Role of the Microbiota in Immune Development

Qing Zhao and Charles O Elson, University of Alabama at Birmingham, Birmingham, AL, USA
Ó 2016 Elsevier Ltd. All rights reserved.

Glossary
Altered Schaedler flora (ASF) A group of eight defined
cultivable bacteria from normal mice gut microbiota that is
used to colonize germ-free mice and to study the effect of
a limited but defined set of gut commensal bacteria in vivo.
Aryl hydrocarbon receptor (AhR) A cytosolic transcription
factor involved in the differentiation of regulatory T cell,
Th17 cell, type 3 innate lymphoid cell, and intraepithelial
cells. After its binding of dietary or microbial products, it
translocates into the nucleus and initiates downstream
gene expression.
C-type lectin A large family of carbohydrate-binding
receptors. Antimicrobial peptide RegIIIg is a C-type lectin.
Defensin A type of antimicrobial peptides produced by
neutrophils and epithelial cells. Defensins can disrupt
microbial cell membrane integrity thus resulting in
microbial killing.
Hygiene hypothesis Hypothesis that the lack of exposure
to symbiotic microbes and pathogens in early childhood
predisposes individuals to increased allergic and
autoimmune diseases later on in life.
Inflammasomes Protein complexes belonging to the innate
immune system to sense damage-associated molecular
patterns. They can activate inflammatory caspases and trigger
the cleavage of proinflammatory IL-1 family cytokines into
their active forms. Subtypes of inflammasomes are named
after the protein that initiates the signaling cascade, including
nucleotide-binding oligomerization domain receptor (NLR)
or AIM2-like receptor (ALR) protein.
Inflammatory bowel disease (IBD) Diseases of immune-
mediated intestinal inflammation caused by environment
and genetic predisposition. Crohn’s disease and ulcerative
colitis are two main types of IBD.
Lamina propria (LP) A layer of loose connective tissue
beneath the epithelium lining the mucosal tissues of the
body. It contains a rich vascular, nerve, and lymphatic
network and various types of immune cells, providing
nutritional and immunological support at the mucosal
surface.
Major histocompatibility complex (MHC) A set of cell
surface molecules that can bind peptide fragments and
display them on antigen-presenting cells for recognition by
corresponding T cells. Class I MHC molecules can be
recognized by CD8þ T cells, while class II MHC molecules
are recognized by CD4þ T cells.
Mesenteric lymph node (MLN) Draining lymph node of
intestinal mucosa that locates in the mesentery.
Microbe-associated molecular patterns (MAMPs) A series
of conserved molecular motifs such as bacterial
lipopolysaccharide and flagellin that are associated with
microbes. They can be recognized by pattern recognition
receptors in both animals and plants.
Microfold cell (M cell) A specialized epithelial cell that
lacks microvilli but has shorter and broader microfolds on
the apical cell surface. In the small intestine, it locates
above Peyer’s patches and can transport antigens from
intestinal lumen to cells in the Peyer’s patches.
Mutualism An ecological relationship that two species live
together and benefit from each other’s activities.
Paneth cell A specialized epithelial cell in the small
intestine that produces a number of antimicrobial
peptides/proteins.
Pattern recognition receptor (PRR) Germ line–encoded
proteins that can recognize microbe-associated molecular
patterns and induce signaling cascade in innate immune
responses.
Peyer’s patch (PP) Isolated secondary lymphoid organs
that locate under the small intestinal epithelium at the side
opposite to mesenteric attachment. It is composed of a dome
area, B cell follicles and interfollicular T cell areas, and
functions as an important inductive site of IgA responses.
Recombination-activating gene (RAG) Encodes enzymes
that are important for rearrangement and recombination of
immunoglobulin and T cell receptor genes. The lack of either
RAG1 or RAG2 will lead to T cell and B cell deficiencies.
Serum amyloid A (SAA) One of a group of highly
conserved proteins associated with high-density
lipoproteins in the serum. The level of SAA proteins
drastically increases during inflammation and participate
in the induction of immune responses.
Severe combined immunodeficiency (SCID) mice A
mouse model broadly used in studies of the immune
system and tissue transplants. These mice lack mature T and
B cells due to a recessive mutation of a gene important for
DNA repair.

Abstract

A well-functioning immune system is required for proper protection from infections and diseases. Vertebrates have coevolved
a comprehensive immune network with the numerous microbes colonizing every surface of their body, particularly in the
digestive tract. As such, the immune system has to be trained to discriminate pathogens from symbiotic microbes as well as
self from nonself. Commensal microbiota in the gut plays a critical role in this development. Investigations have found

Encyclopedia of Immunobiology, Volume 5 http://dx.doi.org/10.1016/B978-0-12-374279-7.19019-8 109

