Epithelial cell contributions to intestinal immunity.

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: [email protected]

Contents 1. Introduction 1.1 Overview of epithelial-microbial interactions in the mammalian intestine 1.2 The intestinal microbiota 1.3 Germ-free mice as experimental tools 2. Cellular Makeup of the Intestinal Epithelial Barrier 2.1 Enterocytes 2.2 Goblet cells 2.3 Paneth cells 2.4 Enteroendocrine cells 2.5 M cells 3. Epithelial Cell Sensing of Intestinal Microbes 3.1 Epithelial detection of microbes by pattern recognition receptors 3.2 Tissue-specific modulation of epithelial cell-specific innate immune responses 4. Mucus Production by the Intestinal Epithelium 4.1 Secretion and assembly of the mucus layer 4.2 Regulation of mucus production 5. Epithelial Antimicrobial Proteins 5.1 Epithelial antimicrobial protein families 5.2 Regulation of epithelial antimicrobial proteins 5.3 In vivo functions of epithelial antimicrobial proteins 6. Intestinal Epithelial Cell Autophagy 6.1 Autophagy as a barrier to bacterial dissemination 6.2 Autophagy-dependent regulation of protein secretion 7. Epithelial Regulation of Adaptive Immunity 7.1 Transcytosis of immunoglobulin A 7.2 Cytokine secretion 7.3 Antigen delivery to subepithelial immune cells 8. Bacterial Stimulation of Epithelial Cell Repair 8.1 MyD88-dependent epithelial repair 8.2 Activation of epithelial repair by reactive oxygen species Advances in Immunology, Volume 126 ISSN 0065-2776 http://dx.doi.org/10.1016/bs.ai.2014.11.003

#

2015 Elsevier Inc. All rights reserved.

130 130 131 132 133 133 134 134 134 135 135 135 138 139 139 139 141 142 145 150 153 153 154 154 155 156 156 157 158 159 129

130

Lora V. Hooper

9. Epithelial Dysfunction in Inflammatory Disease 10. Future Perspectives Acknowledgments References

159 161 162 162

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 function 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 bacteria, 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 enormous 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 indigenous 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 present 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 antimicrobial proteins and activation of autophagy, as well as strategies for shaping 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 mechanisms 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 intestinal 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 predominantly 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, Bacteroides 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 polysaccharides, 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 environment 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 interweaving of many aspects of mammalian and microbial physiology, and thus intestinal 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 signals 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 neurodevelopmental 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 (Ba¨ckhed et al., 2004).

1.3. Germ-free mice as experimental tools Animals raised in microbiologically sterile (germ-free) conditions are important experimental tools for the study of epithelial contributions to intestinal immunity. Such animals are reared in sterile isolators that control their exposure 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


133

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 germfree 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 microbefilled lumen by a single-epithelial layer that is 20 μm thick. Intestinal epithelial 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 epithelial surfaces. This occurs in several ways, each of which will be discussed in detail in subsequent sections. First, enterocytes secrete a variety of antimicrobial 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 resistinlike 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 intestinal 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 epithelium. 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 comprise 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 nutrients, and secrete multiple regulatory factors such as gastric inhibitory


135

peptide, glucagon-like peptide, and vasoactive intestinal peptide that regulate 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 follicleassociated 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 microorganisms, 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 microorganisms 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 components 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 component 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 signaling cascades that trigger the nuclear translocation of the transcription factor 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 MyD88deficient 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


137

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 recognition. 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 subsequently 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 components 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), NOD2dependent 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 fragments in the cell cytoplasm and initiates the signaling cascades that activate NFκB (Keestra & Baümler, 2014; Keestra et al., 2013). Other members of the NLR family are involved in inflammasome activation. 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 inflammasomes 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 detrimental by provoking tumorigenesis. For example, the activation of epithelial 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 possible 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 compartmentalization 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 bacterial 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). Consequently, 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 modulate the concentrations of LPS required to activate epithelial cell inflammatory 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).


139

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 TNFreceptor-associated factor 6 (TRAF6) (Deng et al., 2000) and receptorinteracting protein kinase (Li, Kobayashi, Blonska, You, & Lin, 2006). A20-deficient (Tnfaip3 / ) mice are highly susceptible to intestinal inflammation, 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 modulate 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 quantities 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 resistant 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 consequently have severe intestinal inflammation ( Johansson et al., 2008). Thus, the mucus barrier is essential for maintaining a beneficial symbiotic relationship 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

Lumen

Bacterial killing a-Defensins

RegIIIa/g

Mucus layer

Bacterial membrane

Assembly into mucus layer

Mucins

Invasive bacteria

Epithelium

ATG5

Paneth cell

ATG16L1

Secretory granules

Secretory granules

Lamina propria

MyD88

a-Defensins

Autophagy

Mucins Enterocyte/ paneth cell

Goblet cell

X

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 antimicrobial proteins, such as α-defensins. RegIIIα (human) and RegIIIγ (mouse) are antimicrobial 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).


141

member of the NOD-like receptor (NLR) family senses stress or damageassociated 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 susceptibility 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 susceptible 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 interesting 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 epithelial surfaces to cope with the challenge of being closely associated with a dense microbial community (Fig. 1). These natural antibiotics are an evolutionarily 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 proteins, likely reflecting the complexity of the microbial communities that challenge the intestinal surface. Most antimicrobial peptides and proteins target the cell walls of microorganisms. 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 nutrient 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 evolutionary 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 arrangement 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 particular 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); however, 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


143

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 subset 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 nonenzymatic disruption of bacterial membranes. RegIIIγ and its human ortholog, 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 positive bacteria (Cash et al., 2006). This is consistent with the fact that peptidoglycan 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 interactions. 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 functions. 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 membrane 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, macrophages, and T cells, and induces cytokine secretion by dendritic cells (Davidson et al., 2004).


