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Pathogenesis of Necrotizing Enterocolitis : Modeling the Innate Immune

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DOI: 10.1016/j.ajpath.2014.08.028

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The American Journal of Pathology, Vol. 185, No. 1, January 2015

ajp.amjpathol.org

REVIEW
Pathogenesis of Necrotizing Enterocolitis
Modeling the Innate Immune Response
Scott M. Tanner,*y Taylor F. Berryhill,* James L. Ellenburg,* Tamas Jilling,z Dava S. Cleveland,x Robin G. Lorenz,y and
Colin A. Martin*

From the Departments of Pediatric Surgery* and Pathologyy and the Division of Neonatology,z Department of Pediatrics, University of Alabama at
Birmingham, Birmingham; and the Department of Pediatric Pathology,x Children’s Hospital of Alabama, Birmingham, Alabama

Accepted for publication August 27, 2014.

Address correspondence to Colin A. Martin,
M.D., Department of Pediatric Surgery,
University of Alabama at Birmingham, 300
Lowder Bldg., 1600 7th Ave., South
Birmingham, AL 35233-1711; or Robin G.
Lorenz, M.D., Ph.D., Department of
Pathology, University of Alabama at
Birmingham, 1825 University Blvd., SHEL
602, Birmingham, AL 35294-2182. E-mail:
cmartin7@uab.edu or rlorenz@uab.edu.
Necrotizing enterocolitis (NEC) is a major cause of morbidity and mortality in premature infants.
The pathophysiology is likely secondary to innate immune responses to intestinal microbiota by
the premature infant’s intestinal tract, leading to inflammation and injury. This review provides an
updated summary of the components of the innate immune system involved in NEC pathogenesis.
In addition, we evaluate the animal models that have been used to study NEC with regard to the
involvement of innate immune factors and histopathological changes as compared to those seen in
infants with NEC. Finally, we discuss new approaches to studying NEC, including mathematical
models of intestinal injury and the use of humanized mice. (Am J Pathol 2015, 185: 4e16; http://
dx.doi.org/10.1016/j.ajpath.2014.08.028)

Necrotizing enterocolitis (NEC) is a disorder characterized
by intestinal necrosis in premature infants that results in
significant morbidity and mortality.1 Approximately 7% of
infants with a birth weight between 500 and 1500 g develop
NEC.1 The pathogenesis is characterized by intestinal
inflammation that can progress to systemic infection/
inflammation, multiorgan failure, and death. The bowel is
distended and hemorrhagic on gross inspection. On micro-
scopic examination, signs of inflammation, mucosal edema,
epithelial regeneration, bacterial overgrowth, submucosal
gas bubbles, and ischemic transmural necrosis are seen
(Figure 1, AeE).2
Currently the pathogenesis of NEC is believed to have
multifactorial causes, including intestinal immaturity and
microbial dysbiosis. Intestinal immaturity leads to a compro-
mised intestinal epithelial barrier, an underdeveloped immune
defense, and altered vascular development and tone. The
compromised epithelial barrier and underdeveloped immune
system, when exposed to luminal microbiota that have been
shaped by formula feedings, antibiotic exposure, and Cesarean
delivery, can lead to intestinal inflammation and sepsis.
Despite therapeutic success in animal model systems, there are
relatively few therapeutic strategies that have allowed for
significantly improved outcomes in infants with NEC. Two
hurdles that persist are our incomplete understanding of the
developing immune system in premature infants and our
inability to adequately replicate these complex factors in ani-
mal models.3,4 This review summarizes the complex intestinal
immune system in premature infants and details what is known
about the involvement of innate immune factors in NEC, both
in animal models and in human disease.
The Neonatal Intestinal Ecosystem
The neonatal intestinal ecosystem is extremely fragile. At
birth, the newborn is exposed to the external environment for
Supported in part by grants from the Kaul Pediatric Research Institute of the
Alabama Children’s Hospital Foundation (C.A.M.), NIH grant P01 DK071176
(R.G.L.), Juvenile Diabetes Research Foundation Grant #36-2008-930
(R.G.L.), Crohn’s and Colitis Foundation of American Grant #26971 (R.G.L.),
and the Digestive Diseases Research Development Center, University of
Alabama at Birmingham grant P30 DK064400 (R.G.L.). Aspects of this project
were conducted in biomedical research space that was constructed with funds
supported in part by NIH grant C06RR020136 (R.G.L.).
Disclosures: None declared.
A guest editor acted as Editor-in-Chief for this manuscript. No person at the
University of Alabama at Birmingham was involved in the peer review process
or final disposition for this article.

Copyright ª 2015 American Society for Investigative Pathology.

Published by Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.ajpath.2014.08.028
 Pathogenesis of Necrotizing Enterocolitis

Figure 1 Examples of the various grades of morphological damage in hematoxylin and eosinestained specimens. AeE: Representative samples of premature
infants with necrotizing enterocolitis. A: Age-matched control from patient with jejunal atresia. B: Mild injury with hemorrhagic necrosis of mucosa and loss of villus
tip architecture. C: Progressive injury with inflammatory infiltration of muscularis with complete villus destruction. D: Severe muscular and epithelial damage with
complete loss of mucosa. E: Perforation with transmural necrosis with complete loss of epithelial and muscular architecture. FeJ: Representative samples from
intestinal injury secondary to gavage feeding in the setting of hypothermia and hypoxia in neonatal rats. F: Intact morphology, grade 0. G: Sloughing of villus tips,
grade 1. H: Mid-villus necrosis, grade 2. I: Loss of villi, grade 3. J: Complete destruction of the mucosa, grade 4. Insets in FeJ show higher magnified portions of the
same sections, corresponding to the boxed regions. KeO: Representative images of tissue injury secondary to 60 minutes of intestinal ischemia and 90 minutes of
reperfusion in 2-week-old mice. K: Sham-operated mice (no ischemia). L: Villus tip necrosis. M: Mid-villus necrosis. N: Loss of villus architecture. O: Complete loss of
mucosal architecture. FeJ, reprinted with permission from Nature Publishing Group.28 Scale bars Z 50 mm (AeE, KeO). Original magnification, 20 (AeO, main
images, and FeJ, insets).