110 Physiology of the Immune System j Role of the Microbiota in Immune Development
correlations of gut microbiota with the development and maturation of a number of components of innate and adaptive
immunity. Disruption of this microbiota-host dialog may lead to immune disorders such as allergy and immune-mediated
inflammatory diseases.
Introduction
The complex community of commensal, symbiotic, and path-
ogenic microorganisms colonizing the body surface of all verte-
brates is termed the ‘microbiota,’ with the largest mass residing
in the lower intestine. Normally, a healthy human intestine
harbors 1014 microbes – 10 times greater than the number of
cells in the human body (Gill et al., 2006). These organisms
include mostly bacteria, but also fungi, archaea, and viruses,
which interact with the mucosal surface and play a variety of
physiological roles, such as facilitating host food digestion
via degradation of plant polysaccharide and fibers, producing
vitamins and energy, promoting the development of the intes-
tinal epithelium, the gut vasculature, and the enteric nervous
system (Duerkop et al., 2009). Recently, there are also studies
correlating altered gut microbiota composition with cognitive
disorders such as depression, anxiety, and autism (Schmidt,
2015). But most importantly, a complete set of balanced gut
microbiota helps with the development and regulation of
a healthy, fully functioning mucosal and systemic immune
system. In particular to the gut, the microbiota influences the
structural development of lymphoid tissues, epithelial barrier
function, and the development of the innate and adaptive
immune systems, assisting with the reciprocal challenges that
the intimate contact of gut microbiota and the mucosal surface
brings – intestinal infections and autoimmune diseases
(Rakoff-Nahoum et al., 2004; Macpherson and Harris, 2004).
Microbiota composition is greatly diversified among individ-
uals, but generally similar within the same species. Depending
on the delivery method of the baby (vaginally or with cesarean
section), the founder bacteria of the gut will be different.
Commonly, the gut microbiota composition of unweaned
infants is structured with fewer microbial species and shows
greater variations between individuals (Kurokawa et al., 2007).
The intestinal ecosystem undergoes a vast change when infants
start eating solid food: more microbial species appear due to
the expansion of nutritional niches and a rapidly developing
immune system, and tends to be relatively stable at the end of
early childhood (Dominguez-Bello et al., 2011). In adult gut,
Bacteroidetes and Firmicutes phyla become predominant.
Although highly variable between individuals, the assemblage
of microbial species shows a functional similarity in the gut
regardless of age and sex (Human Microbiome Project, 2012).
The understanding of how intestinal microbiota shape the
gut immune system is largely established via comparing germ-
free (GF) mice or microbial-reduced mice with conventional
mice raised under specific pathogen free (SPF) conditions.
Lack of microbial antigen stimulation results in underdevelop-
ment of the mucosal as well as the systemic immune system,
including impaired development of lymphoid organs, decreased
recruitment of lymphocytes, unbalanced lymphocyte differentia-
tion, and immune cell malfunction. These homeostatic immune
system defects in GF and antibiotic-treated mice can
subsequently lead to altered immune responses, impaired
microbial containment upon pathogen challenge, and increased
risk of immune-mediated inflammatory diseases (Dimmitt et al.,
2010; Hill et al., 2010; Fukuda et al., 2011). In humans, antibiotic
treatment during infancy has resulted in increased risk of allergy,
asthma, and inflammatory bowel disease (IBD) later in life (Hviid
et al., 2011; Kronman et al., 2012; Munyaka et al., 2014).
Thus microbiota colonization and concomitant immune devel-
opment during neonatal life and infancy are crucial for a properly
functioning immune system. In the sections that follow, we will
discuss immune system development and maturation, much of
which occurs during this early-life exposure to the microbiota.
Intestinal Lymphoid Tissue Development
Lymphoid tissue inducer (LTi) cells, which are generated prena-
tally in the fetal liver due to the expression of transcription factor
Rorgt and Id2 (Eberl et al., 2004), are fundamental for the early
development of lymph nodes (LNs), Peyer’s patches (PPs), cryp-
topatches (CPs), and isolated lymphoid follicles (ILFs) (Eberl and
Littman, 2004). LNs and PPs develop in the fetus in utero, in the
absence of microbial stimulation under the influence of a number
of factors (Table 1). However, microbial signals are required for
the postnatal development and maturation of PPs and mesenteric
lymph nodes (MLNs) (Pollard and Sharon, 1970; Round and
Mazmanian, 2009). The development and maturation of micro-
fold cell (M cell), a specialized epithelial cell localized on top of
PPs to facilitate antigen translocation, are also influenced by
bacterial colonization (Randall and Mebius, 2014).
The formation of CPs and ILFs happens postnatally. CPs are
mainly composed of clusters of LTi cells in the LP at the base of
intestinal villi. In mice, over 1500 CPs can be detected about
2 weeks after birth, even in GF mice without microbial coloniza-
tion (Kanamori et al., 1996). Upon microbe-associated molec-
ular pattern (MAMP) recognition through Toll-like receptors
(TLRs), stromal cells become activated by LTi cells through lym-
photoxin-b receptor signaling, and recruit DCs and B cells into
the CPs to form ILFs (Tsuji et al., 2008). Recognition of gram-
negative bacterial peptidoglycan through nucleotide oligomeri-
zation domain 1 (NOD1) expressed in intestinal epithelial cells
(IECs) induces CCL20 and b-defensin 3 and recruits B cells to the
LTi-DC clusters to form ILFs (Bouskra et al., 2008). ILFs are
dynamic reservoirs for the induction of secretory IgA (sIgA) via
both T-dependent and -independent pathways (Figure 1; Tsuji
et al., 2008; Knoop and Newberry, 2012; Maynard et al., 2012).
Yet another type of intestinal lymphoid tissue is tertiary
lymphoid tissue (tLT), which contains B cell follicles and T cell
areas and is generated in the LP during chronic inflammation
(Aloisi and Pujol-Borrell, 2006) but is absent in healthy tissues.
Contrary to PPs, CPs, and ILFs; the development of tLTs is not
dependent on Rorgt, because Rorgt-deficient mice can form
them in the intestine in the presence of a gut microbiota. These
tLTs along with increased serum IgG help contain microbes in
Rorgt/ mice at steady state in compensation for the lack of
 Physiology of the Immune System j Role of the Microbiota in Immune Development 111

