixture failed to impact on behavior in mice, we interpreted
our findings to mean that the altered microbial profile in the gut was responsible for
the changes in behavior and that the latter was mediated by changes in hippocampal
and amygdala BDNF. We investigated underlying mechanisms and found that
neither surgical bilateral sub-diaphragmatic vagotomy, nor chemical sympathec-
tomy altered the anxiolytic behavioral profile induced by antibiotics [59]. We did
not see inflammatory changes in the antibiotic mice nor significant elevations in
cytokines, leading us to conclude that the changes observed in behavior most likely
reflected intestinal bacterial products either acting directly on the brain, or indi-
rectly via host metabolism. Confirmation of the role of the microbiota in altering
behavior was confirmed in subsequent experiments in which we were able to
transfer components of behavior phenotype to germ free mice of a different species
and behavioral profile via microbial colonization. As there are marked differences
in microbiota composition and exploratory behavior between NIH Swiss mice,
which are daring and courageous, and BALB/c mice, which are shy and timid,
we derived these two mouse strains into germ-free conditions and colonized them
with the two sets of microbiota. Colonizing NIH Swiss mice with BALB/c
microbiota made them less daring while the exploratory drive increased in
BALB/c mice colonized with NIH Swiss bacteria [59]. In agreement with previous
experiments with antibiotic-induced dysbiosis, we observed changes in hippocam-

pal BDNF but did not find any signs of immune activation.
Interestingly, a recent study in a single human using a beta-lactam antibiotic
intervention to alter the microbial composition resulted in significant changes in
host metabolomic profiles of the gut with changes in anabolic sugar metabolism, the
production of acetyl donors and the synthesis and degradation of intestinal/colonic
epithelium components being among the most prominent changes [60]. This finding
turns attention towards the strong possibility that the shared host-microbiota
metabolome is the likely sources of molecules that act directly or as precursors to
modify brain function as reflected in the above-described experiments. Taken
together, experimental evidence support the integration of the intestinal bacteria
into the microbiota-gut-brain axis and demonstrates the utility of using a defined
antimicrobial combination to investigate how bacteria access this vitally important
axis. Furthermore, we should consider the possible clinical implications of the
accumulated data in view of fecal transplant therapy, which is being increasingly
used not only for patients with recurrent C. difficile infection but also for patients
with IBD and IBS. In our opinion, healthy stool donors should be assessed beyond
the current recommendations of bacterial and viral pathogen screening, and this
should include psychiatric evaluation.
Summary/Conclusions
Changes in the microbial composition of the gut influence the gut-brain axis. This is
true in the presence or absence of pathogenic bacteria. It is evident from previous
work that the early invasion of the gut by enteric pathogens is monitored by the
brain and generates anxiety-like behavior that may modify host behavior to mini-
mize further exposure to the pathogen. More chronic and insidious infections also
induce not only anxiety-like behavior and changes in cognition, but may also distort
feeding patterns, perhaps in an effort to reduce the chances of further infection.
These interpretations are based on a role of the microbiota-gut-brain axis in
maintaining homeostasis and the health of the host. In the context of promoting
host dysfunction and disease, it is evident that the induction of dysbiosis, either by
an enteric infection, exposure to antibiotics, or dietary change can lead to host
dysfunction that becomes evident not only in the gut, in conditions such as IBS, but
perhaps also in behavioral disorders such as anxiety and depression. This notion
opens a new field of research aimed at developing microbial-directed therapies to
treat this broad spectrum of human conditions.
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Chapter 14
Microbiota, Inflammation and Obesity
Yolanda Sanz and Angela Moya-Pérez
Abstract Interactions between metabolism and immunity play a pivotal role in the
development of obesity-associated chronic co-morbidities. Obesity involves
impairment of immune function affecting both the innate and adaptive immune
system. This leads to increased risk of infections as well as chronic low-grade
inflammation, which in turn causes metabolic dysfunction (e.g. insulin resistance)
and chronic disease (e.g. type-2 diabetes). Gut microbiota has emerged as one of the
key factors regulating early events triggering inflammation associated with obesity
and metabolic dysfunction. This effect seems to be related to diet- and obesity-
associated changes in gut microbiota composition and to increased translocation of
immunogenic bacterial products, which activate innate and adaptive immunity in
the gut and beyond, contributing to an increase in inflammatory tone. Innate
immune receptors, like Toll-like receptors (TLRs), are known to be up-regulated
in the tissue affected by most inflammatory disorders and activated by both specific
microbial components and dietary lipids. This triggers several signaling transduc-
tion pathways (e.g. JNK and IKKβ/NF-κB), leading to inflammatory cytokine and
chemokine (TNF-α, IL-1, MCP1) production and to inflammatory cell recruitment,
causing insulin resistance. T-cell differentiation into effector inflammatory or
regulatory T cells also depends on the type of TLR activated and on cytokine
production, which in turn depends upon gut microbiota-diet interactions. Here, we
update and discuss our current understanding of how gut microbiota could contrib-
ute to defining whole-body metabolism by influencing diverse components of the
innate and adaptive immune system, both locally and systemically.
Y. Sanz (*) • A. Moya-Pérez Microbial

Ecology, Nutrition & Health, National Research Council (IATA-CSIC), Av. Agust´ınEscardino
7, 46980 Paterna, Valencia, Spain
e-mail: yolsanz@iata.csic.es
292 Y. Sanz and A. Moya-Pérez
Abbreviations
BMI Body mass index

ER Endoplasmic reticulum
ERK Extracellular signal-regulated kinase
ERS Endoplasmic reticulum stress
FetA Fetuin-A
HFD High-fat diet
IKK Inhibitory κB kinase
IL Interleukin
IL-1Ra IL-1 receptor antagonist
iNOS Inducible nitric oxide synthase
IR Insulin receptor
IRF Interferon regulatory transcription factor
IRS Insulin receptor substrate
IRS-1 Insulin receptor substrate 1
LTA Lipoteichoic acids
M1 “Classically activated” macrophages
M2 “Alternative activated” macrophages
MAPKs Mitogen-activated protein kinases
M-cells Microfold cells
MCP Monocyte chemotactic protein
MDP Muramyl dipeptide
Meso-DAP D-Glutamyl-meso-diaminopimelic acid
MHC Major histocompatibility complex
NF- κB Nuclear factor-κB
NKT Natural killer T
NLRs Nod-like receptor family
NOD Nucleotide oligomerization domain
NOS2 Nitric-oxide synthase 2
PGN Peptidoglycan
PI3K Phosphatidylinositol 3-kinase
PI3-K Phosphatidylinositol 3-kinase
RHM Recruited hepatic macrophage
ROS Reactive oxygen species
SAA3 Serum amyloid A3 protein
SFA Saturated fatty acid
SOC Suppressor of cytokine signaling
STAT3 Signal transducer and activator of transcription 3
TH1 T helper 1
TLRs Toll-like receptor family
Tregs Regulatory T
ZO Zonula occludens
14 Microbiota, Inflammation and Obesity 293
Introduction
Obesity is associated with immune function impairment, affecting both the innate
and the adaptive immune system. These alterations lead to an increased risk of
infections and to a state of chronic low-grade inflammation, which is a major cause
of metabolic dysfunction (e.g., insulin resistance, metabolic syndrome) and chronic
disease (e.g., type 2 diabetes, fatty liver disease, cardiovascular disease, etc.).
Obesity is characterized by infiltration of macrophages and lymphocytes in the
adipose tissue and other peripheral organs. This is accompanied by an imbalance in
the cytokine network with increased production of pro-inflammatory cytokines,
adipokines, acute-phase proteins and other immune mediators [1, 2]. These inflam-
matory mediators, as well as several transcription factors and kinases, are involved
in inflammation-induced metabolic dysfunction such as insulin resistance [3].
Gut microbiota is likely to be one of the factors influencing our predisposition to
develop obesity and associated comorbidities. Alterations in the gut microbiota
structure have been related to obesity and metabolic dysfunction in murine models
[e.g., 4–6] as well as in human observational studies [7, 8]. Differences in
microbiota composition in obese animal models could be a consequence of diet
and other environmental factors [6, 9–11] and of genotype (e.g., deletion of the
leptin gene or its receptor [4, 5]). Notwithstanding, animals with the same genotype
and under the same dietary influence (high-fat diet [HFD]) can also develop
different metabolic phenotypes (either diabetic or non-diabetic) as a function of
their specific gut microbiota profile. This finding suggests that gut microbiota per se
determines the risk of developing metabolic dysfunction [12]. This relationship is
also supported by studies showing that germ-free mice are protected against diet-
induced obesity and by fecal transplantation experiments showing that when
microbiota from twins discordant for obesity is transplanted in germ-free mice,
these mice develop the corresponding phenotype whether they are fed a low-fat diet
or high-saturated fat diet [11], although opposite results have also been
published [10].
In humans, many studies associate alterations in gut microbiota structure and
function with obesity and markers of metabolic risk, which may also be partly a
consequence of diet [7, 13, 14], while effects of the genotype predisposing to
obesity on the microbiota are largely unknown. Nonetheless, diet-induced gut
microbiota alterations (e.g., an increase in Firmicutes and decrease in
Bacteroidetes) seem to play a role in obesity by, for example, increasing energy
harvest and lipid absorption [15, 16].
Gut microbiota is likely to be involved in body weight regulation by influencing
the host’s metabolic and endocrine network, and the immune system [7]. Coloniza-
tion of the newborn intestine has an enormous impact on the development of
mucosal and systemic immunity, contributing to its ability to discriminate between
harmful and innocuous antigens with important effects during early postnatal life
through adulthood [17]. The innate immune system is one of the key regulators of
the crosstalk between the host and its commensal and pathogenic intestinal bacteria
[18]. Innate immune recognition of specific microbial components is mediated by

families of pattern-recognition receptors (e.g. Toll-like receptor family [TLRs] and
Nod-like receptor family [NLRs]) which, upon ligand binding, activate different
signaling pathways. These can trigger inflammatory responses leading to pathogen
clearance or attenuate intestinal inflammation, depending on the stimulus which
may also vary depending on gut microbiota composition [19]. These receptors are
also activated by dietary lipids and up-regulated in most tissues affected by
inflammatory disorders (e.g. adipose tissue, liver, brain) contributing to the inflam-
matory process leading to insulin resistance [20, 21]. Lymphocyte differentiation
into effector or regulatory T (Tregs) cells also depends on the type of TLR
activation and cytokine production [19]. Therefore, obesity-associated alterations
in lymphocyte distribution and their phenotype may also depend on gut microbiota-
diet interactions [22]. Furthermore, recent animal studies report that the intestine,
which is the tissue most exposed to “noxious” nutrients (saturated fatty acids) and
to a high load of bacterial antigens, is where the inflammatory process associated
with diet-induced obesity originates [23, 24].
Therefore, a growing body of scientific evidence supports the notion that the
crosstalk between the gut microbiota, diet and immune system activates mediators
and signaling pathways, which influence whole body metabolism and disease. Here
we update and discuss our current understanding of the specific role that the gut
microbiota may play in obesity and metabolic dysfunction (insulin resistance) by
influencing host innate and adaptive immunity.
Inflammation Associated with Obesity and Metabolic

Dysfunction
Adipose Tissue Inflammation
Adipose tissue inflammation is likely the main contributor to inflammatory signals

that lead to metabolic dysfunction (e.g. insulin resistance) in obesity. In fact,
expression of pro-inflammatory cytokines in adipose tissue seems to be 100–
1,000 times higher than in the liver of subjects with severe obesity and fatty liver
disease [25]. Inflammation of this tissue is mainly attributed to macrophage infil-
tration, which may represent up to 40 % of all cells. This is accompanied by
inflammatory cytokine and adipokine production (e.g. tumor necrosis factor
(TNF) α, interleukin [IL]-6 and IL-1β and leptin) [1, 2]. Macrophage migration is
promoted by adipose tissue-produced chemokines, particularly monocyte chemo-
tactic protein (MCP)-1. Adipose tissue inflammation is also characterized by an
increased ratio of “classically activated” macrophages (M1) to “alternative acti-
vated” macrophages (M2). M1 are highly inflammatory macrophages via induction
of pro-inflammatory cytokines and other factors (e.g. primarily TNF-α IL-1β, IL-6
and resistin [in humans] and inducible nitric oxide synthase [iNOS]). However, M2,
Proinflammatory cytokines
(IL-1, IL-18 , IL-6, TNFα)
Leptin Adiponectin
mRNA
ObRb TNFR1 IL-18R or IL-1R IL-6Rα AdipoR1/R2
Plasma membrane
SOC
PCR ERK1/2
STAT3 ER stress
MAPK/JNK IKKβ
ERK ROS
NOS2 IRS-1
?
AP-1 NF-kB PPAR
Inflammatory
mediators
Insulin Resistance
Nucleus
Fig. 14.1 Mode of action of the main cytokines and adipokines and transcriptional factors
produced by macrophages and adipose tissue in obesity-associated insulin resistance. TNF-α
induces IKKβ/NF-κB and JNK activation promoting the phosphorylation of IRS-1 at serine sites
that negatively regulate normal signaling through the insulin receptor/IRS-1 axis and suppress the
transcription of adiponectin. IL-1β and IL18 induce insulin resistance by reducing IRS1 expression
at a transcriptional level through an extracellular signal-regulated kinase (ERK) dependent
mechanism and activating IKKβ/NF-κB. IL-6 may contribute to insulin resistance via induction
of SOC proteins and inhibition of adiponectin transcription. Leptin contributes to inflammation by
inducing activation of the MAPKs p38 and ERK and of STAT3, leading to pro-inflammatory
cytokine production (TNF-α, IL-6 etc). Leptin also activates NOS2 production, leading to
increased ROS. Adiponectin, improves insulin sensitivity by suppressing the NF-κB-dependent
synthesis of TNF-α and IFNγ, and inducing the anti-inflammatory mediators IL-10 and IL-1RA, as
well as by activating PPARγ, which inhibits the NF-κB pathway
which are predominant macrophages in lean adipose tissue, exert anti-inflammatory

effects via induction of IL-10 and IL-4 cytokine production [26, 27]. M2 also
produce catecholamines to sustain adaptive thermogenesis, increasing thermogenic
gene expression and contributing to fatty acid mobilization and energy expenditure
in adipose tissue in a macrophage-dependent manner [28].
Figure 14.1 summarizes the mode of action of the main cytokines and
adipokines, and transcriptional factors produced by macrophages and adipose tissue
in obesity-associated insulin resistance. Insulin regulates glucose homeostasis by
activating the insulin receptor (IR). This occurs by IR autophosphorylation, leading
to tyrosine phosphorylation of several substrates, such as the insulin receptor
substrate (IRS)-1 and -2. Subsequent to tyrosine phosphorylation, IRS-1 and
IRS-2 bind and activate phosphatidylinositol 3-kinase (PI3-K), which increases

serine phosphorylation of Akt, leading to glucose transport in muscle and adipose
tissue, glycogen synthesis in muscle and liver and lipogenesis in adipose tissue
[29]. The proper signaling of this pathway may be disrupted by several mecha-
nisms. These include serine phosphorylation of IRS proteins by protein kinases
such as c-Jun N-terminal kinase (JNK) and inhibitory κB kinase (IKK)-β of the
nuclear factor-κB (NF-κB) pathway, and decreased tyrosine phosphorylation of
IRS-1 [29]. The JNK pathway can be activated by endoplasmic reticulum stress
(ERS) activation, which occurs in both adipose tissue and liver [30]. Insulin
signaling can also be impaired by increased secretion of pro-inflammatory cyto-
kines of innate immunity, including TNF-α and IL-6, IL-1 and IL-18 as well as
IL-17 and IFN-γ produced by T cells [30, 31]. Impaired production or action of
adipokines such as leptin and adiponectin may also be involved [30].
TNF-α is overproduced exclusively by activated macrophages in the adipose
tissue, and directly causes insulin resistance by acting locally on insulin target cells
through paracrine mechanisms. Signaling via TNF-α activates intracellular kinases,
such as cJNK and IKK, which inhibit insulin receptor signaling by serine phos-
phorylation of insulin receptor substrate 1 (IRS-1). Activation of transcription
factors AP-1 and NF-κB also exacerbates pro-inflammatory cytokine production
by a feedback loop mechanism, whereby proinflammatory cytokine production is
increased [31]. TNF-α also suppresses the transcription of adiponectin in adipocyte
cell cultures, which explains the reduction in serum adiponectin levels in obese
individuals. TNF-α production in adipose tissue can also contribute to increasing
circulating levels, which can reach other peripheral tissues (e.g., muscle and liver)
and contribute to systemic insulin resistance [31].
Several IL-1 family cytokine members, including pro-inflammatory (e.g., IL-1α,
IL-1β, IL-18) and anti-inflammatory components (IL-1 receptor antagonist [IL-
1Ra]), are produced by both immunocompetent cells and obese adipose tissue,
and play an important role in metabolic inflammation [32]. Neutralization of IL-1
by IL-1Ra can improve hyperglycemia and glycemic control in humans, supporting
a role for IL-1 in diabetes and related insulin resistance. Like other inflammatory
cytokines, IL-1β and IL18 induce insulin resistance by reducing IRS1 expression at
a transcriptional level through an extracellular signal-regulated kinase (ERK)
dependent mechanism activating IKKβ/NF-κB [30]. IL-6 is a pro-inflammatory
cytokine involved in regulating the acute phase response and in insulin resistance
via induction of suppressor of cytokine signaling (SOC) proteins and inhibition of
adiponectin transcription [30]. Although preclinical data are not fully conclusive as
to whether IL-6 is beneficial or detrimental, in the context of hyperglycemia small
clinical trials suggest a beneficial effect of anti-IL-6 therapy. The fact that anti-IL-1
therapies strongly decrease IL-6 levels, also suggests that neutralizing IL-6 is
effective and plays a role in metabolic disease [32]. IL-6 also suppresses
adiponectin transcription in adipocyte cell cultures such as TNF-α.
Adipocytokines also play different immune roles in the monocyte–macrophage
components of the innate immune system, and in T cells of the adaptive immune
system [3]. While adiponectin is generally considered anti-inflammatory, leptin and
resistin are considered pro-inflammatory adipocytokines. Through interaction with

its receptor (AdipoR1/R2), adiponectin suppresses the NF-κB-dependent synthesis
of TNF-α and interferon-γ (IFNγ), and induces IL-10 and IL-1RA production.
Adiponectin also induces apoptosis of monocytes and inhibits phagocytosis by
macrophages. Adiponectin also decreases T-cell proliferation, reducing the poten-
tial allogeneic T-cell response. Nevertheless, adiponectin also has a pro-
inflammatory effect in specific circumstances that could be explained by different
roles played by the different full-length and globular forms of adiponectin in
inflammation and immunity, which are not fully understood yet. In monocytes/
macrophages, the mitogen-activated protein kinases (MAPKs) p38 and
extracellular-signal-regulated kinase (ERK), signal transducer and activator of
transcription 3 (STAT3), are activated by leptin via its OBRb receptor. This
activation leads to pro-inflammatory cytokine production, including TNF-α, IL-6
and IL-12. In addition, leptin activates nitric-oxide synthase 2 (NOS2) production,
leading to increased reactive oxygen species (ROS), and enhances macrophage
phagocytosis, and activation, proliferation and migration of monocytes. Leptin also
influences the adaptive immune system, for example by increasing production of
the T helper 1 (Th1) cytokines IL-2 and IFNγ, and suppressing Th2 cytokine IL-4
production in T-cell proliferation assays with mouse cells; however, its role in
humans is less clear. In monocytes/macrophages, resistin also activates p38, ERK
and phosphatidylinositol 3-kinase (PI3K). This adipocytokine also increases the
production of TNF, IL-1β, IL-6 and IL-12, contributing to inflammation.
PPARγ is an additional transcriptional factor and a genetic sensor of fatty acids,
which is required for fat development and exerts insulin-sensitizing effects. In
adipose tissue, PPARγ is also required for the maturation of M2 macrophages
and induces adiponectin synthesis. PPARγ expressed by macrophages also inhibits
TLR- and IFN-γ-mediated inflammatory responses and is essential for normal
skeletal muscle and liver insulin sensitivity [30].
B cells and T lymphocytes also infiltrate the adipose tissue, which can contribute
to inflammation and metabolic dysfunction. The sequence of recruitment of each
cellular type into adipose tissue is unclear but their functional roles are known to
some extent. B cells [33], CD8+ cytotoxic T cells [34], CD4+ Th1 cells [35] and
CD4+ Th17 [36] may promote insulin resistance, whereas CD4+ regulatory T
(Treg) cells reduce inflammation, likely contributing to improve insulin sensitivity
[37, 38]. Treg cells are drastically reduced in obese adipose tissue paralleled to B
cell increases [34, 38]. Tregs regulate the macrophage phenotype, inhibiting their
polarization into M1-type and preventing macrophage recruitment into tissues [35,
38]. Depletion of Treg cells via administration of diphtheria toxin is also accom-
panied by substantial decreases in insulin-stimulated insulin-receptor (IR) tyrosine
phosphorylation in epididymal fat and liver, supporting a role of this cellular
population in glucose metabolism, at least in these tissues [38]. A recent study in
mice on a HFD also suggests that CD19+ B lymphocytes are quickly recruited into
adipose tissue and activate pro-inflammatory macrophages and T cells, thus
adversely influencing glucose metabolism [33]. Adipose tissue-associated B cells
can induce major histocompatibility complex (MHC)-dependent pro-inflammatory
cytokine release, including IFNγ, from resident CD4+ and CD8+ T cells, which in
turn modulate macrophage polarization. The role of B cells in obesity-associated
metabolic dysfunction is also supported by the increased insulin sensitivity found in
B-cell-deficient mice on a HFD compared with wild-type mouse controls [33]. CD4
+ IFN-γ producing cells can also participate in adipose tissue inflammation and
insulin resistance. IFN-γ promotes insulin resistance by (1) reducing insulin-
stimulated uptake of glucose in adipocytes parallel to reducing serine/threonine-
specific protein kinase Akt phosphorylation and down-regulating the insulin recep-
tor, IRS-1 and the glucose transporter Glut4, which impair insulin signaling;
(2) polarizing, activating and stimulating M1 macrophages in adipose tissue and
up-regulating T-cell and monocyte chemoattractants (e.g. IP-10 and RANTES and
MCP-1 and MCP-2); and (3) inducing STAT1 phosphorylation and SOCS1/3
expression in adipocytes [36]. Increased CD8+ T cell infiltration or increased
CD8+/CD4+ ratio have also been described as another critical event driving
adipose tissue inflammation since it can contribute to producing critical
pro-inflammatory cytokines such as IFN-γ [34, 39]. The CD4+ Th17 cells, produc-
ing IL-17, are also detected in visceral adipose tissue, but at low frequencies. IL-17
produced by Th17 cells is a pathogenic mediator of inflammation in numerous
autoimmune disorders, for example by triggering NF-κB activation and cytokine
release. However, the role of Th17 cells in obesity-related insulin resistance is still
unclear and requires further investigation [36].
Liver Inflammation
Two macrophage populations are identified in liver: a resident macrophage popu-

lation (Kupffer cells) and a recruited hepatic macrophage (RHM) population, which
migrated upon weight gain under the influence of the liver-derived MCP-1 and can
represent 30–70 % of all hepatic macrophages in obesity [40, 41]. Both types of
macrophage populations seem to contribute to chronic hepatic inflammation and
insulin resistance. Natural killer T (NKT) cells, which can respond to lipid antigen,
may be involved in obesity and glucose tolerance, but evidence from animal models
is inconsistent [42, 43]. While mice fed a HFD showed increased expression of
NKT cells (defined by CD3+NK1.1+) in adipose tissue, amounts decreased in the
liver [44]. Hepatic insulin resistance is a driving force in the pathogenesis of type
2 diabetes Mellitus, coupled with excessive fat storage that ensures liver inflam-
mation. Activation of transcription factor NF-κB and downstream inflammatory
signaling pathways systemically and in the liver are considered key events in the
etiology of hepatic insulin resistance and also in β-cell dysfunction, although the
molecular mechanisms involved are only partly understood [45].
Central Nervous System Inflammation
The central control of energy balance and adjustment of food intake and expendi-
ture mainly occurs in the hypothalamus, where there is a complex interplay between
insulin, leptin and other neuro-regulators (e.g. serotonin) partly via IRS/PI3K
signaling, which negatively regulates energy balance, reduces food intake and
improves insulin signaling [46]. Obesity is associated with hypothalamic inflam-
mation and production of pro-inflammatory cytokines that cause central leptin
resistance, leading to reduced central regulation of food intake and energy expen-
diture. Central nervous system inflammation also contributes to systemic insulin
resistance, particularly in the liver, via a brain-liver neuronal signal [47]. In animal
models, inhibition of either TLR4 or TNFα reduces hypothalamic inflammation,
which is accompanied by reduced hypothalamic resistance to leptin, and improved
hepatic insulin signal transduction, reduced steatosis and reduced gluconeogenesis.
All these effects are mediated by parasympathetic signals delivered by the vague
nerve. Circulating IL-6 is also known to activate the hypothalamic-pituitary-adrenal
axis, which is associated with central obesity, hypertension, and insulin
resistance [48].
Decreased Immunological Surveillance Associated

with Obesity
Obesity is also associated with alterations in the immune defense mechanisms, thus
leading to increased risk of infection and decreased response to vaccination. This
constitutes an important cause of morbidity and mortality in obese subjects. Epi-
demiological human studies demonstrate that obese subjects are at a greater risk of
nosocomial infections after surgery [49]. Obesity is also an independent risk factor
for increased morbidity and mortality related to infection by influenza A (H1N1)
virus [49]. Obesity also seems to compromise the efficacy of vaccination against
viral infections, as demonstrated in murine models of obesity [50].
The mechanisms underlying obesity-associated risk of infection have been
studied in murine models of genetically or diet-induced obesity. In leptin-deficient
murine models of obesity (ob/ob or db/db) both innate and adaptive immune
systems are adversely affected. Leptin activates monocytes/macrophage chemo-
taxis, phagocytic activity and cytokine production and, consequently, these func-
tions are impaired in leptin-deficient mice [51]. In fact, ob/ob mice showed
impaired immunological protection against different bacterial pathogens due to
defective phagocytic activity [52]. Leptin deficiency in mice also leads to an
impairment of DC function, characterized by increased production of immunosup-
pressive cytokines and decreased stimulation of allogenic T cells [53, 54]. T cell
reactivity is also impaired in HFD-fed mice that are transgenic for a TRC recog-
nizing a peptide from ovalbumin, indicating that similar defects in immunity occur
in diet-induced obesity. T cells from HFD-fed na¨ıve transgenic mice exhibit an

inflammatory response against in vitro antigen/mitogen stimulation, which could
contribute to chronic obesity-associated low-grade inflammation [55]. In contrast,
antigen-experienced T cells from ovalbumin immunized HFD-fed mice produce a
Th2 cytokine profile and have reduced proliferation capacity. DCs from HFD-obese
mice are also less able to present antigens to T cells, which may influence T cell
polarization [55]. All these findings explain this increased susceptibility to infec-
tions and hypo-responsiveness to vaccination in obese subjects.
Influence of Gut Microbiota on Inflammation Associated

with Obesity and Metabolic Dysfunction via Regulation
of Innate Immunity
Gut microbiota is considered one of the factors contributing to chronic-low grade

inflammation associated with obesity and metabolic dysfunction (e.g. insulin resis-
tance). The mechanisms by which gut microbiota influences this process are not
well understood, but could be related to alterations in gut microbiota composition.
Such changes could increase bacterial components that might activate innate
immunity locally in the gut and systemically, and increase translocation of immu-
nogenic bacterial products via different routes, thus contributing to inflammation.
The innate immune system is one of the key regulators of the crosstalk between
the host and the microbiota (commensal and pathogenic microbes). Innate immune
recognition of specific microbial components (e.g. LPS, DNA, etc.) is mediated by
families of pattern-recognition receptors, like the TLR family and NOD-like recep-
tor family, which are expressed in epithelial cells and antigen presenting cells (DCs
and macrophages). Upon ligand binding, different signaling pathways (e.g. NF-κB,
MAPKs/JNK and the interferon regulatory transcription factor [IRF]) are activated,
leading to the expression of inflammatory genes encoding cytokines, cytokine
receptors, immuno-regulatory proteins, adhesion molecules and stress-associated
proteins [18]. These molecules also induce the recruitment of other immune cells
(T cells, basophils, neutrophils, DCs and NK cells) that trigger inflammatory
responses and can lead to pathogen clearance [18]. These signaling pathways are
also responsible for maintaining tolerance to commensal bacteria which, unlike
pathogens, attenuate intestinal inflammation via different mechanisms
(e.g. inhibiting NF-κB, inducing regulatory T cells, etc.; [19]). TLRs are up-
regulated in most tissues affected by inflammatory disorders and activated by
dietary lipids, and thus mediate the crosstalk between the gut microbiota, the host
innate immune system and whole body metabolism [56]. A schematic representa-
tion of the mechanisms by which gut microbiota and dietary lipids could contribute
to “metabolic” inflammation by activating innate immunity in the gut and system-
ically is shown in Fig. 14.2.
(PGN)
HFD induced-microbiota alterations

Gram- /  Gram+ bacteria?
Dietary
lipids
LPS PGN DNA PGN
LPS TLR
Chylomicrons
M cell
NOD 1/2
Bacteria/ antigens
LPS
LPS Inflammatory mediators
Tight Junction
 ZO-1 (TNFα, IFN , IL6, IL18, MCP1 ,etc.)
DNA
iNOS, COX2
Insulin resistance
PGN Macrophages
 Claudin-1 DCs
 Occludin
MLN
Circulation
Fig. 14.2 Main mechanisms of action for gut microbiota components and their interactions with
dietary lipids in the context of inflammatory processes leading to obesity-associated metabolic
dysfunction (insulin resistance). LPS from Gram-negative bacteria activate TLR4/MyD88/NFκB
and MAPKs/JNK pathways in epithelial and immunocompetent cells activating inflammatory
mediator synthesis, inflammatory cell recruitment and activation of the underlying lymphoid
tissue, thus contributing to inflammation. Dietary lipids (saturated fatty acids) increase the
expression and activation of innate immune receptors (TLR4, TLR2 and inflammasome) and
contribute to translocation of bacterial products (LPS, PGN, etc.) by transcellular and paracellular
pathways that activate immunocompetent cells in the gut-associated lymphoid tissue and in
peripheral tissues. HFD-induced microbiota imbalances leading to inflammatory cytokine produc-
tion also alter tight-junctions between enterocytes, increasing paracellular permeability to bacte-
rial antigenic products. Bacteria and bacterial antigenic products can also translocate via M-cells
and reach peripheral tissues via DCs. TLR2 recognizes lipoteichoic acids from Gram-positive
bacteria and also LPS from Gram-negative bacteria, acting synergically with TLR4 triggering
inflammation via NFκB and JNK pathways. NOD1 and NOD2 proteins recognize bacterial
peptidoglycan (PGN) and mediate insulin resistance in different tissues via activation of common
signaling transduction pathways (MAPKs), expression and production of pro-inflammatory cyto-
kines/chemokines, and impairment of insulin signaling
LPS and TLR4 Signaling
Lipopolysaccharide (LPS) from the cell wall of Gram-negative bacteria is currently

thought to play a role in both immunity and metabolism through the TLR4/MyD88/
NF-κB signaling pathway. Increased LPS plasma levels are associated with an
elevated body mass index (BMI) and high-fat feeding, postprandial inflammation
and risk of type-2 diabetes and atherosclerosis in humans [57, 58]. In animal
models, increased LPS in plasma (termed “metabolic endotoxemia”) has been
causally linked to adiposity and obesity-related insulin resistance and inflammatory
liver diseases, such as non-alcoholic fatty liver disease and non-alcoholic
steatohepatitis [59, 60]. Mice fed a HFD exhibit a significant increase in plasma
LPS, associated with changes in the gut microbiota, obesity, inflammation and
glucose metabolic dysfunction. The role of circulating LPS per se in metabolic
dysfunction is demonstrated by LPS infusion. On reaching the same plasma LPS
levels as those measured in HFD-fed mice they reproduce the same phenotype of
HFD-fed mice. The role of LPS and TLR4 is proven in mice deficient in CD14, a
key molecule in TLR4 signaling activated by LPS, showing that these mice are
resistant to inflammation in adipose deposits, liver and muscles, induced by both
HFD and chronic LPS administration [60]. The fact the gut microbiota is involved
in LPS-induced metabolic dysfunction has been shown by administering antibiotics
(norfloxacin and ampicillin) to two different mouse models of insulin resistance
(genetically and diet induced), which led to gut microbiota depletion parallel to
reduced serum LPS levels, low grade inflammation, obesity and type-2 diabetes
[61]. Comparisons between germ-free and conventional mice have also demon-
strated the direct role of the gut microbiota in triggering colonic serum amyloid A3
protein (SAA3) expression, which could contribute to inflammation via LPS sig-
naling [62]. This effect is demonstrated to be partially mediated via TLR/MyD88/
NF-κB signaling, by comparing wild-type with Myd88 deficient mice, with the
latter gaining less weight on a Western HFD and lower epididymal fat pad and liver
masses [62]. A recent study highlights the importance of diet-gut microbiota
interactions in this process, reporting that a HFD causes deregulation of the gut
microbiota composition (increasing the Firmicutes to Bacteroidetes ratio) leading
to increased fecal endotoxin and colonic inflammation, parallel to increased plasma
LPS and systemic inflammation. This intestinal inflammation was characterized by
increased expression of proinflammatory cytokines, TLR4, iNOS and COX-2,
activation of NF-κB and reduced the expression of tight junction-associated pro-
teins claudin-1 and occludin. This study also demonstrates that TLR4 mediates
inflammation associated with adiposity and obesity induced by a HFD comparing
wild-type with TLR4-deficient mice [24]. Comparing germ-free and conventionally
colonized mice has also demonstrated that gut microbiota colonization leads to
impaired glucose metabolism and increased macrophage accumulation, and polar-
ization towards a pro-inflammatory M1 phenotype in white adipose tissue in mice
fed a standard diet, without HFD feeding [63]. In the same study mice were
colonized with an Escherichia coli strain, producing immunogenic LPS or not,
demonstrating that macrophage recruitment requires LPS, whereas impairment of
systemic glucose metabolism is not exclusively LPS-dependent and may involve an
additional mechanism [63]. The fact that protection of TLR4-deficient mice from
obesity-induced insulin resistance does not require germ-free conditions, also
suggests the microbiota is not the only factor activating this signaling pathway
triggering metabolic disease [64, 65]. Altogether, these findings demonstrate that
LPS-induced TLR4 signaling constitutes one of the links between the gut
microbiota and inflammation that leads to metabolic dysfunction, which is also

influenced by diet.
LPS can impair metabolic functions when reaching tissues involved in glucose
and lipid metabolism, such as the liver and the adipose tissue, by stimulating TLRs
expressed in infiltrated immune cells (e.g., macrophages and dendritic cells) and in
obese adipose tissue. In adipose tissue, LPS-stimulated TLR4 activates p65/p50 and
p68/p52 NF-κB signal transduction pathway, inducing the expression of inflamma-
tory mediators such as IL-6, TNF-α, and SAA3 protein, possibly impairing insulin
sensitivity as explained in a previous section (Fig. 14.1). This signaling pathway
can also induce endoplasmic reticulum (ER) stress and JNK activation accompa-
nied by increased IRS-1 serine 307 phosphorylation in the liver, muscles, and
adipose tissue, leading to a reduction in insulin sensitivity and signaling
[29]. TLR4 signaling also increases expression of iNOS, which reacts with cysteine
residues to form S-nitrosothiol adducts, inducing S-nitrosation/S-nitrosylation of
the insulin signaling pathway, leading to insulin resistance in the liver, muscles, and
adipose tissue. Circulating LPS can also activate monocyte chemo-attractant pro-
tein MCP-1, mediating migration of monocytes to peripheral tissues and contrib-
uting to the inflammatory process [66].
High intake of saturated fat, which results in increased levels of circulating free-
fatty acids and/or lipid accumulation in muscles and liver, is also known to be
directly involved in the inflammatory process leading to insulin resistance [21]. Sat-
urated fatty acids (SFAs) trigger both the expression of TLRs and their activation,
which may contribute together with the microbiota-derived products to the
increased induction of inflammatory cytokines in different tissues, such as adipose
tissue and liver [67]. SFAs activate innate immunity components, such as TLR4 and
2 and the inflammasome, thereby triggering kinase activation (JNK and IKK) and
inflammatory cytokine production, inhibiting insulin signaling and action [68,
69]. A protein called fetuin-A (FetA), which is a major carrier of free-fatty acids
in serum, acts directly as an endogenous ligand of TLR4, thus activating its
signaling pathway, promoting insulin resistance in peripheral tissues [70]. In con-
trast, polyunsaturated free-fatty acids (e.g. Ω-3 fatty acids) can inhibit TLR4
signaling. LPS stimulation also increases cytokine-mediated plasma lipid levels
by increasing VLDL lipoprotein synthesis in the liver and inhibiting lipoprotein
lipase. In fact, mobilization of lipid stores is considered a mechanism to fuel the
host’s response against infections; moreover, lipoproteins also seem to help fight
against infection by binding and neutralizing LPS [71].
It is still unclear which gut microbiota components constitute a source of LPS in
animal models of obesity and in observational human studies. LPS originating from
E. coli is reportedly sufficient to promote glucose and insulin intolerance and
macrophage accumulation in white adipose tissue when mono-colonizing the gut
of germ-free mice [63]. In our own studies, compared with standard-diet-fed mice,
HFD-fed mice showed increased numbers of Enterobacteriaceae, which were
reduced by B. pseudocatenulatum CECT 7765 administration, parallel to amelio-
ration of metabolic dysfunction; however, LPS translocation was not measured
[6]. By contrast, in HFD-fed mice increases in Proteobacteria (which include
enterobacteria) and reductions in Firmicutes and Bacteroidetes by vancomycin

administration have been related to reduced body weight gain, TNF-α production
and metabolic dysfunction [72]. Other animal studies demonstrated that gut
microbiota alterations associated with genetically or HFD-induced obesity do not
involved Gram-negative bacteria, which could contribute to LPS increases [24, 73],
but reductions in Gram-positive bacteria [74]. A couple of recent human studies
support the idea that increased proportions of Proteobacteria are associated with
inflammatory and metabolic disease risk markers [8, 13] while other studies do not
support such an association [7]. This controversy could partly be due to the
influence of confounding factors and differences in methodologies used for
microbiota analyses. Furthermore, gut barrier dysfunction associated with diet-
induced obesity can lead per se to increased LPS translocation without significant
alterations in gut microbial ecology.
Bacterial products may be translocated via different mechanisms, including
transcelluar and paracellular pathways. LPS could translocate via a transcellular
epithelial pathway together with chylomicrons formed to incorporate dietary long-
chain fatty acids in the form of triglycerides, which are finally released into the
mesenteric lymph. This LPS translocation mechanism also requires TLR-4 expres-
sion by epithelial intestinal cells [75]. In blood, LPS-enriched chylomicrons
exchange LPS with other lipoproteins, a process that requires the LPS-binding
protein (LBP) and involves the soluble CD14 receptor, facilitating LPS transport
to different tissues and blood vessels. LPS can also translocate by a transcellular
pathway through intestinal-epithelial microfold cells (M-cells), which are more
permeable and responsible for uptake of bacteria and bacterial antigens by the
underlying lymphoid tissue, with a preference for Gram-negative bacteria
[76]. Murine models of HFD-induced obesity have also demonstrated that live
Gram-negative commensal intestinal bacteria (E. coli) can translocate to the
blood and adipose tissue [77]. This translocation is dependent of innate immunity
pattern-recognition receptors (TLR4 and Nod1) demonstrated by the fact it is
blocked in mice lacking CD14 or Nod1 but increased in Myd88 knockout and
ob/ob mice. This ‘metabolic bacteremia’ is thought to be mediated by DCs and
reversed by administration of Bifidobacterium animalis subsp. lactis 420, which
also improves the animals’ overall inflammatory and metabolic status. This study
also suggests that leptin plays a role in intestinal bacterial adherence and translo-
cation in the intestine since leptin treatment reduces translocation in ob/ob
mice [77].
Alcohol ingestion and HFD, common in obese subjects, can also lead to
increased intestinal permeability, which is reflected in alterations of tight-junction
integrity and related proteins. This might facilitate the translocation of LPS and
other bacterial components by a paracellular pathway [74]. Alterations in gut
microbiota composition could also contribute to increasing paracellular permeabil-
ity via alterations in tight-junctions, which could be secondary to excessive activa-
tion of inflammatory cytokine production (e.g. TNF-α; [78]). Thus, HFD-fed
diabetic mice show alterations in gut bacteria, associated with increased intestinal
permeability, characterized by reduced expression of genes coding for two tight
junction proteins ZO-1 and occludin, while antibiotic-treated mice recover normal
intestinal epithelial integrity. This reveals the specific role of the microbiota, which
seems to be greater than the role of diet in gut permeability [79]. A selective
increase in Bifidobacterium spp. by feeding ob/ob mice with a prebiotic
(oligofructose) also reduces the impact of the HFD-induced metabolic
endotoxaemia, inflammatory tone and metabolic dysfunction and improves intesti-
nal permeability, demonstrating that these effects are partly mediated by gut
microbiota-induced changes [59]. The protective effects of the prebiotic on gut
barrier function could also be explained by the reduction in plasma cytokines,
known to promote tight-junction disruption, including TNFα, IL1β, IL1α, IL6 and
INFγ [59]. These effects could also be attributed to the trophic effect of bacterial
fermentation products (short-chain fatty acids [SCFAs] including butyrate) on the
gut, leading to increased villus height and crypt depth and thickened mucosal layer
[59, 80]. Researchers have also reported that prebiotic-microbiota-induced changes
are associated with increased endogenous production of the glucagon-like peptide-2
(GLP-2), whose production may improve mucosal barrier function by increasing
the rate of crypt cell proliferation and villus elongation, and reduce apoptosis
[59]. In a more recent study, administration of the mucin-degrading bacterium
Akkermansia muciniphila has also been shown to reverse metabolic endotoxemia
and high-fat diet-induced metabolic disorders in mice obesity models, via restora-
tion of gut barrier function and inflammation by increasing the intestinal levels of
endocannabinoids (e.g. 2-arachidonoylglycerol and 2-oleoylglycerol) and mucus
thickness [81].
TLR-2, Lipoteichoic Acids and LPS
TLR2 recognizes lipoteichoic acids (LTA) from Gram-positive bacteria and also
LPS from Gram-negative bacteria, acting synergically with TLR4. Although TLR4-
LPS activation is necessary to trigger an innate immune response, TLR2 partici-
pates in the up-regulation of genes encoding TNF-α and in the connection between
innate and adaptive immunity [82].
In addition, TLR2 can be activated by saturated fatty acids [20]. Thus, TLR2 in
conjunction with TLR4 can synergically contribute to insulin resistance in different
tissues and constitute one of the links between gut microbiota components and
metabolic dysfunction.
The role of TLR2 in metabolic dysfunction was directly evidenced by comparing
effects of HFD on Tlr2(—/—) mice and Tlr2(+/+) mouse controls, showing that
knock-outs were protected from the adverse metabolic effects of diet [83]. Glucose
tolerance, insulin sensitivity, and insulin secretion were markedly improved, par-
ticularly in female Tlr2(—/ —) mice. This was paralleled by increased fat-burning
rates in Tlr2(—/ —) mice as well as reduced tissue inflammation [83]. The specific
role of gut microbiota was shown in studies demonstrating that TLR2-deficient
mice, under germ-free conditions, were protected from HFD-induced insulin
resistance, whereas they were not under conventional conditions. TLR2-deficient

mice conventionally colonized developed metabolic syndrome parallel to a three-
fold increase in Firmicutes and a slight increase in Bacteroidetes, accompanied by
decreased Proteobacteria compared to wild-type controls [84]. This phenotype was
reproduced when microbiota from conventionally reared TLR2-deficient mice was
transplanted to Bacillus-monoassociated wild-type lean mice, and was subse-
quently reversed by antibiotic treatment. These findings prove that gut microbiota
can define a specific phenotype regardless of the predisposing genotype for a
specific condition. Increased LPS plasma levels may induce insulin resistance by
interfering with insulin signaling, as in other models, but insulin resistance in
TLR2-deficient mice has particular characteristics. There was activation of TLR4
in liver, muscles, and adipose tissue, associated with endoplasmic reticulum
(ER) stress and JNK activation, but no activation of the IKKβ-IκB/NFκB pathway,
probably due to lack of TLR4-TLR2 interactions in the knock-outs. While chronic
activation of TLR4 by low doses of LPS is sufficient to increase JNK activation, the
activation of the IKK/IkB/NF-κB pathway may also depend on the interplay of
TLR2 and TLR4 [84].
TLR2 is also involved in regulating intestinal barrier function via modulation of
tight-junctions. TLR2 deficiency leads to barrier dysfunction, reflected in decreased
expression of the tight-junction protein zonula occludens (ZO)-1 in the ileum,
which leads to increased gut permeability and increased LPS translocation and
inflammation even in mice fed standard rodent chow. These effects are paralleled to
Bifidobacterium spp. decreases while their increase leads to reduced gut perme-
ability. Gut microbiota transplantation from TLR2-deficient mice to Bacillus-
monoassociated wild-type mice also reduces ZO-1 expression in the ileum, proving
the role of gut microbe-TLR2 interactions in this phenomenon [84].
TLR5 and Flagellin
TLR5 is expressed in the intestinal mucosa, recognizes flagellin and, upon ligand
binding, induces an inflammatory response with TNFα production, contributing to
defenses against infection. However, TLR5 may protect against metabolic syn-
drome as genetically deficient TLR5 mice exhibit hyperphagia and develop the
main features of metabolic syndrome, including hyperlipidemia, hypertension,
insulin resistance, and increased adiposity [85]. These metabolic dysfunctions
correlated with changes in gut microbiota composition (diversity and phylotypes
related to murine bacteria). Also gut microbiota transferred from TLR5-deficient
mice to wild-type germ-free mice conferred many features of metabolic syndrome
to recipients, demonstrating the role of the microbiota in this particular metabolic
phenotype via interaction with the innate immune system. Metabolic syndrome in
TLR5-deficient mice was exacerbated by a HFD. Food restriction prevented obe-
sity, but not insulin resistance in the TLR5-deficient mice, suggesting that the latter
effect is primarily dependent on TLR5-gut microbiota interactions [85].
TLR9 and DNA
TLR9 recognizes special DNA sequences (unmethylated CpG motifs) and activates
innate immunity. Translocation of bacterial DNA to the blood stream has been
identified in animal models of metabolic dysfunction and also associated with the
onset of diabetes and cardiovascular disease risk in humans [86]. Therefore, TLR9
activation constitutes another possible route by which bacterial components may
contribute to metabolic diseases.
TLR9 is involved in non-alcoholic fatty liver disease, steatohepatitis and fibro-
sis, as shown by comparing wild-type and TLR9-deficient mice. In a nonalcoholic
steatohepatitis murine model, induced by a choline-deficient amino acid-defined
diet, TLR9 signaling induced IL-1β production by Kupffer cells, leading to
steatosis, inflammation, and fibrosis [87]. Steatohepatitis and fibrosis were also
reduced in mice deficient in MyD88, an adaptor molecule for TLR9 and IL-1R
signaling [87]. However, the aforementioned studies did not specifically evaluate
relationships with the gut microbiota.
NOD1/2 and Peptidoglycan
Nucleotide oligomerization domain (NOD) proteins NOD1 and NOD2 are mem-
bers of the NOD-like receptor (NLR) family in mammals. These are cytosolic
pattern recognition receptors, expressed not only in immune but also in metabolic
tissues, which play a role in detecting intracellular microorganisms. These receptors
propagate inflammatory signals in response to bacterial peptidoglycan (PGN).
NOD1 detects D-glutamyl-meso-diaminopimelic acid (meso-DAP)-containing
PGN found principally in Gram-negative bacteria, whereas NOD2 detects muramyl
dipeptide (MDP) present in all bacteria, though more abundant in Gram-positive
bacteria [88]. NOD1 is expressed in all cell types and required for NF-κB activation
by Gram-negative bacterial infection, once the bacteria have bypassed TLR acti-
vation [89]. NOD2 is expressed in monocytes/macrophages and DCs and induced in
intestinal epithelial cells by TNF-α. NOD2 mutations have also been associated
with defective IL-10 production, and Crohn’s disease in humans [90].
PGN levels are lower in the serum of germ-free and antibiotic-treated mice
[91]. Germ-free mice are protected from HFD-induced insulin resistance and anti-
biotic treatment in conventionally colonized mice attenuates the HFD-induced
metabolic dysfunctions. Altogether this suggests that PGN is a potential factor
linking innate immunity and metabolic dysfunction [91]. The fact mice deficient
in NOD1 and NOD2 peptidoglycan receptors are protected from HFD-induced
inflammation and insulin intolerance is evidence of causality. Activation of NOD1
causes acute systemic insulin resistance, as demonstrated in mice injected with
mimetics of meso-diaminopimelic acid-containing PGN or the minimal bioactive
PGN motif, which activate NOD1 and NOD2, respectively. Ex vivo, NOD1 ligand
can cause pro-inflammatory cytokine secretion and impaired insulin-stimulated

glucose uptake in adipocytes and also cause inflammation and insulin resistance
in primary hepatocytes from wild type, but not NOD1( — /—), mice [92]. PGN motifs
acting on NOD2, but not those acting on NOD1, induce muscle cell-autonomous
insulin resistance [91]. NOD1 mediates insulin resistance by acting on adipocytes/
hepatocytes, and NOD2 by acting on myocytes, through mechanisms activating
common pathways such as the MAPKs (p38, JNK, ERK1/2) pathway, expression
and production of proinflammatory cytokines/chemokines, and impairment of insu-
lin signaling at the level of IRS-1. However, we do not know why these metabolic
tissues utilize divergent intracellular innate immune sensors [88]. Overall, it can be
concluded that NOD1-activating PGN causes peripheral insulin resistance, involv-
ing the complex crosstalk between hepatic and adipose tissues, which is indirectly
manifested in skeletal muscles. In contrast, NOD2-activating bacterial PGN motifs
cause a milder insulin resistance that affects skeletal muscle.
NLRP6 and NLRP3 Inflammasomes
Inflammasomes are signaling platforms that sense diverse microbial products as

well as stress and damage-associated endogenous signals. Inflammasome com-
plexes can be formed by members of the NOD-like receptor family or the PYHIN
family AIM2. Upon formation, inflammasomes trigger proteolysis of caspase-1,
which cleaves the cytokine precursors of IL-1β and IL-18 to initiate a pro-
inflammatory and antimicrobial response. Research has linked inflammasome
activation to metabolic disorders, including atherosclerosis, type 2 diabetes, liver
disease and obesity [69].
Inflammasome-deficiency is associated with changes in gut microbiota compo-
sition, parallel to exacerbated hepatic steatosis and inflammation through influx of
TLR4 and TLR9 agonists in the portal circulation, leading to enhanced hepatic
TNF-α expression, which drives disease progression [93]. Co-housing of
inflammasome-deficient mice with wild-type mice, implying microbiota exchanges
by coprophagy, results in exacerbation of hepatic steatosis and obesity in wild-type
mice. These findings demonstrate that defective NLRP3 and NLRP6 inflammasome
sensing alters interactions between the gut microbiota and the host innate immune
system, possibly contributing to metabolic complications [93].
Influence of Gut Microbiota in Macrophage Infiltration

in Peripheral Tissues
Different studies show that gut microbiota, and its modulation by dietary interven-
tion, could influence migration and infiltration of macrophages into peripheral
tissues, this being a major feature of obesity-induced metabolic dysfunction. For
example, in ob/ob mice fed a normal diet, prebiotic administration and the conse-
quent increase in intestinal bifidobacterial numbers, reduced several serum inflam-
matory and anti-inflammatory cytokines (IL-1β, TNF-α, IL-18, and IL-15) as well
as the main chemokine (MCP-1) involved in monocyte/macrophage migration and
infiltration in the adipose tissue [60].
When mice with HFD-induced obesity and gut microbiota imbalances were
administered B. pseudocatenulatum CECT 7765, there was a reduction in serum
inflammatory cytokines and chemokines of the innate immune system (IL-6 and
MCP-1) and a decline in macrophage infiltration in adipose tissue, presumably due
to lowered MCP-1 production [6]. These changes in inflammatory markers were
accompanied by improvements in glucose tolerance and insulin sensitivity in
HFD-fed mice administered B. pseudocatenulatum CECT 7765, as well as partial
restoration of HFD-induced gut microbiota imbalances [94]. These finding reveal
that gut microbiota modulation might help to ameliorate metabolic dysfunction via
regulation of macrophage chemoattractants.
Gut microbiota alterations induced by chronic treatment with olanzapine are also
suspect to be involved in infiltration of macrophages in adipose tissue and meta-
bolic dysfunction associated with the consumption of this antipsychotic. This
hypothesis has been proven by showing that gut microbiota alterations induced
by antibiotic administration (neomycin, metronidazole and polymyxin B) to chron-
ically olanzapine treated female rats reduces metabolic alteration caused by
olanzapine alone, including body weight gain, uterine fat deposition and plasma
free fatty acid levels and macrophage infiltration of adipose tissue [95].
Influence of Gut Microbiota on Adaptive Immunity

Alterations Associated with Obesity and Metabolic
Dysfunction
Fewer studies report the possible influence of the gut microbiota on the adaptive
immune system and its role in the chronic low-grade inflammation associated with
metabolic disorders. However, proof of concept of the role played by gut
microbiota can be found in studies demonstrating the beneficial effects of interven-
tion with specific bacterial strains on adaptive immune function in animal models of
obesity. These beneficial effects seem to be mediated mainly by inducing Tregs,
which express the transcription factor Foxp3 and act by limiting proliferation of
effector CD4+ T cells, which are often critical in regulating intestinal
inflammation [96].
In mice with Western diet-induced obesity (characterized by a CD4(+) Th17-
biased immune profile and changes in microbial communities) the administration of
L. reuteri ATCC 6475 shifted this pro-inflammatory immune cell profile and
prevented abdominal fat pathology and age-associated weight gain [22]. The bac-
terial effects were mediated by induction of Foxp3(+) Tregs and IL-10 in colonic
mesenteric lymph nodes, without significantly influencing gut microbiota compo-

sition. Furthermore, these microbe-related beneficial effects were transferable into
na¨ıve recipients by adoptive transfer of purified L. reuteri-induced CD4(+) Foxp3+
T cells [22].
In mice with HFD-induced obesity, B. pseudocatenulatum CECT 7765 supple-
mentation increased cytokine production of the adaptive immune system, including
the anti-inflammatory cytokine IL-4 [6]. In the context of obesity, this cytokine
together with IL-13 contribute to macrophage differentiation into M2 macrophages,
which secrete the anti-inflammatory cytokine IL-10, thus helping to control inflam-
mation and promote normal insulin sensitivity [97]. IL-4 also mediates Th2 lym-
phocyte differentiation and inhibits production of inflammatory cytokines such as
IL-1β, TNF-α and IL-6.
A. muciniphila, a newly discovered mucus-degrading bacterium of the human
gut, improves glucose tolerance in HFD-fed mice by inducing Foxp3 Tregs in the
white adipose tissue. This effect has been related to reduced gene expression of
pro-inflammatory cytokines (IL-1β and IL-6) but not to changes in M1/M2 or
CD4/CD8 T cell ratios, altered by HFD [39].
A recent study investigating the possible protective effect of H. pylori in diet-
related disorders reported that it favorably modulates glucose metabolism and
suppressed weight gain in db/db mice (lacking the long isoform of the leptin
receptor) and mice with diet-induced obesity, particularly when animals were colo-
nized by a non-pathogenic strain negative for cag PAI (cytotoxin-associated gene
pathogenicity island) [98]. The effects were mediated by up-regulation of gastric
PPAR γ-responsive genes (i.e., CD36 and FABP4) parallel to decreased white
adipose tissue macrophages and increased adipose tissue Tregs, since the effects
were impaired in mice deficient in PPAR γ in immune and epithelial cells [98].
Although the precise mechanisms by which microbiota exerts these Treg induc-
tive effects are unknown, short-chain fatty acids (SCFAs) derived from gut
microbiota fermentative activity are one of the possible actors [96]. SCFAs con-
tribute to regulating the size and function of the colonic Treg pool, specifically
inducing Foxp3+IL-10-producing Tregs but not colonic Foxp3+TGFb+cTregs,
colonic Th17 and Th1 or MLN cell and splenic Tregs [96].
TLR2-deficient mice also have lower Tregs in visceral adipose tissue, suggesting
this pattern-recognition receptor may also contribute to regulate insulin resistance,
an effect that in turn can be influenced by gut microbiota molecules recognized by
this receptor [84].
Influence of Gut Microbiota on Decreased Immunological

Surveillance Associated with Obesity and Metabolic
Dysfunction
There is scarce research into the potential role of gut microbiota in immunological
dysfunction, leading to weakened host responses against infections and vaccination.
Recent studies have demonstrated that when mice with HFD-induced obesity are
fed B. pseudocatenulatum CECT 7765 or Bacteroides uniformis CECT 7771, the

oxidative burst of macrophages, which reflects their role in phagocytosis, is
increased parallel to restoration of HFD-induced microbiota imbalances [6,
94]. Administration of B. pseudocatenulatum CECT 7765 or B. uniformis CECT
7771 to HFD-fed mice also improved the ability of DCs to activate T-lymphocyte
proliferation, a function also adversely affected by HFD-induced obesity in mice [6,
94]. These findings indicate that modifying the gut microbiota may contribute to
restoring host defense mechanisms impaired by diet-induced obesity in mice.
Studies of rodents with genetic deficiency in leptin or leptin receptors, reveal
obesity-related deficits in macrophage phagocytosis via alterations in phospholi-
pase activation and reduced pro-inflammatory cytokine secretion (e.g. TNF-α and
IL-6) in vivo and in vitro. These effects may be due to leptin deficiency as
exogenous leptin up-regulated both phagocytosis and proinflammatory cytokine
production by macrophages [51]. In leptin-deficient models of obesity (ob/ob
mice), DCs and their role in T cell priming is also adversely affected. DCs from
ob/ob mice are less able to activate allogenic T cells in vitro. The impaired
functionality of DCs may be related to increased secretion of the immunosuppres-
sive cytokine TGF-β, rather than to changes in expression of activation markers,
which could be due to the absence of leptin in ob/ob mice [53]. Leptin can improve
DCs functions and survival, driving na¨ıve T cell polarization toward a Th1 type
phenotype by activating NF-κB and exerting an antiapoptotic effect via up-
regulation of gene expression (bcl-2 and bcl-xL). These results demonstrate the
ability of leptin to improve DCs and T cells functions and to promote DC survival
[54], which could be important in defense against pathogens and in
response to vaccination. Decreased leptin plasma concentration in food-deprived
animals or malnourished humans impairs immune functions similarly to those
detected in leptin-deficient mice.
Similar immune defense mechanism dysfunctions have been demonstrated in a
murine model of HFD-induced obesity, the most common form of obesity charac-
terized by hyperleptinemia, presumably due to leptin resistance and, therefore, lack
of adequate leptin functionality [6, 94]. Alterations in macrophage, DCs and T-cell
function, identified in both models of obesity (genetically or diet-induced), may
also be related to obesity-associated alterations in glucose uptake and metabolism,
possibly affecting immune cells which are sensitive to insulin because glucose is
their major energy source [20].
Conclusions and Future Perspectives
Scientific evidence supports a role of gut microbiota in immunological dysfunctions

associated with obesity and metabolic disease, including intestinal and systemic
chronic low-grade inflammation, and diminished responses against infections and
vaccination. The interdependency of diet and gut microbiota is evident in that diet
constitutes a major factor influencing gut microbiota structure and function.
Moreover, both dietary lipids and gut microbes can exacerbate inflammation by
activating similar pattern-recognition receptors and signaling pathways of the
innate immune system. Furthermore, it has been evidenced that intestinal inflam-
mation is an early event preceding obesity and metabolic disease and the fact that
this can be altered by dietary-modulation of the gut microbiota paves the way for
novel preventive dietary intervention strategies, designed to combat these disor-
ders. In this context, it is essential to identify the exact immunological processes
that are sensitive to gut microbiota interactions within a specific dietary context and
to gain a better understanding of the role gut microbiota plays in early responses
particularly of the adaptive immune system to high calorie diets.
Acknowledgements This work was supported by grants AGL2011-25169 and Consolider Fun-
C-Food CSD2007-00063 from the Spanish Ministry of Economy and Competitiveness (MINECO,
Spain). The scholarship of A Moya from MINECO is fully acknowledged.
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Chapter 15
Microbiota, Immunoregulatory Old Friends
and Psychiatric Disorders
Graham A.W. Rook, Charles L. Raison, and Christopher A. Lowry
Abstract Regulation of the immune system is an important function of the gut

microbiota. Increasing evidence suggests that modern living conditions cause the gut
microbiota to deviate from the form it took during human evolution. Contributing
factors include loss of helminth infections, encountering less microbial biodiversity,
and modulation of the microbiota composition by diet and antibiotic use. Thus the
gut microbiota is a major mediator of the hygiene hypothesis (or as we prefer,
“Old Friends” mechanism), which describes the role of organisms with which we
co-evolved, and that needed to be tolerated, as crucial inducers of immuno-
regulation. At least partly as a consequence of reduced exposure to immuno-
regulatory Old Friends, many but not all of which resided in the gut, high-income
countries are undergoing large increases in a wide range of chronic inflammatory
disorders including allergies, autoimmunity and inflammatory bowel diseases.
Depression, anxiety and reduced stress resilience are comorbid with these condi-
tions, or can occur in individuals with persistently raised circulating levels of bio-
markers of inflammation in the absence of clinically apparent peripheral
inflammatory disease. Moreover poorly regulated inflammation during pregnancy
might contribute to brain developmental abnormalities that underlie some cases of
autism spectrum disorders and schizophrenia. In this chapter we explain how the gut
microbiota drives immunoregulation, how faulty immunoregulation and inflam-
mation predispose to psychiatric disease, and how psychological stress drives further
G.A.W. Rook (*)

Centre for Clinical Microbiology, UCL (University College London), Royal Free Campus,
Rowland Hill Street, London NW3 2PF, UK
e-mail: g.rook@ucl.ac.uk
C.L. Raison
Department of Psychiatry, College of Medicine and Norton School of Family and Consumer
Sciences, College of Agriculture and Life Sciences, University of Arizona, Tucson, AZ, USA
C.A. Lowry
Department of Integrative Physiology and Center for Neuroscience, University of Colorado
Boulder, Boulder, CO, USA
320 G.A.W. Rook et al.
inflammation via pathways that involve the gut and microbiota. We also outline how
this two-way relationship between the brain and inflammation implicates the
microbiota, Old Friends and immunoregulation in the control of stress resilience.
Abbreviations
ASD Autism spectrum disorders

BH4 Tetrahydrobiopterin
CD Crohn’s disease
CRH Corticotropin-releasing hormone
CRP C-reactive protein
dACC Dorsal anterior cingulate cortex
DC Dendritic cells
DCreg Regulatory dendritic cells
GABA g-Aminobutyric acid
GCR Glucocorticoid resistance
HPA Hypothalamic-pituitary adrenal axis
IDO Indoleamine-2,3-dioxygenase
IFN-α Interferon-alpha
IL Interleukin
MCP-1 Monocyte chemoattractant protein-1
MS Multiple sclerosis
NO Nitric oxide
Nod1 Nucleotide-binding oligomerization domain-containing protein-1
PBMCs Peripheral blood monocyte cells
PET Positron emission tomography
SCFA Short chain fatty acids
SLE Systemic lupus erythematosus
SNP Single nucleotide polymorphisms
SNS Sympathetic nervous system
SSRI Selective serotonin reuptake inhibitors
T1D Type 1 diabetes
Treg Regulatory T cells
UC Ulcerative colitis
XLAAD X-linked autoimmunity-allergic dysregulation syndrome
15 Microbiota, Immunoregulatory Old Friends and Psychiatric Disorders 321
Introduction
This chapter concentrates on those gut-brain interactions that operate indirectly via
the immune system, rather than via direct neural pathways. At least two sets of
findings underlie this aspect of gut-brain interaction. First, we know that persis-
tently raised levels of inflammatory mediators are associated with several psychi-
atric conditions. This will be discussed with particular reference to depression and
to reduced stress resilience. The regulation of background levels of inflammation is
dependent upon “learning” inputs to the immune system from appropriate microbial
exposures during the prenatal and neonatal periods, and continuing diversity of
input in later life. This concept, initially called the “hygiene hypothesis” is becom-
ing renamed the “Old Friends” mechanism, which places it firmly within the field of
Darwinian and evolutionary medicine. The various mammalian microbiotas, parti-
cularly the gut microbiota, are important components of the Old Friends mecha-
nism, and have a continuing immunoregulatory role in the adult. Secondly, we
know that inflammation during pregnancy can lead to abnormal development of the
central nervous system (CNS). This will be illustrated by considering autism
spectrum disorders (ASD) and schizophrenia, and aspects of epidemiology that
suggest the importance of microbe-dependent immunoregulatory effects during
pregnancy.
The expression “Hygiene hypothesis” was first published in 1989, following the
observation that, when examined at 11 years old, children brought up in families
with many older siblings were less likely to have developed allergic disorders. This
concept was at first a narrow one, focusing on the notion that childhood infections
somehow prevented subsequent allergies. In fact it had been known since the
nineteenth century that the environment could modulate the likelihood of develop-
ing hay fever, which was increasing amongst wealthy townsfolk, while remaining
rare amongst farmers [1]. This protective effect of the farming environment in
allergic disorders has subsequently been rigorously confirmed and found to extend
to juvenile-onset inflammatory bowel disease (IBD) as well [2]. Moreover to reap
protection from these immune-mediated diseases it can be sufficient to expose the
pregnant mother to the farming environment, rather than the infant itself [3]. The
most recent observations indicate that the farm effect is mostly explained by
exposure to increased microbial biodiversity, which was documented by analyzing
the bacterial and fungal taxa present in the dust in children’s bedrooms [4].
Meanwhile sporadic observations in other branches of medicine have confirmed
that allergic disorders are not the only chronic inflammatory conditions that have
been increasing in high-income countries, particularly in urban populations. Inflam-
matory bowel diseases and autoimmune diseases have increased at about the same
rate, and in the same places [5, 6] as allergic conditions. Subsequently large
epidemiological surveys have shown that childhood infections, originally impli-
cated as the protective mechanism behind the hygiene hypothesis, do not protect
against allergic disorders, and may in some cases, such as human rhinoviruses and
respiratory syncytial viruses, actually trigger allergic responses. Considered
together, these observations led to a Darwinian reformulation of the hypothesis as

the Old Friends mechanism, described in the next section.
Old Friends Mechanism
The Old Friends mechanism states that mammals co-evolved with various
microbiotas and commensals (gut, skin, lung etc.), as well as with chronic infec-
tious agents picked up at birth, helminths that persisted for life, and environmental
organisms from animals, mud and untreated water with which we were in daily
contact. Because all of these categories of organism needed to be tolerated, they
took on a role of inducers of immunoregulatory circuits [7, 8]. For example,
helminthic parasites need to be tolerated because, although not always harmless,
once they are established in the host, efforts by the immune system to eliminate
them are typically futile, and merely cause tissue damage [9].
Contact with the “Old Friends” rapidly diminished when industrialization
occurred, and mankind started to inhabit a plastic and concrete environment, to
consume washed food and chlorine-treated water, and to minimize our contact with
mud, animals and feces. This withdrawal of the organisms that drive immuno-
regulatory circuits results in defective immunoregulation that, depending on the
genetic background of any given individual, can manifest itself as a variety of
chronic inflammatory disorders, including allergies, IBD and autoimmunity. We
know that a failure of immunoregulatory mechanisms really can lead to simul-
taneous increases in diverse types of pathology. For example, defects in the gene
encoding the immunoregulatory transcription factor Foxp3 lead to the X-linked
autoimmunity-allergic dysregulation syndrome (XLAAD) that includes aspects of
allergy, autoimmunity and enteropathy [10].
The underlying Darwinian principle of the Old Friends mechanism is illustrated
in Fig. 15.1. The immune system at birth is analogous to a computer with hardware,
some software, but very little data. The minimal data that it does have comes from T
lymphocyte selection in the thymus, and probably from transfer of at least some
environmental and maternal antigenic material across the placenta. After birth the
immune system requires the largest possible exposure to environmental microbial
biodiversity in order to build a very broad repertoire of potential effector lympho-
cytes. Since all life forms are ultimately constructed with similar building blocks,
such diversity of “education” can even provide the system with T cells that
recognize, for example, some obscure viral pathogen that might be encountered
in the future [11].
However in the context of this chapter, still more important than the diverse
effector repertoire is the setting up of appropriate immunoregulation. Just as
exposure early in life to a wide range of microbial and parasitic organisms trains
the immune system regarding what to be on guard against, it also teaches immunity
what to profitably ignore because the organisms in question either confer some
benefit to the host, or confer no danger or despite posing some risk are not easily
Fig. 15.1 The immune system requires “educational” input. The microbiota of others, tolerated
organisms (such as helminths) with which we co-evolved and organisms from the natural envi-
ronment are required to expand the effector and regulatory branches of the immune system. During
subsequent encounters with pathogens, danger signals generated by tissue damage enhance
effector mechanisms and attenuate regulatory pathways to permit an appropriate immune
response. Adequate background levels of regulatory T cells and dendritic cells and other regula-
tory mechanisms are required to maintain suppression of responses to “forbidden targets” and to
switch off inflammation completely when the danger is eliminated, so that proinflammatory
mediators do not continue to circulate
eradicated by immune mechanisms once established. These immunoregulatory

inputs benefit the host by teaching the immune system not to waste precious energy
engaging in futile battles, by reducing the cost to the host of chronic inflammation
and by reducing the risk of destruction of host tissues, either through bystander
effects or via the induction of autoimmunity. Because humans in traditional envi-
ronments were exposed to organisms that dampened, as well as stimulated, immune
function, the Old Friends mechanism implies that inflammation should be better
regulated in low-income than in high-income countries. At first sight this might
seem paradoxical, because the high prevalence of infections in low-income coun-
tries might be expected to cause high levels of inflammation [12]. However recent
work by McDade et al. [13, discussed in 14] has largely resolved this paradox. The
results reveal that in a low-income country where there is still abundant exposure to
the immunoregulation-inducing “Old Friends”, immunoregulation is efficient, and
the inflammatory response is vigorous during an infection, but is terminated when
no longer needed, with the result that “resting” C-reactive protein (CRP) is close to
zero. These longitudinal results illuminate a previous finding by McDade et al. [15]
that high levels of microbial exposure in the perinatal period and in infancy
correlated with low levels of “resting” CRP in adulthood. In contrast, in the USA
and other high-income countries there is often constant low-grade inflammation
which tends to be stable across individuals, manifested as chronically raised CRP or

interleukin (IL)-6, in the absence of any clinically apparent inflammatory stimulus.
Such chronically elevated inflammation greatly increases the risk of subsequent
inflammatory disease and cardiovascular problems and has been shown in some
studies to predict the future development of depression [16].
Inflammation and Psychiatric Disorders
Inflammation is involved not only in chronic inflammatory disorders such as

allergies, autoimmunity and IBD but also in many psychiatric disorders. We have
reviewed this topic in detail elsewhere [17, 18]. Briefly, a large subset of depressed
individuals has persistently raised levels of proinflammatory cytokines and other
downstream inflammatory markers [19, 20], together with a relative deficit in anti-
inflammatory mediators and regulatory T cells [fully referenced in 18, 21]. Inter-
estingly, depressed individuals also show exaggerated release of inflammatory
mediators in response to psychosocial stressors [22], implying altered immuno-
regulation (Fig. 15.2), and epidemiological studies in the United Kingdom
(UK) showed that raised CRP and IL-6 predicts subsequent risk of depression
assessed over a decade later [16].
The possibility that inflammatory mediators might play direct causal roles
in depressive pathogenesis has been confirmed for interferon-alpha (IFN-α),
interleukin 6 (IL-6), and tumor necrosis factor (TNF). When IFN-α is used thera-
peutically (to treat viral hepatitis or some cancers) it causes depression-like symp-
toms in a high percentage of patients. These symptoms have been repeatedly shown
to respond to treatment with standard antidepressants, such as selective serotonin
reuptake inhibitors (SSRI) [20, 23]. Similarly the cytokine antagonist infliximab,
which blocks TNF actions, has been shown to have antidepressant properties, but
only in depressed individuals with evidence of increased peripheral inflammation
prior to treatment [24].
Finally, when IL-6 is administered to pregnant animals it causes abnormal brain
development in the fetus, discussed later in relation to autism [25]. Similarly
increased peripheral levels of IL-6 cause increased production of IL-6 in the
CNS, and affect neurogenesis in the hippocampus [reviewed in 26]. That IL-6 is
directly relevant to the changes seen is supported by the fact that these effects can
be blocked by IL-6 receptor antagonists, and knockout mice with non-functional
IL-6 genes have enhanced working memory compared to wild type mice [27] and
are refractory to peripheral inflammation-induced impairments of spatial memory
[28]. In humans raised levels of IL-6 are associated with diminished cognitive
performance and reduced hippocampal gray matter [26, 29]. Mechanisms for these
effects are discussed in a later section.
Fig. 15.2 Exaggerated and prolonged cytokine release in response to a psychosocial stressor in
individuals with diminished immunoregulation. Populations that have poorly immunoregulatory
gut microbiota and reduced exposure to immunoregulation-inducing “Old Friends” such as
helminths are susceptible to excessive and prolonged cytokine release in response to psychosocial
stressors, which may result in reduced stress resilience and inappropriate triggering of depressive
episodes. Reprinted from Rook et al. (2013) Evolution, Medicine and Pubic Health (1): 46–64,
doi: 10.1093/emph/eot004, by permission of Oxford University Press and the Foundation for
Evolution, Medicine, and Public Health
Immunoregulation and Stress Resilience in Developing