145

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 phospholipids, 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-Go´mez & 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 transcriptional 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 signals (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 microbial 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 family, 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 colonization (Cash et al., 2006; Hooper et al., 2003). As discussed earlier, host pattern recognition receptors direct the expression of some of these bacterially regulated epithelial antimicrobial proteins. For example, stimulation of TLRs is required for RegIIIγ mRNA expression 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). Further, 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

Lumen

Subset of regulated α-defensins MAMP (e.g., LPS)

α-Defensins

RegIIIγ Bacterial stimulation of granule exocytosis

Toll-like receptor

ATG16L1 mutant Defective

MDP

Xexocytosis

Epithelium

MyD88 α-Defensin NOD2 TCF4

RegIIIγ

NFκB

Enterocyte

Lamina propria

Paneth cells

Figure 2 See legend on next page.

Paneth cells

IL-22R

IL-22 Innate lymphoid cell

Secretory granules

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 cytokine 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 produce low levels of IL-22 (Sanos et al., 2008), indicating that intestinal bacteria 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 cellintrinsic 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 bacterial 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. Several mechanisms regulate antimicrobial expression and function in the intestinal epithelium. 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 molecular 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 process of granule formation and secretion (Cadwell et al., 2008). Thus, patients with a single amino acid variant of ATG16L1 that is associated with Crohn's disease have abnormal Paneth cell granule formation (Cadwell et al., 2008).


149

monocytogenes (Kobayashi et al., 2005). These results indicate that NOD2stimulated 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 barrier is established at the mucosal surface by the subset of antimicrobial proteins 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 beneficial 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 expression 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 membranebound 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 peptides (Wilson et al., 1999). In humans, trypsin cleaves α-defensins to their mature forms (Ghosh et al., 2002). RegIIIγ also requires N-terminal proteolytic 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 conditions 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 regulated 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.

151


5.3.1 Protection against pathogens Experiments in mice have yielded key insight into the importance of intestinal antimicrobial proteins in pathogen protection in vivo. MMP7 is required to generate bactericidally active α-defensins in mice, and consequently, 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 colonization by Gram positive intestinal pathogens such as L. monocytogenes

Protection against Control of microbiota Limiting bacteriapathogens epithelial contact composition

S. typhimurium Outer mucus layer

a-defensins

RegIIIg 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 community. Mmp7 / and DEFA5-transgenic mice each show marked α-defensindependent 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 composition. These changes include altered proportions of Firmicutes, the major Gram positive phylum, and Bacteroidetes, the major Gram negative phylum, 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 development 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. Further, most antimicrobial activity is confined to the mucus layer and is essentially 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 bacterial access to the epithelium by forming a diffusion barrier that concentrates 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 positives (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


153

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 cytoplasmic contents during cellular starvation and by recycling damaged organelles and proteins (Rabinowitz & White, 2010). However, autophagy has also been shown to be critical for the recognition and degradation of intracellular 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 functions are discussed in this section.

6.1. Autophagy as a barrier to bacterial dissemination Recent studies have shown that autophagy is an important epithelial cellautonomous mechanism of antibacterial defense that protects against dissemination 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 epithelial cells and remains at baseline levels in response to the microbiota normally 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 noninvasive 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 distinct from classical autophagy involving the targeting of bacteria to autophagosomes (Zhao et al., 2008). As discussed earlier, mice lacking epithelial cell expression of ATG16L1 or ATG5 exhibit defective Paneth cell granule formation and exocytosis, indicating a role for these autophagy proteins 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 underlying 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).

155


Lumen

Bacteria

Epithelium

Luminal antigens

Transcytosis Secretory granules Goblet cell

Lamina propria

Enterocyte TSLP

BAFF

pIgR

APRIL

CD103+ DC Antigen presentation to DC

Dendritic cell B cell

IgA+ plasma cell

IgA

IL-10

Figure 4 Epithelial cell regulation of adaptive immunity. The intestinal epithelial cellderived cytokine TSLP (thymic stromal lymphopoietin) causes dendritic cells and macrophages 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 cellindependent 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 surface 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 confining bacteria to the intestinal lumen (Macpherson et al., 2000), though the exact mechanisms 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 epithelial barrier. IgA is the most abundant immunoglobulin isotype in the intestine 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 IgAsecreting 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 epithelium, and the IgA is deposited on the apical epithelial surface of the epithelium (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 organisms 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 example, 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 family) 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 stromal 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 sampling 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 tissues have been associated with antigen sampling. As discussed in Section 2, these specialized cells mediate the sampling of antigens and microorganisms 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


157

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 intestinal 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, allowing dendrite extension into the lumen for direct sampling of microorganisms 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 bacteria, 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 pathogenic 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 penetration. 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 visible 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 epithelial 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 antibiotictreated mice with commensal bacteria restores normal epithelial repair processes. 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 signaling pathways is essential for colonic tissue repair processes. The DSS injury model has also provided essential clues about the intestinal cell populations that promote microbe-regulated epithelial repair. Analysis of bone marrow chimeric mice revealed that the MyD88dependent signals that drive epithelial repair derive from bone marrowderived 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 localized next to epithelial progenitor cells and express factors that stimulate cellular proliferation (Pull et al., 2005). Together, these results suggest a model in which epithelial repair is driven by bacterial stimulation of mobile


159

subepithelial myeloid cells that migrate to damaged regions and produce factors that promote epithelial restitution.

8.2. Activation of epithelial repair by reactive oxygen species Bacteria also promote epithelial repair by inducing ROS in intestinal epithelial 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 mechanism 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 inflammation of the colon or the distal small intestine, or both. Although the exact causes of IBD remain poorly understood, its pathologic characteristics indicate 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 epithelium (Swidsinski, Weber, Loening-Baucke, Hale, & Lochs, 2005). This suggests that IBD is characterized in part by a failure of mechanisms that normally limit microbiota-epithelial contact. Consistent with this idea, several IBD risk alleles alter epithelial cell function by impairing production of antimicrobial peptides or mucus (Fig. 5). NOD2 was identified as the first genetic susceptibility locus for Crohn’s disease (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 conjunction 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

ATG16L1 Senses MDP and influences α-defensin expression

Required for granule exocytosis and antibacterial autophagy

NOD2 XBP1

Required for ER expansion during stress

Endoplasmic reticulumGolgi 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; PetnickiOcwieja 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 antimicrobial 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).