the first time, and the immune response must begin to
distinguish between self and nonself. In particular, the
recognition of food antigens and commensal microbiota must
be distinguished from that of potential pathogens. This pro-
cess is even more challenging in premature infants, as they
are typically placed on broad-spectrum antibiotics that disrupt
both the timing and the diversity of the initial bacterial
colonization of the intestinal tract.1 In addition, the immature
epithelial barrier appears to be more sensitive to the detection
of bacteria5,6 and more susceptible to bacterial translocation,7
allowing for both an unwarranted inflammatory response to
commensals and the translocation of pathogens, both of
which may contribute to intestinal damage.
The innate immune system is the first line of defense
against infections. Innate immune cells respond in a
nonspecific manner and do not confer long-lasting immunity
to the host.8 The major components are cells (including
macrophages, neutrophils, dendritic cells (DCs), natural killer
cells, B1 B cells, innate lymphoid cells, and gd T cells) and
anatomical barriers (such as the intestinal epithelium and the
gastrointestinal mucus layer). In addition, the presence of
commensal microbiota can serve as a part of the innate im-
mune system by preventing the colonization by pathogenic
bacteria (colonization resistance).
Mucosal Innate Immunity
Several major components of the human mucosal immune
system are in place before birth. These components include
IgMþ B cells; gd T cells found in the intraepithelial
lymphocyte compartment, which are seen as early as 12 to
15 weeks of embryonic age; and Peyer’s patches, which can

The American Journal of Pathology - ajp.amjpathol.org 5

Tanner et al
be seen by about 30 weeks of gestation.9 This development
contrasts with that of the murine mucosal system, in which
the same B cells and intraepithelial lymphocytes are seen
only about 1 day before birth and macroscopic Peyer’s
patches are not seen until at least 1 week after birth.10 These
differences in intestinal immune system development imply
that a newborn mouse may have intestinal immaturity very
similar to that of a premature infantdone reason that murine
models are widely used to study the immune mechanisms
that predispose to NEC development.
Anatomical Barriers
Separation of the intestinal lumen from the rest of the organism
is accomplished through a physical barrier established by the
intestinal epithelial cells. All intestinal epithelial cells,
including enterocytes, Paneth cells, and goblet cells, play an
important role in maintaining barrier integrity. The barrier is
maintained by the presence of tight junctions between the
epithelial cells. In humans, tight junctions are formed by 10
weeks of gestation and are composed of claudins, junctional
adhesion molecules, and zonula occludins.11
Goblet cells, positioned throughout the intestine, are
involved in the secretion of mucin glycoproteins, which
generate the mucus layer of the intestine.12 Goblet cells can
be found as early as 9 to 10 weeks of gestation,12 and mucins
reach adult levels by 27 weeks of gestation.13 Animal model
studies suggest that mucus gene expression is influenced by
bacterial colonization, that mucus protein glycosylation pat-
terns are developmentally regulated, and that glycosylation
alters interactions with bacterial pathogens; however, equiv-
alent studies in humans have not been reported.14e16 Mucus
forms in two distinct layers; the outer, thicker layer prevents
intestinal bacteria from reaching the epithelial layer, whereas
the inner layer is vital for cell signaling if a disruption oc-
curs.12,17 In addition to preventing bacterial interaction with
the epithelium, the mucus layer also provides scaffolding for
antimicrobial peptides (AMPs) and secretory IgA (SIgA),
which are discussed in the following section.
Microbial Sensors and AMPs
Should pathogenic or commensal bacteria penetrate the
mucus layer and reach the epithelial cells, pattern-recognition
receptors (PRRs) are able to sense the presence of the bacteria
and initiate an appropriate response. The most common PRRs
are Toll-like receptors (TLRs) and nucleotide-binding oligo-
merization domains (NODs), which are expressed by both
epithelial cells and immune cells, particularly macrophages
and DCs, throughout the intestinal compartment. The PRRs
detect common bacterial structures, such as lipopolysaccha-
ride (LPS), flagella, and CpG-rich DNA. Current dogma in-
dicates that abnormal TLR4 signaling in the premature
intestinal epithelium is associated with NEC, perhaps by
increasing platelet-activating factor (PAF) levels. However,
on detection of microbial components, protective PRRs can
6 ajp.amjpathol.org
initiate signaling pathways leading to the production of
AMPs, IgA, and epithelial healing factors.
Paneth cellsdfound at the base of the intestinal cryptsd
are yet another secretory cell type that provides protection to the
host. Paneth cells are present at around 13 weeks of gestation
and produce AMPs, which function to disable or kill microor-
ganisms that have entered the digestive tract. Enterocytes,
macrophages, and human neutrophils can also produce AMPs,
including a-defensins, b-defensins, lysozyme, and type IIA
secretory phospholipase A2, with a-defensins and lysozyme
being the most common in the neonatal intestine.17 Type IIA
secretory phospholipase A2 hydrolyzes the phospholipids in
bacterial cell walls, which results in the production of arach-
idonic acid and lyso-PAF.18 As lyso-PAF is converted to the
highly active inflammatory mediator PAF, type IIA secretory
phospholipase A2 provides a mechanism that is at the inter-
section of bactericidal and inflammatory host defenses.
Luminal and Environmental Factors
As the newborn immune system begins its development, SIgA
is transferred via maternal breast milk. Additionally, growth
factors, cytokines, and other immune modulators are compo-
nents of human milk, including IL-6, tumor necrosis factor a,
IL-10, transforming growth factor (TGF)-b, and lysozyme.19
Newborns acquire IgA through passive transfer via the inges-
tion of human milk after birth. The levels of IgA in human milk
remain high throughout the breastfeeding period and help to
provide protection and aid in the colonization of commensal
bacteria.20 Although neonatal IgA production begins at
approximately 2 weeks after birth, it does not reach adult values
until 2 to 6 years of age.21 Maternal IgA in milk is thought to
react with the microbiota present in the maternal gastrointestinal
tract, and the passive transfer of these antibodies not only is
thought to prevent the adherence of bacteria to mucosal surfaces
and bacterial penetration into the mucosa but also is vital for the
formation and composition of the newborn’s intestinal micro-
biota.19 Maternal milk has been shown to promote colonization
with bificobacteria.22 As NEC cases are more highly associated
with formula feeding, lower numbers of bifidobacteria and
bacteroidetes, and higher proportions of proteobacteria and
actinobacteria, it would appear that the shaping of the intestinal
microbiota by the maternal IgA is one of the crucial factors in
preventing NEC.23 Discussion of the multiple studies that have
investigated the dysbiosis seen in NEC are outside of the scope
of this review, but those studies were recently reviewed by
Torrazza and Neu.24
IL-10 and TGF-b are anti-inflammatory cytokines involved
in regulating T-cell responses and in down-regulating proin-
flammatory secretions from macrophages and neutrophils.
Neonatal monocyte and T cellederived IL-10 levels are
significantly lower than are adult levels; however, IL-10 has
been detected in human milk, suggesting an important role for
milk-derived IL-10 in neonatal immune development.25 A
recent study has indicated that a low blood TGF-b level is
associated with a higher risk for NEC.26
- The American Journal of Pathology

