Hooper2015 Paneth

CHAPTER THREE
Epithelial Cell Contributions

to Intestinal Immunity
Lora V. Hooper*,†,1
*Department of Immunology, The University of Texas Southwestern Medical Center, Dallas, Texas, USA
†
The Howard Hughes Medical Institute, The University of Texas Southwestern Medical Center, Dallas, Texas,
USA
1
Corresponding author: e-mail address: lora.hooper@utsouthwestern.edu
Contents
1. Introduction 130
1.1 Overview of epithelial-microbial interactions in the mammalian intestine 130
1.2 The intestinal microbiota 131
1.3 Germ-free mice as experimental tools 132
2. Cellular Makeup of the Intestinal Epithelial Barrier 133
2.1 Enterocytes 133
2.2 Goblet cells 134
2.3 Paneth cells 134
2.4 Enteroendocrine cells 134
2.5 M cells 135
3. Epithelial Cell Sensing of Intestinal Microbes 135
3.1 Epithelial detection of microbes by pattern recognition receptors 135
3.2 Tissue-specific modulation of epithelial cell-specific innate immune
responses 138
4. Mucus Production by the Intestinal Epithelium 139
4.1 Secretion and assembly of the mucus layer 139
4.2 Regulation of mucus production 139
5. Epithelial Antimicrobial Proteins 141
5.1 Epithelial antimicrobial protein families 142
5.2 Regulation of epithelial antimicrobial proteins 145
5.3 In vivo functions of epithelial antimicrobial proteins 150
6. Intestinal Epithelial Cell Autophagy 153
6.1 Autophagy as a barrier to bacterial dissemination 153
6.2 Autophagy-dependent regulation of protein secretion 154
7. Epithelial Regulation of Adaptive Immunity 154
7.1 Transcytosis of immunoglobulin A 155
7.2 Cytokine secretion 156
7.3 Antigen delivery to subepithelial immune cells 156
8. Bacterial Stimulation of Epithelial Cell Repair 157
8.1 MyD88-dependent epithelial repair 158
8.2 Activation of epithelial repair by reactive oxygen species 159
Advances in Immunology, Volume 126 # 2015 Elsevier Inc. 129

ISSN 0065-2776 All rights reserved.
http://dx.doi.org/10.1016/bs.ai.2014.11.003
130 Lora V. Hooper
9. Epithelial Dysfunction in Inflammatory Disease 159

10. Future Perspectives 161
Acknowledgments 162
References 162
Abstract
The epithelial surfaces of the mammalian intestine interface directly with the external
environment and thus continuously encounter pathogenic bacteria, fungi, viruses, and
parasites. The intestinal epithelium is also closely associated with complex communities
of symbiotic microorganisms. Intestinal epithelial cells are thus faced with the unique
challenge of directly interacting with enormous numbers of microbes that include both
pathogens and symbionts. As a result, gut epithelia have evolved an array of strategies
that contribute to host immunity. This chapter considers the various mechanisms used
by epithelial cells to limit microbial invasion of host tissues, shape the composition of
indigenous microbial communities, and coordinate the adaptive immune response to
microorganisms. Study of intestinal epithelial cells has contributed fundamental insights
into intestinal immune homeostasis and has revealed how impaired epithelial cell func-
tion can contribute to inflammatory disease.
1. INTRODUCTION
1.1. Overview of epithelial-microbial interactions in the
mammalian intestine
The epithelial surfaces of the mammalian intestine interface directly with the
external environment. As a result, these tissues continuously encounter bac-
teria, fungi, viruses, and parasites that are ingested in food and water and may
be potential pathogens. The epithelium separating these microorganisms
from internal tissues comprises a single-cell layer and encompasses an enor-
mous surface area (200 m2 in humans) (Neish, 2002). Thus, the intestinal
epithelium is faced with the unique challenge of defending a large surface
area in order to prevent pathogen invasion.
The mammalian intestine is also home to a diverse community of indig-
enous microorganisms numbering in the trillions. This community is known
as the microbiota. These organisms establish symbiotic relationships with
their hosts, making critical contributions to mammalian metabolism while
occupying a protected, nutrient-rich environment. Despite the symbiotic
nature of this relationship, the enormous numbers of intestinal bacteria pre-
sent a continuous threat of host barrier breach, with the single-cell epithelial
layer and large surface area compounding this threat. Such opportunistic
invasion of host tissues by resident bacteria can subvert the symbiotic
host-microbial relationship and lead to pathologies such as bacteremia or
Epithelial Cell Contributions to Intestinal Immunity 131
chronic inflammation. At the same time, the intestinal epithelium must

avoid potentially harmful overreactions that could unnecessarily damage
intestinal tissues or alter the essential metabolic functions of the microbiota.
In order to meet these challenges, epithelial cells have evolved a number
of strategies for managing the dense bacterial loads. These include epithelial
cell-intrinsic innate immune defenses such as secretion of mucus and anti-
microbial proteins and activation of autophagy, as well as strategies for shap-
ing downstream adaptive immune responses. As a consequence, epithelial
cells play a central role in limiting bacterial invasion of host tissues, in shaping
the composition and location of indigenous microbial communities, and in
coordinating the responses of subepithelial immune cell populations. This
chapter explores each of these functions in detail, as well as how these mech-
anisms can become dysregulated in disease.
1.2. The intestinal microbiota

During the past decade, the development of molecular profiling techniques
has allowed the acquisition of a comprehensive view of the composition of
gut microbial communities. Numerous molecular profiling studies using
ribosomal DNA sequencing methods have revealed that the human intesti-
nal microbiota includes hundreds to thousands of distinct bacterial species,
with prominent representation of both Gram positive and Gram negative
bacteria (Eckburg et al., 2005). The species composition of the intestinal
microbiota can vary widely between individuals, and changes in response
to age and diet (Eckburg et al., 2005; Faith et al., 2013; Ley et al., 2005;
Turnbaugh et al., 2006; Yatsunenko et al., 2012). An additional layer of
complexity comes from microbes that gain entry to the intestinal ecosystem
through host intake of food and water, but which are not stable members of
the gut microbiota.
Mammalian intestinal microbial communities are comprised predomi-
nantly of species from two phyla: the Firmicutes and the Bacteroidetes
(Eckburg et al., 2005; Ley et al., 2008). The Firmicutes are Gram positive
bacteria that include species belonging to the Clostridia class, as well as
members of the Enterococcaceae and Lactobacillaceae families and
Lactococcus species. The Bacteroidetes are Gram negative bacteria that
include several Bacteroides species such as Bacteroides thetaiotaomicron, Bacte-
roides fragilis, and Bacteroides ovatus (Eckburg et al., 2005). The remaining
intestinal bacteria account for less than 10% of the population and belong
predominantly to the Proteobacteria and Actinobacteria phyla (Eckburg
et al., 2005).
132 Lora V. Hooper
This microbial community makes several important contributions to

human health and development. A key function of the microbiota is to
increase the efficiency of host digestion (Ley et al., 2008; Wostmann,
Larkin, Moriarty, & Bruckner-Kardoss, 1983). Gut bacterial societies are
metabolically active, degrading dietary substances that would otherwise
be indigestible by the host (Martens et al., 2011; Turnbaugh et al., 2006).
Certain members of the microbiota, such as B. thetaiotaomicron, produce a
diverse repertoire of glycosyl hydrolases that metabolize complex plant poly-
saccharides, thereby liberating simple carbohydrates for uptake by the host
(Gill et al., 2006; Martens et al., 2011; Xu et al., 2003). This effectively
increases the caloric value of the diet and would thus be favorable in an envi-
ronment where nutrients are in short supply.
Enhanced host digestive efficiency is thought to be the primary driving
force behind the evolution of microbiota associations with mammalian
hosts. However, millions of years of co-evolution have led to the interweav-
ing of many aspects of mammalian and microbial physiology, and thus intes-
tinal microbes influence numerous aspects of host physiology and
development. For example, the mammalian microbiota has a profound
influence on the immune system. Intestinal bacteria provide instructive sig-
nals for the development of several lymphocyte subsets, including T helper
17 (TH17) cells (Ivanov et al., 2008, 2009), T regulatory cells (Atarashi et al.,
2011), and B cells (He et al., 2007). Additionally, intestinal bacteria impact
systemic immune responses by influencing the ratio of TH1 and TH2 effector
cells (Mazmanian, Liu, Tzianabos, & Kasper, 2005). The microbiota also
has pronounced effects on the nervous system and brain, with studies
in mice suggesting that intestinal bacteria modulate susceptibility to neu-
rodevelopmental defects such as autism spectrum disorders (Mazmanian
et al., 2005). Finally, the microbiota plays an essential role in regulating host
metabolic processes such as fat storage (Bäckhed et al., 2004).
1.3. Germ-free mice as experimental tools

Animals raised in microbiologically sterile (germ-free) conditions are impor-
tant experimental tools for the study of epithelial contributions to intestinal
immunity. Such animals are reared in sterile isolators that control their expo-
sure to microorganisms, including bacteria, viruses, and eukaryotic parasites.
Germ-free animals can be studied in their microbiologically sterile state or
can be used as living test tubes for the establishment of microbial ecosystems
that range from simple to complex. The technology has come to be known
as “gnotobiotics,” a term derived from Greek roots and meaning “known

life.” Important insights about how resident microbial communities impact
epithelial cell function have come from experimental comparisons of germ-
free and colonized mice (Cash, Whitham, Behrendt, & Hooper, 2006;
Hooper et al., 2001). These include the discovery of microbe-dependent
antimicrobial protein expression (Cash et al., 2006; Hooper,
Stappenbeck, Hong, & Gordon, 2003) and bacterial activation of epithelial
cell autophagy (Benjamin, Sumpter, Levine, & Hooper, 2013). Studies of
gnotobiotic animals have also produced important insights into how the
microbiota shapes the development of adaptive immune cell populations
(Geuking et al., 2011; Ivanov et al., 2008; Mazmanian et al., 2005).
2. CELLULAR MAKEUP OF THE INTESTINAL EPITHELIAL