161

Together, these studies suggest that defects leading to reduced antimicrobial 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 discussed in this chapter highlight the diverse array of strategies used by epithelial 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 surfaces, such as the skin (Grice & Segre, 2011), respiratory tract (Dickson, ErbDownward, & 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 interactions between epithelial cells and intestinal bacteria. However, the intestinal 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 processes and likely profoundly influence epithelial function. These other microbiota components will provide fascinating targets for further exploration of epithelial cell contributions to intestinal homeostasis. Ultimately, such efforts should produce deeper insight into how mammalian hosts manage 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.

REFERENCES Agostini, L., Martinon, F., Burns, K., McDermott, M. F., Hawkins, P. N., & Tschopp, J. (2004). NALP3 forms an IL-1beta-processing inflammasome with increased activity in Muckle-Wells autoinflammatory disorder. Immunity, 20, 319–325. Akira, S., Uematsu, S., & Takeuchi, O. (2006). Pathogen recognition and innate immunity. Cell, 124, 783–801. Alam, A., Leoni, G., Wentworth, C. C., Kwal, J. M., Wu, H., Ardita, C. S., et al. (2013). Redox signaling regulates commensal-mediated mucosal homeostasis and restitution and requires formyl peptide receptor 1. Mucosal Immunology, 7, 645–655. Artis, D., Wang, M. L., Keilbaugh, S. A., He, W., Brenes, M., Swain, G. P., et al. (2004). RELMbeta/FIZZ2 is a goblet cell-specific immune-effector molecule in the gastrointestinal tract. Proceedings of the National Academy of Sciences of the United States of America, 101, 13596–13600. Atarashi, K., Tanoue, T., Shima, T., Imaoka, A., Kuwahara, T., Momose, Y., et al. (2011). Induction of colonic regulatory T cells by indigenous clostridium species. Science, 331, 337–341. Ayabe, T., Satchell, D. P., Wilson, C. L., Parks, W. C., Selsted, M. E., & Ouellette, A. J. (2000). Secretion of microbicidal α-defensins by intestinal Paneth cells in response to bacteria. Nature Immunology, 1, 113–118. Ba¨ckhed, F., Ding, H., Wang, T., Hooper, L. V., Koh, G. Y., Nagy, A., et al. (2004). The gut microbiota as an environmental factor that regulates fat storage. Proceedings of the National Academy of Sciences of the United States of America, 101, 15718–15723. Bals, R., & Wilson, J. M. (2003). Cathelicidins—A family of multifunctional antimicrobial peptides. Cellular and Molecular Life Sciences, 60, 711–720. Bates, J. M., Akerlund, J., Mittge, E., & Guillemin, K. (2007). Intestinal alkaline phosphatase detoxifies lipopolysaccharide and prevents inflammation in zebrafish in response to the Gut microbiota. Cell Host & Microbe, 2, 371–382. Benjamin, J. L., Sumpter, R., Levine, B., & Hooper, L. V. (2013). Intestinal epithelial autophagy is essential for host defense against invasive bacteria. Cell Host & Microbe, 13, 723–734. Biragyn, A., Ruffini, P. A., Leifer, C. A., Klyushnenkova, E., Shakhov, A., Chertov, O., et al. (2002). Toll-like receptor 4-dependent activation of dendritic cells by beta-defensin 2. Science, 298, 1025–1029.


163

Brandl, K., Plitas, G., Mihu, C. N., Ubeda, C., Jia, T., Fleisher, M., et al. (2008). Vancomycin-resistant enterococci exploit antibiotic-induced innate immune deficits. Nature, 455, 804–807. Brandl, K., Plitas, G., Schnabl, B., DeMatteo, R. P., & Pamer, E. G. (2007). MyD88-mediated signals induce the bactericidal lectin RegIIIγ and protect mice against intestinal Listeria monocytogenes infection. The Journal of Experimental Medicine, 204, 1891–1900. Bruno, M. E. C., Frantz, A. L., Rogier, E. W., Johansen, F.-E., & Kaetzel, C. S. (2011). Regulation of the polymeric immunoglobulin receptor by the classical and alternative NF-κB pathways in intestinal epithelial cells. Mucosal Immunology, 4, 468–478. Cadwell, K., Liu, J. Y., Brown, S. L., Miyoshi, H., Loh, J., Lennerz, J. K., et al. (2008). A key role for autophagy and the autophagy gene Atg16l1 in mouse and human intestinal Paneth cells. Nature, 456, 259–263. Cadwell, K., Patel, K. K., Maloney, N. S., Liu, T.-C., Ng, A. C. Y., Storer, C. E., et al. (2010). Virus-plus-susceptibility gene interaction determines Crohn’s disease gene Atg16L1 phenotypes in intestine. Cell, 141, 1135–1145. Cash, H. L., Whitham, C. V., Behrendt, C. L., & Hooper, L. V. (2006). Symbiotic bacteria direct expression of an intestinal bactericidal lectin. Science, 313, 1126–1130. Cerutti, A., Puga, I., & Cols, M. (2011). Innate control of B cell responses. Trends in Immunology, 32, 202–211. Chen, Y., Chou, K., Fuchs, E., Havran, W. L., & Boismenu, R. (2002). Protection of the intestinal mucosa by intraepithelial gamma delta T cells. Proceedings of the National Academy of Sciences of the United States of America, 99, 14338–14343. Christa, L., Carnot, F., Simon, M. T., Levavasseur, F., Stinnakre, M. G., Lasserre, C., et al. (1996). HIP/PAP is an adhesive protein expressed in hepatocarcinoma, normal Paneth, and pancreatic cells. The American Journal of Physiology, 271, G993–G1002. Clevers, H. C., & Bevins, C. L. (2013). Paneth cells: Maestros of the small intestinal crypts. Annual Review of Physiology, 75, 289–311. Colgan, S. P., Hershberg, R. M., Furuta, G. T., & Blumberg, R. S. (1999). Ligation of intestinal epithelial CD1d induces bioactive IL-10: Critical role of the cytoplasmic tail in autocrine signaling. Proceedings of the National Academy of Sciences of the United States of America, 96, 13938–13943. € H., et al. Conway, K. L., Kuballa, P., Song, J. H., Patel, K. K., Castoreno, A. B., Yilmaz, O. (2013). Atg16l1 is required for autophagy in intestinal epithelial cells and protection of mice from salmonella infection. Gastroenterology, 145, 1347–1357. Coombes, J. L., Siddiqui, K. R. R., Arancibia-Carcamo, C. V., Hall, J., Sun, C. M., Belkaid, Y., et al. (2007). A functionally specialized population of mucosal CD103 + DCs induces Foxp3 + regulatory T cells via a TGF- and retinoic acid dependent mechanism. Journal of Experimental Medicine, 204, 1757–1764. Davidson, D. J., Currie, A. J., Reid, G. S. D., Bowdish, D. M. E., MacDonald, K. L., Ma, R. C., et al. (2004). The cationic antimicrobial peptide LL-37 modulates dendritic cell differentiation and dendritic cell-induced T cell polarization. Journal of Immunology, 172, 1146–1156. Deng, L., Wang, C., Spencer, E., Yang, L., Braun, A., You, J., et al. (2000). Activation of the IkappaB kinase complex by TRAF6 requires a dimeric ubiquitin-conjugating enzyme complex and a unique polyubiquitin chain. Cell, 103, 351–361. Deretic, V., & Levine, B. (2009). Autophagy, immunity, and microbial adaptations. Cell Host & Microbe, 5, 527–549. Dickson, R. P., Erb-Downward, J. R., & Huffnagle, G. B. (2013). The role of the bacterial microbiome in lung disease. Expert Review of Respiratory Medicine, 7, 245–257. Dieckgraefe, B. K., Crimmins, D. L., Landt, V., Houchen, C., Anant, S., Porche-Sorbet, R., et al. (2002). Expression of the regenerating gene family in inflammatory bowel disease