Countries
The vicious cycle described in Fig. 15.2, considered against the background of the
Old Friends mechanism, suggests that in developing countries there will be less
release of inflammatory mediators in response to psychosocial stressors, and less
psychiatric consequences of such stressors. Recent data support this hypothesis. In
experimental animals parental deprivation is a potent inducer of long-term changes
to stress responses and immunoregulation [30]. Human studies suggest similar
correlations [31]. However in a recent study performed in a developing country,
parental absence in childhood was a significant predictor of raised CRP in adult-
hood, as it would be in a rich country, but only in a subset of the cohort raised in
hygienic environments [32]. However, adults who had a high level of microbial
exposure in infancy were resistant to the long-term proinflammatory effects of this
severe childhood stressor [32]. The same was true of perceived stress during the
previous month in young adults. CRP correlated with recent perceived stress in
subjects with low microbial exposure in infancy, but not in those with high
microbial exposure. Again, exposure to immunoregulation-inducing “Old Friends”
seemed to provide resistance to the inflammation-inducing effects of psychosocial
stressors [32].
This leads to an obvious question. If psychosocial stressors cause depression at

least partly by triggering the release of proinflammatory mediators (Fig. 15.2), are
inhabitants of developing countries resistant to psychosocial stress-induced depres-
sion? If so the prevalence of depression should be increasing in developed countries
in parallel with the chronic inflammatory disorders [33], and lower in developing
countries than in developed ones. Comparative studies are difficult to do, but this is
indeed what data collected by the World Health Organization indicate [34]. More-
over one study failed to find a correlation between depression and raised CRP in a
developing country whereas this association is routinely found in rich ones [35].
Urbanization and Immigration to High-Income Countries
If a dysregulated immune system resulting from diminished contact with immuno-

regulation-inducing “Old Friends” is partly to blame for the increasing prevalence
not only of chronic inflammatory disorders such as allergies, autoimmunity and
IBD, but also of those psychiatric disorders that can be triggered by inflammatory
mediators, then it should be useful to examine urban-rural differences in disease
prevalence, and the effect of migration from low-income to rich urban environ-
ments. In each case there will be loss of exposure to Old Friends.
Urban Versus Rural
A feature shared by most of the disorders discussed here is a higher prevalence in

urban communities compared to rural ones. For example a meta-analysis of high
quality studies performed in high-income countries since 1985 found that the
prevalence of depression in urban areas was 39 % higher than in rural areas.
Similarly, the prevalence of anxiety disorders was 21 % higher in urban than in
rural areas [36], though a small minority of studies fails to find this urban-rural
difference [37]. Peen et al. [36] also noted an increased urban prevalence of
psychiatric disorders in general (38 % more in urban communities). This agrees
well with another large meta-analysis that found a significantly raised prevalence of
schizophrenia in urban communities [38]. Similarly, a study of all children born in
Denmark between 1 January 1984 and 31 December 1998 found that the degree of
urbanization of place of birth was very significantly correlated to risk of
autism [39].
The urban > rural phenomenon is also well established for chronic inflammatory
disorders, where the etiology is known to involve dysregulation of the immune
system. Contact with the farming environment, whether early postnatal [40] or
prenatal [3, 41] protects against allergic disorders, whereas the prevalence of these
conditions increases with increasing urbanization [42]. The same is true for IBD
[43], and for autoimmune diseases such as multiple sclerosis (MS) [44, 45,
discussed in 46].
Immigrants
Another striking parallel between chronic inflammatory diseases and psychiatric

disorders concerns the effects that immigration has on these conditions. All the
diseases discussed here, whether chronic inflammatory [43, 47–49] or psychiatric
[50–52], tend to be more common in immigrants than in the birth population from
which the immigrant was derived, at least when the migration is from a developing
to a high-income country. Other relevant variables include the age of the individual
at the time of immigration, and whether the prevalence increases in second gene-
ration immigrants, born in the adopted country. A study of these parameters pro-
vides some insight into whether the relevant influences, be they psychosocial or
immunological, need to occur before birth, or in early childhood, or whether they
can still exert their effects on adults.
Immigration and Psychiatric Disorders
Depression is particularly interesting in this respect [53, 54]. Mexicans, Cubans and
African/Caribbean peoples were found to have a two to threefold increase in the
prevalence of depression if immigration to the USA occurred when the individual
was less than 13 years old, or was born in the USA, compared to the prevalence in
those who migrated after the age of 13 [53]. But this is not likely due to psycho-
social stress related to skin color, because white Eastern European immigrants show
the same effect. In sharp contrast, the effect is not seen in immigrants from Western
Europe, or from Puerto Rico, which is closely associated with the USA. (These last
two populations already have a high prevalence of depression that is not increased
by immigrating to, or being born in, the USA) [53]. These findings imply that
influences important for depression occur perinatally, or in the early years of life.
The same is true for psychotic disorders [55]. A large Danish study noted that
immigration into Denmark when less than 4 years old was associated with a
strikingly increased risk for psychotic disorders, whereas the increased risk gradu-
ally decreased with older age at migration and disappeared in those immigrating
when more than 29 years old [56]. Similarly a large meta-analysis confirmed that
schizophrenia was increased amongst first generation immigrants, and further
increased amongst second generation immigrants, particularly when the country
of origin was a developing one [57]. Again, early events seem crucial.
Age at immigration is irrelevant to an early onset condition such as autism, but
autism is strikingly (as much as tenfold) increased in second generation Caribbean
or African immigrants born in the UK, compared to children of white UK-born
mothers [52]. These findings implicate crucial early events in the perinatal period
or early childhood as risk factors for depression, schizophrenia and autism.
Immigration and Chronic Inflammatory Disorders
Migration has clear effects on the prevalence of MS, and the crucial events that
confer increased risk for the disease occur very early in life, as is true for the
psychiatric disorders [reviewed and referenced in 58, 59]. Iranians who migrate to
Sweden have twice the prevalence of MS seen in their birth country [49]. Interest-
ingly, if the second (or later) generation immigrants return to their developing
country of origin, they retain their increased susceptibility to MS, which remains
higher than in the local population that was not born abroad [60]. A similar
phenomenon was seen when people born in the UK (a high MS country) migrated
to South Africa (SA: a low MS country). Migration from the UK to SA was
protective when the migrant was a child, whereas adult migrants retained their
high UK prevalence of MS [61]. Analysis of this and other studies suggests that the
environmental factors that protect from or predispose to MS act during the first two
decades of life [58, 59]. The same is true for type 1 diabetes (T1D). Here the crucial
factor is to have been born in the receiving developed country, again suggesting that
relevant environmental factors act very early, or even in the prenatal period [48].
The role of migration in conferring risk for allergic disorders has been inten-
sively examined. A study of children adopted into Sweden from developing coun-
tries showed that the prevalence of asthma, hay fever and eczema were highest in
those adopted when less than 2 years old [62]. Similarly, for Mexican immigrants to
the USA, the prevalence of asthma was highest for those born in the USA, while in
those not born in the USA, the prevalence of asthma decreased as the age at
immigration increased [63]. This effect of age at the time of childhood immigration
was also seen in immigrants to Israel from the former Soviet Union or Ethiopia who
were assessed when 17 years old [64]. These observations suggest the importance
of early environmental influences for allergy/asthma risk, a conclusion that is
powerfully supported by evidence that prenatal exposure (i.e. of the pregnant
mother) to the farming environment protects the infant against some allergic
manifestations [3, 41]. This is discussed later in another context.
Finally, a definitive study of all first- and second-generation immigrants in
Sweden between January 1, 1964, and December 31, 2007 showed that some first
generation immigrants remain partially protected from both ulcerative colitis
(UC) and Crohn’s disease (CD), presumably by environmental factors encountered
in their countries of origin, but the diseases increased in prevalence in second
generation immigrants, relative to first generation immigrants [65]. Similarly, the
prevalence of UC in South Asian immigrants to Leicester in the UK was higher in
second than in first generation immigrants [66]. This again implicates perinatal
factors as potentially causative of this migration effect.
Thus the influence of immigration, acting via factors that occur perinatally or
very early in life, is equally consistent and highly apparent for both psychiatric and
chronic inflammatory disorders.
Mechanisms of Immunoregulation by Old Friends
Urbanization and immigration from low- to high-income countries cause dimi-

nished contact with Old Friends, and correlate with an increased incidence of
chronic inflammatory disorders, all of which show evidence of failed immuno-
regulation [reviewed in 67]. Moreover adverse outcomes in animal models of all of
these chronic inflammatory conditions can be prevented or treated with “Old
Friends” such as helminths, certain gut commensals or probiotics that induce
immunoregulation [68–70]. What are the mechanisms that enable the “Old Friends”
to exert immunoregulatory effects? This is a vast topic, and here we outline some of
its most studied aspects, particularly those that involve, or occur in, the gut.
Regulation of Innate Immunity
The gut microbiota has been shown to be necessary for priming of innate immunity,
measured as the microbicidal activity of splenic macrophages [71]. This micro-
bicidal activity was increased following a social disruption stressor. However, if the
mice were germ-free no increase in microbicidal activity was seen [71]. Moreover
depletion of microbiota with antibiotics attenuated the stressor-induced macro-
phage activation, and reduced stressor-induced increases in circulating bacterial
cell wall peptidoglycan [71], and eliminated the increases in circulating IL-6 and
monocyte chemoattractant protein-1 (MCP-1) usually seen in stressor-exposed
mice [72]. These observations were in agreement with an earlier finding that
systemic activation of the innate immune system by the gut microbiota involves
recognition of meso-diaminopimelic acid (mesoDAP)-containing peptidoglycan
found predominantly in Gram-negative bacteria, by the pattern recognition receptor
nucleotide-binding, oligomerization domain-containing protein-1 (Nod1) [73].
Similarly abdominal surgery causes systemic release of Nod2-binding bacterial
components and consequent rises in several inflammatory biomarkers [74].
Regulatory Macrophages
Regulatory microorganisms can also operate via macrophages. During helminth

infections there is expansion of the population of alternatively-activated macro-
phages, activated by Th2 rather than Th1 cytokines [75]. Such macrophages secrete
IL-10 and TGF-β rather than IL-12, are able to inhibit lymphocyte proliferation in a
contact-dependent manner [76, 77], and may be responsible for preventing inflam-
mation in mucosal surfaces such as the lung. However, some helminths drive types
of regulatory macrophages that are distinct from alternatively activated macro-
phages [77]. Several species of filarial nematodes secrete cystatin, a cysteine
protease inhibitor that induces macrophages to make IL-10 and IL-12 p40 through
activation of intracellular signaling pathways. These can prevent allergic sensiti-
zation and airway hyperresponsiveness [78]. Similarly a colon-infiltrating macro-
phage population induced by Schistosoma infection was shown to prevent colitis in
mice [79]. The protection was independent of T cells in general and regulatory T
cells (Treg) in particular.
Regulatory B Cells
Helminths also induce regulatory B cells. S. mansoni infection prevented anaphy-

laxis in a mouse model, and this suppression of the effector phase of the allergic
response was mediated by IL-10-secreting B cells [80]. These IL-10-secreting
CD1dhiCD5+ regulatory B cells can also suppress experimental autoimmune
encephalomyelitis [81], and have recently been designated B10 cells [82]. They
act in part by increasing the number of pulmonary CD4+CD25+Foxp3+ regulatory
T cells in the lungs [83]. IL-10+ regulatory B cells are also found in humans
[84]. Depletion of human B-cells using rituximab can occasionally exacerbate
Th1-mediated conditions, suggesting that the rituximab removed a B-cell-mediated
regulatory mechanism [84]. IL-10 production by B cells is increased in multiple
sclerosis patients developing intestinal helminth infections [85], but not in patients
infected by Trypanosoma cruzi [85]. The helminths also increase circulating Treg,
as discussed below.
Regulatory Dendritic Cells (DC)
A particularly important immunoregulatory function is the generation of regulatory

dendritic cells (DCreg) that tend to drive regulatory rather than inflammatory
responses. This is crucial because such DCreg can process gut contents,
autoantigens and allergens, and so downregulate responses to the target antigens
of the major groups of chronic inflammatory disease. It is likely that health requires
the presence of a certain background proportion of DCreg. This could be regarded
as a “Treg adjuvant” function. A mixture of several putative probiotic organisms
(VSL#3; four lactobacilli, three bifidobacteria, and one streptococcal strains) was
found to ameliorate recurrent Th1-mediated murine colitis by inducing IL-10 and
TGF-β+Treg [86]. In vitro this preparation caused human DC to release more IL-10
and inhibited their ability to drive Th1 cells [87]. Other probiotic strains [88, 89],
and a ubiquitous environmental saprophyte often present in untreated or muddy

water [90] also modulate human DC function in vitro so as to induce T cell
responses with a more regulatory bias. In the gut some of these DCreg express
the integrin alpha chain CD103 and have unique immunoregulatory properties
[91]. CD103 is involved in de novo conversion of Foxp3-CD4+ cells to Foxp3+
Treg cells [92]. Conversion of DC to this tolerogenic phenotype is driven locally by
TGF-β and retinoic acid (RA). CD103+ DCs express aldh1a2, the gene encoding
RALDH2. This enzyme is involved in conversion of dietary retinal to RA, which
enhances development of FoxP3+ T cells rather than Th17 cells [reviewed in
93]. Some probiotic Lactobacillus strains, such as L. plantarum WCFS1 induce
migration of these CD103+ DCreg as far as the spleen, and bias the response
towards Treg [94].
An example of a Treg adjuvant effect that must at some stage involve DC is seen
when MS patients become infected with helminths. The disease stops progressing,
and circulating Treg appear in the peripheral blood [95, 96]. These Treg recognize
the major epitope from myelin basic protein. Thus the immunoregulation caused by
the helminths is not merely a bystander effect of IL-10 release, but rather a genuine
Treg adjuvant effect that generates regulation specific for the autoantigen. Although
the mechanism is not elucidated this is an exciting observation that has led to formal
clinical trials [97].
Regulatory T Cells (Treg)
Ultimately many of these immunoregulatory mechanisms result in a relative

increase in the numbers of Treg, whether secondary to changes in macrophages,
B cells or DC. However some Old Friends release molecules that specifically
expand Treg populations. The gut commensal Bacteroides fragilis releases a
polysaccharide antigen that drives expansion of Treg via TLR-2 [98]. The helminth
Heligmosomoides polygyrus drives Treg expansion via the TGF-β receptor
[99]. Treatment with oral Lactobacillus reuteri for 9 days significantly increased
the percentage and total number of CD4+CD25+Foxp3+ T cells in the spleens of
experimental animals [70]. Colonization of mice by commensal Clostridium strains
increased TGF—β levels and numbers of Foxp3+ Treg in the colon [100].
Gut Microbiota Diversity and Regulation of Inflammation
Interestingly the diversity of the gut microbiota appears to have consequences for
immunoregulation, perhaps for the reasons discussed in relation to Fig. 15.1. From
birth our microbiota are constituted by colonization with organisms from our
mothers, from other social contacts [101, 102], and from the environment, and
then further modified by factors such as diet and antibiotics [103–106]. Thus
lifestyle has major effects on an individual’s microbiota and on its diversity. The
gut microbiota of children from traditional villages in Burkina Faso is totally
different from that of Europeans, and shows greater diversity [104]. There is
abundant evidence that diversity of gut microbiota is associated with wellbeing.
Mice exhibit at least two enterotypes (bacterial ecosystems in the gut microbiota),
one of which has low biodiversity, and correlates with biomarkers of inflammation
[107]. In humans reduced biodiversity of the gut microbiota has been associated
with a range of inflammatory states, including allergic disorders [108], inflamma-
tory bowel diseases [109–111] and obesity [112]. Loss of diversity in later life is
associated with increases in circulating CRP and IL-6 levels [113]. This implies that
diversity is associated with effective immunoregulation.
In agreement with this, allergies are less common in children exposed to sources
of microbial biodiversity such as farms [40], dogs [114], or the natural environment
[115, 116]. Similarly, allergies are reduced where there is evidence of social
interactions that promote exchange of microbiota. Indicators of such exchange
include infection with orofecally transmitted organisms such as enteroviruses
[117], H. pylori, T. gondii, and hepatitis A virus [118]. There is some evidence
that these orofecally transmitted pathogens are themselves immunoregulatory.
However it might be much more important that these organisms are markers of
transfer of microbiota between individuals. Interestingly mothers who clean their
baby’s dummy/pacifier by sucking it rather than by sterilizing it have children with
less allergic problems [119].
In sharp contrast, lifestyle events that are likely to restrict the diversity of gut
microbiota are associated with increased risk of chronic inflammatory disorders.
Birth by caesarian section may be a risk factor for allergic disorders [120, 121].
Similarly, excessive antibiotic use during pregnancy [122] or in early childhood is a
risk factor for allergic disorders [123, 124] and IBD [125, 126]. And as discussed
above, living in a high-income rather than in a low-income country is a risk factor
for all of these disorders.
Organisms from the Natural Environment and Microbiota
Clearly microbiota from other people (and animals) can colonize our guts. But do
organisms from the natural environment also colonize, or are these organisms
“pseudocommensals” that impinge on the skin [116], airways and gut, and have
independent immunoregulatory properties? Both mechanisms probably occur,
though there are rather limited data on these issues. An interesting animal experi-
ment compared piglets that were housed in a natural outdoor environment, with
genetically similar piglets that had been reared in a very clean indoor facility.
Firmicutes, in particular Lactobacillus strains were dominant in the gut microbiotas
of the outdoor piglets, whereas the hygienic indoor piglets had reduced Lacto-
bacillus and more potentially pathogenic phylotypes [127]. The indoor piglets also
had less diverse gut microbiota, and a more inflammatory pattern of gene
Fig. 15.3 Environmental organisms and immunoregulation. Microbial biodiversity from the
environment can modulate immunoregulation by (D) directly interacting with the immune system,
or (A, B, C) by leading secondarily to altered microbiota. The environmental organism may cause
secondary changes to the microbiota by (A) colonizing, or (B) antagonizing or competing with
established microbiota or (C) modulating the host immune system-microbiota relationship
expression in ileal biopsies [127]. For example they had increased Type 1 interferon
activity, increased MHC Class 1, and upregulation of many chemokines [127],
again confirming the correlation between reduced gut microbial biodiversity and
poor control of inflammation discussed above.
Were these effects due to direct colonization by immunoregulation-inducing
organisms from the outdoor environment [pathway (A) in Fig. 15.3], or did these
organisms fail to colonize, but exert indirect effects on the immune system? The
answer is unclear, but indirect effects certainly can occur in several ways. Some
organisms compete with, or antagonize established organisms [pathway (B)] and so
alter the microbiota [128]. Others alter the immune system directly [pathway (D)],
or modulate the immune system in ways that lead secondarily to a change in the
host-microbiota relationship, which in turn leads to changes in the microbiota
[pathway (C) in Fig. 15.3].
The last mechanism is well established in experimental models. Genetic manipu-
lations of the innate immune system that have profound effects on immune function
(such as gene knockout) often operate indirectly by altering the gut microbiota. The
phenotypic effects can then be transferred to wild-type mice that have not been
genetically modified, by transferring the altered microbiota [129, 130]. It is the
altered microbiota that is the proximate cause of the altered immunoregulation
[129–133].
Is the Gut Still Involved in Immunoregulation by Organisms

That Do Not Enter the Gut?
Does this mean that all immunoregulation by “Old Friends” operates indirectly by
modulating the immune system, and so secondarily causing changes in the gut
microbiota? It is likely that such indirect effects occur, but there could be direct
effects too. For example the skin microbiota has at least some immunoregulatory
role independent of the gut [134]. Indeed components of the skin microbiota extend
into the subepidermal compartments, suggesting subtle and unexplored mecha-
nisms [135]. Moreover psychological stressors cause increased bacterial trans-
location to lymphoid tissue from both the gut and the skin so the nature of the
organisms present on skin is likely to be directly relevant to subsequent effects on
immune function [136]. Interestingly, in the British field mouse (Apodemus), the
burden of the louse Polyplax serrata correlated with the state of activation of the
innate immune system in the spleen, implying that ectoparasites (fleas, lice, mites,
ticks) might also have immunoregulatory roles [137, 138].
The blood nematodes are also of interest because these do not enter the gut at any
phase of their life cycles, but they are powerfully immunoregulatory [9]. They
cause impaired induction of T-bet and GATA-3 mRNA, Th1/Th2 deficiency and
increased Foxp3, TGF-β, CTLA-4, PD-1, ICOS and IDO. Some blood nematodes
secrete identifiable immunoregulatory molecules [139, 140].
Nevertheless, in view of the experiments listed in the previous section, it is still
possible that in addition to direct effects on the immune system [pathway (D) in
Fig. 15.3] these effects also operate indirectly via secondary modification of the gut
microbiota [pathway (C) in Fig. 15.3].
Genetics and “Inflammatory Overshoot” in High-Income

Countries
In parts of the world where there was a heavy load of organisms that drive potent
immunoregulation (such as helminths) there has been selection for single nucleo-
tide polymorphisms (SNP) or other variants to partially compensate for excessive
immunoregulation, or to combat new infections such as malaria that spread from
gorilla to man about 10,000 years ago [141, 142]. Such proinflammatory SNPs are
seen for several proinflammatory cytokines [143], IgE [144] and STAT6, a tran-
scription factor involved in Th2 responses [145]. There is also an increased
frequency of the short allele of the serotonin transporter promoter that also has a
marked proinflammatory effect [146]. However this results in a dangerous situa-
tion. As soon as the immunoregulation-inducing organisms are withdrawn by the
modern lifestyle, or after immigration to a high-income country, these genetic
variants lead to inflammatory overshoot. The proinflammatory variants become
risk factors for chronic inflammatory disorders [143–146].
This is important because work that identifies proximate “causes” for diseases
that were rare or nonexistent before the late nineteenth or early twentieth centuries,
and that remain rare in low-income countries, may merely be unraveling a gene-
environment interaction that would be irrelevant if the microbial status could be
returned to that seen in the paleolithic age. For instance, the recent claim to have
discovered that the “cause” of Crohn’s disease is a genetically determined defect in
the homing of neutrophils [147] is difficult to reconcile with the fact that 100 years
ago the disease barely existed. But recent environmental changes could conceivably
have caused this phenotype to become a risk factor.
How Does Stress Cause Inflammation?
Two major issues were avoided in the discussion of the role of immunoregulation in
determining stress resilience (Fig. 15.2). First we did not discuss why stress causes
release of inflammatory mediators, and secondly we did not discuss why such
mediators trigger depression. Stress leads to activation of the hypothalamic-
pituitary adrenal axis (HPA) and the sympathetic nervous system (SNS), and to
changes in the microbiota and gut permeability. How do these mechanisms com-
bine to result in raised proinflammatory cytokine levels?
GC Resistance
Depression is commonly associated with hypercortisolaemia and glucocorticoid

resistance (GCR) [148]. A recent analysis has revealed that persistently raised
levels of inflammatory cytokines cause GCR by impairing the function of gluco-
corticoid receptors [148], leading to further loss of control of inflammation. This
was the suggested mechanism in individuals with recent exposure to severe psycho-
social stressors who developed GCR and subsequently released more proinflam-
matory cytokines in response to an inflammatory stimulus (virus challenge to
airways) [149].
Sympathetic Nervous System (SNS)
There is an increase in plasma concentrations of norepinephrine following exposure

to a standardized laboratory stressor, the Trier Social Stress Test. This is accom-
panied by activation of the master regulator of inflammation, nuclear factor-kappa
beta (NF-κB), in peripheral blood monocyte cells (PBMCs) [150]. Blocking the
effects of activation of the SNS with the β-adrenergic receptor antagonist
propranolol blocked the stressor-induced increases in proinflammatory cytokines,

and reduced the development of GCR [151].
Corticotropin-Releasing Hormone (CRH)
Increased expression of corticotropin-releasing hormone (CRH) is found in CSF

and in the limbic brain regions in depression [152, 153], but CRH is also involved in
the control of gut permeability [154–156]. For example, chronic administration of
CRH via minipumps caused colonic barrier dysfunction in rats [155]. Moreover
when released in the periphery by T cells, CRH is not only a regulator of intestinal
permeability [154, 155], but also a potent pro-inflammatory cytokine [157]. In
many cell types CRH activates NF-κB, and stimulates expression of IL-1β, IL-6,
and TNF mRNAs [158], so some of the effects of CRH on permeability are
secondary to the release of proinflammatory cytokines. Paracellular permeability
is controlled by tight junctions, intermediate junctions and desmosomes, which
constitute a size- and cation-selective filter for small molecules. However, TNF,
IL-17, IFN-γ and nitric oxide (NO) increase permeability. IFN-γ disrupts the tight
junctions, and can modify para-epithelial traffic of inflammatory cells. By contrast,
the immunoregulatory cytokine TGF-β decreases permeability [154, 155].
The composition of the microbiota, particularly Lactobacillus strains and hel-
minth “Old Friends” also modulate permeability. When idiopathic chronic diarrhea
in rhesus monkeys was treated with the whipworm Trichuris trichiura, clinical
improvement was accompanied by striking changes in the microbiota attached to
the mucosa [159]. Similarly in a mouse model of IBD, infection with H. polygyrus
caused an increase in lactobacilli.
Indirect Effects of Psychosocial Stress via the Microbiota
Stress induces changes in the composition of the microbiota of rodents [72], and
induces bacterial translocation from gut and skin [136]. The same is true in humans.
When sampled within hours of admission to the emergency room fecal bacterial
counts were decreased 1,000-fold compared to control subjects, and obligate
anaerobes and Lactobacillus species were significantly decreased [160]. Similarly,
a large change in the microbiota after allogeneic bone marrow transfer was iden-
tified as a risk factor for subsequent inflammation and graft-versus host disease
[161], implying that stress alters immunoregulation at least partly by altering the
microbiota.
The gut microbiota is involved in the activation of the HPA axis by stress, and in
the systemic release of cytokines. A social disruption stressor caused increases in
circulating IL-6 and MCP-1 that correlated with changes in the composition of the
microbiota. But this response was greatly attenuated by pre-treatment with an
antibiotic cocktail to deplete the microbiota [72]. Therefore much of the systemic
cytokine response to stress might be secondary to uptake of LPS and other
proinflammatory microbial components. Uptake of LPS was measured in another
study. Animals subjected to restraint stress show increased portal blood LPS,
together with HPA axis activation manifested as increased plasma ACTH and
corticosterone, increased hypothalamic CRF and increased IL-1β, IL-6 and TNF.
All of these manifestations were blocked by treatment with Lactobacillus
farciminis, which blocks leakiness due to HPA axis activation [162].
Stress Resilience and the Microbiota
The microbiota might also be involved in the observation that poor stress resilience
and an exaggerated cytokine response to environmental [31] or laboratory [22]
stressors is characteristic of people who have suffered increased early life stress.
We know that early life stress can modulate the microbiota [30, 163]. This inter-
pretation is in agreement with the observation that in a low-income country
population the adults who had a high level of microbial exposure in infancy were
resistant to the long-term proinflammatory effects of a very severe childhood
stressor [32]. In addition to the role in immunoregulation, the microbiota in the
first weeks of life also modulates the development of the HPA axis and stress
response [164], and the development of the brain [165].
How Does Inflammation Alter Behavior and Cognition?
The mechanisms that cause inflammatory responses to alter behavior and cognition
have been extensively reviewed recently [166] and are summarized briefly here.
From an evolutionary point of view this link between immunity and psychiatry is
explained by the fact that during an acute infection, withdrawal (to conserve
resources, fight infection and heal wounds) and hypervigilance (to detect danger)
are adaptive responses [167]. Withdrawal and hypervigilance probably result from
inflammation-mediated signals to distinct parts of the brain. Positron emission
tomography (PET) and functional magnetic resonance imaging (fMRI) have been
used to identify brain regions affected by inflammatory cytokines, and the list
includes the basal ganglia, the dorsal anterior cingulate cortex (dACC), amygdala,
hippocampus, insula, dorsolateral prefrontal cortex, and subgenual ACC [reviewed
in 166]. It is suggested that involvement of the basal ganglia is important for
withdrawal, while the effects on the dACC are important for hypervigilance.
However these states are not sustainable and if prolonged, withdrawal becomes
depression and hypervigilance becomes anxiety. This relationship between
prolonged inflammatory stimuli and behavioral changes that resemble depression
and anxiety was postulated long ago following observations of “sickness behavior”
in mice [168].
Inflammation-associated depression and anxiety are more likely in situations
where there is poor control of inflammation [169]. This is seen in high-income
countries where persistently high CRP is common [170], as discussed earlier in the
context of the Old Friends mechanism. Similarly, depression and anxiety often
accompany the chronic inflammatory disorders that are increasing in high-income
countries. They are also seen in obesity where fat tissue contains cytokine-secreting
activated macrophages, and in people who underwent traumatic childhoods, per-
haps because of developmental changes in the HPA axis, brain, and microbiota [31,
discussed in 169]. Genetic factors also play a role. For example individuals who
have the short allele of the serotonin transporter-linked polymorphic region were
more likely to get depression after IFN-α treatment [171].
Cytokines and Cellular Infiltration
Recent studies of patients receiving IFN-α have confirmed in humans many of the
mechanisms previously reported in animal studies. For example, recipients of IFN-
α develop raised CSF levels of IL-6 and MCP-1, proving that the inflammatory signal
is transmitted to the brain [172]. Signals from the inflamed periphery enter
the brain via several pathways. First, cytokines can enter the brain in areas such as
the circumventricular organs where there is no blood-brain barrier. Perhaps these
areas should be regarded as sensory organs of which one role is the detection of
inflammation. Signals also pass via activation of endothelial cells within the
cerebral vasculature, leading secondarily to release of prostaglandins and NO in
the CNS. Furthermore brain endothelium expresses specific cytokine transporters.
Cytokines in the periphery can also signal via afferent fibres within the vagus and
other sensory nerves. This has been called the “facsimile” mechanism, because the
cytokine stimulating the peripheral nerve terminals may subsequently be synthe-
sized de novo and released within the brain.
These various inflammatory signals may secondarily activate local CNS cell
populations. The microglia are derived from a subset of CD45+ monocytic cells and
enter the CNS during embryogenesis in utero and during early post-natal life. These
stable long-lived cells form a network with surveillance functions within the brain
parenchyma, and under normal conditions are in a non-activated, non-terminally
differentiated state, with low expression of major histocompatibility complex class
II [173]. However inflammation propagated to the brain by the pathways listed
above can cause these cells to express inflammatory cytokines and to release
reactive oxygen and nitrogen species [166]. Activated microglia may also be a
source of the MCP-1 mentioned earlier, which recruits monocytes into the
brain [174].
Recruited leukocytes can enter the CNS by several routes. In healthy individuals
there is background traffic via the choroid plexus or through postcapillary venules
located in the subarachnoid space [175, 176]. However in inflammatory states cells
can cross the endothelium of parenchymal post-capillary venules, and so enter the
perivascular space. The relevant adhesion molecules are not expressed on these
endothelial cells under resting conditions but they are induced by peripheral
inflammatory signals such as LPS or TNF, facilitating the MCP-1-driven
recruitment [176].
Indoleamine-2,3-Dioxygenase (IDO)
Inflammatory cytokines also activate the enzyme, indoleamine-2,3-dioxygenase

(IDO), converting tryptophan to kynurenine. It has been suggested that this can
deplete tryptophan sufficiently to cause depression. Recent mouse work showed
that inflammation induced by peripheral administration of lipopolysaccharide
(LPS) activated IDO and caused mice to display a depression-like behavioral
syndrome that could be inhibited by blocking IDO [reviewed in 177]. However,
the syndrome could be reproduced by administering kynurenine, which is taken up
into the brain by the large amino acid transporter, where it can be further meta-
bolized in microglia, astrocytes and macrophages to quinolinic acid, kynurenic acid
and other metabolites [178]. This suggested that depletion of tryptophan was not the
primary mechanism of the depression-like syndrome [179] (though this cannot be
ruled out as an additional factor because there is evidence that kynurenine can
compete with tryptophan for transport into the brain [180]). These mouse findings
have been confirmed in patients with hepatitis C who were being treated with
IFN-α, in whom there were depressive symptoms, but no reduction in CSF con-
centrations of tryptophan [181]. The treatment did however cause increased CSF
levels of kynurenine, quinolinic acid and kynurenic acid, and also of IFN-α, soluble
tumor necrosis factor-α receptor 2 and MCP-1 [181]. This suggested that as in the
mouse, the depressogenic effect might involve transport of kynurenine into the
brain followed by local generation of further active metabolites, rather than deple-
tion of tryptophan [181]. There is increasing evidence that these neuro-active
tryptophan metabolites are important in depression and in schizophrenia, and this
topic has been reviewed in detail recently [177].
Transporters and Reuptake
Inflammatory cytokines also increase the expression of the transporters responsible

for reuptake of dopamine, norepinephrine and serotonin. For example, in mice, IL-
1, TNF and LPS all cause increased expression of the serotonin transporter
paralleled by depression-like behavior [182]. In humans administration of IFN-α
increases the reuptake and decreases the release of radiolabeled L-DOPA, the
precursor of dopamine [183].
Tetrahydrobiopterin (BH4)
Inflammation also disturbs the availability of tetrahydrobiopterin (BH4), which is

an essential cofactor for tryptophan hydroxylase and tyrosine hydroxylase. These
are the rate-limiting enzymes for the synthesis of serotonin, dopamine and norepi-
nephrine [166]. It is possible that the BH4 gets used up when cytokines drive NO
synthase to generate NO, but this pathway is less active in man than in mouse.
However BH4 can also be degraded by oxygen radicals and nitrogen radicals.
Evidence for depletion of BH4 in the human brain in the presence of inflammatory
mediators has recently been obtained in patients receiving IFN-α [184]. In the CSF
of treated patients levels of the inactive oxidized form BH2 were increased, while
levels of BH4 were inversely correlated with increased IL-6 [184].
Anti-Inflammatory Neurotransmitter Pathways
Inflammation in the CNS may also interfere with neurotransmitter pathways that
have anti-inflammatory roles, and so weaken negative feedback on the inflamma-
tory response. For example neural signals transmitted to the periphery via the vagus
nerve inhibit cytokine release through a mechanism that requires the α7-subunit-
containing nicotinic acetylcholine receptor [185]. This cholinergic anti-inflam-
matory pathway can be inhibited centrally by mediators such as IL-1 that increase
neuronal acetylcholinesterase activity [185]. Similarly IL-1β might reduce signal-
ing by γ-aminobutyric acid (GABA), and so enhance local inflammation [186]. This
is due to the fact that GABA-ergic tone is anti-inflammatory because it inhibits the
NF-κB and p38 MAPK pathways, and so reduces the response of microglia to LPS
or IFN-γ [186].
Abnormal Brain Development
In addition to the postnatal mechanisms described above, inflammation can also act
in utero to cause developmental defects in the foetal CNS that lead later to
psychiatric problems. The likelihood of this phenomenon occurring will be
influenced by the efficiency of immunoregulation in mother and child during
pregnancy (Fig. 15.4).
The Old Friends mechanism will be one factor that determines this immuno-
regulation, though as discussed below, there are also genetic factors. Interestingly
autism spectrum disorders (ASD) are increased in towns [39] and in second
generation immigrants [52]. These findings parallel the simultaneous increases in
chronic inflammatory disorders, and depression in which the Old Friends mecha-
nism likely plays a role [169].
Fig. 15.4 Reduced efficiency of immunoregulation during pregnancy could predispose to inflam-
matory episodes in utero that lead to neurodevelopmental abnormalities. Such abnormalities are
seen in ASD and schizophrenia, and both disorders are accompanied by evidence of failing
immunoregulation that is most striking in ASD and in family members. The immunological points
listed in the boxes at lower left and right are taken from, and fully explained within, the references
in the main text
Maternal Infection, Immunoregulation, Fetal Inflammation

and ASD
There has been a significant and genuine increase in the prevalence of autism
spectrum disorders (ASD) that cannot be explained only by increased awareness
[187]. There is debate about the relative contribution of genetics and environment.
A very recent study suggested that “Susceptibility to ASD has moderate genetic
heritability and a substantial shared twin environmental component” [188]. Rather
than worrying about the relative importance of genes and environment it is impor-
tant to note that known autism susceptibility genes include a neuronal module and a
module enriched for immune genes and glial markers [189]. Another study of
interactome networks associated with highly expressed ASD-candidate genes
found that immune signaling through NF-κB, TNF, and Jnk were strongly
represented and that these interactomes involved glia in addition to neurons
[190]. Thus the genetics point to inflammation and the immune system, which
could modulate susceptibility to the types of environmental influence discussed in
this chapter. A recent study suggests that there might be an underlying immune
phenotype (whether genetic or environmental): the immune systems of autistic
children and their healthy siblings were found to have similar immune
dysregulation, when compared to the immune systems of matched healthy

children [191].
An obvious link between immunoregulation and ASD is provided by evidence
that maternal infection during pregnancy increases the risk of ASD in the infant. In
one study 13 % of infants developed ASD following exposure to congenital rubella
[192]. However it seems that any infection increases the risk, particularly if severe
enough to require hospitalization during pregnancy [193]. A study of 1.2 million
births in Finland showed that raised maternal CRP early in gestation was associated
with increased risk, whatever the cause [194]. Animal work proves that maternal
inflammation during pregnancy is transmitted to the foetus. TNF-α and IL-1β
expression was upregulated in a dose dependent manner in the fetuses of pregnant
rats exposed to LPS [discussed in 195]. Similarly I125-labelled IL-6 administered
i.v. to pregnant rats was found in the fetal compartment [196].
Experimental animals exposed to maternal immune activation in utero also
display developmental changes measured by MRI, and behavioral changes sugges-
tive of ASD [197]. In pregnant mouse models these effects are dependent upon IL-
6, and can be mimicked by administering IL-6 itself rather than an indirect
inflammatory stimulus [25]. Interestingly these abnormalities are accompanied by a
systemic deficit in CD4+ TCRβ+ Foxp3+ CD25+ T regulatory cells, and by increased
IL-6 and IL-17 production by CD4+ T cells [198]. The behavioural abnormalities
can be attenuated by transplanting normal bone-marrow, implicating the immune
system both in the development of the syndrome, and in its subsequent
maintenance [198].
Inflammation and Faulty Immunoregulation in ASD
The role of inflammation in utero in the development of the CNS abnormalities that
accompany at least some cases of human ASD is not in doubt. But there is
increasing evidence for an ongoing immunoregulatory deficit in human ASD
[199, 200], as in the mouse model mentioned above [198]. ASD patients have
increased circulating levels of proinflammatory cytokines, and reduced levels of
TGF-β [200], and there is an increased prevalence of asthma and autoimmunity in
family members, reinforcing the view that there is an hereditary, or at least familial,
immunoregulatory deficit [191, 199, 200]. This tendency to autoimmunity is
manifested as brain autoantibodies in plasma from children with ASD, and from
their mothers [200].
Studies of autistic brains reveal activation of microglia and astroglia and
increased expression of a range of proinflammatory mediators such as TNF, IFN-
γ, IL-8 and IL-6 [201–203]. IL-6 is normally expressed at very low levels in the
brain, but it is able to cross the placenta [196] and induce an ASD-like state in the
offspring when administered to pregnant mice [25]. Overexpression of IL-6 in
transgenic mice causes neuroanatomical and neurophysiological alterations
associated with neurological disease [204]. Immunohistochemistry studies con-

firmed that IL-6 is raised in the cerebellum of autistic brain [205].
Autoantibodies to Brain in ASD
There is an alternative way of interpreting some of the data. There is no doubt that
autoantibodies present during fetal life [206], or during inflammatory episodes
when blood-brain barrier function is compromised [176], can alter CNS function
[207]. Such antibodies have been shown in autism [206] and, as suggested by a
murine model, might explain the high frequency of learning disorders in the
offspring of mothers suffering from systemic lupus erythematosus (SLE)
[208]. The association between autoimmune disease and psychiatric disease has
been confirmed in a massive recent study [209]. Patients with MDD have not only a
higher frequency of brain-reactive antibodies, but also an increased circulating
Th17/Treg ratio [21]. In short, the relationship between immunodysregulation and
psychiatric disease might involve additional autoantibody-mediated damage when
autoimmunity is one of the forms of chronic inflammatory disorder present in a
given individual, even if subclinical. The evidence for this in some cases of ASD is
strong [206].
GI Symptoms and Immunoregulation in ASD
Gastrointestinal symptoms are common in ASD. These include diarrhea, consti-

pation, vomiting/reflux, abdominal pain/discomfort, gaseousness, and unusually
foul-smelling stools [210] and are similar to the symptoms of irritable bowel
syndrome (IBS), which affects 10–20 % of the US population [discussed in
211]. While symptoms are not in doubt, there is controversy about whether these
correlate with detectable inflammation in the gut mucosa [discussed in 211]. The
observation that low-grade endotoxemia occurs in patients with severe autism tends
to suggest that some gut inflammation might be present [212]. Meanwhile the use of
modern culture-independent methods has revealed that the gut microbiota of
autistics is abnormal [213–215]. Some authors argue that this abnormality might
lead to excessive production and absorption of short chain fatty acids (SCFA;
for example propionic acid) that in experimental animals induce autism-like
states [216].
Brain Development and Schizophrenia
The etiology of schizophrenia is not known but the predominant view is that, like at
least some cases of ASD, it can involve abnormalities in brain development
occurring during fetal/neonatal life (Fig. 15.4) long before manifestation of the
illness in adolescence or early adulthood [173, 217]. There is correlational evidence
for this. Raised maternal levels of IL-8 and TNF (but not of IL-6 and IL-1β) in mid
gestation were associated with psychosis in the children [173, 218, 219]. As in ASD
there is evidence for immunoregulatory problems in adults with schizophrenia,
though these are less extreme than in ASD [199]. Interestingly a relationship
between ASD and schizophrenia has been postulated that attributes the differing
disease manifestations to relative expression of maternal or paternal copies of
imprinted genes [220]. It might be possible to reconcile this hypothesis with the
differing degrees of immunodysregulation characteristic of the two conditions
[199]. It is of particular interest that some genes that predispose to ASD do not
need to be expressed in the fetus, involve the immune system and probably act
during pregnancy [221].
Conclusions
The study of the gut microbiota by modern molecular methods, and the modulation
of the microbiota by modern lifestyle and dietary habits, have led to a vast
expansion of our knowledge, and to a tendency for the study of the microbiota to
be seen as an independent medical discipline. Meanwhile, the original hygiene
hypothesis was seen as a narrow concept dealing mostly with factors affecting
allergic disorders in childhood, while an offshoot of the hygiene hypothesis,
sometimes known as the “helminth hypothesis” has been applied mostly to the
increases in IBD and MS. In this chapter we suggest that these concepts need to be
studied together, or even unified under one heading such as the “Old Friends”, or
“biodiversity” mechanism. They all deal with the issue of the education and
regulation of the immune system by microbial contact. The gut microbiota is
certainly a major component of this system, but it acts in concert with other
environmental inputs that regulate the immune system, including organisms that
never enter the gut. By seeing the whole picture we may be able to determine
whether immunodysregulation due to divergence of our microbial exposure from
that with which we evolved is able to explain the worrying and parallel increases in
chronic inflammatory disorders and inflammation-linked psychiatric disorders in
high-income countries.
Acknowledgements GAWR is supported by the National Institute for Health Research Univer-
sity College London Hospitals Biomedical Research Centre. CLR receives grant support from the
National Center for Complementary and Alternative Medicine (R01AT004698, R01AT004698-
01A1S1), the Depressive and Bipolar Disorder Alternative Treatment Foundation and The Brain
and Behavior Research Foundation. He reports the following activities for the previous 2 years:
advisory board participation and related travel funds for Pamlab, Lilly and North American Center
for Continuing Education; development and presentation of disease state slides for Pamlab, Pfizer
and Johnson & Johnson, as well as related travel funds for these activities; development of
continuing medical education material for North American Center for Continuing Education and
for CME Incite. CAL receives grant support from the National Institute of Mental Health
(R01MH065702, R01MH086539, R01DA019921, R01MH075968), the National Science Foun-
dation (NSF- IOS 0921969), the Depressive and Bipolar Disorder Alternative Treatment Founda-
tion, and is the recipient of an NSF CAREER Award (NSF-IOS 0845550) and a NARSAD, Brain
& Behavior Research Foundation 2010 Young Investigator Award. He reports the following
activities for the previous 2 years: consultant for Enlight Biosciences.
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Chapter 16
Microbiota-Gut-Brain Axis and Cognitive
Function
Mélanie G. Gareau
Abstract Recent studies have demonstrated a clear association between changes in

the microbiota and cognitive behavior. Intestinal dysbiosis, as modeled using GF
mice (containing no microbiota), bacterial infection with an enteric pathogen, and
administration of probiotics, can modulate cognitive behavior including learning
and memory. This chapter will highlight recent findings in both human and animal
studies indicating how changes in the composition and diversity of the microbiota
can impact behavior and brain physiology in both disease states and in health.
Cognitive behavior can not only be affected in cases of intestinal disease, but also
manifests changes in extra-intestinal disease conditions.
Abbreviations
5-HT Serotonin
ANS Autonomic nervous system
BDNF Brain derived neurotropic factor
CD Crohn’s disease
CREB cAMP response element binding protein
CRF Corticotrophin-releasing factor
DA Dopamine
DLPFC Dorsolateral pre-frontal cortex
EPSP Excitatory postsynaptic potential
GF Germ-free
GI gastrointestinal
HE Hepatic encephalopathy
HPA Hypothalamus-pituitary-adrenal
M.G. Gareau (*)

Department of Medicine, University of California, San Diego, 9500 Gilman Dr #0063, La
Jolla, CA 92093, USA
e-mail: mgareau@ucsd.edu
358 M.G. Gareau

MS Maternal separation
NGF Nerve growth factor
PAMPs pathogen associated molecular patterns
PGN Peptidoglycan
SPF Specific pathogen free
UC Ulcerative colitis
Introduction
The gut-brain-microbiota axis has recently been demonstrated to play an important

role in the establishment and maintenance of cognitive function. In laboratory
animals cognition, or learning and memory, is assessed by specific behavioral tests
(Table 16.1) targeting spatial (or working) and non-spatial (or recognition) memory.
Communication between the gut and the brain can occur via neuronal, endocrine and
immunological pathways, highlighting the complexity in deciphering the specific
mechanisms involved in mediating normal physiology and homeostasis [1]. Studies
involving changes in the composition of the microbiota, either following bacterial
infection, administration of antibiotics or probiotics, or in germ-free (GF) mice all
demonstrate that modulating the microbiota can impact behavior and cognition.
These mouse models involving the presence of an altered microbiota can be used
independently or in combination to study the overall impact of pathology, and
chronic disease in changing cognitive behavior.
This review will focus on the role of the gut-brain-microbiota axis in mediating
alterations in cognition in both human and animal studies and comparing normal
and disease states. Specifically, studies utilizing GF mice, enteric infections, and
neonatal stress as systems modeling an altered gut-brain-microbiota axis will be
described in detail. In addition, studies in healthy human controls as well as those
evaluating the effect of intestinal, including inflammatory bowel disease (IBD) and
irritable bowel syndrome (IBS), or extraintestinal disease states, including diabetes
mellitus and hepatic encephalopathy, on cognitive function will be presented.
Microbiota and Cognition
The microbiome has emerged in recent years as a leading factor in establishing

normal physiology and function of the host as well as being a causative factor in
numerous disease states when inappropriately altered [2]. Changes in the intestinal
microbiota, either due to inflammation, infection, or drugs—including administra-
tion of antibiotics—can lead to extraintestinal effects, including changes in the
brain. Alterations in behavior, including anxiety, depression and cognitive defects,
16 Microbiota-Gut-Brain Axis and Cognitive Function 359
Table 16.1 Common tests Test Function tested

for cognitive behavior
Morris water maze Spatial learning
Fear conditioning Memory skills; fear levels
Novel object test Non-spatial memory
T or Y maze Working (spatial) memory
have recently been demonstrated as additional targets of the microbiome, or more

specifically, the presence of dysbiosis [3]. It is well established that the
hypothalamus-pituitary-adrenal (HPA) axis is regulated by the microbiota. As
evidence of this association, GF mice have increased baseline HPA-axis activation
compared to specific pathogen free (SPF) colonized controls, indicated by elevated
levels of serum corticosterone [4]. This supports the notion that the microbiota can
regulate the development of the central response to stress, at least in rodents.
With respect to cognition, GF mice were recently demonstrated to have a deficit
in non-spatial memory and impaired working memory compared to SPF controls at
baseline [5]. Exposure to acute psychological stress in the form of one hour of water
avoidance, which activates the HPA-axis, did not further affect learning and
memory in GF mice [5]. This finding suggests that under GF conditions, the HPA-
axis cannot be stimulated by exposure to stress, creating a neuroendocrine system
that is vulnerable to external threats. This altered cognition in GF mice is
accompanied by reductions in two proteins that play important roles in the regula-
tion of hippocampal-dependent memory, namely brain derived neurotropic factor
(BDNF) and c-fos [5]. BDNF is a potent modulator of synaptic plasticity, particu-
larly in the hippocampus during neurogenesis [6] whereas c-fos is an immediate
early gene that is a target of CREB, which is required for hippocampus-dependent
long-term memory formation [7]. As was observed in protein studies, decreased
BDNF mRNA was also demonstrated in the hippocampus of GF mice compared to
SPF controls [8, 9]. Taken together, these studies suggest a potential association
between the microbiota and BDNF or c-fos levels in regulating brain physiology
and memory. Despite these studies, it is not known at this time whether cognitive
defects can be normalized by colonization of GF mice either in early life or in
adulthood. Numerous subsequent studies have however demonstrated that GF mice
display anxiolytic-like behavior, compared to their SPF counterparts [8, 10, 11]. In
these studies, conventionalization of mice could normalize behavior, but only in
early stages of life [8]. These findings will be discussed in greater detail in other
chapters.
Following the discovery that GF mice have altered behavior compared to SPF
controls, studies using metabolomics to obtain a complete picture of the influence
of the microbiota on brain function were undertaken. Assessment of the cerebral
metabolome in GF versus conventionalized controls was recently performed to
quantify metabolites that might underlie the gut-brain-microbiota axis, with bio-
synthetic pathways for dopamine (DA) and serotonin (5-HT) demonstrating signif-
icant microbiota-associated changes [12]. These pilot studies revealed a change in
brain neurotransmitter levels impacted by the microbiota, which could significantly
360 M.G. Gareau
impact brain health, development and behavior [12]. Serotonin is important in

cognition, with manipulations in the serotonergic system capable of producing
changes in cognitive function independent of changes in overall mood [13]. GF
mice were demonstrated to have elevated hippocampal and plasma 5-HT levels,
suggesting a possible role for the humoral system in the communication of the
microbiota with the central nervous system [9]. A sexual dimorphic effect was also
observed, with only male GF mice demonstrating these elevations in the serotoner-
gic system in contrast to female mice [9]. Colonization of GF mice post-weaning
could reverse the behavioral changes observed, but not the biochemical changes,
suggesting that 5-HT is not the only factor that underlies the changes in behavior
that are produced by the microbiota [9]. The use of GF mice will continue to
provide important knowledge about the role of the microbiota in establishing
learning and memory.
In addition to GF status, studies looking at commensal or non-pathogenic
organisms have also revealed an important role for the microbiota in mediating
cognitive behavior. Mycobacterium vaccae is an aerobic bacterium that is consid-
ered to be a transient commensal organism, due to its inability to effectively
colonize the gastrointestinal tract. In humans, administration of heat killed
M. vaccae to terminal lung cancer patients was able to improve emotional health
and cognitive function, leading to the hypothesis that the immune response to the
bacteria involved neurotransmitters, such as 5-HT, resulting in improved mood
[14]. In mice, administration of M. vaccae decreased maze run time compared to
controls in a Hebbs-Williams style complex maze consisting of a close-field test
apparatus to study intelligence, suggesting an improvement in learning and memory
[15]. Additionally, immunization with M. vaccae altered emotional behavior,
decreasing the time mice spent immobile during a forced swim test, which was
hypothesized to occur as a result of altered serotonergic signaling in the dorsal
raphe nucleus of the brain [16]. These studies highlight a role for the immune
system, in part via the serotonergic system, in mediating a commensal microbe
effect on the brain and cognition.
Stress, Infection and Cognition
Exposure to psychological or physical stress results in activation of the HPA-axis,

which subsequently activates the neuroendocrine system and numerous down-
stream responses. Stress, or the increased perception of stress, has been associated
with precipitation of symptoms in patients with IBD, and decreased overall quality
of life [17]. Animal models of chronic stress cause changes in the microbiota and
intestinal physiology. These include increased macromolecular permeability and an
elevated secretory state [18–20]. Furthermore, chronic stress is associated with
cognitive deficits, including reduced non-spatial memory [21]. It is therefore not
surprising that exposure to stress can also increase susceptibility to a bacterial
infection. Chronic physical stress (prolonged restraint stress) was associated with
increased pathogen load following infection with the non-invasive murine pathogen
Citrobacter rodentium [22]. Exposure to stress can change the composition of the
intestinal microbiota [22] and modify microbial-host interactions, resulting in an
increase in bacterial attachment and internalization in the epithelium [18]. These
altered host-microbe interactions are directly mediated by stress-induced changes
in the microbiota, as administration of probiotics was able to normalize these
changes [23]. Exposure to stress is also associated with precipitating cognitive
impairments. Social defeat stress in mice was sufficient to induce dysfunction in
cognitive behavior, including changes in spatial object recognition, using the Y-
maze, without affecting anxiety or locomotor activity as assessed by the elevated
plus maze [24]. These cognitive effects were mediated in part by the glutaminergic
signaling pathway within the hippocampus [24]. Infection with C. rodentium alone
in wild type mice does not cause changes in memory and cognition, however
exposure to a single session of psychological stress (water avoidance stress) in
infected mice, but not in uninfected controls led to decreases in both non-spatial
recognition memory and working memory [5]. These stress-induced cognitive
defects remained in place well after the pathogen had cleared, demonstrating a
long lasting effect [5]. Administration of probiotics starting 1 week prior to
C. rodentium infection was able to prevent these stress-induced changes in behavior
[5], again highlighting a role for the microbiota in driving these gut-brain axis
effects. The changes in cognition in stressed mice infected with C. rodentium were
associated with reduced expression of hippocampal BDNF and c-fos [5]. Taken
together, this suggests a potential association between stress-induced changes in
intestinal physiology and cognition via hippocampal BDNF and c-fos, although this
remains speculative at this time. Stress, therefore, plays an important role in the
maintenance of the composition of the intestinal microbiota, with profound effects
in the context of infection with a bacterial pathogen and changes in the gut-
brain axis.
Maternal/offspring interactions are important in shaping the HPA-axis of the
offspring and regulating behavior in adulthood. Maternal separation (MS) during
the neonatal period in mice increases stress-induced intestinal dysfunction [25,
26]. Exposure to early life stress in rats, using MS, can change the composition of
the microbiota both in early life [27, 28] and in adulthood [29]. These changes can
increase disease risk and severity in the development of colitis [30] and facilitate
infection with a parasite, Nippostrongylus brasiliensis [31] in adulthood. MS was
also recently demonstrated to cause discordant, biphasic changes in cognition and
learning depending on age. Younger rats (2 months) previously subjected to MS
demonstrated increased neurogenesis, and decreased repressive histone methyla-
tion at the BDNF IV promoter along with increased hippocampal BDNF and
improved spatial learning and non-spatial learning [32]. In stark contrast, adult
(15 month) rats that had undergone the same stressor regime as neonates demon-
strated opposing changes in neurogenesis, epigenetic regulation of BDNF and
behavior compared to what was observed in younger rats [32]. This neurological
decline in middle-aged rats was prevented by administration of anti-depressants
post-exposure to early life stress, suggesting a role for epigenetic changes in
362 M.G. Gareau
modifying BDNF promoter function and behavior [32]. Similar effects of MS were
observed by other groups, who demonstrated an improvement in hippocampal-
dependent memory, but not in the learning ability following anti-depressant treat-
ment in rats [33]. A recent study by Aisa et al. [34] demonstrated cognitive defects
that were accompanied by decreased nerve growth factor (NGF) expression in the
hippocampus. Alternatively, Baudin et al. [35] demonstrated defects in prefrontal
cortex-dependent, but not hippocampal-dependent, cognitive function following
exposure to MS. Taken together, these studies suggest that the effect of MS on
cognition is complex, and may involve different regions of the brain based on the
type of cognition being assessed. While the microbiota was not assessed in these
studies, MS is known to change the microbiota [28, 29], possibly suggesting its role
in mediating the cognitive defects seen in maternally separated rats.
In related studies, epidemiological evidence suggests an association exists
between prenatal maternal infection and the increased risk of neurodevelopmental
brain disorders in rat and mouse pups [36]. Maternal infection of pregnant rats with
E. coli is associated with altered cognitive development in the offspring, and is
associated with increased hippocampal neuronal apoptosis [37]. Similarly,
intracerebellar injection of LPS in neonatal rats results in learning deficits and
reduced hippocampal volume in adulthood [38]. Peripheral neonatal bacterial
infection also caused impaired memory in adulthood, but only following LPS
administration prior to behavioral testing [39]. This phenomenon could be
prevented by daily neonatal handling that altered the basal HPA axis. These data
further highlight a role for the HPA-axis in regulating cognitive development
[39]. Finally, maternal injection of mice with polyI:C during gestation, to mimic
viral infection, significantly impaired non-spatial memory and learning in the pups
at 3 weeks and 9 weeks of age. Thus, the impact of infection or immune responses
to mimetics of infectious agents, including pathogen associated molecular patterns
(PAMPs), has long lasting effects in the offspring [40]. At present, the conse-
quences of maternal infection for the microbiota of the offspring are not known,
as are any links of these changes with future cognitive abnormalities.
Recent evidence suggests that the cumulative effect of exposure to multiple
infectious pathogens, both bacterial and viral, may be associated with changes in
behavior. In humans, an elevated infectious burden, defined as a composite sero-
logic measure of exposure to specific common pathogens (e.g. cytomegalovirus,
Helicobacter pylori and herpes simplex virus), was associated with cognitive
impairment as assessed by the mini-mental state examination in a prospective
cohort of healthy individuals [41]. These past infections may contribute to cognitive
impairments [41]; as a population of home-dwelling elderly individuals who were
seropositive for common bacteria and viruses exhibited cognitive impairment
[42]. As such these studies suggest that exposure to infectious agents over the
course of a lifetime can contribute to determining cognitive function in adults.
While it is tempting to speculate that infection alone, and/or the immune response
to infectious agents, is the causative factor in cognitive decline, there are no clear
data as yet to support precise mechanisms.
IBS/IBD and Memory
Irritable bowel syndrome (IBS) is a prevalent functional gastrointestinal (GI)

disorder associated with an altered gut-brain-microbiota axis [43]. Symptoms are
often precipitated by exposure to stressors [44], or an enteric pathogen, the latter
being termed post-infectious IBS [45]. Patients with IBS, in contrast to healthy
controls or patients with organic gastrointestinal disease, display an increased
recognition memory to words with a negative emotional connotation [46]. In a
separate, more recent study by Gibbs-Gallagher et al., patients with IBS also exhibit
altered recall to words and phrases describing GI symptoms versus those associated
with respiratory symptoms or neutral phrases [47]. In the Gibbs-Gallagher study,
however, patients with organic disease—consisting of patients with asthma—was
also skewed towards higher recall of words associated with respiratory illness,
suggesting that patients with organic disease may also have a memory bias
[47]. This supports the cognitive behavioral theory of IBS, where selective attention
to GI sensations may play a role in decreased pain thresholds and consequently
increased symptom severity [47]. Similarly, depletion of serotonin in patients with
IBS by acute tryptophan depletion induced a significant shift in affective memory
bias towards loss of recall of positive words, from a list of emotionally loaded
stimulus words versus negative or neutral words, in the absence of overall changes
in mood, in contrast to healthy controls [48]. Serotonin plays an important role in
the gut-brain axis, mediating behavior and intestinal physiology including motility,
secretion and visceral sensitivity. Using imaging techniques, patients with IBS
demonstrated latent impairment of cognitive flexibility due to altered activity in
the dorsolateral pre-frontal cortex (DLPFC), insula and hippocampus as well as
impaired connectivity between the DLPFC and pre-supplementary motor area as
determined using the Wisconsin Card Sorting Test [49]. A direct link between
serotonin levels and changes in the composition of the microbiota and changes in
mood and cognition would be of interest in IBS patients.
Inflammatory bowel disease (IBD) is composed of two distinct diseases, Crohn’s
disease (CD) and ulcerative colitis (UC). Patients with IBD often have increased
intestinal permeability, changes in their microbiota and the presence of inflamma-
tion during active disease. A change in the gut-brain-microbiota axis has recently
been described in many patients with IBD that present with co-morbid mood
disorders, including anxiety and depression, that are found in a subset of patients
[50, 51]. These mood disorders not only diminish quality of life directly, but can
also increase disease severity [52]. Cognitive function in adult patients with IBD
was recently found to be decreased compared to healthy controls, as assessed using
a verbal IQ test [53, 54]. Similarly, in an adolescent population, patients with IBD
demonstrated decreased mild verbal memory compared to controls with juvenile
idiopathic arthritis [55]. This could highlight that inflammation alone may not be a
sufficient driver of these functional cognitive impairments. In a pediatric popula-
tion, administration of steroids to CD patients, but not UC patients, was associated
with a negative effect on mood, memory and behavior, compared to non-steroid
364 M.G. Gareau
treated CD controls, although these differences weren’t representative of a marked

dysfunction [56]. It would be of significant interest to assess whether changes in
mood and memory in patients with IBD is associated with inflammation or the
HPA-axis or a combination of the two risk factors.
Diet, Microbiota and Behavior
Dietary habits have been demonstrated to significantly affect the composition of the
intestinal microbiota [57]. In humans dietary influence begins after birth, with the
choice of feeding modality impacting the composition of the infant microbiota. As
evidence of this, changing colonization patterns have been observed in breast milk-
fed compared to formula-fed infants [58]. In mice, supplementing the diet with
50 % beef protein for 3 months increased the diversity of the intestinal microbiota,
which was accompanied by changes in behavior [59]. These changes included
improvements in working and reference memory, along with reduced anxiety in
the diet-supplemented group, compared to mice fed standard chow [59]. A potential
mechanism of action for the protein enriched diet remains to be determined. Clearly
not all dietary alterations are beneficial to host cognition, as feeding mice a
Western-style diet high in fat and refined sugar increased anxiety-like behavior
and decreased memory function in the setting of low-grade inflammation in an IL-
10 deficient mouse model. These deficits could be ameliorated by administration of
Lactobacillus-containing probiotics [60]. At this point it is uncertain what the
complex relationship between the probiotic, inflammation, and diet was on the
microbiota and behavior, and whether this was directly due to diet-associated
effects of the probiotics or reducing inflammation in this model system.
In a human study involving healthy volunteers, and in particular with no
gastrointestinal or psychiatric symptoms, administration of a fermented milk prod-
uct supplemented with probiotic bacteria resulted in alterations of brain intrinsic
connectivity, having effects in the regions that control central processing of emo-
tion and sensation as assessed by neuroimaging using fMRI [61]. In this study,
however, this chronic administration of fermented milk products supplemented
with probiotic organisms had no effect on the composition of the microbiota
compared to the placebo [61]. Whether these changes in brain connectivity are
associated with a beneficial role in modulating pain sensitivity, stress responsive-
ness, mood or anxiety remains to be determined. In contrast, in a group of Viet-
namese school children, supplementation with a milk or an inulin fortified milk
beverage enhanced weight gain, reduced anemia and increased serum zinc levels
compared to the reference control group in a manner that was associated with
microbiota changes [62]. Specifically, levels of Bifidobacteria and Bacteroides spp.
increased in both treatment groups, which was coupled with improved short-term
memory scores and quality of life compared to children in the control group
[62]. However, a third study showed that while administration of a probiotic-
containing beverage to a healthy cohort resulted in improved mood, this occurred
only in subjects who were in the bottom third of overall mood scores; and surpris-
ingly, memory was slightly increased in the placebo group compared to the
probiotic group [63]. Thus, while these studies suggest that dietary modifications
may ultimately be employed as a means of affecting behavior, including cognition,
in patients with intestinal diseases, whether this reflects an impact on the microbiota
is still controversial. Further, studies in relevant patient groups are still lacking, and
it may not be possible to extrapolate from observations in healthy volunteers.
Extraintestinal Impacts of the Gut-Brain-Microbiota Axis
Diabetes mellitus, a metabolic disorder characterized by insulin deficiency or

resistance, is accompanied by moderate disturbances in learning and memory,
due in part to oxidative stress [64]. Administration of a combination of Lactoba-
cillus acidophilus, Bifidobacterium lactis and Lactobacillus fermentum in the
drinking water reversed the behavioral (spatial learning task) and electrophysio-
logical (declined potentiated excitatory postsynaptic potential [EPSP] in the hip-
pocampus) deficits observed in diabetic rats [64]. Performance in the Morris water
maze navigation task for spatial learning and memory was restored to control
levels, and basic synaptic activity in the hippocampus was normalized [64].
In the setting of liver cirrhosis, hepatic encephalopathy (HE) can develop in a
subset of patients, leading to poor cognition and poor survival. HE is thought to
occur in the setting of an altered microbiota in the context of increased intestinal
permeability [65]. Using a systems biology approach, the presence of
Alcaligeneceae, Porphyromonadaceae, and Enterobacteriaceae were found to be
strongly correlated with HE, decreased cognition and the presence of inflammation
[66]. In a follow up study with patients with milder HE (minimal HE), administra-
tion of the antibiotic rifaximin improved cognition in these patients and reduced
endotoxemia [67]. This effect of rifaximin is thought to occur via changes in gut
bacterial linkages with metabolites, rather than changes in overall microbial abun-
dance [68]. Taken together, these findings may provide supportive evidence for
predicting the risk of HE in patients with cirrhosis, as well as a potential role for
probiotics or antibiotics in preventing cognitive deficits and endotoxemia in
patients.
Impact of the Gut-Brain-Microbiota Axis on Cognitive

Function in Health
As demonstrated by studies in GF mice, the microbiota is essential for normal

cognitive development [5]. It is therefore not surprising that changing the
microbiota, for example by administration of beneficial probiotics, could have an
366 M.G. Gareau
BDNF NGF
BRAIN
c-fos
CREB
Serotonin LPS
CRF PGN
ANS polyI:C
Permeability Bacterial -host