BARRIER
The internal tissues of the intestine are separated from the microbe-
filled lumen by a single-epithelial layer that is 20 μm thick. Intestinal epi-
thelial surfaces are composed of several distinct cell lineages, each of which
makes unique contributions to barrier integrity and mucosal immunity.
2.1. Enterocytes
The enterocyte is the most abundant epithelial cell lineage in both the small
and the large intestines. Enterocyte membranes, as well as the tight junctions
that form between the cells, present a significant physical barrier to microbial
invasion. However, enterocytes also assume an active role in defending epi-
thelial surfaces. This occurs in several ways, each of which will be discussed in
detail in subsequent sections. First, enterocytes secrete a variety of antimicro-
bial proteins that directly attack and kill bacteria (discussed in detail in
Section 5). Second, they support cellular processes, such as autophagy
(Benjamin et al., 2013; Conway et al., 2013; Wlodarska et al., 2014), which
defend against invading bacteria (discussed in Section 6). Third, enterocytes
produce cytokines that coordinate responses from subepithelial immune
populations (discussed in Section 7.2). Fourth, they transport secretory
immunoglobulin A from the basolateral epithelial surface to the apical surface
of the epithelium for discharge into the lumen (discussed in Section 7.1). This
IgA plays an essential role in maintaining homeostasis between host tissues and
intestinal microbial communities (Macpherson et al., 2000).
134 Lora V. Hooper
2.2. Goblet cells

Goblet cells are a secretory epithelial cell lineage found in both the small and
the large intestines. A major function of goblet cells is the production of
mucus, which forms a protective gel-like layer over the surface epithelium
and protects against bacterial invasion ( Johansson et al., 2008) (discussed in
detail in Section 4). Other secreted products of goblet cells include resistin-
like molecule β (Artis et al., 2004), which modifies T cell-mediated
immunity (Nair et al., 2008), and trefoil factor, which promotes epithelial
restitution after mucosal injury (Farrell et al., 2002; Mashimo, Wu,
Podolsky, & Fishman, 1996; Playford et al., 1995). Finally, recent studies
have revealed that goblet cells can acquire soluble antigens from the intes-
tinal lumen and deliver them to subepithelial dendritic cells (McDole et al.,
2012). Thus, goblet cells participate in antigen uptake and presentation to
underlying immune cells, which was previously thought to be an exclusive
function of intestinal M cells (discussed later).
2.3. Paneth cells

Paneth cells are an epithelial lineage unique to the small intestine. These
secretory cells are positioned at the base of small intestinal crypts of
Lieberkuhn and contain abundant secretory granules containing a number
of microbicidal proteins including α-defensins, C-type lectins, lysozyme,
and phospholipase A2. As a result, this cell lineage is responsible for a large
proportion of the small intestinal antimicrobial output. Upon detection of
microbial signals, Paneth cells release their microbicidal granule contents
into the gut lumen (Ayabe et al., 2000).
Paneth cells also play a central role in regulating small intestinal epithelial
renewal. Paneth cells are positioned in crypts alongside the multipotent stem
cells that give rise to all of the lineages of the differentiated intestinal epithe-
lium. By secreting factors such as EGF, WNt3, and the Notch ligand Dll4,
Paneth cells sustain proliferating epithelial stem cells and thus contribute to
epithelial renewal (Clevers & Bevins, 2013; Sato et al., 2011).
2.4. Enteroendocrine cells

Enteroendocrine cells are scattered throughout the small intestine and com-
prise about 1% of the epithelial cell population (Sternini, Anselmi, &
Rozengurt, 2008). Like goblet cells and Paneth cells, enteroendocrine cells
are specialized for secretion. They sense luminal contents, particularly nutri-
ents, and secrete multiple regulatory factors such as gastric inhibitory
peptide, glucagon-like peptide, and vasoactive intestinal peptide that regu-

late digestion, intestinal motility, and food intake (Moran, Leslie, Levison,
Worthington, & McLaughlin, 2008). Although enteroendocrine cells are
scattered throughout the intestine, taken together they constitute one of
the largest endocrine systems in the body.
2.5. M cells
Microfold cells, or M cells, are intestinal epithelial cells that are specialized
for antigen sampling. They are found predominantly in the follicle-
associated epithelium overlying the surfaces of intestinal lymphoid tissues
such as Peyer’s patches and isolated lymphoid follicles. M cells function in
transepithelial transport of both luminal antigens and intact microorgan-
isms, which are presented to the immune cells of the lymphoid follicle
in order to generate an immune response (Kraehenbuhl & Neutra,
2000). Although M cells are important for the generation of a strong
immune response, they also represent a weak point in the intestinal
epithelium as many pathogens exploit them as a portal of entry (Tahoun
et al., 2012).
3. EPITHELIAL CELL SENSING OF INTESTINAL MICROBES

3.1. Epithelial detection of microbes by pattern
recognition receptors
Several epithelial cell-intrinsic mechanisms of innate defense are activated by
direct bacterial recognition by epithelial cells. Recognition of microorgan-
isms is mediated by host-encoded receptors, known as pattern recognition
receptors, which recognize conserved microbial molecular patterns unique
to prokaryotes. These molecular patterns include bacterial cell wall compo-
nents such as lipopolysaccharide (LPS) and peptidoglycan, as well as protein
components of specialized bacterial structures such as flagella (Ronald &
Beutler, 2010). Certain pattern recognition receptors recognize viruses,
mainly through the detection of viral nucleic acids (Yan & Chen, 2012).
Ligand binding to pattern recognition receptors activates signaling cascades
that control transcription of defensive or proinflammatory genes (Ronald &
Beutler, 2010).
Toll-like receptors (TLRs) are a family of membrane-bound pattern
recognition receptors that play a central role in microbial pattern recognition
in mammals. Twelve mouse and ten human TLRs have been identified to
date (Ronald & Beutler, 2010). At least four TLRs recognize molecular
136 Lora V. Hooper
patterns of bacteria. TLR2 and TLR4 recognize the bacterial cell wall
components—lipoteichoic acid and LPS, respectively (Ronald & Beutler,
2010; Takeuchi et al., 2002). TLR5 detects flagellin, the major protein com-
ponent of Gram negative flagella (Gewirtz, Navas, Lyons, Godowski, &
Madara, 2001; Hayashi et al., 2001). TLR9 binds to unmethylated CpG
DNA, which is present in bacteria but not in eukaryotic cells (Hemmi
et al., 2000). TLR11 recognizes both profilin, which is a molecular signature
of protozoan parasites (Yarovinsky et al., 2005), and flagellin from Salmonella
typhimurium (Mathur et al., 2012). Upon ligand binding, TLRs initiate sig-
naling cascades that trigger the nuclear translocation of the transcription fac-
tor NFκB, which directs expression of proinflammatory factors such as
tumor necrosis factor and interleukin (IL)-8.
Signaling through several TLRs occurs through an adaptor protein,
MyD88. MyD88 is recruited to the TLR cytoplasmic domain and signals
through IRAK (Akira, Uematsu, & Takeuchi, 2006). Studies of MyD88-
deficient mice have produced insight into the broad role of TLRs in
intestinal epithelial cells, and several studies have identified an epithelial
cell-intrinsic role for MyD88 in epithelial cell-mediated immunity. For
example, MyD88 / mice are deficient in expression of several epithelial
proteins, including the antimicrobial lectin RegIIIγ (Brandl, Plitas,
Schnabl, DeMatteo, & Pamer, 2007; Rakoff-Nahoum & Medzhitov,
2007; Vaishnava, Behrendt, Ismail, Eckmann, & Hooper, 2008;
Vaishnava et al., 2011). Forced expression of a MyD88 transgene in Paneth
cells specifically restored Paneth cell expression of RegIIIγ, indicating that
Paneth cells directly sense bacteria through MyD88-dependent pathways
(Vaishnava et al., 2008). Similarly, mice with MyD88 deleted specifically
in enterocytes had lowered RegIIIγ expression (Vaishnava et al., 2011),
emphasizing the importance of epithelial cell-intrinsic MyD88 signaling
for regulating antimicrobial protein expression. The same epithelial
cell-specific MyD88 / mice revealed a role for epithelial cell-intrinsic
MyD88 in bacterial activation of epithelial cell autophagy (Benjamin
et al., 2013). Finally, the activation of epithelial TLR4 increased expression
of TLR-dependent cytokines, such as CCL20, CCL28, and APRIL, which
promoted increased B cell recruitment and differentiation in the intestine
(Fukata et al., 2011; Shang et al., 2008). Together, these studies demonstrate
the importance of epithelial cell-intrinsic recognition of microbial signals
through TLRs for key immune functions of epithelial cells.
Several studies have analyzed epithelial cell-specific deletions of signaling
molecules that lie upstream of the transcription factor NFκB. These include
the inhibitor of NFκB (IκB) kinase (IKK) complex and the NFκB modulator
NEMO. Epithelial cell-specific deletion of the genes encoding either of
these proteins produced increased susceptibility to induced and spontaneous
colitis in mice (Nenci et al., 2007; Zaph et al., 2007). These studies reveal an
essential role for epithelial cell-intrinsic NFκB signaling pathways in
maintaining immune homeostasis in the intestine.
The nucleotide-binding oligomerization domain-like receptors (NLRs)
are a second major group of proteins involved in microbial pattern recog-
nition. In contrast to the membrane-bound TLRs, NLRs are located in
the host cell cytoplasm. NOD2 was originally identified as the first genetic
susceptibility locus for Crohn’s disease, which is characterized by chronic
inflammation of the distal small intestine or proximal colon, or both
(Hugot et al., 2001; Ogura et al., 2001). NOD1 and NOD2 were subse-
quently found to activate inflammatory signaling pathways that depend
on sensing microbial molecular patterns (Girardin et al., 2003; Inohara
et al., 2003). Both receptors are expressed in intestinal epithelial cells
(Kim, Lee, & Kagnoff, 2004; Kobayashi et al., 2005; Lala et al., 2003;
Ogura et al., 2003), and signal in response to muramyl peptides that are com-
ponents of bacterial peptidoglycan (Girardin et al., 2003; Inohara et al.,
2003). While NOD1-dependent signaling requires muramyl tripeptides that
are unique to Gram negative bacteria (Girardin et al., 2003), NOD2-
dependent signaling requires activation by a specific muramyl dipeptide
common to both Gram positive and Gram negative bacteria (Inohara
et al., 2003). Interestingly, recent findings indicate that NOD1 functions
as part of a multiprotein complex, or “nodosome,” that includes small
Rho GTPases and HSP90. It is this complex that detects peptidoglycan frag-
ments in the cell cytoplasm and initiates the signaling cascades that activate
NFκB (Keestra & Bäumler, 2014; Keestra et al., 2013).
Other members of the NLR family are involved in inflammasome acti-
vation. Inflammasomes are multiprotein complexes that incorporate a sensor
protein such as an NLR family member, an adaptor protein (ASC—
apoptosis-associated speck-like protein containing a caspase activation and
recruitment domain (CARD)), and a caspase. These complexes function
platforms for the activation of caspases, such as caspase-1, which drive
proinflammatory responses by processing the proinflammatory cytokines
IL-1β and IL-18 (Martinon, Burns, & Tschopp, 2002). NLRP6 inflamma-
somes are of particular importance in the regulation of mucus production by
goblet cells (Wlodarska et al., 2014) and likely play other important roles in
gut epithelial biology (Elinav et al., 2011).
138 Lora V. Hooper
Although epithelial cell-intrinsic recognition of microbes can activate