Modeling NEC
Numerous animal models are currently used for studying NEC
disease pathogenesis. Although each model has specific ad-
vantages in mimicking certain aspects of the human disease, it
has limitations,3 including the fact that laboratory animal
models lack genetic diversity, are subject to static environ-
mental conditions, and do not have innate immune and
intestinal development identical to that in humans.27 However,
human tissue is also not optimal, as surgical specimens are
often necrotic and may not reflect the mechanisms that lead to
the development of NEC. An ideal model of NEC would: i) be
based on the mediators thought to contribute to and induce
human disease, ii) take into account the developmental dif-
ferences specific to the neonatal period, iii) have impaired
intestinal restitution and development of necrosis, iv) include
factors shown to play a role in the human disease (prematurity,
hypoxic stress, formula feeding, dysbiosis), and v) have
increased severity in the ileum. As this review will demon-
strate, there is no model that perfectly replicates NEC; how-
ever, each of the following models is a useful tool for
evaluating the specific mechanisms of the disease (Table 1):
gavage/hypoxia (G/H) model, ischemia/reperfusion (I/R)
model, PAF administration, Paneth cell depletion, xenograph
model, infection model, and chemical injury models.
G/H Model
Perhaps the most commonly cited animal model of NEC is
the G/H model, which has been described in mice, rats, and
piglets.28,29 This model links the altered vascular develop-
ment and tone that are thought to be components of intestinal
immaturity with early exposure to cold stress and a nonmilk
diet. Rats are popular for use in these studies because they are
easier to gavage than are mouse pups and are easier and less
expensive to maintain than are piglets. The G/H models are
characterized by sloughing epithelial cells, severe edema in
the submucus and muscle layers, villous damage and
destruction, separation of the mucosa and lamina propria,
and, ultimately, complete destruction and necrosis of all
Table 1 Accuracy of Current Animal Models in Incorporating the Components of Human NEC
Necrotizing enterocolitis models
Components of an optimal animal model G/H
Based on predicted mediators of NEC pathogenesis Yes
Reflects developmental differences specific to the
neonatal period/prematurity
Impaired intestinal restitution Yes
Development of necrosis Yes
Hypoxic stress Yes
Temporal relationship to feeding/formula feeding Yes
Dysbiosis Yes
More severe disease in the ileum Yes
The Institutional Care and Use Committee and the institutional review board of the University of Alabama at Birmingham approved all experiments.
G/H, gavage/hypoxia; I/R, ischemia/reperfusion; NEC, necrotizing enterocolitis; PAF, platelet-activating factor.
The American Journal of Pathology - ajp.amjpathol.org
Pathogenesis of Necrotizing Enterocolitis
epithelial structures, representing a presentation similar to
that of human NEC (Figure 1, FeJ).30 This model has been
adapted and more fully understood over the years, and it is
now known that hypoxia, formula gavage, and intestinal
bacteria are required for the development of NEC-like
pathophysiology and histological factors.31 The mouse G/H
model was originally described with pups delivered by Ce-
sarean section (E20-21) but has recently been simplified to
use newborn pups gavaged with adult commensal bacte-
ria.28,29 This simplified model will encourage the use of the
plethora of available knockout and transgenic mice to further
our understanding of NEC. In addition, the G/H model has also
has been used for linking several components of the innate
immune system to the pathogenesis of NEC. Mucin produc-
tion, SIgA, TLR4 signaling, AMP expression, and epithelial
junctional proteins have all been shown to impact the degree of
intestinal injury in animals that are subjected to the G/H model
(details in Proposed Innate Immune Mechanisms Leading to
NEC); however, the role of innate immune cells in the path-
ogenesis of NEC has not been investigated to date.
Challenges associated with the G/H model include sig-
nificant variability across research groups regarding the
specific age at which the protocol is started, the type of
formula administered, whether the formula is supplemented
with bacteria and/or LPS, and the modality of hypoxia
treatment. These challenges make it difficult to reproduce
the model between laboratories and to unify the knowledge
gained in the multitude of studies. Although the piglet
model is not widely used because of costs and a lack of
reagents, the piglet gastrointestinal system offers the
resemblance perhaps closest to that in humans, particularly
in the neonatal and newborn stages.32 In pigs, the hypoxia
and gavage are not always paired because one insult (either
hypoxia/hypothermia or formula feedings) can result in
NEC-like illness.33
I/R Model
The I/R model of intestinal injury is caused by occluding
the superior mesenteric artery, thus obstructing blood flow
PAF Paneth cell Infection and
I/R administration depletion Xenograft chemical injury
Yes Yes Yes Yes
Yes
Yes Yes
Yes Yes Yes Yes
Yes
Yes Yes Yes
Yes
7
Tanner et al
to the small bowel for a short time, followed by a period of
reperfusion.34 In the intestine, the initial ischemia results in
damage to the epithelial cells, whereas the subsequent