164

Lora V. Hooper

mucosa: Reg Ialpha upregulation, processing, and antiapoptotic activity. Journal of Investigative Medicine, 50, 421–434. Eckburg, P. B., Bik, E. M., Bernstein, C. N., Purdom, E., Dethlefsen, L., Sargent, M., et al. (2005). Diversity of the human intestinal microbial flora. Science, 308, 1635–1638. Elinav, E., Strowig, T., Kau, A. L., Henao-Mejia, J., Thaiss, C. A., Booth, C. J., et al. (2011). NLRP6 inflammasome regulates colonic microbial ecology and risk for colitis. Cell, 145(5), 745–757. Fahlgren, A., Hammarstr€ om, S., Danielsson, A., & Hammarstr€ om, M.-L. (2003). Increased expression of antimicrobial peptides and lysozyme in colonic epithelial cells of patients with ulcerative colitis. Clinical and Experimental Immunology, 131, 90–101. Faith, J. J., Guruge, J. L., Charbonneau, M., Subramanian, S., Seedorf, H., Goodman, A. L., et al. (2013). The long-term stability of the human gut microbiota. Science, 341, 1237439. Faraldo-Go´mez, J. D., & Sansom, M. S. P. (2003). Acquisition of siderophores in Gramnegative bacteria. Nature Reviews Molecular Cell Biology, 4, 105–116. Farrell, J. J., Taupin, D., Koh, T. J., Chen, D., Zhao, C.-M., Podolsky, D. K., et al. (2002). TFF2/SP-deficient mice show decreased gastric proliferation, increased acid secretion, and increased susceptibility to NSAID injury. The Journal of Clinical Investigation, 109, 193–204. Flo, T. H., Smith, K. D., Sato, S., Rodriguez, D. J., Holmes, M. A., Strong, R. K., et al. (2004). Lipocalin 2 mediates an innate immune response to bacterial infection by sequestrating iron. Nature, 432, 917–921. Fukata, M., Chen, A., Vamadevan, A. S., Cohen, J., Breglio, K., Krishnareddy, S., et al. (2007). Toll-like receptor-4 promotes the development of colitis-associated colorectal tumors. Gastroenterology, 133, 1869–1881. Fukata, M., Shang, L., Santaolalla, R., Sotolongo, J., Pastorini, C., Espanã, C., et al. (2011). Constitutive activation of epithelial TLR4 augments inflammatory responses to mucosal injury and drives colitis-associated tumorigenesis. Inflammatory Bowel Diseases, 17, 1464–1473. Gallo, R. L., Kim, K. J., Bernfield, M., Kozak, C. A., Zanetti, M., Merluzzi, L., et al. (1997). Identification of CRAMP, a cathelin-related antimicrobial peptide expressed in the embryonic and adult mouse. The Journal of Biological Chemistry, 272, 13088–13093. Ganz, T. (2004). Antimicrobial polypeptides. Journal of Leukocyte Biology, 75, 34–38. Garabedian, E. M., Roberts, L. J., McNevin, M. S., & Gordon, J. I. (1997). Examining the role of Paneth cells in the small intestine by lineage ablation in transgenic mice. The Journal of Biological Chemistry, 272, 23729–23740. Geuking, M. B., Cahenzli, J., Lawson, M. A. E., Ng, D. C. K., Slack, E., Hapfelmeier, S., et al. (2011). Intestinal bacterial colonization induces mutualistic regulatory T cell responses. Immunity, 34, 794–806. Gewirtz, A. T., Navas, T. A., Lyons, S., Godowski, P. J., & Madara, J. L. (2001). Cutting edge: Bacterial flagellin activates basolaterally expressed TLR5 to induce epithelial proinflammatory gene expression. Journal of Immunology, 167, 1882–1885. Ghosh, D., Porter, E., Shen, B., Lee, S. K., Wilk, D., Drazba, J., et al. (2002). Paneth cell trypsin is the processing enzyme for human defensin-5. Nature Immunology, 3, 583–590. Gill, S. R., Pop, M., Deboy, R. T., Eckburg, P. B., Turnbaugh, P. J., Samuel, B. S., et al. (2006). Metagenomic analysis of the human distal gut microbiome. Science, 312, 1355–1359. Girardin, S. E., Boneca, I. G., Carneiro, L. A. M., Antignac, A., Je´hanno, M., Viala, J., et al. (2003). Nod1 detects a unique muropeptide from Gram-negative bacterial peptidoglycan. Science, 300, 1584–1587. Greten, F. R., Eckmann, L., Greten, T. F., Park, J. M., Li, Z.-W., Egan, L. J., et al. (2004). IKKbeta links inflammation and tumorigenesis in a mouse model of colitis-associated cancer. Cell, 118, 285–296.