Table 1 Requirements for the development of intestinal lymphoid tissues

Lymphoid tissue Rorgt Id2 LTab-LTbR Chemokines and cytokines Age References

MLNs þ þ þ TRANCE Prenatal De Togni et al. (1994), Yokota et al. (1999),

Eberl et al. (2004), and Kim et al. (2000)
Peyer’s patches þ þ þ IL-7, TNF, CXCL13, CCL19, Prenatal De Togni et al. (1994), Yokota et al. (1999),
CCL20, CCL21 Eberl et al. (2004), and Eberl and Lochner (2009)
Cryptopatches þ ? þ IL-7 Postnatal Kanamori et al. (1996)
ILFs þ ? þ CXCL13 and CCL20 needed Postnatal Tsuji et al. (2008), Bouskra et al. (2008),
for small intestine and Baptista et al. (2013)
tLTs  ? LTab-LTbR and CCL19, CCL21, CXCL12, Postnatal Aloisi and Pujol-Borrell (2006) and Lochner
TNFSF14-LTbR CXCL13 et al. (2011)

MLNs, mesenteric lymph nodes; ILFs, isolated lymphoid follicles; tLTs, tertiary lymphoid tissues; Rorgt, retinoid acid receptor–related orphan receptor gt; Id2, inhibitor of DNA
binding 2; LTab, lymphotoxin-ab; LTbR, lymphotoxin-b receptor; TRANCE, tumor necrosis factor–related activation-induced cytokine; TNF, tumor necrosis factor; CXCL13,
chemokine (CeXeC motif) ligand 13; CCL9, chemokine (CeC motif) ligand 9; TNFSF14, tumor necrosis factor superfamily member 14; þ, required.

(a) Prenatal (b) Postnatal

Dendritic cell
Bacteria
Antimicrobial
Intraepithelial peptides
Lumen lymphocyte
Dendritic cell
MAMPs Dimeric IgA
Goblet cell
M cell IgA+ cell
Epithelium
T cell

B cell
Lamina
propria
Peyer’s patch
Cryptopatch
Paneth cell
Peyer’s patch
LTi cell Mature isolated
Mesenteric lymphoid follicle
lymph node

Figure 1 The development of intestinal lymphoid tissues. (a) Prenatally, secondary lymphoid tissues (Peyer’s patches and mesenteric lymph nodes)
and cryptopatches develop by the spatiotemporal recruitment of lymphoid tissue inducer (LTi) cells to sites of the developing intestine and supporting
neurovascular structures. This, in turn, stimulates the recruitment of dendritic cells (DCs), T cells, and B cells in preparation for the immune response to
the microbiota. Intraepithelial lymphocytes seed the epithelium before birth. (b) Postnatally, bacteria colonize the neonatal intestine immediately, initiating
multiple events that affect the development or functional maturation of the mucosa- and gut-associated lymphoid tissues. Shown from left to right:
microbe-associated molecular patterns (MAMPs) sensed by pattern recognition receptors on intestinal epithelial cells and DCs adjacent to cryptopatches
stimulate the further recruitment of B cells and T cells, causing the cryptopatches to develop into mature isolated lymphoid follicles. The isolated
lymphoid follicles release IgA-producing plasma cells – which are formed through T cell–dependent and –independent interactions – into the lamina
propria. Microbes also cross the epithelium and enter Peyer’s patches through M cells, from which they are endocytosed by DCs in the subepithelial
dome. Antigen-loaded DCs in the Peyer’s patch interact with local lymphocytes to induce T cell differentiation and T cell–dependent B cell maturation in
the germinal centre to induce the development of IgA-producing plasma cells that home to the lamina propria, where they release dimeric IgA for trans-
port into the intestinal lumen. Dendritic-cell-mediated luminal sampling of microbial products or transcytosis of bacteria across the epithelium results in
antigen loading of lamina propria dendritic cells, which then migrate through the afferent lymphatics vessels (not shown) to a draining mesenteric lymph
node to induce differentiation of effector T cells that traffic to the lamina propria. Shown on the far right, sensing of MAMPs stimulates the proliferation
of intestinal epithelial cells in crypts, resulting in their increased depth and, in the small intestine, increased density of Paneth cells. This sensing also
arms the intestinal epithelial cells for release of antimicrobial peptides (figure and legend from Maynard et al., 2012).

other lymphoid organs. However, when colonic injury was
induced with chemicals in Rorgt/ mice, such tLTs contributed
to exaggerated disease (Lochner et al., 2011).
Intestinal Barrier Function and Innate Immune
System Development
Microbiota and IECs
Gut epithelium is the most critical physical and physiological
frontline barrier that separates the intestinal microbiota from
the host, while at the same time transporting and absorbing
dietary substances for the host’s needs. The epithelial surface
is constantly shedding but rapidly self-renewing by specialized
stem cells that reside at the base of the intestinal crypt. Five
types of epithelial cells – Paneth cells, goblet cells, M cells,
absorptive enterocytes, and enteroendocrine cells, can all
perform immune functions that either facilitate the physical
clearance of infiltrating microbes and/or orchestrate innate
and adaptive immune machinery to maintain intestinal
homeostasis.
112 Physiology of the Immune System j Role of the Microbiota in Immune Development