reperfusion injury causes significant damage to the rest of
the mucus layer.35 Histologically, this model is typically
evaluated based on the extent of necrosis in the intestine,
ranging from necrosis of the villus tip to transmural
necrosis penetrating the muscle layer (Figure 1, KeO).36
This model attempts to replicate the ischemia that is
thought to occur before NEC; however, it is more accu-
rately classified as a model of intestinal injury rather than as
a true model of NEC. The I/R model is particularly useful
for investigating the role of free-radical damage in intestinal
injury, as high levels of oxidative damage are induced after
reperfusion (mimicking the potential reperfusion after an
ischemic event in newborn humans). The primary strength
of the I/R model is that it leads to defined and reproducible
mucosal injury and barrier dysfunction. Both TLR4 and
junctional protein expression have been shown to impact
intestinal injury in the I/R model.37
PAF Administration
The administration of PAF, combined with bacterial LPS,
has been reported to initiate NEC-like pathologies in both
mice and rats.38,39 PAF has been identified as a proin-
flammatory phospholipid, causing vascular permeability,
enhanced leukocyte degranulation, adhesion, and chemo-
taxis, as well as the production of tumor necrosis factor a,
IL-6, and IL-8. PAF is primarily produced by innate im-
mune cells, such as neutrophils, basophils, and monocytes,
as well as endothelial cells and platelets. To induce NEC-
like necrosis, PAF and LPS are injected directly into the
mesenteric vascular circulation. In rats, the hemorrhagic
lesions of necrosis observed in the jejunum and ileum are
similar to those observed in human NEC. On microscopic
examination, severe cases demonstrate transmural necrosis,
with loss of mucosal architecture and sloughing of epithe-
lial cells.40
Paneth Cell Depletion
Dithizone, when administered i.v. to rats, leads to cell death
in 25% to 30% of Paneth cells.41 This technique was used
in conjunction with the administration of enteric pathogens
(Escherichia coli, Klebsiella pneumonia) to induce NEC-
like pathology in one study.42 That study revealed gross
necrotic lesions as well as damage to the intestinal tissue,
including mucosal edema, loss of villous integrity, areas of
transmural necrosis, and intramural air, all of which are
observed in human patients. It is interesting to note that
when this treatment was performed in mice before mature
Paneth cell development, no disease was observed.43 The
Paneth cell depletion model is useful as it is simpler
to administer dithizone and an enteric pathogen than it
is to perform feeding gavages or ischemia-reperfusion
8 ajp.amjpathol.org
operations in young mice. However, it implies a central
role of Paneth cells in disease initiation, a hypothesis that
has recently been proposed but not proven.44 In adults, it is
clear that Paneth cell dysfunction, secondary to genetic risk
factors, results in dysbiosis and plays a role in intestinal
inflammation; therefore, the evaluation of NEC patients for
similar genetic defects might yield support for the role of
Paneth cells in NEC.45
Xenograft Model of Human Intestinal Development
The xenograft model provides a means of recapitulating the
developmental changes in innate immunity and intestinal
development, both of which may have a role in NEC
pathophysiology. Human fetal intestine is implanted s.c.
into an immunodeficient mouse host, the host is allowed to
heal, and the fetal intestinal tissue matures for a designated
period. The xenograft model allows for the assessment of
ontogenic changes at points in human intestinal develop-
ment that correspond to the second and third trimesters of
pregnancy.46e48 Although this is a potentially accurate
model of human intestinal development, it does not mirror
NEC pathogenesis as other models do.
Infection and Chemical Injury Models
A mouse model of NEC based on Cronobacter sakazakii
infection was recently reported.49,50 The justification of
using this specific pathogen is the relatively common
contamination of various formula preparations by this bac-
teria.51 This model involves the recruitment of innate im-
mune cells (DCs, macrophages, and neutrophils) and has
been used for showing that the depletion of DCs in newborn
mice protects against C. sakazakiieinduced NEC, whereas
the depletion of neutrophils and macrophages exacerbates
inflammation. Macrophages also may have a role in a
chemically induced model of NEC.52 This model is based
on the clinical observation of numerous macrophages and
few neutrophils in tissue samples from NEC patients.
MohanKumar et al52 hypothesized that this observation was
a reflection of the underdeveloped innate mucosal immune
system, and they designed a model that used trinitrobenzene
sulfonic acideinduced inflammation for comparing mucosal
injury in newborn versus adult mice. The neonatal mouse
has a macrophage-predominate infiltrate similar to that in
human NEC. These two new models may serve as tools
useful for the study of innate immune cells in NEC, which
to date have not been adequately investigated.
Proposed Innate Immune Mechanisms Leading
to NEC
Dysbiosis and Barrier Dysfunction
A recent study demonstrated a lack of diversity in fecal
bacteria from patients who developed NEC compared to
- The American Journal of Pathology