GUT
Diet interactions
MICROBIOTA
Antibiotics GF Pathogens Probiotics
Fig. 16.1 Gut-brain-microbiota and cognition. Each component of the gut-brain-microbiota axis
can participate in overall changes in cognitive behavior. Infection with enteric pathogens, admin-
istration of antibiotics, treatment with probiotics or mice in a germ-free (GF) state can impact the
composition of the microbiota (or lack thereof) and subsequently intestinal physiology. Changes in
intestinal physiology can lead to increased intestinal permeability, altered bacterial-host interac-
tions or be modulated by the composition of the diet. Bacterial components, including lipopoly-
saccharide (LPS), peptidoglycan (PGN) and viral components (mimicked by the use of polyI:C)
can all result in changes in the signaling to the brain. Exposure to stress can produce
corticotrophin-releasing factor (CRF), neurotransmitters that can signal via the autonomic nervous
system (ANS) or activation of the serotonergic system, which can each modulate intestinal
function. All these changes can result in alterations in brain, more specifically hippocampus,
including expression of nerve-growth factor (NGF), brain-derived neurotropic factor (BDNF), c-
fos and subsequently cAMP response element binding protein (CREB), resulting in changes in
cognitive function
impact on normal physiology. A recent study by Bravo et al. studied the effect of a
potential probiotic (L. rhamnosus strain JB-1) on behavior, including fear condi-
tioning to assess the cognitive aspects of anxiety behavior. Enhanced memory
consolidation, in the context of decreased hippocampal GABAB1b mRNA, follow-
ing administration of probiotics suggests a potential mechanism by which Lacto-
bacillus species may modulate cognitive behavior [69].
In humans, administration of a probiotic cocktail (composed of L. helveticus and
B. longum) to healthy human volunteers significantly impacted normal behavior.
Behavior was studied using the coping checklist, which measures cognitive efforts
to adapt to external stimuli that extend beyond a subject’s resources [70]. Subjects
receiving probiotics decreased their self-blame score and displayed a higher prob-
lem solving score compared to their baseline values, following 30 days of admin-
istration of probiotics. This suggests that administration of probiotics may
potentially provide benefits on overall mood and cognition in a healthy control
population.
Conclusions
Cognitive behavior and function are maintained in part by the gut-brain-microbiota

axis. While the mechanistic details still remain to be determined, including specific
pathways of microbial communication with various structures in the brain, these
recent advances highlight the importance of microbial colonization and communi-
ties in mediating appropriate behaviors. Numerous factors can contribute to the
brain-gut-microbiota axis at each level, and bi-directional communication can
together add significant complexity to the system (Fig. 16.1). Determining the
precise mechanisms involved in communication between each step may have
important clinical significance in the future. The novel findings that probiotics
may have an impact on normal cognitive behaviors is particularly interesting,
since it suggests that there exists a certain flexibility within the gut-brain-
microbiota axis, well into adulthood and after completion of developmental stages
thought to be the critical time for mediating potential changes. Shifting the com-
position of the microbiota, in part via administration of probiotics, appears to be a
viable therapeutic option for modulating both intestinal physiology and behavior,
and may improve quality of life in certain patient populations, including IBS and
IBD, as well as the general population.
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Chapter 17
The Impact of Microbiota on Brain
and Behavior: Mechanisms &
Therapeutic Potential
Yuliya E. Borre, Rachel D. Moloney, Gerard Clarke, Timothy G. Dinan,

and John F. Cryan
Abstract There is increasing evidence that host-microbe interactions play a key

role in maintaining homeostasis. Alterations in gut microbial composition is asso-
ciated with marked changes in behaviors relevant to mood, pain and cognition,
establishing the critical importance of the bi-directional pathway of communication
between the microbiota and the brain in health and disease. Dysfunction of the
microbiome-brain-gut axis has been implicated in stress-related disorders such as
depression, anxiety and irritable bowel syndrome and neurodevelopmental dis-
orders such as autism. Bacterial colonization of the gut is central to postnatal
development and maturation of key systems that have the capacity to influence
central nervous system (CNS) programming and signaling, including the immune
and endocrine systems. Moreover, there is now expanding evidence for the view
that enteric microbiota plays a role in early programming and later response to acute
and chronic stress. This view is supported by studies in germ-free mice and in
animals exposed to pathogenic bacterial infections, probiotic agents or antibiotics.
Although communication between gut microbiota and the CNS are not fully
Y.E. Borre
Laboratory of NeuroGastroenterology, Alimentary Pharmabiotic Centre,
R.D. Moloney
Laboratory of NeuroGastroenterology, Alimentary Pharmabiotic Centre,
G. Clarke
Department of Psychiatry and Laboratory of NeuroGastroenterology Alimentary Pharmabiotic
Centre, University College Cork, Cork, Ireland
T.G. Dinan
J.F. Cryan (*)
Department of Anatomy and Neuroscience, University College Cork, Cork, Ireland
e-mail: j.cryan@ucc.ie
374 Y.E. Borre et al.
elucidated, neural, hormonal, immune and metabolic pathways have been

suggested. Thus, the concept of a microbiome-brain-gut axis is emerging,
suggesting microbiota-modulating strategies may be a tractable therapeutic
approach for developing novel treatments for CNS disorders.
Abbreviations
APC Antigen presenting cell
ASD Autism spectrum disorder
BDNF Brain derived neurotrophic factor
DC Dendritic cell
DSS Dextran sodium sulphate
EC Enteroendocrine cells
ECC Enterochromaffin cells
EPA Eicosapentaenoic acid
GALT Gut-associated lymphoid tissues
GI Gastrointestinal
HDAC Histone deacetylase
HPA Hypothalamic-pituitary-adrenal axis
IDO Indoleamine-2,3-dioxygenase
NMDAR N-methyl-D-aspartate receptor
NPY Neuropeptide Y
SCFA Short chain fatty acid
SSRI Selective serotonin reuptake inhibitor
TCA Tricyclic antidepressant
TDO Tryptophan 2,3-dioxygenase
Treg The regulatory T cells
Introduction
Though still in its early days, the twenty-first century may be seen as the era of the
microbiota in scientific discovery. This is due to the significant increase in research
investigating the role of the microbiota and in particular the gut microbiota in a
wide spectrum of scientific and medical fields. The human microbiota, a collection
of various microorganisms, comprises about 1–3 % of total body mass, hosting an
impressive 100 trillion bacteria, most of which find their niche in the intestine
17 The Impact of Microbiota on Brain and Behavior: Mechanisms & Therapeutic... 375
[1]. The majority of the microbial cells are comprised of bacteria from 500 to 1,000
different species varying in diversity and stability, adding over eight million genes
to the human genome [2–4]. An individual’s intestinal microbiota outnumbers
somatic cells of the human body by approximately a factor of 10 [3], suggesting
that an individual organism can no longer be identified as a single entity but rather
as a complex ecosystem. Microbes colonize the digestive tract, reaching high
numbers following birth and immediately thereafter [5], evolving and dynamically
changing throughout one’s lifespan [6]. The importance of microbiota for human
health has been known since the early twentieth century, when Metchnikoff, the
father of the modern probiotics, hypothesized that rebalancing bacteria in the gut
with lactic acid could normalize bowel health and prolong life [7].
We now know that the microbiota plays a crucial role in maintaining physio-
logical homeostasis including digestion, metabolism, growth, development and
function of the immune system and resistance to pathogens [8–11]. More recently,
an increasing volume of evidence has supported the relationship between the
enteric microbiota and brain function [11] both in pre-clinical [12–14] and clinical
settings [15–17]. The concept of the gut microbiota contributing to the range of
central nervous system (CNS) disorders opens new avenues not only for developing
novel therapeutic strategies in treating brain-gut axis dysfunctions, but also man-
aging everyday stress responses.
Microbiota Throughout Lifespan
Although a stable core microbiome is shared among individuals, certain gut micro-
bial populations fluctuate over time, depending on several factors such as mode of
delivery, feeding regimen, maternal diet/weight, probiotic and prebiotic use and
antibiotic exposure pre-, peri- and post-natally [18]. Bacterial colonization follows
a relatively consistent pattern, under the influence of a variety of exogenous and
endogenous factors. Exogenous factors include exposure to microorganisms from
maternal origin such as gut, vaginal canal, or skin but also the environment in
general. Endogenous factors encompass the birth delivery mode (vaginally or via
cesarean section), gestational age, the type of feeding (breastfeeding or formula),
and antibiotic or drug use [19].
The human host-microbe symbiosis is initiated in early life and its establishment
is an intriguing and dynamic biological process. The developing microbiome
undergoes its own evolution throughout the host’s lifetime, in particular the first
3 years, during which a stable microbiome is established [20–22]. Despite the
general dogma that a developing fetus is sterile up until birth [20, 23], increasing
evidence suggests that an infant’s initial microbiome might in fact be seeded by its
mother prior to birth [24, 25] and is then supported by the presence of maternal
microbes during birth [26] and breastfeeding [27, 28]. During and shortly after
birth, infants are exposed to microbes mainly originating from the mother [29,
30]. Growing evidence suggests that it is this initial inoculation and subsequent
development of the intestinal microbiota in early life that is crucial for healthy
development, especially neurodevelopment.
The mode of delivery at birth has recently attracted attention from the scientific
community since infants delivered by C-section are more likely to suffer from
allergies, asthma and diabetes later in life [21, 31, 32]. Although reasons for these
correlations are difficult to tease apart, it has been linked to the crucial role of the
early life environment in the development of a healthy microbiome. While the
microbial composition of vaginally delivered infants initially resembles that of their
mother’s vaginal canal, the microbiota of infants delivered via C-section is more
similar to the microbiota of their mother’s skin [26]. Although infants delivered by
C-section exhibit a delayed acquisition of the members (Firmicutes and
Bacteroidetes) which dominate the adult microbiome, their microbiota composition
does eventually match that of their vaginally delivered counterparts in later life
[33]. It is currently unclear if birth mode can influence brain development and
behavior.
In addition to the birth delivery mode, gestational age is thought to contribute to
the microbial composition of the host. For example, the microbiota of the pre-term
infants lacks two of the main bacterial genera, Bifidobacterium and Lactobacillus,
usually present in full-term infants, and instead display a dominance of the
Proteobacteria [34]. However, breastfeeding enriched the microbiota of the pre-
term infants with the absent microbial species, enhancing the ability of the
infant microbiome to utilize human milk oligosaccharides [20]. In addition to the
maternal role in the developing infant’s microbiome [35], genetic and environ-
mental factors play a role in defining the adult core microbiome. For example, twin
studies revealed higher similarities in the microbiota composition between mono-
zygotic and dizygotic twins in comparison to other family members, suggesting a
significance of the environmental factors over genetics [36, 37] and that microbial
ecologies tend to cluster in family members [33]. The contribution of the genetic
background and environmental factors to the microbiota of the host and the
subsequent functional outcomes remains to be fully elucidated.
Knowing that the microbiota can significantly interfere with the human meta-
bolic, cognitive, and immune systems, the initiation of the symbiosis especially
during prenatal, early postnatal, and adolescence phases appears to be a crucial step
for preparing optimal brain development overall and mental health later in life [38–
41]. Consequently, understanding the early interaction between the intestinal
microbiota and the host opens new avenues for therapeutic interventions, parti-
cularly for infants and young children. Unlike our genetic background, our gut
microbiota may be modified in the first 2 years of life and possibly throughout
pregnancy via the prenatal diet.
The gut microbiome evolves throughout the lifespan and the microbiota diver-
sity declines with ageing, shifting in the dominant species but keeping a stable total
number of anaerobic bacteria [42, 43]. It has recently been shown that microbial
composition of aged individuals correlated with and was influenced by their
residential community, dietary regimen and the health status of the individual
[44]. Crucially, the loss of community-associated microbiota correlated with
increased fragility. Because of the geographical and ethnic homogeneity of the

studied population, future investigations in heterogeneous cohorts are needed to
support the importance of the interactions between diet, the microbiota, health and
ageing [45].
The complex ecosystem of the host’s microbiota is established at birth and its
dynamic nature evolves throughout life span, suggesting its role in maintaining
physiological processes potentially via the microbiota-brain-gut axis network. Bi-
directional communication between the gut microbiota and CNS embodies several
key components which converge to form a complex reflex network with afferents
projecting to the CNS structures and efferents innervating the intestinal wall [11].
Interdisciplinary Conceptualization
of the Microbiota-Brain-Gut-Axis
The concept of the microbiota-brain-gut axis is becoming increasingly recognized

in scientific research, creating multidisciplinary collaborations in the fields of
neuroscience, psychiatry, immunology, gastroenterology and microbiology. The
initial concept of the brain-gut axis, which describes the complex bi-directional
communication system linking the CNS and the gastrointestinal tract, stems largely
from studies of the regulation of the digestive tract in the nineteenth century and
preceded any notion that microorganisms residing in the gut played a modulatory
role in brain function and development. The brain-gut axis plays an important role
in maintaining homeostasis and its dysfunction has been implicated in various
psychiatric and non-psychiatric disorders [46–51]. In addition, modulation of the
brain-gut axis is linked to the stress response and altered behavior with the
microbiome being an important factor in the brain-gut axis communication network
[9, 46, 49, 52–54].
The microbiota-brain-gut axis is a complex network of communication between
the gut, microbiota, and the brain which modulates gastrointestinal and CNS
function [49, 52]. It encompasses the CNS, the sympathetic and parasympathetic
branches of autonomic nervous system as well as the enteric nervous system (ENS)
and the neuroendocrine and neuroimmune systems [46]. Afferent fibers which
project from the gut to cortical centers of the brain such as cerebral, anterior and
posterior cingulate, insular, and amygdala cortices and as well as effector fibers
projecting to the smooth muscle of the gut are the major routes for bi-directional
communication along this axis [55]. Most of our knowledge about the microbiota-
brain-gut axis is primarily confined to neuronal communication between the ENS
and the CNS, however, the exact role of the microbiota has yet to be elucidated.
Pathways of Microbiota-Brain-Gut Communication
Neural Pathways
Within the gut, the microbiota-to-neuron signaling has been shown to depend on
signaling within the ENS. The ENS innervation of the gut is a complex network of
neurons comprised of sensory, motor, and interneurons that are capable of inde-
pendently regulating basic GI functions (motility, mucous secretion, and blood
flow). Due to the similarity of ENS to CNS and its autonomous nature, the ENS is
often referred to as the ‘second brain’. The autonomic nervous system interacts
with the ENS via its main neurotransmitters, adrenaline, noradrenaline and acetyl-
choline, and via efferent and afferent neurons, which innervate the gut [56].
Moreover, specific subsets of enteric neurons in the colonic myenteric plexus of
rats have recently been shown to be sensitive to microbial manipulation, specifi-
cally, a Lactobacillus reuteri strain. It was shown to activate calcium-dependent
potassium channels in this subset of ENS neurons, which may lead us to postulate
that these effects on gut motility and pain perception are mediated by a direct link
between microbiota and the ENS [57]. A more recent study has shown electro-
physiological properties of myenteric neurons are altered in germ-free mice specifi-
cally; decreased excitability in myenteric sensory neurons was found in the absence
of intestinal microbiota. Upon colonization of germ-free mice with normal gut
microbiota, excitability of after-hyperpolarization sensory neurons in germ-free
mice was increased [58].
Another neuronal pathway by which the microbiota can communicate with CNS
is via the vagus nerve. The vagus nerve is the major nerve of the parasympathetic
division of the autonomic nervous system, which regulates several vital body
functions, including heart rate, gut motility, and bronchial constriction [59,
60]. Microbiota can elicit signals via the vagal nerve to the brain and vice versa
[61–63] (Fig. 17.1). For example, the behavioral effects mediated by two separate
probiotic strains in rodents were dependent on intact vagal nerve activation
[64]. Specifically, chronic treatment with Lactobacillis rhamnosus induced
region-dependent alterations in GABA receptor expression in the brain and reduced
stress-induced corticosterone and anxiety- and depression-like symptoms via vagus
nerve signaling [13]. Similar effects were observed in an animal model of colitis,
where anxiolytic effect of Bifidobacterium was absent in vagotomized mice
[65]. In contrast to probiotic-mediated effects, antibiotic treatment-induced
microbiota alterations in mice did not show a similar dependence on vagal nerve
activity [66] suggesting that enteric microbiota communicates with the brain by
diverse mechanisms (Fig. 17.1). In addition to neuroanatomical complexity, neuro-
chemistry may play a vital role in modulating microbiota-brain-gut
communication.
Fig. 17.1 Microbiota-brain-gut axis communication in health and disease (left hand side) Under
healthy conditions, the predominance of symbiotic bacteria, an intact intestinal barrier, a healthy
innate immunity controlling pathobiont overgrowth inside the intestinal tract and healthy gut
function support the symbiotic relationship between the CNS function and gut microbiota. (Right
hand side) Under pathological stress and/or disease conditions, intestinal dysbiosis can adversely
influence gut physiology leading to inappropriate brain–gut axis signaling and associated conse-
quences for CNS functions and disease states. Stress at the level of the CNS can also impact on gut
function and lead to perturbations of the microbiota. A change the balance of symbionts and
pathobionts favoring pathobiont overgrowth, results in dysbiosis. Pathobiont overgrowth leading
to perturbations in intestinal microbiota induces inflammation and loss of barrier function (leaky
gut), promoting increased translocation of pathogenic bacterial components from the intestinal
mucosa to the systemic circulation, where they activate innate immunity characterized by pro-
duction of pro-inflammatory cytokines, resulting in systemic inflammation and abnormal gut
function. These mechanisms potentially lead to impaired CNS function such as altered neuro-
chemistry, cognition, behavior, stress response and visceral pain. DC dendritic cells
Serotonin and Tryptophan Metabolism Pathway
Serotonin [5-hydroxytryptamine (5-HT)] is a biogenic amine that functions as a

neurotransmitter in the body, both in the CNS and the gut. Peripheral 5-HT is
involved in the regulation of GI secretion, gut motility, and pain perception
[67, 68], in the brain it plays an important role maintaining mood and cognition
[69]. Alterations in serotonin transmission may underlie the pathological symptoms
of both GI and some psychiatric disorders, and, may explain their high co-morbidity
[70]. Pharmacotherapy modulating serotonergic neurotransmission, such as tricy-
clic antidepressants (TCAs) and selective serotonin reuptake inhibitors (SSRIs),
have shown therapeutic efficacy in the treatment of both affective and GI disorders
[71–73].
Serotonin synthesis in the brain depends on the availability of its precursor,
tryptophan. Within the CNS, tryptophan concentrations are dependent on peripheral
supply derived from the diet. Acute tryptophan depletion using tryptophan-
restricted diets results in decreased tryptophan plasma levels and consequently
decreased CNS serotonin levels in animals [74] and healthy human volunteers
[75]. The enteric microbiota appears to play a role in tryptophan availability and
metabolism, having an indirect effect on serotonin concentrations in the brain
[76]. Further support for the relationship between the gut microbiota and tryptophan
metabolism comes from germ-free mouse studies, where the absence of the
microbiota in early life resulted in increased plasma tryptophan concentrations
and increases in hippocampal serotonin levels in adulthood [41]. Importantly, the
former measures are restored following the introduction of bacteria in germ-free
mice post weaning [77].
Tryptophan metabolism along the kynurenine pathway, the dominant physio-
logical fate for this essential amino acid and one that is increasingly scrutinized in
many disorders of both the brain and gastrointestinal tract [78–80], also warrants
consideration. The initial rate-limiting step in this metabolic route is catalyzed by
either the ubiquitous indoleamine-2,3-dioxygenase (IDO) or tryptophan 2,3-
dioxygenase (TDO) which is of hepatic origin [81]. The activity of these enzymes
can be induced by either mediators of inflammation (IDO) or by corticosteroids
(TDO) and there is evidence of altered enzyme activity in irritable bowel
syndrome (IBS), a disorder associated with altered microbiota profiles [82–84]. The
probiotic Bifidobacterium infantis affects tryptophan metabolism along this path-
way [76] although this does not appear to generalize to all Bifidobacterium strains
as administration of Bifidobacterium longum does not affect kynurenine concen-
trations [85]. The absence of the microbiota in early life, which increases plasma
tryptophan concentrations, also reduces the kynurenine:tryptophan ratio which is
used as an index of either IDO or TDO enzyme activity [41]. Importantly, normal
enzyme activity is restored following the introduction of bacteria in germ-free mice
post weaning.
In summary, the gut microbiota may play a crucial role in tryptophan availability
and metabolism and consequently impact on central serotonin concentrations as
well as kynurenine and downstream neuroactive metabolites. These effects may be
facilitated by indirect immune-mediated or endocrine mechanisms or by a
more direct route by modulating tryptophan metabolism at the level of the gut.
For example, in certain bacteria, indole is produced from tryptophan by the
tryptophanase enzyme [86]. More studies are warranted to elucidate the underlying
processes involved in modulation of this microbiota-brain-gut axis communication
pathway.
Gut Hormonal Response Pathway
In addition to immune and neural pathways, the gut communicates to the brain via
hormonal signaling pathways that involve the release of gut peptides from
enteroendocrine cells, which can act directly on the brain. Gut peptides such as
orexin, galanin, ghrelin, gastrin, and leptin, modulate feeding behavior, energy
homeostasis, circadian rhythm, sexual behavior, arousal, and anxiety [87, 88]. For
example, galanin is suggested to modulate the hypothalamic-pituitary-adrenal axis
(HPA) response to stress and may act as a link between stress, anxiety, and memory
given the established adverse effects of galanin on cognitive function [89, 90]. Sim-
ilarly, ghrelin may be involved in the modulation of the HPA response to stress or
changes in metabolic status [91, 92]. Leptin receptors can be found in limbic
structures, and chronic leptin treatment reverses stress-induced behavioral deficits
[93], suggesting a potential role for this hormone in emotional processes [94]. More-
over, antidepressant effects of leptin have been shown in diabetic mice [95]. The
idea that changes in enteric microbiota composition can alter gut hormone release is
supported by probiotic studies [96, 97]. Furthermore, germ-free studies suggest that
the gut microbiota mediates and regulates the release of gut peptides [98], yet little
is known about the underlying mechanism of the hormonal aspect of the
microbiota-brain-gut communication. Neuropeptide Y (NPY) is another target
thought to be involved in microbiome brain interactions as it is sensitive to
microbiota manipulations and functions both as a neural and endocrine messenger
[99]. NPY is present at numerous locations throughout the microbiota-brain-gut
axis and have a broad array of functions such as regulation of mood, stress
resilience and gastrointestinal motility. The role of the gut hormonal response in
the microbiota-brain-gut crosstalk is clearly an area of research that demands more
attention and may offer novel therapeutic targets for the brain-gut axis disorders.
Immune System Pathway
The immune system plays an important role in maintaining the delicate balance
between the brain and the gut [8, 100]. The intestinal microbiota imprints the
mucosal immune system and modulates the immune activation outside the gastro-
intestinal tract of the host [101]. The gut and the gut-associated lymphoid tissues
(GALT) are the largest immune organ of the human body, providing a defensive
barrier between externally-derived pathogens and the internal environment
[102]. The gut is patrolled by a variety of immune cells such as T-cells (Treg) and
antigen presenting cells (APCs), which can traffic from GALT to other peripheral
lymphoid sites including the CNS. Immune cell produced within the gut could cross
the blood-brain barrier and be reactivated within the CNS by the resident APCs
[10]. During homeostasis, a fine balance is maintained between the microbiota and
the innate mucosal immune system of the host; but perturbations of the intestinal
microbial composition disturb this equilibrium, resulting in activation of toll-like

receptors and consequent alteration of cytokine profiles, which may lead to
impaired behavior and cognition. For example, peripheral administration of pro-
inflammatory cytokines in rodents induces sickness behaviors such as depressive-
like symptoms, disrupted circadian rhythm and reduced appetite [103, 104].
Moreover, the state of the adaptive immune system has been implicated in a range
of psychiatric [105] and neurodevelopmental disorders [106].
Despite a growing appreciation of the role played by the intestinal microbiota in
immune responses, the precise immunomodulatory mechanisms remain to be
elucidated. It has been proposed that immunomodulating effects of probiotic
microorganisms may occur through the generation of Treg cell populations and
the synthesis of the anti-inflammatory cytokines [107, 108]. Furthermore, coloni-
zation of germ-free mice with commensal bacteria promotes T reg and IL-10 pro-
duction [109], suggesting that alterations in the enteric microbiota may impact
behavior by modulating inflammatory responses of the host in the periphery and the
brain (Fig. 17.2).
Bacterial Metabolite Pathways
In addition to the above-mentioned potential pathways, bacterial products or

metabolites from gut commensals, such as short chain fatty acids (SCFAs), may
translocate from the intestinal mucosa to the systemic circulation, where they could
interfere with immune regulation and CNS function. SCFAs such as acetate,
butyrate or propionate are produced via the fermentation of dietary carbohydrates
and have immunomodulatory properties [110]. SCFAs can interact with nerve cells
by stimulating the sympathetic nervous system [111] and butyrate in particular has
been suggested to modulate brain function via histone deacetylase (HDAC) inhi-
bition [112]. Specifically, systemic injection of butyrate exerted antidepressant-like
effects by inducing histone hyperacetylation in mice [113]. Moreover, administra-
tion of propionate resulted in autistic-like symptoms in rats [114, 115]. Increasing
emphasis is thus being placed on SCFAs as key microbial-induced epigenetic
modifiers of brain-gut function [116].
The Microbiome and Behavior
One of the most exciting areas in examining the role of microbiome in health and
disease is the effects of the microbiome on behavior. Several approaches including
the use of germ-free animals, dietary changes, exposure to adverse stressful events,
animals with pathogenic bacterial infections, animals exposed to microbiota-
modulating agents such as pro-, pre- and anti-biotics (Table 17.1) have been used
to tease apart the role of microbiota on brain and behavior. These data have yielded
Fig. 17.2 Potential pathways underlying the communication along the microbiota-brain-gut axis.
Several pathways have been proposed to understand the communication between the intestinal
microbiota and brain function, some of which have been summarized in this figure. These include
neuroendocrine (hypothalamus-pituitary-adrenal axis), immune system (neuromodulating cyto-
kines), enteric nervous systems and autonomic nervous systems (vagus nerve). Gut microbes
produce tryptophan-related metabolites, gut hormones, 5-hydroxytryptamine (5-HT) from
enteroendocrine cells, short chain fatty acids, and neurometabolites GABA, noradrenalin, and
dopamine potentially modulating CNS function. Stress and emotions can influence the microbial
composition of the gut through the release of stress hormones or sympathetic neurotransmitters
that influence gut physiology and alter the microflora balance. DC dendritic cell, EC
enteroendocrine cells, ECC enterochromaffin cells
fascinating results highlighting the importance of the microbiome in both health

and disease. However, the diversity and multifunctionality of the microorganisms
residing in the enteric microbiota add an extra complexity to the equation.
Germ Free Studies
Germ-free animals are powerful tools for examining the relationship of the gut
microbiota and brain function. Germ-free animals are maintained in a sterile
environment in gnotobiotic units, eliminating the chance of the post-natal coloni-
zation of their GI tract, thus, being a ‘microbiota-free’ control group for the
conventionally colonized gut of their counterparts. Despite exaggerated neuro-
endocrine responses to stress as demonstrated by increased basal levels of
plasma corticosterone [117], several independent laboratories have demonstrated
consistent decreases in anxiety-like behavior in germ-free mice when exposed to

novel and aversive environments (elevated plus maze, light/dark box, open field)
[41, 117, 118] (Table 17.1). Decreased anxiety in germ-free mice is normalized
following post-weaning bacterial colonization of germ-free mice. However, this
effect is dependent on the time of colonization, be it in adolescence or adulthood
[10, 41, 118], proposing neurodevelopmental sensitive time periods [119–121].
In addition to decreased anxiety, germ-free mice show social impairments and
increased stereotypical behaviors [122], suggesting that the gut microbiota plays
a role in socially-driven behaviors which may be of relevance to certain psychiatric
and/or neurodevelopmental disorders (see section “Autism”). At the cognitive
level, germ-free mice demonstrate impairments in non-spatial and working memory
tasks (novel object recognition and spontaneous alternation assessed in the T-maze)
[123]. Further assessment of the cognitive and behavioral phenotype of germ-free
animals would allow us to fully appreciate the role of microbiota in the modulation
of behavioral and cognitive function of the host.
At the molecular level, germ-free mice have reduced levels of N-methyl-D-
aspartate receptors (NMDARs), specifically the NR1 and NR2A subunits, in the
hippocampus [124], or NR2B subunits in the amygdala [117]. These molecular
targets have been shown to play a key role in neuropsychiatric disorders
[125]. Moreover, germ-free animals have decreased levels of brain derived
neurotrophic factor (BDNF), a key neurotrophin involved in neuronal growth and
survival [124]. Interestingly, germ-free effects seem to be sex dependent. While a
decrease in hippocampal BDNF mRNA expression was observed in male germ-free
animals, a qualitative increase in BDNF mRNA expression was present in female
germ-free mice [41], suggesting that BDNF expression differences are related to
sex. Moreover, genetic background appears to play a major role in modulating
the microbiome-brain-gut axis. In summary, germ-free studies demonstrate utility
in teasing apart the mechanisms underlying the microbiota-brain-gut axis commu-
nication relevant to brain function.
Antibiotics
Another way to artificially modulate microbiota and induce intestinal dysbiosis is

via the administration of the antimicrobial drugs. Several studies demonstrated that
antibiotic treatment leads to a microbiota perturbation as demonstrated by in vitro
[126] and in vivo experiments [66, 127]. Administration of an antibiotic cocktail in
rodents leads not only to microbiota depletion but also an increased visceral pain
sensitivity [128], hyperlocomotion and altered BDNF levels in the brain [65]. Inter-
estingly, similar antibiotic treatment in germ-free mice showed no change in
behavior, but colonization of the germ-free mice with the microbiota of BALB/C
mice resulted in significant increases in anxiety-like symptoms [66], emphasizing
the role of the host microbiota on behavior. Further evidence comes from clinical
setting, where short-term antibiotic treatment affected evolution of the infant gut
Table 17.1 Studies of microbiota-gut brain axis
17 The Impact of Microbiota on Brain and Behavior: Mechanisms & Therapeutic...

Behavioural Microbiota
Model Basal behaviour test manipulation Treatment effect References
GF (mice) Reduced anxiety, hyperlocomotion OF, LDB, na na Diaz Heijtz et al. [118]
and increased rearing EPM
GF (mice) Reduced anxiety EPM na na Neufeld et al. [117]
GF (mice) Reduced anxiety LDB na Clarke et al. [182]
C. rodentium infec- Increased anxiety OHB na na Lyte et al. [63]
tion (mice)
Naive mice OF, EPM, L. rhamnosus Reduced anxiety Bravo et al. [13]
FST
Cued FC Enhanced fear memory Bravo et al. [13]
Trichuris muris Increased anxiety LDB na na Bercik et al. [85]
(nematode para-
site) (mice)
DSS induced colitis Reduced anxiety SD B. longum Normalized Bercik et al. [65]
(mice)
C. jejuni (food path- Increased anxiety OHB, EPM Goehler et al. [204];
ogen) (mice) Lyte et al. [205]
Naive rat CDB L. helveticus Decreased anxiety Messaoudi et al. [15]
+ B. longum
Maternal separation Depressive-like symptoms FST B. infantis Normalized Desbonnet et al. [12]
(rat)
Myocardial infarc- Depressive-like symptoms & FST, SD L. helveticus Normalized Arseneault-Bréard
tion (mice) impaired social interaction + B. longum et al. [206]; Gilbert
et al. [207]
Diabetes (rats) Impaired spatial memory MWM B. lactus Improved Davari et al. [51]
+ L. fermentum
GF (mice) Impaired (short-term) recognition NOR, TM na na Gareau et al. [123]
memory and working memory
regardless of stress
385
(continued)
386
Behavioural Microbiota
Model Basal behaviour test manipulation Treatment effect References
C. rodentium Impaired (short-term) recognition NOR, TM L. rhamnosus + L Normalized Gareau et al. [123]
infection (mice) memory and working memory helveticus
after stress induction
GF (mice) Avoidance of conspecifics, TCST, na na Desbonnet et al. [122]
increased stereotypical grooming
behavior
GF (mice) Decreased social investigation STFP Desbonnet et al. [122]
GF (mice) No preference for novel TCST Desbonnet et al. [122]
conspecific
Maternal immune Increased anxiety MB/OF B. fragillis Normalized Hsiao et al. [132]
activation (mice) Impaired social interaction TCST No change
Increased stereotypical behaviour Grooming Normalized
Antibiotic treatment Increased anxiety, LDB, SD, OF na na Bercik et al. [53]
& GF (mice) hyperlocomotion
Naive mice HLB 50 % lean ground Increased working and Li et al. [208]
beef diet reference memory
decreased anxiety
Several behavioral parameters were shown to be altered in germ-free mice or rodents treated with either probiotics or infectious bacteria or parasites. Probiotic
treatment effects on the behavioral and cognitive function are shown
na not applicable, NOR novel object recognition, MB marble burrowing, TM T-maze, TCST three-chambered sociability test, STFP social transmission of food
preference, LDB light-dark box, FST forced swim test, FC fear conditioning, OHB open hole board, SD step-down, MWM Morris water maze, CDB
conditioned defensive burying, HLB hole-board
Y.E. Borre et al.

microbiota, disturbing the colonization pattern of Bifidobacterium in the first

months of life [129]. Antibiotic cocktail treatment provides an attractive alternative
to germ-free mice as means to investigate the role of microbiota-brain-gut function.
Although antibiotic-induced dysbiosis and consequent behavioral abnormalities
provide further support for the role of microbiota in gut-brain communication, the
pathways of antimicrobial-based strategies remain unknown and future studies are
warranted to extricate the underlying mechanisms.
Probiotics and Prebiotics
Maintaining homeostasis of the gut ecosystem is essential for health, and dietary
manipulations such as pre-and-probiotics may present a therapeutic strategy to
impact gastrointestinal and CNS-driven disorders. Probiotics are defined as ‘live
organisms which when administered in adequate amounts confer a health benefit on
the host’. Lactobacillus and Bifidobacterium are the main genera of microorgan-
isms used as probiotics and several studies have demonstrated their beneficial
effects on behavior of the host. For example, L. rhamnosus treatment decreased
anxiety and depressive-like symptoms in mice [13]. Similarly, early-life stress-
induced depressive-related behaviors were reduced after treatment with the pro-
biotic B. infantis, an effect similar to that of the antidepressant citalopram
[12]. Interestingly, the status of the host is critical to the efficacy of probiotics
with certain strains exhibiting beneficial effects during pathological states such as
IBS without an effect in healthy controls.
L. helveticus reduced anxiety-like behavior and attenuated memory deficit in
both na¨ıve and western diet fed mice [130]. Treatment with live Mycobacterium
vaccae resulted in reduced anxiety and improved cognitive performance
[131]. Bifidobacterium normalized anxiety-like behavior in the dextran sodium
sulphate (DSS)-induced colitis model [65]. Furthermore Bifidobacterium, but not
L. rhamnosus, normalizes anxiety-like behavior in Trichuris muris infection
[85]. Combining probiotic strains have shown therapeutic efficacy as well.
For example, L. rhamnosus and L. helveticus treatment rescued stress-induced
memory impairment in mice [123]. Most recently, Bacteroides fragilis treatment
in addition to improving GI function and restoring serum metabolites, alleviated
some of the autism-related behavioral traits such as anxiety, sensorimotor gating,
and stereotypical behaviors in a mouse model of autism [132], providing further
support for the potential of probiotic therapy in combating neurodevelopmental
disorders. In addition to the ameliorating effects of probiotics on behavior and
cognition, probiotics have also proved efficacious in alleviating visceral pain
responses [128, 133–135].
Although the use of probiotics in animal studies has demonstrated therapeutic
efficacy on anxiety- and depressive-like behaviors, data on the effects of probiotics
on depression or anxiety symptoms in humans is rather sparse to date. In a double-
blind, placebo-controlled, randomized parallel group clinical trial, combinational
treatment of a mixture of probiotics containing L. helveticus and Bifidobacterium in

a healthy subjects for 30 days resulted in a significantly less psychological distress
compared to the placebo group [15]. In a similar study, treatment with a probiotic-
containing milk drink resulted in improved mood and cognition in health subjects
when compared to the placebo group [136]. In a pilot study, patients suffering from
the chronic fatigue syndrome receiving Lactobacillus casei daily for 2 months
showed significant reduction in anxiety compared to the placebo group [16].
Specifically, treatment with a fermented milk product with probiotic reduced the
response of healthy volunteers and altered activity of the brain regions, controlling
central regions of emotion and sensation to an emotional faces attention task
[137]. Although these beneficial effects have yet to be demonstrated in pathological
anxiety, combination of L. helveticus and Bifidobacterium alleviated psychological
distress in healthy subjects [138].
Unlike probiotics, prebiotics have not received as much attention, but have
demonstrated promising results [139]. Prebiotics are non-digestible food ingredi-
ents that selectively stimulate the growth of Lactobacilli and Bifidobacteria in the
gut [140]. Increasing the proportion of these bacteria with prebiotics has many
beneficial effects on the gut, the immune system [141–143], and most recently on
the brain function, specifically, increased BDNF expression and NMDAR signaling
[14], providing initial support for further investigations of the utility of prebiotics in
mental health and potential treatment of psychiatric disorders.
Taken together, studies examining the impact of pro-and-prebiotics on cognition
and behavior (see Table 17.1) are in the early stages, yet these preliminary results
offer novel therapeutic potential for treating mood and anxiety disorders with
psychobiotic-based approaches [107]. Despite promising evidence that certain
pre- and/or pro-biotic strains are able to modulate brain function and behavior,
caution is warranted when translating and generalizing the pre-clinical data into the
clinical domain. Clinical validation and identifying underlying mechanisms of the
microbe-based therapeutic effects are essential prior to assessing the actual value of
these microbiota-modulating agents in treating disorders of the microbiota-brain-
gut axis.
Disorders of the Microbiome-Brain-Gut Axis
In healthy individuals, the normal dominant microbiota is relatively stable and is in

a symbiotic rapport with the host. Perturbations in the delicate symbiotic host-
microbiota relationship may have serious consequences resulting in various psy-
chiatric and non-psychiatric disorders, collectively known as the disorders of the
brain-gut axis.
Stress
Despite the well-established association between stress and psychiatric disorders,

the struggle to understand the complex processes by which stress mediates patho-
logical changes that increase vulnerability to disease is on-going [144]. Although
stress is a natural occurrence, chronic, severe, and uncontrollable stressors can
trigger abnormal changes in brain structure and function with long-term physical
and mental stress [145–147]. Chronic stress has been a common denominator in
several GI disorders and a key player in microbiota-brain-gut axis dysregulation of
the stress-related CNS diseases [9, 11, 38, 148–151]. Animal studies have shown
that emotional stressors, such as maternal separation, restraint conditions,
crowding, heat stress and acoustic stress negatively impact on the composition of
microbiota [152–159]. The association between microbiota and stress-response is
further supported by experiments with germ-free mice and rodents treated with
probiotics and/or antibiotics. Sudo and colleagues [124] demonstrated an enhanced
HPA axis activity in germ-free mice following an acute psychological stress,
providing first convincing evidence of the essential role played by microbiota in
programming of the stress response. Moreover, increased hippocampal 5-HT and
5-HIAA as well as plasma tryptophan was found in germ-free males [41]. Several
independent germ-free studies are summarized in Table 17.1. Furthermore, mono-
amine neurotransmission has been shown to be increased in the colonized germ-
free mice compared with germ-free mice [119], resulting in an altered behavioral
phenotype compared to germ-free mice.
Another line of evidence comes from the studies in the maternal separation
model, one of the best-characterized animal models in relation to the long-term
effects of stress in early life on microbiota [157]. In light of the mutual relationship
that exists between the stress response and microbiota, it is not surprising that the
period most critical to HPA axis development and programming of the neuroendo-
crine stress response, early postnatal life, is also an important time-point for the
initial establishment of the core gut microbiota. This model and others have been
employed to investigate numerous aspects of brain gut interactions more recently
the effects of probiotic treatment on fatty acid metabolism of the host [160–
162]. This data is particularly relevant in the context of depression with an increas-
ing body of evidence highlighting the role of fatty acids such as eicosapentaenoic
acid (EPA) and docosahexaenoic acid (DHA) in depressive disorders [163].
One of the principal mechanisms proposed to underlie stress-induced alterations
is the “leaky gut” phenomenon, which has also been described in major depression
[164] (Fig. 17.1). It is hypothesized that the epithelial barrier of the gastrointestinal
tract is compromised as a result of either a stressful event or microbiota dysbiosis,
leading to increased intestinal permeability and the consequent translocation of
pathobionts across the mucosal lining to sites where direct interaction with immune
cells and the ENS can occur [165]. This leads to activation of an immune response
characterized by increased production of pro-inflammatory mediators in circulation
and eventually the CNS. In support of this hypothesis, pre-treatment with the
probiotic Lactobacillus farciminis attenuated the effects of acute restraint stress on

intestinal permeability and HPA axis response [166] (Fig. 17.2).
Depression and Anxiety
Depression and general anxiety are disorders with well-established etiological links
to the traumatic life events, particularly when experienced in early life and during
periods of chronic stress [167, 168]. Although current clinical literature contains
few reports that describe the state of the gut microbiota in depressed individuals,
data from associated illnesses, such as IBS, which are often accompanied by
depressive symptoms, have revealed reduced Bacteroidetes and increased
Firmicutes content in the fecal samples of patients with these disorders [169–
171]. These findings emphasize that the microbiota is an attractive target for
potential therapeutic strategies whereby altering the host’s gut microbiota may
infer health benefits to the host.
Indeed, animal studies focusing on the effects of chronic probiotic treatment,
both in na¨ıve and stress-related animal models of CNS disorders, have also served
as invaluable and informative tools in ascertaining the specific contributions made
by gut bacteria to anxious and depressive disorders. Probiotic treatment during the
postnatal stress period in maternally separated rat offspring has been shown to
normalize basal corticosterone levels [172]. When administered to na¨ıve rodents,
L. rhamnosus reduced stress-induced corticosterone, which was paralleled by
region-dependent alterations in GABA receptor gene expression levels in the
brain [13]. Moreover, the neurochemical effects were not found in vagotomized
mice, identifying the vagus nerve as a major modulatory communication pathway
between the bacteria exposed to the gut and the brain [13]. B. infantis altered
peripheral cytokine levels and concentrations of the serotonin precursor, trypto-
phan, which may allude to the development of possible protective mechanisms
prior to stress exposure [76]. The therapeutic potential of probiotics in psychiatric
conditions has been the topic of intense discussion and additional investigations are
required to fully elucidate the role of probiotics in brain function [173, 174].
Despite the lack of clinical data to support the idea to utilize probiotics in
treatment of mood disorders, there are sufficient pre-clinical data to support this
view. Potential psychobiotics are delivery vehicles for neuroactive compounds, and
have a capacity to reduce inflammatory response and reduce HPA activity, a much
broader profile than of existing antidepressant treatment options [107]. As not all
probiotics are equal in their effects and may not have psychobiotic potential, a
careful examination of their efficacy is warranted. There is no doubt that many
patients, particularly those with milder symptom profiles, would value the rise of
nonconventional antidepressants in the form of psychobiotics.
Taken together, the enteric microbiota has a significant impact on the behavioral,
neurochemical and immunological measures relevant to the brain-gut axis disorders
with psychobiotics as a promising emerging treatment [107] (Fig. 17.2).
Irritable Bowel Syndrome (IBS)
IBS is a disorder of the brain-gut axis and in addition to gastrointestinal symptoms

is associated with frequent comorbidities of depression and anxiety [82]. Although
the precise mechanisms of the underlying pathology remains unclear, the role of
brain-gut communication in the etiology of IBS has become widely recognized with
altered CNS control of visceral pain and inflammatory responses being integral
pathophysiological features [175]. Recent neuroimaging studies demonstrated thin-
ning of the anterior cingulate and insular cortex of the IBS patients [176, 177]
increased activation of the thalamus, anterior cingulated and prefrontal cortex and
microstructural reorganization in the IBS patients which was correlated with
symptoms of visceral hypersensitivity, providing further evidence of the abnormal
brain function in disease pathology.
Chronic low-grade inflammation is a common feature in many IBS patients and
studies have identified several susceptibility genes for IBS involved in innate
immunity and recognition of bacteria [178]. Subgroups of IBS patients may have
an altered microbiota composition relative to healthy individuals based on the
analysis of fecal microbiota [170, 179]. Altered microbiota diversity [180, 181]
has been reported in IBS patients, indicating a loss of homeostasis of the intestinal
bacterial ecosystem [44]. Although the specific mechanisms by which changes in
the gut microbiota lead to IBS symptoms remain unclear, it is hypothesized that an
influx of certain microbes such as Lactobacilli and Veillonella in IBS patients result
in a high level of organic acids such as acetic and propionic acid, which in turn may
contribute to intestinal discomfort and anxiety [170]. In light of these promising
preliminary findings it is not surprising that a positive effect of treatment with
microbial-based therapeutics (both non-absorbable antibiotics such as rifaximin
and probiotics) has been demonstrated in IBS [140, 182–185].
Autism
Neurodevelopmental disorders are characterized by impaired brain development

and behavioral, cognitive, and/or physical abnormalities. Several share behavioral
abnormalities in sociability, communication, and/or compulsive activity. Autism
spectrum disorders (ASDs) are neurodevelopmental disorders characterized by
presence of stereotypical behavior and social interaction deficits [186]. Although
the ASD etiology remains unknown, genetic and environmental factors are thought
to play a role in the development of ASD [187]. Among several comorbidities in
ASDs, gastrointestinal (GI) distress is of particular interest, given its high pre-
valence and correlation with symptom severity [188, 189]. GI abnormalities in
ASDs have been linked to alterations in microbiota composition and function [6,
132, 190–192]. Despite these correlational studies, clinical data interpretation is
compromised by high rates of antibiotic use and dietary variations in ASD patients,
which makes it difficult to draw definite conclusions about ASD-related microbiota

changes [11]. Studies in germ-free mice demonstrated robust and reproducible
social deficits and increases in repetitive behaviors similar to that observed in
ASD [122], suggesting that the microbiota is a critical factor in the development
of social behavior and the etiology of ASDs. Most recently, studies demonstrated
that autism-like behavioral and GI phenotypes are associated with altered
microbiota in two separate mouse models of ASDs [132, 190]. Both clinical and
pre-clinical studies provide promising evidence indicating an important role for the
gut microbiota in the pathogenesis of ASDs, creating opportunities for developing
novel therapeutic strategies in managing neurodevelopmental disorders via
microbiome-based treatment. Indeed, B. fragilis given in early adolescence has
been shown to ameliorate some but not all of the behavioral dysfunction in a mouse
model of autism [132]. Moreover, whether other neurodevelopmental disorders
such as schizophrenia or attention deficit hyperactivity disorder are associated
with microbiota changes have yet to be investigated either in animal models or
human populations.
Cognition
Cognition, which is a general term used to describe thought processes that contri-
bute to decision making, problem solving, and executive function, is affected in a
number of CNS disorders including depression, schizophrenia, autism and
Alzheimer’s disease. Despite the advances in our understanding of the cognitive
processes in the brain [193, 194], cognitive deficits remain a difficult symptom to
address mainly due to the lack of efficacious treatments. It is recognized that
chronic or uncontrollable stress in early life has a profound effect on the develop-
ment of cognitive neuronal pathways, which also has a negative impact on cogni-
tive function in later life [195]. Although our knowledge in regards to the role of
microbiota in cognition is rather limited, recent studies demonstrated impaired
non-spatial memory and social cognition (the ability to distinguish between a
novel and previously-encountered mouse) in germ-free mice [122, 123], suggesting
a potential link between cognitive processes which may depend on the presence of
gut microbiota. Further support of this hypothesis comes from studies demon-
strating that treatment with probiotics in naive rodents enhances fear memory
[13] and reverses memory deficits observed in infected Citrobacter rodentium-
infected mice after acute stress exposure [123]. The role of microbiota in cognitive
function has also become a topic of interest in IBS, a disorder associated with
varying degrees of cognitive impairment [196, 197]. Further investigations into the
effects of microbiota, antibiotic and probiotic-based therapies on cognitive perfor-
mance in both the clinical and preclinical domain are warranted.
Sex Differences
Another important feature of psychiatric conditions is the different prevalence rates

reported in males and females. For instance, whereas autism occurs more frequently
in males (4:1 male to female ratio [198]) depression and anxiety are more prevalent
in females [199]. To date, a limited number of studies have focused on the impact
sex of the host may have on the microbiota-brain communication in the context of
brain development [41]. Interestingly, the most robust changes in neurochemistry
(serotonin), hippocampal neurobiology (BDNF) and behavior (social deficits) have
been observed in males [41, 122], which is in line with the neuropathology and
symptoms observed in autism [200]. Further studies are needed to explore sex-
related factors underlying the conflicting outcomes in brain neurochemistry and
function as a result of microbiota-host interactions in males and females. Although
establishing the underlying mechanisms of sex differences in microbiota- brain-gut
communication may provide insights into the pathogenesis of these disorders,
these findings should be addressed with caution when assessing their translational
value, requiring more investigations.
Implications and Future Perspectives: Towards Novel

Therapies for Brain Disorders
Despite the field of the microbiome-brain-gut research being in its infancy, both
pre-clinical and clinical evidence suggest that the gut microbiota plays a key role in
the development of various aspects of brain function including anxiety, mood,
cognition, and more recently in social behavior. Early pre-weaning and adolescence
periods appear to be critical periods for modifying enteric microbiota and potential
modulation of abnormal behaviors. Environment in early postnatal life has a crucial
impact in the neurodevelopment, thus establishing and targeting vulnerable periods
for the microbiota-brain-gut axis will allow identification of critical windows of
opportunity, in which restoration of the “normal” core microbiota may have
therapeutic value in psychiatric and neurodevelopmental disorders.
Existence of a core healthy microbiota profile remains debatable. Advances in
high-throughput screening techniques allow characterization of the microbial com-
munity at a genome-level in health and disease, shedding more light on the
composition, diversity and functions of the human microbiome. Identifying specific
combinations and/or subsets of microorganisms, which are essential for optimal
health of the host, will create opportunities for preventive, diagnostic and thera-
peutic approaches for disorders of the microbiota-brain-gut axis. In line with this
idea, advances in fecal transplant strategies have already been attempted to treat
metabolic and GI disorders, providing convincing evidence for the benefits of the
microbiota-based therapies [201–203]. Despite this progress, fecal microbiota
therapy in CNS disorders, especially neurodevelopmental and mood disorders,
have yet to be explored. More pre-clinical research is needed to determine bacterial

populations in the gut microbiota that are altered and could be of benefit in CNS
disorders prior to introducing this innovative microbial-based therapy into the
clinic. Currently, pro- and-prebiotic-driven research offers a potentially safer and
more explored alternative to fecal transplantation.
As the century continues and technologies to investigate microbiome-gut-brain
interactions improve and are applied to the study of CNS disorders, we will see the
advent of psychobiotic-based therapies for a variety of neuropsychiatric disorders.
Although the vast majority of studies examining the role of microbiota in brain
development and function have yielded promising results, they require validation in
the clinical domain. Further studies are warranted to examine the physiological
impact of the microbiota on behavior, and consequently the contribution of the gut
microbiome in the pathogenesis of psychiatric disorders. With rapid advancements
in metagenomic techniques, non-invasive techniques to monitor brain structure,
function and signaling, and the development of multidisciplinary collaborations the
fast-evolving and exciting field of host-microbiota research area will make signi-
ficant progress, creating new avenues for microbial-based therapeutics that benefi-
cially influence healthy and pathological brain function.
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Chapter 18
Neuroimaging the Microbiome-Gut–Brain
Axis
Kirsten Tillisch and Jennifer S. Labus
Abstract The brain is the most complex organ in the human body, interacting with
every other major organ system to continuously maintain homeostasis. Thus it is
not surprising that the brain also interacts with our microbiota, the trillions of
bacteria and other organisms inhabiting the ecosystem of the human being. As we
gather knowledge about the way that our microbiota interact with their local
environments, there is also increasing interest in their communication with the
brain.
Abbreviations
BOLD Blood oxygen level dependent

DTI Diffusion tensor imaging
FA Fractional anisotropy
MGBA Microbe-gut-brain axis
MRI Magnetic resonance imaging
PET Positron emission tomography
K. Tillisch (*) • J.S. Labus

Division of Digestive Diseases, Department of Medicine, Gail and Gerald Oppenheimer
Family Center for Neurobiology of Stress, David Geffen School of Medicine at UCLA, 10833
LeConte Ave, CHS 42-210 MC737818, Box 957378, Los Angeles, CA 90095-7378, USA
e-mail: ktillisch@mednet.ucla.edu
406 K. Tillisch and J.S. Labus
Introduction
The brain is the most complex organ in the human body, interacting with every
other major organ system to continuously maintain homeostasis. Thus it is not
surprising that the brain also interacts with our microbiota, the trillions of bacteria
and other organisms inhabiting the ecosystem of the human being. As we gather
knowledge about the way that our microbiota interact with their local environ-
ments, there is also increasing interest in their communication with the brain.
Brain-Gut Communication
Bidirectional communication between the brain and gut has been well described
(Fig. 18.1) [1–4]. The brain communicates with the gut via the autonomic nervous
system (particularly the vagus nerve) and the hypothalamic-pituitary adrenal axis.
Descending monoaminergic pathways also act on the dorsal horn and can regulate
gut-related sensations. Gastrointestinal motility, secretion, local blood flow, and
immune regulation are modulated by the brain, generating stereotypic patterns of
gut response which are context specific, such as the classic gastrointestinal stress
response of nausea and/or fecal urgency. Thus the local environment of gastroin-
testinal microbes is continuously adjusted by central influences. These interactions
provide a partial explanation for the differences in gut bacterial populations
between healthy persons and those with gastrointestinal illness [5–7] or prolonged
psychological stress [8]. Similarly, preclinical studies have identified altered fecal
bacteria after experimental pre and post-natal stress [9–12].
Completing the bidirectional loop, the brain receives afferent input from the gut,
likely from a variety of pathways, as described below. With a surface area far
exceeding that of the skin, the gut is the largest interface between the body and the
external environment, and contains the body’s most numerous population of
microbes. The gut also has a vast immune system and complex nervous system
through which the microbiota can communicate with the brain. Biologically active
compounds such as serotonin, histamine [13], catecholamines [14], gamma-
aminobutyric acid (GABA) [15], and others can be produced in various amounts
by specific bacteria. Additionally, organisms can stimulate the release of these
compounds by gut enterochromaffin cells, leading to central signaling and clini-
cally apparent symptoms [16]. An example of this is the central nausea induced at
the nucleus tractus solitarius after rotavirus-stimulated gastrointestinal serotonin
release [17]. An alternate pathway by which information may reach the brain from
the gut is via neurochemicals secreted into the portal venous system, as is seen in
hepatic encephalopathy [18, 19].
The vagus nerve has been shown to be essential in some but not all preclinical
studies of microbe-brain interactions and likely plays a key role in the microbe-gut-
brain axis (MGBA) in humans [20, 21]. Interoceptive (internal) signals of body
18 Neuroimaging the Microbiome-Gut–Brain Axis 407
Fig. 18.1 The microbiota-gut-brain axis (MGBA). The traditional gut-brain axis consists of the
brain with bidirectional connections to the enteric nervous system of gastrointestinal tract via the
autonomic nervous system (sympathetic and parasympathetic branches) and hypothalamic-
pituitary-adrenal (HPA) axis. Here the expanded MGBA network is shown. The gastrointestinal
microbiota communicate with the brain via enteric nervous system and via metabolic products.
The immune system interacts with each member of the MBGA bidirectionally
state are relayed from vagal and spinal afferent nerves to the brain stem and then for
further processing in higher cortical centers [22, 23]. It has been proposed that
interoceptive input has relevance beyond merely reporting the homeostatic “status”
of the body. In the model proposed by Craig and others, interoceptive signals
appear to be integrated with emotional and cognitive input primarily in the anterior
insula. This combined input is used continuously to create a sense of momentary
“self” which can be consciously interpreted as happy, sad, healthy, ill, etc. [24,
25]. Since visceral feedback from the gut and other body sites contributes to our
conscious state of wellbeing, it then follows that the gut’s luminal organisms also
have the opportunity to influence mood states like anxiety or depression [26,
27]. Given the difficulty of gaining access to the cellular workings of the brain in
humans, neuroimaging has emerged as a tool to increase our understanding of the
MGBA. In the section below, several of the key imaging modalities will be
reviewed and their integration into analyses of the MGBA will be discussed.
Neuroimaging in Humans
Functional Neuroimaging Techniques
One of the most common research techniques used to image changes in brain
function between groups or after a treatment intervention is functional magnetic
resonance imaging (fMRI). This technique is non-invasive, safe, and easy to
perform. Functional MRI measures changes in the percentage of oxygenated versus
deoxygenated hemoglobin, taking advantage of the differing magnetic properties of
the molecules. During an experimental task, when a brain region is more active
compared to a baseline or control task, blood flow increases and thus a higher
proportion of oxygenated hemoglobin is observed in that area. This change in the
regional magnetic properties is measured as the blood oxygen level dependent
(BOLD) signal by the scanner and provides an indirect measurement of a change
in brain activity. Functional MRI has fairly good spatial resolution of 2–4 mm but
does not have the precision of post-mortem studies in animals. Functional MRI has
been used successfully to identify differences in brain function in gastrointestinal
disease states, such as irritable bowel syndrome and inflammatory bowel disease, as
well as in healthy people before and after chronic ingestion of probiotics [28–30].
The other common mode of functional neuroimaging is Positron Emission
Tomography (PET). Radiolabeled chemicals are injected into the blood stream
and PET measures the emissions regionally throughout the brain. PET has the
advantage of measuring physiologic processes more directly via the use of radio-
labeled ligands; however it has the drawback of being more invasive and requires
radiation exposure. Radioligand PET can be used to explore baseline interactions
between regional brain distribution of a variety of signalling systems (including
dopamine [31, 32], serotonin [33], substance P/neurokinin-1 [34, 35]) with gut
microbiome and metabolomic profiles, as well as assess pre- to post-intervention
changes in the MGBA after intervention with specific probiotics. While PET
imaging is more invasive and difficult to perform, it has the advantage over fMRI
of isolating specific biological processes or pathways for measurement.
The Functional Imaging of the Gut-Brain Axis
The brain-gut axis has been examined using fMRI and PET in humans, particularly
in the setting of evoked pain, or anticipation to pain in the esophagus and distal
colon. Alterations in resting brain function have also been described in patients with
functional gastrointestinal disorders, which are believed to involve brain-gut axis
dysfunction [36–38]. Whether these resting brain signal changes represent ongoing
gastrointestinal input to the brain or persistent changes in the function of neural
circuitry due to chronic disease is not yet known.
Functional MRI has been extensively used to observe changes in brain response
after a treatment intervention, most commonly using pharmaceuticals or behavioral
interventions, but little has been done to image the effects of antibiotics, probiotics,
or dietary interventions in humans [39–41]. Only one study to date has described
functional brain changes in response to a probiotic intervention [29]. In this study
healthy, normal weight women without any gastrointestinal symptoms, pain or
psychiatric disorder, were randomized to treatment with a probiotic, a placebo
dairy product or no treatment. The response to an emotional attention task was
measured with fMRI before and after the treatment period and the probiotic group
showed reductions in response to the emotional task, suggestive of reduced vigi-
lance to negative emotional stimuli. This difference in brain activity was not
correlated to any subject reports of mood or gastrointestinal symptoms. Evaluation
of the microbiota in that study confirmed that the experimental probiotic could be
identified in the stool of the probiotic ingesting subjects but did not show group
specific changes in the overall architecture of the microbiota. This is consistent with
other studies and suggests that microbial metabolites rather than overall microbial
configuration may be the salient result of probiotic ingestion [42]. This initial study
suggests that subtle changes in the gut contents can lead to measureable changes in
brain function, even in the absence of a conscious awareness of the change. Future
studies, which may be able to use microbiome composition, along with
metabolomic and metagenomic measurements from stool to correlate with brain
function at baseline or after a probiotic intervention, will lead to a better under-
standing of how the MGBA can be modulated in health and disease.
Structural Neuroimaging
In addition to functional neuroimaging, advances in MR imaging of gray and white

matter structure have proven valuable in describing group differences in psychiatric
illness and chronic pain syndromes compared to healthy populations. Differences in
both white matter and gray matter have been identified in irritable bowel syndrome
and functional dyspepsia, both of which are considered to be disorders of the brain-
gut axis and which likely are accompanied by alterations in the gut microbiota [43–
51]. High resolution structural brain images can be used to produce global (whole-
brain), regional, and voxel-level indices of gray matter density and volume as well
as cortical thickness, surface area and mean curvature (Fig. 18.2). Network analysis
from graph theory has recently been applied to gray matter morphometry to
demonstrate alterations in regional topology, providing strong evidence for exten-
sive structural reorganization of cortical and subcortical regions previously impli-
cated in altered brain responses to visceral pain stimuli and their expectation
[43]. The biological substrate underlying grey matter changes may involve
increased or decreased glial cells, changes in dendritic spines or synapses or less
likely, neural degeneration. Gray matter has been shown to remain quite plastic
even during adulthood [53–55]. The effects of peripheral factors such as the
Fig. 18.2 Multimodal neuroimaging. (a) White matter tracts in the brain can be visualized with
diffusion tensor imaging (DTI). (b) The gray matter structure can be viewed with magnetic
resonance imaging (MRI) and parcellated into structural or functional regions, measuring charac-
teristic features including volume, cortical thickness and regional curvature. (c) Visualization
subcortical and cortical brain architecture is depicted using a ‘connectogram’ [52]. The outer ring
shows the brain regions represented by location. The next inner four rings depict the gray matter
volume, surface area, cortical thickness, and degree of connectivity. Connectivity between regions
was determined using DTI and probabilistic tractography. The color of the links represents the
distribution of fractional anisotropy. The number of fiber tracks between regions is represented by
the transparency of the line
microbiota on gray matter structure is likely most profound during development,

and has been shown in rodent models [56]. However, given that alterations in brain
function and behavioral symptom changes occur in response to probiotic interven-
tions in adults, it is likely that structural changes will follow.
Another MRI-based modality of assessing brain structure is diffusion tensor
imaging (DTI), which allows the evaluation of white matter integrity and anatomy.
DTI can assess the connectivity between gray matter regions via white matter tracts,
measuring the fiber pathways that support functional networks. Two main types of
DTI analyses are frequently performed [57]. In the first, white matter tract integrity
is measured, most commonly expressed as fractional anisotropy (FA), although
additional measurements, such as radial or mean diffusivity are also used. This
technique assesses the diffusivity of water in the brain tissue. Water molecules
unconstrained by cellular architecture, such as in the CSF, freely move in all

directions (isotropic) and thus have a FA value of 0. However, water molecules
in dense, parallel white matter tracts containing axons are constrained and have
high FA values. Decreases in the FA of white matter tracts can indicate decreased
axonal number, myelin integrity, or axonal cytoskeleton integrity. The other DTI
analysis method, tractography, allows quantification of fiber density between brain
regions, and is commonly used to describe limited or whole brain networks.
It has yet to be clearly defined whether the differences in brain structure in
disorders of the brain-gut axis are a result of the chronic condition or a predisposing
factor, though there is a great likelihood that both pathways occur. Associations
between brain structure and microbiota profiles have not yet been described but
provide an opportunity to better understand the interactions between the luminal
contents and the brain.
Neuroimaging in Animals
Imaging the brain in animals is also achieved with MRI and PET, as well as more
direct radiotracer studies. Rodent fMRI and PET provide fair spatial and temporal
resolution but require restraint and/or sedation of the animal to avoid movement,
which may confound the interpretation of the functional results. Autoradiography
allows neuroimaging in non-sedated, nonrestrained animals. A radiotracer is
injected and after the experiment the animal is sacrificed and the brain is
cryosectioned to identify regional tracer uptake, allowing a very detailed view of
the involved neural circuitry [58]. Using animal imaging in parallel with modula-
tion of the microbiota is likely to inform human studies as animal studies allow for
the control of more variables and ability to perform post-mortem studies of the
brain.
Incorporation of Behavioral and Gastrointestinal

Measurements to Neuroimaging Studies
Preclinical studies have been useful in identifying potential behavioral and periph-
eral measures that are of particular relevance in examining the MGBA. Modulation
of gastrointestinal flora in rodents by using specific bacterial strains, antibiotics, or
by using germ-free animals has shown associations with anxiety-like behavior
across multiple paradigms [20, 21, 56, 59, 60]. Rodent models of anxiety-like
behavior are well developed and show responses to pharmacological agents, such
as selective serotonin reuptake inhibitors, indicating the presence of relevant shared
core neural circuitry with humans. In humans, measures of anxiety and depression
including clinical diagnosis, trait measures and psychological symptoms correlate
with brain structure and function [61–63]. Similar to the findings in rodent models,
the ingestion of a Bifidobacterium and Lactobacillius containing probiotic in
healthy humans showed diminished psychological symptoms, including anxiety
symptoms in a placebo controlled randomized clinical trial [64]. The central
mechanisms through which these symptoms change can be probed with neuroim-
aging, using symptom measures as covariates. In addition to looking at the inter-
actions between psychological symptoms and brain function when modulating the
microbiota in clinical trials, additional gastrointestinal measures such as intestinal
permeability, immune activation, motility and visceral sensitivity will be useful in
better elucidating gut to brain communication.
Evaluating the MGBA in the Era of Big Data
The ability to analyze the large datasets produced by neuroimaging studies and
microbiota profiling has been advancing rapidly [65]. While studies evaluating
effects of single organisms or probiotic consortia on the brain will continue to be
of great interest; the emerging use of systems biology approaches to the under-
standing of the relationship between complex structural and functional neural
networks and the microbiome is likely to advance our understanding of the
MGBA tremendously [66]. Both the microbiome and the brain act within integrated
networks for which classical hypothesis driven analytic approaches are not ideal.
Agnostically applied multivariate analysis techniques are being used to identify
neural networks to develop biomarkers of complex diseases, such as chronic pain,
anxiety and depression. These approaches can be utilized to combine complex
imaging datasets with genomic, metagenomic and metabolomic data to study the
interaction between neural and microbial networks [67]. Since current evidence
suggests that the gastrointestinal microflora are likely to play a role in the devel-
opment and persistence of these disorders, it will be important to look at the
interactions between brain phenotypes and the gut microbiome.
Limitations in Neuroimaging of the MGBA
In both the imaging of animal and human MGBA there are a number of limitations.
In animals, we have the ability to meticulously manage the presence or absence of
specific microorganisms, we are able to image the brain in both direct and indirect
ways, and we can observe the effects of various environmental pressures on the
developing animal. However, we are faced with the difficulty of translating the
relevance of behavior from rodent models to humans, and must deal with the clear
differences in the brain between species. As stated by Craig, “A rat is not a monkey
is not a human” [68]. He and others [69] have described the difficulties of the bench
to clinical translation with a particular focus on interoception and pain processing,
but similar arguments can be made for the study of the stress response, emotion and
cognition. If an animal model, as Craig describes in the case of the rodent, lacks the
anterior insular cortex, the site in which our subjective sense of physical wellbeing
may arise, and if the basic pathways through which the visceral afferents commu-
nicate with emotional and cognitive centers vary, then our animal models of
complex phenomena must be interpreted with caution.
In humans on the other hand, we have great limitations in our ability to study all
three branches of the MGBA precisely. Our access to the gut is limited and most
data samples are collected non-invasively, via the stool. This allows us to examine
the gut microbiome in broad strokes, but does not differentiate between the luminal
and mucosal environment, much less local microenvironments or regional differ-
ences throughout the gut [70, 71]. In humans the effects of diet, medications, and
external stressors on microbiota content, gastrointestinal motility and immune
function are difficult to account for even in the most carefully controlled experi-
ments. Additionally, it is likely that many of the MGBA pathways affected by the
microbiota are established early in life, while the brain has its most rapid and
dramatic remodeling [72]. Despite these concerns, the combination of human and
animal imaging, using a translational or reverse-translational model [73–75] may
prove to be the most effective and flexible strategy in evaluating the role of the gut
microbiome in brain function, mood and cognition.
Conclusion
Neuroimaging of the MGBA is in its infancy but will clearly be an important

modality on the road to understanding the role of microbes in many aspects of
health and disease. The current focus on disorders of gastrointestinal disease, such
as inflammatory or function bowel diseases, is already shifting to the study of
anxiety and depression, metabolic diseases and neurologic disease. With this shift,
incorporation of neuroimaging techniques will allow us to measure the rich con-
nectivity between three complex systems: the microbiota, gut and brain.
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Chapter 19
The Future of Probiotics for Disorders
of the Brain-Gut Axis
Eamonn M.M. Quigley and Fergus Shanahan
Abstract Probiotics, or at the very least products that might have probiotic prop-
erties, have been with us for decades, if not centuries, but it has only been in recent
years that they have been subjected to serious scientific study. This surge in interest
in probiotics has coincided with the era of the microbiome; as more and more is
understood about the gut microbiota in health and disease, the therapeutic option of
modulating the microbiota through the administration of probiotics has gained a
more secure foundation. Regrettably, while a vast literature attests to the beneficial
impact of probiotics in a variety of animal models and the mechanisms underlying
such positive effects have been dissected in great detail, the data base on probiotics
in man remains pretty slender.
To make progress, a number of basic issues need to be addressed: strain
characterization and other aspects of quality control need to be rigorously applied
and additional steps such as dose optimization, definition of desired site of effect
and tailoring of formulation accordingly accomplished before large scale trials,
based on appropriately selected study endpoints and employing a clinically mean-
ingful study duration, are embarked upon. Meantime, it is to be hoped that
the regulatory climate will have been clarified and appropriate guidelines for the
evaluation of probiotics, whether as food or drug, developed. Ultimately, the
current terminology may have to be abandoned as evidence for biological and
clinical activity for dead bacteria, bacterial components and bacterial products
accumulates.
E.M.M. Quigley (*)

Division of Gastroenterology and Hepatology, Medicine Department, Houston Methodist
Hospital, 6550 Fannin Street, SM 1001, Houston, TX 77030, USA
e-mail: equigley@tmhs.org
F. Shanahan
Department of Medicine, Alimentary Pharmabiotic Centre, University College Cork, Cork,
Ireland
418 E.M.M. Quigley and F. Shanahan
Abbreviations
EFSA European Food Safety Authority

FGID Functional gastrointestinal disorder
IL Interleukin
SIBO Small intestinal bacterial overgrowth
Introduction
The reader may be excused for hesitating when confronted by a piece that purports
to address the future of an issue or a concept that has been around for almost
100 years; surely the future of such an entity is not in question and certainly, you
will say, its course should by now be clearly set? Yet so capricious has been the
story of probiotics over this century that it has been only in very recent years that
the true potential of this field has come to be appreciated and a few glimpses of the
future fleetingly snatched. Before we embark on that most risky and, some would
say, doomed, of tasks, namely, predicting the future, let us take stock, firstly, of
where we are and, secondly, of the issues that still confront us. Perhaps, if we can
crystallize the latter; the future may take care of itself.
Disorders of the Gut-Brain Axis
The list of disorders wherein that bidirectional channel of communication, the

brain-gut axis, may play some role is a long one and extends from situations
where the brain, the gut and their linkage, through the autonomic nervous system,
are affected by the same pathologic process, as in Parkinson’s disease [1], to those
instances where neurologic symptoms are a consequence of a primarily gastroin-
testinal pathology, as in the malabsorption syndromes [2], and, finally, to those
common gastrointestinal symptoms that afflict us all when stressed and reflect the
impact of signals of central origin on a number of gastrointestinal functions
[3]. This paradigm is also invoked to explain symptoms in a broad spectrum of
common gastrointestinal disorders of uncertain etiology, the functional gastroin-
testinal disorders (FGIDs) [4]. Several entities have been included under this
umbrella ranging from functional heartburn and non-cardiac chest pain, at one
end of the gastrointestinal tract, to functional constipation, at the other. Of these
disorders, irritable bowel syndrome (IBS) is the best characterized and most
thoroughly investigated. While the microbiota has been implicated in the patho-
physiology of other FGIDs; the role of Helicobacter pylori in functional dyspepsia
[5] and descriptions of alterations in the fecal microbiota in chronic constipation [6]
19 The Future of Probiotics for Disorders of the Brain-Gut Axis 419
being two examples, investigations of microbiome-host and microbiome-gut-brain

interactions have been far more extensive in IBS.
Clinical Evidence for a Role of the Microbiota in Disorders

of the Gut-Brain Axis; Lessons from IBS
Irritable bowel syndrome (IBS) is one of the most common gastrointestinal ail-
ments worldwide affecting anywhere from 5 to 15 % of adults in the general
population [7]. Despite considerable effort, a biomarker(s) specific for IBS has
not been identified [8] and its definition remains entirely clinical, based on the
presence of abdominal pain/or discomfort associated with altered bowel habit, often
accompanied by symptoms of bloating and distension [9]. The spectrum of symp-
tom severity in IBS is broad with the majority of those affected never seeking
medical advice but self-medicating or instituting dietary or life-style measures to
control symptoms. At the other end, are a smaller number of individuals whose
symptoms are so debilitating that they impose a very significant impact on quality
of life. Interestingly, IBS is commonly associated with other FGIDs and has also
been linked with extra-intestinal disorders ranging from fibromyalgia and chronic
fatigue syndrome to depression; leading to the speculation that there may some
common etiological factors for all of these disorders.
Over the years, altered gut motility, visceral hypersensitivity, aberrant brain
perception of visceral events, dysregulated stress responses and altered autonomic
function have all been invoked to explain the genesis of symptoms in IBS; phe-
nomena that can be collectively incorporated into the concept of the gut-brain axis
[10]. Thus, within the spectrum of clinical presentation, symptom severity and
natural history that is IBS once can begin to visualize individuals in whom
symptoms are primarily of gut origin and, at the other end, where abdominal pain
and altered bowel habit are primarily driven by central factors, such as stress or
psychopathology.
That the microbiota might be a factor in IBS was first suggested by the obser-
vation, in several large series, that IBS could develop de novo in the aftermath of
acute enteric bacterial, viral and parasitic infections [11]. More recently, modern
sequencing technology has been applied to the study of the microbiota in IBS, in
general, and relationships between a variety of clinical and demographic parame-
ters and the microbiota investigated. To date, studies have been largely based on the
assessment of fecal samples, despite evidence that fecal and colonic mucosal
populations may be quite different in IBS subjects [12–15]. While data remains
limited, it is evident that IBS patients have an altered microbiota relative to healthy
individuals. Bacterial diversity is reduced [12] and more detailed analyses have
identified differences at species and strain level [13–26] among both children [19,
20] and adults [13–18, 21–26] with IBS. Not surprisingly, given the heterogeneity
of the IBS phenotype, these results have not been consistent and the sizes of the
study populations involved have not been large enough to encompass the entire
symptom and demographic spectrum that is IBS.
More controversial has been the suggestion that small intestinal bacterial over-
growth (SIBO) plays a role in IBS [27, 28]. There are several problems with this
proposal; firstly, SIBO, per se, is difficult to define [29]; secondly, the methodology
primarily employed in studies supporting a role for SIBO, the lactulose breath
hydrogen test, is subject to considerable problems in relation to sensitivity and
specificity [29, 30]; and, finally, studies of the prevalence of SIBO in IBS have been
highly variable [31]. Nonetheless, if SIBO is indeed a factor in IBS, the potential for
SIBO to modulate gut-brain axis communication has already been amply
documented in the context of hepatic encephalopathy [32].
Probiotics in IBS
Other clinical evidence apart from the aforementioned supports the potential role
of interventions that could modulate the microbiota in IBS. Firstly, modulation of
the microbiota could impact on two important functions of colonic bacteria, bile
acid metabolism and fermentation and, thereby, alter stool consistency and flatus
production, respectively. Secondly, and though its precise mechanism of action
remains unclear, the broad spectrum, poorly absorbed, antibiotic rifaximin has been
shown, in large clinical trials, to ameliorate the cardinal symptoms of IBS
[33]. Thirdly, and most germane to this volume, is evidence for efficacy of certain
probiotics in IBS [34].
Probiotics have, indeed, been used on an empirical basis by IBS sufferers for
decades and remain popular among those who self-medicate. During the latter half
of the last century a number of clinical trials evaluated the efficacy of probiotics in
IBS on a more formal basis. While there are many shortcomings with these studies
(including but not limited to the clinical definition of the study population, non-
randomization, absence of placebo control and small sample sizes) not to
mind variations in strain, dose, and method of delivery, when taken together they
do suggest a trend towards benefit for probiotics in IBS [35]. Indeed, recent meta-
analyses have concluded that probiotics, in general, do benefit patients with IBS
[34, 36–40]. What are more difficult to define are the relative benefits of different
species or strains. In one of these meta-analyses, for example, it was concluded that
Bifidobacterium spp. were effective in IBS while Lactobacilli were not [34]. A
major problem facing any analyst of the literature in this field continues to be the
poor quality of many studies: small study populations, variable end-points, and the
use of various organisms bedevil their interpretation. Indeed, Brenner and col-
leagues went so far as to state that only one organism, Bifidobacterium infantis
35624, had support for efficacy in IBS based on clinical studies of adequate quality
[38]. Since that publication, another strain, Bifidobacterium lactis DN-173-010A,
has shown promise among IBS subjects with constipation-predominant IBS and
prominent bloating [41]. Indeed, the clinical effects of this strain on constipation
Table 19.1 Proposed mechanisms of action of probiotics in irritable bowel syndrome