165

Grice, E. A., & Segre, J. A. (2011). The skin microbiome. Nature Reviews Microbiology, 9, 244–253. Gudmundsson, G. H., Agerberth, B., Odeberg, J., Bergman, T., Olsson, B., & Salcedo, R. (1996). The human gene FALL39 and processing of the cathelin precursor to the antibacterial peptide LL-37 in granulocytes. European Journal of Biochemistry, 238, 325–332. Gum, J. R., Hicks, J. W., Toribara, N. W., Siddiki, B., & Kim, Y. S. (1994). Molecular cloning of human intestinal mucin (MUC2) cDNA. Identification of the amino terminus and overall sequence similarity to prepro-von Willebrand factor. The Journal of Biological Chemistry, 269, 2440–2446. Hampe, J., Franke, A., Rosenstiel, P., Till, A., Teuber, M., Huse, K., et al. (2007). A genome-wide association scan of nonsynonymous SNPs identifies a susceptibility variant for Crohn disease in ATG16L1. Nature Genetics, 39, 207–211. Harder, J., & Schroder, J.-M. (2002). RNase 7, a novel innate immune defense antimicrobial protein of healthy human skin. The Journal of Biological Chemistry, 277, 46779–46784. Harwig, S. S., Tan, L., Qu, X. D., Cho, Y., Eisenhauer, P. B., & Lehrer, R. I. (1995). Bactericidal properties of murine intestinal phospholipase A2. The Journal of Clinical Investigation, 95, 603–610. Hase, K., Eckmann, L., Leopard, J. D., Varki, N., & Kagnoff, M. F. (2002). Cell differentiation is a key determinant of cathelicidin LL-37/human cationic antimicrobial protein 18 expression by human colon epithelium. Infection and Immunity, 70, 953–963. Hayashi, F., Smith, K. D., Ozinsky, A., Hawn, T. R., Yi, E. C., Goodlett, D. R., et al. (2001). The innate immune response to bacterial flagellin is mediated by Toll-like receptor 5. Nature, 410, 1099–1103. He, B., Xu, W., Santini, P. A., Polydorides, A. D., Chiu, A., Estrella, J., et al. (2007). Intestinal bacteria trigger T cell-independent immunoglobulin A(2) class switching by inducing epithelial-cell secretion of the cytokine APRIL. Immunity, 26, 812–826. Heller, F., Fuss, I. J., Nieuwenhuis, E. E., Blumberg, R. S., & Strober, W. (2002). Oxazolone colitis, a Th2 colitis model resembling ulcerative colitis, is mediated by IL-13-producing NK-T cells. Immunity, 17, 629–638. Hemmi, H., Takeuchi, O., Kawai, T., Kaisho, T., Sato, S., Sanjo, H., et al. (2000). A Tolllike receptor recognizes bacterial DNA. Nature, 408, 740–745. Hochmuth, C. E., Biteau, B., Bohmann, D., & Jasper, H. (2011). Redox regulation by Keap1 and Nrf2 controls intestinal stem cell proliferation in Drosophila. Cell Stem Cell, 8, 188–199. Hood, M. I., & Skaar, E. P. (2012). Nutritional immunity: Transition metals at the pathogenhost interface. Nature Reviews Microbiology, 10, 525–537. Hooper, L. V., Stappenbeck, T. S., Hong, C. V., & Gordon, J. I. (2003). Angiogenins: A new class of microbicidal proteins involved in innate immunity. Nature Immunology, 4, 269–273. Hooper, L. V., Wong, M. H., Thelin, A., Hansson, L., Falk, P. G., & Gordon, J. I. (2001). Molecular analysis of commensal host-microbial relationships in the intestine. Science, 291, 881–884. Hornef, M. W., P€ utsep, K., Karlsson, J., Refai, E., & Andersson, M. (2004). Increased diversity of intestinal antimicrobial peptides by covalent dimer formation. Nature Immunology, 5, 836–843. Hugot, J. P., Chamaillard, M., Zouali, H., Lesage, S., Ce´zard, J. P., Belaiche, J., et al. (2001). Association of NOD2 leucine-rich repeat variants with susceptibility to Crohn’s disease. Nature, 411, 599–603. Iliev, I. D., Funari, V. A., Taylor, K. D., Nguyen, Q., Reyes, C. N., Strom, S. P., et al. (2012). Interactions between commensal fungi and the c-type lectin receptor dectin-1 influence colitis. Science, 336, 1314–1317.