that from unaffected patients.53 A further study
described an increase in proteobacteria and a decrease
in firmicutes in NEC patients relative to healthy
neonatal controls.54 These studies clearly demonstrate a
difference in intestinal microbiota in NEC patients.
Because these alterations were detected at the initial
colonization phase in the patients, it is possible that this
dysbiosis may contribute to the development of NEC
and is not just a reflection of the disease state. Several
factors may result in a bacterial dysbiosis after birth,
including Cesarean instead of vaginal delivery, feeding
of breast milk instead of formula, and broad-spectrum
antibiotic exposure.
It is becoming increasing clear that dysbiosis can drasti-
cally impact intestinal immune defenses by altering
apoptotic and inflammatory signaling, barrier function, and
bacterial detection mechanisms in the intestine.55 Increased
intestinal permeability and epithelial damage, coupled with
deficient epithelial healing, have been identified in NEC,
although it is unclear whether these aspects precede or result
from NEC. Alterations in the tight junctions and the mucus
layer would provide a mechanism for dysbiotic bacteria to
reach and cross the epithelial barrier and initiate altered
immune responses. Conversely, full development of a
functional mucosal immune system requires the normal
bacterial colonization that occurs after birth, as newborn
pups raised by dams on broad-spectrum antibiotics have
increased bacterial translocation, decreased numbers of
CD4þ T cells in mesenteric lymph nodes, and altered
cytokine production (a pattern that is similar to that in in-
fants with NEC).17,37,56
Several G/H studies have demonstrated a loss of mucin
2, which might allow for increased bacterial translocation
from the lumen (Table 2).59,73 In addition, rat pups sub-
jected to G/H and subsequently treated with epidermal
growth factor (EGF) showed an amelioration of experi-
mental NEC that was correlated with increases in mucin 2,
occludin, and claudin-3 and decreased epithelial cell
apoptosis.69,73 These studies demonstrate the importance
of the epithelial barrier and imply its role in preventing
bacterial translocation that may be crucial for the pre-
vention of NEC.
Also controlling microbial colonization in the intestines
of neonates are AMPs, several of which are produced by
Paneth cells. Studies have demonstrated that AMP secre-
tions are altered in infants with NEC; specifically, lyso-
zyme is decreased, whereas the a human defensins 5 and 6
are increased.57,58 Human NEC samples have been re-
ported to have both an increase and a decrease in Paneth
cell numbers relative to control samples.17,57 Because
Paneth cell numbers increase during gestation and are
regulated by inflammatory mediators, these discrepancies
may be attributed to differences in gestational age at birth
or they might reflect the heterogeneity of samples. As the
dithizone model certainly mimics some of these observa-
tions, it should be useful for further studies on the
The American Journal of Pathology - ajp.amjpathol.org
Pathogenesis of Necrotizing Enterocolitis
importance of dysbiosis and the innate immune compo-
nents that shape the microbiota in NEC.84
TLRs
One potential initiating factor of NEC is the response of
epithelial and immune cells to the microbiota that colonize
infants after birth. The expression of TLR4, a sensor of the
Gram-negative bacterial component LPS, is increased in
human NEC as well as fetal intestine, and this increase
appears to be dependent on microbial colonization
(Table 2).37,47 PAF has also been shown to induce TLR4
expression in intestinal epithelial cells, and PAF levels are
elevated in newborns with advanced NEC, whereas levels
of acetylhydrolase (the PAF-inactivation enzyme) are
decreased in NEC patients.68 Mouse G/H studies have
demonstrated that the changes in TLR4 expression precede
histological injury and that epithelial expression of TLR4
is required for NEC development.5,60,61 Also, treatment
with polyunsaturated fats to reduce TLR4 expression or
with amniotic fluid to inhibit TLR4 signaling reduces the
severity of G/H-induced NEC, and the absence of TLR4
protects from disease.5,64,65 TLR4 signaling during bacte-
rial colonization results in inhibited proliferation, migra-
tion, and survival and increased endoplasmic reticulum
stress and apoptosis of enterocytes in the intestine.62,63
These findings suggest that TLR4 activation leads to
impaired enterocyte repair and migration, therefore inhib-
iting the effective repair of the intestinal epithelium and
subsequently resulting in increased bacterial translocation,
inflammation, and NEC.
Recently, Yazji et al85 described a novel role for TLR4 on
endothelial cells in the regulation of intestinal perfusion.
Intestinal ischemia has been thought to be a potential trigger
of NEC; this study shows the importance of innate immune
recognition outside the intestinal mucosa in the develop-
ment of NEC. The role of TLR4 in NEC has also recently
been linked to other PRRs, including TLR9 and NOD2. The
administration of bacterial CpG DNA (the ligand of TLR9)
or the activation of NOD2 inhibited TLR4 signaling and
greatly reduced the incidence of murine NEC.66,67 The
dysbiosis seen in infants predisposed to NEC may
contribute to the disease through differential ligation of
TLR4 versus TLR9/NOD2.
Using the xenograft model of intestinal development,
studies indicate that immature xenograft and NEC intestinal
epithelial cells have increased mRNA expression of TLR2,
TLR4, myeloid differentiation primary response 88 (MyD88),
NF-kB, and IL-8 and decreased expression of the TLR and
NF-kB negative regulators, toll-interacting protein, and single
Ig IL-1erelated receptor compared with that in mature con-
trols.47 Additionally, reports describe that the treatment of
immature xenografts with media containing probiotic factors
led to the down-regulation of TLR2 and TLR4 as well as
the up-regulation of toll-interacting protein and single Ig
IL-1erelated receptor comparable to those in mature
9
Tanner et al