IECs express a series of pattern recognition receptors (PRRs) to
sense microbial antigens, referred to as MAMPs. TLRs are a family
of cell surface PRRs, which are indispensible for sensing intestinal
microbiota to control IEC homeostasis and protect from epithe-
lial injury (Rakoff-Nahoum et al., 2004). Shortly after birth,
IECs become activated due to the stimulation of TLR4 by lipo-
polysaccharide (LPS), which is a major component of the outer
membrane of gram-negative bacteria. This spontaneous activa-
tion is microbial signal driven, since it is not seen in cesarean
section–delivered mice or TLR4-deficient mice. Within hours of
initial microbial exposure, the posttranscriptional downregula-
tion of a protein essential for TLR4 signaling, interleukin-1 (IL-
1) receptor–associated kinase 1, leads to the lifelong loss of
responsiveness of IECs to LPS stimulation. This programmed
negative-regulatory mechanism helps the host adapt to the rapid
microbial colonization of the intestine (Lotz et al., 2006). IEC
expression of TLR3 also shows an age-dependent increase due
to microbial colonization (Pott et al., 2012).
Intestinal antimicrobial peptides/proteins (AMPs) are
a group of natural antibiotics produced by multiple types of cells
lining the gut epithelium, but primarily by intestinal enterocytes
and Paneth cells. AMPs are predominantly present in the mucus
layer above the epithelial surface, either directly killing or inac-
tivating bacteria and thus creating a relatively sterile zone that
greatly reduces the direct contact of microbes and the epithelium
(Gallo and Hooper, 2012). Microbial signals are involved in the
regulation of the expression and secretion of most AMPs other
than a-defensins. One example is Paneth cell production of
angiogenin-4 which rapidly increases to adult levels during
weaning due to microbial colonization; this upregulation can
also be induced by monocolonization with Bacteroides thetaio-
taomicron (Hooper et al., 2003). Another example is the expres-
sion of C-type lectin RegIIIg, which primarily targets gram-
positive bacteria in the intestine, and requires microbial sensing
through TLR and MyD88 (Vaishnava et al., 2011).
Goblet cells contribute to immune defense by the production
of highly glycosylated mucin proteins that form a viscous layer
outside the epithelial surface to restrict the penetration of resident
microbes. The thickness of the mucus layer correlates with bacte-
rial load in the intestine (Petersson et al., 2011). MUC2/ mice
develop colitis spontaneously (Van der Sluis et al., 2006), and
following Citrobacter rodentium infection, MUC2 deficiency results
in exaggerated colonic pathology due to impaired clearance and
increased translocation of this intestinal pathogen (Bergstrom
et al., 2010). GF mice have fewer numbers and smaller-sized
goblet cells in the epithelium (Kandori et al., 1996), and corre-
spondingly the inner mucus layer is substantially diminished
compared to conventionally reared mice. These defects can be
restored with LPS or peptidoglycan exposure (Petersson et al.,
2011), indicating the importance of microbial stimulation on
goblet cell development and mucin production (Figure 2).
Furthermore, microbiota are involved in the regulation of
an array of other barrier pathways. For example, epithelial
cell expression of polymeric immunoglobulin receptors
(PIgR), which can bind IgA and IgM produced in the LP and
facilitate their transport across the epithelium into the intes-
tinal lumen, is strongly induced by microbial colonization
(Hooper et al., 2001; Hapfelmeier et al., 2010). Microbiota
also induce the glycosylation of IECs by stimulating group 3
innate lymphoid cell (see below) production of IL-22 and