(IBS)
1. Prevention of post-infectious IBS through anti-bacterial or anti-viral actions
2. Normalizing an abnormal microbiota which, in turn, could alleviate symptoms through:
a. Direct effects of bacteria on mucosal functions
b. The elimination of inflammatory/immunogenic stimuli
c. Alterations in microbial metabolism
i. Fermentation of carbohydrates
ii. Deconjugation of bile acids
iii. Production of short-chain fatty acids
d. An impact on motility
e. Modulation of visceral sensation
f. Effects mediated through the microbiome-gut-brain axis
3. Reducing/eliminating small intestinal bacterial overgrowth
and bloating have been supported by evidence that this bacterium accelerates colon
transit and reduces abdominal distension [41]. Other strains have shown benefits for
specific symptoms, such as bloating [42, 43] or flatulence [44]. While most studies
of probiotics in IBS either did not examine relative effects according to IBS sub-
type of failed to power adequately for such a sub-group analysis, some have
shown benefit exclusively in diarrhea-predominant IBS [45]. How probiotics exert
these beneficial actions in IBS is unclear. A number of proposals have been made
and these are summarized on Table 19.1. While the proposal that probiotics could
influence the central nervous system is based primarily on animal studies [46], a
recent brain-imaging study in human volunteers suggests that orally administered
probiotics can modulate brain responses [47].
Probiotics 2013; Where Are We?
The concept of probiotics has been with us since the observations of Metchnikoff
among Bulgarian peasants in the first decade of the last century. For much of the
intervening time, however, the concept has languished in the realm of “alternative”
or “natural” medicine and scarcely attracted the interest of either science or
conventional medicine. Several factors have, of late, conspired to dramatically
change the profile of probiotics and the probiotic concept. These include, firstly,
rapid progress, now aided by constantly evolving molecular techniques, in our
appreciation of the vital role of the gut microbiota and its interactions with the
host in health and disease and, secondly, the application of modern science to the
study of probiotics per se. This has resulted in the accurate classification of
individual probiotic organisms, as well as detailed descriptions of their genetic,
microbiological and immunological properties, and has led to extensive in vivo and
in vitro studies of the impact of various probiotics on a variety of biological systems
and, most recently, to well conducted clinical trials of probiotics in specific clinical
scenarios in man and domestic animals.
Despite all of this progress several problems persist in relation to these areas that
continue to sully the image of probiotics and muddy the field. It is important at this
stage to reflect on the most widely accepted (FAO/WHO) definition of a probiotic:
Live microorganisms which when administered in adequate amounts confer a health benefit
on the host [48].
Two issues deserve special emphasis: the focus on “live” organisms and the
insistence on conferring “a health benefit on the host”. Firstly, while it is readily
acknowledged that studies in a number of animal models have demonstrated
efficacy for killed bacteria, or even bacterial products or components [49–51], in
generating a number of anti-inflammatory and anti-infective effects, this strategy
has not, as yet, been explored or validated in man. It seems improbable that effects
of probiotics in man will be confined to live organisms so this aspect of the
definition will ultimately have to be refined or the term abandoned completely.
Secondly, it is obvious from the latter part of this definition that clinical claims in
man be they in the augmentation of health or in the treatment of disease, must be
supported by credible clinical trial data. Up until the last few years, probiotics were
not regulated as drugs and had been able to come on to the market as food
supplements or under other designations that have allowed them, to a greater or
lesser extent, to make a variety of “health” claims in the absence of supporting data.
Recent deliberations from and decisions by the European Food Safety Authority
(EFSA) demonstrate a seismic shift in attitude to the extent that the very use of the
term probiotic has been deemed to represent a tacit health claim and its use
restricted to those “probiotics” that can support such a claim. To date no “probiotic”
product, despite their safety and considerable clinical trial data, has received the
imprimatur of EFSA. It seems that those who developed and widely promulgated
the current definition of a probiotic have now been hoist on their own petard; truly a
case of man bites dog. A similar level of scrutiny is now being leveled on these
products in North America and elsewhere.
This impasse should have been foreseen. At present the consumer is not being
served, not only by the aforementioned issues relating to “health” claims, but also
by major problems with quality control. Firstly, it is not unusual for the benefits of a
given species or organism to be touted based on evidence derived from studies
involving other organisms and species, despite the fact that detailed studies have
demonstrated that, in terms of a probiotic property, be it immune modulation [52–
55] or anti-bacterial activity [50, 53, 56], there are tremendous differences between
different lactobacilli and bifidobacteria, not to mind between lactobacilli and
bifidobacteria, for example. No two probiotics are the same and extrapolations
from one to another should be resisted at all times. Secondly, an individual who is
about to consume a given probiotic preparation should know exactly what he or she
is about to take: is it live (if that is necessary for its benefit), what is it’s concen-
tration, will the organism survive as it makes contact with acid, bile and digestive
enzymes as it transits the gut and what will be the actual concentration of the
organism at its desired site of action? Few probiotic preparations have been
characterized and formulated with sufficient rigor to allow the manufacturer to
provide answers to these critical questions. Of further concern, critical examina-
tions of the actual constituents of commercially-available probiotic preparations
have, in the past, revealed worrying deviations from those included in the product
label [57–61].
Nevertheless, evidence for efficacy for specific probiotics in certain clinical
conditions continues to accumulate. Most notable have been studies in diarrheal
illnesses. Several studies have reported that probiotics may be effective in short-
ening of the duration of acute diarrheal illnesses in children, such as that related to
rotavirus infection [62]. Probiotics also appear to be effective in antibiotic-
associated diarrhea [63–65], pouchitis [66, 67], some instances of inflammatory
bowel disease [68, 69], and, as already described above, irritable bowel syndrome
[55, 70, 71].
The Future of Probiotics
Rather than make wild speculations regarding the future, or even risking modest
predictions, we will now attempt to identify those areas where, we believe, the
greatest challenges persist and the most important questions remain unanswered.
Quality Control and Regulation
If the field of probiotics is to progress further and gain acceptance within the
hallowed halls of science, quality control and appropriate regulation must occur.
Inevitably, this will take place on a nation-by-nation basis but, however accom-
plished, must ensure that the consumer or the prescriber is sufficiently informed of
the nature of any given product and assured of the accuracy of its label, including its
shelf life, and the validity of health claims. It is incumbent on the medical and
scientific communities to actively engage in these processes and to thereby ensure
that new requirements and regulations in relation to quality control have scientific
credibility and validity. This is a matter of great urgency; failure may result in a
gradual ebbing away of confidence in the entire area and the loss of valuable
products because the public simply cannot differentiate them from impostors.
Probiotic Characterization
As individual probiotic organisms are subjected to genomic analysis [72] the stage
is set for both the accurate definition of each individual organism and the
identification, on the genome, of areas of interest in relation to a particular property

or action. This must be the way forward for both the definition of individual
organisms and the comparison of their individual characteristics. Parallel develop-
ments such as the various collaborative projects defining the human microbiome in
health and disease will ultimately lead to a complete description of the microbiome
and its metabolic properties and in so doing will facilitate a complete delineation of
the interactions (good and bad) between bugs and the host. In so doing, considerable
progress should be made in defining the basis for the beneficial actions of probiotic
bacteria.
Mechanism of Action
While genomics and metabolomics may suggest certain roles for certain probiotics,
these must, ultimately, be further elucidated in appropriate biological systems,
including man. Indeed, a further component of the characterization of a probiotic
must be the definition of it effects, if any, in a variety of contexts. Does the
organism exert anti-bacterial or anti-viral properties, what are its effects on immune
responses or metabolic processes? Again a standardized and validated approach to
the interrogation of a given organism in relation to a particular use must be
developed, where possible. Currently, the methodologies and test systems to be
employed to assess the efficacy of an organism against, say Clostridium difficile, are
well characterized but how does one evaluate the potential impact of an organism in
IBS, a disorder whose pathogenesis remains unknown? With regard to the latter,
one can only do what the pharmaceutical industry has done for decades, test the
organism in relation to putative pathophysiological mechanisms such as, in the case
of IBS, dysmotility [73] or visceral hypersensitivity [51, 74, 75]. Proposals to use a
probiotic in man must have a plausible scientific rationale; hype and appeals to
“being natural” should no longer be sufficient.
Waking the Dead
As emphasized at the outset of this chapter, the current definition of probiotics

insists on the inclusion of live organisms. This will undoubtedly change; bacteria
are metabolically active organisms that produce a variety of molecules with bio-
logical activity [51, 75]. As already mentioned, bacterial DNA has been demon-
strated to exert anti-inflammatory activity on certain systems [49, 50]; it seems
reasonable to assume that other bacterial components, such as the cell wall or its
outer coat, may prove effective in certain contexts. The whole area of bacterial
components and bacterial products will be an exceptionally active one in the
coming years. In clinical terms, this approach has already shown dividends through
the isolation of probiotic products with specific anti-bacterial activities [76,

77]. There is much more to come.
More Trials!
Performing clinical trials with probiotics is not easy. Quite apart from the afore-
mentioned issues in relation to strain selection for a given indication, the clinical
investigator is faced with significant obstacles in choosing formulation, dose and
duration of study. Dose is, for the most part, a “black box” in this field, very few
dose ranging studies have been attempted and extrapolations from animal studies
must always remain mindful of the fact that, weight for weight, probiotic doses used
in the mouse or the rat exceed by several orders of magnitude those used in man.
We must attempt to get our doses right! Here, however, we encounter the issue of
formulation; what may be most acceptable to the patient (e.g. a once a day capsule)
may not permit the inclusion of an optimal dose of the organism. These challenges
must and will be met; our obligation then is to ensure the performance of clinical
trials whose design is optimal for the given indication. Only then can we recom-
mend probiotics to our patients.
Probiotics could, in the future act as vehicles for the targeted delivery of
therapeutic molecules to the gut. It has already been shown that probiotics can be
genetically engineered to deliver Interleukin (IL)-10 to the intestinal mucosa using
an ingenious system which ensures that the organism will not survive outside of the
host [78–80].
One great advantage that probiotics currently enjoy in the clinical arena, and in
comparison to conventional pharmaceuticals, is that of safety. We must remain
vigilant in this area and perform the same rigorous and extensive phase IV, post-
marketing, surveys that have become the norm elsewhere. Here again genomic
analysis will provide an important supportive role by identifying pathogenicity
islands or features that suggest the potential for transference of antibiotic
resistance [72].
The changing regulatory climate may alter the approach to clinical trials and
pose challenges for potential investigators; specifically who will fund the trials
which will be required to satisfy the demands of authorities such as EFSA [81, 82]?
If it is decided that a given probiotic product is to be regarded as a food, profit
margins will be slim and the target population will, by definition, be the healthy
population. Such trials will by virtue of their endpoints require very large numbers
of participants and be very expensive. For the food industry, ideal endpoints should
be validated biomarkers of risk, of which there are few (e.g. cholesterol for heart
disease), not biomarkers of early disease (which will not be applicable in this
category). Both issues, size of study population and need for validated biomarkers
of risk, pose huge problems for the food industry which is unlikely to be in a
position to fund such trials. In other words, it may be cheaper to study probiotics as
drugs within the pharmaceutical sector (paradoxically lower costs and higher
margins on licensable product) unless new microbial biomarkers of risk emerge.
New Horizons; Moving Beyond the Gut!
For obvious reasons, including their source and the well-documented interactions
between the microbiota and the gut, studies of probiotics have, to date, concentrated
in large part on intestinal disorders. Hints to suggest efficacy for probiotics in
disorders beyond the gut accumulate with studies illustrating the ability of orally
administered probiotics to modulate systemic cytokine patterns in man towards an
anti-inflammatory phenotype being of particularly interesting [83, 84]. Experimen-
tal and limited clinical data suggest the potential for probiotics to impact on such
extra-intestinal disorders as non-alcoholic fatty liver disease [85, 86], arthritis [87],
allergy [88], obesity [89] and, as has been amply illustrated in this volume,
symptoms arising from and disorders of the central nervous system. The latter is
in keeping with very recent and exciting data on the ability of orally administered
probiotics and other modifications of the microbiota to influence behavior, mood,
cerebral function and morphology in experimental animals [90–92]. Indeed, some
preliminary data suggests that probiotics may be able to modulate brain activity in
man [47]. Given the pro-inflammatory phenotype associated with such psychiatric
disorders as depression and schizophrenia [93, 94] the aforementioned anti-
inflammatory actions of some probiotics may be relevant here also. Here, as
elsewhere, the development of clinical trial endpoints will be a challenge; psycho-
social and behavioral endpoints are symptoms driven and thus prone to consider-
able placebo responses; objective endpoints are relatively few and those that have
been studied, such as brain imaging, are not widely available and expensive.
Ongoing studies of the microbiome-gut-brain axis may reveal, not only more
objective targets for intervention, but also identify those bacterial components or
products that may have optimal biological activity. As our understanding of
microbiota-host interactions increases, new applications for probiotics will arise.
As the true importance of the microbiota in human homeostasis comes to be
recognized the therapeutic potential of probiotics and the broader category of
pharmabiotics [95] can begin to be realized.
Conclusion
Having languished for years in the nether world of the “alternative”, probiotics have
enjoyed a very recent and very rapid acceleration in scientific investigation and
clinical application [96]. In some instances the latter has, regrettably, preceded the
former, an approach that, coupled with continuing issues with quality control and
regulation, continues to dog the credibility of this area. These hurdles can and will
be overcome and will allow scientifically-based and rigorously tested probiotic

products to assume their rightful place in the therapeutic armamentarium.
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Index
A Biosynthesis, 165
Adaptive immunity, 86, 291, 294, 305, Brain-gut, 95, 143, 382, 388, 406–407
309–310 Brain-gut axis, 73–96, 115–127, 135–149,
ANS. See Autonomic nervous system (ANS) 195–197, 205, 222, 224, 375, 377, 379,
Antibiotics, 19, 85, 120, 127, 201, 203, 244, 381, 388–393, 408, 411, 417–427
248, 256, 261, 268, 279–286, 302, 306,
309, 329, 331, 332, 337, 358, 365, 366,
375, 378, 384–387, 389, 391, 392, 409, C
411, 420, 425 Catecholamine, 5, 8–10, 16, 18, 88, 139, 145,
Anxiety, 92, 96, 117, 118, 120, 122, 125, 146, 146, 178, 222, 224, 226–227, 230, 242,
147, 179–182, 191, 197, 201, 202, 204, 248, 259, 295, 406
205, 208, 211, 224, 225, 228, 280, 281, Cell biology, 172
285, 286, 326, 337, 338, 358, 361, 363, Central nervous system (CNS), 11, 15, 17, 18,
364, 366, 381, 384–388, 390, 391, 393, 41, 42, 48–49, 56, 57, 62, 63, 75, 87, 88,
407, 411–413 95, 96, 116, 120, 139, 140, 145, 159,
Autism, 92, 96, 202, 230, 321, 324, 326–328, 182, 195, 198, 199, 203, 205, 222, 226,
340, 341, 343, 384, 387, 391–393 227, 229, 231, 232, 280–282, 284, 299,
Autoinducers, 9, 242–245, 247 321, 324, 328, 340, 342, 343, 360, 375,
Autonomic nervous system (ANS), 75, 86, 136, 377–383, 389–394, 421, 426
137, 147, 257, 258, 366, 377, 378, 383, Citrobacter rodentium, 263, 361, 392
406, 407, 418 Clinical trial, 35, 118, 296, 331, 387, 412, 420,
422, 425, 426
CNS. See Central nervous system (CNS)
B Cognition, 5, 12, 15, 17, 140, 142, 145, 203,
Bacterial adrenergic receptor (BARs), 205, 226, 282, 286, 337–338, 358–367,
245–247, 249 379, 382, 387, 388, 392, 393, 413
Bacterial gene expression, 241–249 Cognitive behavior, 358–361, 363, 366, 367
Bacterial infection, 88, 199, 307, 358, 360, Cognitive function, 227, 228, 281, 282,
362, 382 357–367, 381, 384, 386, 392
Bacterial virulence, 178, 224, 244–247 Colitis, 77, 78, 80, 85, 92, 93, 96, 119, 125,
BARs. See Bacterial adrenergic receptor 126, 145, 147, 203, 207, 210, 232, 263,
(BARs) 266, 267, 271, 280, 328, 330, 361, 363,
Behavior, 4, 9–19, 142, 147, 161, 178, 190, 378, 385, 387
264, 265, 280–286, 337–339, 358–367, Colonic physiology, 88, 259, 285
373–394, 411, 412, 426 Cytokines, 11, 75–77, 79, 80, 84, 85, 87, 90, 93,
Bioinformatics, 27, 28, 35 117–121, 138, 141, 143, 145–147, 172,
DOI 10.1007/978-1-4939-0897-4, © Springer New York 2014
434 Index
197–200, 207, 225, 263–268, 270, 271, G

281–282, 285, 293–304, 308–311, 324, Gamma aminobutyric acid (GABA), 6, 9, 11,
325, 329, 334–340, 342, 379, 382, 383, 12, 125, 183, 188–190, 200, 222–225,
390, 426 268, 283, 340, 378, 383, 390, 406
Gastrointestinal (GI), 10, 19, 39–63, 74, 75,
87–89, 92, 95, 136, 139, 143–145,
D 157–172, 197–201, 203, 204, 209, 210,
DCreg. See Regulatory dendritic cell (DCreg) 223, 227, 243, 248, 282–283, 343, 363,
Depression, 92, 96, 117–121, 125, 126, 143, 391, 406, 408, 409, 411–412, 418, 419
146, 147, 181, 182, 190, 191, 197, motility, 89, 123, 161, 210, 258, 381, 406,
199–201, 203, 207–211, 224, 225, 280, 413
281, 286, 321, 324, 326–328, 335–340, tract, 7, 9, 15, 17, 19, 41–50, 53, 55, 56, 62,
363, 378, 387, 389–393, 407, 411–413, 63, 81–83, 86, 88, 89, 94, 117, 118, 124,
419, 426 159–162, 164, 167, 170, 178, 183, 184,
Diet, 12, 14, 16–17, 94, 120, 121, 141, 148, 196, 197, 206, 211, 223, 227, 229, 231,
231, 256, 261, 263, 293, 294, 299, 300, 245, 247–249, 256, 258, 259, 360, 377,
302–305, 307, 309, 311, 331, 364–366, 380, 381, 389, 407, 418
375–377, 380, 386, 387, 413 GBA. See Gut brain axis (GBA)
Diffusion tensor imaging (DTI), 410, 411 Growth factors, 75, 91, 93, 157, 159–163, 172
Digestion, 57, 63, 91, 158, 170, 172, 178, Gut brain axis (GBA), 149, 196–200, 203, 205,
197, 198, 200, 375 206, 209–210, 280, 282–284, 286, 361,
Disease, 4, 5, 7–9, 13, 20, 26–29, 31, 32, 35, 363, 408–409, 418–420
47–49, 78, 81, 85, 86, 93–95, 119, 120, Gut ecosystem, 25–35, 387
135–149, 181, 197, 200, 211, 223–226, Gut hormones, 41, 54, 117, 159, 160, 162, 164,
228, 245–247, 259, 268, 280–282, 286, 166, 167, 170–172, 197, 198, 203, 207,
293, 294, 296, 302, 304, 307, 308, 311, 209–211, 381, 383
312, 324, 326–328, 330, 332, 335, 336, Gut microbiota, 125, 178–183, 185, 198–211,
343, 344, 358, 361, 363, 365, 379, 382, 223, 228, 231, 232, 248, 258–268, 270,
383, 389, 391–393, 408, 409, 412, 413, 293, 294, 300–312, 321, 329, 331–334,
418, 421–426 336, 343, 344, 367, 374–381, 383, 384,
389–394, 409, 421
Gut-to-brain, 10, 14–18
E
Endocrine cells, 50, 55, 159, 167, 169–171, H
196, 198 Health, 7, 26, 35, 49, 89, 91, 135–149, 197,
Enteric nervous system (ENS), 6, 7, 11, 15, 17, 211, 223, 225, 226, 228, 230, 248, 269,
18, 39–63, 75, 76, 89, 120, 122–124, 280, 286, 325, 326, 330, 360, 365–367,
138, 197, 200, 222, 223, 228, 283, 285, 375–377, 379, 382, 383, 387, 388, 390,
377, 378, 383, 389, 407 393, 409, 413, 421–424
Evolution, 5, 8–9, 19, 163, 164, 269, 325, Helminth, 322, 323, 325, 329–331, 334,
375, 384 336, 344
Hormone(s), 5–11, 18, 19, 41, 43, 44, 54,
55, 62, 82, 86–91, 139, 140, 146,
F 157–172, 177–191, 197, 198, 202–204,
Fatty acids, 59, 121–123, 144, 201, 223, 224, 207, 209–211, 241–249, 257, 259, 266,
229–231, 294, 297, 301, 303–305, 383, 268, 381, 383
389, 421 genes, 164–166, 168
Food intake, 117, 137, 148, 161, 172, 198, 205, receptors, 171, 172
210, 299 Hygiene, 120, 321, 344
Functional MRI (fMRI), 147, 337, 364, 408, Hypothalamus-pituitary-adrenal axis, 75, 139,
409, 411 202, 381, 383
Index 435
I M
IBD. See Inflammatory bowel disease (IBD) Macrophage, 7, 82–84, 90, 92, 119, 120, 138,
Immune system, 10, 41, 50, 74–76, 82–86, 137, 145, 146, 206, 207, 232, 242, 245–247,
141, 144, 178, 198–200, 204–207, 209, 266–270, 281, 293–300, 302, 303,
271, 281, 283, 293, 294, 296, 297, 300, 307–311, 329–331, 338, 339
306, 309, 310, 312, 321–323, 326, 329, Mast cells, 55, 75, 79, 82–85, 87–90, 93–96,
333, 334, 341, 342, 344, 360, 375–377, 145, 270, 271
381–383, 388, 406, 407 Metabolome, 14, 17, 286, 359
Immunoregulation, 322–326, 329–337, Metagenomics, 14, 28, 33–35, 394, 409, 412
340–343 MGBA. See Microbiota-gut-brain axis
Infection, 7–9, 11, 16, 48, 78, 85–88, 91, 117, (MGBA)
124, 148, 199, 207, 208, 242, 243, Microbial metabolites, 199, 409
245–248, 261, 263–265, 267, 271, Microbial organ, 6, 17
279–286, 293, 299, 303, 306, 307, 310, Microbiology, 4, 10, 35, 377
311, 321, 323, 329, 330, 332, 334, 336, Microbiome, 5, 12, 14, 27, 30–35, 115–127,
337, 341–342, 358, 360–362, 366, 382, 177–191, 223, 226, 227, 231, 247, 248,
385–387, 419, 423 261, 284, 358, 359, 375–377, 381–394,
Inflammation, 26, 46, 75, 77, 78, 80, 84, 85, 405–413, 419, 421, 424, 426
88–90, 92, 93, 95, 96, 118–121, 124, Microbiota, 3–20, 26–35, 39–63, 73–96,
126, 140, 141, 145–148, 162, 181, 115–127, 135–149, 157–172, 177–191,
207–210, 229, 259–268, 270, 271, 195–211, 221–232, 241–249, 255–271,
279–286, 291–312, 321–325, 279–286, 291–312, 319–345, 357–367,
330–333, 335–344, 358, 363–365, 373–394, 405–413, 417–427
379, 380, 391 Microbiota-gut-brain axis (MGBA), 3–20, 191,
Inflammatory bowel disease (IBD), 26, 84, 195–211, 222, 279–286, 357–366, 385,
85, 89, 92, 96, 119, 145–147, 197, 406–409, 411–413
207, 225, 263, 280, 286, 321, 324, Motility, 48, 51, 52, 56, 57, 60, 62, 75, 76, 89,
326, 332, 336, 344, 358, 360, 90, 95, 96, 122, 123, 137, 161, 162, 170,
363–364, 367, 408, 423 183, 188, 201, 205, 210, 223, 244, 248,
Inflammatory cytokine, 80, 119, 120, 146, 258, 270, 363, 378, 379, 381, 406, 412,
263, 264, 266–268, 294, 301, 303, 413, 419, 421
304, 308–310, 335, 337–339 Myenteric ganglia, 43–47, 49, 51–53, 62, 140
Innate immunity, 83, 296, 300–308, 329,
379, 391
Insulin resistance, 231, 293–298, 301–303, N
305–308, 310 Neuroactive, 5, 6, 9–11, 13–19, 122, 123,
Interkingdom communication, 243–244 190, 200–201, 221–232, 281, 284,
Intestinal barrier function, 73–96, 306 380, 390
Intrinsic primary afferent neuron (IPAN), 42, Neurobiology, 4, 393
51, 52, 59, 60, 122, 123, 140 Neuroendocrine (NE) stress hormones, 5–11,
Irritable bowel syndrome (IBS), 26, 81, 18, 19, 241–249, 259, 268
87–89, 95, 96, 144–145, 147, 197, Neuroimaging, 12, 364, 391, 405–413
280, 286, 343, 358, 363–364, 367, Neuropeptides, 5, 22, 75, 90, 143, 159,
380, 387, 390–392, 408, 409, 418–421, 161–163, 166, 168, 172, 195–211
423, 424 Neuropeptide Y (NPY), 53, 140, 142, 161, 197,
203–211, 381
Nutrition, 12, 14, 17, 120, 121, 198, 282
L
Lactobacillus, 6, 11, 12, 88, 92, 122, 123, 125,
126, 190, 200, 203, 204, 223–226, 228, O
229, 232, 258, 260, 261, 264, 265, 269, Obesity, 32, 120, 148, 202, 231, 291–312, 332,
270, 331, 332, 336, 337, 364–366, 376, 338, 426
378, 387, 388, 390 Old friends mechanism, 321–325, 340, 344
436 Index
P Regulatory T (Treg), 119, 294, 297, 300,

Pain, 46–49, 62, 87, 88, 95, 96, 137, 139, 143, 309, 310, 323, 324, 330, 331, 343,
144, 146–148, 197, 198, 204, 205, 208, 381, 382
209, 343, 363, 364, 378, 379, 384, 387, RNA-seq, 33–34
394, 408, 409, 412, 418, 419
Pathogen, 5, 7, 8, 10, 11, 15, 26, 28, 49, 56,
60, 61, 81, 83–86, 91, 124, 141, 178, S
179, 226, 241–249, 259, 261, 263, 264, Satiety, 42, 43, 141, 148, 162, 201, 202, 210
266–268, 270, 271, 283, 286, 294, 299, Schizophrenia, 321, 326–328, 339, 341, 344,
300, 311, 322, 323, 332, 361–363, 366, 392, 426
375, 381 16S sequencing, 27–30, 33, 35
Peptides, 19, 55, 76, 78, 81–85, 89, 90, 117, Strain characterization, 417
123, 138, 141, 144, 148, 159–170, 172, Stress, 5, 7, 8, 14, 86–92, 95, 96, 136, 143,
196, 198, 199, 201–207, 210, 211, 222, 145–147, 178–182, 191, 203, 205,
244, 246, 247, 381 208–210, 242, 243, 245–247, 249,
Peptide YY (PYY), 55, 82, 117, 161, 163, 201, 257–262, 265, 268, 269, 280, 282, 292,
202, 204, 206–208, 210, 211 296, 300, 303, 306, 308, 319, 321,
Peroxynitrite, 269 325–327, 335–337, 358–362, 364–366,
Pharmabiotic, 426 375, 377–379, 381, 383, 385–387,
Probiotics, 11–13, 88, 92, 203, 222–224, 228, 389–390, 392, 406, 413, 419
231, 232, 264, 267, 268, 283, 329–331, Stress resilience, 181–182, 191, 205, 209, 321,
358, 361, 364–367, 375, 378, 380–382, 325–326, 335, 337, 381
386–392, 408–410, 412, 417–427 Structural MRI, 409–411
Psychobiotic, 221–232, 388, 390, 394 Submucosal ganglia, 45–47, 50–53, 55, 62
Psychological stress, 87–90, 95–96, 280,
359–361, 389, 409
Psychobiotics, 221–232, 388, 390, 394 T
PYY. See Peptide YY (PYY) Treg. See Regulatory T (Treg)
Q V
Quality control, 422, 423, 426 Vagus nerve, 18, 54, 57, 58, 62, 67, 116–121,
124–127, 142, 199, 280, 281, 338, 340,
378, 383, 390, 406
R Visceral hyperalgesia, 147
Regulation, 4, 60, 75, 79–81, 89, 90, 92–95, Visceral pain, 95, 137, 143, 204, 209, 379, 384,
117, 119, 120, 147, 148, 158, 161, 162, 387, 391, 409
167, 172, 178, 180, 197, 198, 200, 204, Visceral sensation, 117, 145, 421
205, 207–211, 225, 226, 229, 230, 243–
245, 247–249, 271, 281, 283, 285, 293,
299–311, 313, 329, 331–333, 344, 359, W
361, 377, 379, 381, 382, 406, 423, 426 Whole genome metagenomic sequencing,33–
Regulatory dendritic cell (DCreg), 330–331 34
Advances in Experimental Medicine and Biology 902
Andreas Schwiertz Editor
Microbiota of
the Human
Body
Implications in Health and Disease
Advances in Experimental Medicine
and Biology
Volume 902
Editorial Board
Irun R. Cohen, The Weizmann Institute of Science, Rehovot, Israel
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Andreas Schwiertz
Editor
Microbiota of the
Human Body
Implications in Health and Disease
Editor
Andreas Schwiertz
Institute of Microecology
Herborn, Germany
ISSN 0065-2598 ISSN 2214-8019 (electronic)

Advances in Experimental Medicine and Biology
ISBN 978-3-319-31246-0 ISBN 978-3-319-31248-4 (eBook)
DOI 10.1007/978-3-319-31248-4
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Preface
Microbes can now be found in nearly every niche the human body offers.
However, the complexity of the microbiota of a given site depends on the
particular environmental condition thereof. Only microbes that are able to
grow under these conditions will prevail. Recent publications imply that the
microorganisms do not only have multiple critical consequences for host
physiological processes, such as postnatal development, immunomodulation
and energy supply, but also effects on neurodevelopment, behaviour and
cognition.
Within this book we will focus on the techniques behind these develop-
ments, epigenomics and on the various parts of the human body, which are
inhabited by microorganisms, such as the mouth, the gut, the skin and the
vagina. In addition, chapters are dedicated to the possible manipulations of
the microbiota by probiotics, prebiotics and faecal transplantation.
I would like to express my gratitude to all chapters’ authors for their con-
tribution to this book and hope that it will be appreciated by readers as well
as it occurred to me as an editor.
Herborn, Germany Andreas Schwiertz
v
Contents
1 A Short Definition of Terms ........................................................ 1

Andreas Schwiertz and Volker Rusch
2 Studying the Human Microbiota ................................................ 5
Alan W. Walker
3 The Gut Microbiota and their Metabolites:
Potential Implications for the Host Epigenome .............................. 33
Mona Mischke and Torsten Plösch
4 The Oral Microbiota ......................................................................... 45
Nicole B. Arweiler and Lutz Netuschil
5 The Microbiota of the Human Skin................................................. 61
Markus Egert and Rainer Simmering
6 Vaginal Microbiota ........................................................................... 83
Werner Mendling
7 The Human Gut Microbiota ............................................................ 95
Hermie J.M. Harmsen and Marcus. C. de Goffau
8 Manipulation of the Microbiota Using Probiotics........................ 109
Verena Grimm and Christian U. Riedel
9 How to Manipulate the Microbiota: Prebiotics ............................ 119
Petra Louis, Harry J. Flint, and Catherine Michel
10 How to Manipulate the Microbiota: Fecal Microbiota
Transplantation............................................................................... 143
Susana Fuentes and Willem M. de Vos
Index ........................................................................................................ 155
vii
A Short Definition of Terms
1
Andreas Schwiertz and Volker Rusch
Keywords
Definition • Microbiota • Microbiome
We humans are colonized by myriads of micro- The estimated mass of the microbiota (1–2 kg in
organisms in various parts of the body, such as an adult body (Forsythe and Kunze 2013)) is
the skin, the mouth, the vagina and the gastroin- comparable to the weight of the adult human
testinal tract. Even the lung and other hitherto brain (ca. 1.5 kg, Parent and Carpenter 1996).
thought to be sterile parts, as the placenta, are As of today our knowledge on the human
now considered to be colonized. Furthermore, microbiota is due to the fast evolution of
our microbiota is not only comprised of bacteria, sequencing. On the 14th of April 2003 the com-
but also of archaea and eukaryotes such as proto- pletion of the human genome sequencing pro-
zoa, fungi and nematodes. Even viruses, collec- cess was announced and in 2004 the quality
tively termed the virome, can be found in the assessment of the human genome sequence
microbiota (Virgin 2014). It has been estimated finally published. Since then huge efforts have
that the human-associated microbiota, consists been undertaken to sequence other important
of at least 40,000 bacterial strains in 1800 genera genomes like that of the rat (rattus norvegicus),
(Luckey 1972; Frank and Pace 2008; Forsythe the honey bee (apis mellifera) and even the
and Kunze 2013), which collectively harbor at Neanderthal. In 2008 the national institute of
least 9.9 million non-human genes (Li et al. health decided to fund the Human Microbiome
2014). They encode for approximately 500 times Project (HMP). Goal was the “characterization
the human protein-coding genes which are cur- of the human microbiome and analysis of its role
rently annotated (http://www.ensembl.org). in human health and disease” (http://hmpdacc.
org/) (Turnbaugh et al. 2007; Human Microbiome
A. Schwiertz (*) Project Consortium 2012). In parallel the
Institute of Microecology, MetaHIT project financed by the European
Auf den Lueppen 8, 35745 Herborn, Germany Commission under the 7th FP program was
e-mail: andreas.schwiertz@mikrooek.de launched. Its aim was to “establish associations
V. Rusch between the genes of the human intestinal micro-
Institute of Integrative Biology, biota and our health and disease” (http://www.
35745 Herborn, Germany
e-mail: rusch@rusch-chateau.ch metahit.eu/) (Qin et al. 2010).
© Springer International Publishing Switzerland 2016 1

A. Schwiertz (ed.), Microbiota of the Human Body, Advances in Experimental
Medicine and Biology 902, DOI 10.1007/978-3-319-31248-4_1
2 A. Schwiertz and V. Rusch
Interestingly, as seen in the two mentioned (Goetz 1950). In the context of human health the
projects the terms microbiome and microbiota are term microbiota was first used to describe the gin-
used analogical even if their scientific definition is gival crevice (Socransky et al. 1953), while it did
dissimilar. The term “microbiota” describes the not appear before 1966 for the description of the
total collection of organisms of a geographic biggest accumulation of bacteria within the
region or a time period. Searching Google scholar human body the gastrointestinal microbiota
and Pubmed the first appearance of the term (Dubos 1966). The term “microbiome” was origi-
microbiota in connection with bacteria was a pat- nally used to refer to the collection of the genomes
ent application by Alexander Goetz from 1945 of the microbes in a particular ecosystem and
Fig. 1.1 Definitions of terms Microbiota

(all present microbes)
posses
Microbiom
(all present genes)
Transcriptom
(all transcripted genes)
Proteom
(all proteins present)
Cell may be alive or dead
Cell has to be alive

Metabolom
(all metabolic substances)
1 A Short Definition of Terms 3
termed by Nobel laureate Joshua Lederberg Forsythe P, Kunze WA (2013) Voices from within: gut
microbes and the CNS. Cell Mol Life Sci 70:55–69.
(1925–2008) (Hooper and Gordon 2001).
doi:10.1007/s00018-012-1028-z
Therefore, the term microbiota would be correct Frank DN, Pace NR (2008) Gastrointestinal microbiology
in the case of 16S rRNA studies and the term enters the metagenomics era. Curr Opin Gastroenterol
microbiome in genome studies. To study the 24:4–10. doi:10.1097/MOG.0b013e3282f2b0e8
Goetz A (1950) Method for producing a microbicidal
microbiota of a given habitat and the therein pres-
composition of matter. Google Patents
ent genes the microbes of interest may be dead or Hooper LV, Gordon JI (2001) Commensal host-bacterial
alive. However, the applied techniques do not relationships in the gut. Science 292:1115–1118
allow for the discrimination between a living or a Li J, Jia H, Cai X, Zhong H, Feng Q, Sunagawa S et al
(2014) An integrated catalog of reference genes in the
dead cell, in contrast to the determination of the human gut microbiome. Nat Biotechnol 32:834–841.
transcriptom, proteome or the metabolom of a doi:10.1038/nbt.2942
habitat (Fig. 1.1). Luckey TD (1972) Introduction to intestinal microecol-
Only the determination of the latter three will ogy. Am J Clin Nutr 25:1292–1294
Parent A, Carpenter MB (1996) Carpenter’s human neuro-
allow us insights on the implication and impor- anatomy. Williams & Wilkins, Baltimore
tance of a specific microbe in a habitat and not Qin J, Li R, Raes J, Arumugam M, Burgdorf KS,
only an ordinary number. Manichanh C et al (2010) A human gut microbial gene
As we humans are not only determined by our catalogue established by metagenomic sequencing.
Nature 464:59–65. doi:10.1038/nature08821
genes, but by the transcribed proteins, so is not Socransky SS, Gibbons RJ, Dale AC (1953) The micro-
the sole microbe of importance, but its liaison to biota of the gingival crevice area of man. I. Total
other microbes and us. microscopic and viable counts of specific microorgan-
As Louis Pasteur once stated:"Le microbe, isms. J Arch Oral Biol 8:275–280
The human microbiome project consortium (2012)
c’est rien, le milieu, c’est tout! – The microbe is Structure, function and diversity of the healthy human
noting, it’s the environment". microbiome. Nature 486:207–214
Turnbaugh PJ, Ley RE, Hamady M, Fraser-Liggett CM,
Knight R, Gordon JI (2007) The human microbiome
References project. Nature 449:804–810. doi:10.1038/
nature06244
Virgin HW (2014) The virome in mammalian physiology
Dubos R (1966) The microbiota of the gastrointestinal
and disease. Cell 157:142–150. doi:10.1016/j.
tract. Gastroenterology 51:868–874
cell.2014.02.032
Studying the Human Microbiota
2
Alan W. Walker
Abstract
There are a range of methodologies available to study the human micro-
biota, ranging from traditional approaches such as culturing through to
state-of-the-art developments in next generation DNA sequencing tech-
nologies. The advent of molecular techniques in particular has opened up
tremendous new avenues for research, and has galvanised interest in the
study of our microbial inhabitants. Given the dazzling array of available
options, however, it is important to understand the inherent advantages and
limitations of each technique so that the best approach can be employed to
address the particular research objective. In this chapter we cover some of
the most widely used current techniques in human microbiota research
and highlight the particular strengths and caveats associated with each
approach.
Keywords
Microbiota • Techniques • Sequencing • PCR • FISH • Stable isotope •
Metabolomics • Proteomics
2.1 Introduction 1980) and this sentiment has undoubtedly been

well exemplified in the field of microbiota
The Nobel prize winning biologist Sydney research. Study of the human microbiota can be
Brenner once remarked that “progress in science traced back to Antonie van Leeuwenhoek’s late
results from new technologies, new discoveries Seventeenth Century description of “animal-
and new ideas, probably in that order” (Robertson cules” in scrapings from the human mouth (Porter
1976), a discovery that was made possible by van
Leeuwenhoek’s ground-breaking work with
A.W. Walker (*) microscopes. From the pioneering endeavours of
Microbiology Group, Rowett Institute of Nutrition Cohn, Pasteur, Koch and others in the Nineteenth
and Health, University of Aberdeen, Century, through to developments in anaerobic
Foresterhill, Aberdeen AB25 2ZD, UK
e-mail: alan.walker@abdn.ac.uk microbiology and molecular biology in the

6 A.W. Walker
Fig. 2.1 Overview of some of the most common tech- depth in the laboratory or in animal hosts. Recently, the
niques used to study the human microbiota term “culturomics” has been applied to high-throughput
(a) The functional activities of the microbiota can be stud- culturing of microbes in multi-welled plates containing
ied by monitoring transcription (using RNA-seq/meta- highly nutritious growth media. Cultured organisms can
transcriptomics), protein production (metaproteomics) or also have their genomes sequenced, providing further
metabolite production (metabolomics). (b) DNA information about their potential activities in vivo. These
sequence-based techniques are used to determine the techniques can be used in combination to generate more
composition of the microbiota (e.g. 16S rRNA gene sur- comprehensive understandings of the human microbiota.
veys) and the functional encoding capabilities of the Reprinted in unmodified form from: Pham and Lawley
microbiome (shotgun metagenomics). (c) Culture remains (2014) (Pham and Lawley 2014) under Creative Commons
highly relevant as cultured organisms can be studied in Attribution (CC BY) license
second half of the Twentieth Century, and the 2.2 Classical Microbiological
Twenty-first Century’s own breakthroughs in Methods
genomics and DNA sequencing technologies
(McPherson 2014), subsequent developments in 2.2.1 Culture
the field of microbiota research have been simi-
larly driven by successive waves of technological For well over a 100 years microbiologists have
and methodological advances. As a result, today’s used the classical approaches of cultivating
microbiota researcher has the benefit of a stag- microbes in the laboratory, isolating individual
gering array of tools at their disposal (Fig. 2.1). colonies and then studying these isolated strains
This chapter gives a broad overview of the many in order to describe their phenotypic characteris-
techniques that are now available, and attempts to tics and metabolic capabilities (see Lagier et al.
describe the inherent advantages and limitations (2015a) for a recent overview of the techniques
of each of these techniques. used). As a result of these extensive efforts, it has
2 Studying the Human Microbiota 7
been estimated that over 1000 distinct microbial the laboratory (Rajilic-Stojanovic et al. 2007).
species have been cultured from the human gas- This problem is particularly acute for bodily sites
trointestinal tract alone (McPherson 2014), and such as the colon, where the majority of the con-
characterisation of microbes and gene function stituent bacteria are strict anaerobes. As such,
discovery in the laboratory remains the bedrock culture alone cannot address the sheer complex-
upon which many of the more modern molecular ity of the human microbiota.
techniques that will be described in later sections Nonetheless, there are many reasons to be
of this chapter rely upon. A further advantage of optimistic that cultured coverage of the human
having a strain in culture is that it allows potential microbiota can be greatly improved. DNA-
exploitation for therapeutic purposes should it sequence based surveys of the gut microbiota, for
turn out to have beneficial properties (Walker example, commonly show that many of the most
et al. 2014). abundant sequences map to cultured species, and
The simplest form of microbial cultivation is that it is the rarer sequences that are less likely to
to incubate samples or individual strains in batch be derived from a cultured isolate (Walker et al.
culture in nutritious or selective growth media. 2014). This suggests that it is insufficient cultur-
Batch culture studies allow selective enrichment ing effort rather than an inherent “unculturabil-
of bacterial groups of interest, comparisons to be ity” that is the main barrier to successful novel
made between growth rates and metabolite pro- isolations. Furthermore, unlike environments
duction on different substrates, and interactions such as soil, which can harbour very slow grow-
between specific species to be observed and mea- ing microbes, bacteria living in the human body
sured (Belenguer et al. 2006). Many microbial are often provided with relatively stable environ-
inhabitants of humans are obligately anaerobic mental conditions, and a generally reliable sup-
and therefore exquisitely sensitive to oxygen. As ply of growth nutrients, and must therefore be
a result, some species can be killed by even very capable of multiplying quickly or else face being
brief exposure to air (Flint et al. 2007), making rapidly outcompeted. Provided the correct condi-
them much more difficult to grow. To permit lab- tions can be supplied in artificial growth media it
oratory cultivation of these species, culturing can be assumed therefore that these species will
must therefore be carried out under strictly anaer- be relatively more amenable to culture. Indeed,
obic conditions, for example by using anaerobic novel species continue to be regularly isolated
cabinets or Hungate roll tubes (Eller et al. 1971). from the human microbiota, and there have been
Cultivation of particularly fastidious gut species some impressive recent examples of successful
can also be enhanced by using media containing high-throughput culturing programmes (Lagier
rumen fluid, filtered stool extracts, or mixtures of et al. 2015b; Goodman et al. 2011). Such efforts
short chain fatty acids, which can be utilised by have been dubbed “culturomics”, and have con-
some gut bacteria as growth substrates (Duncan tributed to a reinvigorated interest in the use of
et al. 2002; Lagier et al. 2015b). culture-based techniques to better characterise
A limitation of batch culture is that results can the human microbiota. Information gleaned from
only be obtained over relatively short periods of modern genomics methods can also be used to
time before the supply of nutrients in the growth design improved culture media that support the
medium is exhausted or toxic by-products accu- growth of previously uncultivated species (Bomar
mulate and lead to cessation of microbial growth et al. 2011).
(Ferenci 1999). A further, and key, disadvantage
to using culture is that it is highly labour inten-
sive, and a range of complex growth media are 2.2.2 Continuous Culture
typically required to recover as wide a diversity
of organisms from a sample as possible. It is also A more sophisticated method to cultivate
known that many of the microbial species that microbes in the laboratory is the use of continu-
inhabit the human body have yet to be grown in ous culture model systems such as fermentors
8 A.W. Walker
Fig. 2.2 Continuous culture fermentor system CO2 or N2 to ensure that they remain anaerobic, and can
Fermentors are continuous culture model systems, which be maintained at defined pH and temperatures, which are
allow long term cultivation of microbes. (A) An example constantly monitored. (B) a modified fermentor vessel,
of a single vessel fermentor system (the culture vessel is incorporating a nylon bag containing insoluble particulate
labelled with “X”), inoculated with human faeces and fed substrates (labelled “Z”), developed to identify fibre-
a constant supply of nutritious growth medium (labelled degrading gut bacteria
“Y”). The contents of the culture vessel are gassed with
(Fig. 2.2). In contrast to the batch approach, con- length of the gastrointestinal tract (Van den
tinuous culture is carried out in an open system, Abbeele et al. 2010). While these model systems
which is continually supplied at one end with are an advance over simple batch culture it should
fresh growth medium/nutrients, and overflow is be noted, however, that they still have important
allowed to drain from the vessel at the other end, limitations. For example, they lack an immune
diluting out toxic metabolic by-products and system, and metabolites such as short chain fatty
dead cells. Systems such as these reach a “steady acids (SCFAs) produced by the bacteria are not
state” equilibrium, allowing the researcher to absorbed, meaning results may not necessarily be
exert an enhanced level of control over prevailing directly translatable to the situation in vivo.
environmental conditions within the culture ves-
sel, and can therefore be run over relatively long
time periods (Miller and Wolin 1981). These sort 2.2.3 Animal Models
of systems have been commonly used to study
colonic microbes, and a number of research Microbes of interest can also be cultivated and
groups have made fermentors more advanced by maintained in animal models. Until relatively
incorporating distinct sequential stages, which recently, for example, the only way to grow seg-
aim to mimic the sort of environmental changes mented filamentous bacteria, which have been
microbes are exposed to as they pass along the shown to have important pro-inflammatory
effects in mice, was in animal models (Klaasen concerns as to whether or not rodent models are
et al. 1991). One disadvantage of using animal most appropriate for studies investigating links
models is that, while the microbiota composition between the microbiota and, for example, diet-
at the phylum level generally appears to be simi- dependent diseases such as obesity (Salonen
lar between humans and other animals, at the et al. 2014). A recent review by Nguyen et al
species and strain level there is considerable (2015) extensively documents the inherent
divergence, likely to due to underlying differ- advantages and disadvantages of using mouse
ences in host anatomy/physiology, and dietary models, and discusses the translatability of find-
regimes (Nguyen et al. 2015). However, recent ings in mice to humans.
work has shown that it may be possible to miti-
gate this issue somewhat as a significant propor-
tion of human-associated bacterial species appear 2.3 Sequence-Based Approaches
to be able to successfully colonise the intestines
of animal models following faecal microbiota While culture remains an important tool, human
transfer (Ellekilde et al. 2014). Germ-free, or microbiota research has been completely revolu-
gnotobiotic, mice are another appealing option as tionised over the last decade by molecular meth-
these mice can be specifically inoculated with ods, and in particular by the falling costs and
microbial strains of interest (Goodman et al. vastly increased throughput of DNA sequencing
2011; Seedorf et al. 2014). This permits a more technologies (Fig. 2.3). This rapidly moving, and
reductionist approach to study host-microbe and highly innovative, field continues to produce
microbe-microbe interactions, separated from the exciting and novel technologies, with the latest
potentially perplexing background complexity of generation of sequencing machines capable of
the wider microbiota. A further particular advan- generating data at a depth of billions of individ-
tage of using mouse models is that extensive ual sequence reads (Illumina HiSeq), or at com-
genotyping analyses have been carried out, and paratively long read lengths (PacBio), or even via
there are a range of knockout mouse lines avail- miniaturised devices that can be plugged into the
able to allow the study of interactions between USB port of a laptop (Oxford Nanopore’s
specific host genetic components and the micro- MinION) (Reuter et al. 2015).
biota (Kostic et al. 2013). The key advantage to sequence-based
There are, however, a number of important approaches is that, by circumventing the require-
limitations to using animal models, particularly ment to grow microorganisms in the laboratory,
rodent models. For example, co-housing, and the they generally give much more comprehensive
practice of coprophagy, generally leads to rapid overviews of the species present in a sample.
transfer of microbiota between cage mates, and They are also typically far less labour intensive
this can confound results by being a stronger than classical microbiological techniques, and as
determinant of intestinal microbiota composition a result it is now possible to carry out experi-
than either host genotype or experimental vari- ments at a scale that would have been unthink-
ables (Lees et al. 2014; Ericsson et al. 2015). able just a decade ago. Indeed, recent global
Furthermore, recent work has indicated that research initiatives such as the Human
rodents who are handled by male experimenters Microbiome Project (HMP) and MetaHIT, for
are likely to be more stressed than those handled example, have taken advantage of these new
by females (Sorge et al. 2014), and it is possible sequencing technologies to produce staggering
that stress may impact microbiota structure amounts of freely available data (Human
(Cryan and Dinan 2012). Finally, emerging evi- Microbiome Project Consortium 2012a; Li et al.
dence suggests that host diet may have a greater 2014). There are a number of ways in which the
impact on microbiota structure and composition power of DNA sequencing can be used to study
in rodents than in humans (explaining around the human microbiota, which are detailed in the
60 % of variance vs 10 % respectively), raising following text. In addition, Table 2.1 summarises
10 A.W. Walker
Fig. 2.3 DNA sequencing approaches have revolution- Pubmed (search date Dec. 1st, 2014) for the terms “gut
ised microbiota research flora” OR “gut microflora” OR “gut microbiota” OR “gut
Chart showing the meteoric rise in publications mention- microbiome” OR “intestinal flora” OR “intestinal micro-
ing the gut microbiota since the advent and market release flora” OR “intestinal microbiota” OR “intestinal microbi-
of next generation sequencing platforms such as 454 ome” OR “colonic flora” OR “colonic microflora” OR
pyrosequencing and Illumina. Data collected by searching “colonic microbiota” OR “colonic microbiome”
the most common uses, and outlines the inherent microbial groups or genera (Woese and Fox
advantages and limitations of each approach. 1977). Following DNA extraction from the
human tissue sample, the SSU rRNA genes are
typically PCR-amplified using primers targeted
2.3.1 Marker Gene Surveys towards highly conserved regions of the gene.
The aim here is to generate a mixed pool of PCR
One common sequence-based approach is to amplicons that are derived from as many of the
carry out surveys of universal marker genes, bacterial species present in the original sample as
which provide a broad census of the microbial possible, which are then sequenced en masse.
species present within a sample. While these sort Typically, the resulting data is then clustered by
of surveys have been carried out since the 1980s sequence similarity into Operational Taxonomic
recent developments in next generation sequenc- Units (OTUs), with the assumption being that
ing technologies mean it is possible to survey these OTUs will be a reasonable approximation
microbial communities at previously unimagina- of the underlying species content of a given sam-
ble depth and scales (Tringe and Hugenholtz ple. It should be noted though that, due to the
2008; Caporaso et al. 2011). The most widely wide variation in 16S rRNA gene operon copy
used universal marker genes are the small subunit numbers between individual strains, results are
ribosomal RNA (SSU rRNA) genes (16S rRNA not truly quantitative (Vetrovsky and Baldrian
gene for bacteria and archaea, 18S rRNA gene 2013). Furthermore, the chosen OTU sequence
for eukaryotes). Within these genes there are similarity threshold is both artificial and subjec-
regions of DNA sequence that are highly con- tive and will not be able to accurately capture
served, but there are also other regions that are diversity correctly across the full range of genera
more variable, and which are unique to certain present in a sample. Nonetheless, when the full
Table 2.1 Comparison between different sequence-based approaches used to study the human microbiota
Method Advantages Limitations
Species profiling via Provides overview of species present Relatively insensitive – can be impossible to
marker gene surveys in a sample derive species-level classifications for some
(e.g. 16S rRNA gene) genera
Much cheaper than other sequencing Only provides information on community
methods composition, does not provide direct functional
capability data
Analysis requires less computational Single marker genes such as 16S rRNA genes
power typically only describe the bacterial/archaeal
fraction of microbial communities. Does not
describe viruses, fungi etc. that may also be
present.
Larger sample sets increase statistical Results can be heavily impacted by sampling,
power storage, PCR and DNA extraction biases
Broad functional capabilities can 16S rRNA gene is usually multi-copy, and the
often be inferred from 16S rRNA number of copies is variable between species,
gene sequences by comparing to meaning results are not truly quantitative
closely related isolates with fully Usually does not discriminate between active
sequenced genomes and inactive/dead cells
Whole genome Provides information on the Usually requires that the organism be cultivated
sequencing complete coding potential of an prior to sequencing the genome
organism
Draft bacterial genomes can now be Modern, short read, sequencing technologies
generated very quickly and cheaply will typically generate draft, not complete,
genomes
Data generated can be used for Many constituent genes will be of unknown
epidemiological purposes, e.g. for function
strain typing
Metagenomics Allows simultaneous profiling of Can require very deep sequencing to achieve
both the functional capabilities and reasonable genome coverage, making it
species composition of microbial comparatively expensive
communities
Can simultaneously obtain genomic Often limited to small numbers of samples,
data of bacterial, archaeal, eukaryotic which reduces statistical power
and viral origin
Complete genomes of constituent Data analysis may require large computational
species, including uncultured resources.
organisms, can be assembled
No PCR bias Assembling genomes can be challenging
Gaps in reference databases mean that large
proportions of the genomic data are of unknown
function
Biases introduced during sampling, storage and
DNA extraction can impact results
Usually does not discriminate between active
and inactive/dead cells
(continued)
12 A.W. Walker

Method Advantages Limitations
Single-cell genomics Provides genomic data from Isolating single cells typically requires access to
uncultured species expensive equipment (e.g. flow cytometry,
micromanipulators)
Allows placement of genomic data in Genome amplification step introduces biases,
a phylogenetic context making complete genome assembly challenging
Data generated from uncultured Sensitivity of the genome amplification step
species can improve reference means that contamination is a constant concern
databases for metagenomic analyses. and must be mitigated against
DNA extraction can impact results
Cannot discriminate between active and inactive/
dead cells
Metatranscriptomics Gives data on the functional activity Short half-life of mRNA is an important
of microbial communities limitation; sample selection and preservation are
key concerns
Focusses on the active members of Can be technically challenging, often need to
the microbiota, results not as deplete the far more abundant rRNA before
impacted by dead/inactive cells as sequencing mRNA
other sequencing methods
Can often attribute source organisms Gaps in reference databases mean that large
to transcripts proportions of the genomic data are of unknown
function
RNA extraction can impact results
1500 bp sequence of the 16S rRNA gene is avail- available, such as mothur, QIIME, VAMPS and
able, clustering into OTUs with 98.7–99 % GUSTA ME (Schloss et al. 2009; Caporaso et al.
sequence similarity appears to best fit species- 2010; Huse et al. 2014; Buttigieg and Ramette
level designations derived from culture work 2015) which allow the researcher to carry out all
(Stackebrandt and Ebers 2011). However, as next of the stages involved in processing marker gene
generation sequencing technologies typically survey data, from quality control steps to statisti-
generate comparatively short read lengths, which cal comparisons and visualisation of results.
are focussed on hyper-variable regions of the While broad marker-gene surveys using uni-
gene, slightly less stringent clustering is required versal markers such as the 16S rRNA gene are
and it is now most common to cluster OTUs with the most commonly applied variation of this
97 % sequence similarity (Schloss and Westcott technique it is also possible to carry out focussed
2011). surveys of functional genes that have more lim-
Regardless of sequence similarity used, OTUs ited dissemination throughout the microbiota
can be mapped against comprehensive reference (Walker et al. 2014). The principle here is simi-
databases such as SILVA, RDP, EzTaxon and lar; degenerate PCR primers are targeted towards
Greengenes in order to assign taxonomic classifi- conserved regions of these functional genes, cre-
cations to them (Quast et al. 2013; Cole et al. ating a mixed pool of amplicons, which are then
2014; Chun et al. 2007; DeSantis et al. 2006). sequenced. This approach has been used, for
This provides information about which taxa were example, to identify novel groups of butyrate/
present in the original sample, and allows the propionate producing bacteria from the human
researcher to monitor differences in microbiota colon, and cellulolytic bacteria from the rumen
composition between samples and between study (Louis et al. 2010; Reichardt et al. 2014; Brulc
cohorts. A range of software options are now et al. 2011). A disadvantage of this targeted
approach is that the PCR primers may not effi- associated microbes. The Human Microbiome
ciently amplify all of the functional genes of Project alone, for example, aims to have gener-
interest in a given sample. Untargeted approaches ated over 3000 draft genomes once the first phase
such as metagenomics (see “Metagenomics” sec- is complete (Human Microbiome Project
tion below) may circumvent this issue, but at the Consortium 2012b). Genome sequence data pro-
cost of having to generate far greater amounts of vides critical information on the putative func-
data, which is considerably more expensive to tional capabilities of a given species, although it
produce and more difficult to analyse (Prakash should be acknowledged that there are often a
and Taylor 2012). large number of unannotated genes due to pau-
city of close, well-characterised matches in refer-
ence databases. Indeed, even with E. coli K-12,
2.3.2 Whole Genome Sequencing which has been extensively studied and used as a
model organism over many decades, around a
The first bacterial genome to be completely quarter of the constituent genes remain unanno-
sequenced was that of Haemophilus influenzae, tated (Conway et al. 2014). Nonetheless, as refer-
in 1995 (Fleischmann et al. 1995). Then, sequenc- ence databases expand, and techniques for high-
ing was carried out using the traditional Sanger throughput, genome-wide, functional probing
method (Sanger et al. 1992) and it took many such as transposon insertion sequencing are
years, and hundreds of thousands of dollars, to developed (van Opijnen and Camilli 2013), this
complete a whole bacterial genome. Advances in situation will improve. A further, and flourishing,
DNA sequencing technology since then mean use for whole genome sequencing is in the field
that draft bacterial genomes can now be gener- of epidemiology, and there are now numerous
ated in a matter of hours, and at a cost that is examples of using whole genome sequence data
thousands of times cheaper (Loman et al. 2012; to trace both global and local dissemination of
Koser et al. 2012). Given the extremely high- microbes within human populations (Parkhill and
throughput nature of next generation sequencing Wren 2011; Eppinger et al. 2014), and to monitor
platforms such as Illumina it is now common to evolutionary changes in genomic content (He
simultaneously sequence many microbial et al. 2010; Schuenemann et al. 2013).
genomes on a single sequencing run. This is done
by multiplexing samples via the addition of a
unique sequence “tags” and then bioinformati- 2.3.3 Metagenomics
cally separating reads from each of the combined
samples post-sequencing (Lennon et al. 2010). An important limitation of whole genome
“Shotgun” sequencing, whereby DNA is ran- sequencing is that it typically requires the organ-
domly fragmented prior to sequencing and then ism to be grown in culture first, so that enough
the resulting overlapping sequence data is pieced DNA can be extracted for subsequent sequenc-
together bioinformatically into contiguous ing. 16S rRNA gene-based surveys have revealed,
stretches (contigs), is the standard method however, that the majority of human microbiota
(Fleischmann et al. 1995). Genomes are typically species have yet to be cultivated in the laboratory
pieced together by either mapping data on to an (Eckburg et al. 2005). As a result complementary
existing reference genome (if one is available) or methods such as metagenomics, which can pro-
by assembling the data de novo. There is a wide vide genomic insights into this uncultured major-
range of software available for the genome ity, are attractive options, and have gained
assembly step, with the optimal choice of assem- increasing favour in recent years. With metage-
bler depending on the sequencing platform used nomics, the researcher directly shotgun sequences
(Loman et al. 2012). DNA extracted from an environmental sample.
There are now a large, and constantly increas- They then either attempt to bioinformatically
ing, number of genomes available from human- piece together the resulting sequence data, which
14 A.W. Walker
will be comprised of fragments of DNA derived will likely improve though as sequencing costs
from the range of different species that were fall and the use of cloud computing facilities
present in the original sample, into contiguous becomes more wide-spread (Angiuoli et al.
stretches of sequence data derived from each 2011). As with other DNA-based approaches, the
individual constituent species, or use the unas- sample storage, preparation, and processing
sembled sequence data directly as a means to methodologies used will also have significant
assess the functional capabilities of the microbial impacts on the quality of the final metagenomics
community as a whole entity (Handelsman data (see section “Common pitfalls of sequence
2004). based approaches” below).
Metagenomic sequencing in this manner was The task of assembling genomes from such a
first applied to samples from the human gut in complex collection of microbes, where there will
2006 (Gill et al. 2006), and has since been used also be great divergences in genome coverage
numerous times to study the human microbiota. depth based on the relative abundance of each
This technique can be hugely powerful, and it is species in the original sample, is also daunting,
possible to generate in depth profiles of the func- particularly when trying to assemble genomes
tional potential of a given microbial community, from closely related strains and species, or highly
including uncultured constituents. It is important fragmented genomes where there is only limited
to note, however, that the high complexity of coverage (Nielsen et al. 2014). Although these
many human-associated microbial habitats, such issues have still not been completely surmounted,
as the colon, means that very deep sequencing is there have been great improvements in this area
often required in order to generate sufficient in recent years, and various bioinformatics tools
sequence data from a representative cross-section have been developed to aid the genome assembly
of the microbes that are present. Luckily, the and species assignment processes (Peng et al.
development of next-generation sequencing plat- 2011; Namiki et al. 2012; Bankevich et al. 2012;
forms such as Illumina mean that this is now pos- Alneberg et al. 2014).
sible, and large-scale metagenomics studies A further concern is that the current reference
incorporating many individual samples are now databases that are routinely used to classify the
being carried out (Hu et al. 2013). Metagenomics DNA sequences are not comprehensive enough.
is also the only technique that allows effective, in As a result, a large fraction of metagenomics data
depth, monitoring of the viral communities (or often goes uncharacterised as there are simply no
“viromes”) that are present in the human body as close matches in the reference database to base a
there are no marker genes equivalent to SSU classification on (Thomas et al. 2012). This also
rRNA that are universally detected in all viruses means that results tend to be heavily weighted
and so can be used for sequence-surveys (Minot towards well characterised housekeeping genes,
et al. 2011). Further key advantages of metage- which are comparatively well covered in refer-
nomics over other sequence-based techniques are ence databases (Walker et al. 2014). This situa-
outlined in Table 2.1. tion will improve, however, as novel gene
There are, however, some important limita- functions and pathways are continually eluci-
tions to the use of metagenomics. For example, dated, and reference databases incorporate
this sort of study is far more expensive than genomes from a more phylogenetically diverse
marker gene surveying, and comes with a require- array of isolates (Walker 2014).
ment for appropriate computational infrastruc-
ture and expertise in order to be able to process
the data effectively. Unfortunately, these factors 2.3.4 Single-Cell Genomics
mean that sample sizes tend to be quite small, and
large-scale metagenomics studies are currently Single cell genomics (SCG) is an emerging and
out of reach for many laboratories. This situation complementary technique to metagenomics, and
is a more targeted approach to generating 2.3.5 Metatranscriptomics

genomes from uncultured microbes. With this
technique, individual microbial cells are isolated A further emerging sequence-based technique
from environmental samples, and their genomic with applicability to the human microbiota is
DNA subsequently amplified by a whole genome metatranscriptomics (also termed RNA-seq).
amplification technique (typically multiple dis- Transcriptomics is the study of the RNA tran-
placement amplification) (Walker and Parkhill scripts produced by a given species, whereas
2008). This exquisitely powerful amplification metatranscriptomics is the study of combined
step generates sufficient DNA from just a single transcripts from an entire microbial community.
cell that subsequent shotgun sequencing becomes Thus, in contrast to metagenomics, metatran-
feasible (Blainey 2013). Moreover, combining scriptomics allows insights into the functional
SCG with a form of targeted cell selection, such activity of the microbiota at a given time and
as fluorescent in situ hybridisation (Amann and under prevailing environmental conditions, not
Fuchs 2008), stable isotope probing or Raman just the functional potential. Typically, this
microspectroscopy, allows the researcher to technique involves isolating RNA from environ-
potentially recover specific cells that are derived mental samples and using this to create reverse
from a particular phylogenetic background, or transcribed cDNA libraries, which can then be
that carry out a function of interest. As such, shotgun sequenced using modern high through-
SCG complements metagenomics by allowing put sequencing platforms such as Illumina
recovery of genomic information from species (Reck et al. 2015). Shotgun sequenced data is
that may be rare in the microbial community, and then typically assembled by either mapping
allows the researcher to understand which organ- back to reference genomes, or by carrying out
isms are capable of carrying out a particular func- de novo assembly. Recent RNA-seq develop-
tion, even if the genes that are responsible for ments now allow strand-specific identification
carrying out this function are unknown or miss- of transcripts, permitting enhanced detection of
ing from reference databases (Walker et al. 2014). both messenger and non-coding RNAs, and pro-
There are some important limitations to this viding new insights into the roles that the latter
technique however (see Table 2.1), which have so may play in cellular function (Croucher and
far hindered wide-scale implementation. Of par- Thomson 2010).
ticular relevance are the issues of contamination Metatranscriptomics is considerably more
(with such a small starting DNA input, any technically challenging than metagenomics as it
amount of contaminating DNA can easily requires additional processing steps such as cre-
overwhelm the sequence data that is derived from ating cDNA and depleting host and bacterial
the cell of interest), and of biases introduced dur- rRNAs, which typically make up the vast major-
ing the amplification step, which can confound ity of RNA present in a sample (Giannoukos
genome assembly software, and typically mean et al. 2012). Furthermore, transcriptomics is also
that only partial genome coverage can be achieved commonly used in combination with reference
(Raghunathan et al. 2005). Nonetheless, SCG has genomes, as mapping transcripts back to a refer-
been used to characterise novel human-associated ence allows the researcher to understand how a
bacteria from rare and understudied phyla such as given species responds to changes in environ-
TM7 and Chloroflexi (Marcy et al. 2007; mental conditions. Metatranscriptomic analyses
Campbell et al. 2014) and holds great promise for of human microbiota samples are therefore ren-
wider future application. Results generated can dered more complex by the fact that there are
also greatly aid metagenomics-based analyses by often no reference genomes available for many
broadening reference databases and providing members of the microbial community. As such,
reference genomes to aid with the assembly steps the raw data may require complex de novo assem-
(Rinke et al. 2013). bly prior to analyses, a process which has been
16 A.W. Walker
improved in recent years with the advent of novel between techniques that sequence-based
software programmes (Tjaden 2015). approaches commonly “miss” a significant frac-
A key limitation of metatranscriptomics is tion of species present in a sample due to their
that, due to the very short half-life of mRNA mol- inherent biases (Shade et al. 2012; Lagier et al.
ecules (typically measured in minutes (Reck 2012). Awareness of these inherent limitations
et al. 2015)), it may not always be entirely repre- and biases is therefore important to ensure that
sentative of microbial activities in situ. For exam- erroneous conclusions are not drawn from
ple, microbial transcriptional activities measured sequence data (Degnan and Ochman 2012).
in faecal samples may not be reflective of gene Sample preservation is a critical, and often
expression occurring in areas such as the proxi- under looked, first step. Emerging evidence sug-
mal colon. A further limitation is that, as with gests that prior freezing of faecal samples can
metagenomics, many of the transcribed genes lead to systematic distortions in molecular profil-
will be of unknown function due to extensive ing results. Specifically, it appears that
gaps in reference databases. Bacteroides-derived DNA may be gradually
Given these inherent complexities and limita- depleted if samples have been previously held in
tions, metatranscriptomics has yet to be applied long term frozen storage (Maukonen et al. 2012;
to human microbiota samples to the same extent Bahl et al. 2012). Furthermore, evidence suggests
as metagenomics, although uptake of this tech- that bacterial community profiles obtained from
nique is increasing (Jorth et al. 2014; Leimena sputum samples may be perturbed by being kept
et al. 2013; Maurice et al. 2013; Macklaim et al. for greater than 12 h at room temperature prior to
2013). Moreover, direct comparisons between being placed in long-term frozen storage, and
metagenomic and metatranscriptomic datasets also by repeated freeze-thaw cycles prior to DNA
demonstrate the worth of this approach, as highly extraction and sequencing (Cuthbertson et al.
significant differences between the two datasets 2014, 2015).
are detected, reflective of the fact that microbes DNA extraction is another key step, and it is
are constantly altering their gene expression pro- known that choice of extraction kit/method can
files in response to prevailing environmental con- have major impacts on the final sequencing
ditions (Franzosa et al. 2014). results obtained (Ferrand et al. 2014; Kennedy
et al. 2014). If the chosen DNA extraction
method is not robust enough to break open the
2.4 Common Pitfalls cell walls of certain microbes then DNA from
of Sequence Based these species will not be recovered and so will
Approaches not be observed in the final sequencing libraries.
For this reason kits with only chemical-based
While sequence-based approaches have undoubt- extraction are not recommended, as they typi-
edly revolutionised the field of microbiota cally generate results with an over-abundance of
research there are a number of key caveats, par- the more easily extracted Gram negative organ-
ticularly in the areas of sample handling and pro- isms present in a sample compared to the more
cessing, that should be considered when applying recalcitrant Gram positive organisms, which
them. Analyses of mock bacterial communities have a stronger cell wall that is less likely to be
prepared for the Human Microbiome Project, for broken down by chemical lysis only (Walker
example, showed that samples clustered together et al. 2015). DNA extraction kits with a mechan-
based upon which of four sequencing centres ical lysis, or bead-beating, step, which is far
generated the data, illustrating the impact that more effective at breaking open Gram positive
sample processing steps can have on final cell walls, are therefore typically recommended
sequencing results (Schloss et al. 2011). (de Boer et al. 2010). However, it should be
Furthermore, it is clear from comparisons noted that some bead-beating kits are more
effective than others (see Fig. 2.4a) (Kennedy cell genomics can confound genome assembly
et al. 2014). software (Lasken and Stockwell 2007). Errors
For sequence-based approaches requiring generated during the sequencing process itself
prior amplification of specific genes, such as 16S can also vastly inflate diversity measures if steps
rRNA genes, PCR primer design is a further crit- are not taken to account for their impact (Huse
ically important consideration. It is known that et al. 2010). Repeated PCR cycling may also lead
certain groups, for example the Actinobacteria, to an over-representation of some groups and the
are systematically under-represented in studies under-representation of others. For this reason it
using the commonly used 27f primer (Frank has been recommended that the number of PCR
et al. 2008). An example of this is the cycles should be kept as low as is feasible (Bonnet
Bifidobacterium genus, typically the dominant et al. 2002).
member of the gut microbiota in breast fed A further potential pitfall is the presence of
infants, which has three mismatches to 27f (Fig. contamination. Sequence-based approaches are
2.4b), therefore this primer should not be used exquisitely sensitive, which means they are an
with infant faecal samples as results will not attractive means with which to investigate areas
reflect the true microbiota content (Walker et al. of the body traditionally thought of as “sterile”,
2015). Incorporating degenerate bases into or that have very low abundance of colonising
primer design is one way to effectively widen the microbes that are difficult to grow. Unfortunately,
range of target organisms (Fig. 2.4b). Sim et al contaminating DNA or cells can be introduced
(2012), for example, were able to show that to the sample of interest at many processing
improved primers resulted in far better recovery stages, including from reagents in common lab-
of bifidobacterial sequences from infant faecal oratory DNA extraction and PCR kits (Tanner
samples (Sim et al. 2012). et al. 1998) (Fig. 2.4d). Recent work by Salter
Primer choice is also important if there are et al has indicated that, when sequencing is
specific groups of bacteria that a researcher is applied to low biomass samples (i.e. sample
interested in. Next generation sequencing plat- containing less than 104 cells), background con-
forms currently generate relatively short reads, tamination effectively “swamps” the targeted
meaning that it is typical to target sub-sections of DNA from the sample and becomes the domi-
the 16S rRNA gene. Unfortunately, no specific nant feature of sequencing results (Salter et al.
variable region, or combination of variable 2014). Therefore, any researcher working with
regions, is able to fully capture the diversity that low biomass samples should ideally make use of
can be described with full-length 16S rRNA gene copious “negative” sequencing controls. This
sequences. It is therefore prudent to ensure that involves running “blank” DNA extractions and
the species of interest can be differentiated using PCR reactions with no sample or template
the variable regions targeted prior to initiating a added, and then sequencing these alongside the
study (Fig. 2.4c). samples of interest. Any contaminating species
A further complicating factor with detected in the negative controls can then be
amplification-based approaches such as marker removed from the sequencing results from the
gene surveys and single-cell genomics is that chi- actual samples.
meric molecules can be created during the ampli- The choice of DNA sequencing platform is a
fication step (Edgar et al. 2011). Indeed, it is further important consideration. A recent com-
estimated that a significant proportion of DNA parative analysis between the Illumina MiSeq
sequences submitted to 16S rRNA gene data- and Ion Torrent platforms, for example, indi-
bases, for example, may in fact be chimeric in cated that a peculiarity of the Ion Torrent
nature (Ashelford et al. 2005). Chimeric mole- sequencing process can lead to premature trun-
cules inflate microbial diversity estimates cation of sequence reads derived from certain
(Schloss et al. 2011), and in the case of single- microbial groups. The effect of this would be to
18 A.W. Walker
Fig. 2.4 Importance of optimising sample processing seen that the yield from a MoBio kit-based protocol is
protocols much lower than that from two variations of the FastDNA
(a) DNA extraction methodology can impact recovery of kit-based protocols. This panel is reprinted in unmodified
DNA from microbiota samples. In this example it can be form from: Kennedy NA et al. The impact of different
bias the results against these groups, and to been used since they have the potential to have a
therefore give misleading estimates of their greater influence on results obtained than any
presence and/or abundance in the original sam- underlying experimental variable (Wesolowska-
ples. Furthermore, error rates appear to be Andersen et al. 2014).
higher on the Ion Torrent platform, which
would artificially inflate measures of diversity
(Salipante et al. 2014). The common practise of 2.5 Other Community Profiling
multiplexing many samples together on a single Approaches
DNA sequencing run can also introduce bias to
the PCR step (Berry et al. 2011) and lead to 2.5.1 Community Fingerprinting
problems with misidentification of barcoded Techniques
samples (Esling et al. 2015).
Finally, as DNA can persist in the environ- Due to their falling costs and increased output
ment after the death of the host organism, sequence-based approaches have become the most
sequencing results (aside from perhaps meta- widely adopted microbial community profiling
transcriptomics, due to the short half-life of techniques in recent years. Nonetheless, there are
RNA compared to DNA) are unable to distin- other molecular techniques, such as temperature/
guish between live and dead/inactive microbes. denaturing gradient gel electrophoresis (T/DGGE)
Results may not therefore accurately represent (Muyzer et al. 1993), terminal restriction fragment
the active microbiota at the site of interest. length polymorphism (T-RFLP) (Marsh 1999) and
However, pre-treatment of samples with agents automated ribosomal intergenic spacer analysis
such as propidium monazide, which can bind to (ARISA) (Popa et al. 2009), that allow rapid pro-
free DNA, and DNA contained within dead or filing of human-associated microbial communi-
damaged cells, make it possible to make ties. These approaches are termed community
sequencing results more representative of the fingerprinting techniques since they usually give
living or active populations within the micro- representative overviews of the species present in
biota (Rogers et al. 2013). a sample, without providing direct detailed infor-
The combined influence of all of these poten- mation about the actual species present. Thus,
tially confounding factors should be particularly although these approaches are relatively quick and
borne in mind when conducting meta-analyses cheap, the resolution and sensitivity is often much
incorporating data generated across many differ- lower than that obtained with direct DNA sequenc-
ent studies where different methodologies have ing (Kovacs et al. 2010; Kisand and Wikner 2003).
Fig. 2.4 (continued) DNA extraction kits and laboratories V3 region allows differentiation of two Neisseria species
upon the assessment of human gut microbiota composi- (N. meningitidis and N. lactamica) but the sequences from
tion by 16S rRNA genesequencing. (Kennedy et al. 2014) both species are identical over the V6 region, meaning dif-
under Creative Commons Attribution (CC BY) license ferentiation would not be possible. Therefore, if the
(b) Primer sequence can impact the recovery of species researcher was particularly interested in distinguishing
in 16S rRNA gene surveys. In this example it can be these two species, primers targeting the V6 region could
seen that the commonly used 27f primer has three mis- not be used
matches with the important intestinal genus (d) Contamination in laboratory reagents. The panel shows
Bifidobacterium. As a result this genus is often under- the qPCR quantification of a serial dilution of a pure cul-
represented in DNA sequence libraries. The bottom ture of Salmonella bongori. The bacterial quantification
configuration shows the same primer with four degen- should reduce in a linear manner as the number of target
erate bases, which widens the specificity of the primer cells reduces. Instead, the quantification plateaus after
and improves coverage of groups such as the bifidobac- three dilutions, indicating the presence of background for-
teria (Walker et al. 2015) eign contamination in the DNA extraction. This panel is
(c) Choice of 16S rRNA gene variable region can impact reprinted in unmodified form from: Salter et al. (2014)
species-specificity of sequence results. In this example the under Creative Commons Attribution (CC BY) license
20 A.W. Walker
Although these techniques are gradually falling A potential advantage that the microarray
out of favour, recent work suggests that, while they approach has over other profiling techniques is
are not as sensitive as modern next-generation that it typically allows the researcher to simul-
sequencing, they can still generate broadly robust taneously detect the presence of even quite low
results (van Dorst et al. 2014). It should also be abundance organisms, which may not be
noted that, as they are all DNA extraction and detected reliably with even a sequence-based
PCR-dependent, and typically make use of marker approach unless very deep sequencing is car-
genes such as 16S rRNA genes, they share many ried out. One major limitation though is that,
of the limitations and biases of the sequence-based unlike random sequencing approaches, detec-
approaches outlined previously in the “Common tion is of course limited to the organisms that
pitfalls of sequence based approaches” section, are targeted by the range of probes that are
and in Table 2.1. included on the initial array. Fortunately, there
are now comprehensive custom arrays for a
range of human-associated habitats such as the
2.5.2 Microarrays gut (Rajilic-Stojanovic et al. 2009; Ladirat
et al. 2013; Tottey et al. 2013), vaginal tract
A microarray is a grid-like collection of micro- (Gautam et al. 2015) and oral cavity (Crielaard
scopic spots of DNA that are anchored to a solid et al. 2011), and the range of oligonucleotide
surface. These can be used to probe for the pres- probes that are included in these can be
ence of complementary stretches of DNA extracted expanded as novel species are detected using
from a sample of interest by hybridising against the sequence-based approaches (Rajilic-Stojanovic
array. Microarrays can therefore be designed to be et al. 2009). It can also be difficult to design
used in a number of different ways, for example to arrays where the hybridisation conditions are
monitor changes in gene expression, or to mine for standardised for all of the probes included. As
the presence of particular functional or marker such it is prudent to control for potential false
genes (Paliy and Agans 2012; Tu et al. 2014). positives/negatives by including more than one
Phylogenetic microarrays (sometimes also referred probe for each taxonomic group targeted (Roh
to as phylochips) are a profiling method used in et al. 2010). Microarrays also share the same
human microbiota research. This technique typi- methodological limitations associated with the
cally involves creating custom arrays seeded with DNA extraction and PCR steps as other DNA-
short oligonucleotides (usually targeting the SSU based techniques (see “Common pitfalls of
rRNA genes) that are selected so that they collec- sequence based approaches” section, and Table
tively encompass the taxonomic range of organ- 2.1).
isms expected to be present within a given
environmental sample type (Loy et al. 2010). DNA
is extracted from the sample of interest, the SSU 2.6 Quantitative Approaches
rRNA genes PCR amplified and labelled with a
fluorescent marker and then hybridised against the There are two widely used molecular methods,
microarray. When particular DNA spots on the namely quantitative PCR and fluorescent in situ
array retain a positive fluorescent signal post- hybridisation, that allow the enumeration or
hybridisation, this indicates that the targeted taxo- quantification of dominant groups of microbes
nomic group is present in the original sample. By within the microbiota. For both of these tech-
measuring the relative strength of the signal niques 16S rRNA gene sequences are typically
obtained for each positive spot post-hybridisation it the underlying basis, with different variable
may also be possible to semi-quantitatively assess regions targeted with oligonucleotide probes and
the abundance of different taxa in a sample (Rajilic- primers that are specific for particular phyloge-
Stojanovic et al. 2009). netic groups.
2.6.1 Quantitative PCR monly designed to target regions of rRNA that

are specific for chosen phylogenetic groups of
Quantitative PCR (qPCR), sometimes also bacteria (Amann and Fuchs 2008). Probes may
referred to as real-time PCR, is a technique based be targeted towards a broad range of bacteria by
on measuring fluorescence released during PCR selecting a highly conserved section of the 16S
amplification (Malinen et al. 2003). The amount rRNA gene or towards a narrower range by tar-
of fluorescent signal generated, and the rate at geting more specific stretches of the gene
which it accumulates, as the number of PCR (Amann and Ludwig 2000). After entering the
cycles increases allows the researcher to quantify fixed cell, the probes hybridise to any sequence
the amount of targeted DNA present in a given of rRNA that is complementary to that of their
extraction. This approach is often used to quan- own. As ribosomes are highly abundant, and
tify total bacterial cell numbers in a sample, but it distributed throughout the bacterial cell, the tar-
can also be used to concurrently quantify the geted cell fluoresces, which allows direct visu-
population levels of a number of different bacte- alisation and enumeration by epifluorescent
rial groups by using a range of targeted primer microscopy (Harmsen et al. 2002). FISH there-
sets (Ramirez-Farias et al. 2009). This is a highly fore, as well as being a quantitative approach,
sensitive method and cell densities as low as 101 has the singular advantage that it allows obser-
to 103 cells per sample may be accurately detected vation of cells of interest in situ. For example, it
(Ott et al. 2004). One limitation of qPCR, how- is possible to determine the composition of spe-
ever, is that it only allows monitoring of groups cific consortia of microbes present on mucosal
that have been specifically targeted by the chosen surfaces, or on the surfaces of particles (Fig.
PCR primers. As a result, untargeted groups will 2.5). A further strength of this approach is that it
not be observed in the results, and extensive mon- can be used to link phylogeny to function by
itoring of microbial communities typically employing it in conjunction with techniques
requires the use of multiple different primer sets. such as microautoradiography (MAR-FISH)
Recent efforts have therefore been made to make (Nielsen et al. 2010), Raman microspectroscopy
this approach more high-throughput (Hermann- (Raman-FISH) (Wagner 2009) or Secondary Ion
Bank et al. 2013). Primers must also be exten- Mass Spectrometry (FISH-SIMS) (Musat et al.
sively tested first, to rule out non-specific binding 2012).
to non-target DNA. As with all other DNA-based However, there are some important limitations
approaches, qPCR is also highly dependent on to the use of FISH. It is a far less sensitive quan-
the choice of DNA extraction methodology. titative technique than qPCR because a critical
mass of bacterial cells (typically around 106 cells/
ml of sample) is required per microscopic field of
2.6.2 Fluorescent in situ view for accurate visual enumeration. As a result,
Hybridisation (FISH) FISH is most often used to monitor bacterial pop-
ulations at broader taxonomic levels as individual
FISH is another widely used quantitative tech- species only rarely reach the required density for
nique, with the added advantage that it does not accurate monitoring (Harmsen et al. 2002). As
require a DNA extraction step so is free from with qPCR, it should also be noted that a further
some of the biases associated with DNA-based limitation is that FISH only allows monitoring of
methodologies. With FISH, bacterial cells are the microbial groups specifically targeted with
first fixed using chemicals such as paraformal- oligonucleotide probes, and results can be con-
dehyde and then permeabilised to allow access founded by false positive/negative results. It is
of fluorescently-labelled oligonucleotide therefore imperative that all newly designed oli-
probes. These oligonucleotides are typically gonucleotides be tested for specificity prior to
between 15 and 30 bases in length and are com- use with samples.
22 A.W. Walker
Fig. 2.5 Fluorescent in situ hybridisation