Table 2 Changes in Innate Immune Mechanisms Observed in Animal Models of Necrotizing Enterocolitis and Related Observations in
Human Studies
General alteration Specific physiological changes Species Model Links to human disease
Dysbiosis/barrier Altered Gram-negative bacterial Mouse G/H Changes in microbiota24,53
dysfunction populations24,28,29
Altered bacterial populations49,50 Mouse Infection
Paneth cell depletion41,43 Rat Dithizone Altered Paneth cell numbers17,57
Decreased antimicrobial activity41,43 Rat Dithizone Decreased lysozyme and
a-defensin58
Decreased mucin 259 Rat G/H Decreased Goblet cells2
Altered microbial Increased epithelial TLR4 expression5,60,61 Rat G/H Increased epithelial TLR447
sensing Mouse
Increased TLR2, TLR4, myeloid Rat Xenograft
differentiation primary response 88, NF-
kB, and IL-8; decreased toll interacting
protein and single Ig IL-1e
related receptor47
Increased epithelial damage after TLR4 Mouse G/H
signaling62,63
Decreased TLR4 after polyunsaturated fat Mouse G/H
treatment64
Decreased TLR4 after amniotic fluid Mouse G/H
treatment (via EGF)65
Decreased TLR4 after TLR9 and NOD2 Mouse G/H
stimulation66,67
PAF induction of TLR468 Rat PAF/LPS Elevated PAF in NEC patients68
EGF Decreased epithelial cell apoptosis/ Rat G/H Low EGF levels in NEC patients71
autophagy after EGF treatment69,70
Decreased NEC after HB-EGF treatment72 Rat G/H
Increased mucin 2, occludin, claudin-3 after Rat G/H
EGF treatment69,73
External modulators Increased intestinal damage on PAF/LPS Rat PAF/LPS Increased PAF levels68
administration38,39 Mouse Decreased acetylhydrolase levels68
Increased disease in IL-10/ mice74 Mouse G/H Differences in IL-10 in breast milk,
IL-10 administration ameliorates Rat G/H serum75,76
disease77,78
Decreased disease on increased IL-10 Mouse PAF/LPS
signaling induced by Lactobacillus
rhamnosus GG79
IGF-1 protects intestinal epithelium from Rat Cell line Low IGF levels in NEC patients81
oxidative stress80
Decreased TGF-b signaling leads to Mouse PAF/LPS Low TGF-b levels in NEC patients and
increased disease40 preterm milk82
Innate immune cells Depletion of neutrophils and macrophages Mouse Infection
increases disease50
Increased macrophage infiltrate52 Mouse Chemical injury Macrophage predominant
infiltrate52
Depletion of DCs protects from disease49 Mouse Infection Decreased plasmacytoid DC function
in preterm infants83
Physiological changes indicated in italics have been reported to be protective.
DC, dendritic cells; EGF, epidermal growth factor; G/H, gavage/hypoxia; HB-EGF, heparin bound-epidermal growth factor; IGF, insulin growth factor; LPS,
lipopolysaccharide; NEC, necrotizing enterocolitis; NOD, nucleotide-binding oligomerization domains; PAF, platelet-activating factor; TGF, transforming
growth factor; TLR, toll-like receptors.