166

Lora V. Hooper

Inohara, N., Ogura, Y., Fontalba, A., Gutierrez, O., Pons, F., Crespo, J., et al. (2003). Host recognition of bacterial muramyl dipeptide mediated through NOD2. Implications for Crohn’s disease. The Journal of Biological Chemistry, 278, 5509–5512. Inoue, J., Nishiumi, S., Fujishima, Y., Masuda, A., Shiomi, H., Yamamoto, K., et al. (2012). Autophagy in the intestinal epithelium regulates Citrobacter rodentium infection. Archives of Biochemistry and Biophysics, 521, 95–101. Ivanov, I. I., Atarashi, K., Manel, N., Brodie, E. L., Shima, T., Karaoz, U., et al. (2009). Induction of intestinal Th17 cells by segmented filamentous bacteria. Cell, 139, 485–498. Ivanov, I. I., Frutos, R. de L., Manel, N., Yoshinaga, K., Rifkin, D. B., Sartor, R. B., et al. (2008). Specific microbiota direct the differentiation of IL-17-producing T-helper cells in the mucosa of the small intestine. Cell Host & Microbe, 4, 337–349. Jaensson, E., Uronen-Hansson, H., Pabst, O., Eksteen, B., Tian, J., Coombes, J. L., et al. (2008). Small intestinal CD103+ dendritic cells display unique functional properties that are conserved between mice and humans. Journal of Experimental Medicine, 205, 2139–2149. Jang, M. H., Kweon, M.-N., Iwatani, K., Yamamoto, M., Terahara, K., Sasakawa, C., et al. (2004). Intestinal villous M cells: An antigen entry site in the mucosal epithelium. Proceedings of the National Academy of Sciences of the United States of America, 101, 6110–6115. Johansen, F.-E., & Kaetzel, C. S. (2011). Regulation of the polymeric immunoglobulin receptor and IgA transport: New advances in environmental factors that stimulate pIgR expression and its role in mucosal immunity. Mucosal Immunology, 4, 598–602. Johansson, M. E. V., Phillipson, M., Petersson, J., Velcich, A., Holm, L., & Hansson, G. C. (2008). The inner of the two Muc2 mucin-dependent mucus layers in colon is devoid of bacteria. Proceedings of the National Academy of Sciences of the United States of America, 105, 15064–15069. Johansson-Lindbom, B., Svensson, M., Pabst, O., Palmqvist, C., Marquez, G., F€ orster, R., et al. (2005). Functional specialization of gut CD103 + dendritic cells in the regulation of tissue-selective T cell homing. The Journal of Experimental Medicine, 202, 1063–1073. Jones, R. M., Luo, L., Ardita, C. S., Richardson, A. N., Kwon, Y. M., Mercante, J. W., et al. (2013). Symbiotic lactobacilli stimulate gut epithelial proliferation via Nox-mediated generation of reactive oxygen species. The EMBO Journal, 32, 3017–3028. Kaser, A., Lee, A.-H., Franke, A., Glickman, J. N., Zeissig, S., Tilg, H., et al. (2008). XBP1 links ER stress to intestinal inflammation and confers genetic risk for human inflammatory bowel disease. Cell, 134, 743–756. Keestra, A. M., & Baümler, A. J. (2014). Detection of enteric pathogens by thenodosome. Trends in Immunology, 35, 123–130. Keestra, A. M., Winter, M. G., Auburger, J. J., Fra¨ssle, S. P., Xavier, M. N., Winter, S. E., et al. (2013). Manipulation of small Rho GTPases is a pathogen-induced process detected by NOD1. Nature, 496, 233–237. Kim, J. G., Lee, S. J., & Kagnoff, M. F. (2004). Nod1 is an essential signal transducer in intestinal epithelial cells infected with bacteria that avoid recognition by toll-like receptors. Infection and Immunity, 72, 1487–1495. Kinnebrew, M. A., Ubeda, C., Zenewicz, L. A., Smith, N., Flavell, R. A., & Pamer, E. G. (2010). Bacterial flagellin stimulates Toll-like receptor 5-dependent defense against vancomycin-resistant Enterococcus infection. The Journal of Infectious Diseases, 201, 534–543. Kobayashi, K. S., Chamaillard, M., Ogura, Y., Henegariu, O., Inohara, N., Nun˜ez, G., et al. (2005). Nod2-dependent regulation of innate and adaptive immunity in the intestinal tract. Science, 307, 731–734. Koprivnjak, T., Peschel, A., Gelb, M. H., Liang, N. S., & Weiss, J. P. (2002). Role of charge properties of bacterial envelope in bactericidal action of human group IIA phospholipase A2 against Staphylococcus aureus. The Journal of Biological Chemistry, 277, 47636–47644.


167

Koslowski, M. J., K€ ubler, I., Chamaillard, M., Schaeffeler, E., Reinisch, W., Wang, G., et al. (2009). Genetic variants of Wnt transcription factor TCF-4 (TCF7L2) putative promoter region are associated with small intestinal Crohn’s disease. PLoS One, 4, e4496. Kraehenbuhl, J. P., & Neutra, M. R. (2000). Epithelial M cells: Differentiation and function. Annual Review of Cell and Developmental Biology, 16, 301–332. Krikos, A., Laherty, C. D., & Dixit, V. M. (1992). Transcriptional activation of the tumor necrosis factor alpha-inducible zinc finger protein, A20, is mediated by kappa B elements. The Journal of Biological Chemistry, 267, 17971–17976. Kuballa, P., Huett, A., Rioux, J. D., Daly, M. J., & Xavier, R. J. (2008). Impaired autophagy of an intracellular pathogen induced by a Crohn’s disease associated ATG16L1 variant. PLoS One, 3, e3391. Lala, S., Ogura, Y., Osborne, C., Hor, S. Y., Bromfield, A., Davies, S., et al. (2003). Crohn’s disease and the NOD2 gene: A role for paneth cells. Gastroenterology, 125, 47–57. Larrick, J. W., Lee, J., Ma, S., Li, X., Francke, U., Wright, S. C., et al. (1996). Structural, functional analysis and localization of the human CAP18 gene. FEBS Letters, 398, 74–80. Lassen, K. G., Kuballa, P., Conway, K. L., Patel, K. K., Becker, C. E., Peloquin, J. M., et al. (2014). Atg16L1 T300A variant decreases selective autophagy resulting in altered cytokine signaling and decreased antibacterial defense. Proceedings of the National Academy of Sciences of the United States of America, 111, 7741–7746. Lee, W.-J. (2009). Bacterial-modulated host immunity and stem cell activation for gut homeostasis. Genes & Development, 23, 2260–2265. Lee, E. G., Boone, D. L., Chai, S., Libby, S. L., Chien, M., Lodolce, J. P., et al. (2000). Failure to regulate TNF-induced NF-kappaB and cell death responses in A20-deficient mice. Science, 289, 2350–2354. Lee, J., Mo, J.-H., Katakura, K., Alkalay, I., Rucker, A. N., Liu, Y.-T., et al. (2006). Maintenance of colonic homeostasis by distinctive apical TLR9 signalling in intestinal epithelial cells. Nature Cell Biology, 8, 1327–1336. Lehotzky, R. E., Partch, C. L., Mukherjee, S., Cash, H. L., Goldman, W. E., Gardner, K. H., et al. (2010). Molecular basis for peptidoglycan recognition by a bactericidal lectin. Proceedings of the National Academy of Sciences of the United States of America, 107, 7722–7727. Leoni, G., Alam, A., Neumann, P.-A., Lambeth, J. D., Cheng, G., McCoy, J., et al. (2013). Annexin A1, formyl peptide receptor, and NOX1 orchestrate epithelial repair. The Journal of Clinical Investigation, 123, 443–454. Levine, B., Mizushima, N., & Virgin, H. W. (2011). Autophagy in immunity and inflammation. Nature, 469, 323–335. Ley, R. E., Ba¨ckhed, F., Turnbaugh, P., Lozupone, C. A., Knight, R. D., & Gordon, J. I. (2005). Obesity alters gut microbial ecology. Proceedings of the National Academy of Sciences of the United States of America, 102, 11070–11075. Ley, R. E., Hamady, M., Lozupone, C., Turnbaugh, P. J., Ramey, R. R., Bircher, J. S., et al. (2008). Evolution of mammals and their gut microbes. Science, 320, 1647–1651. Li, H., Kobayashi, M., Blonska, M., You, Y., & Lin, X. (2006). Ubiquitination of RIP is required for tumor necrosis factor alpha-induced NF-kappaB activation. The Journal of Biological Chemistry, 281, 13636–13643. Lichtenstein, A., Ganz, T., Selsted, M. E., & Lehrer, R. I. (1986). In vitro tumor cell cytolysis mediated by peptide defensins of human and rabbit granulocytes. Blood, 68, 1407–1410. Ma, B., Forney, L. J., & Ravel, J. (2012). Vaginal microbiome: Rethinking health and disease. Annual Review of Microbiology, 66, 371–389. Macpherson, A. J., Gatto, D., Sainsbury, E., Harriman, G. R., Hengartner, H., & Zinkernagel, R. M. (2000). A primitive T cell-independent mechanism of intestinal mucosal IgA responses to commensal bacteria. Science, 288, 2222–2226. Macpherson, A. J., & Uhr, T. (2004). Induction of protective IgA by intestinal dendritic cells carrying commensal bacteria. Science, 303, 1662–1665.