A key advantage of FISH is that it allows
direct visualisation of bacteria in
environmental samples. In this example we
can see groups of bacteria colonising an
insoluble fibre particle recovered from a
human faecal sample. Cells coloured green
belong to the Lachnospiraceae family, those
labelled red belong to the Ruminococcus
genus and those in blue are labelled with the
universal DAPI stain and do not belong to
either of the these bacterial groups. Thus it
can be seen that the majority of cells
attaching to this fibre are derived from the
Lachnospiraceae and Ruminococcaceae
2.7 Functional Analyses activity by growing the transformed host species

on agar plates containing a substrate of interest.
Community profiling techniques can only pro- Where functional activity is observed, the cloned
vide an overview of the microbial composition in gene can then be sequenced to provide support-
a given sample or, in the case of shotgun metage- ing genomic data. This approach has been used,
nomics, can only provide an overview of the for example, to identify complex-carbohydrate
encoding potential of a microbial ecosystem. degrading enzymes derived from the human gut
Indeed, while we now have a much clearer pic- (Tasse et al. 2010). Functional metagenomics is
ture of the kind of microbes that inhabit the vari- therefore a potentially hugely powerful approach,
ous niches associated with the human body we with the key advantage that it allows the
know comparatively far less about the roles that researcher to simultaneously identify novel genes
each individual species plays. Fortunately, there encoding specific functions from a broad range
are now a number of complementary techniques, of bacterial species, including those that may not
beyond the culture-based and metatranscrip- be amenable to culture in the laboratory
tomics methods described previously in this (Uchiyama and Miyazaki 2009). A further advan-
chapter, that can be used to assess the functional- tage is that the functional annotation of previ-
ity of the microbiota. ously unknown genes enhances reference
databases, which can then be used to improve
classification success rates and accuracy for
2.7.1 Functional Metagenomics sequence-based shotgun metagenomics studies.
There are, however, a number of important
In contrast to whole shotgun metagenomics, limitations, which has so far limited the use of
where the aim is to generate deep sequencing- functional metagenomics in comparison to the
based profiles of the entire functional capability sequence-based shotgun metagenomics
of the microbiota, with functional metagenomics approach. For example, it is typically highly
the aim is instead to identify specific functional laborious and inefficient; millions of random
genes by cloning and expressing them in a sur- DNA fragments may need to be cloned in order
rogate bacterial species (Handelsman et al. 1998). to identify activities of interest. Furthermore,
Typically this involves large-scale cloning of ran- there are important technological barriers that
dom environmental DNA fragments into a host impinge upon the effectiveness of the approach.
species such as E. coli and then screening for Many of the cloned fragments will be poorly
expressed by foreign hosts such as E. coli, mean- previously in that, by measuring proteins rather
ing that alternative hosts/approaches may need than mRNA, it provides a broader, more repre-
to be considered (Liebl et al. 2014). In addition, sentative picture of the functional activity of the
the DNA extraction step is crucially important as microbiota as it also accounts for the impact of
the researcher must reach a balance between processes such as post-translational modifica-
using a protocol that is stringent enough to tions (Cain et al. 2014). Proteins are also typi-
extract DNA from as wide a range of species in cally more stable than mRNA molecules,
the original sample as possible, but is not so meaning that results obtained may not be so
stringent that it shears the resulting DNA to the dependent on the speed with which the samples
extent that many of the cloned genes and gene are processed. A particular advantage over DNA-
clusters are disrupted (Kakirde et al. 2010). A based metagenomics is that metaproteomics is
further limitation is that, while this approach faster, and cheaper (Verberkmoes et al. 2009).
may identify products formed from individual The relatively untargeted nature of metapro-
genes or relatively simple contiguous gene clus- teomics also means that it may be possible to
ters, it is unlikely to be able to identify gene identify marker proteins that are indicative of a
products that result from complex metabolic healthy or diseased human host status.
pathways (Walker et al. 2014). However, although the technology involved
in metaproteomics is rapidly improving, there
are a range of important limitations, and this
2.7.2 Metaproteomics technique is currently far less commonly applied
in comparison to DNA-based approaches.
Metaproteomics is the study of the complement Although resolution is improving, metapro-
of proteins produced by mixed microbial com- teomics can only currently characterise thou-
munities (Wilmes and Bond 2009). As such, it sands out of the millions of proteins/peptides
provides functional information by allowing the that might be present in a complex microbiota
researcher to monitor changes in protein expres- sample at one time (Kolmeder and de Vos 2014).
sion by the entire microbiota in response to As such, only proteins produced by the most
changes in prevailing environmental conditions. dominant members of the microbiota can be
With this technique, proteins must first be expected to be captured with reasonable cover-
extracted from the environmental sample of inter- age (Verberkmoes et al. 2009). It can also be dif-
est, and then separated prior to characterisation ficult to differentiate similar proteins or ascribe
with mass spectrometry and subsequent them to particular phylogenetic groups
bioinformatics-based comparisons with refer- (Lichtman et al. 2015), and, as with metagenom-
ence databases (Hettich et al. 2012). Until ics studies, a large proportion of the data recov-
recently proteins (or peptides) were most com- ered will have no close matches to available
monly separated by using gel electrophoresis reference databases (Verberkmoes et al. 2009).
approaches (Magdeldin et al. 2014), but they are The methodology chosen during the protein
now increasingly separated by using liquid chro- extraction step will also have significant impacts
matography instead. Recent technological on the representativeness of the protein comple-
advances in the field mean that it is now possible ment recovered, and it is important to extract
to carry out very high throughput liquid proteins with reasonable efficiency from both
chromatography-mass spectrometry based analy- Gram positive and Gram negative constituents
ses, where many thousands of different proteins/ (Tanca et al. 2014). Human-derived proteins will
peptides can be separated and characterised also be present, and can be a highly significant
(Hettich et al. 2013). component in samples such as biopsies, meaning
Metaproteomics offers some key advantages it is sometimes necessary to carry out selective
over the metatranscriptomics approach described steps to enrich for microbial proteins (Kolmeder
24 A.W. Walker
and de Vos 2014). There are also issues sur- founded by the presence of DNA derived from
rounding reproducibility between samples, par- dead or inactive species in the sequence-based
ticularly when using gel electrophoresis to results, and by the fact that there can be consider-
separate proteins (Magdeldin et al. 2014). able metabolic flux within complex ecosystems,
such that metabolites associated with taxa that
are dominant in sequence surveys may not actu-
2.7.3 Metabolomics ally be produced by them (Abram 2015).
Furthermore, many metabolites, for example
Metabolomics is the study of the metabolites/ short chain fatty acids, are rapidly absorbed by
small molecules present within a given sample at the host, meaning that production levels cannot
the time of sampling. As with the metaproteomics be accurately defined or ascribed to particular
approach outlined above, metabolomics there- species (Kolmeder and de Vos 2014). An addi-
fore offers distinct advantages over other functional important disadvantage is that reference
tional approaches such as metatranscriptomics as databases are generally lacking, even more so
it allows the direct monitoring of the end prod- than those for DNA and proteins, meaning that
ucts of bacterial metabolism (Ursell et al. 2014). only a small fraction of metabolomics data can
With metabolomics, metabolites are typically currently be assigned to known metabolites
isolated from bodily samples such as urine, fae- (Baker 2011). Finally, as with metaproteomics,
ces and blood and measured using technologies resolution limits (even with the most modern
such nuclear magnetic resonance (NMR) micros- instruments) mean that it is only possible to accu-
copy or mass spectrometry (Nicholson and rately monitor a small subset of the wide range of
Lindon 2008). The end result of these approaches metabolites that may be present in a complex
are a series of characteristic spectra or peaks sample such as faeces (Goedert et al. 2014).
derived from the range of metabolites that are It can be seen, therefore, that all four key mod-
present within the original sample (Savorani ern “omics” technologies (metagenomics for
et al. 2013). Depending on the approach used, DNA, metatranscriptomics for RNA, metapro-
metabolomic screens can either be carried out in teomics for proteins, and metabolomics for
a targeted way for particular groups of metabo- metabolites) have distinct strengths and limita-
lites (for example, short chain fatty acids), or on tions. As a result, there is increasing interest in
a more global basis (Griffiths et al. 2010). In the integrating the output from each of these
latter case, the main challenge is to assign par- approaches in order to enhance their overall
ticular spectra from the complex mixture of peaks power and provide a more comprehensive, sys-
to specific compounds, and then to attempt to tems biology-based, overview of the human
correlate presence/absence of these compounds microbiota. Effective integration of these com-
with markers of host health (Lenz and Wilson plex datasets remains to some extent an unful-
2007). By simultaneously capturing both host filled ambition, but one that is being rapidly
and microbial-derived metabolites, metabolo- guided by improvements in computing infra-
mics has particular appeal as an approach to char- structure, bioinformatics, mathematical model-
acterise host-microbe interactions (Wikoff et al. ling and statistical approaches (Abram 2015).
2009).
A key limitation of this technique is that it can
be difficult to accurately determine which micro- 2.7.4 Stable Isotope Probing
bial species are producing particular metabolites.
While attempts are often made to correlate One final functional approach with strong appli-
metabolite production with microbial composi- cability to the study of the human microbiota is
tion data generated in tandem by sequence survey stable isotope probing (SIP). With this technique,
or metagenomic approaches, these can be con- mixed microbial communities are incubated with
labelled substrates containing heavy stable iso- thus far. SIP is far more technically challenging
topes such as 13C, 15N, and 18O. Species that are than approaches such as SSU rRNA gene sur-
able to grow on the labelled substrate incorporate veys or metagenomics, and the modern single-
the isotope markers into cellular biomass, which cell resolution techniques such as Raman
can then be studied by looking at components microspectroscopy and SIMS can be prohibi-
such as DNA (DNA-SIP), RNA (RNA-SIP), pro- tively expensive (Wagner 2009). Similarly, use
teins (protein-SIP) or phospholipid-derived fatty of SIP is limited by the supply and cost of
acids (PFLA-SIP). Approaches like density gra- labelled substrates (Uhlik et al. 2013). Recent
dient ultracentrifugation (Dunford and Neufeld innovations though, such as the use of the cheap
2010) or advanced single-cell resolution tech- and readily available heavy water (D2O) as a
niques such as Raman microspectroscopy and general marker of cellular growth, allows SIP to
Secondary Ion Mass Spectrometry (SIMS) are be carried out without specific labelled carbon or
used to distinguish the active microbes from spe- nitrogen sources (Berry et al. 2015). A further
cies that did not incorporate the marker (Eichorst limitation is that SIP requires microbes to be
et al. 2015). Regardless of the actual cellular grown in the presence of the labelled tracer so
components targeted SIP is therefore an attrac- that it can be incorporated into active cells. Often
tive basis for uncovering which microbes within this means growing mixed communities under
complex microbial communities carry out par- artificial laboratory conditions, meaning that
ticular functions (Uhlik et al. 2013). results may not entirely reflect the activity of the
SIP is an emerging means with which to microbiota in vivo (Uhlik et al. 2013).
unravel the complex activities of the human Nonetheless, impressive new innovations have,
microbiota. Early studies used this technique in for example, allowed researchers to identify
tandem with community profiling approaches microbes growing in vivo that forage host-
like T-RFLP and FISH to characterise the derived proteins for growth (Berry et al. 2013).
microbes that were able to actively utilise labelled Finally, there is considerable metabolic flux
substrates such as resistant starch and oligofruc- within complex microbial communities, with
tose (Kovatcheva-Datchary et al. 2009; Reichardt cross feeding between species a common fea-
et al. 2011). When used in combination with ture. This means that stable isotopes such as 13C
more modern “omics” techniques SIP has the may “flow” from the primary degrader of a
potential to be particularly powerful. For exam- labelled substrate to many other species that are
ple, fractionated DNA or RNA containing the present within the community, potentially
stable isotopes can then be sequenced using impeding the ability to detect the initial utilising
marker gene surveys, metagenomics or metatran- species (Dumont and Murrell 2005).
scriptomics in order to identify the species that
were active during incubation with the labelled
substrate (Chen and Murrell 2010). Similarly, 2.8 Conclusions
advances in micro-manipulation technologies
such as optical tweezers mean that whole cells There are now many different ways in which the
that have been shown by techniques like Raman human microbiota can be studied, and each meth-
microspectroscopy to have incorporated the sta- odology has inherent advantages and limitations.
ble isotopes can then be isolated from the sample Ultimately, the best technical approach for a
and either cultured or, if that is not possible, put given situation will clearly depend on the ques-
forward for genome sequencing via single cell tion that the researcher wishes to address.
genomics (Berry et al. 2015). Although each technique has largely been con-
While SIP approaches can be hugely power- sidered in isolation in this review it should be
ful there are important caveats, which have lim- emphasised here that, where possible, the syner-
ited widespread application of these techniques gistic use of multiple methodological approaches
26 A.W. Walker
offers perhaps the greatest power with which to sequence records currently held in public repositories
is estimated to contain substantial anomalies. Appl
uncover novel insights.
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The Gut Microbiota and their
Metabolites: Potential Implications 3
for the Host Epigenome
Mona Mischke and Torsten Plösch
Abstract
The gut microbiota represents a metabolically active biomass of up to 2 kg
in adult humans. Microbiota-derived molecules significantly contribute to
the host metabolism. Large amounts of bacterial metabolites are taken up
by the host and are subsequently utilized by the human body. For instance,
short chain fatty acids produced by the gut microbiota are a major energy
source of humans.
It is widely accepted that microbiota-derived metabolites are used as
fuel for beta-oxidation (short chain fatty acids) and participate in many
metabolic processes (vitamins, such as folic acid). Apart from these direct
metabolic effects, it also becomes more and more evident that these
metabolites can interact with the mammalian epigenetic machinery. By
interacting with histones and DNA they may be able to manipulate the
host’s chromatin state and functionality and hence its physiology and
health.
In this chapter, we summarize the current knowledge on possible inter-
actions of different bacterial metabolites with the mammalian epigenetic
machinery, mostly based on in vitro data. We discuss the putative impact
on chromatin marks, for example histone modifications and DNA meth-
ylation. Subsequently, we speculate about possible beneficial and adverse
consequences for the epigenome, the physiology and health of the host, as
well as plausible future applications of this knowledge for in vivo transla-
tion to support personal health.
M. Mischke
Nutricia Research,
Uppsalalaan 12, 3584 CT Utrecht, The Netherlands
e-mail: mona.mischke@danone.com
T. Plösch (*)
Department of Obstetrics and Gynaecology,
University Medical Center Groningen,
Hanzeplein 1, 9700 RB Groningen, The Netherlands
e-mail: t.plosch@umcg.nl;
http://www.epigenetic-programming.nl

34 M. Mischke and T. Plösch
Keywords
Bacterial metabolites • Epigenetics • (early life) programming • (early life)
nutrition • DNA methylation • Histone modification • Breast feeding
3.1 Introduction studies in humans was the transfer of the gut

microbiota from lean to obese volunteers with
It has been known for a long time that the mam- insulin resistance that resulted in restored insulin
malian gut microbiota is indispensable for the sensitivity in the recipients (Vrieze et al. 2012).
production of vitamins and the fermentation of The obvious control experiment – transfer from
otherwise indigestible components of the diet obese to lean volunteers – could not be performed
(Albert et al. 1980; Cummings and Macfarlane because of ethical reasons.
1997; Ramotar et al. 1984). In ruminants for These and similar studies show that the gut
example, the microbiota produces the majority of microbiota plays an important role in our health
fatty acids used by the organism as an energy and survival. One relevant link between nutrition,
source, and also humans make good use of the microbiota, and health outcome that has to be
energy and specific metabolites produced by the explored in more detail, is the metabolite mix
gut microbiota (Cook and Sellin 1998). produced by the microbiota, the so called metab-
In the last decade it became clear that the olome, which forms a specific entity together
microbiota is not only a producer of nutrients and with the microbiota (McHardy et al. 2013).
vitamins, but also closely interacts with the host. Specific microbiota-mediated metabolite profiles
Experiments in rodents and humans have shown can be associated with the predisposition to met-
that the composition of the gut microbiota is abolic impairments, such as impaired glucose
related to the nutritional, but also to the metabolic homeostasis and non-alcoholic fatty liver disease
and health status of the host (David et al. 2014; (NAFLD) (Dumas et al. 2006). Very recently, we
Ridaura et al. 2013; Tremaroli and Backhed have put forward the hypothesis that the gut
2012; Remely et al. 2013; Le Chatelier et al. microbiota by means of the associated metabo-
2013). So, it was observed that the metabolic lome may influence the host’s long-term physiol-
adaption to a high-fat diet is associated with ogy via modulating its epigenome (Mischke and
changes in the gut microbiota in mice (Serino Plosch 2013). Here, we will extend and underline
et al. 2012). Moreover, the gut microbial phylo- this notion on the basis of reviewing a number of
types in hundred different inbred mouse strains relevant bacterial metabolites and their docu-
correlate with the level of obesity in response to a mented effects.
high-fat/high-sucrose diet (Parks et al. 2013), and
the status of gut colonization is linked with
immune response and longevity (Tazume et al. 3.2 Epigenetics - the Mediator
1991; Round and Mazmanian 2009). In humans, Between the Genome
similar associations between diet, microbiota, and Physiology
and health were detected (Tremaroli and Backhed
2012; Claesson et al. 2012). Several studies link Functionally, epigenetics explains how a multi-
the composition of the gut microbiota to obesity cellular, differentiated organism can be con-
in humans, although their partly conflicting structed from a uniform genome. This implies
results highlight that the exact role of the gut that during differentiation the activity of genes
microbiota and causality remains to be elucidated can be specifically increased or silenced depend-
(Duncan et al. 2008; Jumpertz et al. 2011; Ley ing on whether or not they are needed in a certain
et al. 2006; Schwiertz et al. 2010; Zhang et al. cell type – and to which extent. This regulation
2009). Nevertheless, one of the most prominent basically takes place on the level of DNA
3 The Gut Microbiota and their Metabolites: Potential Implications for the Host Epigenome 35
modification and DNA accessibility, achieved by tone modifications often closely interact with
the close interaction of DNA methylation and each other, together determining the transcrip-
histone modifications, which form the epigenome tional activity of a locus and shaping the epigen-
(Cedar and Bergman 2009; Bernstein et al. 2007). etic landscape (Cedar and Bergman 2009).
On the level of DNA, the activity of a gene can
be regulated by specific methylation of CpG
dinucleotides in a certain gene region. DNA 3.3 Interaction of Bacterial
methylation of gene promoters or enhancers Metabolites and the Host’s
often blocks the transcriptional activity of the Metabolic Regulation
appertaining gene (Jaenisch and Bird 2003). In
general, the combined methylation of multiple Obviously, both DNA methylation and histone
CpG dinucleotides contributes to the resulting modifications require substrates. For example,
activity state. Once methylated, the methylation the availability of C1 metabolites is necessary for
is in principle stable including subsequent cell methylation reactions as is ATP for phosphoryla-
generations. This is achieved by faithful mainte- tion (Jimenez-Chillaron et al. 2012). Although it
nance methylation of the new DNA strands is not plausible that such important regulatory
formed during semiconservative replication, reactions are exclusively regulated on the level of
according to the template DNA strand (Sharif substrates, severe or long-lasting fluctuations in
et al. 2007; Hermann et al. 2004). Thus, DNA substrate availability may influence the level of
methylation of specific CpG positions can be modifications. Such fluctuations in substrate
fully conserved and therefore carried over to the availability may occur in extraordinary physio-
daughter cells as transcriptional label. Removal logical situations, including undernutrition dur-
of the DNA methylation label requires either ing pregnancy, as seen in the Dutch Hunger
DNA synthesis without sufficient maintenance Winter cohort. In this study cohort, DNA hypo-
methylation, which gradually dilutes the DNA methylation of some genes was observed, which
methylation marks, or selective oxidation of may partially be related to substrate availability.
methylcytosine to hydroxymethylcytosine (Wu However, in the same persons other genes have
and Zhang 2014). been shown to be hypermethylated, which argues
The accessibility of DNA for transcription is against one single mechanism of action (Heijmans
mainly regulated by covalent modifications at et al. 2008). Moreover, less extraordinary, more
specific amino acid residues of the histone tails. naturally occurring variations in substrate avail-
These modifications determine the steric confor- ability, as for example seen during the cycle of
mation of the histones and therefore the packag- the seasons or due to regional dietary prefer-
ing density of the chromatin. On the contrary to ences, were also identified to have a specific
DNA methylation, where only a few enzymes are impact on epigenetic modifications (Waterland
involved in methylation and de-methylation of et al. 2010).
the DNA, the situation for histone modification is Numerous epigenetically relevant substances
far more complex: numerous modifications of the are not only provided by the diet, but are also pro-
different histones are known, including but not duced by the gut bacteria. Thus, apart from direct
limited to acetylation, methylation, and phos- nutritional variation as described above, also
phorylation of different amino acid residues. This changes in the gut microbiota have the potential
requires a plethora of enzymes from several to contribute to substrate fluctuations through the
classes to attach and remove the functional associated metabolome. Eventually, not only epi-
groups to and from the amino acid residues of the genetically active substances from the diet, but
histone tails (Plass et al. 2013; http://www.cell- also significant levels of epigenetically active
signal.com/contents/resources-reference-tables/ bacterial metabolites reach cells of the host
histone-modification-table/science-tables- organism (Table 3.1). For example, C1 groups for
histone). Ultimately, DNA methylation and his- methylation reactions are provided by the
Table 3.1 Bacterial metabolites that are indicated to have an epigenetic function
Metabolite Model Epigenetic action
Short-chain fatty acids (SCFA), general In vitro, human HDAC inhibition (Waldecker et al. 2008)
Associated to LINE-1 DNA methylation
(Worthley et al. 2011)
Acetate (C2:0) In vitro HDAC inhibition; histone (H3, H4) hyper-
acetylation (Sealy and Chalkley 1978)
Propionate (C3:0) In vitro HDAC inhibition; histone (H3, H4) hyper-
acetylation (Sealy and Chalkley 1978;
Takenaga 1986)
Butyrate (C4:0) In vitro, in vivo HDAC inhibition of HDAC class I, IIa, and IV
(Davie 2003)
Regulation of transcription factor availability
(Blottiere et al. 2003)
Valerate (C5:0) In vitro HDAC inhibition (Ortiz-Caro et al. 1986)
Branched-chain fatty acids (BCFA), general
Isobutyrate In vitro HDAC inhibition (Waldecker et al. 2008)
Increased histone acetylation, probably via
HDAC inhibition (Suzuki-Mizushima et al.
2002)
Isovalerate In vitro HDAC inhibition (Waldecker et al. 2008)
Increased histone acetylation, probably via
HDAC inhibition (Suzuki-Mizushima et al.
2002)
Organic acids, general In vitro, in vivo HDAC inhibition by low pH (Latham et al.
2012)
Lactate (D-, L-lactate) In vitro, in vivo (weak) HDAC inhibition (Latham et al. 2012)
Phenolic compounds In vitro, in vivo HDAC inhibition (Waldecker et al. 2008)
Bacterial break down of dietary polyphenols
(quercetin, curcumin, catechin) redering them
unavailable for affecting HDAC activity
(Rajendran et al. 2011)
Phenylbutyrate In vitro HDAC inhibition (Lea and Tulsyan 1995; Lea
et al. 2004)
Phenylacetate In vitro HDAC inhibition (Waldecker et al. 2008; Lea
and Tulsyan 1995)
Reduction of reactive oxygen species, which
otherwise affect HAT and HDAC activity and
increase DNA methylation (Beloborodova
et al. 2012)
4-hydroxyphenylacetate In vitro HDAC inhibition (Waldecker et al. 2008)
Reduction of reactive oxygen species, which
otherwise affect HAT and HDAC activity and
increase DNA methylation (Beloborodova
et al. 2012)
Phenylpropionate In vitro HDAC inhibition (Waldecker et al. 2008)
4-hydroxyphenylpropionate In vitro HDAC inhibition (Waldecker et al. 2008)
(continued)

p-cresol In vitro, mouse Expression induction of DNA
methyltransferases 1, 3a, and 3b (Sun et al.
2012)
CpG hypermethylation of Klotho gene;
decreased Klotho expression (Sun et al. 2012)
Sulfur compounds In vitro, in vivo Histone modifications (Canani et al. 2011)
Hydrogen sulfide (H2S) In vitro, rat Inhibition of cell proliferation by epigenetic
mechanism reducing recruitment of Brg1 to
relevant promoter regions (Li et al. 2013)
Reduction/neutralization of reactive oxygen
species, which otherwise affect HAT and
HDAC activity and increase DNA methylation
(Afanas’ev 2014)
Cell wall components
Lipopolysaccharide (LPS) In vitro Chromatin modification at IL-8 gene, including
histone H3 acetylation and methylation; IL-8
activation (Angrisano et al. 2010)
Peptidoglycan (PGN) In vitro Modulation of chromatin structure and
transcriptional activity at Foxp3 locus (Lal
et al. 2011)
Lipoteichoic acid (LTA) Mouse, in vitro Potential epigenetic regulation of genes in
colorectal cancer (Lightfoot et al. 2013)
Vitamins
Thiamine (vitamin B1) In vitro, in vivo As coenzyme involved in generation of ATP;
critical for phosphorylation reactions (Hill
1997)
Riboflavin (vitamin B2) In vitro, in vivo As cofactor involved in one-carbone
metabolism; critical for methylation reactions
(Anderson et al. 2012)
Niacin (vitamin B3) In vitro, in silico SIRT (Class III HDAC) inhibitor (Avalos et al.
2005; Denu 2005)
Pantothenate (vitamin B5) In vitro, in vivo As coenzyme A substrate for acylation and
acetylation reactions; signal transduction,
enzyme activity regulation (Marmorstein 2001)
Histone hypoacetylation and fragile DNA by
impaired Coenzyme A synthesis (Cai et al.
2011)
Pyridoxine (vitamin B6) In vitro, in vivo As cofactor involved in one-carbone
(Anderson et al. 2012)
Biotin (vitamin B7) In vitro, Substrate for histone biotinylation; gene
drosophila activity regulation and transposable element
repression (Hassan and Zempleni 2008)
Decreased histone biotinylation associated
with life span and stress resistance in
Drosophila (Zempleni et al. 2008)
(continued)

Folate (vitamin B9) In vitro, in vivo, As methyl donor involved in one-carbone
rat metabolism; critical for methylation reaction
(Kalhan 2013; Anderson et al. 2012)
Activity reduction of DNA methyltransferase
(Ly et al. 2011)
Cobalamin (vitamin B12) In vitro, in vivo As cofactor involved in one-carbone
(Kalhan 2013; Anderson et al. 2012)
Choline In vitro, mouse Methyl donor; loses availability through
break-down by human gut microbiota (Dumas
et al. 2006)
DNA methylation and gene expression changes
in colitis (Schaible et al. 2011)
Broken down to TMAO and betaine (Wang
et al. 2011)
Betaine In vitro, in vivo, As methyl donor involved in one-carbone
human metabolism; critical for methylation reaction
(Kalhan 2013; Canani et al. 2011)
Alterations of DNA methylation associated
with abnormalities of DNA methyltransferases
in human cancers (Kanai and Hirohashi 2007)
Trimethylamin-N-oxid (TMAO) In vitro, mouse Break down product of methyl donor choline
(Wang et al. 2011)
Equol In vitro Ppromoter CpG island hypomethylation of
breast cancer susceptibility genes BRCA1 and
BRCA2; increase of BRCA1 and BRCA2
proteins (Bosviel et al. 2012)
Ammonium (NH4) Human Inverse association of fecal NH4 and rectal
LINE-1 methylation (Worthley et al. 2011)
α-ketoglutarate In vitro, human Effect on histone and DNA (de)methylation;
co-factor of HDM and TET protein family
memebers (Wang et al. 2013; Hou and Yu
2010)
Conjugated linoleic acids (CLA) In vitro, in vivo Increased SIRT1 deacetylation activity by
trans-10, cis-12 CLA treatment via reciprocal
activation of AMPK (Jiang et al. 2012)
Decreased histone phosphorylation by CDK2
inhibition (Cho et al. 2006)
HDM histone demethylase, TET Ten-eleven translocation protein
one-carbon metabolism in the form of the co- 2013; Anderson et al. 2012). Dietary choline, on
substrate S-adenosyl methionine (Dominguez- the other hand, which contributes as methyl
Salas et al. 2013). Known bacterial metabolites donor to the one-carbon metabolism, can be
contributing to a well-functioning one-carbon metabolized by bacteria to trimethylamin-N-oxid
metabolism include various vitamins and their (TMAO) and betaine, effectively rendering this
metabolites, such as folate (vitamin B9), cobala- methyl donor unavailable for the host (Schaible
min (vitamin B12), pyridoxine (vitamin B6), et al. 2011; Wang et al. 2011, 2014). Other bacte-
riboflavin (vitamine B2), and betaine (Kalhan rial metabolites that are known to function as
substrates for epigenetic modifications are biotin be products of the bacterial metabolism (Sun
(vitamin B7) for biotinylation of histones (Hassan et al. 2012; Ly et al. 2011; Kanai and Hirohashi
and Zempleni 2008; Zempleni et al. 2008) and 2007; Wang et al. 2013).
pantothenic acid (vitamin B5) to form acetyl-
CoA for acetylation reactions (Marmorstein
2001; Nakamura et al. 2012; Cai et al. 2011). 3.4 Putative Targets of Bacterial
Another level of complexity is added by the Metabolites - in Time
fact that several metabolites produced by the gut and Space
microbiota can influence epigenetic regulation,
for example by inhibiting modifying enzymes. As for metabolites of the gut microbiota, the cells
The most prominent example is short chain fatty of the intestinal epithelium, including their stem
acids, which are known to inhibit mammalian cells, are most likely to be reached by epigeneti-
histone deacetylases (HDAC). Extensive in vitro cally relevant concentrations of these metabo-
studies have shown that HDAC inhibition by lites. In the second line, immunocompetent cells
these short chain fatty acids links to an increase that are in proximity of the gut, but also the liver
in the average histone acetylation level of the are putatively exposed to these metabolites. It is
cell, in turn affecting gene activity (Blottiere therefore conceivable that the epigenome of these
et al. 2003; Davie 2003; Ortiz-Caro et al. 1986; cells may be influenced by different fluxes of
Sealy and Chalkley 1978; Takenaga 1986; bacterial metabolites under different metabolic
Waldecker et al. 2008). In this context, butyrate is circumstances. However, for most of the metabo-
considered the most active HDAC inhibitor, fol- lites discussed the fluxes have not yet specifically
lowed by propionate (Table 3.1). Remarkably, been measured.
both butyrate and propionate are highly promi- At least for DNA methylation it is known that
nent in the gut (Cook and Sellin 1998), and their the organism is especially vulnerable during crit-
abundance is influenced among others by the ical windows in development, including the intra-
host’s diet and the bacterial population present uterine and early post-natal life, which refers to
(Cummings 1984; Cummings and Englyst 1987; the “first 1,000 days of life” concept (Arenz et al.
McBurney and Thompson 1990). Apart from 2004; Zhang et al. 2012). One of the underlying
short chain fatty acids, also other bacterial metab- reasons may be the fact that this is a period where
olites have been indicated to influence the activ- dividing cells require relative high levels of
ity of the epigenetic machinery, either directly or methyl donors to methylate CpG positions at the
indirectly: organic acids such as lactate are newly formed DNA strand to maintain the estab-
known to be weak HDAC inhibitors via lowering lished methylation patterns. In the absence of a
the pH (Latham et al. 2012); specific phenolic sufficient supply of methyl donors, methyl marks
and sulfur compounds are indicated to impact may then be purely lost by dilution (Wu and
DNA methylation and histone acetylation via Zhang 2014).
influencing cellular levels of reactive oxygen During pre- and postnatal development the
species (Afanas’ev 2014; Li et al. 2013; body is formed in an orchestrated sequence of
Beloborodova et al. 2012); and niacin (vitamin steps, forming different cell types and tissues.
B3) as well as conjugated linoleic acids affect the Epigenetically vulnerable periods vary for the
activity of sirtuins, which are class III HDACs different organs. Organs like the heart which are
(Avalos et al. 2005; Denu 2005). The majority of formed early in embryogenesis, when exposure
these examples relates to histone modifications, to bacterial metabolites occurs only indirectly via
but there are also a few indications that the activ- the mother, may therefore be almost inert against
ity of DNA methylating enzymes can be regu- these factors. On the other hand, organs being
lated similarly by metabolites, such as folate, formed and differentiated in a later period, like
betaine, α-ketoglutarate, or p-cresol, which can the brain, or with high cell division rates, like the
intestine, might be more susceptible to the influ- One possibility to test if early-life influences
ence of metabolites from the microbiota. This on the microbiota might affect the host’s long-
may explain the relationship between timing of term health via associated changes of the micro-
the stimulus and the observed physiological out- biota’s metabolome and the host’s epigenome is
come (Rueda-Clausen et al. 2011). Another wave the exploration of the metabolic and epigenetic
of epigenetic events may then occur during consequences of Caesarean sections. Future stud-
puberty, when extensive remodeling processes ies should specifically address how the well-
take place, possibly linked with epigenomic re- characterized differences in the microbiota upon
arrangements. Caesarean sections (Dominguez-Bello et al.
Apart from certain developmental stages that 2010) affect the microbial metabolome and epig-
require sufficient abundance of substrates, such enome of the host.
as methyl donors, to satisfy increased substrate One of the most relevant health problems in
expenditure, it should be noted that also situa- the twenty-first century is the increasing inci-
tions of unbalanced nutrient supply might lead to dence and severity of obesity. It has been shown
a relative substrate shortage with associated in animal experiments, but also clinical studies,
effects on the epigenome. It is conceivable that in that the microbiota of lean and obese subjects dif-
situations with a low intake of micronutrients and fers significantly. Moreover, manipulation of the
high intake of fat and proteins, as often seen in microbiota has been shown to improve metabolic
obesity, the delicate balance between supply and parameters as well as body weight directly, lead-
demand of necessary substances is most likely ing to the idea that the gut microbiota could pose
disturbed. On the one hand this could be due to a novel target for weight control. Specifically, the
direct effects of low quality diets and higher first human trials of fecal transplants from lean to
requirements of a bigger body volume, on the obese people initiated a debate whether this tech-
other hand specific dietary effects on the micro- nology might be a novel treatment strategy for
biota and its metabolome might contribute to obesity. This adds to the long lasting discussion
this. over possible dietary or supplementary interven-
tions with pre- and probiotics to improve human
health.
3.5 Health Implications - Sooner On the background of our ideas, the situation
or Later gets even more complex. Bacterial metabolites,
impacting the host epigenome, could not only
We have recently proposed that infant nutrition have direct but especially long-lasting physiolog-
may have long-term physiological consequences, ical and health consequences. For any interven-
mediated by the epigenome (Mischke and Plosch tion aimed at the gut microbiota, this would
2013). We speculated that early-life nutrition imply that changing the microbiota might initiate
may determine the composition of the early gut long-lasting effects, which need to be considered
microbiota and therefore the absolute amounts before these interventions can be viewed as safe.
and ratios of bacterial metabolites, including On the other hand, (early-life) microbiota-related
butyrate and folate. As a consequence, tissues strategies may not only have tremendous impact,
exposed to high concentrations of these metabo- but consequently also offers enormous opportu-
lites, like the intestinal epithelium or the liver, nities regarding prevention of disease and modu-
may undergo epigenetic changes, which might lation of long-term health. A first, rather simple
program their long-term metabolic set points and step to neutralize negative effects of the delivery
regulation. This, in turn, may alter organ plastic- mode on microbiome, metabolome and epig-
ity linked to the health status of the host. enome of the infant might be the inoculation of
Obviously, this hypothesis needs to be thoroughly infants after Caesarean section with a natural
tested. bacterial population. Beyond this, it may be
Fig. 3.1 Proposed mechanism

how epigenetic actions that
potentially impact long-term
physiology and health are
linked to (early-life) factors
via the microbiota and its
metabolites (Modified from
(Mischke and Plosch 2013))
helpful comments and stimulating discussions and

feasible one day to integrate our knowledge of
Charlotte Snethla for supporting the data compilation.
the host-microbiota interaction into novel nutri-
tional strategies for infants. Apart from benefi-
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The Oral Microbiota
4
Nicole B. Arweiler and Lutz Netuschil
Abstract
The oral microbiota represents an important part of the human microbiota,
and includes several hundred to several thousand diverse species. It is a
normal part of the oral cavity and has an important function to protect
against colonization of extrinsic bacteria which could affect systemic
health. On the other hand, the most common oral diseases caries, gingivitis
and periodontitis are based on microorganisms. While (medical) research
focused on the planktonic phase of bacteria over the last 100 years, it is
nowadays generally known, that oral microorganisms are organised as
biofilms. On any non-shedding surfaces of the oral cavity dental plaque
starts to form, which meets all criteria for a microbial biofilm and is sub-
ject to the so-called succession. When the sensitive ecosystem turns out of
balance – either by overload or weak immune system – it becomes a chal-
lenge for local or systemic health. Therefore, the most common strategy
and the golden standard for the prevention of caries, gingivitis and peri-
odontitis is the mechanical removal of this biofilms from teeth, restora-
tions or dental prosthesis by regular toothbrushing.
Keywords
Biofilm • Health-disease-relationship • Periodontitis • Dental plaque
The oral microbiota represents an important part thousand diverse species. This diversity com-
of the human microbiota, and includes, according prises several facets:
to different references, several hundred to several
1. Variety: It is estimated, that a minimum of
700 species occur in the human cavity, from at
N.B. Arweiler (*) • L. Netuschil least 12 phyla (Wade 2013), including even
Department of Periodontology, University of Archaea;
Marburg, Georg-Voigt-Str., 35039 Marburg, Germany 2. Diverse locations: Saliva; soft tissues like
e-mail: arweiler@med.uni-marburg.de;
netuschi@med.uni-marburg.de mucosa and the surface(s) of the tongue; hard

46 N.B. Arweiler and L. Netuschil
tissues (teeth) where the dental biofilm (dental et al. 2005), and in this context Wade (2013)
plaque) is located in fissures or supra- or sub- stresses the topic of “uncultivable oral bacteria”.
gingival, as well as on hard materials like den- This problem touches a serious discussion lasting
tures and, more recently, oral implants; now for more than a century concerning the via-
3. Intra-oral dislodging: While quite a lot of bility of (marine and other) bacteria (Winterberg
the “700 species” prefer specific niches as a 1898; Ziegler and Halvorson 1935; Postgate
habitat, some are found at different locations. 1969; Davey 2011; Netuschil et al. 2014).
For example Streptococcus mutans is detected However, while marine microorganisms seem to
in saliva, in dental (fissure and supragingival) be “unculturable” due to dormancy, low tempera-
plaque as well as on the tongue. These strepto- ture or substrate depletion, the problems in cul-
cocci, as well as other species, dislodge from turing oral bacteria are based on their need for
one location in the oral cavity to others with a very specific nutrients, in part extreme oxygen
certain mutual relationship. sensitivity, and, finally, dependence on other
4. Age-related microbiological changes: two neighboring organisms (Wade 2013). For exam-
different “points of view” are to distinguish: ple, some species of the periodontitis-associated
(i) truly age-related changings, and (ii) altera- microbiota are influenced by the levels of human
tions due to the emergence of teeth, i.e. natu- sexual hormones (Kornman and Loesche 1980,
ral hard surfaces, or to the incorporation of 1981; Jensen et al. 1981). Table 4.1 (from Marsh
artificial hard surfaces like orthodontic et al. 2009) lists some properties of the oral
devices, implants, or dentures. microbiota contributing to the difficulty in deter-
5. Succession of the oral microbiota – biofilm mining its composition.
formation: This is the change in the composi- More recent genetic analyses disclosed even
tion of the oral microbiota on dental hard sur- 10.000 species-level biotypes (Keijser et al. 2008),
faces between day 1 (streptococcal, facultative) for example using ‘454 pyrosequencing’
up to day 7 (Gram-negative rods, spirochetes (Voelkerding et al. 2009; Zaura et al. 2009). In spite
etc., anaerobes), including the development of of the fact that those numbers should be seen with
sub-systems and so-called “complexes”. caution, it is a fact that more than tenfold of the
6. Biofilm structure: The dental plaques on oral species numbers were detected via conventional
hard surfaces represent unique examples of cultivation (Zaura et al. 2009). As an example
microbial biofilms. Table 4.2 lists only those genera which were found
7. “Health-disease-relationship” – signifi- and described since 1990 (from Wade 2013).
cance of oral flora for systemic health: The
normal protective microbiota as compared to
(i) caries-related, (ii) periodontitis-related 4.2 Locations of the Oral
bacteria and (iii) eventually denture stomatitis Microbiota
(Candidiasis) as well as immune stimulation
Figure 4.1 depicts the diverse habitats of the oral
microbiota. The saliva represents the “planktonic
4.1 Variety phase” of the oral microbiota. Similar to bacterial
laboratory fluid cultures saliva contains up to 109
About 700 bacterial species inhabit the human microorganisms per milliliter, which are swal-
mouth, and this quantity seems to be a magic lowed continuously. In this way, about 5 g of bac-
number in quite a lot of articles (Moore and teria ‘disappear’ into the stomach daily. Therefore,
Moore 1994; Aas et al. 2005; Bik et al. 2010; Liu saliva is not considered to have its own resident
et al. 2012; Jakubovics and Palmer 2013 microbiota, and that bacterial numbers in saliva,
[Preface]). in contrast to dental plaque, do not multiply
About 50 % of these species or phylotypes within the mouth (Marsh et al. 2009). However,
have not even cultured yet (Marsh 2005; Aas saliva is the primary source for the continuous
4 The Oral Microbiota 47
Table 4.1 Properties of the oral microflora that contribute to the difficulty in determining its composition (From Marsh
et al. 2009)
Property Comments
High species diversity The oral microflora, and especially dental plaque,
consists of a diverse number of microbial species, some
of which are present only in low numbers
Surface attachment/coaggregation (coadhesion) Oral microorganisms attach firmly to surfaces and to
each other and, therefore, have to be dispersed without
loss of viability
Obligate anaerobes Many oral bacteria lose their viability if exposed to air
for prolonged periods
Fastidious nutrition/unculturable Some bacteria are difficult to grow in pure culture and
may require specific cofactors etc. for growth
Some groups (e.g. certain spirochaetes; TM7 group)
cannot as yet be cultured in the laboratory
Slow growth The slow growth of some organisms makes
enumeration time consuming (e.g. they may require
14–21 days incubation)
Identification The classification of many oral microorganisms still
remains unresolved or confused; simple criteria for
identification are not always available (particularly for
some obligate anaerobes)
Table 4.2 Recently described bacterial genera with oral Moreover, shedding surfaces, where only
representatives (since 1990) (Adapted from Wade (2013)) monolayers of bacteria originate and which are
Phylum Genera regularly desquamated (cheek, palate) have to be
Actinobacteria Actinobaculum, Atopobium, discriminated from the tongue with its ‘stable’
Cryptobacterium, Kocuria, Olsenella, multilayers of biofilm-like bacteria. It is estimated
Parascardovia, Scardovia, Slackia,
Tropheryma that the tongue harbours the majority of the micro-
Bacteroidetes Bergeyella, Prevotella, Tannerella bial burden of the oral cavity, and supports a higher
Firmicutes Abiotrophia, Anaerococcus, bacterial density and a more diverse microbiota
Aneroglobus, Bulleidia, Catonella, than the other mucosal surfaces; 30 % of the bacte-
Dialister, Filifactor, Finegoldia, rial population detectable by molecular studies
Granulicatella, Johnsonella,
Mogibacterium, Parvimonas,
were found only on the tongue (Marsh et al. 2009).
Peptoniphilus, Pseudoramibacter, On any non-shedding surfaces dental plaque
Schwartzia, Shuttleworthia, starts to form, which meets all criteria for a micro-
Solobacterium bial biofilm (cf. paragraph 6), and is subject to the
Proteobacteria Lautropia, Suttonella so-called ‘succession’ (cf. paragraph 5). Such bio-
Synergistetes Jonquetella, Pyramidobacter film formation is found at different locations:
bacterial (re)colonization of the diverse oral soft • Fissure biofilm (in cavities inside the teeth,
and hard surfaces. approaching the dental pulp) is dominated by
Regarding microbial settlement shedding facultative species, especially streptococci,
surfaces (mucosal sites) like lips, cheek, palate causing fissure caries and eventually endodon-
and tongue have to be differentiated from non- tic problems;
shedding surfaces, the natural teeth as well as • Supragingival biofilm (on the dental enamel
artificial materials surfaces of fissure sealings, adjacent to the gingiva) contains, related to its
tooth fillings, orthodontic appliances, dentures maturation and thickness, a mixture of facul-
and also oral implants (Fig. 4.1, Table 4.3; Marsh tative and anaerobe species, causing an unspe-
et al. 2009; Zaura et al. 2009). cific gingival inflammation (gingivitis);
Fig. 4.1 Colonizing strategy of oral bacteria on the different oral surfaces, and mutual transfers. Blue: saliva, plank-
tonic phase; red: shedding surfaces; white: non-shedding (hard) surfaces
(Fig. 4.2 shows plaque grown on teeth and 4.3 Intra-oral Dislodging: Mutual
stained red with disclosure solution) Transfer
• Only when supragingival plaque exists for
quite a long time harming the gingival crevice, While quite a lot of the “700 species” prefer
periodontitis may occur due to development specific niches as a habitat, some are found at
of subgingival plaque. This type of biofilm multiple locations. For an example Streptococcus
contains mainly anaerobe species. mutans, a streptococcal species causing caries,
• Plaque on artificial surfaces (e.g. dental fill- is detected in saliva, in dental (fissure and supra-
ings) resembles mainly the supragingival gingival) plaque as well as on the tongue
entity. Denture plaque may harbour Candida (Schlagenhauf et al. 1995) with a certain mutual
spp., which may cause ‘denture stomatitis’. relationship (Van Houte and Green 1974). A
The microbiota relevant for peri-implant corresponding common semi-quantitative
mucositis (analogous to gingivitis) and ‘Caries Risk Test’ is said to rely on the concen-
eventually peri-implantitis (analogous to peri- tration of S. mutans in saliva (Beighton 1986).
odontitis) is not yet well understood (for some However, the clinician conducting the test is
further details cf. paragraph 4). asked to place and turn the test stick on the dor-
sal surface of the tongue (Schlagenhauf et al.
While Table 4.3 describes the diverse habitats 1995), and therefore the test results reflect rather
of the oral microbiota in general, Table 4.4 (both the amount of S. mutans of the tongue biofilm.
tables taken from Marsh et al. 2009) summarizes After suppression of S. mutans with chlorhexi-
the various bacterial species and groups found at dine a specific recolonisation pattern could be
different locations in a normal human oral cavity. observed (Emilson et al. 1987). Moreover, it is
known that S. mutans cannot be eradicated from saliva, the periodontal pockets and moreover the
its oral habitat. oro-pharyngeal area (Quirynen et al. 2001). A
The same holds true regarding periopathogens specific example represents Aggregatibacter
(mainly Gram-negative anaerobes). Most of these actinomycetemcomitans, which is detected not
species colonize various niches within the oral only in supra- and subgingival plaque, but also in
cavity, e.g. the oral mucosa, the tongue, the saliva and on mucous membranes (Petit et al.
1994). Similar to S. mutans these findings have
Table 4.3 Distinct microbial habitats within the mouth implications for therapeutic measures because
(Adapted and complemented from Marsh et al. 2009) the intra-oral translocation of periopathogens
Habitat Comments may jeopardize the outcome of periodontal ther-
Saliva Planktonic phase apy (Quirynen et al. 2001).
Continuously swallowed One study tried to evaluate a sibling relation-
Lips, cheek, palate Biomass restricted by ship on the periodontal conditions. Significant
(shedding, desquamation sibship effects were found, among others, for spi-
monolayer) Some surfaces have rochetes on the tongue and in the pockets, for
specialized host cell types Porphyromonas gingivalis on the gingiva and in
Tongue (shedding, Highly papillated surface saliva and for Prevotella intermedia in saliva,
multilayer) Acts as a reservoir for demonstrating that all those habitats contribute to
obligate anaerobes
the overall picture (Van der Velden et al. 1993).
Natural (teeth) and Non-shedding surfaces
artificial hard enabling large masses of
surfaces (dental microbes to accumulate
materials) (non- Teeth have distinct surfaces 4.4 Inter-oral Transmission
shedding, for microbial colonization; of Bacteria and Age-Related
multilayers) (dental each surface (e.g. fissures, Microbiological Changes
plaque biofilms) smooth surfaces, approximal,
gingival crevice) will support
a distinct microflora because Teo different “points of view” are associated with
of their intrinsic biological age-related changes: (i) truly age-related changes,
properties and (ii) alterations due to the emergence of teeth,
Fig. 4.2 Dental biofilm on

teeth visualized by dental
disclosure solutions
Table 4.4 Relative proportions of some cultivable bacterial populations at different sites in the normal oral cavity
(From Marsh et al. 2009)
Bacterium Saliva Buccal mucosa Tongue dorsum Supragingival plaque
Streptococcus sanguinis 1 6 1 7
S. salivarium 3 3 6 2
S. oralis/S. mitis 21 29 33 23
Mutans streptococci 4 3 3 5
Actinomyces naeslundii 2 1 5 5
A. odontolyticus 2 1 7 13
Haemophilus spp 4 7 15 7
Capnocytophaga spp <1 <1 1 <1
Fusobacterium spp 1 <1 <1 <1
Black-pigmented anaerobes <1 <1 1 +a
a
Detected on occasions
i.e. natural hard surfaces, or to the implementation appliances, Micheelis et al. 2006) such new mate-
of artificial hard surfaces like orthodontic devices, rial surfaces contribute to a tremendous increase
implants, or dentures. At first glance the microbi- in the salivary levels of caries-related bacteria
ota does not change over decades in age-cohorts like S. mutans. Last not least when dentures are
between 20 and <70 years. Percival et al. (1991) used, Candida spp. may increase (Gendreau and
detected no age-related changes in caries associ- Loewy 2011; Salerno et al. 2011). The corre-
ated mutans streptococci and in periodontitis- sponding literature reflects an ambiguous picture.
related spirochetes. However, differences were On the one hand it is described that Candida
found in age groups >70 years differences were overgrowth is not associated with denture-
found regarding lactobacilli, staphylococci, and wearing (Percival et al. 1991; Kraneveld et al.
yeasts, which increased significantly. These 2012), while on the other hand “denture stomati-
increases were not related to denture-wearing or tis” has become a standard term regarding this
disease. It has been assumed that age-dependent aspect in dentistry of the elderly.
multi-morbidity and multifold usage of medica- It is also well established that transmission of
ments, which are well known to diminish saliva anaerobic bacteria, in part periopathogens, occurs
production, and the subsequent acidification of during the first year in children (Könönen 1999),
the oral milieu are responsible for this change where Veillonella spp. and P. melaninogenica
(Kraneveld et al. 2012). However, these studies were found even after 2 months, while A.
did not consider children and adolescents. actinomycetemcomitans seems to be a ‘late colo-
Moreover, the caries-related and the periodontitis- nizer’ emerging between the ages of 4–7 years
associated microbiota have to be distinguished. (Alaluusua and Asikainen 1988; Könönen 1999).
Concerning caries alterations due to point (ii) For a short overview see Table 4.5.
are of importance. An obvious example repre- Similar to S. mutans, these species are mostly
sents the emergence of teeth occurring during the transferred via the maternal saliva (Könönen
6th-7th month on. Only then streptococci like S. et al. 1992); in one specific case even the trans-
mutans and S. sobrinus are able to colonize the mittance of A. actinomycetemcomitans from a
hard enamel surfaces. Surprisingly, it is not until dog to a child was reported (Preus and Olsen
1 year later that a corresponding “window of 1988). The pattern of colonizing of these anaer-
infectivity” can be found and designated obes is even influenced by the emergence of the
(Caufield et al. 1993) between 19 and 31 month primary dentition (Könönen et al. 1994) in spite
of age, with a median of 26 month. of the fact that these species have no impact on
In later years, when orthodontic appliances diseases of intraoral hard tissues like enamel. In
are incorporated (e.g. in Germany, 45 % of 12 2- to12- year-old children periopathogens are fre-
years old and 58 % of 15 years old wear such quently detected (Okada et al. 2001).
Table 4.5 The effect of tooth eruption on the composi- els. However, no such relationship could be
tion of the cultivatable oral microflora in young children
established.
(From Marsh et al. 2009)
A specific and in part still neglected problem
Percentage isolation
is the peri-implant mucositis and peri-implantitis
frequency
(which is called “the periodontitis of the
At mean age
implant”). Implant surfaces are as easily and as
Bacterium 3 months 32 months
Prevotella melaniogenica 76 100
rapidly colonized by bacteria as teeth (Fürst et al.
Non-pigmented Prevotella 62 100
2007), however, different colonization patterns
Prevotella loescheii 14 90 seem to exist on implants as compared to teeth
Prevotella intermedia 10 67 (Salvi et al. 2008). In general, only few investiga-
Prevotella denticola Not detected 71 tions exist assessing the microbiota of implants
Fusobacterium nucleatum 67 100 when compared to the bulk of literature regarding
Fusobacterium spp. Not detected 71 periodontitis – contributing to a confusing pic-
Selenomonas spp. Not detected 43 ture. The peri-implant microbiota is described to
Capnocytophaga spp. 19 100 be quite similar to that of periodontitis (Leonhardt
Leptotrichia spp. 24 71 et al.1993), but with some relevant differences
Campylobacter spp. 5 43 (Persson and Renvert 2013). For example
Eikenella corrodens 5 57 Staphylococcus aureus is more common in peri-
Veillonella spp. 63 63 implant plaque (Rams et al. 1990), because this
germ is attracted by titanium surfaces (Harris and
Richards 2004). Figure 4.3 shows a SEM picture
Intrafamilial transmission has been shown of a biofilm on an implant which had to be
(Alaluusua et al. 1991; Petit et al. 1994), espe- explanted due to periimplantitis.
cially between spouses, where disease-related These local bacterial changes are also a sys-
bacteria are transferred between (clinically) temic challenge and stimulation for the immune
healthy and periodontally diseased family mem- system. This aspect will be discussed in Sect. 4.7.
bers (Offenbacher et al. 1985; Saarela et al. 1993).
It was evident that horizontal transmission
between spouses ranged between 14 % and 60 % 4.5 Succession of the Oral
for A. actinomycetemcomitans, and between 30 % Microbiota: Biofilm
and 75 % for P. gingivalis, while vertical trans- Formation
mission was estimated between 30 % and 60 % for
A. actinomycetemcomitans, but only rare vertical Because Saliva is frequently swallowed, no suc-
transmission of P. gingivalis was observed (Van cession can occur. Salivary bacteria regularly
Winkelhoff and Boutaga 2005). Thus, it is a mat- colonize the mucosal cells (Slots and Gibbons
ter of discussion whether this phenomenon ham- 1978). In spite of the fact that the soft tissues rep-
pers the treatment outcome (Von Troil-Linden resent 80 % of the surfaces prone to bacterial
et al. 1997; Kleinfelder et al. 1999). colonization (Collins and Dawes 1987) no patho-
After early childhood there seems no further genetic problems arise thereof, because the
change till puberty (Könönen 1999), while dur- mucosal cells desquamate and are swallowed,
ing puberty alterations are found regarding similar to saliva. Therefore, only a non-pathogen
Veillonella spp., Prevotella denticola and monolayer consists on mucosal surfaces.
Prevotella melaninogenica (Gusberti et al. 1990; In contrast hard tissues are immediately cov-
Moore et al. 1993). Because, as previously men- ered by the ‘pellicle’ (Sönju 1987; Hannig 1997)
tioned, some members of the periodontitis- (for example after meticulous tooth cleaning
associated microbiota are influenced by the levels measures) and concomitantly colonized by bac-
of human hormones (Kornman and Loesche teria (Rönström et al. 1977; Kolenbrander and
1980; Jensen et al. 1981), Moore et al. (1993) London 1993). Following this first phase the
looked for an association with testosterone lev- plaque biofilm microbiota changes steadily
Fig. 4.3 (a, b): SEM picture of a (highly diverse) biofilm on an implant which had to be explanted due to periimplan-
titis (Thanks also to Prof. S. Nietzsche, University of Jena)
from day to day, a process called succession Induction: This first phase is characterized by
(Theilade et al. 1966; Ritz 1967; Marsh 1990). the formation of the aforementioned pellicle
An immediate streptococci-dominated ‘primary as a ‘conditioning film’ or ‘linking film’
flora’ changes during 7 days of development to (Busscher and van der Mei 1997), but the first
an anaerobic ‘climax community’, characterized bacteria are also sometimes already visible
by Gram-negative rods (Morhardt and Fitzgerald (Marsh and Bradshaw 1995; Hannig 1999)
1980). Due to different localizations as well as (see Fig. 4.4a).
diverse exogenous influencing factors, plaque of Accumulation: This second step includes differ-
different thickness and bacterial composition ent topics like bacterial adhesion, bacterial
develops, not only on a macroscopic scale but growth (“planar colonisation”) and so called
also at the micro-ecological level, as related to ‘quorum sensing’ (cf. Chap. 6).
O2-tension, local pH, matrix structure and avail- Existence: The third phase, when occurring, is
ability of nutritive substances (Marsh et al. characterized by equilibrium between growth
2009). Thus diverse sub-systems develop and concomitant decomposition via so called
(Garlichs et al. 1974; Morhardt and Fitzgerald “biofilm erosion” and “biofilm sloughing”,
1980; van Palenstein Helderman 1981; whereby cells and cell clusters are teared off
Babaahmadi et al. 1998). One example is the for settling on other surfaces.
site-specific distribution pattern of periodontitis-
relevant micro-organisms as described by Tanner The event of a succession in the oral microbi-
et al. (1998). ota was confirmed for subgingival plaque by
Socransky et al. (1998) and Haffajee et al. (1999).
Using molecular biology techniques the authors
4.6 Phases of Biofilm assessed and grouped diverse bacterial “inhabit-
Development ants” of about 13,000 plaque samples in so called
‘complexes’. One ‘yellow complex’ comprised
Several different phases are characterized during mainly of streptococci, which in accordance to
succession. This holds true for dental but also for other authors (Theilade et al. 1966; Kolenbrander
all other natural occurring biofilms – such as et al. 1999) were shown to represent the earliest
medical or environmental biofilm. Dental biofilm colonizers. In a second ‘orange complex’ differ-
formation can be visualized for example by con- ent species were grouped, the most important
focal laser scanning microscopy (CLSM) on being Fusobacterium nucleatum. This species has
enamel slabs, presented in Fig. 4.4a–d. a high ability to coaggregate with other bacteria
Fig. 4.4 (a–d): Biofilm formation on dental enamel tracked by confocal laser scanning microscopy (gray modus)
(Kolenbrander et al. 1989), thus “bridging” spe- responsible for the etiology of gingivitis and peri-
cies of the earlier ‘yellow’ with those of the late odontitis (Theilade et al. 1966; van Palenstein
‘red complex’. This last complex comprising of P. Helderman 1981; Socransky et al. 1998; Haffajee
gingivalis, Tannerella forsythus and Treponema et al. 1999).
denticola was strongly associated with the main-
tenance and worsening of periodontitis.
Interestingly, F. nucleatum proved to be the most 4.7 Dental Plaque: A Typical
frequent anaerobe species in infants’ mouths at 1 Biofilm
year of age (Könönen 1999), a finding perfectly
fitting with the concept of the crucial role of F. There are several definitions of the term biofilm,
nucleatum in intergeneric coaggregation and bio- for example “matrix enclosed bacterial popula-
film formation (Kolenbrander et al. 1989). In sum tions adherent to each other and/or to surfaces or
a succession from mainly facultative species (in interfaces” (Costerton et al. 1995; Costerton and
part involved in dental caries) to a more and more Lewandowsky 1997); “a biofilm will form on any
anaerobe community occurs, which is finally surface that is exposed to microbes, water, and a
small amount of nutrient” (Wimpenny 1997); or, butes such as altered growth rate and antibiotic
somewhat more detailed “A biofilm is defined as resistance: biofilm organisms transcribe genes
bacterial aggregates, usually existing as closely that planktonic cells do not. Moreover, bacteria
associated communities, that adhere to assorted bound in the environment of biofilms exert a sub-
natural or artificial surfaces, usually in aqueous stantial resistance against detergents, antibiotics
environment that contains a sufficient concentra- or other antibacterial compounds, and phagocy-
tion of nutrients to sustain the metabolic needs of tosis due to “persisters” (Donlan and Costerton
the microbiota” (Listgarten 1999). In this respect 2002; Obst et al. 2006; Anwar et al. 1992). The
dental plaque shows the general characteristics of underlying mechanisms are complex and multi-
biofilms (Wimpenny et al. 2000; Costerton et al. factorial (del Pozo and Patel 2007). A modern
1999); moreover, as mentioned, it harbours a definition thus needs to contain the facets that
plethora of bacterial species, and thus, is biofilm organisms exhibit an altered phenotype
extremely heterogeneous (Costerton et al. 1999; with respect to growth rate, gene transcription
ten Cate 2006; Paster et al. 2006; Zaura et al. and ‘quorum sensing’. This latter phenomenon,
2009). For a short overview see Table 4.6. also called ‘biofilm signaling’, describes the
In spite of the fact that the biofilm phase inter-generic bacterial communication (signal
worldwide comprises 80–90 % of micro- transduction), which depends on cell density and
organisms, the (medical) research focused on the occurs in maturating biofilms (Kaiser and Losick
planktonic phase of bacteria over the last 100 1993; Fuqua et al.1996; Costerton et al. 1999;
years. Biofilms contain 1000-fold more bacteria Prosser 1999).
per gram than the planktonic phase; on the other Concerning methods in biofilm research
hand they are by factor 500–1000 more resistant there are some crucial prerequisites when evalu-
against antibacterial compounds. It is notewor- ating oral (plaque) biofilms: (1) Intraoral splint
thy, that both of these factors are equal in their systems, which enable the undisturbed accumu-
importance. However, the higher density is not lation of dental biofilms on the surface(s) of
the main reason for the increased resistance. A native enamel slabs (Auschill et al. 2004;
few general examples are on the enormous Arweiler et al. 2004) or dental materials (Auschill
importance of biofilms in ecology, medicine and et al. 2002); including (2) the concomitant forma-
industry are presented in Table 4.6. tion of a native pellicle (Hannig 1997, 1999).
Donlan and Costerton (2002) state that not Traditionally, the oral tooth-related microbi-
only the architecture and other readily observable ota was and still is assessed either by conven-
characteristics like adherence and extracellular tional microbiological methods (cultivation;
matrices, are important for differences in com- Theilade et al. 1966; Theilade and Theilade 1970;
parison to planktonic cells, but also inherent attri- Mikkelsen 1993) or by electron microscopy
Table 4.6 Characteristics of biofilms

Parameter Planktonic phase Biofilm
General definition Free floating, non-adherend, not localized Adherend, localized
Density 108–109 Bacteria/mL = 108–109 Bacteria/ 1011–1012 Bacteria/cm3 = 1011–1012
gram Bacteria/gram
Occurence 10–20 % 80–90 %
Research focus 1880–1980 1960–to date
Resistance against antibac+terial Generally low Generally high or very high
compounds
(Medical) importance E.g. Legionella; E. coli Med. catheters, lenses
Oral diseases
Food/paper/oil industries
Navigation
(TEM and SEM; Listgarten 1965; Theilade and stomatitis (Candidiasis). Most people are carriers
Theilade 1970; Saxton 1973; for review cf. of candida but oral candidiasis is very rare.
Newman and Wilson 1999). Furthermore, vital The role of periodontal disease as a risk
(fluorescence) staining techniques were used to factor in the development and/or progression
elucidate the portion of vital or dead bacteria in of systemic diseases such as diabetes, rheuma-
the dental biofilm (Netuschil et al. 1989, 2014), toid arthritis, cardiovascular disease, adverse
which can also visualize the effect of antibacte- pregnancy outcomes, head-and-neck cancer)
rial agents by CLSM (Fig. 4.5, vitalfluorescence has been subject of many investigations in
(VF); Fig. 4.6, VF combined with CLSM). More recent years (Han et al. 2014).
recently the FISH technology (Fluorescence in
situ hybridization) was introduced to plot specific
bacterial species and to depict the distribution of 4.9 Prospects
them in a biofilm network (Al-Ahmad et al. 2007;
Fig. 4.7). Thus, different “visualizing” methods While vaccination is due to the high diversity of
were combined with CLSM to reveal the three- the oral biofilm not an alternative, brand new
dimensional architecture of oral biofilms concepts aim to strengthen the normal, protective
(Netuschil et al. 1998, 2014; Auschill et al. 2002, microbiota. Probiotics are known as promoter of
2004; Arweiler et al. 2004, 2013; Zaura et al. a natural microbiota, e.g. following the adminis-
2001; Al-Ahmad et al. 2007, 2009, 2010). tration of antibiotics. Meanwhile, numerous pro-
biotic products containing bacteria such as
lactobacilli or E. faecalis are commercially avail-
4.8 “Health-Disease- able and have been widely-used for their health
Relationship” benefits. From a dental point of view, coloniza-
and Significance of Oral tion of the oral cavity and particularly the oral
Flora for Systemic Health biofilm with probiotic bacteria is feared, since
they are able to ferment sugars. This results in
As mentioned, highly diverse oral microbiota is acid production, which causes dissolution of the
a normal part of the oral cavity. It has an impor- enamel and caries development. This factor and
tant function to protect against colonization of also the question if probiotic bacteria can reside
extrinsic bacteria which could affect systemic and persist in the oral biofilm, have to be answered
health. in future research. Most recent research provide
Former recommendations aimed on a high an indication that probiotic bacteria such as lac-
standard of oral hygiene (of parents) to prevent tobacilli will not prevail within the oral biofilm
microbial colonization of the infantine oral but can significantly suppress S. mutans spp. (Al
cavity. Newer findings show that parental suck- Ahmad et al. 2014).
ing of pacifiers or spoons leads to a risk reduction
of allergy development possibly via immune
stimulation by the transferred microbes 4.10 Summary
(Hesselmar et al. 2013).
On the other hand, the most common oral dis- The microbiota is an important part of our oral
eases caries, gingivitis and periodontitis are cavity. However, when this sensitive ecosystem
based on microorganisms. Bacteria are a neces- turns out of balance – either by overload or weak
sary but not sufficient requirement for the devel- immune system – it becomes a challenge for local
opment of these diseases. It is generally assumed or systemic health. Therefore, the most common
that ecological conditions (especially of the host) strategy and the golden standard for the preven-
play a key role in the development of these dis- tion of caries, gingivitis and periodontitis is the
eases. The same hold true for infection with can- mechanical removal of this biofilms from teeth,
dida species and the formation of a denture restorations or dental prosthesis by regular tooth-
Fig. 4.5 Vital fluorescence staining

to elucidate the portion of vital
(green) and dead (red) bacteria in
dental biofilm
Fig. 4.6 Three dimensional

architecture of oral biofilm and
distribution of vital and dead
bacteria after regular rinsing
with chlorhexidine (antiseptic
agent). Still a “vital core” is
left which shows the highly
resistant nature of biofilms
Fig. 4.7 Confocal micrographs of 3-day-old dental Veillonella spp.-specific probe; (D) Green, eubacteria-
plaque biofilm hybridized with four different specific specific Probe – all bacteria. The distribution of specific
probes (Multiplex-FISH) as described in Al-Ahmad et al. bacteria in the biofilm network can be determined by com-
(2007). (A) yellow, F. nucleatum-specific probe; (B) parison with eubacteria using a software program. (By
magenta, Streptococcus spp.-specific probe; (C) red, courtesy of Prof. Dr. Ali Al-Ahmad)
brushing, daily interdental cleaning and by dental situ investigated using fluorescence in situ hybridiza-
professionals on a regular basis. This has to be tion. J Med Microbiol 58:1359–1366
Al-Ahmad A, Wiedmann-Al-Ahmad M, Faust J, Bächle
trained and adapted from childhood. It also guar- M, Follo M, Wolkewitz M, Hannig C, Hellwig E,
antees that biofilms which start to form within a Carvalho C, Kohal R (2010) Biofilm formation and
few minutes after removal only develop into the composition on different implant materials in vivo.
J Biomed Mater Res B Appl Biomater 95:101–109
phase of an early, thin and not anaerobe biofilm.
Al-Ahmad A, Hellwig E, Follo M, Auschill TM, Arweiler
NB (2014) Probiotic lactobacilli do not integrate into
oral biofilm in situ. In: The ESCMID Study Group for
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The Microbiota of the Human Skin
5
Markus Egert and Rainer Simmering
Abstract
The aim of this chapter is to sum up important progress in the field of
human skin microbiota research that was achieved over the last years.
The human skin is one of the largest and most versatile organs of the
human body. Owing to its function as a protective interface between the
largely sterile interior of the human body and the highly microbially con-
taminated outer environment, it is densely colonized with a diverse and
active microbiota. This skin microbiota is of high importance for human
health and well-being. It is implicated in several severe skin diseases and
plays a major role in wound infections. Many less severe, but negatively
perceived cosmetic skin phenomena are linked with skin microbes, too. In
addition, skin microorganisms, in particular on the human hands, are cru-
cial for the field of hygiene research. Notably, apart from being only a
potential source of disease and contamination, the skin microbiota also
contributes to the protective functions of the human skin in many ways.
Finally, the analysis of structure and function of the human skin microbi-
ota is interesting from a basic, evolutionary perspective on human microbe
interactions.
Key questions in the field of skin microbiota research deal with (a) a
deeper understanding of the structure (species inventory) and function
(physiology) of the healthy human skin microbiota in space and time, (b)
the distinction of resident and transient skin microbiota members, (c) the
distinction of beneficial skin microorganisms from microorganisms or
communities with an adverse or sickening effect on their hosts, (d) factors
shaping the skin microbiota and its functional role in health and disease,
(e) strategies to manipulate the skin microbiota for therapeutic reasons.
M. Egert (*)
Faculty of Medical and Life Sciences, Institute of
Precision Medicine, Microbiology and Hygiene R. Simmering
Group, Furtwangen University, Jakob-Kienzle-Str. Corporate Scientific Services, Henkel AG & Co.
17, 78054 Villingen-Schwenningen, Germany KGaA, Henkelstr. 67, 40589 Düsseldorf, Germany
e-mail: Markus.Egert@hs-furtwangen.de e-mail: Rainer.Simmering@henkel.com