protective immune responses, overstimulation of these pathways can be det-
rimental by provoking tumorigenesis. For example, the activation of epithe-
lial cell TLR4 can promote the development of colorectal tumors that are
associated with colitis (Fukata et al., 2007). Similarly, chronic epithelial
cell-intrinsic activation of IKKβ, which lies directly upstream of NFκB, leads
to tumorigenesis that is secondary to colitis (Greten et al., 2004).
3.2. Tissue-specific modulation of epithelial cell-specific innate

immune responses
The abundance of intestinal bacteria and their proximity to intestinal tissues
provokes the question of how the intestine avoids overactive inflammatory
responses to bacterial signals. Several mechanisms appear to limit activation
of intestinal immune responses. First, there is an additional physical barrier
imposed by the mucus layer, which limits bacterial access to the intestinal
epithelium ( Johansson et al., 2008; Vaishnava et al., 2011). Thus, it is pos-
sible that epithelial cell TLRs are stimulated only when the numbers of
surface-associated microbes become large enough to pose a significant
threat. Second, epithelial cells are polarized, allowing compartmentaliza-
tion of certain pattern recognition receptors. For example, TLR5 is
restricted to the basolateral surface of epithelial cells and thus can only sense
bacteria that have invaded host tissues (Gewirtz et al., 2001). The polarity of
epithelial cells also allows differential responses depending on whether bac-
terial signals are detected on the apical or the basolateral epithelial surface.
For example, basolateral detection of ligands by epithelial TLR9 leads to
activation and nuclear translocation of NFκB, while apical detection of
ligands inhibits NFκB activation by stabilizing IκB (Lee et al., 2006). Con-
sequently, the apically activated cells become refractory to further microbial
stimulation.
A third mechanism that limits overactivation of epithelial inflammatory
pathways is expression of factors that modulate pattern recognition receptor
signaling. Studies in zebrafish have shown that intestinal alkaline phosphatase
(IAP) alters bacterial LPS and thus reduces its proinflammatory potential
(Bates, Akerlund, Mittge, & Guillemin, 2007). In this way, IAP may mod-
ulate the concentrations of LPS required to activate epithelial cell inflamma-
tory signaling. This threshold concentration would be governed both by the
affinity of LPS binding to its receptor(s) and by the rate at which IAP
dephosphorylates LPS (Vaishnava & Hooper, 2007).
Another protein that modulates inflammatory signaling pathways is A20,

a zinc-finger protein whose expression is controlled by NFκB (Krikos,
Laherty, & Dixit, 1992). A20 is an ubiquitin-modifying enzyme that inhibits
NFκB activation by downregulating key polyubiquitination-dependent
inflammatory mediators (Wertz et al., 2004). A20 targets include TNF-
receptor-associated factor 6 (TRAF6) (Deng et al., 2000) and receptor-
interacting protein kinase (Li, Kobayashi, Blonska, You, & Lin, 2006).
A20-deficient (Tnfaip3 / ) mice are highly susceptible to intestinal inflam-
mation, suggesting that A20 plays an essential role in regulating the immune
activation threshold in the intestine (Lee et al., 2000; Turer et al., 2008).
By expressing factors such as IAP and A20, intestinal epithelia may mod-
ulate the threshold bacterial density that is required to elicit an innate
immune response. Such strategies may contribute to the relative tolerance
of intestinal surfaces to the presence of high bacterial loads.
4. MUCUS PRODUCTION BY THE INTESTINAL

EPITHELIUM
4.1. Secretion and assembly of the mucus layer
Goblet cells, found in both the small and large intestines, secrete large quan-
tities of mucin proteins (Fig. 1). Mucins are highly glycosylated proteins that
assemble to form a protective layer of viscous mucus that acts as an additional
physical barrier between the epithelial surface and the bacterial communities
in the intestine. The mucus layer extends up to 150 μm from the epithelial
surface and is composed of two distinct strata ( Johansson et al., 2008). The
outer layer is heavily colonized with bacteria, while the inner layer is resis-
tant to bacterial penetration, resulting in a protected zone directly adjacent
to the epithelial surface ( Johansson et al., 2008). Bacterial penetration of the
inner mucus layer is also controlled by antibacterial proteins that are secreted
by epithelial cells and retained in the mucus layer (Meyer-Hoffert et al.,
2008; Vaishnava et al., 2011). Mice lacking the mucin glycoprotein
MUC2 are unable to limit bacterial contact with the epithelium and con-
sequently have severe intestinal inflammation ( Johansson et al., 2008). Thus,
the mucus barrier is essential for maintaining a beneficial symbiotic relation-
ship with the luminal microbiota.
4.2. Regulation of mucus production

Inflammasomes are multiprotein complexes that regulate the processing and
secretion of proinflammatory cytokines. Their assembly is triggered when a
140 Lora V. Hooper
RegIIIa/g
Peptidoglycan
Pore
formation
Bacterial
membrane
Assembly into
Bacterial mucus layer
Mucus layer
killing
Lumen
a-Defensins RegIIIa/g Invasive

Mucins
bacteria
ATG5
a-Defensins MyD88
Epithelium
ATG16L1
Secretory
Secretory granules Autophagy
granules Mucins
Enterocyte/
Paneth cell Goblet cell
X
Lamina propria
paneth cell
Bacterial
dissemination
Figure 1 Epithelial cell-intrinsic mechanisms of innate immune defense. Epithelial cells
perform several cell-intrinsic innate immune functions that regulate interactions
between luminal microorganisms and host tissues. Paneth cells secrete numerous anti-
microbial proteins, such as α-defensins. RegIIIα (human) and RegIIIγ (mouse) are antimi-
crobial lectins that are secreted by Paneth cells and enterocytes (Cash et al., 2006). The
RegIII lectins bind to peptidoglycan on Gram positive bacteria and kill the bacteria by
forming a hexameric pore in the bacterial membrane (Cash et al., 2006; Lehotzky et al.,
2010; Mukherjee et al., 2014). Goblet cells secrete mucin glycoproteins that assemble
into a viscous mucus layer that limits bacterial interactions with the epithelial surface
(Gum, Hicks, Toribara, Siddiki, & Kim, 1994; Johansson et al., 2008). Autophagy is
activated in response to invasive bacteria and prevents bacterial dissemination to
deeper tissues (Benjamin et al., 2013; Conway et al., 2013). Epithelial antibacterial
autophagy depends on the TLR signaling adaptor MyD88 (Benjamin et al., 2013) as well
as the essential autophagy factors ATG5 and ATG16L1 (Benjamin et al., 2013; Conway
et al., 2013).
member of the NOD-like receptor (NLR) family senses stress or damage-

associated molecular patterns (Schroder & Tschopp, 2010). This leads to
recruitment of the adaptor protein ASC into a multiprotein complex that
regulates the activity of caspase-1, a protease that cleaves and activates
proinflammatory cytokines such as IL-1β and IL-18 (Agostini et al., 2004;
Martinon et al., 2002).
Recent findings have revealed that inflammasome formation regulates
mucus secretion by goblet cells. The NLR family member NLRP6 is an
important regulator of inflammasome formation in the intestine. Genetic
deletion of NLRP6 resulted in reduced intestinal IL-18 production and
an altered microbiota. These phenotypes were associated with spontaneous
intestinal hyperplasia, inflammatory cell recruitment, and enhanced suscep-
tibility to chemically induced colitis (Elinav et al., 2011). Subsequently,
NLRP6-dependent inflammasomes were found to regulate mucus secretion
by goblet cells. Deletion of NLRP6 led to defective autophagy, which
resulted in defective mucin granule exocytosis and made the mice suscepti-
ble to persistent infection by Citrobacter rodentium (Wlodarska et al., 2014).
Although it is not yet clear whether epithelial cell-intrinsic inflammasome
formation is responsible for this phenotype, these findings reveal an interest-
ing connection among inflammasome activation, autophagy, and mucus
production by the epithelium.
Goblet cell mucin secretion is also regulated by proteins that govern the
autophagy pathway. In colonic goblet cells, proteins essential for
autophagosome formation, such as ATG5, are required for efficient mucus
secretion (Patel et al., 2013). This process is dependent on the endosomal
pathway and the generation of reactive oxygen species (ROS). Perturbation
of these pathways leads to an abnormal accumulation of mucin granules in
goblet cells (Patel et al., 2013). Future studies will be required to assess the
impact of mucin granule accumulation on the formation of the intestinal
mucus layer and its ability to control interactions with the intestinal
microbiota.
5. EPITHELIAL ANTIMICROBIAL PROTEINS