168

Lora V. Hooper

Martens, E. C., Lowe, E. C., Chiang, H., Pudlo, N. A., Wu, M., McNulty, N. P., et al. (2011). Recognition and degradation of plant cell wall polysaccharides by two human gut symbionts. PLoS Biology, 9, e1001221. Martinon, F., Burns, K., & Tschopp, J. (2002). The inflammasome: A molecular platform triggering activation of inflammatory caspases and processing of proIL-beta. Molecular Cell, 10, 417–426. Mashimo, H., Wu, D. C., Podolsky, D. K., & Fishman, M. C. (1996). Impaired defense of intestinal mucosa in mice lacking intestinal trefoil factor. Science, 274, 262–265. Mathur, R., Oh, H., Zhang, D., Park, S.-G., Seo, J., Koblansky, A., et al. (2012). A mouse model of Salmonella typhi infection. Cell, 151, 590–602. Mazmanian, S. K., Liu, C. H., Tzianabos, A. O., & Kasper, D. L. (2005). An immunomodulatory molecule of symbiotic bacteria directs maturation of the host immune system. Cell, 122, 107–118. McDole, J. R., Wheeler, L. W., McDonald, K. G., Wang, B., Konjufca, V., Knoop, K. A., et al. (2012). Goblet cells deliver luminal antigen to CD103 + dendritic cells in the small intestine. Nature, 483, 345–349. Meyer-Hoffert, U., Hornef, M. W., Henriques-Normark, B., Axelsson, L.-G., Midtvedt, T., Putsep, K., et al. (2008). Secreted enteric antimicrobial activity localises to the mucus surface layer. Gut, 57, 764–771. Miki, T., Holst, O., & Hardt, W. D. (2012). The bactericidal activity of the C-type lectin RegIII against Gram-negative bacteria involves binding to lipid A. Journal of Biological Chemistry, 287, 34844–34855. Moran, G. W., Leslie, F. C., Levison, S. E., Worthington, J., & McLaughlin, J. T. (2008). Enteroendocrine cells: Neglected players in gastrointestinal disorders? Therapeutic Advances in Gastroenterology, 1, 51–60. Mukherjee, S., Zheng, H., Derebe, M. G., Callenberg, K. M., Partch, C. L., Rollins, D., et al. (2014). Antibacterial membrane attack by a pore-forming intestinal C-type lectin. Nature, 505, 103–107. Mundy, R., MacDonald, T. T., Dougan, G., Frankel, G., & Wiles, S. (2005). Citrobacter rodentium of mice and man. Cellular Microbiology, 7, 1697–1706. Nair, M. G., Guild, K. J., Du, Y., Zaph, C., Yancopoulos, G. D., Valenzuela, D. M., et al. (2008). Goblet cell-derived resistin-like molecule beta augments CD4 + T cell production of IFN-gamma and infection-induced intestinal inflammation. Journal of Immunology, 181, 4709–4715. Neish, A. S. (2002). The gut microflora and intestinal epithelial cells: A continuing dialogue. Microbes and Infection, 4, 309–317. Nenci, A., Becker, C., Wullaert, A., Gareus, R., van Loo, G., Danese, S., et al. (2007). Epithelial NEMO links innate immunity to chronic intestinal inflammation. Nature, 446, 557–561. Ogura, Y., Bonen, D. K., Inohara, N., Nicolae, D. L., Chen, F. F., Ramos, R., et al. (2001). A frameshift mutation in NOD2 associated with susceptibility to Crohn’s disease. Nature, 411, 603–606. Ogura, Y., Lala, S., Xin, W., Smith, E., Dowds, T. A., Chen, F. F., et al. (2003). Expression of NOD2 in Paneth cells: A possible link to Crohn’s ileitis. Gut, 52, 1591–1597. Olszak, T., Neves, J. F., Dowds, C. M., Baker, K., Glickman, J., Davidson, N. O., et al. (2014). Protective mucosal immunity mediated by epithelial CD1d and IL-10. Nature, 509, 497–502. O’Neil, D. A., Cole, S. P., Martin Porter, E., Housley, M. P., Liu, L., Ganz, T., et al. (2000). Regulation of human beta-defensins by gastric epithelial cells in response to infection with Helicobacter pylori or stimulation with interleukin-1. Infection and Immunity, 68, 5412–5415. O’Neil, D. A., Porter, E. M., Elewaut, D., Anderson, G. M., Eckmann, L., Ganz, T., et al. (1999). Expression and regulation of the human β-defensins hBD-1 and hBD-2 in intestinal epithelium. Journal of Immunology, 163, 6718–6724.