62 M. Egert and R. Simmering
Keywords
Skin microbiota • Bacteria • Archaea • Fungi • Actinobacteria • Firmicutes
• Proteobacteria • Homeostasis • Skin diseases • Skin defense and immu-
nity • Cosmetics • Perspiration
5.1 Introduction skin of humans (cutis, dermis); the microbiota

residing on epithelia inside the human body
The human skin is one of the largest and most (mouth, GI tract etc.) will be addressed in other
versatile organs of the human body. Owing to its chapters. In addition to this chapter, the reader is
function as a protective interface between the also referred to several excellent recent review
(mostly sterile) interior of the human body and articles on the structure and function of the
the (unsterile) outer environment, it is densely human skin microbiota (Grice and Segre 2011;
colonized with a diverse and active microbiota. Kong 2011; Kong and Segre 2012; Rosenthal
With respect to the number of microbial cells, the et al. 2011; Schommer and Gallo 2013). A brief
human skin is ranked fourth place among the historical view on the skin microbiota is given by
various niches of the human body, that are colo- Grice and Segre (2011); for more comprehensive
nized with microorganisms, outnumbered just by overviews on the more classical literature (based
the human gastrointestinal tract, the oral cavity mainly on culture-dependent studies) the reader
and the vagina (Wilson 2008). The skin microbi- is referred to the respective chapter in (Wilson
ota is implicated in several severe skin diseases 2008) or the reviews by Roth and James (1988)
(e.g., acne, psoriasis, atopic dermatitis etc.) and or Bojar and Holland (2002).
plays a major role in skin wound infections. Key questions in the field of skin microbiota
Several negatively perceived cosmetically rele- research deal with (a) a deeper understanding of
vant skin phenomena are linked with skin the composition and distribution of the human
microbes, too, e.g. impure skin, dandruff and skin microbiota in space (across the human body
body odor. The skin microbiota is also of high and the different niches of the human skin) and
relevance for the wide field of hygiene (clinical time (age of the host), (b) the distinction of resi-
hygiene, production hygiene, personal hygiene dent and transient skin microbiota members, and
etc.). Here, the microbiota of the human hands is (c) the distinction of beneficial (symbiotic) mem-
of particular importance. However, apart from bers of the skin microbiota or even beneficial
being a potential source of disease and contami- microbial communities from (parasitic) microor-
nation, the skin microbiota is also crucial for the ganisms or communities with an adverse or sick-
protective function of the human skin, e.g. by ening effect on their hosts. Finally, (d) abiotic
contributing to the skin acid mantle, by triggering and biotic factors shaping the skin microbiota
the skin immune system, or by preventing skin composition in space and time and (e) strategies
colonization with pathogenic microorganisms (preventive, therapeutic, cosmetic etc.) to manip-
(colonization resistance). Finally, the analysis of ulate the skin microbiota are of high medical but
structure and function of the human skin micro- surely also of commercial interest.
biota is also interesting from an evolutionary per-
spective on human microbe interactions.
In conclusion, the human skin microbiota, is 5.2 The Human Skin as a Habitat
without doubt of high importance for human for Microorganisms
health and well-being. The aim of this chapter is
to sum up important progress in the field of The human skin is a complex organ with many
human skin microbiota research achieved over important functions. As an interface between
the last years. The focus of the text is on the outer host and environment it provides a mechanical
5 The Microbiota of the Human Skin 63
and biological barrier against chemical, physical on human skin. Some areas of the skin are a
and pathogenic threats. Furthermore, it partici- rather sebaceous environment, resulting in a
pates in the process of thermoregulation and sup- microbiota dominated by lipophilic bacteria such
ports immunological functions. Melanogenesis is as propionibacteria. These areas are found on the
as well a process the skin is involved in, as it pro- scalp, the forehead, the neck and partly on the
tects against the effects of UV-radiation. To be upper part of the back. Other parts of the body are
able to fulfill all these important functions, the moist and warm, like the armpit, the genital area,
skin comprises many different structures and a and the feet. Finally, there areas which are rela-
diversity of cells with different properties. tively dry, such as the forearms or the legs and
Anatomically, the skin comprises two distinct lower part of the back. These differences are
compartments (Fig. 5.1): the epidermis, an avas- caused by the uneven distribution of sweat and
cular layer mainly composed of keratinocytes, sebaceous glands over the body.
and the dermis, a fibroblast-rich network of col- As mentioned above, an important role of the
lagen and elastin fibers that provides the skin human skin is the regulation of homeostasis, i.e.
with strength and elasticity. The dermis also con- body temperature and water content (Percival
tains capillary and lymphatic vessels, which et al. 2012). Organ failure due to denaturation of
serve as the entry and exit portals for immune proteins and subsequent cell death is caused by
cells. Additional skin appendages such as hair perpetual elevation of the body core temperature
follicles, sebaceous glands and sweat glands, as above 40 °C. Therefore, regulation of body tem-
well as nerve endings are also found in the der- perature represents a fundamentally important
mis. For a deeper understanding of the histology process (Wilke et al. 2007). This regulation is
of the normal and healthy human skin, the reader achieved by perspiration, i.e. the secretion of
is referred to (Urmacher 1990). sweat. Sweat is secreted by sweat gland, i.e. spe-
Due to their very versatile physiology, micro- cialized exocrine glands that are appendages of
organisms are able to colonize many of the differ- the skin (Fig. 5.1). Sweat glands can be catego-
ent niches (micro-ecosystems) that are realized rized into eccrine and apocrine glands. Sweat
Fig. 5.1 Layers of the human

skin and associated glands and
vessels. Microbial life was
long-believed to be restricted
to the epidermis and its
appendages (hair follicles and
glands). However, recent
studies suggest skin
microorganisms also to be
present in deeper layers, where
they can interact with the
skin’s immune system (Picture
credit: Don Bliss; National
Cancer Institute, USA)
glands exhibiting attributes of both types are until the onset of puberty and androgen stimula-
named apoeccrine sweat glands. The distribution tion (Wilke et al. 2007; Noël et al. 2012).
of these cutaneous glands varies over the body. In Following activation during puberty, apocrine
addition, each gland type is regulated by different glands are then stimulated by hormones.
stimuli and has different functions (Wilke et al. Apocrine sweat appears milky and viscous, and
2007; Noël et al. 2012). is known to comprise lipids, lactate, nitrogen,
Eccrine sweat glands are the most abundant electrolytes, steroids, proteins and vitamins
sweat glands. On average, 100–200 glands per besides other ions (Noël et al. 2012; Fredrich
cm2 are distributed virtually all over the body sur- et al. 2013). It is also suspected to contain phero-
face. The palms and soles exhibit higher densities mones (Wilke et al. 2007). However, the exact
of about 600 glands per cm2. In contrast, body composition of apocrine secretion has yet to be
parts such as the lips and nail bed are depleted of elucidated due to the lack of pure samples. In
eccrine glands (Noël et al. 2012). Eccrine sweat addition, while apocrine glands are suspected to
glands are effective from birth on in executing be involved in emotional sweating, their distinct
their main function, i.e. thermoregulation. function remains unidentified (Wilke et al. 2007;
Thermoregulatory perspiration is affected by Fredrich et al. 2013). Whereas eccrine sweat
environmental parameters, such as temperature, glands contribute only slightly to human body
humidity, skin and body temperature in general, odor, apocrine sweat is at least well accepted to
but also by physical fitness, circadian rhythm and contain substances that can be transformed into
the menstrual cycle. While temperature is the odorous molecules upon bacteriolysis (Wilke
major onset of eccrine glands, these glands are et al. 2007; Kelly and Wood 2010; Grice and
also activated by pain, stress, fear, and anxiety Segre 2011; Noël et al. 2012; Fredrich et al.
resulting in emotional sweating. Digestion is also 2013).
speculated to induce eccrine sweating. However, An important feature of skin and mucosa is –
the underlying mechanisms are not well under- in contrast to abiotic surfaces – the ability to con-
stood to date (Wilke et al. 2007). tinuously (re)generate new cells and to create
Another crucial function of eccrine sweat is response reactions which provide protection
the prevention of bacterial colonization and against microbial infection and degradation. This
growth through acidification of the skin surface innate immune system has to be considered as an
(Grice and Segre 2011). Sweat secreted from additional and important factor influencing the
eccrine sweat glands is clear and composed of microbial equilibrium on human skin. Langerhans
mainly water containing sodium and potassium cells in the epidermis as well as dendritic cells,
salts as well as amino acids, sugars, lactate and macrophages, mast cells, T and B cells, plasma
glycoproteins (Kelly and Wood 2010). Its exact cells and natural killer cells in the dermis partici-
composition differs depending on hormonal pate in immune responses within the skin. Many
activity, physical condition, acclimatization to cell types that permanently reside in the skin pro-
environmental conditions as well as secretion duce anti-microbial peptides, including keratino-
rate (Noël et al. 2012). cytes, sebocytes, eccrine glands, and mast cells
Apoeccrine sweat glands were first intro- (Schauber and Gallo 2008).
duced by (Sato et al. 1989). They are far less Thus, an important part of the antimicrobial
abundant on skin than eccrine glands. About defense system of the human skin constitutes of
2,000 of them are distributed around the eyes and small cationic peptides, i.e. human ß-defensins
ears as well as in the breast skin, while the high- hBD-1, hBD-2, hBD-3, hBD-4 (García et al.
est densities are reported for axillae and groin. 2001; Harder et al. 2001; Schauber and Gallo
Apocrine glands release their secret into hair 2008), of the human cathelicidin LL-37 (Frohm
canals, and are consequently limited to hairy et al. 1997), of antimicrobial enzymes like lyso-
body surfaces (Wilke et al. 2007; Kelly and Wood zyme and RNAse 7 (Harder and Schroder 2002)
2010; Noël et al. 2012). They are not activated and of several other molecules exhibiting
antimicrobial potential (for a review see Braff 5.3 Structure and Variability
et al. 2005). of the Healthy Skin
The active form of Cathelicidin LL-37 is Microbiota
derived from the precursor protein hCAP18 by
cleavage by serine proteases. It consists of 37 The skin microbiota of humans comprises bacte-
amino acids, exhibits an α-helical form and is ria, fungi (mostly yeasts), viruses and – which is
active against bacteria, fungi and viruses (Braff only known since recently – also archaea, i.e. all
and Gallo 2006). three domains of life. In addition, higher, para-
ß-defensins are small peptides (4–5 kDa) sitic eukaryotes (mostly arthropods) can occur
with a characteristic set of three disulfide bonds. (e.g., mites) but will not be discussed here, as
In general, hBD-1 is regarded as being constitu- they do not represent microorganisms sensu
tively expressed in the epithelium, while hBD-2 stricto. Recent investigations on the structure,
and hBD-3 are only induced by inflammation. In i.e. the species inventory, of the human skin
acne lesions, a strong induction of hBD-2 was microbiota were significantly influenced by the
observed in highly inflammated pustules, while application of high throughput next generation
hBD-1 was only moderately expressed with the sequencing technologies, which allow the analy-
strongest signal in the papules (Philpott 2003). In sis of millions of nucleic acid sequences (e.g.,
contrast, hBD-2 was not upregulated in atopic 16S rRNA or 18S rRNA genes) in a single study.
dermatitis (Ong et al. 2002), while both hBD-2 It is an advantage that molecular methods also
and hBD-3 were expressed in inflamed psoriatic detect microbes that are difficult to culture or
lesions (Harder and Schroder 2002; Nomura even have not been cultured yet. However, they
et al. 2003). In keratinocytes exposed to are usually inappropriate to differentiate living
Staphylococcus aureus, the expression of hBD-2 from dead microbes and can be affected by well
was strongly induced, while hBD3 and LL-37 know biases, such as incomplete nucleic acid
showed only moderate, and hBD-1 virtually no extraction from the samples or discrimination of
induction (Midorikawa et al. 2003). Malassezia sequences types due to mismatches with the
furfur was shown to induce the expression of primers used for PCR amplification (Forney et al.
hBD-2 via protein kinase C, but not of hBD-1 in 2004). Nonetheless and interestingly, (older)
keratinocytes (Donnarumma et al. 2004). RNAse cultivation-based and (more recent) large scale
7 was found to be induced in keratinocytes by molecular studies on the skin micobiota yielded
contact with heat-inactivated cells of bacterial rather similar results with regard to the dominant
pathogens like Pseudomonas aeruginosa, groups of microbial species on human skin.
Staphylococcus aureus, Escherichia coli and
Streptococcus pyogenes (Harder et al. 2001).
These antimicrobial peptides represent one 5.3.1 The “normal” Skin Microbiota
element, among others, of the innate immune of Healthy Adults
system of the skin. Their role is by far not only
restricted to the direct inactivation of skin micro- In order to define a “normal” skin microbiota
organisms but also includes stimulation of further healthy adults were studied. An adult and healthy
cellular reactions. As an example, a complex cas- human being is approximately colonized by 10 8–
cade involving IL-6, IL-10 and other cytokines is 1010 skin microbes in total, which are distributed
activated by cathelicidin LL-37 via cell-surface unevenly across the different niches of the human
receptors (for an overview see Schauber and skin. Cell numbers range from approximately
Gallo 2008). 102 cm−2 (fingertips, back) to 106 cm−2 (forehead,
Among the constitutive properties of skin that axilla). Owing to its physico-chemical conditions
help preventing colonization and infection by (see previous paragraph), the skin is typically
microorganisms are finally its relatively low tem- colonized by mesophilic, xerophilic, acido-
perature and the acidic pH (Grice and Segre 2011). philic, osmotolerant, and facultative aerobic
microorganisms. However, depending on the of the sequences corresponded to known and yet
respective niche, also microbes with other physi- cultivated species. Analysis of 817 clones
ological traits can occur. While it was long obtained 8–10 months later from four subjects
believed that on healthy skin microbial life is showed two additional phyla, 28 additional gen-
restricted to the epidermis and appendages such era and 65 additional species. Only four (3.4 %)
sebaceous and sweat glands (see Fig. 1 in (Grice of the 119 genera (Propionibacterium,
and Segre 2011)), recent analyses (Nakatsuji Corynebacterium, Staphylococcus,
et al. 2013) suggest microbial life also to occur in Streptococcus) were observed in each subject
deeper skin layers, i.e. the dermis and underlying tested twice, albeit these genera represented
fatt tissue (Fig. 5.1). This finding is of high 54.4 % of all clones.
importance from an immunological point of In a subsequent landmark study, Grice and
view, because it suggests direct communication colleagues (2009) analyzed more than 112,000
between host and microbial cells in a tissue pre- nearly full length bacterial 16S rRNA gene
viously thought to be sterile. sequences obtained from 20 different body sites
of 10 healthy adults, half of them were sampled a
5.3.1.1 Bacteria second time 4–6 months after the first sampling.
Bacteria represent by far the most abundant Nineteen bacterial phyla were detected, but most
and best studied group of living microorgan- sequences were assigned to just four phyla:
isms on healthy human skin. The vast majority Actinobacteria (52 %), Firmicutes, (24 %),
of them are affiliated with three phyla (typical Proteobacteria (17 %), and Bacteroidetes (6 %).
and abundant genera in brackets): Of the 205 identified genera represented by at
Actinobacteria (Corynebacterium, least five sequences, three were associated with
Propionibacterium, Micrococcus, more than 62 % of the sequences: Corynebacteria
Brevibacterium), Firmicutes (Staphylococcus, (23 %, Actinobacteria), propionibacteria (23 %;
Streptococcus) and Proteobacteria Actinobacteria), and staphylococci (17 %;
(Acinetobacter, Methylobacterium). However, Firmicutes). Interestingly, the diversity and tem-
recent studies using high throughput sequenc- poral stability of the microbial community were
ing technologies have shown, that the skin dependent on the specific characteristics of the
microbiota is highly diverse and comprises skin site. Sebaceous sites were dominated by pro-
members affiliated with more than 25 different pionibacteria and staphylococci, Corynebacteria
phyla, albeit mostly at low abundances. Such and (to a lower extent) staphylococci predomi-
investigations also revealed that the composi- nated in moist sites. A mixed population of bac-
tion of the skin microbiota is highly individual teria resided in dry sites, with a greater prevalence
and changes significantly over time. of ß-Proteobacteria and Flavobacteriales. In
In a pioneering – and according to today’s view of the fact that Gram-negative bacteria are
standards now small scale – study, Gao and col- usually restricted to rather moist habitats, the last
leagues (2007) sampled the superficial volar left finding was unexpected. Species diversity, mea-
and right forearms in six healthy adult subjects sured as Shannon-diversity index, was side-
by means of swabbing. Based on approximately dependent; it was lowest for the back,
1,200 cloned and sequenced partial 16S rRNA retroauricular crease and toe web space samples
genes they identified 182 operational taxonomic and highest for the popliteal fossa, plantar heel
units (“species”) belonging to 91 genera and 8 and antecubital fossa samples. Longitudinal (i.e.,
phyla. On average, 48 species were detected per temporal) stability of the skin microbiota was
human individual. Actinobacteria, Firmicutes, also site-dependent: it was high for protected
and Proteobacteria accounted for ~95 % of the sites such as the nares and the external auditory
clones. Interestingly, and in sharp contrast to canal and low for samples from more exposed
many other microbial ecosystems on earth, 85 % sites, such as buttock and popliteal fossa. In sum-
mary, the study impressively proved the strong albeit with a focus on molecular studies, is given
influence of the respective skin niche on the com- by (Edmonds-Wilson et al. 2015).
position of the skin microbiota (Fig. 5.2), which A fascinating study on the microbiota in
is, in fact, more dependent on the investigated human belly buttons (Hulcr et al. 2012) showed
skin site than on the individual (Grice and Segre that also this “frequently overseen” habitat is
2011). Rightly, the authors considered their study microbiologically as diverse as other skin habi-
as an important baseline for further studies on the tats. On average, 67 bacterial phylotypes were
role of the skin microbiota in human health and found per belly button. However, the communi-
disease. ties were strongly dominated by a few taxa: only
Another landmark study was conducted by six phylotypes occurred on 80 % of all humans.
Fierer and colleagues (2008) on the microbiota Abundant phylotypes were affiliated with staphy-
of human hands. Here, more than 350,000 par- lococci, corynebacteria, and several genera of
tial 16S rRNA gene sequences were obtained Actinobacteria (e.g., Micrococcus) and
from left and right hands (palmar surface) of 27 Clostridiales (e.g., Anaerococcus, Finegoldia,
healthy men and 24 healthy women. Across all Peptinophilus), bacilli, and, to a lesser extent,
samples they determined more than 4,700 bacte- Gamma-Proteobacteria (e.g., Acinetobacter).
rial phylotypes (“species”); on average, every The authors hypothesize that the macroecologi-
hand was colonized by 158 different phlyotypes. cal concept of “oligarchs” can be applied to skin
More than 25 phyla were detected, however, microbiomes. “Oligarchs” are groups of organ-
Actinobacteria, Firmicutes, and Proteobacteria isms that are closely related and dominate the
accounted for 94 % of the sequences, and again, community composition in many, but (in contrast
themostabundantgenerawere Propionibacterium to “core species”) not all samples. They represent
(31.6 % of all sequences), Streptococcus the organisms that are evolutionary best adapted
(17.2 %), Staphylococcus (8.3 %); to the respective habitat.
Corynebacterium (4.3 %;) and Lactobacillus
(3.1 %). These genera were found on virtually all 5.3.1.2 Fungi, Archaea and Viruses
palm surfaces sampled. This pronounced micro- In comparison to bacteria, relatively little is
bial diversity – being in the order of magnitude known about other members of the human skin
of the human intestinal tract – was not expected. microbiota, such as fungi, archaea and viruses.
However, it can easily be explained with the Cultivation-based and earlier molecular studies
function of the human hand as central “grabbing agreed that the human skin fungal microbiota
organ”, that daily gets in contact with many (the skin mycobiome) is dominated by yeasts, in
unsterile surfaces and is exposed to many factors particular of species affiliated with the genus
with a strong influence on microbial community Malassezia. Findley and colleagures (2013)
composition. analyzed fungal communities of 14 skin sites in
The authors also observed pronounced intra- 10 healthy adults by means of Next Generation
and interpersonal variation in bacterial commu- Sequencing (NGS) of PCR-amplified 18S rRNA
nity composition. Hands from the same individual genes and ITS regions. Eleven core-body and
shared only 17 % of their phylotypes, with differ- arm sites were dominated by fungi of the genus
ent individuals sharing only 13 %. Interestingly, Malassezia, with only species-level classifica-
women had a significantly higher diversity than tions revealing fungal-community composition
men, and community composition was signifi- differences between sites. By contrast, three
cantly affected by handedness, time since last foot sites showed a considerably high fungal
hand washing, and an individual’s sex. The resi- diversity, comprising also other genera (in addi-
dent and transient microbiota of the human hand tion to Malassezia) such as Aspergillus,
is of high practical relevance from an hygienic Cryptococcus, Rhodotorula, Epicoccum and
point of view. A very recent review on this topic, others, and a lower stability over time. Fungal
Fig. 5.2 Diversity map of major bacterial groups on phy- explained with the fact that data from a single individual
lum and family-level from a human individual. The figure are shown (Picture credit: Darryl Leja, National Human
is based on data from the landmark study by (Kong and Genome Research Institute, USA; http://www.genome.
Segre 2012). Some results are unusal, such as the domi- gov/dmd/img.cfm?node=Photos/Graphics&id=85320)
nance of proteobacteria under the axilla, but can be
communities were also strongly influenced by 5.3.2 Factors Shaping

skin topography, but – in comparison to bacte- the Composition of the Skin
rial skin communities –, less clearly by skin Microbiota
physiology. Interestingly, Malazessia species
were by far not the most abundant fungi in a Several factors have been identified that influence
study on the fungal microbiota on healthy scalps the composition of the skin microbiota of the
and scalps with dandruff (Park et al. 2012). human body in space and time. In general, these
Based on PCR-NGS-based analyses of 26 rRNA factors can be divided into intrinsic (host) and
genes, Acremonium spp. represented ca. 62 % of extrinsic (environmental) factors (Grice and
all sequences on healthy scalps while Malazessia Segre 2011). Table 5.1 provides a selection of
spp. did only 0.07 %. recent studies dealing with various factors that
Maybe with exception of the human intesti- shape the overall composition of the skin micro-
nal tract, the role of Archaea in the human biota. A more detailed view on the relationship
microbiota is still very unclear and under debate between certain skin diseases and changes in skin
(Horz and Conrads 2010; Horz 2015). Until microbial community composition is given in
recently, they could not be detected in human paragraph 5.4.2. An up-to-date review on host
skin samples, neither by cultivation nor by PCR- factors that interact with the human skin micro-
based methods (Gao et al. 2008), and conse- biota can be found in (SanMiguel and Grice
quently, their presence was questionable (Grice 2015).
and Segre 2011). However, a recent report In addition to the factors mentioned in Table
(Probst et al. 2013) suggested that archaea can 5.1, several others are likely to influence the com-
represent more than 4 % of the human prokary- position of the human skin microbiota, such as
otic skin microbiota. Phylotypes detected by diet, climate, solar/UV-radiation, occupation,
PCR and FISH were mostly affiliated with non-topical use of antibiotics, stress etc., how-
Thaumarchaeota and to a lesser extent also with ever, appropriate studies are still missing.
Euryarchaeota. The physiological role of these Finally, interactions among the different
skin archaea is absolutely unclear, a role in skin members of the skin microbiota are another
ammonia metabolism is suggested, following exciting, yet also still poorly investigated fac-
the function of Thaumarchaeota in many envi- tor with influences on the overall composition
ronmental ecosystem. of the skin microbiota. Staphylococci can be
While it is widely accepted that the human regarded as the best investigated bacteria in
skin, in particular the skin of the human hands, this respect. Staphylococcus epidermidis, a
can transmit viruses (Julian et al. 2010), very typical commensal of the human skin, was
little is known about a potentially commensal or shown to produce antimicrobial peptides, e.g.
resident viral microbiota of the human skin, i.e. phenol-soluble modulins, that selectively
the skin virome. In a landmark study, Foulongne inhibit the growth of skin pathogens such as
and colleagues (2012) used a highthroughput Staphylococcus aureus and group A strepto-
metagenomic approach to study the skin micro- cocci (Cogen et al. 2010). In addition, Iwase
biota of five healthy patients and one patient with and colleagues (2010) showed that some com-
skin cancer. Significant proportions (up to more mensal strains of. S. epidermidis can protect
than 87 %) of the obtained DNA sequences per their host from colonization with S. aureus by
patient represented DNA viruses, mostly affili- means of a serine protease (Esp) that destroys
ated with polyomaviruses, papillomaviruses and biofilms, for instance in the anterior nares.
circoviruses, even on healthy appearing skin. The Much less in known about interactions includ-
physiological role of these viruses, partly repre- ing fungi and/or viruses, although – facing the
senting new species, in human health and disease situation in the human gastrotintestinal tract
still remains to be elucidated. (Minot et al. 2011) – it appears likely that for
Table 5.1 Summary of various host (h) and environmental (e) factors shaping/influencing the composition of the
human skin microbiota
Factor Effects/observations References
Skin siteh Differences in physiological parameters at Findley et al. (2013), Grice
different skin sites significantly shape et al. (2009)
skin microbiota, leading to a higher
intrapersonal than interpersonal
variability
Sexh Skin microbiota of males and females Fierer et al. (2008), Giacomoni
differs significantly, presumably due to et al. (2009)
physiological and/or behavioral reasons
Early agee Diversity of skin microbiota increases Capone et al. (2011)
within first year of living; influence of
delivery mode disappears
Delivery modee Skin flora of newborns delivered Dominguez-Bello et al. (2010)
vaginally is dominated by vaginal
bacteria; cesarean section leads to skin
microbiota dominated by typical skin
bacteria. Implications for early infections
of newborns suspected
Personal hygiene/use of cosmeticse Axillary cosmetics modify the microbial Callewaert et al. (2014)
community and can stimulate odor-
producing bacteria
Life style/sportse Skin to skin contact shaped skin Meadow et al. (2013)
microbiota composition of roller derby
players
Hand washinge Hand washing altered relative abundances Fierer et al. (2008)
of bacterial groups on human hands but
not overall bacterial diversity
Genetic predispositionh Mutations in the Filaggrin gene disturb McAleer and Irvine (2013)
the skin barrier function and are
correlated with atopic dermatitis and
significant changes in skin microbiota
composition
Immune statush Patients with primary immunodeficiencies Oh et al. (2013)
show a significantly altered skin
microbiota
Skin diseasesh Most skin diseases go along with See paragraph 5.4.2 and studies
significant changes in skin microbiota cited therein
composition. It is largely unclear whether
this is cause or effect of the respective
disease. Antibiotic treatments can
sometimes alleviate disease symptoms
Underlying (non-skin) diseasesh Patients with diabetes were more likely to Redel et al. (2013)
carry S. aureus even on their forearms; an
altered skin microbiota might play a role
in wound-infections
Geographye The microbiota of long uncontacted Clemente et al. (2015)
people might serve as a basis for a
“natural” microbiota, not/less affected by
industrialization
instance bacteriophages also shape the compo- they found negative correlations between
sition of the skin microbiota. Interactions Actinobacteria and feet Ascomycota and
between fungi and bacteria are suggested by Basidiomycota and positive correlations with
Findley and coworkers (2013). For human feet, Firmicutes and Proteobacteria.
The previous brief discussion of microbe- status of microbiological skin homeostatis) is

microbe interactions leads to a more functional difficult. Moreover, assigning distinct beneficial
view of the human skin microbiota in human functions to individual members of the complex
health and disease (Paragraph 5.4). A deeper skin microbiota, comprising also yet-uncultured
knowledge of microbe-microbe interactions is species, is even more challenging. Nevertheless,
also the prerequisite for rational manipulation recent years have also brought considerable prog-
(prevention, curation) strategies of skin microbi- ress here.
ota related diseases, in particular probiotic strate- The skin “acid mantle” of ~pH5 represents an
gies, i.e. strategies involving living (beneficial) important line of defense against pathogenic
bacteria (Paragraph 5.5). A promising pilot study microorganisms. Facultative anaerobes, such as
(Harris et al. 2009) was performed with amphib- Propionibacterium acnes, reside under the anaer-
ians: Addition of violacein-producing obic conditions of sebaceous glands, where they
Janthinobacterium lividum to the skin of frogs release free fatty acids from sebum onto the skin,
prevented them from the (fatal) fungal skin dis- which subsequently contribute to the acidic pH
ease chytridiomycosis. (Grice and Segre 2011). The capability of
Staphylococcus epidermidis to produce antimi-
crobial peptides against skin pathogens such as S.
5.4 Functional Aspects aureus and group A streptococci was already dis-
of the Human Skin cussed above.
Microbiota Close interactions between the microbiota and
the human immune system are well known from
Due to the rapid progress in DNA sequencing the human intestinal tract (recently reviewed by
technologies, recent years have brought consider- (Min and Rhee 2015). There, the presence of a
able progress in unraveling the diversity of the gut microbiota is regarded important for the
human skin microbiota in many niches of the development and regulation of innate and adap-
human body as well as major influencing factors. tive immune systems and maintenance of gut
In comparison, considerably less is known about homeostasis. The same obviously applies to the
the functionality of the human skin microbiota microbial human skin (Belkaid and Segre 2014;
and its role in human health and disease, and for Nakamizo et al. 2015).
human well-being. For recent review articles on For instance, Lai and co-workers (2009)
that particular topic the reader is referred to showed that the skin microbiota can modulate
(Rosenthal et al. 2011; Sanford and Gallo 2013; Toll-like-receptor (TLR-) dependent cutaneous
Zeeuwen et al. 2013). inflammatory responses. S. epidermidis cell wall-
derived lipoteichoic acid was shown to prevent
skin injury-based skin inflammation by inhibition
5.4.1 Protective Functions of inflammatory cytokines release from keratino-
of the Human Skin Microbiota cytes as well as TLR2-based immune-responses
caused by RNA, deliberated from the injured skin
Nowadays it is well accepted that colonization of cells. So far, skin bacteria were rather believed to
the human skin with a normal, balanced or cause than to reduce skin inflammation. In addi-
healthy microbiota is in general beneficial for tion, it was shown that S. epidermids can aug-
humans, because it protects them from skin infec- ment skin defense mechanisms against infection
tions and other skin diseases/disorders, a phe- by enhancing gene expression of antimicrobial
nomenon also known as “colonization resistance”. peptides such as human beta defensins (Lai et al.
However, in view of pronounced temporal, intra- 2010). Finally, it was shown that S. epidermids
personal as well as interindividual variations in can also tune the function of resident skin T-
microbial diversity, the definition of a “healthy”, lymphocytes and thereby contribute to protec-
“normal” or “balanced” skin microbiota (i.e. as tive immunity against skin pathogens (Naik et al.
2012). For instance, germ-free mice produced cles are virtually exclusively colonized by P.
less interleukin-17A and interferon-γ than acnes (Bek-Thomsen et al. 2008), which under-
specific-pathogen-free mice. However, monoas- lines the importance of this single species in acne
sociation of the skin of germ-free mice with S. pathology. Consequently, complete genome anal-
epidermidis restored the production of IL-17A in ysis of P. acnes contributed much to the under-
skin T-cells. While the skin of germ-free mice standing of the variety of virulence factors
reacted immunologically abnormally against an causing acne, e.g. enzymes such as hyaluroni-
infection with Leishmania major, a protozoan dases, lipases and proteases (Brüggemann et al.
parasite, monoassociation with S. epidermidis 2004). However, it also became clear that the
rescued protective immunity. Notably, these virulence of different P. acnes strains can differ
effects were not dependent on the intestinal flora considerably, despite high identities on the
of the investigated mice (Naik et al. 2012). genome level, suggesting more pronounced dif-
In summary, these results suggest that skin ferences in gene expression among these strains
commensals such as S. epidermidis and P. acnes (Brüggemann 2005). Notably, the most important
are important drivers and amplifiers of human basic pathomechanism of acne is hormone-
skin immunity (Nakamizo et al. 2015) and a fine induced increased sebum production, providing
example that the human body comprises several P. acnes with ideal living conditions in an anaero-
niches (in addition to the intestinal tract), were bic and lipid-rich environment.
immunosurveillance systems are locally fine- In the case of Psoriasis and Atopic dermati-
tuned by a commensal microbiota (Belkaid and tis, genetic and environmental patho-factors
Naik 2013). A nice overview on skin commen- appear to be involved (Schommer and Gallo
sals as “host guardians” with a special emphasis 2013). Gao and co-workers (2008) showed higher
on the two probably best studied skin commen- frequencies of Firmicutes and lower frequencies
sals so far (P. acnes and S. epidermidis) was of Actinobacteria, in particular of propionibacte-
recently given by (Christensen and Brüggemann ria, in psoriatic lesions compared to normal skin
2014). Consequently, disturbance of microbio- stretches of patients and of skin from healthy
logical skin homeostatis might lead to skin dis- patients. Based on the analysis of 51 triplet sam-
functions and diseases. ples from diseased, unaffected and healthy (con-
trol) skin, Alekseyenko and coworkers (2013)
concluded that psoriasis induces physiological
5.4.2 Role in Skin Disorders changes both at the lesion site and (!) at the sys-
and Skin Diseases temic level. Lesions were characterized by a
reduced microbial species diversity. Psoriasis
Many human skin disorders and diseases have patients were characterized by higher combined
been linked with changes in skin microbial com- relative abundances of the major skin genera
munity composition, however, in most cases it is Corynebacterium, Propionibacterium,
still not clear, whether these observed changes Staphylococcus, and Streptococcus (Firmicutes-
are cause or effect of the underlying disease, Actinobacteria microbiota cutaneotype), while
which is, however, an important basis to chose an genera such as Cupriavidus, Methylobacterium,
adequate therapy. and Schlegelella were significantly less abundant
In the case of acne (Acne vulgaris), a chronic (Proteobacteria-microbiota cutaneotype).
inflamatory disease of the pilosebaceous unit Although the underlying, selecting patho-factors
which can be regarded as the most prevalent remain unclear, the authors conclude that these
human skin disease on earth, Propionibacterium findings may have important diagnostic, preven-
acnes is for long known as the primary disease- tive, and potentially therapeutic implications.
associated bacterium. Despite the pronounced Atopic dermatitis is characterized by an
diversity of the human skin microbiota, it has impaired barrier function of the skin, leading to
been shown that healthy and diseased hair folli- increased bacterial colonization and more
frequent infections with S. aureus. Lower pro- such as diabetes (Tomic-Canic et al. 2014).
duction of skin antimicrobial peptides and a dis- Interestingly, using germ-free mice, Canesso and
turbed skin cornification due to a mutation-based co-workers (2014) could show that in the absence
disturbed filaggrin production have been identi- of a commensal microbiota, skin wound healing
fied as potential pathological reasons here is accelerated and scarless, partially because of
(Schommer and Gallo 2013). AD-patients also reduced accumulation of neutrophils, increased
showed a significantly altered skin microbiota at accumulation of alternatively activated healing
sites of disease predilection, in particular higher macrophages, and better angiogenesis at wound
shares of staphylococci (Kong et al. 2012; Seite sites.
et al. 2014). These differences could for instance In addition to the above-mentioned (severe)
be reversed by an emollient treatment (Seite et al. skin disorders and diseases, the skin microbiota
2014). Interestingly, Stenotrophomonas species also affects several cosmetic skin problems,
were significantly more abundant in the commu- such as impure skin, sensitive skin, dandruff, or
nities of patients that responded to emollient body odor. In fact, many of these problems are
therapy, suggesting a possible role of this genus just milder/less severe forms of the above-
in restoration of the skin microbiota in patients mentioned diseases. Over the last years, consid-
with AD. erable progress has been made in the field of
Rosacea and Serborrheic dermatitis are skin body odor formation, recently reviewed in
disease, where non-bacterial members of the skin (James et al. 2013). In the armpit, which repre-
microbiota are thought to play a major role, i.e. sents one of the most densely colonized parts of
Demodex mites and Malassezia fungi, respec- the human skin, the resident microbiota metabo-
tively, and take favor of an (immunological and/ lizes odorless sweat to malodorous compounds
or prokaryotic) dysbiosis of the skin ecosystem using hydrolytic enzyme activities, such as ami-
(Schommer and Gallo 2013). In the case of noacylase and c-s lyase. While it was long
Rosacea, Microbiota-associated changes on the believed that mainly corynebacteria produce
skin (and simultaneously the small intestine!) body odor, recent cultivation-independent studies
have recently been summarized elsewhere (Troccaz et al. 2015) suggest that also other
(Picardo and Ottaviani 2014). Serborrheic der- groups are involved here, which is corroborated
matitis of the scalp is also known as dandruff, by studies on anaerococii (Fujii et al. 2014) and
affects ~50 % of the global population and is staphylococci (Egert et al. 2013, 2014; Bawdon
mainly caused by M. restricta and M. globosa. So et al. 2015). As also shown for other skin habi-
far unknown factors might switch them into a tats, the composition of the armpit microbiota is
pathogenic state, in which they produce/secret gender-specific (Egert et al. 2014; Troccaz et al.
irritant fatty acids leading to skin hyperprolifera- 2015). Moreover, it is characterized by an asym-
tion and scaling (Schommer and Gallo 2013). metric (left vs. right) activity, which was shown
The skin microbiota also plays an important using a differential 16S rRNA gene vs. 16S rRNA
role in wound healing. A culture-independent sequencing approach, that allows discrimination
pilot study (Hannigan et al. 2014) of open frac- of active from less active microbial populations
ture wounds showed pronounced differences in (Egert et al. 2011). Microbially-caused body
microbial community composition between odor formation and suitable prevention and treat-
wound center (significantly enriched with pseu- ment strategies are not only an economically, but
domonds) and adjacent skin, which was charac- also medically important field of research, as
terized by a normal skin microbiota. These strong body odor (bromidrosis) is a pathological
differences disappeared during healing. In gen- phenomenon (Mao et al. 2008). Moreover, body
eral, the microbiota of wounds appears to be odor triggers a person’s attractiveness for mos-
depended on several factors such as wound type quitoes and might play a role in the transmission
(blunt, penetrating, chronic, acute), affected part of infectious diseases such as malaria (Verhulst
of the body, and underlying (chronic) diseases, et al. 2011).
5.5 Manipulation of the Human larly important that the members of the protect-
Skin Microbiota ing/beneficial part of the microbiota are kept in a
suitable balance. One way to reach and preserve
The human skin microbiota is a dynamic envi- a balanced skin microbiota could be by using the
ronment, which changes over time and space. prebiotic concept. The concept of prebiotics was
Latest research indicates that the differences introduced by Gibson and Roberfroid in 1995
between subjects and/or between microbial colo- (Gibson and Roberfroid 1995). They defined pre-
nized regions are higher than the inter-individual biotic actives as “non-digestible food ingredients
differences over time (Grice and Segre 2011). that beneficially affect the host by selectively
However, besides the “natural” dynamics occur- stimulating the growth and/or activity of one, or a
ring in those ecosystems, also artificial, i.e., limited number of, bacteria in the colon”. This
treatment-based changes in the microflora com- concept was adapted by Bockmühl in 2004 to the
position and cell density do occur. Literally, one field of cosmetic skin products (Bockmühl 2004),
can divide them into two principals: by introducing the prebiotic coefficient, and sub-
sequently used by Carolan and co-workers
(a) Antimicrobial manipulation, aiming at a (2008).
significant reduction of the numbers of all An increasing amount of prebiotic products
skin microorganisms. was developed in recent years. Different plant
(b) Manipulation aiming at an increase of the extracts were investigated on their effect on
number of selected, potentially beneficial Propionibacterium acnes, involved in acne gen-
microorganisms using special nutrients (pre- eration, as well as on the commensal bacterium
biotics) or even living microorganisms (pro- Staphylococcus epidermidis (Bockmühl et al.
biotics) (Fig. 5.3). 2006). It was possible to identify plant extracts
that demonstrate inhibitory effects on P. acnes
The overall beneficial and often life-saving and stimulating effects on S. epidermidis. In par-
effect of antibiotics against microbial infections ticular, a mixture of pine and black currant was
is undisputed. Nevertheless, in particular studies very effective, whereas black currant extract
on the human intestinal tract, such as (Dethlefsen alone did not work as well. These effects were of
and Relman 2011), have shown that the use of particular interest since Propionibacterium acnes
broad-spectrum antibiotics can have a pervasive is seen as a major reason for the development of
and long-lasting effect on structure and (poten- inflamed skin conditions (see paragraph 5.4.2).
tially the) function of this microbial ecosystem, Therefore, a reduction of this bacterial species
and thereby uncouple many of the mutualistic appears as a suitable cosmetic treatment for
host-microbe relationships that have been unrav- inflamed and acne prone skin.
eled in the recent years (Modi et al. 2014). In a human study, the efficacy of the above
Although similar intervention studies with antibi- mentioned substances in cosmetic formulations
otics are – to our knowledge – still missing, the was proven. It was observed that twice daily
same probably also applies to the human skin and application of a cosmetic product containing
the equilibrium of its microbiota. Consequently, 0.5 % of selected plant extracts of pine, black cur-
therapies that make use of the variety of benefi- rant and ginseng to human skin for a total of 3
cial host–skin microbiota interactions and an weeks was effective in inhibiting the growth of
intact microbiological equilibrium are increas- P. acnes, whereas coagulase-negative staphylo-
ingly investigated (for reviews see Krutmann cocci (CNS), such as S. epidermidis, were not
2009; Scharschmidt and Fischbach 2013; affected (Bockmühl et al. 2006). The relative
Al-Ghazzewi and Tester 2014; Grice 2014). abundance of P. acnes was reduced while the
The composition of the skin microbiota relative abundance of CNS was increased. In a
depends on several factors that can easily disturb clinical study (Janssen and Waldmann-Laue
the microbial equilibrium. It is, however, particu- 2008), a cosmetic formulation containing a wash
Prebiotic strategy
Addition of actives or nutrients
that promote beneficial & inhibit harmful bacteria
cfu
cfu
time time
Unbalanced skin microbiota Balanced skin microbiota

S. epidermidis < P. acnes S. epidermidis > P. acnes
Probiotic strategy
Addition of beneficial bacteria
Fig. 5.3 Pre- and probiotic concepts to rebalance the composition of the skin microbiota, e.g. in the case of impure skin
(Adapted from (Krutmann 2009; Simmering and Breves 2009). Cfu = colony forming units, i.e. bacteria
gel, a toner and a skin fluid were tested on 30 titis (Akiyama et al. 2000; Katsuyama et al.
volunteers with a mild form of impure skin. All 1997; Ogawa et al. 1994; Williams et al. 1990).
formulas contained in total 1 % of the above men- Katsuyama and colleagues (2005) described the
tioned prebiotic actives. A significant improve- use of farnesol and xylitol to balance the skin
ment for papules, pustules, comedones and microflora of patients with atopic dermatitis.
sebum production was detected. They were able to remove and prevent the adhe-
Another approach to balance the skin micro- sion of biofilm-producing S. aureus species on
biota was used for people suffering from atopic the skin.
dermatitis (AD) and dry skin, respectively. Applying beneficial bacteria as probiotic
Although genetic factors determine the predis- agents directly to the skin represents another way
position to AD, also environmental factors of achieving a rebalanced microbiota situation.
influence the severity of that disease. Ouwehand and colleagues (2003) proposed the
Furthermore it is known that Staphylococcus use of propionibacteria for cosmetic products.
aureus produces a number of toxins and Food grade strains were chosen as candidates,
enzymes that often seriously worsen the state of because cutaneous isolates might be correlated
the skin (Kozuka 2002). It was reported that S. with skin infections. An antimicrobial activity
aureus was detected and the microbiota was against the skin pathogens M. furfur, C. albicans
unbalanced on not only severely diseased, but and S. aureus, was demonstrated, presumably
also dry-type skin of patients with atopic derma- due to the secretion of organic acids and the
interference with the attachment of these target of binding to receptors and the influence on cyto-
germs to keratin. kine expression was also discussed.
A rather therapeutic approach was followed in An even more complex reaction was induced
a pilot study with inactivated Lactobacillus aci- by the probiotic strain Streptococcus salivarius
dophilus, which was used as a treatment against K12, which was shown to stimulate an anti-
mild to moderate vernal keratoconjunctivitis, an inflammatory response (Cosseau et al. 2008).
allergic eye disease. After giving the probiotic The main task in further development will be
containing eye-drops for 2–4 weeks, a reduction to stimulate the antimicrobial defense system
of symptoms as well as of the molecular markers without causing an overreaction that might lead
ICAM-1 and TLR-4, proteins of the innate to allergic reactions, severe inflammation or even
immune system, was observed (Iovieno et al. sepsis (Finlay and Hancock 2004). This chal-
2008). lenge was addressed in a screening system based
Preparations of different lactic acid bacteria, on keratinocytes, in which the induction of
i.e. L. paracasei, L. brevis or L. fermentum, were hBD-2 and hBD-3 by a series of natural product
proposed as ingredients for skin care products. extracts was investigated (Pernet et al. 2005).
Cultures of lactobacilli were investigated in vitro Nine extracts were found to be positive without
and in vivo on the skin of individual volunteers inducing pro-inflammatory cytokines such as IL-
for their probiotic potential and were found to 8, IL-1α or MIP-3α. Thus, these extracts such as
promote S. epidermidis and to block S. aureus, E. Arnica, Betel, Black elder and Mugwort were
coli or M. luteus (Lang et al. 2006). discussed as suitable for cosmetic or therapeutic
While the probiotic approach in principal applications.
can be done in live form, especially in cosmetics Interestingly, the expression of the antimicro-
the dosage of the beneficial microbes as inacti- bial peptide cathelicidin LL-37 is regulated by a
vated biomass preparations is more widespread. vitamin D3 responding element in the promoter
This will lead to an approach of stimulating the region. The oral supplementation with vitamin
skin’s immune defence by microbial preparations D3 has been discussed as beneficial in atopic der-
or other suitable actives. The effects on the skin matitis as well as the direct topic dosage, although
caused by prebiotic and probiotic actives presum- the latter is being hampered by skin irritation
ably are not restricted to a direct promotion or effects found in experiments with mice (Schauber
restriction of microorganisms on the skin and and Gallo 2008).
oral cavity, but must be seen in the triangular In a more recent double-blind, placebo-
relationship between the active substances, the controlled study, oral intake of Lactococcus lac-
microorganisms and the epithelial cells. tis strains improved some skin properties and
Especially probiotic applications using microbial body characteristics in women, like skin elastic-
preparations probably involve the skin’s innate ity (Kimoto-Nira et al. 2012). This and other,
immune system. Thus, the idea to stimulate the partly unpublished studies have led to an increas-
skin defense directly by actives and therapeutic ing amount of pre- and probiotic cosmetic
agents appears very attractive (Finlay and products.
Hancock 2004).
Donnarumma and colleagues (2004) have
shown that an extract from avocado was able to 5.6 Outlook: Trends
influence the adherence of M. furfur to keratino- and Challenges
cytes and to induce the production of human ß-
defensin 2 (hBD-2). The extract mainly con- The microbiota of the human skin represents a
sisted of two rare sugars, i.e. mannoheptulose major part of the human microbiome, which
and perseitol. The observed activity of these sub- undoubtedly plays a significant role for human
stances might be due to a structural similarity to health and well-being. A deeper and more mech-
constituents of the cell wall of yeasts. The mode anistic (rather than descriptive) understanding of
the complex interaction-network of skin micro- Community differentiation of the cutaneous microbi-
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Vaginal Microbiota
6
Werner Mendling
Abstract
The knowledge about the normal and abnormal vaginal microbiome has
changed over the last years. Culturing techniques are not suitable any
more for determination of a normal or abnormal vaginal microbiota. Non
culture-based modern technologies revealed a complex and dynamic sys-
tem mainly dominated by lactobacilli.
The normal and the abnormal vaginal microbiota are complex ecosys-
tems of more than 200 bacterial species influenced by genes, ethnic back-
ground and environmental and behavioral factors. Several species of
lactobacilli per individuum dominate the healthy vagina. They support a
defense system together with antibacterial substances, cytokines, defen-
sins and others against dysbiosis, infections and care for an normal preg-
nancy without preterm birth.
The numbers of Lactobacillus (L.) iners increase in the case of
dysbiosis.
Bacterial vaginosis (BV) – associated bacteria (BVAB), Atopobium
vaginae and Clostridiales and one or two of four Gardnerella vaginalis –
strains develop in different mixtures and numbers polymicrobial biofilms
on the vaginal epithelium, which are not dissolved by antibiotic therapies
according to guidelines and, thus, provoke recurrences.
Aerobic vaginitis seems to be an immunological disorder of the vagina
with influence on the microbiota, which is here dominated by aerobic bac-
teria (Streptococcus agalactiae, Escherichia coli). Their role in AV is
unknown.
W. Mendling (*)
Deutsches Zentrum für Infektionen in Gynäkologie
und Geburtshilfe, St. Anna Hospital,
Vogelsangstrasse, 106, 42109 Wuppertal, Germany
e-mail: w.mendling@t-online.de;
http://www.werner-mendling.de

84 W. Mendling
Vaginal or oral application of lactobacilli is obviously able to improve

therapeutic results of BV and dysbiosis.
Keywords
Vaginal microbiota • Dysbiosis • Bacterial vaginosis • Aerobic vaginitis •
Lactobacilli • Probiotics
6.1 A Historic Perpective described as “a replacement of lactobacilli by

characteristic groups of bacteria accompanied by
Albert Döderlein (1860–1941) was the first one changed properties of the vaginal fluid” (Mardh
to discover the importance of lactic acid produc- et al. 1984). With the advent of molecular and
ing bacteria in the vagina (Döderlein 1892). genetic technologies we had to reconsider our
Krönig (1895), a co-worker of Döderlein, view and definition of a normal vaginal microbi-
described lactobacilli as anaerobic and curved ota. New bacteria were discovered and the con-
rods, which were later cultured by Curtis (1913) cepts of a bacterial biofilm and of vaginal
and named Mobiluncus curtisii (Spiegel and microbiota types was introduced.
Roberts 1984). Finally, Stanley Thomas (1928)
coined the term Lactobacillus acidophilus. In the
1980s Lauer, Helming and Kandler were able to 6.2 Normal Vaginal Microbiota
distinguised several Lactobacillus species previ-
ously termed L. acidophilus by DNA-DNA The outer and inner surfaces of a child born by
hybridisation. vaginal delivery are primarily colonised by the
At the beginning of the last century first vaginal microbiota of the mother. Futher colonis-
attempts were made to grade the vaginal micro- ers are aquired from the skin and mouth micro-
biota. Manu af Heurlin (1914) characterised the biota of the mother. In the last years it became
vaginal microbiota of children, pregnant and not evident that mothermilk harbours a unique micro-
pregnant women and women of old age and tried biota, mainly dominated by Lactobacilli, which
to establish grades of healthines. Robert Schröder is transfered to the suckling child (Martin et al.
(1921) was the first one to define three bacterio- 2003). Before the menarche the vagina microbi-
logically different vaginal microbiota types ota is a unsteady mix of skin and gut microbes,
termed “Reinheitsgrade” (grades of purity). Otto which may harbor some lactobacilli (Fettweis
Jirovec (Jirovec et al. 1948) distinguished et al. 2012). The environmental conditions for
between six vaginal microbiota types (normal, lactobacilli become improved by estrogens and
abnormal, abnormal with many leucocytes, gon- progesterone with the start and during the repro-
orrhea, trichomoniasis, candidosis). ductive phase of women. Estrogens support the
In 1955 Herman Gardner and Charles Dukes proliferation of the vaginal epithelium and the
(1955) described Haemophilus vaginalis, later development of intraepithelial glycogen, while
renamed as Gardnerella vaginalis (Greenwood progesterone supports the cytolysis of epithelial
and Pickett 1980) as the main causative for bacte- cells, which release glycogen. Lactobacilli and
rial vaginosis (BV), the most common distur- other bacteria are able to metabolise this glyco-
bance of the vaginal microbiota and considered it gen to glucose and maltose and further to lactic
as a sexually transmitted disease. Furthermore, acid. This leads to a vaginal pH of 3.8–4.4 which
they emphasised the importance of microscopy is defined as normal.
and defined “clue cells” as diagnostic marker. Until now more than 120 lactobacillus species
By the end of the last century the hitherto have been described (de Vos et al. 2012). Within
valid definition of bacterial vaginosis was the vaginas of women of reproductive age more
6 Vaginal Microbiota 85
than ten different species can be found. However, Within the human microbiome project, the
a single women is usually dominated by one or vaginal microbiome project investigated the rela-
two species, of which the most frequent are L. tionship between the vaginal microbiota and vari-
crispatus, L. gasseri, L. jensenii and L. iners ous physiological and infectious conditions
(Vasquez et al. 2002). Several of these lactoba- (Fettweis et al. 2012). Various “vagitypes” have
cilli are able to produce bacteriocines, biosurfac- been identified of which many are dominated by
tants and coaggregating molecules to inhibit the a single bacterial taxon, others by a broad spec-
adhesion of pathogens (Reid 2001). Another trum of different bacteria. Interestingly the ethnic
property of lactobacilli found within the vagina is background of women has an influence on the
the ability to produce hydrogen peroxid (H 2O2). vaginal microbiota, as white/caucasian women
Lactobacilli are by definition strict anaerobic are dominated by L. iners, asian women by L.
bacteria. However, they are often found in niches crispatus and black and hispanic women by L.
enriched with oxygen. To detoxify the otherwise jenseni. However, a significant group of women
toxic oxygen, several but not all lactobacilli are harbored no lactobacilli in the vagina (Ravel
able to produce H2O2. The presence of H2O2 pro- et al. 2011, Hickley et al. 2012).
ducing lactobacilli is negatively associated with Jespers et al. (2012) identified in Antverpen/
the formation of BV (Eschenbach et al. 1989). Belgium similarly three types of vaginal micro-
biota in healthy premenopausal women and in
women at risk for BV of a STD clinic.
6.2.1 The Normal Vaginal One group of women was dominated by L.
Microbiota: A Mixture of Many crispatus, L. iners, L. jensenii and L. vaginalis
Bacteria in a Balance with lower counts (<30 %) of L. gasseri and
Atopobium vaginae. A second group harbored
Currently, the dogma, that a healthy vaginal preferentially L. gasseri and L. vaginalis, but less
microbiota is dominated by lactobacill is falter- L. jensenii, L. iners or L. crispatus. The third
ing as by genomic sequencing over 250 species group was dominated by L. gasseri, A. vaginae
of bacteria have been identified in the vagina (Li and L. iners. Whithin the third group were mainly
et al. 2012). Besides Lactobacilli many other bac- african and asian women. These flora types
teria can be found in the normal or abnormal underly dynamic variations during the menstrual
vaginal microbiota, such as Actinomyces, cycle and are influenced by external circum-
Aerococcus, Allisonella, Alloscardovia, stances, for instance sexual behavior. But they
Anaerococcus, Arcanobacterium, Atopobium, seem to be in a rather stable balance, and a
Bacteroides, Balneimonas, Bifidobacterium, healthy vaginal system can obviously be strong
Blastococcus, Blautia, Bulleidia, Campylobacter, enough to correct disturbences from outside, as
Citrobacter, Coriobacteriacea, Corynebacterium, Gajer et al. (2012) demonstrated in a longitudinal
Enterobacter, Escherichia, Facklamia, study. Women were grouped according to Ravel’s
Faecalibacterium, Finegoldia, Gardnerella, et al. (2011) “community state types” and vaginal
Gemella, Haemophilus, Lachnospiracea, swaps were taken for 16 successive weeks.
Massilia, Megasphera, Mobiluncus, Mollicutes, Furthermore, menstruation, tampon use, vaginal,
Moryella, Olsinella, Parvimonas, Peptinophilus, anal or oral sex, sex toys, digital penetration and
Peptostreptococcus, Prevotella, Porphyromonas, lubricants were documented. It was evident, that
Proteobacteria, Providencia, Rhizobialis, the vaginal microbiota of several women became
Ruminococcaceae, Salmonella, Shigella, heavily disturbed by some of these actions, how-
Shuttleworthia, Sneathia, Solobacterium, ever other microbiotas showed no disturbences
Staphylococcus, Streptococcus, Veillonella, despite very frequent manipulations. Once again,
Ureaplasma, and many lactobacilli species black women were significantly different in their
(Gajer et al. 2012). “community state types”.
86 W. Mendling
The vaginal flora is influenced by the anal and binding lectin (MBL), defensins, secretory leuco-
the oral flora. Petricevic et al. (2012) found in cyte protease inhibitor, nitric oxid, and
around 80 % of 30 pregnant women and in 40 % membrane-associated factors, the TLR (11 TLR
of 30 postmenopausal women one or more have been identified) and phagocytes. Different
Lactobacillus ssp. in the vagina and in the rec- TLR recognize lipoproteins and peptidoglucan in
tum, and they were in 80 % resp. 40 % of the the surface of Gram-positive bacteria, the lipo-
same identity. These women were also in 50 % polysaccharid of Gram-negative bacteria, flagel-
(pregnant) and in 30 % (postmenopausal) colo- lins, and others (Linhares et al. 2010; Mirmonsef
nised by one or more Lactobacillus ssp. in their et al. 2011). Vaginal cells release defensins with
mouth. A healthy vaginal, balanced microbiota a non-specific antimicrobial activity. The produc-
protects not only against ascending infections or tion of special defensins is stimulated by estro-
HIV acquisition, but also against prematurity gens and inhibited by progesterone. Bacterial
(Hoyme and Hübner 2010; Donders et al. 2011; vaginosis in pregnant women was associated
Lamont et al. 2011; Martin 2012; Mendling et al. with lower vaginal concentrations of defensin 3
2013). (Mitchell et al. 2013). Women with MBL defi-
On the other hand, too many vaginal lactoba- ciency due to a polymorphism are more suscep-
cilli (Cibley and Cibley 1991) or abnormal long tible to recurrent Candida albicans vaginitis
lactobacilli (Horowitz et al. 1994) can cause ves- (Babula et al. 2003).
tibular pruritus, itching and dysuria. This “cyto- Toll-like receptor ligands and fatty acids,
lytic vaginosis” or “lactobacillosis” can be which are produced by many vaginal bacteria,
misdiagnosed clinically as candidosis (Demirezen have dramatic effects on the vaginal immune
2003). function: the anaerobes of BV produce bad smell-
ing amines (putrescin, cadaverin and others), suc-
cinate, sialidases, and immunomodulatory
6.2.2 Gene Polymorphisms substances such as lipopolysaccharides, lipotei-
and Vaginal Immunity choid acids and peptidoglycans with many influ-
ences on cytokines and other immune responses
The vaginal microbiota is not only influenced by (Mirmonsef et al. 2011).
the ethnic background, but also by gene polymor-
phisms: the individual capacity to produce low or
high levels of anti- or pro-microbial factors influ- 6.3 Abnormal Vaginal Flora
ences the composition of the vaginal microbiota.
Polymorphisms in the interleukin-1 receptor A disturbed vaginal microbiota may be the cause
antagonist gene or the Toll-like receptor (TLR) 4, for various diseases. However, within this chapter
which acts in the innate recognition of Gram- only two will be discussed, as only they are
negative bacteria, influence the quantity of vagi- directly connected to a dysbiotic vaginal
nal bacteria (Rodriguez et al. 1999) and can microbiota.
influence individual susceptibility to pregnancy
complications (Genc and Onderdonk 2011). Such
polymorphisms vary between different racial 6.3.1 Bacterial Vaginosis (BV)
groups and may be associated with the different
ecosystems between different populations Gardner and Dukes (1955) named the vaginal
(Linhares et al. 2010). Interestingly, periodontal disorder Haemophilus vaginalis vaginitis and
disease and BV are influenced by gene polympr- described “clue cells”. It was later characterized
phisms and are both associated with preterm as bacterial vaginosis and is defined by a replace-
birth (Sanu and Lamont 2011). ment of lactobacilli with characteristic groups of
The innate immune system of the vagina is bacteria accompanied by changed properties of
represented by soluble factors like mannose- the vaginal fluid (Weström et al. 1984).
The first diagnostic criteria for BV were pub- diversity of vaginal lactobacilli and are in some
lished by Amsel et al. (1983): grey-white milky women a risk factor for urogenital infections.
discharche, pH >4.5, bad “fishy” smell, espe- Sexual practices, especially receptive oral sex
cially if 10 % KOH solution is added, and at least and digital vaginal penetration are significant risk
20 % “clue cells”. Later, Eschenbach et al. (1989) factors for BV (which is perhaps an explanation
determined the lack of H2O2-producing lactoba- for a higher risk of BV in lesbian women
cilli, an overgrowth of G. vaginalis and anaerobic (Marrazzo et al. 2012)), and also cigarette smok-
Gram – negative rods and anaerobic Gram – posi- ing, black race, receptive anal sex before vaginal
tive cocci as essential factors for the presence of intercourse (Cherpes et al. 2008; Manhart et al.
BV. To improve the diagnostic analyis, Nugent 2012). It should be kept in mind, that Gardner
et al. (1991) poposed a score (Nugent score): 0–3 and Dukes (1955) could not cause BV by trans-
= normal, 4–6 = intermediate, 7–10 = BV. It is ferring cultivated G. vaginalis from a woman
solely based on Gram-staining criteria. However, with BV to a healthy woman, but if they trans-
it has been reported, that roughly 20 % of preg- ferred the discharge of a woman with BV to a
nant women in Germany have BV by definition, healthy vagina, this woman got BV. Hence, not
but not all suffered from symptoms (Mendling single bacteria, albeit in high numbers, is impor-
et al. 2013). tant, but a critical mixture of BVAB together with
The development of BV was long associated special lactobacilli, and a lack of other lactoba-
with the presence of G. vaginalis. Currently, four cilli seem to play a role in the development of BV
different G. vaginalis strains have been (Lamont et al. 2011). Lamont et al. (2011) dis-
described, of which only two produce the BV cussed, “that it is whether or not the strain/spe-
marker sialidase and only one predominated in cies of Lactobacillus produces H2O2 that dictates
women with BV (Jayaprakash et al. 2012). whether BV is present or absent. However, given
Hence, the existance of G. vaginalis in the vagina that H2O2 – producing L. gasseri are found in BV
is no precondition of BV. In the last years it patients, albeit at lower incidence, one might also
became evident, that no single strain alone is the argue that in vitro production of H2O2 is only a
cause of BV. Recently, BV associated bacteria biomarker of a protective species of Lactobacillus,
(BVAB) 1, 2 and 3 have been described. Nearly not an active factor in limiting the growth of vagi-
all of these bacteria are unknown in clinical nal anaerobes.”
practice. Women, who harbor BVAB, especially
G. vaginalis and Leptotrichia/Snethia or
Megasphera in higher concentrations, develop 6.3.2 Polymicrobial Bacterial
significantly more BV (p = 0.001) (Marrazzo Biofilms in BV and Sexual
et al. 2012; Hillier et al. 2010). Additionally, Transmission
Fredricks et al. (2005) demonstrated that the
presence of L. iners is strongly associated with A biofilm is defined as any group of microorgan-
BV. L. iners, which belongs to the normal micro- isms in which cells stick to each other on a sur-
biota, but seems to be a “poisened apple in the face. The first biofilm in gynecology was
basket”, because its presence is strongly con- described in women with BV by Swidsinski et al.
nected with a shift of normal to abnormal micro- (2005). The epithelial cells of the vagina of
biota. On the other hand, Women, who harbor L. healthy pre- or postmenopausal women or of
crispatus are significantly less at risk to develop children are free of bacteria. But BV is character-
BV than others (p = 0.02). ized by structured polymicrobial biofilms adher-
BV is influenced by environmental and genetic ent to epithelial cells of the vagina. “Clue cells”,
factors. Thus, gene polymorphisms influence the which Gardner and Dukes (1955) have seen
occurrence of G. vaginalis and A. vaginae microscopically on vaginal epithelial cells of the
(Verstraelen et al. 2009). Furthermore, decreas- discharge, have their origin from this biofilm –
ing estrogen levels influence the number and coat on the vaginal wall. The biofilm consists in
88 W. Mendling
its majority of G. vaginalis (>50 % up to 90 %) scopic field. High levels of interleukin – 1 beta, –
and of A. vaginae (10–40 %), but also of lactoba- 6, – 8 and leukaemia inhibiting factor in contrast
cilli and other bacteria. G. vaginalis formes to BV. Severe cases resemble to desquamative
in vitro significantly stronger biofilms, if inflammatory vaginitis, which is discussed to be
Fusobacterium nucleatum or Prevotella bivia are an early form of Lichen ruber of the vagina. AV
added (Machado et al. 2013). No BV is equal in is a higher risk factor for preterm labour and pre-
the composition of different bacteria and no bio- term birth than BV (Donders et al. 2011). Some
film of BV is equal. believe, that AV is primarily an immunologic dis-
It is unknown, whether the lactobacilli found order with secondary abnormal microbiota, or a
in the biofilm are L. iners or other species. If dermatological disease in the vagina (Edwards
treated with metronidazole, it does not get 2010). Women with AV are at risk for low grade
disrupted and, thus, seems to be the reason for the cervical squamous intraepithelial cell lesions
high recurrence rates of about 30 % after 3 (Jahic et al. 2013). About 5 % of women in repro-
months and 60 % after 6 months following ther- ductive age are suffering from AV, but some diag-
apy, respectively. nosed it in a much higher frequence of 23 % (Fan
In addition to be present in the vagina, the et al. 2013). But partner treatment is without ben-
BV-typical biofilm can be found on epithelial efit for the woman with BV.
cells in the urine of females with BV and in the
urine of their partners. Sometimes it may be
found in cryopreserved donor semen, in the endo- 6.4 Prophylaxis and Therapy
metrium of non-pregnant women and in tissue of with Probiotics
missed abortion/abortion (Swidsinski et al.
2013). If men were asked to void their urine after Probiotics are microorganisms that provide a health
having pulled back the preputium, no biofilm was benefit to the host. They act in the gastrointestinal
found, which confirms the observation that male tract and influence in various ways the immune
circumcision reduces the risk for ulcerations, system (Sherman et al. 2009). Although lactoba-
trichomoniasis and BV (Gray et al. 2009). cilli are in use for prophylaxis or treatment of vagi-
Circumcision is associated with a significant nal discharge since decades, probiotic research
change in the microbiota and with a significant devoleped rapidly over the last 30 years within the
decrease in putative anaerobic bacteria, espe- field of gynaecology (Spurbeck and Arvidson
cially Clostridiales and Prevotellaceae (Price 2011; Reid 2012). One of the first clinical studies
et al. 2010). Women with treated BV have a proposed the daily oral application of about 250 g
higher risk for recurrence, if they have inter- Yoghurt containing L. acidophilus for 6 months to
course with the same partner without using con- women with recurrent candida vulvovaginitis. The
doms (Marrazzo et al. 2012; Guédou et al. 2013). mean rate of recurrences in the control arm was 2.5
versus 0.38 in the yoghurt arm (p = 0.001) (Hilton
et al. 1992). Since then, several species have been
6.3.3 Aerobic Vaginitis (AV) tested in various studies. L. rhamnosus Lcr 35
(Coudeyras et al.2008a, b) showed increased abil-
In 2002 Donders et al. (2002) characterised a new ity to metabolise glycogen to lactic acid and in vitro
type of vaginitis. It is in contrast to BV domi- growth inhibition of G. vaginalis and C. albicans.
nated by aerobic bacteria, mainly Streptococcus The effect was higher after the manufactering pro-
agalactiae and Escherichia coli and named aero- cess than compared to three other L. rhamnosus
bic vaginitis (AV). The patients suffer from strains (Nivoliez et al. 2012). Lcr 35 adheres to cer-
yellow-green discharge, the vagina is red by vicovaginal cells and is an antagonist of BVAB
inflammation, the pH is 5.5–6.5, many toxic leu- (Coudeyras et al. 2008a, b).
cocytes, parabasal cells and a sparse coccoid The strain L. rhamnosus GR-1 causes signifi-
flora without lactobacilli dominate the micro- cant killing of E. coli in vitro and is able to
cause bacterial death in BV biofilms in vitro (2012) showed in a pilot study, that oral adminis-
(McMillan et al. 2011). The two strains L. rham- tration of L. rhamnosus Lcr35 is able to improve
nosus GR-1 and L. reuteri RC-14 (formerly L. the Nugent score to normal values.
fermentum), which are traded in a vaginal tablet, In addition to the improvement of BV symp-
inhibited in vitro the growth of C. albicans and toms the recurrences of vulvovaginal candidosis
upregulated inflammatory Interleukin levels in a can be influenced by probiotics (Homayouni
human vaginal epithelial cell line (Martinez et al. 2014, Huang et al. 2013). Ehrström et al.
et al. 2009a, b). C. albicans lost it’s metabolic (2010) showed improved treatment rates for
activity, showed increased expression of stress- women with BV and vulvovaginal candidosis by
related genes and lower expression of genes additional administration of L. gasseri LN40,
involved in fluconazole resistance (Köhler et al. L. fermentum LN99, L. casei subsp. rhamnosus
2012). Similar results were demonstrated by LN113 and P. acidilactici LN23 for 5 days in
Sanchez et al. (2008) with a different strain, L. vaginal capsules. Martinez et al. (2009a, b)
rhamnosus GG, which showed in a monolayer improved the clinical treatment results of vulvo-
cell culture protection against damage by C. vaginal candidosis with oral fluconazole,
albicans, modulation of immune responses and L. rhamnosus GR-1 and L. reuteri RC-14 similar
immune conditioning of the mucosal surfaces to Kern et al. (2012).
(Schaller 2012). Probiotics, here administered Prebiotics, such as inulin, glycogen, or others,
as a daily probiotic drink for 6 months, can also which support the metabolism of probiotics are
enhance the clearance of human papillomavi- sometimes added to probiotic tablets. However,
rus-related cervical lesions significantly against within the field of gyneacolgy hitherto clinical
placebo (Verhoeven et al. 2013). studies to assess their superiority over probiotics
Clinical studies had been performed with dif- are missing.
ferent probiotics administered vaginally or orally.
L. crispatus CTV-05 is one of the new probiotics
in gynecology and well tolerated (Hemmerling 6.5 Summary and Conclusion
et al. 2010). Vaginal intercourse (seminal fluid),
and the presence of lactobacilli of the same spe- The normal and the abnormal vaginal microbiota
cies during vaginal administration inhibit the is not yet fully understood. It is an ecosystem,
colonisation (Antonio et al. 2009). There seems which is influenced by genetic, ethnic, environ-
to be a competition of one’s own and the foreign mental and behavioral factors. More than 100 to
lactobacilli. 200 bacterial species, commensal, transient and
Oral administration of lactobacilli to influence endogenous, colonise the vagina and are influ-
the vaginal microbiota seems to be effective. The enced by the oral, rectal and penile microbiota.
first to demonstrate this were Hilton et al. (1992) Cultural methods for the determination of normal
against Candida vaginitis and Shalev et al. (1996) or abnormal microbiota are insufficient and
against Candida and/or BV. Rectal lactobacilli detect only a small, mostly aerobic, not represen-
with vaginal tropism can colonise the vagina and tative and clinically unimportant spectrum.
vice versa. Oral application of a mixture of 108 L. Lactobacilli mainly dominate the vaginal micro-
fermentum 57A, L. plantarum 57B and L. gasseri biota and are responsible, with other bacterial
57C daily for 60 days was able to colonise the species, for the creation of a pH value between
rectum and the vagina between day 20 and 70 and 3.8 and 4.5, which is considered as normal, at
decreased the vaginal pH, while the Nugent score least in caucasian or asian women. Together with
improved (Strus et al. 2012). The oral administra- their antibacterial properties and immunological
tion of L. rhamnosus GR-1 and L. reuteri RC-14 factors lactobacilli create a defense system
for 30 days following treatment of BV with oral against dysbiosis and infections within the
metronidazole improved the cure rate (Anukam vagina. This system is responsible for a healthy
et al. 2006). Furthermore, Bohbot and Cardot outer and inner genital tract, for a balanced resti-
90 W. Mendling
tution after intercourse and for a normal preg- the Lactobacillus casei group. Appl Environ Microbiol
74:2679–2689
nancy and childbirth on time.
Coudeyras S, Jugie G, Vermerie M, Forestier C (2008b)
Adhesion of human probiotic Lactobacillus rhamno-
sus to cervical and vaginal cells and interaction with
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The Human Gut Microbiota
7
Hermie J.M. Harmsen and Marcus. C. de Goffau
Abstract
The microbiota in our gut performs many different essential functions that
help us to stay healthy. These functions include vitamin production, regu-
lation of lipid metabolism and short chain fatty acid production as fuel for
epithelial cells and regulation of gene expression. There is a very numer-
ous and diverse microbial community present in the gut, especially in the
colon, with reported numbers of species that vary between 400 and 1500,
for some those we even do not yet have culture representatives.
A healthy gut microbiota is important for maintaining a healthy host.
An aberrant microbiota can cause diseases of different nature and at differ-
ent ages ranging from allergies at early age to IBD in young adults. This
shows that our gut microbiota needs to be treated well to stay healthy. In
this chapter we describe what we consider a healthy microbiota and dis-
cuss what the role of the microbiota is in various diseases. Research into
these described dysbiosis conditions could lead to new strategies for treat-
ment and/or management of our microbiota to improve health.
Keywords
Microbiota • Development • Obesity • Aberrant microbiota • Diabetes
7.1 Introduction that are essential for us, such as vitamin produc-
tion, detoxification of toxins, regulation of cho-
The microbiota in our gut normally helps us to lesterol metabolism, bile deconjugation,
stay healthy (Hooper and Gordon 2001; Sekirov providing colonization resistance to pathogens,
et al. 2010). It performs many different functions SCFA production as fuel for epithelial cells and
regulation of gene expression (Walter and Ley
H.J.M. Harmsen (*) 2011). However, the main function of the microbe
Department of Medical Microbiology, University of itself is to survive and thrive. The microbe is
Groningen, University Medical Center Groningen, there because, maybe by coincidence, it has
30001, 9700 Groningen, The Netherlands found a niche to grow in. Whatever these
e-mail: H.J.M.Harmsen@med.umcg.nl
microbes do, they need to be able to colonize our
M.C. de Goffau gut, to grow on the nutrition provided and to
Wellcome Trust Sanger Institute,
Hixton, CB10 1SA Cambridge, UK reproduce in a rapid way, because in, let’s say