Epithelial antimicrobial proteins play a central role in allowing epithe-
lial surfaces to cope with the challenge of being closely associated with a
dense microbial community (Fig. 1). These natural antibiotics are an evolu-
tionarily ancient defense system that is present in virtually all plants and
animals. Mammalian antimicrobial proteins rapidly kill or inactivate
142 Lora V. Hooper
microorganisms and are members of a diverse group of protein families. The

epithelial cells lining the intestine produce a rich array of antimicrobial pro-
teins, likely reflecting the complexity of the microbial communities that
challenge the intestinal surface.
Most antimicrobial peptides and proteins target the cell walls of micro-
organisms. As discussed later, several groups of antimicrobial proteins kill
bacteria by disrupting their membranes. Other antimicrobial factors are
enzymes that kill bacteria by digesting specific cell wall structures. Finally,
some antimicrobial proteins work through other mechanisms such as nutri-
ent sequestration. The presence of multiple antimicrobial protein families
and the use of diverse killing strategies is likely important for limiting the
evolution of resistance to multiple antimicrobial factors. In addition, the
targeting of essential cell wall or cell membrane structures might promote
the continued effectiveness of endogenous antimicrobial proteins over evo-
lutionary timescales, as bacteria cannot readily alter these structures without
compromising fitness.
5.1. Epithelial antimicrobial protein families

5.1.1 Defensins
A key mechanism by which antimicrobial proteins kill bacteria is through
nonenzymatic disruption of microbial membranes. Defensins constitute
the major family of membrane-disrupting peptides in mammals and are
one of the most diverse and highly expressed protein families in the intestinal
epithelium. The defensins are small peptides (2–3 kDa) with a conserved
three-dimensional structure that has a characteristic amphipathic arrange-
ment of cationic and hydrophobic amino acids (Zasloff, 2002). This
arrangement produces a positively charged surface that is spatially separated
from neighboring hydrophobic regions, thus facilitating insertion into
negatively charged microbial membranes.
Defensins are classified into three major groups - α, -β and -θ - that vary
in their disulfide bond arrangements and cysteine residue spacing (Selsted &
Ouellette, 2005). The spectrum of antimicrobial activity varies for each par-
ticular protein, but in general, defensins exhibit a broad spectrum of activity
against both Gram positive and Gram negative bacteria and in some cases are
active against fungi, viruses, and protozoa (Selsted & Ouellette, 2005); how-
ever, individual defensins have marked differences in their activity spectrum
and expression patterns (Ouellette, 2011).
The expression of α-defensins in the gastrointestinal tract is restricted to
Paneth cells in small intestinal crypts and is lacking from other epithelial cell
lineages (Selsted & Ouellette, 2005). Because of their localization in the

crypt, mouse α-defensins are termed “cryptdins.” In addition to cryptdins,
mice encode a family of diverse cryptdin-related sequence (CRS) peptides.
CRS peptides have four intramolecular disulfide bridges and further form
covalent dimers by an additional intermolecular disulfide bridge (Hornef,
P€utsep, Karlsson, Refai, & Andersson, 2004). These dimeric peptides
exhibit potent antimicrobial activity against both Gram positive and Gram
negative bacteria (Hornef et al., 2004). CRS peptides can form both
heterodimers and homodimers, thus increasing their combinatorial
diversity.
In contrast to α-defensins, β-defensins are expressed in enterocytes of the
large and small intestines (O’Neil et al., 1999). The human genome contains
at least 28 β-defensins, 8 of which are expressed (Schutte et al., 2002). A sub-
set of these is expressed in intestinal epithelial cells (Fahlgren, Hammarstr€
om,
Danielsson, & Hammarstr€ om, 2003; O’Neil et al., 2000; Wehkamp et al.,
2002). There are reports that β-defensins also may help to recruit immune
cells such as dendritic cells and T cells (Biragyn et al., 2002).
5.1.2 Lectins
Soluble lectins are a second group of antibacterial proteins that kill by non-
enzymatic disruption of bacterial membranes. RegIIIγ and its human ortho-
log, RegIIIα (also known as hepatocarcinoma-intestine-pancreas/
pancreatic-associated protein, or HIP/PAP), are expressed in multiple small
intestinal epithelial lineages, including enterocytes and Paneth cells (Fig. 1)
(Cash et al., 2006; Christa et al., 1996). Both proteins bind to peptidoglycan
and have intrinsic bactericidal activity (Cash et al., 2006; Lehotzky et al.,
2010). In contrast to defensins, the RegIII lectins are selective for Gram pos-
itive bacteria (Cash et al., 2006). This is consistent with the fact that pepti-
doglycan is accessible for binding on the outer surfaces of Gram positive
bacteria, but it is buried in the periplasmic space of Gram negative bacteria.
The bactericidal action of the RegIII lectins is mediated by membrane
disruption (Fig. 1) (Mukherjee et al., 2014). Similar to defensins, RegIIIα
interacts with the charged bacterial membrane through electrostatic interac-
tions. Upon contact with the lipid bilayer, RegIIIα oligomerizes to form a
hexameric, membrane-penetrating pore (Mukherjee et al., 2014). The
closely related lectin RegIIIβ is usually coexpressed with RegIIIγ in mice.
RegIIIβ also binds to peptidoglycan (Lehotzky et al., 2010), although recent
studies suggest that it can also bind to carbohydrate moieties on LPS and thus
kill Gram negative bacteria (Miki, Holst, & Hardt, 2012; Stelter et al., 2011).
144 Lora V. Hooper
As several other members of the Reg family of C-type lectins are expressed
in gastrointestinal tissues (Dieckgraefe et al., 2002), it seems likely that the
Reg lectins represent a general mechanism of antibacterial defense at the
mucosal surface.
Members of the galectin family of lectins also have antibacterial func-
tions. Galectin-4 and galectin-8 are expressed in the gastrointestinal tract
and specifically recognize and kill Escherichia coli that express carbohydrate
structures that mimic human blood group antigens. Bacterial killing is
accompanied by disruption of the bacterial membrane (Stowell et al.,
2010), although the mechanism of membrane disruption remains to be
defined. It is possible that these bactericidal lectins evolved as an outcome
of a host-microbial arms race, as mimicry of host antigenic structures
is a mechanism of pathogen evasion. By specifically recognizing such
structures, these galectins may promote killing of microorganisms that
are especially prone to evade the adaptive immune system (Stowell
et al., 2010).
5.1.3 Cathelicidins
Cathelicidins are a third general class of epithelial antimicrobial peptides that
are expressed in the intestinal epithelium and kill microorganisms by mem-
brane disruption (Hase, Eckmann, Leopard, Varki, & Kagnoff, 2002). They
are cationic, α-helical peptides with a conserved 14-kDa N-terminal
“cathelin” (cathepsin L inhibitor)-like domain and a variable C-terminal
region. The single cathelicidin gene (CAMP in humans) encodes a precursor
protein (hCAP18) (Larrick et al., 1996). This protein can be cleaved at an
alternate site to generate several active antimicrobial proteins, including
the 37 amino acid peptide LL-37 (Gudmundsson et al., 1996) and the
murine peptide CRAMP (cathelin-related antimicrobial peptide) (Gallo
et al., 1997).
Cathelicidins exhibit biological activities similar to those of the defensin
family. They kill bacteria by first binding to bacterial membranes via
charge–charge interactions, followed by membrane insertion and disruption
(Bals & Wilson, 2003). Both LL-37 and CRAMP exhibit antimicrobial
activity against Gram positive and Gram negative bacteria as well as fungi
(Bals & Wilson, 2003). Like β-defensins, LL-37 has biological functions that
are independent of its bactericidal activity. For example, it has been shown
to be chemotactic in vitro for immune cells, including monocytes, macro-
phages, and T cells, and induces cytokine secretion by dendritic cells
(Davidson et al., 2004).
5.1.4 Lysozyme and phospholipase A2

Enzymes that kill bacteria through enzymatic attack on microbial cell walls
constitute another key group of antimicrobial proteins. One such enzymatic
protein, lysozyme, is abundantly expressed and secreted by Paneth cells.
Lysozyme is a glycosidase that hydrolyzes the 1,4-β-glycosidic linkages of
peptidoglycan. Lysozyme is more effective against Gram positive bacteria,
whose peptidoglycan is on the outer cell wall surface and therefore more
easily accessible than the peptidoglycan that is present in the periplasmic
space of Gram negative bacteria (Ganz, 2004). Like lysozyme, secretory
phospholipase A2 (sPLA2) is expressed in Paneth cells (Harwig et al.,
1995). sPLA2 rapidly kills bacteria by hydrolyzing bacterial membrane phos-
pholipids, thus compromising the integrity of the microbial membrane
(Koprivnjak, Peschel, Gelb, Liang, & Weiss, 2002).
5.1.5 Lipocalin
A limited subset of antimicrobial factors function by depriving bacteria of
essential nutrients, thus promoting what is known as “nutritional immunity”
(Hood & Skaar, 2012). During infection, bacteria acquire much of their iron
from the host through the production of siderophores that transport iron
into the pathogen (Faraldo-Gómez & Sansom, 2003). Lipocalin binds and
sequesters iron-bound siderophores, such as enterocalin, and thus inhibits
bacterial growth (Flo et al., 2004).
5.1.6 RNases
The molecular mechanisms underlying the antibacterial activity of other
intestinal microbicidal proteins remain unclear. Angiogenin-4 (Ang4) is a
member of the ribonuclease family and is expressed exclusively in Paneth
cells. Ang4 has broad-spectrum bactericidal activity against Gram positive
and Gram negative bacteria (Hooper et al., 2003) and is thus similar to other
bactericidal RNases, including RNase 7 (Harder & Schroder, 2002) and
eosinophil cationic protein (Rosenberg, 1995). Although Ang4 has RNAse
activity, it remains unclear whether this enzymatic activity is required for
bactericidal function.
5.2. Regulation of epithelial antimicrobial proteins