xenografts.48 These data suggest that differences in the
maturation of the TLR-induced NF-kB signaling pathway may
be a part of the mechanism of NEC development, and that
these pathways may be controlled by interaction with probiotic
microorganisms.
Together, these data highlight the importance of proper
bacterial detection by the intestinal epithelial barrier
(particularly through TLR4 and TLR9) and their subsequent
signaling cascades to induce a proper immune response.
When this sensing is disrupted through antibiotic treatment,

10 ajp.amjpathol.org - The American Journal of Pathology


or occurs prematurely in neonatal infants, the physical
barrier is breached and bacteria are exposed to the infant’s
immune system. In contrast to the increased epithelial
expression of TLR4 in premature infants, it has been re-
ported that infants born at <28 weeks of gestation have very
low levels of innate immune receptors (CD14, TLR2,
TLR4) on their core blood leukocytes and have severely
impaired leukocyte inflammatory responses to bacteria.86
This impaired innate immune function likely leads to an
inability to control infection due to the impaired barrier, and
subsequently aberrant immune activation results, leading to
NEC. However, further studies are needed to specifically
investigate the contributions of PRRs on leukocytes in the
development of NEC.
EGF
Studies in both animal models and human patients have
identified EGF as a potential biomarker for NEC develop-
ment and as a future treatment of NEC. EGF is found in the
amniotic fluid and is produced by the salivary glands,
Brunner’s glands in the duodenum, Paneth cells, and mac-
rophages. EGF is important for cell survival (reduced auto-
phagy), division, and migration in the intestine.70,71 Human
studies have revealed that decreased cord blood EGF levels
are predictive of NEC in veryelow-birth-weight infants and
that heparin-binding EGF (HB-EGF) treatment in rat pups
subjected to the G/H NEC model protects the pups from
injury (Table 2).72,87 In addition, in the G/H model, the
administration of mesenchymal stem cells along with HB-
EGF results in a synergism that reduces injury and im-
proves survival.88 These studies demonstrate that impaired
epithelial healing secondary to reduced EGF/HB-EGF may
be a major component in the pathogenesis of NEC.
External Modulators
Full-term infants acquire several immunomodulatory com-
ponents from the breast milk of their mothers, including
PAF acetylhydrolase; growth factors such as EGF, HB-
EGF, insulin-like growth factor (IGF), and TGF-b; and
immune factors such as SIgA and IL-10 (Table 2).89 The
immaturity of a preterm infant’s immune system may make
the presence of these molecules even more vital for pre-
venting intestinal damage during the first weeks of life.
Due to the nearly absent levels of IgA in newborn infants
and the importance of IgA in regulating the commensal
microbiota, it has been suggested that a lack of IgA may be
involved in NEC, particularly in formula-fed infants.17 To
address this issue, several human studies have attempted to
supplement IgA levels, as well as other immunoglobulins, in
newborns; however, there has not been a striking
improvement in outcomes.90 In interpreting these data, it
should be noted that in all studies in which formula was
supplemented with immunoglobulins, serum immunoglob-
ulins and not SIgA was used. IgA arrives in the intestinal
The American Journal of Pathology - ajp.amjpathol.org
Pathogenesis of Necrotizing Enterocolitis
lumen and breast milk by transcytosis through the epithelial
cells. During this process, it acquires a secretory component
that protects the IgA antibodies from degradation by the
gastric acids and enzymes of the digestive system. There-
fore, SIgA has properties that are profoundly different from
those of the serum IgA used so far in studies. Further in-
vestigations are necessary to elucidate the role of SIgA in
protection from NEC, as none of the current animal models
have evaluated this aspect of innate immunity in the disease.
Studies involving IL-10 have revealed slightly more in-
formation and do suggest an important role of IL-10 in
protection from NEC. It has been reported that the per-
centage of mothers with undetectable IL-10 in their milk
was significantly greater in mothers of infants who devel-
oped NEC.25 In addition, levels of SIgA and IL-10 in
human milk are lower after very preterm delivery (<30
weeks of gestation) compared with preterm (30 to 37 weeks
of gestation) and term (38 to 42 weeks of gestation) de-
livery.75 Conversely, a recent study measured increased
levels of IL-10 in the serum of NEC patients relative to
healthy controls.76 This increase could be explained by a
lack of IL-10 in the maternal milk, requiring increased IL-10
production by the newborn in an attempt of the young im-
mune system to control the disease; however, IL-10 pro-
duction by the infant may be too delayed to effectively
control the immune response. The G/H model has been used
for investigating the role of IL-10 in the development of
NEC. IL-10/ mice develop more severe histological as-
pects, characterized by increased epithelial cell apoptosis
and decreased organization of tight junction proteins.74
Additionally, the administration of IL-10 ameliorates dis-
ease, and ileal IL-10 levels are increased in rats fed with rat
milk versus a cow-milk substitute.77,78 The PAF/LPS mouse
model shows that the probiotic Lactobacillus rhamnosus
GG protects mice from NEC by increasing IL-10 receptor
transcription, thereby increasing the signaling abilities of
both maternal milkederived and endogenous IL-10.
Increased IL-10 signaling in these studies resulted in
lower levels of colonic production of tumor necrosis factor
a and the neutrophil chemoattractant macrophage inflam-
matory protein 2, indicating that IL-10 signaling in the
context of NEC leads to decreased inflammatory cytokine
production, and the modulation of IL-10 may be possible
through the administration of probiotics.79
A low IGF level has been reported to be associated with
NEC.81 IGF-1 can protect intestinal epithelial cells from
oxidative stresseinduced apoptosis and promote the devel-
opment and cytotoxic activity of natural killer cells, whereas
IGF-2 induces TGF-b release.80,91,92 NEC is associated with
decreased tissue levels of TGF-b, and in mice, the disrup-
tion of TGF-b signaling results in more severe NEC-like
mucosal injury in the PAF/LPS model.40 As TGF-b nor-
mally suppresses macrophage inflammatory responses in the
developing intestine, its absence (from either a lack of
breastfeeding or decreased IGF levels) can clearly impact
the function of the innate immune system in premature
11
Tanner et al

Prematurity
Figure 2 The innate immune response in
Formula Feeding necrotizing enterocolitis (NEC. Top: hematoxylin
and eosin stain of normal neonatal human small
Antibiotics bowel. Middle: Mechanistic diagram of the fac-
tors that have been associated with the increased
Delivery Method incidence of human NEC and the potential innate
immune cells and molecules involved in the dis-
ease. In this diagram, prematurity, antibiotic use,
Dysbiosis method of delivery (vaginal versus Cesarean), and
Proteobacteria formal feeding can result in dysbiosis, altered
Firmicutes barrier function, and altered growth factors and
cytokines. The dashed arrows represent a recip-
rocal vicious cycle between dysbiosis and barrier
function, and dysbiosis and altered growth fac-
Barrier Dysfunction Altered Growth tors and cytokines. All of these factors can lead to
TLR4 Factors and Cytokines increased bacterial translocation, decreased
Permeability Acetylhydrolase enterocyte migration and to increased inflamma-
Epithelial damage EGF/HB-EGF tion and necrosis, as evidenced in the hematox-
Epithelial healing IL-10 ylin and eosin panel (bottom), showing severe
Mucus layer SIgA
Altered AMP function muscular and epithelial damage with complete
IGF
TGF-β loss of mucosa. EGF, epidermal growth factor; HB-
EGF, heparin bound-epidermal growth factor; IGF,
insulin-like growth factor; SIgA, secretory immu-
Bacterial Translocation noglobulin A; TLR4, Toll-like receptor 4; and TGF,
Enterocyte Migration transforming growth factor.
Inflammation/Necrosis