96 H.J.M. Harmsen and M.C. de Goffau
24–36 h (Heaton et al. 1992), the bowel is emp- Linnaeus shows first the genus name Escherichia,
tied and the cycle starts again. The microbiota in followed the species name coli. It belongs to the
our gut performs many different essential func- family of Enterobacteriaceae, part of the order of
tions that help us to stay healthy. These functions Enterobacteriales, which is part of the class of
include vitamin production, regulation of lipid Gammaproteobacteria belonging to the phylum
metabolism and short chain fatty acid production of Proteobacteria of the kingdom Bacteria
as fuel for epithelial cells and regulation of gene (Whitman and Parte 2009; Moore et al. 1987).
expression. There is a very numerous and diverse There are four numerically important bacterial
microbial community excisting in the gut, espe- phyla present in the adult human gut:
cially in the colon, with reported numbers of spe- Bacteriodetes (Gram-negative anaerobes) and
cies that vary between 400 and 1500, for some Firmicutes (Gram positives) are most dominant,
those we even do not yet have culture followed by Actinobacteria and Proteobacteria.
representatives.A healthy gut microbiota is Major Bacteriodetes genera in the gut are
important for maintaining a healthy host. An Bacteroides and Prevotella, major Firmicutes
aberrant microbiota can cause diseases of differ- genera are Clostridium, Blautia,
ent nature and at different ages ranging from Faecalibacterium, Eubacterium, Roseburium,
allergies at early age to IBD in young adults. This Ruminococcus, Streptococcus and Lactobacillus.
shows that our gut microbiota needs to be treated Actinobacteria are represented by the genera
well to stay healthy. In this chapter we describe Bifidobacteria, Atopobium and Collinsella, the
what we consider a healthy microbiota and dis- Proteobacteria by Enterobacteriaceae like the
cuss what the role of the microbiota is in various genus Escherichia (Tap et al. 2009; Walker et al.
diseases. Research into these described dysbiosis 2010). An additional phylum the Verrucomicrobia
conditions could lead to new strategies for treat- is represented by one species, Akkermansia
ment and/or management of our microbiota to muciniphila (Derrien et al. 2004). This is a very
improve health. simplistic overview, in reality the families pres-
The presence of a very numerous and diverse ent in the gut are subdivided by a long list of
microbial community excisting in the gut, espe- well-known genera, but also new genera, such as
cially in the colon, is thus quite amazing. The Christensenella (Morotomi et al. 2012) and
numbers of different species that are mentioned reclassifiedgenera, suchas Erysipelatoclostridium
in the current studies vary between 400 and 1,500 ramosum, a reclassification of Clostridium ramo-
species (Qin et al. 2010; Turnbaugh et al. 2010; sum (Yutin and Galperin 2013). Despite the cur-
Gill et al. 2006). These species are mostly rent advances in detection and identification
expressed as operational taxonomic units techniques, it will take some time until a stable
(OTU’s), these are unique sequence types that taxonomic list is created which contains a proper
should represent a species but do not always have naming for all the species present in the gut.
a specific name yet, since there is no culture rep- Furthermore, next to bacteria there are also mem-
resentative yet or the taxonomy is lacking behind bers of the kingdom Archaea present, predomi-
(Rajilić‐Stojanović and Vos 2014). The most nantly Methanobrevibacter species that produce
dominant species present in healthy individuals methane in the gut (Samuel et al. 2007), and
are illustrated in Fig. 7.1 with an indication of the Eukarya, such as the yeast Candida, microbial
substrate utilization and the relative abundance in parasites, such as Entamoeba (Parfrey et al.
human feces as detected in different next genera- 2011) and macro parasites, for example hel-
tion sequencing studies (Flint et al. 2014; Qin minths (Weinstock and Elliott 2009) . Finally,
et al. 2010; David et al. 2013). also viruses and bacteriophages play an signifi-
The bacteria taxonomy can be quite confusing cant role in the ecology and maintenance of a
some way. As an example we will explain the tax- healthy balance in the gut (Kernbauer et al. 2014).
onomy of the well-known gut bacterium The driving force for the maintenance of the
Escherichia coli: the double name according complex microbiota in our gut should be sought
7 The Human Gut Microbiota 97
Host substrates Sugars Dietary fiber Proteins and lipids

Bile Lactose Cellulose Amino acids
Mucus and saliva Sucrose Inulin Peptides
Digestive fluids Fructose Resistant starch Saturated Lipids
Oligosaccharides Unsaturated lipids
Redox
+ Enterococcus Escherichia coli
potential Enterobacteria
Streptococcus
Lactobacillus
Veillonella Anaerotruncus
Bacteriodes Prevotella Dialister Catenibacterium
Bifidobacterium Bryantella Butyrivibrio
Alistipes Collinsella Holdemania
Atopobium
Eggerthella Gordonibacter
Parabacteroides
Fusobacterium
Rum. gnavus
et rel.
Cl. ramnosum
Desulfovibrio Blautia
Akkermansia Eubacterium Cl. perfringens
Subdoligranulum Roseburia
Anaerostipes Cl. dificile
Faecalibacterium Dorea
Ruminococcus Methanobrevibacter Coprococcus
-
Fig. 7.1 Diagram with dominant bacterial genera and dual (blue) or unclear role (purple). Size of the circles
species in relation to the preferred redox potential and the reflect the average abundance in the gut. Rum is
substrate utilization in the human gut. Colors correspond Ruminococcus, Cl is clostridium
to the associations with health (green), disease (red) or a
in the complexity of substrate availability and, large spectrum of carbohydrolases (Ouwerkerk

perhaps even more importantly, the (mutualistic) et al. 2013; Benjdia et al. 2011). Akkermansia
interactions between the many different species muciniphila is a bacterium specialized in degrad-
(Flint et al. 2012a; Ley et al. 2008; Arumugam ing mucins and will be part of this niche (Derrien
et al. 2011). Each species in the gut fills up a spe- et al. 2004). Cross feeding and syntrophic inter-
cific niche, which could be best described by the actions will enable other microbes to utilize the
substrates these bacteria utilize. If no external carbohydrates liberated from these mucins, and
nutrition would enter the gut, the only substrate the various proteins and amino acids from the
bacteria would have are host excreta, such as excreta. For instance, Faecalibacterium prausnit-
saliva, mucus, gastric and pancreatic juices and zii is able to utilize the N-acetyl-D-glucosamine
bile. Most of the excreta create harsh conditions, liberated from mucins (Wrzosek et al. 2013;
like a low pH, contain proteolytic enzymes, or Ouwerkerk et al. 2013; Lopez-Siles et al. 2012).
anti-microbial substances. Mucins from saliva Enterobacteria will also be able to grow on left
and mucus on the intestinal epithelium, will most over peptides and carbohydrates, and will utilize
likely be the most dominant substrates for bacte- the low amount of oxygen and other electron
ria to utilize (Arumugam et al. 2011). The phy- acceptors penetrating from the epithelial lining
lum Bacteroidetes including Prevotella and (Walter and Ley 2011). The growth on excreta
Bacteriodes species are versatile bacteria that alone will already sustain a diverse population in
have the capabilities to utilize mucins with their gut. This diversity will multiply when nutrition
comes in that escaped host’s digestion of the diet. malfunctioning of the gut microbiota. The main
Especially, soluble and insoluble dietary fiber diseases that are clearly linked to an aberrant gut
will enable the growth of many typical gut anaer- microbiota are inflammatory bowel diseases
obes that ferment these into short chain fatty (IBD) like Crohn’s disease and ulcerative colitis.
acids (Flint et al. 2012b). These will be Prevotalla Another category of diseases are the auto-
and Bacterodes species, ruminococci, bifidobac- immune diseases like atopy, eczema, asthma,
teria, lactic acid bacteria, eubacteria and clos- celiac disease and type 1 diabetes. A last category
tridia as well as fungi like saccharomyces and that will be discussed here are related to an
candida (Martens et al. 2009; Ze et al. 2012; Flint increasing welfare and age: obesity, metabolic
et al. 2008). disease and type 2 diabetes.
Figure 7.1 shows presumptive utilization of
the main nutrients available for the gut microbi-
ota. From this we can see that there is a lot of 7.2 The Normal Microbiota
redundancy in the metabolic capacities of the
microbes. This may explain the variation and The normal gut microbiota is any microbiota that
diversity of microbiota in different individuals. is present in the gut of a healthy individual.
In 2011 the concept of enterotypes was intro- Various studies have shown that there is a large
duced, which proposed that the gut microbiota of inter-individual variation, no gut microbiota is
all human could be divided in at least three the same. There is a relation with genetics as
enterotypes (Arumugam et al. 2011). These identical twins have a more similar microbiota
enterotypes are Bacteroides type, the Prevotella than siblings, which in turn share more similari-
type and the Ruminococcus type according to the ties with one another than not related people
central microbe in an association network. The (Turnbaugh et al. 2010; Zoetendal et al. 2001;
authors state that “the enterotypes are in fact Tims et al. 2012). There is some relation with
driven by groups of species that together contrib- cultural habits and diets in different regions of the
ute to the preferred community compositions”. World. However, a lot of variation is not explained
This is based on the core consortium of microor- and this will have to do with co-incidental coloni-
ganisms that breaks down the main complex sub- zation and stabilization of an ecosystem in an
strates and especially mucin degradation seems existing niche that is formed by genetics, diet,
to be an important determinant (Arumugam et al. age and habits. Despite this variation there is a
2011). This study was followed by the study of common functionality of all these microbiota’s,
Wu which showed that enterotypes were strongly being the breakdown of the available nutrients.
associated with long-term diets, particularly pro- This commonality will cause a division of the
tein and animal fat (Bacteroides) versus carbohy- microbiota’s in different types, defined as entero-
drates (Prevotella) (Wu et al. 2011) which could types before (Arumugam et al. 2011). In this
be influenced by diet (David et al. 2013). chapter we would like to share a few thoughts on
We believe that a well-functioning gut micro- these enterotypes based on what we see in various
biota helps to ensure that the gut remains healthy studies described in literature combined with our
and is therefore important in maintaining the experiences. We see four types of microbiota:
health of the host. When there is in a unbalanced
state (dysbiosis of microbiota), the functioning of The first type is the common type in the North-
the gut microbiota will be impaired and this may West European regions. It seems to be related
lead to disease. Current developments in gut with an omnivore type diet, based on grains,
microbiota analysis techniques have opened up fruits and vegetables, and meat. This is a type
the possibility to show clear associations in dominated by bacteria of Clostridium clusters
between particular gut microbial compositions IV and XIVa, clusters containing many butyr-
(or the lack thereof) and the development of vari- ate- and other SCFA-producing bacteria.
ous diseases. In this chapter we want to show Especially the number of Faecalibacterium are
how diseases can be link with and dysbiosis and high in this type. Furthermore, there is a fair
amount of bifidobacteria and mucus degrading colonization starts with a diverse microbiota of
Akkermansia. Bacteroides are present in rea- the mother, where Bacteroides/Prevotella, lacto-
sonable numbers but not very dominant and bacilli, streptococci, staphylococci, and
there is a low level of E. coli and relatives (de Enterobacteriaceae species grow out quickly to
Goffau et al. 2014; Harmsen et al. 2002). dominant numbers (Penders et al. 2006; Isolauri
The second type is more related to a fibre rich (veg- 2012; Dominguez-Bello et al. 2010; Harmsen
etarian) diet with little or no animal products. et al. 2000). When nutrition starts the microbiota
Found in a part of the western population, but will change towards those species that are
also in for instance, in rural Africa (de Vrieze selected by the nutrition. In most cases the nutri-
2014; De Filippo et al. 2010). This type is domi- tion will be breastfeeding that contains bacteria
nated by Prevotella and Dialister-Veillonella as well to colonized the baby (Harmsen et al.
bacteria. There are also members of Clostridium 2000; Martin et al. 2009). Components of breast
clusters IV, especially ruminococci, and a little milk are strongly bifidogenic. This is not primar-
XIVa. It characterizes itself by the very low ily lactose but also specific oligosaccharides for
amount of Bacteroides and low E. coli. which bifidobacteria have specific enzymes (Sela
The third type is a type found especially in eaters et al. 2008; Zivkovic et al. 2011). This normally
of an western type diet high in animal protein results in a dominance of bifidobacteria within 1
and fat. This type is dominated by Bacteroides, week after birth in some cases this can be almost
and high levels of Clostridium clusters IV up to 100 % bifidobacteria in the gut microbiota.
Faecalibacterium and of Clostridium clusters As the child gets older the bifido-dominated
XIVa Ruminococcus gnavus (David et al. microbiota gets more diversified. It is not clear
2013). This type has virtually no E. coli. what is driving this but gastrointestinal infections
The last type we find is a type that seems to be in and antibiotic use will play a role in this. The spe-
dysbiosis, since it is often found in relation to cies of bifidobacteria that colonize the gut may
inflammatory diseases and diarrhea. It is charac- vary a lot between individuals, although B.
terized by a high amount of enterobacteria, such longum seems to be most common combined
as E. coli. Furthermore, R. gnavus is relatively with colonization of either B. bifidum, B. breve,
high and there are Bacteroides present. There is and at a later time point B. pseudocatenulatum or
a low amount of other Clostridium clusters B. adolescentis (Bergstrom et al. 2014). Between
XIVa species, and a low amount of Clostridium 3 and 6 months introduction of foods other than
clusters IV (Harmsen et al. 2012; Willing et al. milk usually starts. Often the solid foods are
2009; Morgan et al. 2012). This very rough fruits and roots, such as carrot, followed by peas
description seems to be useful if we relate it to and beans etc. This means a nutrition driven
function and disease. The first two seem to be diversification of the microbiota. With the intro-
healthy microbiota’s, the third Bactoroides type duction of these solid foods also dietary fibre
seems a risk microbiota and the last type a clear degrading bacteria will colonize, such as rumino-
microbiota “out of order”. Although the latter cocci, Bacteroides, Prevotella and Clostridium
may be a temporarily state, for instance caused clusters IV and XIVa (Favier et al. 2002). Also
by enteroviral infection, it does seem to be a non- other firmicutes species and different enterobac-
stable type vulnerable to chronic dysbiosis. teria will become introduced. This weaning
period is a critical phase in the colonization of the
children as it coincides with a period in time of
7.3 Gut Microbiota immune system training (Grönlund et al. 2007).
Development: The Young At the age of 1 year the microbiota appears to
Microbiota have the major functions that an adult microbiota
should have, however the microbiota seems to
The gut of an unborn child is in principle sterile increasingly diversify until the age of 3 years
in the womb and microbiota development starts where it starts to stabilize (Yatsunenko et al.
as soon as a child passes the birth channel. The 2012). This stabilization leads to a phase where
the microbiota does slowly develops further. The 7.4 The Role of Gut Microbiota
microbiota of young children and adolescents is in Disease
still different from that of adults and the micro-
biota continues to develop and change until old As stated before, gut microbiota is involved in
age, where there are again significant differences health and when it is in a state of dysbiosis, the
with the microbiota of e.g. 30 year old adults gut microbiota may lead to disease, although a
(Claesson et al. 2011). Many factors play a role in causal relationship is often hard to show. Current
the early development of the microbiota. developments in gut microbiota analysis revealed
Breastfeeding vs. formula feeding and Caesarian the involvement of microbiota in various dis-
section vs. natural delivery are logical important eases. The main diseases that are clearly linked to
determinants of the gut microbial development an aberrant gut microbiota are inflammatory
which seem to have a long term effect (Lozupone bowel diseases (IBD) like Crohn’s disease and
et al. 2013; Adlerberth and Wold 2009; Van ulcerative colitis. Another category of diseases
Nimwegen et al. 2011). Less obvious factors are the auto-immune diseases like atopy eczema,
such as maternal factors expressed in breast milk, asthma, celiac disease and type 1 diabetes. A last
host factors like genetics and innate immunity are category that will be discussed here are related to
however also important (Martín et al. 2007; an increasing welfare and age: obesity and type 2
Zivkovic et al. 2011). Environmental factors and diabetes.
choice and timing of nutrition subsequently con-
tinue to steer the development of the microbiota.
There are of course a lot of environmental factors 7.4.1 IBD
that can influence the microbiota development.
Environment can be determined by conditions Inflammatory bowel diseases were already asso-
that favor encounters with different micro- ciated with an aberrant gut microbiota for a long
organisms, such as hygiene conditions, number time ((Xavier and Podolsky 2007). Bacteria such
of siblings, daycare centers and school, animals as mycobacterium, adherent invasive E. coli
in the house, rural or urban lifestyle (Azad et al. (AIEC) and viruses have been associated with
2013). These condition will determine the com- IBD (Fiocchi 1998). Today it seems apparent that
position of the gut microbiota, just as later in life IBD is not caused by a single micro-organism but
meat, sugar and fat consumption, smoking and that it is an delicate interplay between genetics,
alcohol consumption (David et al. 2013). All immunology, environmental factors and gut
these factors will be important in relation to dis- microbiota. Principal component analysis of data
eases where the gut microbiota is believed to be from metagenomic sequencing clearly showed
involved (Lozupone et al. 2013). For example, that Crohn’s disease patients had a microbiota
atopy and eczema, which are correlated with a that was different from healthy controls and UC
dysbiosis of the microbiota, are also negatively patients, who’s microbiome seems only moder-
correlated with the number of siblings (Penders ately different from healthy patients (Qin et al.
et al. 2007). 2010). It shows first of all that CD and UC are
These relations can be explained by the different diseases, of which the microbiota of CD
hygiene hypothesis that state that we have to patients is clearly more affected than that of UC
encounter sufficient microbes for the program- patients. What it doesn’t show is what causes CD
ming of our immune system. Extra siblings bring but merely what the effect of inflammation is on
extra microbes with them that helps to diversify the microbiota and vice versa. Patients in remis-
the microbiota. The hygiene hypothesis is cur- sion still show a high number of enterobacteria
rently tested in several studies, to find or exclude and a reduced number of faecalibacteria and
a relation with autoimmune diseases that are cur- other butyrate producing species (Sokol et al.
rently on the rise like atopy, asthma and type 1 2009; Miquel et al. 2013). An inverse relation-
diabetes (Vaarala et al. 2008; Dunne et al. 2014). ship between the first two groups is seen in many
of the studies on IBD (Willing et al. 2009; thelial lining to regenerate itself and hence
Harmsen et al. 2012). Bacteroides numbers are becomes susceptible to new inflammatory trig-
usually also higher in CD patients, although this gers (Sokol et al. 2008). This vicious cycle is
may have to do with a lowering of other bacteria considered in the concept of a leaky gut (Fasano
like Clostridium groups IV and XIVa. It seems 2012). A possible cure to break this cycle could
that due to the inflammation, the gut lumen is less on one hand be immunological, such as to steer
reduced so that facultative anaerobes and oxygen the inflammatory signaling by improving the
tolerant bacteria get more room to grow, hence autophagy or regulate (anti)-inflammatory cyto-
the increase in enterobacteria and along with that, kines. Or on the other hand, try to improve the
enterococci. On the other hand, strict anaerobes dysbiosis so that a balanced microbiota produces
like faecalibacteria and other Clostridium group the right SCFA and anti-inflammatory signals,
IV and XIVa will see their numbers diminish in for a well-functioning gut barrier with a low
such conditions. The mechanism behind this is grade of inflammation, properly responding to
the production of reactive oxygen species (ROS) normal triggers. The focus is now on the use of
and nitric oxide (NO·) during intestinal inflam- anaerobic butyrate producers, such as faecalibac-
mation (Winter et al. 2013). For instance, NO- teria, as probiotics to restore the dysbiosis
production by inducible nitric oxide synthase (Miquel et al. 2013). The role of the microbiota
(iNOS) leads to the formation of nitrite formation in UC patients may be more difficult to under-
in the gut. This will be used for anaerobic respira- stand. With UC the gut is mildly inflamed but
tion by locally present facultative over a larger area compared to CD the gut micro-
Enterobacteriaceae that in this way have a com- biota is usually less affected. There seems to be a
petitive advantage over the strict anaerobes that crucial role for the interplay between the adaptive
are troubled by the oxidative stress anyway. This immune system (neutrophyles) and the microbi-
effect also explains the loss of colonization resis- ota (Fries and Comunale 2011; Chen et al. 2014).
tance after antibiotic treatment. Experiments Both IBD diseases affect usually young adults,
with streptomycin showed that antibiotic these patients often had a normal gut function in
increased the epithelial inflammation and thus their childhood. Whether their gut microbiota
stimulated growth of enterobacteria by nitrate was effected before the disease set in, remains to
respiration rather than changing the microbial be seen.
populations themselves (Spees et al. 2013).
A changed microbiota in CD patients would
be a result of the chronic inflammation and not 7.5 Abberant Gut Microbiota
necessarily the cause. The cause may be purely Development and Type 1
coincidental, a viral infection, or infection with Diabetes
pathogenic Enterobacteriaceae or even a trauma
such as disturbance of the intestinal integrity Auto-immune diseases correlate, unlike IBD,
(Xavier and Podolsky 2007; Strober 2011; with an aberrant microbiota development.
Liverani et al. 2014). This results in inflammation Various studies have been performed in humans
of the gut epithelium and in genetically- and in rodents in relation to the development of
predisposed individuals this would lead to type 1 diabetes (T1D) and its association with the
chronic inflammation, maybe because they may gut microbiota. Many of the associations found
be impaired in autophagy or their anti- between the gut microbiota and T1D overlap
inflammatory signaling (Sadaghian Sadabad between the different studies and they impor-
et al. 2014). Periods of inflammation could be tantly do not contradict one another. A coherent
followed by a dysbiosis of the gut microbiota picture in which the gut inflammatory state is
which is unable to restore its balance. In that reversely coupled to the abundance of butyrate-
case, the microbiota does not produce the right producing bacteria, emerges consistently out of
SCFA and anti-inflammatory signals for the epi- all these studies. Sufficient butyrate production
keeps the gut healthy as it is the main energy rier where the immune system subsequently
source for colonic epithelial cells (Hague et al. comes into contact with it by indirectly hamper-
1996), regulates the assembly of tight junctions ing the production of butyrate by other bacteria
(reduces permeability) (Peng et al. 2009) and it via competition and directly by inducing inflam-
importantly reduces intestinal inflammation mation via LPS.
(Hamer et al. 2009). Several bacterial genera Rather large differences exist between coun-
from the Clostridium clusters IV and XIVa, such tries in regards to the prevalence of T1D. Part of
as Faecalibacterium and Roseburia, are well this can be explained genetically but a large part
known butyrate producers and are usually under- is likely due to lifestyle and nutrition patterns.
represented in the microbiota of people that either The effects of diet on the prevalence of
have T1D or that are developing it (de Goffau Bacteroides have especially been well docu-
et al. 2013). On the other hand, diabetogenic bac- mented as a western diet high in protein and ani-
terial groups are also identified and interestingly, mal fat consumption (think hamburgers and fast
the Bacteroides genus, is found to be associated foods) leads to a high prevalence of Bacteroides
with T1D without exception in basically every (David et al. 2013). People in developing coun-
study (de Goffau et al. 2014; Murri et al. 2013; tries eating traditional foods on the other hand
Brugman et al. 2006; Giongo et al. 2010; Brown usually have a microbiota which is dominated by
et al. 2011). Several explanations exist for this Prevotella instead (De Filippo et al. 2010) as
particular association with Bacteroides. The first Prevotella are mainly associated with a carbohy-
reason is that Bacteroides is not a butyrate pro- drate rich diet (roots, tubers, corn, sorghum, etc.).
ducer as it producers propionate and acetate; an In western countries a healthy gut microbiota
overabundance of Bacteroides will leave less composition would be considered to either con-
room for actual butyrate producers in the gut sist out of a composition which is largely domi-
(competition for resources). Secondly, nated by bacteria from Clostridium clusters IV
Bacteroides, like Escherichia coli, are Gram- and XIVa, bifidobacteria and just small amounts
negative bacteria of which particular cell wall of Bacteroides or a composition which is more
components, namely the lipopolysaccharides centered around Prevotella (Prevotella entero-
(LPS), can induce inflammation by acting as type), in which Clostridium cluster IV is still
endotoxins (inducing an inflammatory response always present in high abundance but where
via the immune system). A third possible link Bacteroides is largely absent. Both these healthy
with T1D, is that Bacteroides (and streptococci) types of microbiota composition are likely estab-
produces glutamate decarboxylase (GAD), which lished by a diet rich in dietary fiber and a moder-
might be a trigger for GAD autoimmunity ate consumption of animal products and limited
(GADA) via molecular mimicry. Glutamate amounts of (processed) sugars. An interesting
decarboxylase is also produced in humans example of the above is a study by Mejía-León
(GAD65) but only in the insulin producing beta et al. done in Mexico, where the incidence of
cells of the pancreas and in neuron cells. When T1D is higher in people living close to the border
the human body raises autoantibodies against with the USA. Patients who just developed T1D
bacterial GAD it might accidentally subsequently in this study had higher Bacteroides numbers
attack its own GAD producing cells, hence result- while the healthy controls had a gut microbiota
ing in T1D as the insulin producing cells are which was dominated by Prevotella. Bacteroides
destroyed. Interestingly, Alzheimer’s disease is is far more dominant in the USA than in Mexico
also considered to be a type of diabetes (Type 3), and T1D rates are indeed much higher there
or a result of it (Vignini et al. 2013), and this (Mejía-León et al. 2014). Similar kinds of pat-
might in part also be due to the destruction of terns can be discerned when comparing Finland
GAD producing neurons. Bacteroides can thus with Estonia (De Goffau, EU project
accommodate its translocation past the gut bar- Diabimmune, unpublished).
7.6 Obesity and Metabolic mice, Cani and his colleagues showed recently
Diseases that this signal-transduction protein partially
protects against diet-induced obesity, diabetes
There is currently a lot of interest in research for and inflammation (Everard et al. 2014). These
obesity, a metabolic disease that became epi- mice spend more energy and had improved glu-
demic in the Western world (Ley et al. 2005). cose homeostasis, reduced liver fattening, fat
One of the first success stories of next-generation mass and inflammation. This partial protection
sequencing is the work of the lab of Gordon and could be transferred to germ-free mice by gut
co-workers on the role the microbiota in this dis- microbiota transplantation. Remarkably, the
ease. Their research suggested that next to a MyD88 deletion increased anti-inflammatory
chronic energy surplus also other factors such as endocannabinoids, restored antimicrobial pep-
the indigenous gut microbiota could play a role tides production and increases intestinal regula-
(Backhed et al. 2004). Even more, that the energy tory T cells during diet-induced obesity. These
overshoot could be caused by an overproduction experiments show that the intestinal epithelial
of SCFA in the colon and that there is a low grade MyD88 acts like a sensor switching the host
of inflammation that often symptomizes obesity metabolism according to the nutritional status.
and related metabolic diseases (Fleissner et al. Obesity and related disorders may therefore be
2010; Backhed et al. 2007). again a result of an unbalanced relationship
Obesity can lead to serious comorbidities, between microbiota and epithelial innate immu-
such as type 2 diabetes and liver cirrhosis caused nity, regulating metabolic functions normally
by alcoholic or nonalcoholic liver diseases. needed for optimal energy harvest for nutrient
Recent studies show a dysbiosis in both type 2 limited diet.
diabetes and liver cirrhosis, although the nature In metabolic diseases the microbiota is out of
and intensity of the dysbiosis is different. In type balance, maybe due to diet or all kinds of differ-
2 diabetes there is only a mild dysbiosis, and ent co-morbidities, maybe due to excessive
fecal samples of the patients seem to be domi- behavior, such as over eating, alcohol abuse or
nated by firmicutes other than the butyrate- sleep deprivation (Cronise et al. 2014). Abnormal
producing Roseburia and F. prausnitzii, and E. production of SCFA and other metabolites as
coli (Qin et al. 2012; Khan et al. 2014). In liver well as an altered lipid metabolism seem to play
cirrhosis the samples were dominated by a major role in the etiology of these diseases.
Bacteroides and buccal microbiota (Qin et al. Propionate over production by Bacteroides spe-
2014). In this case the buccal microbiota may be cies and over production of acetate and butyrate
able to colonize the ileal and colonic gut since by different firmicutes may lead to an energy sur-
there is a compromised bile production and less plus (Holmes et al. 2012; Turnbaugh and Gordon
bile-resistant bacteria can survive. 2009; Schwiertz et al. 2010). Different microbial
Recent studies showed that lipopolysaccha- metabolites influence the satiety of the host and
rides (LPS) cause the low grade inflammation regulate the adipose tissue formation (Roelofsen
in obesity, a metabolic endotoxemia that dys- et al. 2010). However, the effect of the gut micro-
regulates the inflammatory tone and triggers biota may be secondary. Improving the obesity
body weight gain and diabetes (Cani et al. by special protein rich diets may change the
2007). The scientists concluded that the system microbiota, but not always for the better (Walker
of CD14 immune cells reacting on LPS set the et al. 2010), since they lead to a more Bacteroides
tone of insulin sensitivity and the onset of dia- type of microbiota (David et al. 2013). A more
betes and obesity. At this moment it is clear that sustainable diet with low calories and high fibre
obesity is somehow associated with altered gut intake will lead to a more balanced microbiota,
microbiota, low-grade inflammation and a clus- that can further improve the metabolic disease
ter of metabolic disorders. By inducible intesti- and will have a positive effect on the insulin
nal epithelial cell-specific deletion of MyD88 in resistance (Walker et al. 2010).
Fecal transplantation as a way to improve the may become leaky, with different diseases as
obesity and type 2 diabetes has led to improved consequence. In this healthy microbiota there is
insulin resistance, but not immediately improves therefore a role for butyrate producing bacteria.
the BMI of the patients (Vrieze et al. 2012). F. prausnitzii is a species which seems to have
Controlled diets may improve both, since this co-evolved with humans, since it is present in
will automatically improve the gut microbiota as most humans at high numbers. Next to this spe-
well. Furthermore, it seems unlikely that obesity cies there is a role for Roseburia, Clostridium
is caused by microbes, but merely shows the group XIVa bacteria that, like faecalibacteria use
effect of diet on the microbiota. New metabolo- the butyryl-CoA pathway to produce butyrate
mics and metagenomic studies will give a more from glucose and acetate. Also other types of
clearer picture on the relationship between obe- butyrate producers seem to be important in this
sity and gut microbiota. respect, such as Coprococcus, Anaerostipes, and
Eubacterium halii, bacteria that produce butyrate
from acetate and lactate.
7.7 Concluding Remarks Supplementing the microbiota with these
micro-organisms may not be enough since these
A healthy gut microbiota is important for main- bacteria require low redox potentials to grow and
taining a healthy host. An aberrant microbiota imbedding in a complex trophic network. This
seems to slowly cause diseases of different nature may not always be provided by the host and the
and at different ages. From allergies at early age dysbiosed gut microbiota.
to IBD in young adults, from autisms to colorec- For most diseases where a relation with a dys-
tal cancer, a large variety of effects are reported. biosed microbiota is demonstrated, still the
This results in an obvious need for gut microbiota chicken and egg question has not been answered:
management. Fortunately, it automatically goes a causal relationship has not been shown. What is
alright in most of the people that have a healthy, the trigger of the deteriorating situation. Viruses
regular lifestyle. But in case of dysbiosis and dis- may certainly have a role as trigger, just like yeast
ease, modulation of the microbiota is wanted by and parasites, which are not discussed in this
means of pro- and prebiotics or in extreme cases chapter. The host may have undergone immuno-
by fecal transplantation. There are still some logical changes that makes it susceptible to trig-
essential questions to be answered on the gut gers from microbiota to start aberrant immune
microbiota. We still do not really know what is a reactions.
healthy microbiota, nor do we know what the Much needs to be investigated and this makes
influence of our genetic makeup is on the micro- us realize that we are just in the beginning of
biota. What is a healthy diet? Are we not apes understanding the microbial world within us an
that should eat nuts, roots and fruits and occa- its relation the health and disease. Fortunately,
sionally a piece of meat? How would our gut new system biology and meta-omics approaches
microbiota look like if we would eat just that, provide the tools to unravel this complex system,
would it look like ape microbiota and would that that we call our body.
be healthy (Ellis et al. 2013)?
There are some clear generalities, such as: for
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Manipulation of the Microbiota
Using Probiotics 8
Verena Grimm and Christian U. Riedel
Abstract
A number of diseases are associated with alterations in the composition of
the microbiota of various niches of the human body. Although, in most
cases, it is unclear if these alterations are the cause or the consequence of
disease, they provide a rationale for therapeutic or prophylactic manipula-
tion of a dysbiotic microbiota. Approaches to manipulate the microbiome
include administration of either live bacteria, which are underrepresented
in the diseased individual, substances that aim at increasing the popula-
tions of these bacteria, or a combination of the two. This chapter summa-
rizes the available data in therapeutic manipulation of a various diseased
states including irritable bowel syndrome, inflammatory bowel disease,
necrotizing enterocolitis, atopic and allergic diseases, and antibiotic-
associated and infectious diarrhoea.
Keywords
IBS • IBD • Allergy • Necrotising enterocolitis • Diarrhea
8.1 Introduction (Human Microbiome Project Consortium 2012;

Faith et al. 2013; Grice and Segre 2011; Gajer
The various epithelial interfaces of the human et al. 2012). Nevertheless, characteristic changes
body with the outside environment harbour in the composition of the microbiota have been
unique and characteristic microbial communities observed in a number of diseases including
that are specific for an individual and relatively inflammatory bowel disease (IBD), irritable
stable over time under normal circumstances bowel syndrome (IBS), atopic and allergic, peri-
odontitis, infectious diseases, metabolic syn-
V. Grimm (*) • C.U. Riedel drome, and cancer (Sears and Garrett 2014;
Institute of Microbiology and Biotechnology, Schwabe and Jobin 2013; Sekirov et al. 2010;
University of Ulm, Gerritsen et al. 2011; Sommer and Bäckhed
Albert-Einstein-Allee 11, 89069 Ulm, Germany 2013; Russell and Finlay 2012). Interestingly,
e-mail: verena.grimm@uni-ulm.de;
christian.riedel@uni-ulm.de dysbioses in the microbiota have also been

110 V. Grimm and C.U. Riedel
observed in psychiatric disorders including The analysis of the composition of the micro-
autism and depression (Sekirov et al. 2010; Dinan biota in diverse habitats of the human body as
and Cryan 2013). The majority of studies link well as therapeutic approaches using prebiotics,
changes in the intestinal microbiota with disease synbiotics or microbiota transplantation will be
(Sekirov et al. 2010; Sommer and Bäckhed 2013) discussed elsewhere in this book. In this chapter,
with less but increasing data available on disease- we will focus the clinical data available for probi-
related changes in the vaginal (Ma et al. 2012; otics treatment in a number of important diseases
Brotman 2011), oral (Pihlstrom et al. 2005; Wang and, where appropriate, we will discuss altera-
et al. 2013), or skin (Grice and Segre 2011; tions in the microbiota that are reported for these
Zeeuwen et al. 2013) microbiota. diseases. Due to the large number of clinical tri-
In most cases, the question whether an altered als performed on probiotic treatments, we will
microbiota is the cause or the consequence of dis- focus on available review articles and meta-
ease is not definitively answered. Nevertheless, analyses on the efficacy of probiotic treatments.
these changes are the rationale for therapeutic Since the microorganisms used as probiotics gen-
or prophylactic interventions attempting to erally belong to the group of GRAS organisms,
manipulate or restore the microbiota for which a we will not elude to studies, reviews, or meta-
dysbiosis has been observed. The approaches to analyses dealing with the safety aspects of probi-
manipulate the microbiota for therapeutic otic treatments. We will not discuss individual
reasons include the administration of either probiotics or formulations in a specific disease.
(potentially) beneficial microorganisms (probiot- Instead, general trends in the efficacy of probiot-
ics), substrates to boost specific populations of ics in these diseases found in the meta-analyses
microorganisms (prebiotics), or a combination of will be summarized. For results on specific probi-
both (synbiotics). otics, the reader is referred to individual studies
The term “probiotic” was initially coined by included in these meta-analyses.
Lilly and Stillwell describing an unidentified
factor produced by one ciliate (Colpidium
campylum) that promoted growth of another 8.2 Irritable Bowel Syndrome
(Tetrahymena pyriformis) (Lilly and Stillwell
1965). Today, probiotics are defined as ‘live IBS is a group of complex intestinal disorders
microorganisms which when administered in that are characterized by a range of very diverse,
adequate amounts confer a health benefit on the sometimes contradictory symptoms including
host’ according to guidelines set by the World chronic abdominal pain, bloating and altered
Health Organisation and the Food and Agriculture stool frequency (constipation, diarrhoea, or both
Organization of the United Nations (FAO/WHO with alternating episodes). Due to the lack of
Working Group 2002). Most of the organisms validated genetic, biochemical or physiological
used and marketed as probiotics belong to the markers, diagnosis of IBS is based on intensive
genera Lactobacillus and Bifidobacterium with a examination and exclusion of other diseases.
few strains of other lactic acid bacteria, Bacillus About 10–20 % of adults and adolescents display
sp., Saccharomyces sp., and E. coli (Gareau et al. symptoms of IBS with a dominance in female
2010; Bron et al. 2012; Foligné et al. 2013). patients. Affected persons are classified into dif-
Probiotic bacteria are marketed and administered ferent patients subgroups according to the ROME
as naturally fermented or non-fermented food criteria based their symptoms (Longstreth et al.
products in various matrices, food supplement 2006; Chang and Talley 2011). The pathogenesis
powders, tablets etc. and these products either of IBS is multifactorial but there is strong evi-
contain single strains or mixes of several probi- dence for a contribution of the GIT microbiota.
otic bacteria (Gareau et al. 2010; Foligné et al. Changes in the composition of both the faecal
2013). and the mucosal microbiota have been observed
8 Manipulation of the Microbiota Using Probiotics 111
and these changes appear to be specific for IBS that have undergone colectomy. By contrast, in
subgroups (Gerritsen et al. 2011; Jeffery et al. CD inflammation is transmural, can affect any
2012; Kerckhoffs et al. 2009; Rajilić-Stojanović site of the GIT, and healthy and inflamed sites
et al. 2011). However, a unifying feature of all can alternate.
IBS subtypes seems to be a reduction in bifido- IBDs are multifactorial diseases with a con-
bacteria (Kerckhoffs et al. 2009; Rajilić- siderable contribution of genetic predisposition,
Stojanović et al. 2011; Rigsbee et al. 2012). environmental factors and the intestinal microbi-
Given the (likely) contribution of the micro- ota involved. In both UC and CD, prominent, a
biota in pathogenesis and the reduced abundance reduced diversity and changes the abundance of
of bifidobacteria in the GIT of patients, probiot- prominent bacterial groups of the microbiota
ics represent an logical therapeutic intervention have been observed (Gerritsen et al. 2011;
of IBS. In recent years, a number of reviews and Peterson et al. 2008; Kostic et al. 2014). Principal
meta-analyses have summarized the clinical data component analysis shows that the microbiota
available for probiotic treatments in IBS profiles of UC, CD and healthy controls are
(Hoveyda et al. 2009; McFarland and Dublin clearly different from each other (Qin et al.
2008; Moayyedi et al. 2010; Brenner et al. 2009; 2010).
Whelan 2011; Whelan and Quigley 2013; Ritchie For CD, extensive 16S rRNA gene sequencing
and Romanuk 2012). These analyses included a of faecal and mucosal samples obtained from 447
total of 30 clinical trials, in which patients were children with on-set, untreated CD and 221
treated with various probiotics containing differ- healthy controls revealed an overall reduction in
ent strains of bifidobacteria, lactobacilli, strepto- diversity in diseased individuals (Gevers et al.
cocci, Saccharomyces boulardii, and E. coli as 2014). Moreover, an increase in
single strain preparations, probiotic mixes or syn- Enterobacteriaceae, Pasteurellaceae,
biotics. While some of the trials were criticised Veillonellaceae, and Fusobacteriaceae, and a
for suboptimal study design, the overall outcome reduction in Erysipelotrichaceae, Bacterdoidales,
was that most probiotics alleviate the symptoms and Clostridiales were observed in mucosal but
of IBS with some strains being more effective not faecal samples. This suggests that changes in
than others. the mucosal microbiota are more prominent and
important for development of CD than changes
in the composition of the luminal microbiota.
8.3 Inflammatory Bowel Disease In other studies similar observations were made
(IBD) with an increase in Proteobacteria and a reduc-
tion in Firmicutes and Bacteroidetes and poten-
Inflammatory bowel disease (IBD) is a group of tially beneficial or anti-inflammatory bacteria
chronic gastrointestinal disorders characterized including Faecalibacterium prausnitzii or bifido-
by relapsing and remitting inflammation of the bacteria (Kostic et al. 2014; Franks et al. 1998;
GIT (Bouma and Strober 2003; Cho 2008; Xavier Sokol et al. 2008; Schwiertz et al. 2010; Joossens
and Podolsky 2007). The two by far most wide- et al. 2011).
spread forms of IBDs are ulcerative colitis (UC) Meta analyses on the effect on probiotics in
and Crohn’s disease (CD). Both UC and CD UC and pouchitis uniformly suggest that probiot-
share most of the symptoms but differ in the ics are more effective than placebo in maintain-
extent and anatomic location of the inflamed tis- ing remission (Shen et al. 2014; Holubar et al.
sues. UC is limited to the mucosa of the large 2010; Jin et al. 2007). However, probiotics did
intestine and extends from the distal to the proxi- not show a positive effect on recurrence in post-
mal colon in varying degrees. One form of UC is operative CD compared to placebo (Doherty
pouchitis, which represents a chronic inflamma- et al. 2009, 2010; Van Loo et al. 2012).
tion of the artificial rectum created by surgical Nevertheless, it was noted that the effect of probi-
formation of a pouch from ileal tissue in patients otics in the management of CD merits further
investigation (Doherty et al. 2009, 2010; Van Loo to revise and generalize the hygiene hypothesis
et al. 2012). In the study by Gevers et al. (2014), (Brooks et al. 2013; Wills-Karp et al. 2001).
in a subgroup of 57 diseased children that already Nevertheless, the microbiota plays a crucial role
had received antibiotics at the time point of sam- in the development of atopic diseases as demon-
pling the dysbiosis was more pronounced com- strated by profound shifts in the composition of
pared to the other, non-treated samples. the GIT and skin microbiota observed in allergic
This indicates that use of antibiotics for treatment patients (Grice and Segre 2011; Russell and
of CD might actually increase dysbiosis ques- Finlay 2012; Zeeuwen et al. 2013). Also, the
tioning the use of antibiotics for treatment development of the microbiota is heavily influ-
of CD. Thus, in light of problems associated with enced by the mode of delivery and early infant
antibotic use in increasing dysbiosis and the rec- feeding (Matamoros et al. 2013). Moreover, cae-
ommendations of the meta analyses, probiotics sarean section and bottle feeding have been asso-
might become an alternative strategy in the man- ciated with an increased risk to develop atopic
agement of CD but need further investigation. diseases (Bager et al. 2008; Yang et al. 2009).
In consequence, probiotics have come into
focus as an alternative or supplementary thera-
8.4 Atopy/Allergy peutic strategy in the management of atopic dis-
eases. Several meta analyses have studied the
Atopy and allergic diseases such as asthma, hay impact of probiotic administration to prevent of
fever, food allergy, and atopic dermatitis (AD) treat allergies. Two analysis on prevention of
are hypersensitivity disorders caused by an aber- asthma and wheeze concluded that there is not
rant immune response to environmental antigens sufficient evidence to recommend administration
(Kay 2000). Affected persons react to normally probiotics (neither prenatally to mothers nor
harmless substances abundant in the environment postnatally to infants) as a protective treatment
such as food antigens, pollen, and commensal (Azad et al. 2013; Elazab et al. 2013). However,
bacteria. The aberrant immune response in aller- one of the studies reported an association between
gic patients is characterized by a predominant probiotics and a reduced risk for sensitisation and
Th2 response resulting in excessive activation of decreased serum levels of IgE (Elazab et al.
mast cells, basophils, and IgE producing B cells. 2013). The authors of both studies suggested to
The symptoms of allergy are the result of the follow-up on existing trials and further clinical
release of local pro-inflammatory mediators and basic research on specific probiotic strains in
including histamines from these cells. Standard childhood asthma. Interestingly, probiotics seem
therapies include treatment with antihistamines to be more effective for prevention of AD. Of
in the acute phase and hypersensitisation for three independent meta analysis only one con-
prophylaxis. cluded that probiotics can not be recommended
Since atopic and allergic diseases have seen an as a treatment option to prevent AD but indicated
enormous increase in the last five decades coin- that the results could be biased by the high het-
ciding with improved sanitary and hygiene stan- erogeneity in the probiotic strains and formula-
dards one of the possible explanations for this tions and specific probiotics may still be
increase is the so-called hygiene hypothesis protective (Boyle et al. 2009). Two more recent
(Brooks et al. 2013). The hygiene hypothesis analyses indicated that prenatal administration of
states that the aberrant immune responses to probiotics to mothers during pregnancy as well as
environmental antigens in allergy is the result of postnatal to infants reduced the risk for AD in
an insufficient exposure to antigen early in life. both the general population and at-risk groups
Recent data particularly on allergic asthma has (Doege et al. 2012; Panduru et al. 2014).
raised scepticism and thus it has been suggested
8.5 Necrotizing Enterocolitis gained considerable interest in the management

of NEC.
Necrotizing enterocolitis (NEC) is an acute In several independent meta-analyses, probi-
inflammatory disorder of the intestinal mucosa otics were found to have beneficial effects in
and one of the leading causes for neonatal mor- NEC patients (Mihatsch et al. 2012; Deshpande
bidity and mortality (Neu and Walker 2011; et al. 2010; Alfaleh and Anabrees 2014). One of
Gephart et al. 2012). While NEC primarily these analyses stated that some probiotics may
affects low birth-weight (less than 1,500 g), pre- reduce the severity and mortality of NEC
term infants it occasionally also occurs in full- (Mihatsch et al. 2012). By contrast, the other two
term infants. Symptoms of NEC include feeding analyses were more enthusiastic noting signifi-
intolerance, bloody stool, abdominal distention cant effects of probiotics containing lactobacilli
and heavy inflammation and necrosis of the intes- alone or in combination with bifidobacteria in the
tinal tissue starting at around 8–10 days post prevention or treatment of NEC (Deshpande
partum. et al. 2010; Alfaleh and Anabrees 2014). Thus,
NEC is thought to be mainly the result of an the available data supports the use of probiotics
immature and highly immunoreactive intestinal in pre-term infants to prevent NEC or reduce the
mucosa (Neu and Walker 2011; Gephart et al. severity and mortality of the disease. In the
2012). Reduced expression of mucins, immuno- future, further comparative studies should be per-
globulin A, and tight junction proteins by the formed to identify the most effective probiotic
immature intestinal epithelium result in a leaky preparations and therapeutic conditions in terms
barrier and an increased exposure of cells of the of dose, timing, and duration.
underlying mucosal immune system to microbial
antigens. Additionally, an increased expression
in Toll-like receptor 4 and a reduced expression 8.6 Diarrhoeal Diseases
of the inhibitor of NF-κB suggests abnormal
sensing and signalling of lipopolysaccharide Globally, there are approximately 1.7 billion
(LPS), a molecule abundant in the outer mem- diarrhoeal episodes per year accounting for
brane of Gram-negative bacteria with potent pro- almost 760,000 deaths of children under 5 years
inflammatory activity. Furthermore, there is of age. Diarrhoeal diseases are thus the second
strong evidence for an altered composition of the leading cause of childhood mortality (WHO fact
intestinal microbiota in NEC patients compared sheet No. 330, April 2013). Diarrhoea is defined
to full-term, vaginally delivered, breast-fed as three or more loose or watery stools per day. In
infants (Carlisle and Morowitz 2013; Grishin case of acute infectious diarrhoea, the diseases is
et al. 2013). The most commonly found differ- caused by a (diagnosed) infectious agent includ-
ences include a reduced diversity and an over- ing viruses, bacteria and eukaryotic parasites
abundance of Proteobacteria, a group of LPS- (Thielman and Guerrant 2004). The vast majority
containing, Gram-negative bacteria. Collectively of these infections are the result of consumption
these factors lead to an excessive inflammatory of water contaminated with human or animal fae-
response ultimately causing necrosis of the ces or poor hygiene standards and a shortage in
intestinal tissue. safe cooking resulting in contaminated food
The treatment options for diagnosed NEC products (WHO fact sheet No. 330, April 2013).
include bowel decompression, discontinued The pathogens most frequently found to cause
enteral feeding, intravenous antibiotics, and sur- diarrhoea are rotavirus, norovirus, Salmonella sp.,
gical intervention. Since abnormal LPS/TLR4 Shigella sp., Campylobacter jejuni, pathogenic
signalling, altered microbial colonisation and E. coli strains, Cryptosporidium sp., and Giardia
other factors strongly suggest a role of the intes- sp. (Thielman and Guerrant 2004; DuPont 2014).
tinal microbiota in the pathology, probiotics have Traveller’s diarrhoea is a form of infectious
diarrhoea and a frequent problem in persons 2012; Goldenberg et al. 2013; Johnson et al.
travelling from geographic zones of low risk 2012). The overall results indicated that probiot-
(Europe, USA, Canada, Japan, and Australasia) ics are protective in preventing antibiotic-
to areas with high risk (northern Africa, Latin associated diarrhoea. Moreover, probiotics
America, Middle East and Southeast Asia) (Al- reduce the stool frequency and shorten the dura-
Abri et al. 2005). Another form of infectious tion of infectious diarrhoea. However, further
diarrhoea is observed in 5–25 % of patients clinical trials are needed to identify the most
treated with a range of antibiotics (Bartlett 2002). effective probiotic strains, doses, and treatment
While C. difficile is responsible for only 15–20 % regimes.
of the cases, it is the most severe form of antibi-
otic-associated diarrhoea since infections often
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How to Manipulate the Microbiota:
Prebiotics 9
Petra Louis, Harry J. Flint, and Catherine Michel
Abstract
During the last century, human nutrition has evolved from the definition of
our nutritional needs and the identification of ways to meet them, to the
identification of food components that can optimise our physiological and
psychological functions. This development, which aims to ensure the wel-
fare, health and reduced susceptibility to disease during life, gave birth to
the concept of “functional foods”. In this context, there is an increasing
interest in the physiological effects induced by the dense and diverse
microbiota which inhabits the human colon and whose development
depends on the fermentation of undigested food residues. Thus, much
research aims at identifying ways to guide these impacts in order to benefit
the health of the host. It is in this context that the concept of “prebiotics”
was developed in the 1990s. Since then, prebiotics have stimulated exten-
sive work in order to clarify their definition, their nature and their physio-
logical properties in accordance with the evolution of knowledge on the
intestinal microbiota. However many questions remain open about their
specificities, their mechanism(s) of action and therefore the relevance of
their current categorisation.
Keywords
Resistant starch • Oligosaccharides • Fermentation • Health effect • Non-
digestible carbohydrates • Dietary fibre • Microbiota composition •
Competition for substrates • Short-chain fatty acids • Mineral absorption •
Appetite regulation • Intestinal barrier • Immune functions
C. Michel
P. Louis (*) • H.J. Flint UMR Physiologie des Adaptations Nutritionnelles,
Microbiology Group, Rowett Institute of Nutrition Université de Nantes, INRA, HNB1- CHU-Hotel
and Health, University of Aberdeen, DIEU, Place Alexis Ricordeau, 44093 NANTES
Foresterhill, Aberdeen AB25 2ZD, UK Cedex 1, France
e-mail: p.louis@abdn.ac.uk; h.flint@abdn.ac.uk e-mail: catherine.michel@nantes.inra.fr

120 P. Louis et al.
9.1 Introduction ecology of the dominant human colonic anaer-

obes is in its infancy, but we are beginning to gain
The human gastrointestinal tract supports a an understanding of certain key functions, such
diverse collection of resident microorganisms butyrate production by Firmicutes (Louis and
(the gut microbiota). Microbial communities in Flint 2009) and glycan utilization by Bacteroidetes
the stomach, small intestine and large intestine (Martens et al. 2009). Here we will discuss the
all have important impacts on human health, but prebiotic concept in the context of our current
by far the greatest concentration of microorgan- knowledge of the gut microbiota and provide a
isms (predominantly anaerobic bacteria) is found brief overview of the main prebiotics and prebi-
in the large intestine. Microbial growth depends otic candidates currently being investigated for
in part on utilization of endogenous host secre- their effect on the microbiota and associated
tions such as mucin, but diet-derived substrates health effects.
are generally thought to provide the major energy
sources for gut microbes. Modification of the
diet, especially non-digestible carbohydrates that 9.2 What Is a Prebiotic?
reach the large intestine, should therefore provide
a highly effective approach for modifying the A prebiotic was originally defined by Gibson and
composition and function of the resident gut Roberfroid (1995) as “a non-digestible food
microbiota. Recent evidence shows that faecal ingredient that beneficially affects the host by
microbiota composition is indeed modified by selectively stimulating the growth and/or activity
changing the major non-digestible energy source of one or a limited number of bacteria in the
in the diet (Walker et al. 2011) while the impact colon, and thus improves host health”. The
of prebiotics on specific groups is well docu- knowledge of the microbial diversity within the
mented (Bouhnik et al. 2004; Flint et al. 2012a). gut as well as the relative abundance of different
Molecular analysis of the healthy human fae- members of the microbiota was limited at the
cal microbiota, mainly via 16S rRNA gene time, as it was based almost exclusively on
sequencing, shows a high degree of diversity and culture-based approaches. Bifidobacteria and lac-
inter-individual variation at the species level. tobacilli (designated collectively here as lactic
Nevertheless 50–60 species occur at high abun- acid bacteria) were generally regarded as the
dance in most healthy individuals and generally main beneficial components of the microbiota.
account for more than 50 % of the bacteria pres- The best-studied prebiotics, the fructose-based
ent (Tap et al. 2009; Walker et al. 2011; Flint carbohydrates inulin and fructooligosaccharides,
et al. 2012b). Most of these dominant species are showed a strong selective stimulation of bifido-
representatives of the two most abundant phyla, bacteria as assessed by culturing, thus a ‘prebi-
the Firmicutes and Bacteroidetes, but certain otic effect’ became synonymous with a
Actinobacteria (Collinsella aerofaciens and ‘bifidogenic effect’ (e.g., Cummings and
Bifidobacterium spp.) and Verrucomicrobia Macfarlane 2002). However, the application of
(Akkermansia muciniphila) also show high abun- emerging molecular tools for microbiota charac-
dance when data from alternative detection meth- terisation revealed that culture-based approaches
ods such as FISH microscopy (that avoids biases had vastly under-represented some of the more
due to PCR and DNA extraction) are considered. fastidious and most abundant bacteria, and it also
The other significant bacterial phylum is the provided a better understanding of the level of
Proteobacteria, which include a number of patho- diversity in the microbiota.
gens. In the past, detailed microbiological work According to the current definition, prebiotics
has been focussed on very few of these organ- are “selectively fermented ingredients that result
isms, with an obvious emphasis on pathogens in specific changes in the composition and/or
and on lactic acid bacteria considered to have activity of the gastrointestinal microbiota, thus
probiotic potential. In contrast, the microbial conferring benefit(s) upon host health” (Gibson
9 How to Manipulate the Microbiota: Prebiotics 121
et al. 2010). Thus, prebiotic effects are no longer edge in microbiota composition and activity it
limited to the colon but may happen anywhere in has to be questioned however whether this view
the gastrointestinal tract. can be upheld. The following questions in par-
To date, all known prebiotics are carbohy- ticular need to be addressed.
drates that may also be classed as dietary fibre, so
what distinguishes prebiotics from fibre? In order
for a compound to be classed as a prebiotic, it has 9.2.1 Are Gut Bacteria Either
to fulfil three criteria (Gibson et al. 2010): Beneficial or Detrimental
for Health?
1. It is resistant to gastric acidity and hydrolysis
by mammalian enzymes and gastrointestinal It is increasingly being recognised that it is too
absorption. simplistic to categorise bacterial species or
2. It can be fermented by intestinal microbiota. groups as either beneficial or detrimental for
3. It selectively stimulates the growth and/or human health. Thus, a bacterial strain may carry
activity of intestinal bacteria associated with both positive and negative traits and its overall
health and wellbeing. effect on the host may vary depending on the
specific gut conditions. For example,
There are several definitions of dietary fibre Faecalibacterium prausnitzii produces butyrate
that are based on different criteria, but a consen- (Duncan et al. 2002), which exerts health-
sus is emerging that carbohydrates with a degree promoting effects on the colonic wall, and also
of polymerisation (DP) of ≥3 that are not hydro- has anti-inflammatory properties that appear to
lyzed by the endogenous enzymes in the small be mediated by factors other than butyrate (Sokol
intestine constitute dietary fibre, regardless of et al. 2008). On the other hand, specific strains of
their solubility or fermentability (Slavin 2013; this species also carry high β-glucuronidase
Howlett et al. 2010). They include carbohydrates activity, which is associated with an increased
(and lignin) occurring naturally in food as con- colorectal cancer risk (McIntosh et al. 2012).
sumed, obtained from food raw material by phys- The level of generation of carcinogenic com-
ical, enzymatic, or chemical means, or synthetic pounds originating from gut microbial β-
in origin, however, the latter two categories must glucuronidase activity is, however, dependent on
be shown to have a physiological health benefit the level of exposure of the host to the respec-
(Howlett et al. 2010). Thus, all dietary fibre ful- tive precursor molecules, and the level of butyr-
fils the first prebiotic criterion of non-digestibility ate produced relative to other fermentation
in the upper gastrointestinal tract, but some do products may vary depending on the gut condi-
not fulfil one or both of the other two prebiotic tions and activities of other members of the gut
criteria. Note that, despite the fact that it is com- microbiota that interact with F. prausnitzii in tro-
monly claimed that all prebiotics are dietary phic webs. Lactobacilli and bifidobacteria are
fibres (e.g., Slavin 2013), according to the current generally regarded as carrying no traits detri-
consensus on dietary fibre there are carbohy- mental to health, however, it is conceivable that
drates generally accepted as prebiotics that an increase in lactic acid bacteria could be detri-
strictly speaking do not constitute dietary fibre, mental for certain individuals. Some patients
such as the synthetic disaccharide lactulose (DP with Ulcerative Colitis, for example, have been
<3). shown to have very high faecal levels of lactate
In the current prebiotic definition, changes in at the expense of the more health-promoting fer-
the gut microbiota are more loosely defined than mentation acids (Vernia et al. 1988), which may
before, however, the mainstream view still per- be further exacerbated by the stimulation of lac-
sists that prebiotics are directed at genus level tate producers. Accumulation of D-lactic acid in
changes and target bifidobacteria and lactobacilli short bowel syndrome can be life-threatening
(Gibson et al. 2010). Based on the current knowl- (Ewaschuk et al. 2005).
122 P. Louis et al.
9.2.2 How Selective Are Prebiotics? butyrate producers (Duncan et al. 2004). It is also
being discussed that if gas production accompa-
Prebiotics may be less selective than originally nies prebiotic intake, the carbohydrate is not act-
thought and directly stimulate non-lactic acid ing as an authentic prebiotic, as bifidobacteria
bacteria. It has for example been found that sev- and lactobacilli cannot produce gas (Gibson et al.
eral butyrate producers also have the capacity to 2010), but again, these bacteria do not exist in
degrade certain prebiotics (Scott et al. 2013), isolation and their activities may stimulate other
and for some of those a significant increase in gas-producing bacteria. Any prebiotic is there-
response to prebiotic intake has been demon- fore likely to lead to effects that are an indirect
strated in human intervention trials (Ramirez- consequence of stimulation of lactic acid bacteria
Farias et al. 2009; Louis et al. 2010; Dewulf and it appears arbitrary not to reject a prebiotic
et al. 2013). This can easily be missed if micro- that leads to positive associated effects (such as
biota analysis is restricted to a few target groups, butyrate production) but reject one that leads to
or is performed only at a broad phylogenetic negative ones (such as gas production). In fact,
scale. Thus, a change in overall abundance of gas production is commonly found to co-occur
the Lachnospiraceae within Firmicutes may not for carbohydrates generally accepted as prebiot-
be found in response to prebiotic intake, while ics and it appears unfeasible to define a prebiotic
specific sub-groups or species within this based on the intake levels that don’t lead to such
diverse family may show a response. We there- effects, especially as this differs widely between
fore require further studies that determine different individuals.
microbiota changes at a fine scale (see eg.
Chung et al. 2016). Based on the current evi-
dence it seems likely that few if any prebiotics 9.2.4 Might the Beneficial Effects
will be entirely specific to bifidobacteria or lac- of a Prebiotic
tobacilli, or any other bacterial species or genus. Be Due to Microbiota Changes
This is further complicated by the fact that dif- Other than Stimulation
ferent individuals may show different responses of Lactic Acid Bacteria?
to the same prebiotic (Figs. 9.1 and 9.2), which
is often masked by the fact that many studies If prebiotics are found to stimulate more than
only report mean data. one group of bacteria, then it becomes difficult
to assign their health benefits to a single group of
‘beneficial’ bacteria. This dilemma is highlighted
9.2.3 Should Prebiotic Effects for example by work on the effects of inulin. In
Be Limited to Specific Species the study of Ramirez-Farias et al. (2009), dietary
or Genera inulin was found to stimulate both bifidobacteria
Within the Microbiota? and F. prausnitzii in stool samples from healthy
subjects. Since F. prausnitzii is a butyrate-
Gut bacteria exist within a complex community producer with claimed anti-inflammatory action
with extensive interactions between the individ- (Sokol et al. 2008) it could easily be argued that
ual members, thus their actions cannot be seen in the increase in its population, rather than that of
isolation. Higher butyrate production has been bifidobacteria, would explain any health benefit.
reported in response to prebiotic consumption Indeed, many currently obscure, but numerically
(Gibson et al. 2010), but neither lactobacilli nor significant, anaerobes could become candidates
bifidobacteria produce this fermentation acid. for delivering health benefits once we know
They may, however, indirectly increase butyrate more about their biology and their interactions
production by feeding lactate to lactate-utilising with the host.
10 baseline control inulin

Bifidobacterium spp.
[% of total bacteria]
9
8
7
6
5
4
3
2
1
0
A B C D E F H I J K L
30
25
F. prausnitzii
20
15
10
5
0
4
3
A. hadrus
0
Subject
Fig. 9.1 Three bacterial groups showing a significant response is shown for each subject individually. Level of
mean increase in response to inulin intake in a human vol- significance across all volunteers: Bifidobacterium spp.
unteer trial, determined by quantitative PCR against the P < 0.001, F. prausnitzii P = 0.019, A. hadrus P = 0.003
16S rRNA gene as a percentage of all bacteria. The (Ramirez-Farias et al. 2009; Louis et al. 2010)
9.2.5 Does Variation in Microbiota hand, overall microbiota composition was driven
Responses more by the individual than by the diet (Walker
Between Individuals Need et al. 2011; Salonen et al. 2014). Furthermore,
to Be Considered? individuals showed variation in the response of
their microbiota to the dietary change that
There is substantial inter-individual variation in appeared to be related to the species composition
the species composition of the gut microbiota. of the microbiota at the start of the intervention
Walker et al. (2011) found significant responses to period (Walker et al. 2011). There is evidence that
controlled dietary manipulation for a subset of such variation applies equally to prebiotic inter-
‘diet-responsive’ bacterial species. On the other ventions. Ramirez-Farias et al. (2009) found that,
124 P. Louis et al.
10 B. bifidum
B. pseudo-
9 catenulatum gr.
8 B. longum
Bifidobacterium spp.
B. adolescentis
7
6
5
4
3
2
1
0
bi c bi c bi c bi c bi c bi c bc i bc i bc i bc i bci
Intervention phase and subject
Fig. 9.2 Response of four different Bifidobacterium spe- centage of all bacteria. The response is shown for each
cies to inulin intake in a human volunteer trial, determined subject individually. Intervention phase b baseline, c con-
by quantitative PCR against the 16S rRNA gene as a per- trol period, i inulin period (Ramirez-Farias et al. 2009)
despite a significant average increase in bifido- 9.2.6 Is Microbiota Diversity Itself

bacteria following inulin supplementation, certain an Indicator for Gut Health?
individuals who showed very low initial bifido-
bacterial numbers did not exhibit a bifidogenic The overall microbiota diversity may also be an
response (Fig. 9.1). Two Firmicutes groups found important factor in considering health effects of
to be significantly increased after inulin consump- prebiotics. A more diverse ecosystem is gener-
tion also did not reveal a clear trend in relation to ally regarded as more resilient due to functional
initial relative abundance (Fig. 9.1). Other studies redundancy of different members of the commu-
however have reported a greater bifidobacterial nity, and reduced diversity has been observed in
response when the initial population is low certain disease states (Lozupone et al. 2012).
(Gibson et al. 2010). Ramirez-Farias et al. (2009) Two studies determining bacterial richness by
found that Bifidobacterium adolescentis showed quantitative metagenomics recently found that
the greatest mean response, but some individuals human populations broadly fall within two cate-
showed an increase in other Bifidobacterium spe- gories of low or high faecal gene count, with vol-
cies (Fig. 9.2). If these bacterial changes have unteers in the reduced gene richness category
consequences for health (as is implied by the pre- showing higher levels of anthropometric and bio-
biotic definition) then we must expect there to be chemical phenotypes associated with disease, as
inter-individual variation in health benefits. It well as microbial metabolic pathways more asso-
should also be noted in this context that prebiotics ciated with an inflammation-associated status.
do not always elicit the same effects in patients as Intriguingly, the two states appeared to correlate
they do in healthy people (Whelan 2013). It may with abundance changes in relatively few bacte-
be that such variation would be less extreme when rial species (Cotillard et al. 2013; Le Chatelier
effects are mediated through common metabolic et al. 2013). More work is clearly necessary to
products than when they depend on interactions fully understand the implications of microbiota
of cell components with the immune system, diversity and its effect on human health, but it has
which may even be strain-specific. recently been suggested to include the stimula-
tion of ecological biodiversity in the definition of dependent on the evolution of knowledge about
prebiotics (Van den Abbeele et al. 2013). If high the nature and the possible benefit of interactions
diversity is indeed a health-promoting factor, between bacterial species that constitute the
high-level intake of a specific prebiotic may actu- intestinal microbiota and the host. Accordingly, it
ally be more detrimental to health than consum- is highly probable that this definition will change
ing fibre from a variety of sources. further in the future.
The above considerations make it clear that
the complexity of the microbiota and its interac-
tions with the host, as well as differences between 9.3 Mechanisms of Action
individuals, make it difficult to define what con- of Microbiota Modulation
stitutes a healthy gut microbiota. This has by Prebiotics (Fig. 9.3)
recently been pointed out by the European Food
Safety Authority (EFSA). Their guidance docu- The most obvious mechanism by which a prebi-
ment on scientific requirements for health claims otic can alter gut microbiota composition is by
related to gut function states that “based on cur- providing an energy source that can only be
rent scientific knowledge, it is not possible to accessed by certain members of the microbial
define the exact numbers of the different micro- community. This would be expected to lead to
bial groups which constitute a normal microbi- enhanced growth of selected microorganisms and
ota” and that the evidence available “does not to their increased representation within the com-
establish that increasing the number of any munity (Flint et al. 2007). Growth tests using cul-
groups of microorganisms, including lactobacilli tured strains show that phylogenetically distant
and/or bifidobacteria, is in itself a beneficial bacterial species often share the ability to utilize
physiological effect” (EFSA panel on dietetic a given prebiotic (Scott et al. 2013). This was
products, nutrition and allergies 2011). also demonstrated recently by a functional
Furthermore, recent developments illustrate how metagenomics approach that identified genes
much the definition of prebiotics is closely from a human microbiota metagenomic library
lactate
Bifidobacterium
adolescentis
oligo-/mono- Eubacterium
Prebiotic saccharides pH Bacteroidetes
hallii
Firmicutes
Faecalibacterium Roseburia
prausnitzii hominis
butyrate
Cross-feeding of Cross-feeding of
Direct Prebiotic breakdown fermentation Changes in gut environment
stimulation intermediates products (e.g. via SCFA production)
Fig. 9.3 Possible mechanisms of prebiotic action on the gut microbiota, illustrated with the example of butyrate gen-
eration in response to prebiotic intake. For further detail see main text
126 P. Louis et al.
for breakdown of several prebiotics in the heter- be explained by the efficient consumption of lac-
ologous host Escherichia coli (Cecchini et al. tate (Belenguer et al. 2006) with lactate accumu-
2013). Clones originating from several different lating only when the activity of the lactate-utilizers
Firmicutes, Actinobacteria and Bacteroidetes is compromised, for example by reduced pH
were able to degrade fructooligosaccharides, (Belenguer et al. 2007). Thus a stimulation of
xylooligosaccharides, galactooligosaccharides or lactate-producing species might lead to increased
lactulose. At the same time, utilization can be populations of lactate-utilizing species such as
species-specific within a given genus, and strain- Eubacterium hallii (Belenguer et al. 2007), and to
specific within a given species. This is clearly increased production of butyrate or propionate.
seen for Bifidobacterium spp. in respect of utili- Another form of cross-feeding involves the
zation of starch (Belenguer et al. 2006; Ryan release of partial breakdown products from com-
et al. 2006) and fructans (Rossi et al. 2005). plex substrates by one species, making them
Chain length is an important factor in determin- available for utilization by other species
ing selectivity, e.g., among fructans very few spe- (Belenguer et al. 2006; Falony et al. 2006). For
cies are able to utilize long chain inulin, whereas example it has been demonstrated recently that
utilisation of short chain fructooligosaccharides several other species can benefit from the ability
is widespread (Scott et al. 2013). Growth in pure of Ruminococcus bromii to degrade particulate
culture however does not equate to competitive resistant starches (Ze et al. 2012). Stimulation of
success within the complex gut community fol- specific members of the microbiota may also have
lowing addition of a prebiotic. Carefully moni- antagonistic effects on others, such as enhanced
tored human dietary trials are therefore crucial in production of bacteriocins.
defining prebiotic outcomes (Ramirez-Farias Prebiotics may also alter the gut environment.
et al. 2009). In time, genome sequence informa- Most obviously, their fermentation will tend to
tion should help to explain and predict inter- decrease the pH of the gut lumen as a result of
species and inter-strain differences in increased production of fermentation acids. In
carbohydrate utilization, but current understand- vitro work with pH-controlled, continuous flow
ing of the mechanistic basis for substrate utiliza- fermentors has shown that a one unit shift in pH
tion in most human colonic anaerobes is between 5.5 and 6.5 can greatly alter the species
surprisingly limited (Flint et al. 2012b). composition of the gut microbiota and conse-
The selective stimulation of certain microbial quently its metabolic products (Walker et al. 2005;
populations by a prebiotic means that their meta- Duncan et al. 2009). Thus another possible expla-
bolic products are likely to be produced at an nation for the butyrogenic effect of a prebiotic is
increased rate. Indeed it is theoretically possible that a decrease in gut pH promotes particular
for a prebiotic to cause increased product forma- butyrate-producing Firmicutes by decreasing
tion without an increase in the relevant bacterial competition with the more acid-sensitive
population, through simple mass action. There is Bacteroides spp. (Walker et al. 2005). In addition
therefore considerable potential for indirect stim- to pH, prebiotics might also affect the gut environ-
ulation of other organisms within the microbial ment through other effects, eg. viscosity, gut tran-
community that may be able to benefit from these sit and interactions with other food components.
metabolites. Lactate is a major product of fermen-
tation for many gut bacteria, including the lactic
acid bacteria, lactobacilli and bifidobacteria, 9.4 Ingredients with Confirmed
when grown in pure culture. Lactate can also be Prebiotic Action
used as a growth substrate by certain other bacte- and Candidate Prebiotics
rial species that are abundant in the human colon,
to produce butyrate (Duncan et al. 2004; Morrison There are several comprehensive reviews on pre-
et al. 2006) or propionate (Bourriaud et al. 2005). biotic effects of various carbohydrates, especially
The relatively low concentrations of lactate that fructans and galactooligosaccharides, which are
are detected in healthy human faecal samples may generally regarded as carbohydrates with con-
firmed prebiotic properties (for example, ota has not been established in sufficient detail.
Macfarlane et al. 2008; Gibson et al. 2010; GOS based on lactose, and containing an extra
Roberfroid et al. 2010; Whelan 2013). Here we galactose residue at C3, C4 or C6, are present in
will provide a brief overview of the structure of human milk. GOS is also produced by enzymatic
the main classes of (candidate-) prebiotics cur- transglycosylation of lactose, which results in
rently being investigated as well as their natural mixtures of mostly tri- to pentasaccharides with
occurrence in food ingredients and/or commer- galactose in β(1→6), β(1→3) and β(1→4) link-
cial synthesis, and review some recent results on age (Fig. 9.4), with the exact composition
their effects on the gut microbiota. depending on the enzymes used and reaction
conditions. These transgalactooligosaccharides
are also known as TOS (Macfarlane et al. 2008;
9.4.1 Fructans Gibson et al. 2010). Stable isotopically labeled
GOS was used recently to examine the selectivity
Inulin and fructooligosaccharide (FOS, alterna- of this prebiotic in an in vitro model of the proxi-
tively named oligofructose) consist of linear fruc- mal colon (Maathuis et al. 2012). Several bifido-
tose chains in β(2→1) linkage, usually with a bacteria and lactobacilli showed the highest label
terminal glucose unit in β(2↔1) linkage as in incorporation, with infant-type bifidobacteria
sucrose (Fig. 9.4). Inulin is normally present as a seemingly stimulated most strongly. Other bacte-
variety of chain lengths with a DP of up to 60, ria, including members of the Enterobacteria,
whereas FOS has a DP of less than 10. FOS can Bacteroidetes and Firmicutes also incorporated
be manufactured from inulin by partial hydroly- the label to a lesser degree.
sis or synthesized enzymatically (Gibson et al. Lactulose is a synthetic disaccharide produced
2010). Inulin-type fructans are present in rela- by the isomerisation of lactose (Fig. 9.4). With a
tively high quantities in various vegetables (e.g., DP of two it does not fulfil the criteria as a dietary
chicory root, Jerusalem artichoke), but they are fibre, but it is generally regarded as a prebiotic
also found in smaller amounts in cereals such as (Gibson et al. 2010). Lactulose-derived GOS is
wheat. Due to high intake levels of cereal prod- more resistant to upper gut digestion than lactose-
ucts (e.g., bread), they are the major contributor derived GOS. A comparison of both types of
to fructan intake in Western societies (Whelan GOS in a rat model revealed a significant stimu-
2013). While originally thought to specifically lation of the Firmicutes Eubacterium rectale/
stimulate lactic acid bacteria, there is now evi- Clostridium coccoides group in caecum and
dence from several independent studies that other colon, whereas bifidobacterial stimulation
bacterial species may also be stimulated either reached significance only for lactulose-derived
directly or indirectly, as detailed in the sections GOS in both intestinal compartments, with lacto-
above. Chain length also appears critical for bacilli being significantly increased on lactose-
determining which bacteria can act as primary derived GOS in the colon only (Marín-Manzano
degraders of fructans (Scott et al. 2013). et al. 2013). The differences seen between the
two types of GOS may be due to their difference
in indigestibility and/or structural differences
9.4.2 Galactooligosaccharides (such as types of glycosidic linkages).
Pulses are rich in natural galactooligosaccharides