Many antimicrobial proteins are toxic to mammalian as well as microbial cell
membranes. Thus, the expression, secretion, and activity of most epithelial
antimicrobial proteins are tightly controlled. This can occur through tran-
scriptional and posttranslational regulation mechanisms (Fig. 2).
146 Lora V. Hooper
5.2.1 Transcriptional regulation of epithelial antimicrobial protein

expression
Studies of germ-free mice have shown that some intestinal antimicrobial
proteins are expressed independently of the microbiota whereas others
require bacterial signals for their expression. For example, the majority of
intestinal α-defensins require the Wnt pathway transcription factor TCF4
(van Es et al., 2005) but are expressed independently of the microbiota
(Fig. 2) (Putsep et al., 2000). Similarly, expression of lysozyme, sPLA2,
and certain members of the β-defensin family does not require microbial sig-
nals (Hooper et al., 2001, 2003; O’Neil et al., 1999). The cathelicidin LL-37
is expressed in human epithelial cells independently of the microbiota,
although it is modestly upregulated by invasive microorganisms (Hase
et al., 2002).
The expression of other epithelial antimicrobial proteins requires micro-
bial stimulation. Members of the CRS family of peptides show increased
levels of expression in conventionally raised mice compared with germ-free
mice (Putsep et al., 2000). Similarly, members of the human β-defensin fam-
ily, including hBD2, are expressed under the control of bacterial signals
(O’Neil et al., 1999). Finally, the expression of both Ang4 and RegIIIγ is
virtually absent in germ-free mice and is increased upon microbial coloni-
zation (Cash et al., 2006; Hooper et al., 2003).
As discussed earlier, host pattern recognition receptors direct the expres-
sion of some of these bacterially regulated epithelial antimicrobial proteins.
For example, stimulation of TLRs is required for RegIIIγ mRNA expres-
sion by intestinal epithelial cells (Fig. 2). Studies of mice-lacking MyD88, an
adaptor molecule common to several TLRs, have revealed that RegIIIγ and
RegIIIβ are expressed under the control of TLRs in vivo (Brandl et al., 2007;
Rakoff-Nahoum & Medzhitov, 2007; Vaishnava et al., 2008, 2011). Fur-
ther, the MyD88 dependence is intrinsic to epithelial cells (Brandl et al.,
2007; Vaishnava et al., 2008, 2011). This suggests that epithelial cells,
including enterocytes and Paneth cells, directly sense bacteria through TLRs
and upregulate expression of RegIIIγ and RegIIIβ in response. Expression
of these antimicrobial proteins is likely triggered by any of several TLRs, as
mice deficient in individual TLRs do not have defects in RegIIIγ or RegIIIβ
expression (Vaishnava et al., 2008). This is consistent with the fact that both
LPS and flagellin (which bind TLR4 and TLR5, respectively) are sufficient
to trigger RegIIIγ expression (Brandl et al., 2007; Kinnebrew et al., 2010;
Vaishnava et al., 2008).
Regulation of antimicrobial gene expression Regulation of granule exocytosis
Bacteria
Subset of regulated
α-defensins
Lumen
MAMP RegIIIγ
(e.g., LPS)
α-Defensins Bacterial stimulation
Toll-like receptor of granule exocytosis ATG16L1 mutant
MDP Defective
MyD88
Xexocytosis
Epithelium
α-Defensin
NOD2
NFκB RegIIIγ
TCF4 Secretory
granules
Paneth cells Enterocyte Paneth cells
Lamina propria
IL-22R
IL-22
Innate
lymphoid cell
Figure 2 See legend on next page.
148 Lora V. Hooper
Intestinal epithelial cell expression of RegIIIγ also requires signals from at

least one subepithelial immune cell lineage. Innate lymphoid cells (ILCs)
reside in the lamina propria and phenotypically resemble natural killer cells
(Sanos, Vonarbourg, Mortha, & Diefenbach, 2011). ILCs produce the cyto-
kine IL-22, which binds to IL-22 receptors on epithelial cells to modulate
epithelial cell function (Wolk et al., 2004). ILCs from germ-free mice pro-
duce low levels of IL-22 (Sanos et al., 2008), indicating that intestinal bac-
teria drive IL-22 expression in these cells. Interestingly, ILC-derived IL-22
is required for epithelial cell expression of RegIIIγ mRNA (Fig. 2) (Sanos
et al., 2008). Thus, RegIIIγ expression is dependent on both epithelial cell-
intrinsic TLR signaling through MyD88 and IL-22 produced by ILCs. It
may be possible to reconcile these disparate observations by proposing that
IL-22 functions as an environmental cue that licenses epithelial cells to
express RegIIIγ. Epithelial cells must then receive an additional direct bac-
terial signal through TLRs in order to express RegIIIγ. Further studies will
be required to unravel this regulatory network.
The expression of other intestinal antimicrobial proteins is regulated by
NOD2, an intracellular pattern recognition receptor that is expressed in
Paneth cells (Ogura et al., 2003). MDP has been shown to control the
production of certain α-defensins (Kobayashi et al., 2005) and to enhance
the bactericidal activity of Paneth cells (Fig. 2) (Petnicki-Ocwieja et al.,
2009). Additionally, Nod2 / mice have alterations in the composition of
their small intestinal microbiota (Petnicki-Ocwieja et al., 2009), as well as
increased susceptibility to oral challenge with the pathogen Listeria
Figure 2—Cont'd Regulation of antimicrobial protein expression and secretion. Sev-

eral mechanisms regulate antimicrobial expression and function in the intestinal epithe-
lium. In the small intestine, the transcriptional control of α-defensin expression requires
the transcription factor TCF4 (van Es et al., 2005). The cytoplasmic pattern recognition
receptor NOD2 also controls expression and secretion of antimicrobial activities in the
small intestinal crypt (Kobayashi et al., 2005; Petnicki-Ocwieja et al., 2009). RegIIIγ mRNA
expression in enterocytes and Paneth cells is controlled by microbe-associated molec-
ular patterns, such as lipopolysaccharide (LPS), through Toll-like receptors (TLRs) and is
dependent on the TLR signaling adaptor MyD88 (Brandl et al., 2007; Vaishnava et al.,
2008, 2011). RegIIIγ mRNA expression also requires interleukin-22 (IL-22) from innate
lymphoid cells in the lamina propria (Sanos et al., 2011). The process of antimicrobial
protein secretion is controlled by bacterial signals through an unknown mechanism
(Ayabe et al., 2000). Autophagy proteins such as ATG16L1 are also involved in the pro-
cess of granule formation and secretion (Cadwell et al., 2008). Thus, patients with a sin-
gle amino acid variant of ATG16L1 that is associated with Crohn's disease have
abnormal Paneth cell granule formation (Cadwell et al., 2008).
monocytogenes (Kobayashi et al., 2005). These results indicate that NOD2-

stimulated antimicrobial defenses shape microbiota composition and protect
the epithelial barrier from pathogen invasion.
Together, these findings reveal that different subsets of antimicrobial
proteins are regulated via distinct mechanisms. A constitutive chemical bar-
rier is established at the mucosal surface by the subset of antimicrobial pro-
teins that are expressed independently of bacterial signals. The regulated
expression of other proteins through TLR and NOD2 activation suggests
that a subset of antimicrobial responses may be more precisely titrated in
response to microbial numbers or the composition of the intestinal microbial
community. Strict regulation of certain antimicrobial responses by bacterial
signals could also protect against overproduction of antimicrobial proteins
that could interfere with intestinal ecology and thus undermine the benefi-
cial contributions of the microbiota.
5.2.2 Developmental regulation of antimicrobial protein expression

At least two epithelial antimicrobial proteins are developmentally regulated.
Both Ang4 and RegIIIγ are strongly induced in the small intestine during
early postnatal life. In conventionally raised mice, the level of Ang4 expres-
sion increases approximately 20-fold during weaning (day 17–21 in mice)
and remains at adult levels thereafter (Hooper et al., 2003). The level of
expression of RegIIIγ in mice increases by a remarkable 3000-fold during
the same period (Cash et al., 2006). These findings suggest that Ang4 and
RegIIIγ could function in part to maintain mucosal homeostasis in the face
of the changing microbial ecology and withdrawal of maternal passive
immunity that is associated with weaning.
5.2.3 Posttranslational regulation of antimicrobial protein function

Because membrane-toxic antimicrobial proteins can also target mammalian
cell membranes (Lichtenstein, Ganz, Selsted, & Lehrer, 1986), the activities
of many antimicrobial proteins are suppressed during storage in membrane-
bound secretory granules. α-Defensins are stored in Paneth cell granules as
inactive propeptides and are processed at their N-termini by matrix
metalloproteinase-7 (MMP7) to produce mature bactericidally active pep-
tides (Wilson et al., 1999). In humans, trypsin cleaves α-defensins to their
mature forms (Ghosh et al., 2002). RegIIIγ also requires N-terminal proteo-
lytic processing by trypsin to yield a bactericidally active protein (Mukherjee
et al., 2014). Similarly, β-defensins are expressed as propeptides, but the
processing mechanism remains to be established (Schutte & McCray, 2002).
150 Lora V. Hooper
The distinctive reducing environment of the intestinal lumen provides

another mechanism of posttranslational regulation that likely protects host
cells during storage of antimicrobial proteins. Under the oxidizing condi-
tions present inside host cells, the antimicrobial protein human β-defensin
1 (hBD1) has weak antimicrobial activity. However, under the reducing
conditions that are characteristic of the intestinal lumen, hBD1 undergoes
marked structural changes that unmask a potent antimicrobial activity
(Schroeder et al., 2011).
5.2.4 Regulation of antimicrobial protein secretion

The process of antimicrobial protein secretion is also controlled by bacterial
signals. As discussed earlier, Paneth cells produce most of the antimicrobial
proteins in the small intestine, including a diverse array of α-defensins, Ang4,
and lysozyme. Paneth cells secrete their granule contents in response to
exposure to live bacteria or to bacterial molecules such as LPS (Fig. 2)
(Ayabe et al., 2000). Thus, antimicrobial protein release is precisely regu-
lated in response to bacterial signals, though the mechanisms of bacterial
sensing that control Paneth cell secretion are not yet clear.
More recently, proteins of the autophagy pathway have been found to
play an important role in regulating granule exocytosis in Paneth cells.
ATG16L1 and ATG5 are proteins that are each essential for autophagy
(Cadwell et al., 2008). Mice-lacking epithelial cell expression of ATG16L1
or ATG5 exhibits defective Paneth cell granule packaging and exocytosis,
indicating a role for these autophagy proteins in the Paneth cell secretory
pathway (Cadwell et al., 2008). Interestingly, a single coding variation
(T300A) in the ATG16L1 protein is strongly associated with the risk of
Crohn’s disease, and patients homozygous for the risk allele also show
abnormal Paneth cell granule formation and exocytosis (Fig. 2) (Cadwell
et al., 2008). This suggests that defective Paneth cell granule secretion
may be one factor that leads to Crohn’s disease pathogenesis.
5.3. In vivo functions of epithelial antimicrobial proteins