Small intestine Large intestine

Intestinal
lumen

~50μm 50-200μm

Epithelium

Paneth Goblet Enterocytes

cell cell

AMPs Outer loose mucus

sIgA Inner mucus

Commensal bacteria

Figure 2 IECs contribute to intestinal barrier functions. Intestinal epithelial cells secrete mucin and a series of antimicrobial peptides (AMPs) to
create a spatial segregation of the commensal microbiota and the epithelium. MUC2 mucin is the major mucin protein in the intestine. It forms
a loose, nonadherent single mucus layer in the small intestine, and a two-layered mucus complex in the large intestine where the inner layer is
compact and firmly attached to the epithelium. The inner layer is 50–200 mm thick and hardly permeable by commensal bacteria. It converts to the
outer loose mucus layer at the luminal border, where commensal bacteria are densely colonized. In addition to mucin proteins, enterocytes and Pan-
eth cells (present only in the small intestine) can secrete AMPs to separate the microbiota from the epithelial surface. In the small intestine where the
inner mucus layer is absent, AMPs can form a  50 mm barrier to limit direct association of luminal bacteria with the epithelium.
 Physiology of the Immune System j Role of the Microbiota in Immune Development
lymphotoxin (Goto et al., 2014a). This benefits certain micro-
biota species that can use epithelial glycans as a nutritional
substrate and thus maintains colonization (Hooper et al.,
1999).
Microbiota and Innate Cells
IECs constantly interact with the innate cells present in the intes-
tinal lamina propria (LP) at steady state. Innate cells such as
dendritic cells (DCs), macrophages, and innate lymphoid cells
(ILCs), are responsible for antigen uptake and presentation,
degradation of cell debris and pathogens, as well as secreting
cytokines and chemokines in response to MAMP recognition,
linking microbiota, the epithelium, and adaptive immune
system all together to form a homeostatic system maintaining
mutualism. Intestinal microbiota plays an important role in
the regulation of innate cell phenotype and functions.
DCs are derived from a common progenitor in the bone
marrow, and migrate directly via blood or afferent lymph into
the intestine to develop into resident DCs and migratory DCs,
respectively (Persson et al., 2013). Colonization of bacteria trig-
gers the recruitment of LP CD103þ DCs to the epithelium for
antigen uptake and subsequent presentation to T cells in the
MLNs (Farache et al., 2013). Commonly, TLR recognition
induces DC production of IL-12 and subsequently promotes
Th1 cell differentiation. However, the abundance of TGFb and
retinoic acid (RA) in the gut environment facilitates the differen-
tiation of tolerogenic DCs, which produce IL-10 instead of IL-12
and promote the differentiation of regulatory T (Treg) cells, there-
fore contributing to the maintenance of gut homeostasis (Stein-
man et al., 2003; Coombes et al., 2007; Cording et al., 2014).
Macrophages have phenotypic overlap with DCs but distinct
functions. Both resident and inflammatory microphages in the
gut are derived from circulating Ly6Chi monocytes (Bain et al.,
2013), and their recruitment is CCR2-dependent in mice (Bain
et al., 2014). Although highly phagocytic and bactericidal, human
mucosal macrophages exhibit nonresponsiveness to a broad
range of microbial antigens and inflammatory stimuli at steady
state (Smythies et al., 2005; Lotz et al., 2006), exerting immune
surveillance functions at mucosal borders as well as contributing
to immune tolerance. Systemic macrophages isolated from
antibiotic-treated adult mice show defective antiviral-related
gene expression, impaired type I and II IFN production, weakened
antiviral functions, and increased susceptibility to influenza infec-
tion (Abt et al., 2012), indicating the role of microbiota in main-
taining macrophage function systemically as well as mucosally.
Microbiota also regulate the homeostasis of systemic
neutrophils via NOD1 recognition of microbial peptidoglycan
(Clarke et al., 2010). Neutrophils are normally not present in
the gut, but are rapidly recruited via chemokines in response
of intestinal pathogens and inflammation. Lacking the priming
by intestinal microbial signals, GF and antibiotic-treated mice
develop reduced numbers of bone marrow and circulating
neutrophils, as well as impaired killing functions against path-
ogens (Deshmukh et al., 2014).
During recent years, an important cell type belonging to
the innate immune system, termed ILCs, have been
discovered and are beginning to be characterized (Colonna,
2009; Spits and Di Santo, 2011; Spits and Cupedo,
2012; Sonnenberg and Artis, 2012; Walker et al., 2013;
113
Sonnenberg et al., 2013; Robinette et al., 2015). ILCs are
lymphocytes that lack antigen receptors but respond rapidly
to microbiota and cytokine signals. They develop from
a common precursor in an IL-7Ra- and Id2-dependent
manner, and differentiate into three groups depending on
the tissue and the environmental stimuli. In the gut, the major
type is Rorgtþ group 3 ILCs, which resemble their adaptive
counterpart Th17 cells by the production of IL-17A and
IL-22. Microbial induction of IL-22 production by ILC3 can
subsequently stimulate epithelial AMP and mucin expression,
as well as epithelial fucosylation (Satoh-Takayama et al.,
2008; Sonnenberg et al., 2011).
It has been controversial whether microbiota plays a role on
the generation of ILC3s. Studies show that the absolute number
of ILC3 subset NKp46þ cells is decreased in GF mice and can be
restored with bacterial colonization (Satoh-Takayama et al.,
2008; Sanos et al., 2009). However, others have found no
difference of either total or NKp46þ ILC3 numbers in GF,
antibiotic-treated, or SPF mice. Also, microbiota can suppress
ILC3 production of IL-22 by inducing epithelial cell IL-25
production, consistent with a negative regulatory loop for
ILC3 cells at steady state (Sawa et al., 2011).
ILC3s can produce GM-CSF in response to microbiota-
induced IL-1b secretion by intestinal macrophages. Impaired
GM-CSF production can lead to altered cell functions of mono-
nuclear phagocytes and Treg cells, and therefore disturbed gut
homeostasis (Mortha et al., 2014). A proper balance of this
macrophage-ILC3 loop is needed because excessive IL-1b
during Helicobacter hepaticus infection promotes an IL-17A
response in both CD4þ T cells and ILCs, leading to an exagger-
ated colonic inflammation (Coccia et al., 2012).
Microbiota and the Development of Adaptive Immune
System
Due to the active interactions with intestinal microbiota and
the mucosal immune system, the majority of lymphocytes
reside in the healthy intestine. In fact, based on observations
from immune-deficient mouse models such as severe
combined immunodeficiency mice and recombination-
activating gene-1/2 (RAG-1/2) knock out mice that have no
mature T and B cells, innate immune pathways are sufficient
to provide host protection in response to commensal micro-
biota stimulation. However, the vulnerability of such mice to
pathogens demonstrates the importance of the adaptive
immune system. Similar to the innate components of the gut
mucosa, the development and maturation of gut adaptive
immune system are actively driven by the microbiota as well.
Microbiota and T cells
Distinct subsets of T cells populate different parts of the intes-
tinal wall – intraepithelial lymphocytes (IELs) are mainly