infants. Of note, recent data indicate that low blood TGF-b
levels are associated with a higher risk for NEC.26
Innate Immune Cells
Both of the newly described chemical and infection models
of NEC have suggested that there may be a defective
response by innate immune cells, leading to disease
(Table 2).49,50 The data suggest a destructive role for DCs in
coordinating a response to an NEC-like disease and a pro-
tective role for neutrophils and macrophages. Further
investigation of the phenotypes of macrophages will reveal
whether they are predominantly of the IL-10esecreting M2
phenotype, and study of the subsets of DCs will determine
whether there may be defects in DC subtypes that are known
to control T-cell homeostasis.93,94 However, in contrast to
these murine models, reports show that numerous macro-
phages are seen in NEC lesions and that macrophages from
the human preterm intestine produce inflammatory cyto-
kines, whereas macrophages from full-term neonates and
adults do not.40,52 These macrophages may play a role in
disease, as they can be suppressed by the regulatory cyto-
kine TGF-b2, which is decreased in both patients with NEC
and in preterm milk.40,82 In addition, the trinitrobenzene
sulfonic acideinduced NEC model is driven by pro-
inflammatory macrophages. These differences between the
infection and chemical models, as well as human disease,
show the difficulty in modeling a disease with an unknown
etiology and that each model must be fully investigated to
determine its strengths and weaknesses.
More studies of other innate immune cells are still needed
in both human disease and animal models. One recent

12 ajp.amjpathol.org - The American Journal of Pathology


publication looking at plasmacytoid DCs reported a pre-
mature morphology and decreased functional capacity,
whereas another study reported diminished levels of natural
killer cell activity in the peripheral blood of preterm in-
fants.83,95 To our knowledge, there are no reports regarding
the presence or function of B1 B cells (which produce
natural serum antibody and T-independent antibodies) or the
newly described innate lymphoid cells in the term versus the
premature neonate. Because these innate cells are present in
neonatal murine models and are involved in intestinal ho-
meostasis, they may also play an important role in NEC and
should be studied further.96
Future NEC Models
Humanized Mouse Models
One potential avenue that has not been explored in NEC
research to date is the utilization of humanized mouse
models. Due to species-specific differences in protein
structure and expression and differences in various ligand
specificities between species,97 humanized mouse models
may provide a bridge to further enhance NEC research.
Humanized TLR4 mice have recently been developed98 and
may prove to be a useful model in future NEC studies. In
addition, the recently developed ability to colonize germ-
free mice with human fecal microbiota is a new opportu-
nity for developing models of the disease that are based on
the fecal communities of NEC and control patients, and
these models may result in a dramatic new understanding of
the disease while avoiding the current confounding factors
of the differing microbial communities of each institution’s
animal facility.99
Novel Transgenic Technologies
One of the main reasons scientists that gravitate toward the
mouse as a model organism is the proficiency of generating
genetically modified animals. The availability of this useful
tool often overrides legitimate concerns regarding devel-
opmental, anatomical, and physiological differences
between mice and humans. The availability of mice with
cell- or tissue-specific knockouts of receptors for IgA,
TGF-b, IGF, and IL-10 make the urgency for an appropriate
mouse model of NEC crucial for testing the importance of
these molecules in the disease. However, the increasing
availability of genetically modified pigs, as well as newly
described enterocolitis models in zebrafish, may enhance
interest in using these novel models for further investigation
of NEC.100,101
Mathematical Modeling of NEC
Mathematical modeling is developing as a method useful for
uncovering the intricacies of human diseases. A mathematic
model of NEC is appropriate because the severity of NEC
The American Journal of Pathology - ajp.amjpathol.org
Pathogenesis of Necrotizing Enterocolitis
depends on a complex combination of intestinal immaturity,
inflammation, injury repair, and microbial communities.102
Several mathematical models of NEC have been devel-
oped, including an effort to combine independent areas of
NEC research, including the inflammatory response in NEC,
the dynamics of epithelial healing, the protective factors
supplied by breastfeeding, and the role of pathogenic bacte-
ria.103 However, these models have yet to translate effec-
tively into clinical practice and must be further optimized.
Future Perspectives
Our understanding of NEC has increased slowly during
recent years. Despite this increase in knowledge, NEC
diagnosis and treatment remain challenging, indicating that
the scientific and medical communities are far from opti-
mally understanding NEC pathogenesis. The animal models
described herein have proven to be invaluable resources in
achieving our current level of understanding; however, the
difficulty in replicating all aspects of the human disease, the
lack of spontaneous disease development, and the lack of
data on the involvement of the innate immune system are
drawbacks common to all models (Table 1). While a shift in
microbiota, breakdown in the intestinal barrier, altered
sensing of and response to bacterial components, and dis-
rupted immune factors and cells may all be involved in
NEC, it is difficult to say which, if any, is the primary insult
leading to disease (Figure 2). It is clear that an NEC model
that more accurately replicates human disease will be
required for effectively studying the disease and for
improving patient outcomes.
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