Studies of genetically engineered mouse models have offered insight into
how epithelial antimicrobial proteins function in vivo. These proteins not
only protect against pathogen colonization but also allow mammalian hosts
to control the composition and location of their resident microbial
communities.
5.3.1 Protection against pathogens

Experiments in mice have yielded key insight into the importance of intes-
tinal antimicrobial proteins in pathogen protection in vivo. MMP7 is
required to generate bactericidally active α-defensins in mice, and conse-
quently, Mmp7 / mice have elevated susceptibility to oral challenge with
the intestinal pathogen S. typhimurium (Wilson et al., 1999). A second mouse
model that has illuminated the in vivo function of α-defensins is a transgenic
mouse overexpressing human α-defensin-5 (DEFA5) in Paneth cells.
DEFA5-expressing mice show greater resistance to oral infection with
S. typhimurium than wild-type mice, demonstrating an essential role for
α-defensins in limiting pathogen colonization (Fig. 3) (Salzman, Ghosh,
Huttner, Paterson, & Bevins, 2003). Finally, antibody-mediated inactivation
of RegIIIγ shows that this antimicrobial lectin is important for limiting col-
onization by Gram positive intestinal pathogens such as L. monocytogenes
Protection against Control of microbiota Limiting bacteria-

pathogens composition epithelial contact
S. typhimurium
Outer mucus layer
RegIIIg
a-defensins
Inner mucus layer
DEFA5
Paneth cells Enterocytes/

Paneth cells
Figure 3 Functions of antimicrobial proteins in the intestine. Forced expression of a
human α-defensin 5 (DEFA5) transgene in Paneth cells limits colonization by
S. typhimurium (Salzman et al., 2003) and controls microbiota composition in the small
intestine (Salzman et al., 2010). The antimicrobial lectin RegIIIγ helps to confine bacteria
to the outer mucus layer, thus limiting bacterial interactions with the epithelial surface
of the small intestine (Vaishnava et al., 2011).
152 Lora V. Hooper
(Brandl et al., 2007) and vancomycin-resistant Enterococcus faecalis (Brandl

et al., 2008).
5.3.2 Shaping microbiota composition

α-Defensins also regulate the composition of the intestinal bacterial commu-
nity. Mmp7 / and DEFA5-transgenic mice each show marked α-defensin-
dependent changes in the composition of their microbial communities
compared with wild-type mice (Fig. 3) (Salzman et al., 2010). Further,
the defensin-deficient Mmp7 / mice and the defensin-complemented
DEFA5-transgenic mice show reciprocal differences in community compo-
sition. These changes include altered proportions of Firmicutes, the major
Gram positive phylum, and Bacteroidetes, the major Gram negative phy-
lum, of the mouse intestine (Salzman et al., 2010). The DEFA5-transgenic
mice also showed a loss of segmented filamentous bacteria (SFB) relative to
wild-type mice. Consistent with the fact that SFB promotes the develop-
ment of intestinal IL 17-producing TH17 cells (Ivanov et al., 2009),
the DEFA5-transgenic mice had lower frequencies of lamina propria
TH17 cells compared with wild-type mice (Salzman et al., 2010). Thus,
α-defensins shape the intestinal microbiota composition and control the
level of immune stimulation.
5.3.3 Limiting bacterial-epithelial cell contact

A key mechanism by which the mammalian intestine maintains homeostasis
with its associated bacterial communities is to minimize contact between the
bacteria and the host tissues. As discussed in Section 4, the mucus layer plays
an essential role in limiting direct contact between microbiota and host. Fur-
ther, most antimicrobial activity is confined to the mucus layer and is essen-
tially absent from the luminal content (Meyer-Hoffert et al., 2008). Thus, in
addition to acting as a physical barrier, the mucus layer may also limit bac-
terial access to the epithelium by forming a diffusion barrier that concen-
trates antimicrobial proteins near the epithelial cell surface.
Consistent with this idea, studies of RegIIIγ / mice exhibit suggest that
RegIIIγ interacts with the mucus layer to limit bacterial contact with the
intestinal epithelial cell surface (Fig. 3). RegIIIγ / mice are characterized
by increased colonization of the small intestinal epithelial surface by Gram
positive bacteria, consistent with the specificity of RegIIIγ for Gram posi-
tives (Cash et al., 2006). Interestingly, these differences do not extend to the
luminal bacterial communities, which are similar when comparing
RegIIIγ / and wild-type littermates. This suggests that the antibacterial
effects of RegIIIγ are confined to the inner mucus layer. The niche-specific
activity of RegIIIγ could arise from restricted diffusion of RegIIIγ through
the mucus barrier, from binding interactions between RegIIIγ and mucus
glycoproteins or because RegIIIγ requires environmental conditions that
are unique to the mucosal surface niche.
6. INTESTINAL EPITHELIAL CELL AUTOPHAGY

Autophagy is an evolutionarily ancient process in which cytoplasmic
materials are targeted to the lysosome for degradation. Portions of the
cytoplasm are sequestered into double-membrane structures, called
autophagosomes, which fuse with lysosomes, delivering their contents for
degradation by lysosomal enzymes (Deretic & Levine, 2009). The process
involves the concerted action of several cytoplasmic proteins. A primary
function of autophagy is to maintain cellular homeostasis by degrading cyto-
plasmic contents during cellular starvation and by recycling damaged organ-
elles and proteins (Rabinowitz & White, 2010). However, autophagy has
also been shown to be critical for the recognition and degradation of intra-
cellular pathogens, thus functioning as an innate barrier to infection
(Benjamin et al., 2013; Conway et al., 2013; Deretic & Levine, 2009;
Levine, Mizushima, & Virgin, 2011).
In the mammalian intestine, the autophagy pathway mediates at least two
distinct functions. First, it acts as an innate barrier to the dissemination of
invasive bacteria, such as S. typhimurium. Second, the proteins that regulate
classical autophagy activation are also essential for proper granule formation
and protein secretion in intestinal secretory cell lineages such as goblet cells
and Paneth cells (Cadwell et al., 2008; Patel et al., 2013). Both of these func-
tions are discussed in this section.
6.1. Autophagy as a barrier to bacterial dissemination

Recent studies have shown that autophagy is an important epithelial cell-
autonomous mechanism of antibacterial defense that protects against dissem-
ination of intestinal bacteria. Epithelial autophagy is activated in the mouse
intestinal epithelium by a pathogen, S. typhimurium, as well as by E. faecalis,
an opportunistically invasive commensal (Benjamin et al., 2013; Conway
et al., 2013). Autophagy is specifically triggered by bacterial invasion of epi-
thelial cells and remains at baseline levels in response to the microbiota nor-
mally present in specified pathogen-free mice. Epithelial cell autophagy is
also tightly regulated, requiring epithelial cell-intrinsic MyD88 signaling
154 Lora V. Hooper
(Benjamin et al., 2013). Finally, epithelial autophagy activation depends on

the presence of the autophagy factors ATG5 and ATG16L1, as deletion of
either of these factors leads to increased extraintestinal spread of
S. typhimurium (Fig. 1) (Benjamin et al., 2013; Conway et al., 2013).
There may also be a role for autophagy in limiting colonization of non-
invasive pathogens. C. rodentium is an attaching and effacing pathogen which
forms lesions on the apical surface of the colonic epithelium but does not
enter epithelial cells in large numbers (Mundy, MacDonald, Dougan,
Frankel, & Wiles, 2005). Epithelial cell expression of the autophagy protein
ATG7 confers protection against luminal colonization by C. rodentium,
although it is not yet clear whether bona fide autophagy is involved in limiting
C. rodentium intestinal colonization and pathogenesis (Inoue et al., 2012).
6.2. Autophagy-dependent regulation of protein secretion

The autophagy machinery is also involved in cellular functions that are dis-
tinct from classical autophagy involving the targeting of bacteria to
autophagosomes (Zhao et al., 2008). As discussed earlier, mice lacking epi-
thelial cell expression of ATG16L1 or ATG5 exhibit defective Paneth cell
granule formation and exocytosis, indicating a role for these autophagy pro-
teins in the Paneth cell secretory pathway (Cadwell et al., 2008). Mice with
an epithelial cell-specific deletion of ATG5 also exhibit defective exocytosis
of goblet cell granules, leading to altered mucus secretion (Patel et al., 2013).
A point mutation (T300A) in the critical autophagy gene ATG16L1 is
associated with a predisposition to Crohn’s disease in humans (Hampe
et al., 2007; Rioux et al., 2007; Wellcome Trust Case Control
Consortium, 2007). The T300A mutation both reduces antibacterial
autophagy (Kuballa, Huett, Rioux, Daly, & Xavier, 2008; Lassen et al.,
2014) and disrupts granule packaging and protein secretion in Paneth cells
(Cadwell et al., 2008, 2010). Such defects could lead to inflammation
through reduced antimicrobial protection at the epithelial surface.
7. EPITHELIAL REGULATION OF ADAPTIVE IMMUNITY

In addition to epithelial cell autonomous functions that kill bacteria
and control the microbiota, epithelial cells also communicate with underly-
ing immune cells to regulate and coordinate adaptive immune responses.
These functions include transcytosis of immunoglobulin A, secretion of
cytokines that direct adaptive immune responses, and delivery of antigen
to the adaptive immune system (Fig. 4).
Bacteria
Lumen
Luminal
antigens
Epithelium
Transcytosis
Secretory
granules
Goblet cell
Enterocyte
pIgR
Lamina propria
TSLP BAFF APRIL