composed of CD8þ T cells, while CD4þ T cells are the major
cell type in the LP. The recruitment and function of gut T cells
are microbiota driven, because GF or antibiotic-treated mice
have reduced numbers of IELs and LP CD4þ T cells in the
gut, as well as altered immune functions (Ichinohe et al.,
2011; Chung et al., 2012). Interestingly, although exposed to
114 Physiology of the Immune System j Role of the Microbiota in Immune Development
rapidly colonizing microbiota, LP CD4þ T cells remain imma-
ture throughout the postnatal period (Torow et al., 2015).
Microbial recognition through nucleotide oligomerization
domain 2 (NOD2) plays a critical role in IEL homeostasis
(Jiang et al., 2013). Functionally, unlike peripheral CD8 T cells,
gut IELs freshly isolated from conventional mice are constitu-
tively cytolytic. However, IELs isolated from GF mice have
substantially reduced expression of Thy-1 and have almost no
lytic function, revealing the crucial role of microbiota on IEL
activation (Lefrancois and Goodman, 1989).
Effector T Cell Development
Lack of microbial exposure in early life leads to a skewed
LP T cell development toward Th2 phenotype (Mazmanian
et al., 2005; Cahenzli et al., 2013), most likely due to the
impaired development of other CD4þ T cell subsets including
Th1, Th17, and Treg cells. Helminth infection greatly induces
proliferation and activation of gut Th2 cells as well as Treg cells,
which in turn inhibit the differentiation of Th1 cells, thus
pig worm ingestion has been proposed as a therapeutic
intervention for inflammatory bowel disorders (Elliott and
Weinstock, 2012).
The normal adult mouse LP shows a Th1/Th17 dominant
effector phenotype. Both Th1 and Th17 cells contribute to
normal intestinal homeostasis but can also mediate inflamma-
tion in IBD and other autoimmune diseases by their cytokine
production and their ability to recruit neutrophils (Strober
and Fuss, 2011; Feng et al., 2011; Neurath, 2014; Harbour
et al., 2015). Several microorganisms have been shown to
participate in the induction of Th1 cells mucosally and system-
ically (Hsieh et al., 1993; Mazmanian et al., 2005; Jakobsson
et al., 2014). Although pathogenic during inflammation, gut
Th1 cells exert important immune protective functions against
enteric pathogens such as Listeria monocytogenes and interact
with the innate immune system to maintain gut homeostasis.
It is shown that Th1-associated cytokine IFNg, induced in
wild-type mice by bacterial colonization, contributes to the
inhibition of innate IL-23 expression, which can function as
a pathogenic factor of intestinal inflammation (Sheikh et al.,
2010). IFNg also participates in the upregulation of major
histocompatibility complex (MHC)-II molecules on IECs
(Thelemann et al., 2014) and in the control of T cell expansion
in lymphopenic mice (Do et al., 2014), contributing to preven-
tion of intestinal inflammation.
Th17 cells are dependent on transcription factors Rorgt
and aryl hydrocarbon receptor (AhR) for their development
(Ivanov et al., 2006; Yang et al., 2008b), and secrete IL-17A,
IL-17F, and IL-22 as their main cytokines. Th17 cells share
the developmental requirement of microbiota-driven TGFb
with induced Tregs (Mangan et al., 2006; Qin et al., 2009;
Lee et al., 2012), but in vitro Th17 cell differentiation also
needs IL-6. The differentiation of intestinal Th17 cells is
dependent on microbial signals, in that GF animals rarely
have them (Ivanov et al., 2008, 2009). It is shown that at
steady state, the induction of intestinal Th17 cells needs
microbiota-dependent IL-1b, but not IL-6 (Shaw et al.,
2012). In addition, other microbiota-induced factors, such
as ATP (Atarashi et al., 2008) and IL-23, are shown to help
with intestinal Th17 cell development or maintenance, respec-
tively. Among the microbiota, segmented filamentous bacteria
(SFB) appear to be potent stimulators of the Th17 subset (Iva-
nov et al., 2008, 2009). SFB is a gram-positive, spore-forming
anaerobic organism that is classified as a unique member of
the Clostridiales, which in the vegetative state forms typical
long filaments. SFB is an auxotroph and interacts closely
with the epithelium and PPs of the terminal ileum. Coloniza-
tion of SFB induces a moderate upregulation of a broad range
of proinflammatory cytokines with mechanisms still not
known, although recently it is postulated that TLR2 recogni-
tion might be involved in this process (Gaboriau-Routhiau
et al., 2009; Schnupf et al., 2015). SFB specifically induces
the differentiation of endogenous Th17 cells in the small
intestine by MHC-II-dependent antigen presentation through
intestinal DCs (Goto et al., 2014b) and stimulation of epithe-
lial production of serum amyloid A (SAA) (Ivanov et al.,
2009). The function of Th17 cells in the gut remains contro-
versial. Th17 cells help maintain gut homeostasis under steady
state conditions and provide host protection against certain
gut pathogens by facilitating epithelial production of AMPs,
B cell production of IgA, and upregulation of PIgR. However,
Th17 cells have been implicated in the pathogenesis of IBD
and other autoimmune diseases. During colitis, Th17 cells
differentiate into an IL-17AþIFNgþ double-positive CD4 T
cell phenotype, and then into IFNgþ single positive ex-Th17,
Th1 cells. The latter cell has recently been identified as the
effector cell driving experimental colitis (Harbour et al.,
2015). Interestingly, regarding antigen specificity, the majority
of SFB-induced endogenous Th17 cells in the gut react
strongly to SFB antigen stimulation, suggesting that SFB
does not work as an adjuvant in inducing Th17 cells to other
commensals (Yang et al., 2014). How SFB-reactive Th17 cells
migrate to organs other than the gut and contribute to autoim-
mune diseases remain to be elucidated (Wu et al., 2010).
Regulatory T Cell Development
The differentiation of naïve T cells in the gut occurs in the PPs
and MLNs. When CD103þ DCs sample intestinal antigens and
migrate to the draining MLNs, they present the processed
peptides to activate the naïve T cells (Pabst and Mowat,
2012). Intestinal CD103þ DCs express high levels of aldh1a2,
which can convert the vitamin A metabolite retinal into RA.
RA together with TGFb favors Treg development in the gut
(Coombes et al., 2007; Sun et al., 2007). Gut Treg cells are
required for the establishment of oral tolerance and are respon-
sible for suppression of effector T cell responses and the resolu-
tion of intestinal inflammation (Hadis et al., 2011). They are
also indispensable for the maintenance of gut immune homeo-
stasis with the microbiota. The pathogenesis of IBD is proposed
to be uncontrolled immune responses to commensal antigens
in the gut in a genetically susceptible host (Maloy and Powrie,
2011). A number of current experimental mouse models are
due to genetic targeting of the Treg pathway. Treg cells perform
their immunosuppressive function with mechanisms such as
the production of anti-inflammatory cytokines IL-10, TGFb,
and IL-35 and consumption of IL-2; they can also inhibit
Ca2þ signaling and subsequently inhibit the activation of
NF-kB and NFAT pathways in conventional T cells (Schmidt
et al., 2012). A number of studies have shown that Treg cells
stimulate the production of sIgA to promote host-microbiota
mutualism (Kawamoto et al., 2014). One possible pathway is
 Physiology of the Immune System j Role of the Microbiota in Immune Development 115