(GOS), including raffinose family oligosaccha- 9.4.3 Resistant Starch, Starch-
rides (RFO), which are based on the extension of and Glucose-Derived
sucrose with galactose residues (Fig. 9.4) Oligosaccharides
(Johnson et al. 2013; Whelan 2013). Raffinose
and stachyose are also known as soybean oligo- Resistant starch (RS) is the fraction of starch
saccharides, but their impact on the gut microbi- (Fig. 9.4) that resists digestion in the upper gut
128 P. Louis et al.
Inulin-type fructans Galacto-OS: Raffinose family oligosaccharides

β(2→1) linked fructose units α-(1→6)-galactosyl derivatives of sucrose
usually with a terminal glucose in (α-D-glucopyranosyl-(1→2)-β-D-fructofuranoside)
α(1→2) linkage
CH2
OH O
CH2OH OH
O
OH O
OH
sucrose
OH O CH2
HOCH OH OH
2 O O
HO OH
OH CH2 OHO
HOCH CH
O O
verbascose
2 2
HO OH O
OH CH2 OH
stachyose
HOCH O n O
2 O OH
HO CH2
raffinose
CH2OH O
OH OH
sucrose
OH O
OH
HOCH2 O
HO
Galacto-OS: transgalacto-oligosaccharides CH2OH
Synthetic products from transglycosylation of lactose OH
(β-D-galactopyranosyl-(1→4)-D-glucose)
β-(1→6)-, β-(1→3)-, β-(1→4)-linkages, mostly tri-to pentasaccharides
CH2OH
O
OH OH
CH2OH CH2OH CH2OH
OH O OH O O O O
O OH OH
OH
OHCH2 O
OH O OH OH
OH
lactose
OH
Lactulose HOCH2 O OH
Synthetic disaccharide from isomerisation of HO
lactose (glucose → fructose) CH2OH CH2OH
Lactulose-based GOS produced as from lactose
OH O O
OH
OH
Starch CH2OH
O
α(1→4)- and α(1→6)-linked D-glucose OH
O O
Precursor for isomaltooligosaccharides
OH
(mainly contain α(1→6)-linkages) CH2 CH2OH CH2OH
O O O
OH OH OH
O O O O
OH OH OH
Fig. 9.4 Carbohydrate structures of fructans, galactooligosaccharides and starch

due to its physicochemical properties. The 9.4.4 Other Oligosaccharides

amount of RS varies between different foods
(such as legumes, cereals and potatoes), and food Pectic oligosaccharides (POS) are derived from
processing can also affect RS levels. While not pectin, a polysaccharide present in various fruits
currently regarded as a prebiotic universally (e.g., and vegetables (e.g., citrus fruit, apple, sugar
Gibson et al. 2010), its classification as a prebi- beet). The polysaccharide backbone consists of
otic has been proposed, particularly as RS intake galacturonic acid residues (homogalacturonan)
is associated with health-promoting effects, such that may alternate with rhamnose residues (rham-
as high butyrate formation (Fuentes-Zaragoza nogalacturonan I). The carboxyl groups may be
et al. 2011). Selective stimulation of several bac- modified by methyl esterification, and C2 or C3
terial groups belonging to the Firmicutes in positions may be acetylated. Sidechains contain-
response to a diet high in RS has recently been ing various sugars (arabinose, galactose, xylose
demonstrated (Walker et al. 2011). Pure culture etc.) are often present and may be substituted
work in vitro confirmed the starch-degrading with ferulic acid (Yoo et al. 2012). In vitro and
ability of R. bromii and B. adolescentis, and to a in vivo studies to establish a prebiotic effect lead
lesser degree of E. rectale and Bacteroides the- to mixed results, which may be complicated by
taiotaomicron. However, in mixed bacterial and the fact that POS from different sources show
faecal incubations, the presence of R. bromii was structural variation (Gullón et al. 2013). Known
crucial for efficient RS breakdown (Ze et al. pectin degraders include several Bacteroides spp.
2012). This was in agreement with the observa- as well as the Firmicutes Eubacterium eligens
tion that human volunteers with very low levels and F. prausnitzii (Lopez-Siles et al. 2012).
of R. bromii were not able to digest dietary resis- The production and characterisation of novel
tant starch completely (Walker et al. 2011). oligosaccharides from non-digestible
Isomaltooligosaccharides (IMO) are found carbohydrate fractions of biomass, such as xylo-,
naturally in some fermented foods and honey, arabino- and mannooligosaccharides is an active
and they are commercially produced from starch research area and complex foodstuffs are also
by enzymatic hydrolysis, resulting in glucans being investigated as sources of novel prebiotics
containing mainly α(1→6) linkages, although (e.g., Otieno and Ahring 2012; Yoo et al. 2012).
other linkages are also present, and other types of The increasing use of genomic and metagenomic
glucooligosaccharies (GOS) are also considered data should enable a more rational design of pre-
as IMOs (Goffin et al. 2011). IMOs are widely biotic compounds in the future (Candela et al.
used in the Asian market. They are partially 2010).
digestible, with digestibility depending on DP
and type of linkages present (Goffin et al. 2011).
There is some indication that IMOs selectively 9.4.5 Non-carbohydrate
stimulate lactic acid bacteria (Gibson et al. 2010; Compounds
Goffin et al. 2011), however, studies investigat-
ing changes in the whole microbiota are required The definition of prebiotics is not restricted to
to fully evaluate their effect on the microbiota. carbohydrates per se (Gibson et al. 2010), how-
Polydextrose, a highly branched glucan con- ever, carbohydrates appear most likely to fulfil
taining a range of glycosidic linkages, is synthe- the criteria of non-digestibility and selective fer-
sized from glucose. There is some evidence that mentability. Cocoa-derived flavanols have also
it is selectively fermented by bifidobacteria, how- been proposed as prebiotics, as they elicited a
ever, a human intervention study that looked at a stimulatory effect on lactic acid bacteria in vivo
wider range of bacterial groups using molecular and in vitro (Tzounis et al. 2011). It is unlikely,
methods did not confirm this (Costabile et al. however, that this effect is due to fermentation of
2012) and further work is required to establish its those compounds, in the sense that the corre-
effects. sponding bacteria achieve a substantial energy
130 P. Louis et al.
gain, for the following reasons: intake levels are increase in the total bacterial mass, stimulation/
generally much lower than for carbohydrate- inhibition of some particular bacterial strains,
based prebiotics and a proportion of the pheno- production of numerous bacterial metabolites,
lics will be absorbed by the host either directly or among which organic acids predominate and – as
after biotransformation by the microbiota. a consequence of the latter – acidification of the
Furthermore, the underlying biochemistry of the luminal contents. All these features can exert bio-
bacterial metabolic transformations (cleavage of logical effects (Macfarlane and Macfarlane 2012;
complex to more simple phenolics, hydrogena- Russell et al. 2013), which, according to their
tion, demethylation, dehydroxylation, etc., nature and combination, can result in different
Russell and Duthie 2011) is unlikely to result in a physiological effects. However, for most of the
major energy gain for the microbes performing physiological effects induced by prebiotics, the
the transformation. Phenolic compounds may, exact contribution of each of these events and the
however, exert antimicrobial effects, the potency precise nature of bacterial factors and of mecha-
of which may differ between bacterial species nistic pathways involved are still to be
(Louis and O’Byrne 2010), which can lead to deciphered.
selective stimulation of certain bacterial groups. Finally, whether these physiological effects
The microbial transformation of phenolics may translate into actual improvements in terms of
indeed function as a detoxification mechanism prevention and/or curing of diseases related to
for the bacteria. This raises the question whether the consumption of prebiotics is still a matter of
the second prebiotic criterion, fermentability by debate. When assessing health effects of prebiot-
the intestinal microbiota, should be retained, or ics it also has to be kept in mind that a correlation
whether it ultimately does not matter by which between gut microbial changes and health
mechanism selective changes within the micro- markers may be by mere association rather than
biota are elicited. by a causal relationship. Some prebiotics may
exert their health-promoting effects independent
of a promotion of beneficial bacteria, for example
9.5 Health Effects of Prebiotics by direct stimulation of the immune system via
on the Host host receptors, or by pathogen binding, which
reduces pathogen adherence (Licht et al. 2012).
By definition, prebiotics are supposed to exert
beneficial impacts on host health and/or well-
being. This criterion has motivated many studies 9.5.1 Physiological Effects
on the ability of prebiotics to induce physiologi- and Underlying Mechanisms
cal effects, many of which are now proven, at
least in the case of inulin and/or FOS, on which 9.5.1.1 Improvement of Intestinal
the majority of these studies had focused (see Functions (Stool Bulking, Stool
summary in Table 9.1 and, for extensive review: Regularity, Stool Consistency)
British Journal of Nutrition Vol. 93, Suppl. 1 In babies and possibly (but with contradictory
2005; Journal of Nutrition Vol. 137 Suppl. 2007; results in this case) in infants, supplementation
Roberfroid et al. 2010). The transferability of with a GOS/inulin mix increases stool frequency,
these findings to other candidate prebiotics is soften stools, acidifies stool pH, and modulates
being evaluated. the SCFA pattern similar to that of breast-fed
The definition of prebiotics also assumes that infants (Gibson et al. 2010; Roberfroid et al.
the health benefits result from their impact on the 2010; Tabbers et al. 2011). This was also observed
composition and/or activity of the gastrointesti- with polydextrose, which is a prebiotic candidate
nal microbiota, i.e., their fermentation. Prebiotic (Ashley et al. 2012). However, in adults inulin
fermentation in the large bowel is expected to has little effect on stool weight (Slavin 2013).
result in changes in the whole ecosystem, namely FOS can cause symptoms, including bloating,
9
How to Manipulate the Microbiota: Prebiotics
Table 9.1 Hypothetical mechanisms underlying the biological and physiological impacts of fructans
Suggested mechanism
Biological impact Expected impact on Linked to modification of the Linked to stimulation of SCFA
(evidence level) Physiological impact health microbiota composition production Other
Increased calcium Increased bone density Osteoporosis Stimulation of colonocyte Trophicity Increased calcium
absorption (+, particularly in teenagers) prevention Ca2+ capture by some bacteria Increased mucosal expression solubility due to
(+++) of calbindin fermentation-induced
luminal acidification
Increased production Appetite regulation Obesity treatment Stimulation of the mucosal
of satietogenic gut (+, few studies) expression of proglucagon
peptides (GLP-1) (→GLP-1)
(++)
Decreased plasma Lipidemia regulation Cardiovascular disease Chelation and/or bile salt Through impact on GLP-1? Decreased reuptake of
triglycerides and (+ in hyperlipidemic humans; prevention hydrolysis by some bacteria Suppression of hepatic bile salts due to
cholesterol +/− in healthy subjects) lipogenesis by propionate acidification of
(+ in hyperlipidemic colonic contents
humans; +/− in healthy
subjects)
Trophicity of the Intestinal barrier homeostasis Inflammatory bowel Stimulation of mucus Main energy source for
colonic mucosa diseases (+ colorectal production by some bacteria colonocytes
Increased mucus cancer, obesity and Stimulation of proliferation
production diabetes?) treatment and cell differentiation
Decreased intestinal Regulation of tight-junctions
permeability
Stimulation of the mucosal
(+)
expression of proglucagon
(→GLP-2)
Increased IgA and Immunomodulation Miscellaneous: Immunomodulatory Immunomodulatory Direct interaction
IL-10 production, Treatment of properties of some bacteria properties of butyrate between fructans and
phagocytosis and infections Allergy immune cells
NK activity prevention, IBS/IBD
Decreased prevention and
inflammatory treatment colorectal
response cancer prevention
(++)
131
132 P. Louis et al.
flatulence, and soft stools in adults when fed at have been obtained in adults. In postmenopausal
high doses (>10 g/day) (Brownawell et al. 2012; women it appears to depend on the number of
Slavin 2013). years past the onset of menopause, with no effect
From knowledge accumulated on dietary in early postmenopausal women, but calcium
fibre, improvement of stool bulking is the result absorption improvement in women who are in
of increased stool weight due to either the physi- the late postmenopausal phase (Roberfroid et al.
cal presence of the fibre and to the water held by 2010). The beneficial impact of fructans on cal-
the fibre, or an increased bacterial mass, with the cium absorption is evidenced only if the calcium
former phenomenon being much more effective intake is sufficient (Scholz-Ahrens and
than the latter one (Brownawell et al. 2012; Schrezenmeir 2002) and appears to depend on
Slavin 2013). In this context, one has to assume the fractional calcium absorption at baseline,
that the different stool bulking effects between with those individuals with lower absorption
infants and adults result from differences in fer- before treatment showing the greatest benefit
mentation intensity (related to microbiota imma- (Griffin et al. 2002). Finally, the prebiotic dosage
turity in infants): the stool bulking capability of also influences the benefit and according to
prebiotics in infants thus appear to stem from the Roberfroid et al. (2010), a minimum level of 8 g/
fact that they may increase the stool water con- day seems to be required to elicit an improve-
tent by osmolarity, because they are likely to be ment on both calcium absorption and bone
incompletely fermented. In agreement with this mineralisation.
assumption, residual unfermented prebiotics Whether or not such effects are expected for
have been detected in infants stools (Moro et al. all prebiotics is not clear. On the one hand, GOS
2005), whereas prebiotics are totally degraded in appears promising for improving calcium absorp-
adults. tion in both animal and human studies (van den
Heuvel et al. 2000; Weaver et al. 2011) and vari-
9.5.1.2 Stimulation of Mineral ous (candidate-) prebiotics such as soyaoligosac-
Absorption and Improvement charides, lactulose, or resistant starch have also
of Bone Density provided evidence of a positive effect on calcium
Numerous studies carried out in animals have absorption, at least in the rat (Roberfroid et al.
demonstrated that linear fructans, particularly a 2010). On the other hand, fructans with different
mixture of inulin and FOS, improve mineral (and degrees of polymerisation (average DP = 3–4 vs
especially calcium) absorption. Some have also average DP>23 vs mix of the two), have different
shown that this results in increases in whole body effects on calcium retention, femoral bone den-
bone mineral content and in bone density mass sity and bone calcium content in rats (Griffin
(Scholz-Ahrens et al. 2007; Roberfroid et al. et al. 2002; Kruger et al. 2003).
2010). Similarly, generalization of these benefits to
In humans, these conclusions must be quali- other minerals is not possible, even if the impact
fied according to the age of the subjects, their of prebiotic consumption on the metabolism of
hormonal status, their calcium intake and of phosphorous, magnesium, iron, copper, and zinc
course the prebiotic dosing. Indeed, the few stud- has occasionally been considered. A beneficial
ies investigating the impact of baby formulae impact on magnesium absorption seems likely
supplemented with prebiotics (mixture of GOS/ (Yap et al. 2005; Scholz-Ahrens and Schrezenmeir
inulin, 9/1) on calcium absorption or other mark- 2002; Roberfroid et al. 2010; Legette et al. 2012),
ers of bone mineral metabolism did not reveal even if available data are very limited. Iron as
any change (Yap et al. 2005; Hicks et al. 2012). well as zinc absorption may also be improved,
By contrast, in teenagers, a mix of inulin and while retention of phosphorous appears not to be
FOS not only enhanced calcium absorption, but affected (Scholz-Ahrens and Schrezenmeir
also calcium accretion in bones (Abrams et al. 2002).
2005; Roberfroid et al. 2010). Contrasting results
The exact mechanisms involved in the related to an increase in satietogenic and/or a

improvement of calcium absorption are not fully decrease in orexigenic (ghrelin) peptides
deciphered. They may involve acidification of the (Delzenne al. 2013).
lumen content which increases calcium solubility The capability of other prebiotics to reduce
(Scholz-Ahrens and Schrezenmeir 2002). Short- energy intake and modulate gut peptides is poorly
chain fatty acids (SCFA) also have a trophic described, however, a recent study suggests that
effect on the mucosa (Hamer et al. 2008), which this property is shared by GOS and by oligosac-
may lead to enlargement of the absorption sur- charides derived from arabinoxylan (AXOS):
face. Enhanced butyrate or propionate production rats consuming GOS presented reduced energy
also stimulates calbindin-D9k expression by intake and increased gene expression of PYY and
colonocytes (Fukushima et al. 2012), a feature proglucagon, the precursor for GLP-1 (Overduin
which is observed in rats consuming FOS (Ohta et al. 2013) and similar results were obtained in
et al. 1998). Other hypotheses are related to stim- mice fed AXOS (Neyrinck et al. 2012).
ulation of particular bacterial species which may The underlying mechanism for these effects is
improve either calcium bioavailability (Bergillos- supposed to be a stimulation of L-endocrine cells
Meca et al. 2013) or calcium absorption by colo- in the intestine, either through triggering differ-
nocytes (Gilman and Cashman 2006), or which entiation of these cells or through stimulated
may stimulate the colonic production of equol, a expression of gut peptides by these cells
phytooestrogen which is associated with bone (Delzenne et al. 2013). SCFA and particularly
health (Coxam 2007). butyrate possibly mediate such a stimulation,
since this bacterial metabolite seems most
9.5.1.3 Regulation of Appetite effective in stimulating GLP-1 production in vitro
and Stimulation of Gut Peptide (Zhou et al. 2008). The free fatty acid receptors
Secretion FFAR2 (GPR43) and FFAR3 (GPR41), which
Numerous studies have shown that fructan con- recognize SCFA, may be involved in the stimula-
sumption decreases energy intake in rodents tion of GLP-1 secretion (Tolhurst et al. 2012).
(Roberfroid et al. 2010; Delzenne et al. 2013), Finally, a new finding suggests that FOS con-
although this was only seen in male animals but sumption results in changes in the neuronal acti-
not females at a specific time point in a long-term vation of the arcuate nucleus, an hypothalamic
study investigating the effect of lifelong interven- structure which contributes to the control of food
tion (Rozan et al. 2008). Fructan intake is usually intake (Anastasovska et al. 2012) but this obser-
accompanied by an increase in glucacon-like vation could result from gut peptide stimulation,
peptide 1 (GLP-1) and, although less docu- since functional receptors for GLP-1 are also
mented, by a increase in peptide YY (PYY), two expressed by hypothalamic neurons (Dalvi et al.
anorexigenic peptides secreted by the intestine 2012). It should be noted that different types of
(Roberfroid et al. 2010; Delzenne et al. 2013). dietary fibre not currently classed as prebiotics
The crucial role of GLP-1 has been demonstrated also regulate appetite, which may partially be due
using GLP-1 receptor knockout mice (GLP- to physicochemical effects in the gastrointestinal
1R(−/−)) in which the FOS-induced decrease of tract (Slavin and Green 2007).
energy intake was abolished (Cani et al.
2006a). 9.5.1.4 Improvement of Intestinal
In humans, pre-adaptation to FOS induced Barrier Integrity
changes in satiety and reduced total energy intake In adult animals, fructan fermentation has been
per day (Cani et al. 2006b; Parnell and Reimer shown to affect the mucus layer and the intestinal
2009) but this does not appear to hold up for mucosal morphometry by increasing the height
acute fructan supplementation (Peters et al. 2009; of villi and the depth of the crypts (Kleessen and
Hess et al. 2011). Anyhow, when occurring, Blaut 2005). FOS supplementation decreases
decreased energy intake in humans was also intestinal permeability and improves tight-
134 P. Louis et al.
junction integrity in mice (Cani et al. 2009) and 9.5.1.5 Regulation of Lipid and Glucose
this improvement in intestinal permeability has Metabolism
been confirmed for humans (Russo et al. 2012). Fructans improve glucose homaeostasis in sev-
These effects appear to be specifically induced by eral rodents (see for review Roberfroid et al.
fructans since the few studies dedicated to other 2010). With regard to lipid metabolism, fructans
prebiotics (GOS or mix of different OS) failed to have been shown to decrease total plasma choles-
demonstrate any particular effect on the intestinal terol in mice or rats and to decrease triglyceride
barrier (Meslin et al. 1993; Barrat et al. 2008; plasma concentration (and, although not system-
Westerbeek et al. 2011). atically, triglyceride hepatic concentration) in
Low-grade systemic inflammation (also called rats or hamsters (Roberfroid et al. 2010).
metabolic endotoxemia) with elevated serum lev- Improvement of glycemia has also been
els of bacterial lipopolysaccharides (LPS) is observed in healthy adults, but not in diabetic
often observed in obese patients, especially when patients (Cani et al. 2009; Roberfroid et al. 2010).
they also present with metabolic disorders. An Fructans decrease the plasma concentration of
improvement of the intestinal barrier is likely to low density lipoprotein (LDL) cholesterol and
contribute to the reduction in metabolic endotox- the LDL to high density lipoprotein (HDL) ratio
emia that has been reported in response to prebi- in hyperlipidemic adults, but do not seem to
otic intake in animal models (Delzenne et al. induce any particular change in normolipidemic
2013). individuals (Roberfroid et al. 2010; de Luis et al.
Numerous biological mechanisms may 2011; Brownawell et al. 2012). Fructans also
participate in the beneficial impact of fructans decrease the hepatic capacity of triglyceride
on intestinal barrier. First, specific fructan- synthesis in human volunteers (Roberfroid et al.
stimulated bacteria may be involved since 2010).
numerous bacterial strains used as probiotics Despite some discrepancy (Boucher et al.
have been shown in vitro to increase mucin 2003), GOS and a GOS and inulin mix also
expression and to enhance tight junction stabil- induce decreases in cholesterol in human micro-
ity, which decreases epithelial permeability biota associated rats (Djouzi and Andrieux 1997),
(Ohland and Macnaughton 2010). Second, in total cholesterol and LDL levels in infants
butyrate may also contribute since it improves (Alliet et al. 2007) or in total cholesterol (TC),
mucosal trophicity and it can also regulate triglycerides, and the TC to HDL cholesterol
mucin production and cellular permeability ratio in obese adults (Vulevic et al. 2013).
in vitro and ex vivo (Hamer et al. 2008). Third, Again numerous mechanisms are supposedly
glucagon-like peptide 2 (GLP-2), which is co- involved in these physiological effects: improve-
secreted with GLP-1 by endocrine L cells, ment of glycemia could stem from stimulation of
appears to be a key factor in the regulation of the GLP-1 production (Delzenne 2003). A decrease
intestinal barrier, since the effects of FOS on in de novo hepatic lipogenesis could be the result
intestinal barrier were reproduced by pharmaco- of the enhancement of SCFA, particularly propi-
logical GLP-2 treatment and abolished in the onate, which is reported to inhibit fatty acid syn-
presence of a GLP-2 antagonist (Cani et al. thesis in vitro (Delzenne and Kok 2001), but also
2009, 2012). Furthermore, prebiotics may also acetate, which also exerts an inhibitory effect on
exert an influence on the endocannaboid system lipogenesis, despite the fact that is a precursor for
with consequent effects on gut barrier function lipogenesis (Roberfroid et al. 2010).
(Delzenne et al. 2013). Again these mechanisms
can be interconnected, considering that the 9.5.1.6 Modulation of Immune
stimulatory effect of butyrate on GLP-1 secre- Functions
tion mentioned above is likely to occur for GLP- The immuno-modulatory potential of prebiotics
2 as well, since both derive from the same has motivated a few studies. As a reflection of the
proglucagon gene. complexity of the immune response (innate vs
adaptive, mucosal vs systemic, pro-inflammatory IL-10 production by peripheral blood mononu-

vs anti-inflammatory), numerous markers have clear cells (PBMC) together with a decrease in
been considered (vaccine-specific serum anti- ex vivo IL-6, tumor necrosis factor alpha (TNFα)
body production, delayed-type hypersensitivity and IL-1β production by PBMC (Vulevic et al.
response, vaccine-specific or total secretory 2008).
immunoglobulin A (sIgA) in saliva, the response Prebiotic effects may impact the immune
to attenuated pathogens, NK cell activity, phago- response indirectly as a result of intestinal fer-
cytosis, T-cell proliferation, cytokine concentra- mentation and promotion of growth of certain
tions) (Roberfroid et al. 2010). members of the gut microbiota (Macfarlane et al.
Several studies have demonstrated an 2008; Roberfroid et al. 2010). SCFA have direct
increased intestinal sIgA response, an increase in immunomodulatory properties. G-protein-
B cell numbers in Peyer’s patches, and an coupled receptors (GPR41 and GPR43), which
enhanced intestinal interleukin 10 (IL-10) protein have been identified as receptors for SCFA, are
secretion in intestinal tissues, as well as a expressed on leukocytes, as well as on entero-
decreased mRNA expression and protein concen- cytes and enteroendocrine cells in the human
tration of pro-inflammatory cytokines resulting colon (Roberfroid et al. 2010). Interestingly,
from the use of FOS in animal models (Macfarlane GPR43-deficient (Gpr43(−/−)) mice showed
et al. 2008; Roberfroid et al. 2010). Furthermore, exacerbated inflammatory responses to various
functional activities of Natural Killer (NK) cells pathological situations (Maslowski et al. 2009).
and phagocytes isolated from various immune Butyrate modulates chemokine expression in
tissues were significantly increased, but depend- intestinal epithelial cells, differentially affects
ing on the source of immune cells (Peyer’s pro-inflammatory IL-2, IFN-γ and immuno-
patches, mesenteric lymph nodes, intraepithelial regulatory IL-10 production by rat lymphocytes
lymphocytes) the prebiotic effects may differ in vitro, through regulation of the transcription
(Roberfroid et al. 2010). factor NF-κB (Macfarlane et al. 2008; Roberfroid
In humans, where infants and elderly were et al. 2010). Acetate increases peripheral blood
mainly investigated, fructans on their own seem antibody production and NK activity when
poorly effective in stimulating a response to vac- administrated intravenously (Macfarlane et al.
cination, while a mix of GOS and inulin (or even 2008). Lastly, SCFA could improve the immune
with a stronger response, a mix of GOS, inulin response through an indirect effect: because they
and AOS (acidic oligosaccharides derived from are used by epithelial cells as energy substrates,
pectin)) appears effective in enhancing T helper their increased production allows for the sparing
cells type 1 (Th1) responsiveness (Jeurink et al. of glutamine. As glutamine is used by immune
2013). A GOS and inulin mix also enhanced fae- cells in the body, this thereby enhances immune
cal sIgA concentrations in infants (Roberfroid system reactivity (Jenkins et al. 1999).
et al. 2010). Both commensal and exogenous bacteria
A similar impact of prebiotics seems to occur interact with both the innate and adaptive immune
in older people: FOS induced a decrease in system through pattern-recognition receptors,
phagocytosis and IL-6 mRNA expression in such as the toll-like receptors (TLR), in a strain-
peripheral blood mononuclear cells in healthy dependent manner. Thus, the presence of
elderly people (Guigoz et al. 2002). However, an increased numbers of a particular microbial
inulin and FOS mix had no impact on sIgA, genus or species, or a related decrease of other
serum titers after vaccination (influenza A and B microbes, may change the collective immuno-
and Pneumococcus), secretion of IL-4 and inter- interactive profile of the microbiota, thus result-
feron gamma (IFN-γ) or on lymphocyte prolifer- ing in a variety of downstream events. This may
ation (Bunout et al. 2002). GOS consumption, on eventually lead to cytokine production steering
the other hand, resulted in increases in ex vivo towards an appropriate immune response for the
NK cell activity, ex vivo phagocytosis and ex vivo microbial event (Roberfroid et al. 2010). For
136 P. Louis et al.
example, some lactobacilli can enhance both the et al. 2013), irritable bowel syndrome (Whelan
innate and adaptive immune defences, resulting 2011) and for the prevention of eczema in infants
in increased production of phagocytes and (Osborn and Sinn 2013). For most other cases
immune effector molecules such as sIgA, and (gut inflammatory diseases, colorectal cancer,
some bifidobacteria are able to induce formation metabolic diseases, osteoporosis, other allergies)
of large amounts of IgA in Peyer’s patches of there are promising results from preclinical stud-
mice (Macfarlane et al. 2008). As a possible ies (i.e., in animal models of disease) or from
illustration of such potential, a positive correla- small pilot studies, but appropriate human inter-
tion between numbers of bifidobacteria in faecal vention studies are too scarce to conclude on the
samples and both NK cell activity and phagocy- real impact of prebiotics (Roberfroid et al. 2010;
tosis was observed in the study of Vulevic et al. Brownawell et al. 2012). For example, although
(2008) mentioned above. However, lactobacilli prebiotics such as FOS, inulin and GOS appear to
and bifidobacteria are far from being the sole favourably alter biomarkers of cardiovascular
bacteria showing this potential, as illustrated by diseases, including low-density lipoprotein cho-
recent studies which have highlighted, for exam- lesterol (LDL-C) (see above) and C-reactive pro-
ple, the immunomodulatory effects of other bac- tein (CRP) (De Luis et al. 2011; Dehghan et al.
teria such segmented filamentous bacteria 2014; Vulevic et al. 2013), whether they actually
(Gaboriau-Routhiau et al. 2009) or F. prausnitzii decrease the risk of CVD is unclear as yet
(Sokol et al. 2008). (Brownawell et al. 2012; Slavin 2013).
Finally, it has been postulated that prebiotics Similarly, prebiotic intervention decreases
could interact directly with epithelial cells by numerous hallmarks of obesity and diabetes such
specific lectin-like receptors (Seifert and Watzl as food intake, fat storage in adipose tissue and in
2007). This remains only theoretical today since the liver (steatosis), glycemia and hepatic insulin
the existence of specific receptors for FOS has resistance, endotoxemia and systemic inflamma-
not been demonstrated to date, although some tion, in several models of obese rodents (Delzenne
indirect evidence obtained in vitro argues in its and Cani 2011; Everard and Cani 2013).
favour, such as the inhibition of phagocytosis of However, from the limited number of interven-
bacteria by granulocytes due to the addition of tion studies which have investigated the role of
fructose (Speert et al. 1984) or the activation of fructans in humans, the actual weight loss (a few
NK activity of peripheral blood mononuclear kg) in obese individuals supplemented with fruc-
cells due to the addition of a plant extract con- tans remains modest (Delzenne et al. 2013) or not
taining FOS (Thakur et al. 2012). It has been statistically significant (Dewulf et al. 2013).
noticed that if such a mechanism is proven true, Furthermore, no long-term benefit on diabetes
this will challenge the prebiotic definition, which has been demonstrated yet (Everard and Cani
supposes a causal relation between the physio- 2013) and preliminary results from acute impact
logical effect of the prebiotic and its impact on are contradictory (Dehghan et al. 2014; Dewulf
the composition and/or activity of the gastroin- et al. 2013).
testinal microbiota. Also with respect to osteoporosis, it is too
early to establish whether the beneficial impact
of fructans on calcium metabolism actually trans-
9.5.2 Current Evidence for Disease lates into reduction of the risk of bone disease,
Prevention or Treatment even if bone-sparing effects have been found in
ovariectomized rats (an animal model for osteo-
Whether the physiological effects outlined above porosis) (Coxam 2007). Such a risk reduction
actually translate into reduction of the risk and/or would require proof of the persistance of the ben-
treatment possibilities of diseases is not estab- efits of prebiotics on calcium absorption
lished yet. Prebiotics appear effective for the (Roberfroid et al. 2010). In this respect, the fact
treatment of infectious diarrhea (Vandenplas that improvement of calcium metabolism was
still detectable after 1 year (Abrams et al. 2005) unique profiles showed major changes (Louis
and that inulin-based fibres exert chronic effects et al. 2010). While this will have to be confirmed
on calcium utilization in a postmenopausal rodent by larger studies, it indicates that some individu-
model (Legette et al. 2012) appear promising, als may benefit more from prebiotic intake than
and prebiotic-induced improvement of calcium others. Some evidence also exists that prebiotic
metabolism may have important implications for intake may actually be harmful for specific con-
future preventative strategies for osteoporosis. ditions, such as certain gut infections (Licht et al.
The diseases covered here are not an exhaus- 2012). When considering overall microbiota
tive list, as it becomes ever clearer that the actions diversity, a diet rich in a variety of different fibres
of the microbiota have far-reaching conse- may actually be more beneficial for some indi-
quences. For example, there appears to be a link viduals or disease states. As different fibres are
between the microbiota and the nervous system, likely to exert different health effects, this may
and psychiatric diseases may become a target for lead to a better overall improvement in health and
therapy via microbiota modulation (for a recent disease prevention (Slavin 2013; Raninen et al.
review see Collins et al. 2012). 2011). Nevertheless, in view of the recent evi-
dence of how far beyond the intestine microbial
activties are exerting their effects, targeting
9.6 Conclusions specific changes in the microbiota looks like an
increasingly promising concept to improve
We have seen a vast expansion of our knowledge human health.
of the microbial community resident in the intes-
tine in recent years, and it is becoming clear that
the prebiotic concept as it currently stands may References
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Walker AW, Duncan SH, McWilliam Leitch EC, Child J 6:1535–1543
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cally alter bacterial populations and short-chain fatty KL, Shen L, Danna SC, Tripathy S, Hegsted M,
acid ratios within microbial communities from the Keenan MJ (2008) Dietary resistant starch upregulates
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Ze X, Brown D, Stares MD, Scott P, Bergerat A, Louis Endocrinol Metab 295:E1160–E1166
How to Manipulate the Microbiota:
Fecal Microbiota Transplantation 10
Susana Fuentes and Willem M. de Vos
Abstract
Fecal microbiota transplantation (FMT) is a rather straightforward therapy
that manipulates the human gastrointestinal (GI) microbiota, by which a
healthy donor microbiota is transferred into an existing but disturbed
microbial ecosystem. This is a natural process that occurs already at birth;
infants are rapidly colonized by a specific microbial community, the com-
position of which strongly depends on the mode of delivery and which
therefore most likely originates from the mother (Palmer et al. 2007;
Tannock et al. 1990). Since this early life microbial community already
contains most, if not all, of the predominantly anaerobic microbes that are
only found in the GI tract, it is reasonable to assume that early life coloni-
zation is the ultimate natural fecal transplantation.
Keywords
Gastrointestinal microbiota • Microbial ecology • Donor • Clostridium dif-
ficile • IBD • Regulation • Safety
10.1 Microbiota Transplantation: a natural process that occurs already at birth;

Concept and History infants are rapidly colonized by a specific micro-
bial community, the composition of which strongly
Fecal microbiota transplantation (FMT) is a rather depends on the mode of delivery and which there-
straightforward therapy that manipulates the fore most likely originates from the mother
human gastrointestinal (GI) microbiota, by which (Palmer et al. 2007; Tannock et al. 1990). Since
a healthy donor microbiota is transferred into an this early life microbial community already con-
existing but disturbed microbial ecosystem. This is tains most, if not all, of the predominantly anaero-
bic microbes that are only found in the GI tract, it
is reasonable to assume that early life colonization
S. Fuentes (*) • W.M. de Vos is the ultimate natural fecal transplantation.
Laboratory of Microbiology, Wageningen University, The work of Eiseman, documented over 50
Dreijenplein 10, 6703 Wageningen, The Netherlands
e-mail: susana.fuentes@wur.nl; willem.devos@wur.nl years ago, is considered the first study of FMT

144 S. Fuentes and W.M. de Vos
Fig. 10.1 Applications of FMT. GI gastrointestinal, IBD difficile infection, CDAD C. difficile associated diarrhea,
inflammatory bowel disease, UC ulcerative colitis, CD NAFLD non-alcoholic fatty liver disease
Crohn’s disease, IBS irritable bowel syndrome, CDI C.
practice in modern medicine (Eiseman et al. (CDAD)). FMT has, however, also been applied
1958). However, historic data indicates that this or is currently under investigation in many other
therapy dates back to the Djong-ji dynasty over intestinal disorders, including inflammatory
1700 years ago, and accounts of its use can be bowel disease (IBD), irritable bowel syndrome
found throughout history (van Nood et al. 2014; (IBS) and many more (Fig. 10.1). Moreover,
Zhang et al. 2012; Nieuwdorp 2014; de Vos FMT is also being studied as potential therapy in
2013). The FMT therapy has been termed in non-intestinal conditions such as metabolic syn-
many different ways, from fecal bacteriotherapy, drome, chronic fatigue syndrome, multiple scle-
duodenal infusion, fecal transfusion, to even rosis or even certain types of cancer, as recently
human probiotic infusion or the more recent term reviewed (Borody et al. 1989, 2011, 2012a, b;
“rePOOPulating” (Petrof et al. 2013). Most of Andrews and Borody 1993; Vrieze et al. 2012;
the published data on FMT refers to its use on Rossen et al. 2015a; Xu et al. 2015).
recurrent Clostridium difficile infection (CDI) Not in all cases FMT leads to successful and
(also referred to as C. difficile associated diarrhea long-term health improvements, and variable
10 How to Manipulate the Microbiota: Fecal Microbiota Transplantation 145
outcomes have been reported in some diseases Firmicutes) that may produce toxins and carry
other than recurrent CDI. A detailed analysis of one or more antibiotic-resistance determinants.
the changes in the intestinal microbiota as illus- CDI is an antibiotic-induced severe form of diar-
trated recently (Fuentes et al. 2014) is expected rhea and colitis, mainly caused by toxin-
to be instrumental in further advancing the producing C. difficile. A stable, healthy gut
understanding of response to the therapy. microbiota can be “tipped” by certain triggers
Whether early diagnosed diseases, states where (such as antibiotic use) leading to a temporal and
the microbiota is greatly disturbed or those unstable state (Lahti et al. 2014). These critical
caused by antibiotic use may respond better, are transitions have been also found to be associated
some of the hypotheses that remain to be con- with disease-related conditions such as obesity or
firmed by properly designed clinical trials. in the elderly. C. difficile overgrowth is usually
reinforced by a disturbed, low diversity GI micro-
biota (Fig. 10.2) and, in many occasions, by reit-
10.2 Applications erated antibiotic treatments.
The appearance of more virulent and
10.2.1 Clostridium difficile Infection antibiotic-resistant strains have led to an increase
in the incidence of CDI, mostly in compromised
Clostridium difficile infection (CDI or CDAD) is individuals such as the elderly or patients suffer-
thus far, of all diseases treated by FMT, the most ing from GI disorders, but also in populations
studied and with best outcomes. C. difficile is a typically considered as healthy (Kelly and
Gram-positive and sporulating anaerobe belong- LaMont 2008). In view of this increasing mor-
ing to the Clostridium cluster XI (phylum bidity and associated mortality there is an urgent
Fig. 10.2 Cycle of C. difficile infection and use of FMT (*Unstable state as described by Lahti et al. (2014))
need for fast and more effective treatments. 10.2.2 Inflammatory Bowel Disease
Currently, several therapies exist for the treat-
ment of CDI, ranging from the more drastic mea- Inflammatory bowel disease (IBD) is a lifelong
sures such as extensive antibiotic treatments to GI disorder characterized by a series of relapses
more mild and adjuvant therapies such as probi- and remissions without a permanent cure. Recent
otics (Na and Kelly 2011; Tannock et al. 2010; reviews show that the success of FMT in IBD is
Lo Vecchio and Zacur 2012). However, FMT is very variable, and to date very limited data is
currently the treatment with highest success rate available to investigate its efficacy and safety
especially for patients suffering from longstanding (Rossen et al. 2015a; Anderson et al. 2012).
recurrent forms of the disease (van Nood et al. Reports of FMT in IBD, including ulcerative
2013) (Table 10.1). colitis (UC) and Crohn’s disease (CD), begin in
The first randomized clinical trial of FMT on the 1980s, when researchers described a remis-
CDI was published in 2013 (van Nood et al. sion of UC for 6 months after FMT (Bennet and
2013). In this study, a total of 42 patients with Brinkman 1989). Since then, results from ran-
recurrent CDI were randomized into three arms: domized clinical trials in over 100 IBD patients
FMT, Vancomycin (standard treatment for CDI) (in some cases concurrent with CDI) have shown
and Vancomycin plus bowel lavage (treatment an improvement of symptoms and remissions
routinely used prior to FMT therapy). Recovery that range from 6 months up to 13 years, with
was 81 % for the FMT arm (94 % after second CD generally being less responsive to treatment
infusion), 31 % for the Vancomycin arm and 23 % than UC, and where very frequently several
for the Vancomycin plus bowel lavage arm. treatments are needed (Borody et al. 2012a,
Differences in recovery rates between the study 2014; Kao et al. 2014; Greenberg et al. 2013)
groups was so large that the non-FMT arms of the (Table 10.2).
trial were stopped for ethical reasons after an In a very recent study on FMT in pediatric
interim analysis. After the therapy, patients patients with Crohn’s disease (Suskind et al.
receiving FMT showed a raise in their GI micro- 2015), it was found via analysis of the gut micro-
biota diversity, approaching levels similar to biota that a stable transfer of healthy donor
those found in healthy donors. An increase in the microbiota in seven out of nine patients had
relative abundance of members of the occurred. According to the pediatric Crohn’s dis-
Bacteroidetes and Clostridium clusters IV and ease activity index, these seven patients were in
XIVa, and a decrease in Proteobacteria species remission at 2 weeks and five out of nine patients
were also found. Based on these findings, it was were still in remission after 12 weeks with no
concluded that long-term restoration of a healthy additional therapy. However, no or very minor
microbiota by FMT creates a sustainable homeo- effect was seen in those patients where the
static status that can prevent future recurrences engraftment of the healthy donor microbiota was
(Fig. 10.2) (Fuentes et al. 2014). not successful.
In UC, remission rates of FMT vary from 0 %
to 68 %. In a recent double-blind, randomized
Table 10.1 Recovery rates for (recurrent) C. difficile clinical trial, 37 patients with mild to moderately
infection treatments from randomized controlled trials a active UC received FMT with either healthy
Recurrences Treatment Recovery (%) donor feces or their own (autologous infusion as
Original infection Vancomycin 70 a control) (Rossen et al. 2015b). Recovery rates
Fidaxomicin 82 were for the per protocol analysis of 41.2 % for
First recurrence Vancomycin 59 the donor feces and 25 % for the autologous
Fidaxomicin 76 transplantation, however, with no significant dif-
>1 recurrences Vancomycin 4 ference mostly due to low numbers (p = 0.29).
FMT 94 Analysis of the fecal microbiota of patients 12
a
Modified from Keller and Kuijper (2015) weeks after treatment revealed that the microbi-
Table 10.2 Clinical outcome data on FMT in IBD

Clinical Clinical
Author Year N Diagnose Aged improvement remission Reference
Angelberger 2013 5 Refractory UC 27 (22–51) 20 % NR Angelberger et al.
(2013)
Borody 2012 62 Active UC M: 42.3 ± 11.5 92 % 68 % Borody et al.
F: 48.45 ± 16.49 (2012a)
Greenberg 2013 16 Refractory CD (2) 39 (20–75) 63 % NR Greenberg et al.
UC (14)b (2013)
Kump 2013 6 Refractory UC 36 (17–52) 33 % 0% Kump et al. (2013)
Kunde 2013 10a Active UCc 7–20 70 % 30 % Kunde et al. (2013)
Vermeire 2012 4 Refractory CD 37.5 (29–50) 0% 0% Vermeire et al.
(2012)
Rossen 2015 48 Active UC 30–56 NR 30.4– Rossen et al.
41.2 %e (2015b)
Modified from Rossen et al. (2015a)
NR not reported
a
1 subject did not tolerate treatment, this subject was considered to be a treatment failure. Endpoint data of the study was
adjusted in this table
b
Concurrent CDI in four UC patients
c
Active disease diagnosed by colonoscopy <6 months before the enrolment
d
Mean ± SD or median, range/IQR
e
For “intention to treat” and “per protocol” analysis respectively
ota of patients responding to the therapy in the from neurological disorders to metabolic syn-
FMT group was similar to that of their healthy dromes (Fig. 10.1).
donors. Remission could also be associated with In some cases, colitis can develop not only as a
increased proportions of Clostridium clusters IV complication of C. difficile overgrowth, but as a
and XIVa, similar to what has been observed in result of other triggers such as antibiotic use.
CDI. FMT has proven to be of help for these cases as
These results highlight one of the limitations well. A recent case study reported the follow up of
of FMT that lies in the importance of case by case a 46-year-old man for over a year (Satokari et al.
targeted therapies rather than undefined mixtures 2014). Contrary to CDI, symptoms were not
of bacteria. There are currently several clinical accompanied by an apparently disturbed fecal
trials designed to investigate FMT on IBD. In microbiota prior to the therapy; the patient showed
clinicaltrials.gov, open studies in recruiting phase high Bacteroidetes levels but within the normal
for FMT in IBD include six in UC (one of them range observed in healthy individuals. These lev-
in UC associated pouchitis), two on CD and four els were further increased 2 days after FMT, but
on IBD in general. Results from these trials will the microbiota composition quickly shifted to one
in time shed some more light on this issue. that was more dominated by Firmicutes 2 weeks
after infusion. Microbiota analysis revealed an
increased bacterial diversity in the rectal mucosa
10.2.3 Other Applications and a stable fecal microbiota up to 3 months after
treatment. In this case of non-infectious colitis
Potential applications of FMT are unlimited, as FMT was able to resolve the symptoms and
the GI microbiota is known to play an important restore normal GI-function, possibly by acting
role in numerous physiological processes. As against a persistent low-grade inflammation.
previously mentioned FMT is under investigation FMT therapy has also been used in cases of
for many (non-) intestinal disorders, ranging irritable bowel syndrome (IBS), as well as
chronic constipation (concomitant to IBS) where 10.3 Methodology and Donor

up to 76 % of the treated patients reported Selection
improvement of symptoms including abdominal
pain or bloating, among others, with a follow up Many different methodologies for FMT have
period of up to a year (Andrews and Borody been published (Gough et al. 2011; van Nood
1993; Pinn et al. 2013). Positive results were also et al. 2009; Owens et al. 2013). The route of
reported in cases of constipation associated to infusion can vary from retention enema, colono-
UC (Rossen et al. 2015a). scope and nasoduodenal tube, and results of
In addition, FMT has an important therapeutic these (so far only compared in the treatment of
potential in non-intestinal disorders. The intesti- CDI) are comparable. In most of the cases, the
nal microbiota has been shown to play an impor- patient’s GI tract is prepared by bowel lavage or
tant role in local and systemic inflammation, key use of laxatives. Cleaning of the bowel has been
factor for obesity and insulin resistance (Verdam shown to have an impact on the GI microbiota
et al. 2013; Turnbaugh et al. 2006; Udayappan composition (Jalanka et al. 2014). In a recent
et al. 2014). A few reported studies evaluated the study, 23 healthy individuals were given a bowel
effect of manipulating the microbiota by means of lavage either in two separate doses of 1 l or in a
fecal transplantation in diseases associated to obe- single 2 l dose. This treatment had an important
sity, such as type 2 diabetes or metabolic syn- effect on the microbiota, by loss of both micro-
drome (Vrieze et al. 2012, 2014; Hartstra et al. bial load and diversity as well as subject specific
2015). In a recent study on a cohort of 18 patients composition. Cleaning by means of a double
diagnosed with metabolic syndrome, a significant dose had a less disturbing effect on the microbi-
increase was observed in insulin sensitivity after ota than the single dose, and it was associated
reception of healthy donor feces as compared to with increased levels of Proteobacteria among
the autologous placebo group (i.e. those patients others.
who received their own fecal material) (Vrieze Stool samples are screened for various para-
et al. 2012). These patients showed an enrichment sites and bacterial pathogens. A shortened ver-
of butyrate-producing species (able to produce sion of donor and patient screening as well as
butyric acid from lactate and acetate) such as therapy protocol for FMT (for CDI) is shown in
Roseburia intestinalis or Eubacterium hallii. Table 10.3. More detailed protocols and ques-
Butyrate is a short chain fatty acid of great impor- tionnaires can be found in van Nood et al. (sup-
tance in colonic function, and known to be related plementary material) (van Nood et al. 2013).
to obesity and pain sensation (Duncan et al. 2007; Donor selection has been hypothesized to be
Hamer et al. 2008; Louis and Flint 2009; essential for success of FMT, although key char-
Vanhoutvin et al. 2009). Butyrogenic bacteria acteristics are still unknown. Increasing evidence
have been shown to be depleted from patients suf- shows that there is a need for donor-patient
fering from CDI (Antharam et al. 2013). matching, and that some donors work better than
Therapies including neurological diseases others (Fuentes et al. 2014; Borody et al. 2014)
such as Parkinson’s disease or multiple sclerosis (Rossen et al. 2015b). As mentioned above, donor
are also being investigated (Borody et al. 2011; selection criteria are based on extensive question-
Borody and Khoruts 2011; Ananthaswamy naires that lead to exclusion following thorough
2004). Anecdotal observations indicated that examination of (non-) GI diseases as well as
after treating CDI patients who suffered from other parameters. Blood samples are also
Parkinson’s disease by FMT, symptoms of screened for numerous antibodies against poten-
Parkinson’s were reduced (Borody et al. 2013). tially transmittable diseases, such as hepatitis
Autoimmune disorders, certain tumours or even (A/B/C), HIV or parasites. If multiple samples
lymphoma are some of the additional applica- are required from selected donors, questionnaires
tions that have been investigated for treatment and screenings are frequently repeated. Donors
with FMT (Xu et al. 2015). that have led to better outcomes in previous FMT
Table 10.3 Shortened Amsterdam protocol for FMT for et al. 2008; Everard et al. 2013; Kang et al. 2013;
CDI
Berry and Reinisch 2013).
Donor Patient
1. Screening 1. Selection criteria for
FMT
10.4 Synthetic Communities
Questionnaire for risk >2 CDI recurrences
factors and potentially Severe therapy
transmittable disorders resistant CDI
Besides infusion of diluted and filtered feces,
Exclusion criteria: other preparations have also been used as “fecal
Antibiotic use (<3 material”, including frozen preparations or selec-
months) tions of several bacterial species grown in vitro,
GI symptoms most of them isolated from fresh fecal samples
(diarrhea, constipation (Petrof et al. 2013; Hamilton et al. 2013;
or IBS symptoms)
Youngster et al. 2014; Tvede and Rask-Madsen
Recent travel to areas
of endemic GI 1989). These preparations have been shown to
pathogens provide similar outcomes as freshly prepared
IBD stools without additional side effects, becoming a
GI malignancies or practical alternative to FMT (Satokari et al. 2015;
polyposis Tvede et al. 2015). Current research aims to iden-
2. Laboratory tests: 2. Pre-treatment of tify specific communities associated with differ-
patient
ent diseases (as well as a defined “healthy”
Blood tests/serology Vancomycin treatment
>4 days (stopped 1
microbiota). This would allow for targeted treat-
day before FMT) ment therapies for specific diseases with well-
Feces Bowel lavage (day characterized strains or active ingredients that are
before FMT) now administered “blindly” by fecal transplanta-
Placement of tion. To this end, several public and private sec-
nasoduodenal tube tors are investigating defined synthetic
(day of FMT)
communities. These communities can be con-
3. 1 day before donation
trolled and reproduced as regular treatments
Questionnaire for recent
health related issues without the need of human donors, and under
FMT more controlled conditions.
Preparation of donor feces: One of the first studies involving a defined
Dilution of feces in sterile saline mixture of strains as treatment for recurrent CDI
Filter through unfolded gauzes and collect in a was published in The Lancet in 1989 (Tvede and
closed bottle Rask-Madsen 1989). A group of six patients was
Infusion: treated by an enema with either stool samples or
Infuse through nasoduodenal tube the donor feces with a mixture of ten cultured bacteria
solution (Clostridium innocuum, C. ramosum, C. bifer-
Flush the nasoduodenal tube with water mentans, Bacteroides ovatus, B. vulgatus, B. the-
Remove tube 30 min after infusion taiotaomicron, Peptostreptococcus productus,
Offer lemonade to patient after removal
Streptococcus faecalis, and two strains of
More information in van Nood et al. (2013) and Keller Escherichia coli). After treatment, the response
and Kuijper (2015)
was rapid, and all patients were asymptomatic
and showed no C. difficile toxin after 24 h.
trials could be preselected as high efficiency Interestingly one of the patients responded better
donors, as well as those with higher levels of the to the mixture than to the FMT. It was not until
already mentioned butyrate producing bacteria or 2013 that a mixture of bacterial strains was used
health related strains such as Faecalibacterium again for the treatment of recurrent CDI in two
prausnitzii or Akkermansia muciniphila (Sokol patients, in this case containing 33 strains isolated
from one healthy donor (Petrof et al. 2013), coin- (Smith et al. 2014). In order for stool banks to
ing the term already mentioned, “rePOOPulat- succeed, the FDA would need to either reclas-
ing” of the gut. sify FMT as suggested by experts, or approve
its use in disorders different from CDI without
the burden of bureaucracy.Very recently, the
10.5 Regulations and Safety Netherlands Donor Feces Bank (NDFB) was
initiated to enable safe and cost-effective FMT
There is an increasingly pressing need to regulate for patients with recurrent CDI, and to support
fecal transplants due to the remarkable interest further research.
that this therapy generates, with publications not Among the limitations of FMT one can list the
properly documented or even videos online for observation that for some therapies (e. g. in
self-infusion. In 2013 the US Food and Drug patients suffering from IBD) several repeated
Administration (FDA) classified human feces (for FMT applications are necessary (Ratner 2014).
medical use) as a drug, in hopes of regulating and This will make the procedure rather costly as
standardizing its use. This implied the require- compared to standard treatments, and as the
ment to submit an IND (investigational new drug) effect is variable, not an attractive alternative.
application when using this therapy. However, This is not the case for CDI, where cost-
after physicians, researchers and patients raised effectiveness has been evaluated, and FMT was
their concern about access to this treatment estimated to save costs when compared to stan-
becoming growingly constrained, the FDA dard antibiotherapy (van Nood 2015).
decided to not enforce the IND for its use on Although the main advantage of FMT is that it
recurrent CDI. Top physicians in the field indi- has limited adverse effects, these are still an
cated that reclassifying human feces as a tissue issue. Although the most frequent are mild
product or a different category (such as blood) adverse effects often self-limiting, especially
would facilitate it’s application while maintaining those related to abdominal discomfort (such as
safety regulations (Smith et al. 2014). pain, bloating, nausea or even vomiting), severe
While in the UK fecal transplants are adverse effects have also been reported includ-
accepted as therapy for CDI, where it is consid- ing, among others, intestinal perforation, post-
ered safe and effective, in the US current regu- transplant sepsis or bacteraemia, although in
lations are somewhat stricter and require that many cases causal relation to the therapy could
donors (and their stools) must be known and not be established (Rossen et al. 2015a). In sev-
screened by the physicians involved in the eral of the FMTs, a careful analysis of the patients
treatment. This rule led to the closing of stool health status has been reported, which included
banks such as OpenBiome, a non-profit organi- the observation that a transient and small increase
zation from the Massachusetts Institute of in CRP levels was observed after 2–3 days both
Technology. OpenBiome carried out 17 blood for the autologous (own microbiota) as healthy
and stool screening assays and supplied sam- donor transplantation (Vrieze 2013).
ples to several US hospitals (more than 100 Furthermore, only few studies include long
treatments in its first 3 months) (Smith et al. term follow up of patients. Long-term conse-
2014). Stool banks, similarly as blood banks quences, although yet unreported and mostly
do, could be a source for thoroughly screened unknown, are still a risk. Studies that would allow
donor material. Not only would this have an evaluating the potential risk of FMT with respect
impact on the safety of the procedure, avoiding to possible future infections, auto-immune or met-
desperate, risky and largely uncontrolled treat- abolic diseases or even cancer are still required. At
ments at home, but also it would make it present, the main fear is that fecal material can
cheaper and more accessible. However, as of carry viruses or other infectious diseases, although
2015 the FDA allows for FMT for CDI, while to date there are no cases of transmission of infec-
for other applications an IND is still mandatory tious diseases directly related to FMT. Additionally,
Fig. 10.3 Evolution of FMT

therapy
changes in an individual’s GI microbiota composi- to 500 g (Rossen et al. 2015a). This would prove
tion could lead to a shift in phenotype to one of challenging when wanting to translate an infu-
higher susceptibility for microbiota related dis- sion of fecal material into a pill format.
eases, such as obesity and other conditions. A Up to date, evidence shows that FMT is an
recent case study reports a successfully treated efficient, simple therapy with rather positive out-
patient who 16 months after therapy developed comes. Modulation of the gut microbiota by
new-onset obesity, after receiving FMT from an FMT can lead to the establishment of a stable
overweight donor (Alang and Kelly 2015). microbiota composition that tends to remain in a
healthy state of homeostasis. However, results
from well controlled, randomized, double blind
10.6 Microbiota Transplantation: clinical trials are urgently needed, and will be key
The future to fine tune this therapy.
Research in the field of fecal transplantation is Acknowledgements We would like to thank all our col-
rapidly growing, with countless applications. laborators, especially those involved in the mentioned
Results from the last decade are showing a rapid clinical trials. This work was supported by CVON-IN
CONTROL (Cardiovascular research, The Netherlands,
evolution of how we will approach the applica- www.cvon.eu), Spinoza Awards and Gravitation
tion of this therapy (Fig. 10.3). Whether the Programmes from the Netherlands Organisation for
future of FMT will be in the form of diluted Scientific Research (NWO).
healthy donor feces, or defined consortia of cul-
tured species in a pill, remains to be assessed.
Identification of essential microbes (and/or References
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Index
A Development, 5, 6, 10, 14, 15, 39, 40, 46, 52, 55, 74, 76,
Aberrant microbiota, 96, 101, 104 77, 87, 98–102, 111, 112, 125
Actinobacteria, 17, 47, 66, 67, 70, 72, 96, 120, 126 de Vos, W.M., 143–150
Aerobic vaginitis, 88 Diabetes, 55, 70, 98, 100, 101, 103, 131, 136, 148
Al-Ahmad, A., 57 Diarrhea, 99, 136, 144, 145, 149
Alekseyenko, A.V., 72 Dietary fibre, 99, 121, 127, 132, 133
Allergy, 55, 112, 131 DNA methylation, 35–39
Appetite regulation, 131 Dominguez-Bello, M.G., 70
Archaea, 1, 10, 11, 65, 67–69, 77, 96 Donders, G.G., 88
Arweiler, N.B., 45–57 Donlan, R.M., 54
Donnarumma, G., 76
Donor, 38, 39, 88, 143, 146–151
B Dukes, C., 84, 87
Bacteria, 1, 2, 7, 8, 10, 12, 15, 17, 21, 22, 35, 38, 41, Dysbiosis, 96, 98–101, 103, 104, 110, 112
46–56, 65, 66, 69–71, 74–77, 84, 85, 87, 88,
96–104, 110, 112, 113, 120–124, 126, 127,
129–131, 134–136, 147–149 E
Bacterial fermentation, 121, 122, 126 Early life nutrition, 40
Bacterial metabolites, 34–40, 130 Early life programming, 40
Bacterial vaginosis (BV), 84–89 Egert, M., 61–77
Biofilm, 46–49, 51–57, 69, 75, 84, 87–89 Ehrström, S., 89
Bohbot, J.M., 89 Epigenetics, 34–35
Breast feeding, 99, 100
F
C Fermentation, 34, 121, 122, 126, 130–133,
Callewaert, C., 70 135, 137
Canesso, M.C.C., 73 Fierer, N., 67, 70
Capone, K.A., 70 Findley, K., 67, 70
Cardot, J.M., 89 Firmicutes, 47, 66, 67, 72, 96, 103, 111, 120, 122,
Carolan, H., 74 124–127, 129, 145, 147
Clemente, J.C., 70 Flint, H.J., 119–137
Clostridium difficile, 144–146 Fluorescent in situ hybridisation (FISH), 21, 22, 55, 57,
Competition for substrates, 126 69, 87, 120
Cosmetics, 62, 70, 73–77 Foulongne, V., 69
Costerton, J.W., 54 Fuentes, S., 143–150
Curtis, A.H., 84 Fungi, 11, 65, 67–70, 73, 77, 98
D G
Definition, 1–3, 53, 54, 71, 77, 84, 87, 120, 121, 124, Gajer, P., 85
125, 130, 136 Gao, Z., 66, 69, 72
Dental plaque, 46, 47, 49, 53, 54, 57 Gardner, H.L., 84, 86, 87

Medicine and Biology 902, DOI 10.1007/978-3-319-31248-4
156 Index
Gibson, G.R., 120 Microbiota, 1–3, 5–26, 34–41, 45–57, 61–77, 83–89,
Goetz, A., 2 95–104, 109–114, 119–137, 143–151
Grice, E.A., 62, 66, 67, 69, 70 Microbiota composition, 9, 19, 62, 70, 102, 120, 121,
Grimm, V., 109–114 131, 137, 147, 148, 151
Mineral absorption, 132–133
Mischke, M., 33–41
H Moore, L.H.V, 51
Haffajee, A.D., 52
Harmsen, H.J.M., 95–104
Health-disease-relationship, 46, 55 N
Health effect, 120, 130, 137 Necrotizing enterocolitis (NEC), 113
Heurlin, M., 84 Netuschil, L., 45–57
Hilton, E., 89 Nguyen, T.L., 9
Histone modification, 35, 37, 39 Non-digestible carbohydrates, 120, 129
Holland, S.M., 62 Nood, E., 149
Homeostasis, 34, 63, 71, 103, 131, 151
O
I Obesity, 9, 34, 40, 98, 100, 103–104, 131, 136,
IBS. See Irritable bowel syndrome (IBS) 145, 148, 151
Immune functions, 86, 134–136 Oh, J., 70
Inflammatory bowel diseases (IBD), 96, 100–101, 104, Oligosaccharides, 99, 126–129, 132, 133
109, 111–112, 144, 146, 147, 149, 150 Ouwehand, A.C., 75
Intestinal barrier, 131, 133–134
Irritable bowel syndrome (IBS), 109–111, 131,
144, 147, 149 P
Irvine, A.D., 70 Percival, R.S., 50
Iwase, T., 69 Periodontitis, 46, 48, 50–53, 55, 109
Perspiration, 63, 64
Petricevic, L., 86
J Probiotics, 26, 40, 55, 71, 74–77, 88–89, 101, 110–114,
James, A.G., 62 120, 134, 144, 146
Jespers, V., 84 Proteobacteria, 47, 66–68, 70, 85, 96, 111, 113,
120, 146, 148
Proteomics,
K
Katsuyama, M., 75
Keller, J.J., 146, 149 R
Kern, A.M., 89 Ramirez-Farias, C., 122–124
Krönig, B., 84 Ravel, J., 85
Redel, H., 70
Regulation, 34–37, 39, 40, 63, 71, 95, 96, 131, 133–135,
L 150–151
Lactobacilli, 50, 55, 76, 84–89, 99, 111, 113, Riedel, C.U., 109–114
120–122, 125, 127, 136 Roberfroid, M.B., 120, 132
Lai, Y., 71 Roth, R.R., 62
Lamont, R.F., 87 Rusch, V, 1–3
Louis, P., 119–137
S
M Safety, 110, 125, 146, 150–151
Martinez, R.C., 89 Salter, S., 17, 19
McAleer, M.A., 70 Sanchez, D., 89
Meadow, J.F., 70 SCFA. See Short-chain fatty acids (SCFA)
Mendling, M., 83–90 Schröder, R., 84
Metabolomics, 6, 24, 104 Schwiertz, A., 1–3
Michel, C., 119–137 Segre, J.A., 62
Index 157
Sequencing PCR, 10, 13 T

Shalev, E., 89 Tanner, A., 52
Short-chain fatty acids (SCFA), 8, 36, 98, 101, 103, 125, Techniques, 6, 7, 9, 12, 14–16, 19–26,
130, 131, 133–135 52, 55, 96, 98
Sim, K., 17 Thomas, S., 84
Simmering, R., 61–77
Skin defense and immunity, 71
Skin diseases, 62, 69–72, 77 V
Skin microbiota, 62, 65–68, 71–77, 112 Vaginal microbiota, 83–89
Socransky, S.S., 52
Stable isotope, 15, 24–25
Starch, 25, 126–129, 132 W
Swidsinski, A., 87 Walker, A.W., 5–26, 123

Artigos I

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ARTIGOS

Advances in Experimental Medicine and Biology 817

IRUN R. COHEN, The Weizmann Institute of Science, Rehovot, Israel

For further volumes:

ISSN 0065-2598 ISSN 2214-8019 (electronic)

© Springer New York 2014

Printed on acid-free paper

Springer is part of Springer Science+Business Media (www.springer.com)

Abilene, TX, USA Mark Lyte

Abilene, TX, USA Mark Lyte

Part I Basic Concepts Underlying the Microbiota-Gut-Brain Axis

1 Microbial Endocrinology and the Microbiota-Gut-Brain Axis .............. 3

7 Gastrointestinal Hormones and Their Targets ..................................... 157

13 The Effects of Inflammation, Infection and Antibiotics on the

Carmen Alonso, MD, PhD Neuro-Immuno-Gastroenterology Group, Digestive

Department of Medicine, Universitat Autònoma de Barcelona, Centro de

Michael T. Bailey, PhD Institute for Behavioral Medicine Research, College of

Premysl Bercik, MD Medicine, McMaster University, Hamilton, ON, Canada

John Bienenstock, MBBS, FCRP, FCRP(C) Pathology and Molecular Medicine,

Yuliya E. Borre, PhD Neurogastroenterology Lab, Alimentary Pharmabiotic

Brid P. Callaghan Anatomy and Neuroscience, University of Melbourne,

Hyun-Jung Cho, PhD Anatomy and Neuroscience, University of Melbourne,

Stephen M. Collins, MD Medicine, McMaster University, Hamilton, ON, Canada

John F. Cryan, PhD Department of Anatomy and Neuroscience, University

Aitak Farzi, MD Institute of Experimental and Clinical Pharmacology, Medical

Gerald F. Fitzgerald, PhD, DSc Microbiology and Alimentary Pharmabiotic

Anthony Fodor, PhD Bioinformatics and Genomics, UNC Charlotte, Charlotte,

Paul Forsythe, PhD Medicine, McMaster University, Hamilton, ON, Canada

John B. Furness Anatomy and Neuroscience, University of Melbourne, Parkville,

Mélanie G. Gareau, PhD Department of Medicine, University of California San

Peter Holzer, MSc, PhD Experimental Neurogastroenterology, Institute of Exper-

Wolfgang A. Kunze, PhD Psychiatry and Behavioural Neuroscience, St. Joseph

Jennifer S. Labus, PhD Division of Digestive Diseases, Department of Medicine,

Beatriz Lobo, MD, PhD Neuro-Immuno-Gastroenterology Group, Digestive Dis-

Department of Medicine, Universitat Autònoma de Barcelona, Centro de

Christopher A. Lowry, PhD Department of Integrative Physiology, Center for

Mark Lyte, PhD, MS, MT(ASCP) Department of Immunotherapeutics and

Rachel D. Moloney, PhD Laboratory of NeuroGastroenterology, Alimentary

Department of Psychiatry, University College Cork, Cork, Ireland

Angela Moya-Pérez, BSc Microbial Ecology, Nutrition & Health, National

Marc Pigrau, MD, PhD Neuro-Immuno-Gastroenterology Group, Digestive

Department of Medicine, Universitat Autònoma de Barcelona, Centro de

Charles L. Raison, MD Department of Psychiatry, College of Medicine and

Jens F. Rehfeld, MD, DMSc, DSc Department of Clinical Biochemistry,

R. Paul Ross, PhD, DSc Alimentary Pharmabiotic Centre, Teagasc Moorepark

Javier Santos, MD, PhD Neuro-Immuno-Gastroenterology Group, Digestive

Department of Medicine, Universitat Autònoma de Barcelona, Centro de

Fergus Shanahan, MD, FRCP, FCCP, FRCPI Department of Medicine, Alimen-

Catherine Stanton, PhD, DSc Alimentary Pharmabiotic Centre, Teagasc

Nobuyuki Sudo, MD, PhD Department of Psychosomatic Medicine, Kyushu

Kirsten Tillisch, MD Division of Digestive Diseases, Department of Medicine,

Mar´ıa Vicario, PhD Neuro-Immuno-Gastroenterology Group, Digestive Diseases

Department of Medicine, Universitat Autònoma de Barcelona, Centro de

Rebecca Wall, PhD Alimentary Pharmabiotic Centre, Teagasc Moorepark Food

Abstract Microbial endocrinology is defined as the study of the ability of micro-

CNS Central nervous system

M. Lyte and J.F. Cryan (eds.), Microbial Endocrinology: The Microbiota-Gut-Brain 3

Microbial Endocrinology: Conceptual Framework

Microbial endocrinology represents the intersection of two seemingly disparate

do bacteria produce neuroendocrine hormones? In large part, most reports of

Origins of Microbial Endocrinology: Evidence from the 1930s

phenylethanolamine-N-methyltransferase which is needed for conversion of nor-

Evolution of Current Microbial Endocrinology-Based