CD103+ DC
Antigen presentation
Dendritic cell to DC
B cell IgA+ plasma cell IgA
IL-10
Figure 4 Epithelial cell regulation of adaptive immunity. The intestinal epithelial cell-
derived cytokine TSLP (thymic stromal lymphopoietin) causes dendritic cells and mac-
rophages to adopt tolerogenic phenotypes and secrete cytokines such as IL-10 (Rimoldi
et al., 2005). BAFF (B cell-activating factor of the TNF family) is a cytokine-like factor that
is secreted by epithelial cells in response to bacterial signals (Xu et al., 2007) and
regulates B cell maturation, survival, and function (Cerutti et al., 2011). APRIL
(a proliferation-inducing ligand) is a cytokine-like factor that is closely related to BAFF.
APRIL is secreted by epithelial cells in a MyD88-dependent manner and drives T cell-
independent IgA2 class switching (He et al., 2007). Epithelial cells also contribute to
adaptive immunity through transport of secretory immunoglobulin A (IgA). Much of this
IgA is specific to commensal bacteria and is produced by plasma cells that develop in
lymphoid tissues and then home to the lamina propria. The plasma cells secrete dimeric
IgA that binds to the polymeric immunoglobulin receptor (pIgR) on the basolateral sur-
face of epithelial cells. The pIgR–IgA complex is transcytosed across the epithelium, and
the IgA is deposited on the apical epithelial surface. IgA plays an essential role in con-
fining bacteria to the intestinal lumen (Macpherson et al., 2000), though the exact mech-
anisms remain unclear. Goblet cells deliver small soluble antigens from the intestinal
lumen to lamina propria DCs (McDole et al., 2012).
7.1. Transcytosis of immunoglobulin A

One important way in which epithelial cells contribute to immune defense is
through transcytosis of secretory immunoglobulin A (IgA) across the epithe-
lial barrier. IgA is the most abundant immunoglobulin isotype in the intes-
tine and is essential for maintaining luminal compartmentalization of
intestinal bacteria and preventing their penetration into host tissue
(Macpherson et al., 2000; Macpherson & Uhr, 2004; Suzuki et al., 2004).
Much of this IgA is specific to intestinal bacteria and is produced by IgA-
secreting plasma cells that develop in lymphoid tissues and then home to
156 Lora V. Hooper
the lamina propria. The plasma cells secrete dimeric IgA that is then bound
to the polymeric immunoglobulin receptor (pIgR), which is positioned on
the basolateral surface of epithelial cells. pIgR is itself expressed under the
control of microbiota signals that are transmitted through MyD88 and
NFκB (Bruno, Frantz, Rogier, Johansen, & Kaetzel, 2011; Johansen &
Kaetzel, 2011). The pIgR–IgA complex is transcytosed across the epithe-
lium, and the IgA is deposited on the apical epithelial surface of the epithe-
lium (Fig. 4). The exact mechanisms by which IgA confines symbiotic
bacteria to the intestinal lumen remain unclear but may involve retention
of bacteria in the mucus layer or promoting phagocytic clearance of organ-
isms that have breached the epithelial barrier.
7.2. Cytokine secretion

Several cytokines produced by intestinal epithelial cells under the control of
microbiota signals promote adaptive immune responses (Fig. 4). For exam-
ple, APRIL (a proliferation-inducing ligand) is secreted by epithelial cells in
a MyD88-dependent manner and drives T cell-independent IgA2 class
switching (He et al., 2007). BAFF (B cell-activating factor of the TNF fam-
ily) is another cytokine-like factor that is closely related to APRIL, is
secreted by epithelial cells in response to bacterial signals, and regulates
B cell maturation, survival, and function (Cerutti, Puga, & Cols, 2011;
Xu et al., 2007). Another epithelial-derived cytokine is TSLP (thymic stro-
mal lymphopoietin), which has a critical immunoregulatory role in both
inflammatory settings and in response to pathogenic worm infections
(Rimoldi et al., 2005; Taylor et al., 2009; Zeuthen, Fink, & Frokiaer,
2008; Ziegler & Artis, 2010). TSLP causes dendritic cells and macrophages
to adopt tolerogenic phenotypes and to secrete cytokines such as IL-10
(Rimoldi et al., 2005).
7.3. Antigen delivery to subepithelial immune cells

A key function of intestinal epithelial cells is participating in antigen sam-
pling and presentation to the adaptive immune system. This results in
directed adaptive immune responses to commensal or pathogenic bacteria.
Classically, M cells that overlie Peyer’s patches and isolated lymphoid tis-
sues have been associated with antigen sampling. As discussed in Section 2,
these specialized cells mediate the sampling of antigens and microorgan-
isms from the intestinal lumen, with presentation to immune cells that
underlie the epithelium (Kraehenbuhl & Neutra, 2000). M cells have also
been reported to be distributed in the villus epithelium and may thus
provide an alternate route of antigen entry in the intestinal epithelium

( Jang et al., 2004).
Goblet cells have recently been shown to participate in luminal antigen
sampling (Fig. 4). These cells deliver small soluble antigens from the intes-
tinal lumen to CD103+ dendritic cells (DCs) in the underlying lamina
propria (McDole et al., 2012). These CD103+ DCs are a specialized DC
subset that promote IgA production, imprint gut homing on lymphocytes,
and induce the development of T regulatory cells (Coombes et al., 2007;
Jaensson et al., 2008; Johansson-Lindbom et al., 2005; Sun et al., 2007;
Uematsu et al., 2008). Although more work is required to understand this
process and its consequences, goblet cell antigen presentation is likely to
have important effects on mucosal immunity.
Enterocytes participate in antigen presentation to the immune system in
at least two ways. First, enterocytes facilitate the extension of the dendrites of
subepithelial mononuclear phagocytes by expressing tight junction proteins
that “pry” open the tight junctions between intestinal epithelial cells, all-
owing dendrite extension into the lumen for direct sampling of microorgan-
isms at the epithelial apical surface (Rescigno et al., 2001). A second way that
enterocytes participate in antigen presentation is through the expression of
CD1d. CD1d presents lipid antigens, derived from either “self” or from bac-
teria, to natural killer T (NKT) cells (Colgan, Hershberg, Furuta, &
Blumberg, 1999; Heller, Fuss, Nieuwenhuis, Blumberg, & Strober, 2002;
Wingender & Kronenberg, 2008). NKT cells play important roles in the
development of intestinal inflammatory responses and are involved in path-
ogenic inflammation in both animal models and human IBD (Heller et al.,
2002). Epithelial CD1d expression suppresses proinflammatory NKT cell
functions and thus reduces intestinal inflammation (Olszak et al., 2014). This
is in contrast to CD1d-mediated antigen presentation through bone
marrow-derived cells, which stimulates proinflammatory functions of
NKT cells (Olszak et al., 2014).
8. BACTERIAL STIMULATION OF EPITHELIAL

CELL REPAIR
The epithelium is a critical physical barrier against microbial penetra-
tion. This function can be perturbed when there is epithelial damage from
environmental insults such as toxins or pathogenic bacteria. The presence of
large indigenous bacterial populations leads to a significant risk for bacterial
invasion, inflammation, and sepsis following intestinal epithelial damage.
The intestinal epithelium must therefore be able to recognize and repair
158 Lora V. Hooper
damage rapidly and efficiently. Interestingly, bacteria play a central role in

triggering repair processes in the epithelium.
8.1. MyD88-dependent epithelial repair

The mechanisms underlying intestinal epithelial repair have relied largely on
the analysis of the dextran sulfate sodium (DSS)-induced model of epithelial
injury. In this model, epithelial injury is initiated in the colons of mice
through administration of DSS in drinking water. Epithelial damage is vis-
ible through the appearance of focal colonic lesions after a few days of DSS
administration, is accompanied by increased mucosal permeability, and can
be detected prior to the ensuing inflammatory response. After removal of
DSS, a complex tissue repair process is initiated, resulting in vigorous epi-
thelial cell proliferation and restoration of an intact epithelial barrier (Chen,
Chou, Fuchs, Havran, & Boismenu, 2002).
Efficient colonic epithelial repair requires the presence of resident gut
bacteria. Mice lacking most of their intestinal microbiota due to antibiotic
treatment are more susceptible to DSS-induced epithelial injury than fully
colonized mice (Rakoff-Nahoum, Paglino, Eslami-Varzaneh, Edberg, &
Medzhitov, 2004). The effect is reversible, as recolonization of antibiotic-
treated mice with commensal bacteria restores normal epithelial repair pro-
cesses. Mice lacking the TLR signaling adaptor MyD88 exhibit defective
epithelial repair in response to DSS-induced mucosal damage, showing that
TLR signaling is required for efficient epithelial repair (Rakoff-Nahoum
et al., 2004). These findings indicate that bacterial activation of TLR signal-
ing pathways is essential for colonic tissue repair processes.
The DSS injury model has also provided essential clues about the intes-
tinal cell populations that promote microbe-regulated epithelial repair.
Analysis of bone marrow chimeric mice revealed that the MyD88-
dependent signals that drive epithelial repair derive from bone marrow-
derived cells (Rakoff-Nahoum, Hao, & Medzhitov, 2006). This finding
was further refined by studies of mice-lacking specific immune cell
populations, which showed that macrophages are required for the colonic
epithelial proliferative response (Pull, Doherty, Mills, Gordon, &
Stappenbeck, 2005). After DSS-induced injury, colonic macrophages are
recruited to sites of active epithelial proliferation where they become local-
ized next to epithelial progenitor cells and express factors that stimulate cel-
lular proliferation (Pull et al., 2005). Together, these results suggest a model
in which epithelial repair is driven by bacterial stimulation of mobile
subepithelial myeloid cells that migrate to damaged regions and produce fac-
tors that promote epithelial restitution.
8.2. Activation of epithelial repair by reactive oxygen species

Bacteria also promote epithelial repair by inducing ROS in intestinal epithe-
lial cells. Bacteria stimulate the formyl peptide receptor on epithelial cells,
activating NADPH-oxidase 1 (NOX1) and enhancing ROS generation
(Alam et al., 2013). Through the inactivation of redox-sensitive tyrosine
phosphatases, ROS promote the formation of focal matrix adhesions that
are necessary for the repair of epithelial damage (Swanson et al., 2011). This
in turn stimulates the migration and proliferation of enterocytes that are
adjacent to sites of epithelial damage (Leoni et al., 2013). Similar pathways
operate in Drosophila, suggesting that this is an evolutionarily ancient mech-
anism of epithelial restitution (Hochmuth, Biteau, Bohmann, & Jasper,
2011; Jones et al., 2013; Lee, 2009).
9. EPITHELIAL DYSFUNCTION IN INFLAMMATORY