Clostridium
SFB IV and XIVa B. fragilis ASF

Intestinal
lumen

Epithelium
IEL

• SAA
• DC activation • SCFA PSA TLR activation
• Bacteria-derived • GPR43 activation
Lamina ATP
propria

Th17 Treg
RORgt+ Foxp3+

T
Naïve

Figure 3 Microbiota induction of intestinal Treg/Th17 cell differentiation. Commensal bacteria such as Clostridium cluster IV and XIVa, Bacteroides
fragilis and altered Schaedler flora (ASF) promote the differentiation and expansion of Treg cells in the intestine through various mechanisms,
whereas microbial-derived ATP and segmented filamentous bacteria (SFB) stimulate the induction of intestinal Th17 cells. Th17 and Treg cells are
both plastic and can convert into each other and into other T cell subsets under certain conditions.

that Treg cells can downregulate Foxp3 expression in the PPs
and convert into follicular helper T (Tfh) cells that facilitate B
cell synthesis of IgA (Tsuji et al., 2009). Treg cells also provide
a survival signal for IgAþ B cells in the LP. Depletion of CD25þ
Treg cells results in decreased IgAþ B cells in the LP and corre-
spondingly reduced total and commensal-specific sIgA levels in
the gut (Cong et al., 2009).
Microbiota play an important role in the differentiation and
expansion of Treg cells. Due to the high density of microbes
and microbial metabolic products in the large intestine, it
becomes a primary location for the induction of peripherally
derived Treg cells and the expansion of thymus-derived Treg
cells. There are a number of different microbiota products
that can induce Treg cells. For example, the immunomodula-
tory molecule PSA derived from commensal bacteria Bacteroides
fragilis drives the induction of colonic Treg cells and IL-10
expression in a TLR2-dependent manner (Round and
Mazmanian, 2010; Round et al., 2011). Colonization of GF
mice with 46 strains of Clostridium that primarily belong to
IV and XIVa clusters specifically induced IL-10þCTLA4þHelios
Tregs and TGFb production in the colon (Atarashi et al., 2011).
Colonization of GF mice with altered Schaedler flora (ASF)
stimulated both Heliosþ and Helios Treg populations, which
is shown to be dependent on TLR-mediated sensing (Geuking
et al., 2011). Most recently, several groups showed that gut
bacterial fermentation products, short-chain fatty acids, exclu-
sively induce Treg cells, but not other T cell subsets, in the colon
in a G protein–coupled receptor 43 (GPR43)-dependent
manner (Figure 3; Arpaia et al., 2013; Furusawa et al., 2013;
Smith et al., 2013). GPR43 deficiency leads to increased inflam-
matory responses in different disease models (Maslowski et al.,
2009). Gut microbiota also regulate the expression of another
G protein receptor – GPR15, which can promote homing of
Treg cells into large intestinal LP and contribute to gut homeo-
stasis (Kim et al., 2013). In addition, microbiota and their
metabolic products impact the epigenetic regulation of Treg
cells as well (Furusawa et al., 2013; Obata et al., 2014).
Microbiota-driven Treg cells are functionally protective in gut
inflammation and in allergic disease models.
LP T cells show high potential of plasticity due to the
complexity of the gut environment. The most reported plas-
ticity occurs between Treg/Th17 and Th17/Th1 conversions
(Yang et al., 2008a; Lee et al., 2009; Zhou et al., 2009; Brown
et al., 2015; Gagliani et al., 2015; Harbour et al., 2015; Liu
et al., 2015). Treg and Th17 cells can convert into Tfh cells in
the PPs and facilitate high-affinity intestinal IgA production
(Tsuji et al., 2009; Hirota et al., 2013). Whether microbial
signals are involved in plasticity still remains to be investigated.
Microbiota and B Cells
IgA, as a noninflammatory immunoglobulin, does not possess
the ability to bind common Fc receptors and activate the
complement system, and represents a unique mechanism of
mucosal immune defense. It is produced in the largest quanti-
ties of any antibody isotypes (Macpherson et al., 2008), and
can be made through T-independent and -dependent manners
by B1 and B2 B cells, respectively (Fujihashi et al., 1996;
Macpherson et al., 2000). After being produced by plasma cells
in the LP, IgA dimers are transcytosed by PIgR expressed on
epithelial cells from the LP into the intestinal lumen, where
they bind to the surface of commensal bacteria and limit
them from penetrating the epithelium. Microbial colonization
predominantly drives the production of intestinal IgA, because
GF mice have substantially impaired IgA production in the
intestinal mucosa whereas serum IgA levels are less affected
116 Physiology of the Immune System j Role of the Microbiota in Immune Development
(Macpherson et al., 2008). Commensal bacteria such as Morga-
nella morganii or SFB can strongly stimulate germinal center
reactions and IgA secreting B cells in the PPs (Shroff et al.,
1995). SFB can also induce de novo IgA production in tLTs in
the LP in mice lacking PPs and ILFs (Klaasen et al., 1993;
Lecuyer et al., 2014). Microbiota can also contribute to IgA
catabolism, as commensal bacteria belonging to the Sutterella
genus can degrade secretory component and IgA in the intes-
tinal lumen, leading to exaggerated intestinal damage upon
chemical challenge, and this pattern is transmissible from
mother to offspring (Moon et al., 2015).
B cell differentiation and function is regulated by gut micro-
biota. For example, in response to microbial stimulation at
steady state, mucosal B cells can produce tumor necrosis factor
(TNF) and inducible nitric oxide synthase (iNOS) for optimal
intestinal IgA production and maintenance of microbiota
diversity (Fritz et al., 2012). During inflammation, B cells can
acquire a regulatory phenotype and start to produce IL-10;
microbiota-driven IL-1b and IL-6 production is required to
promote this process (Rosser et al., 2014). In the absence of
diversified microbial exposure, neonatal mice develop more
IgE switching B cells at the mucosal sites and correspondingly
higher serum IgE levels, in a CD4 T cell– and IL-4-dependent
manner, leading to increased surface-bound IgE on mast cells
and exaggerated oral-induced allergic reaction with ovalbumin
(Cahenzli et al., 2013). MyD88-dependent basophil activation
is also involved in this process (Hill et al., 2012).
Microbiota and Immunological Diseases
With the profound impact on the development of gut immune
system, an unbalanced composition of gut microbiota or ‘dys-
biosis’ can lead to disturbed immune responses and has been
linked to a variety of autoimmune and allergic diseases in
recent years (Berer et al., 2011; Horai et al., 2015). One
example is depletion of transcription factor T-bet in Rag/
mice resulting in the development of spontaneous colitis,
which can be treated with broad-spectrum antibiotics, indi-
cating a microbiota-driven pathology. Cohousing of colitic
Tbet/Rag/ with wild-type mice results in colitis develop-
ment in the latter as well, suggesting that pathogenic micro-
biota is sufficient to induce inflammation in the normal gut
(Garrett et al., 2007). Similarly, perturbation of inflammasome
NLRP6 or IL-22 pathway also causes a shift in gut microbiota
and subsequently leads to either colonic inflammation or
a greater risk to develop colonic inflammation, respectively
(Elinav et al., 2011; Zenewicz et al., 2013).
In accordance with findings in animal studies, human IBD
has been associated to dysbiosis. A subset of patients with IBD
develops an altered gut microbiota with greatly increased Proteo-
bacteria and reduced Bacteroidetes and Firmicutes (Frank et al.,
2007; Gevers et al., 2014). Children living in developing coun-
tries with greater intestinal bacterial diversity have lower frequen-
cies of inflammatory disorders in adulthood including IBD. Also,
early-life exposure to antibiotics has been shown highly associ-
ated with IBD development later in life (De Filippo et al.,
2010; Hviid et al., 2011; Kronman et al., 2012; Lin et al., 2013).
The increasing incidence of autoimmune and allergic
diseases in industrialized countries occurs concurrently with
the cleanliness of environment and the broad use of antibiotics
(Bach, 2002; Noverr and Huffnagle, 2004; Okada et al., 2010),
consistent with the concept of the hygiene hypothesis, which
was first brought up by Strachan in the late 1980s (Strachan,
1989). The vast environmental transition from the sterile
uterus to the outside world full of microbes greatly stimulates
immune development and maturation, making the early child-
hood an important period for the establishment of a well-
functioning immune system. The human data linking altered
early-life microbial exposure with impaired immune develop-
ment and long-term susceptibility of autoimmune and allergic
diseases demonstrate the significance of proper microbiota
colonization during this transitional period (Lundell et al.,
2011, 2012; Olszak et al., 2012; Cahenzli et al., 2013; Munyaka
et al., 2014). As we progress in the application of comprehen-
sive metagenomic and metatranscriptomic analysis, a better
understanding of how microbiota impact immune develop-
ment will provide us with new insights in immune disease
prevention and treatment.
See also: Allergy: The Hygiene Hypothesis of Allergy and
Asthma. Anatomy and Microanatomy of the Immune System:
Tertiary Lymphoid Tissues. Cells of the Innate Immune System:
Dendritic Cells and Dendritic Cell Subsets; Macrophage
Activation and Polarization; Neutrophils – Role in Innate
Immunity. Cytokines and Their Receptors: Inflammasomes.
Development of T Cells and Innate Lymphoid Cells:
Development of Regulatory T Cells in the Thymus; Innate
Lymphoid Cells Type 3. Immune Deficiency: Combined T Cell
and B Cell Deficiency – SCID Forms: TBþ; Severe
Combined Immune Deficiency with Absence of B and T
Lymphocytes (TBNKþSCIDs): The Key Function of V(D)J
Recombination for Lymphocyte Development. Immunity to
Bacterial, Parasitic and Fungal Infections: The Hygiene
Hypothesis and Immunity to Parasitic Helminths. Molecular
Aspects of Innate Immunity: The Mucins. Structure and
Function of Diversifying Receptors: Structure and Function of
IgA. T Cell Activation: Th17 and Th22 Cells; Treg Cells.
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Further Reading
Abreu, M.T., 2010. Toll-like receptor signalling in the intestinal epithelium: how
bacterial recognition shapes intestinal function. Nat. Rev. Immunol. 10, 131–144.
Hooper, L.V., Littman, D.R., Macpherson, A.J., 2012. Interactions between the
microbiota and the immune system. Science 336, 1268–1273.
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immunity and inflammatory disease. Nat. Rev. Immunol. 13, 321–335.
Mowat, A.M., Agace, W.W., 2014. Regional specialization within the intestinal immune
system. Nat. Rev. Immunol. 14, 667–685.
Peterson, L.W., Artis, D., 2014. Intestinal epithelial cells: regulators of barrier function
and immune homeostasis. Nat. Rev. Immunol. 14, 141–153.
Saleh, M., Elson, C.O., 2011. Experimental inflammatory bowel disease: insights into
the host-microbiota dialog. Immunity 34, 293–302.
Underhill, D.M., Iliev, I.D., 2014. The mycobiota: interactions between commensal
fungi and the host immune system. Nat. Rev. Immunol. 14, 405–416.