DISEASE
Inflammatory bowel disease (IBD) is characterized by severe inflam-
mation of the colon or the distal small intestine, or both. Although the exact
causes of IBD remain poorly understood, its pathologic characteristics indi-
cate that disease arises in part from dysregulated bacterial interactions with
the intestinal epithelial surface. As evidence of this, IBD patients frequently
show increased numbers of bacteria in direct contact with the intestinal epi-
thelium (Swidsinski, Weber, Loening-Baucke, Hale, & Lochs, 2005). This
suggests that IBD is characterized in part by a failure of mechanisms that nor-
mally limit microbiota-epithelial contact.
Consistent with this idea, several IBD risk alleles alter epithelial cell func-
tion by impairing production of antimicrobial peptides or mucus (Fig. 5).
NOD2 was identified as the first genetic susceptibility locus for Crohn’s dis-
ease (Hugot et al., 2001; Ogura et al., 2001). Patients with NOD2 defects
exhibit reduced α-defensin antimicrobial peptide expression in Paneth cells,
coincident with severe intestinal inflammation (Wehkamp et al., 2005). One
possible model to explain the inflammatory phenotype is that reduced
α-defensin production leads to increased numbers of epithelium-associated
bacteria, which could contribute to uncontrolled inflammation in conjunc-
tion with other genetic defects. As discussed in Section 6, ATG16L1 is a
Crohn’s disease risk allele that disrupts antibacterial autophagy and impairs
160 Lora V. Hooper
Bacteria
Autophagosome
MDP
Required for granule exocytosis

ATG16L1
and antibacterial autophagy
NOD2
Senses MDP and influences
α-defensin expression
XBP1 Required for ER
expansion during stress
Endoplasmic reticulum-
Golgi
TCF4 α-Defensins
Required for α-defensin

transcription
Figure 5 Dysregulation of epithelial cell function in inflammatory disease. NOD2, TCF4,

XBP1, and ATG16L1 each promote antimicrobial protein expression and secretion by
Paneth cells (Cadwell et al., 2008; Kaser et al., 2008; Kobayashi et al., 2005; Petnicki-
Ocwieja et al., 2009; van Es et al., 2005), and ATG16L1 is also critical for antibacterial
autophagy (Conway et al., 2013; Lassen et al., 2014). Polymorphisms in the corresponding
genes are associated with an increased incidence of inflammatory bowel disease
(Cadwell et al., 2008; Hugot et al., 2001; Kaser et al., 2008; Koslowski et al., 2009; Ogura
et al., 2001; Wehkamp et al., 2005). This could be due to reduced production of antimi-
crobial proteins that normally control the microbiota and limit bacterial contact with the
intestinal epithelium, as well as impaired antibacterial autophagy.
packaging and exocytosis of Paneth cell secretory granules, thus inhibiting

antimicrobial protein release (Cadwell et al., 2008). Defective antibacterial
autophagy as well as impaired Paneth cell secretion of antimicrobial proteins
could diminish the capacity of the epithelium to manage interactions with
bacteria, thereby increasing the likelihood of bacterial penetration and
mucosal inflammation.
Finally, the transcription factor XBP1 participates in the response of the
endoplasmic reticulum to stress and is required for normal development
of Paneth cells and goblet cells (Kaser et al., 2008). Xbp1 / mice, which
lack Paneth cells and show reduced goblet cell numbers, exhibit spontaneous
intestinal inflammation. Moreover, hypomorphic XBP1 variants are linked
to IBD in humans (Kaser et al., 2008).
Together, these studies suggest that defects leading to reduced antimicro-

bial protein or mucus production may increase the likelihood of bacterial
invasion of the epithelial barrier with consequent inflammation. However,
it is important to note that epithelial cell defects, such as genetic Paneth cell
ablation, are insufficient to produce inflammation in mice (Garabedian,
Roberts, McNevin, & Gordon, 1997). This suggests that the development
of inflammatory disease in humans may require additional genetic defects
that impact, for example, the ability of phagocytic cells to remove bacteria
that breach the epithelial barrier. Thus, multiple genetic lesions that target
different levels of immune control of the microbiota may be required before
inflammatory disease is manifested.
10. FUTURE PERSPECTIVES

The mammalian intestinal epithelium is faced with a complex and
dynamic microbial challenge that is unique among tissues. The studies dis-
cussed in this chapter highlight the diverse array of strategies used by epithe-
lial cells to maintain homeostasis with the enteric microbiota and to prevent
pathogen invasion. These strategies include epithelial cell-intrinsic functions
such as antimicrobial protein production, mucus secretion, and autophagy
activation. At the same time, the intestinal epithelium plays a central role
in stimulating and coordinating adaptive immune responses to intestinal
microorganisms. Finally, it is clear that disrupting epithelial cell functions
can have profound consequences for host health.
Most of our current understanding of epithelial cell immune function is
derived from studies of the gastrointestinal tract. However, other body sur-
faces, such as the skin (Grice & Segre, 2011), respiratory tract (Dickson, Erb-
Downward, & Huffnagle, 2013), and urogenital tract (Ma, Forney, & Ravel,
2012), are also home to diverse communities of indigenous microorganisms
that are in close contact with epithelial cells. These tissues are thus also likely
to be sites where epithelial immune functions have profound effects on host
health. Future studies of other body surfaces will therefore be critical for
obtaining a comprehensive picture of epithelial cell contributions to
immunity.
Finally, the majority of studies performed to date have focused on inter-
actions between epithelial cells and intestinal bacteria. However, the intes-
tinal microbiota also includes eukaryotic viruses (Virgin, 2014),
bacteriophage (Reyes et al., 2010), and eukaryotic organisms such as fungi
(Iliev et al., 2012). These other elements of the microbial community are
162 Lora V. Hooper
undoubtedly also important targets of epithelial cell-intrinsic immune pro-

cesses and likely profoundly influence epithelial function. These other
microbiota components will provide fascinating targets for further explora-
tion of epithelial cell contributions to intestinal homeostasis. Ultimately,
such efforts should produce deeper insight into how mammalian hosts man-
age interactions with diverse microbial communities and provide new
opportunities to improve human health.
ACKNOWLEDGMENTS
L. V. H. is supported by the Howard Hughes Medical Institute, the National Institutes of
Health (R01 DK070855), and the Burroughs Wellcome Foundation (Investigators in the
Pathogenesis of Infectious Diseases Award). The author has no conflicting financial interests.
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CHAPTER THREE

Epithelial Cell Contributions

Advances in Immunology, Volume 126 # 2015 Elsevier Inc. 129

9. Epithelial Dysfunction in Inflammatory Disease 159

chronic inflammation. At the same time, the intestinal epithelium must

1.2. The intestinal microbiota

This microbial community makes several important contributions to

1.3. Germ-free mice as experimental tools

as “gnotobiotics,” a term derived from Greek roots and meaning “known

2. CELLULAR MAKEUP OF THE INTESTINAL EPITHELIAL

2.2. Goblet cells

2.3. Paneth cells

2.4. Enteroendocrine cells

peptide, glucagon-like peptide, and vasoactive intestinal peptide that regu-

3. EPITHELIAL CELL SENSING OF INTESTINAL MICROBES

Although epithelial cell-intrinsic recognition of microbes can activate

3.2. Tissue-specific modulation of epithelial cell-specific innate

Another protein that modulates inflammatory signaling pathways is A20,

4. MUCUS PRODUCTION BY THE INTESTINAL

4.2. Regulation of mucus production

a-Defensins RegIIIa/g Invasive

member of the NOD-like receptor (NLR) family senses stress or damage-

5. EPITHELIAL ANTIMICROBIAL PROTEINS

microorganisms and are members of a diverse group of protein families. The

5.1. Epithelial antimicrobial protein families

lineages (Selsted & Ouellette, 2005). Because of their localization in the

5.1.4 Lysozyme and phospholipase A2

5.2. Regulation of epithelial antimicrobial proteins

5.2.1 Transcriptional regulation of epithelial antimicrobial protein

Intestinal epithelial cell expression of RegIIIγ also requires signals from at

Figure 2—Cont'd Regulation of antimicrobial protein expression and secretion. Sev-

monocytogenes (Kobayashi et al., 2005). These results indicate that NOD2-

5.2.2 Developmental regulation of antimicrobial protein expression

5.2.3 Posttranslational regulation of antimicrobial protein function

The distinctive reducing environment of the intestinal lumen provides

5.2.4 Regulation of antimicrobial protein secretion

5.3. In vivo functions of epithelial antimicrobial proteins

5.3.1 Protection against pathogens

Protection against Control of microbiota Limiting bacteria-

Outer mucus layer

Paneth cells Enterocytes/

(Brandl et al., 2007) and vancomycin-resistant Enterococcus faecalis (Brandl

5.3.2 Shaping microbiota composition

5.3.3 Limiting bacterial-epithelial cell contact

6. INTESTINAL EPITHELIAL CELL AUTOPHAGY

6.1. Autophagy as a barrier to bacterial dissemination

(Benjamin et al., 2013). Finally, epithelial autophagy activation depends on

6.2. Autophagy-dependent regulation of protein secretion

7. EPITHELIAL REGULATION OF ADAPTIVE IMMUNITY

TSLP BAFF APRIL

B cell IgA+ plasma cell IgA

7.1. Transcytosis of immunoglobulin A

7.2. Cytokine secretion

7.3. Antigen delivery to subepithelial immune cells

provide an alternate route of antigen entry in the intestinal epithelium

8. BACTERIAL STIMULATION OF EPITHELIAL

damage rapidly and efficiently. Interestingly, bacteria play a central role in

8.1. MyD88-dependent epithelial repair

8.2. Activation of epithelial repair by reactive oxygen species

9. EPITHELIAL DYSFUNCTION IN INFLAMMATORY

Required for granule exocytosis

Required for α-defensin

Figure 5 Dysregulation of epithelial cell function in inflammatory disease. NOD2, TCF4,