169

Ouellette, A. J. (2011). Paneth cell α-defensins in enteric innate immunity. Cellular and Molecular Life Sciences, 68, 2215–2229. Patel, K. K., Miyoshi, H., Beatty, W. L., Head, R. D., Malvin, N. P., Cadwell, K., et al. (2013). Autophagy proteins control goblet cell function by potentiating reactive oxygen species production. The EMBO Journal, 32, 3130–3144. Petnicki-Ocwieja, T., Hrncir, T., Liu, Y.-J., Biswas, A., Hudcovic, T., TlaskalovaHogenova, H., et al. (2009). Nod2 is required for the regulation of commensal microbiota in the intestine. Proceedings of the National Academy of Sciences of the United States of America, 106, 15813–15818. Playford, R. J., Marchbank, T., Chinery, R., Evison, R., Pignatelli, M., Boulton, R. A., et al. (1995). Human spasmolytic polypeptide is a cytoprotective agent that stimulates cell migration. Gastroenterology, 108, 108–116. Pull, S. L., Doherty, J. M., Mills, J. C., Gordon, J. I., & Stappenbeck, T. S. (2005). Activated macrophages are an adaptive element of the colonic epithelial progenitor niche necessary for regenerative responses to injury. Proceedings of the National Academy of Sciences of the United States of America, 102, 99–104. Putsep, K., Axelsson, L.-G., Boman, A., Midtvedt, T., Normark, S., Boman, H. G., et al. (2000). Germ-free and colonized mice generate the same products from enteric prodefensins. The Journal of Biological Chemistry, 275, 40478–40482. Rabinowitz, J. D., & White, E. (2010). Autophagy and metabolism. Science, 330, 1344–1348. Rakoff-Nahoum, S., Hao, L., & Medzhitov, R. (2006). Role of toll-like receptors in spontaneous commensal-dependent colitis. Immunity, 25, 319–329. Rakoff-Nahoum, S., & Medzhitov, R. (2007). Regulation of spontaneous intestinal tumorigenesis through the adaptor protein MyD88. Science, 317, 124–127. Rakoff-Nahoum, S., Paglino, J., Eslami-Varzaneh, F., Edberg, S., & Medzhitov, R. (2004). Recognition of commensal microflora by toll-like receptors is required for intestinal homeostasis. Cell, 118, 229–241. Rescigno, M., Urbano, M., Valzasina, B., Francolini, M., Rotta, G., Bonasio, R., et al. (2001). Dendritic cells express tight junction proteins and penetrate gut epithelial monolayers to sample bacteria. Nature Immunology, 2, 361–367. Reyes, A., Haynes, M., Hanson, N., Angly, F. E., Heath, A. C., Rohwer, F., et al. (2010). Viruses in the faecal microbiota of monozygotic twins and their mothers. Nature, 466, 334–338. Rimoldi, M., Chieppa, M., Salucci, V., Avogadri, F., Sonzogni, A., Sampietro, G. M., et al. (2005). Intestinal immune homeostasis is regulated by the crosstalk between epithelial cells and dendritic cells. Nature Immunology, 6, 507–514. Rioux, J. D., Xavier, R. J., Taylor, K. D., Silverberg, M. S., Goyette, P., Huett, A., et al. (2007). Genome-wide association study identifies new susceptibility loci for Crohn disease and implicates autophagy in disease pathogenesis. Nature Genetics, 39, 596–604. Ronald, P. C., & Beutler, B. (2010). Plant and animal sensors of conserved microbial signatures. Science, 330, 1061–1064. Rosenberg, H. F. (1995). Recombinant human eosinophil cationic protein. Ribonuclease activity is not essential for cytotoxicity. The Journal of Biological Chemistry, 270, 7876–7881. Salzman, N. H., Ghosh, D., Huttner, K. M., Paterson, Y., & Bevins, C. L. (2003). Protection against enteric salmonellosis in transgenic mice expressing a human intestinal defensin. Nature, 422, 522–526. Salzman, N. H., Hung, K., Haribhai, D., Chu, H., Karlsson-Sj€ oberg, J., Amir, E., et al. (2010). Enteric defensins are essential regulators of intestinal microbial ecology. Nature Immunology, 11, 76–83. Sanos, S. L., Bui, V. L., Mortha, A., Oberle, K., Heners, C., Johner, C., et al. (2008). RORγt and commensal microflora are required for the differentiation of mucosal interleukin 22– producing NKp46+ cells. Nature Immunology, 10, 83–91.

170

Lora V. Hooper

Sanos, S. L., Vonarbourg, C., Mortha, A., & Diefenbach, A. (2011). Control of epithelial cell function by interleukin-22-producing RORγt+ innate lymphoid cells. Immunology, 132, 453–465. Sato, T., van Es, J. H., Snippert, H. J., Stange, D. E., Vries, R. G., van den Born, M., et al. (2011). Paneth cells constitute the niche for Lgr5 stem cells in intestinal crypts. Nature, 469, 415–418. Schroder, K., & Tschopp, J. (2010). The inflammasomes. Cell, 140, 821–832. Schroeder, B. O., Wu, Z., Nuding, S., Groscurth, S., Marcinowski, M., Beisner, J., et al. (2011). Reduction of disulphide bonds unmasks potent antimicrobial activity of human β-defensin 1. Nature, 469, 419–423. Schutte, B. C., & McCray, P. B. (2002). β-defensins in lung host defense. Annual Review of Physiology, 64, 709–748. Schutte, B. C., Mitros, J. P., Bartlett, J. A., Walters, J. D., Jia, H. P., Welsh, M. J., et al. (2002). Discovery of five conserved beta-defensin gene clusters using a computational search strategy. Proceedings of the National Academy of Sciences of the United States of America, 99, 2129–2133. Selsted, M. E., & Ouellette, A. J. (2005). Mammalian defensins in the antimicrobial immune response. Nature Immunology, 6, 551–557. Shang, L., Fukata, M., Thirunarayanan, N., Martin, A. P., Arnaboldi, P., Maussang, D., et al. (2008). Toll-like receptor signaling in small intestinal epithelium promotes B-cell recruitment and IgA production in lamina propria. Gastroenterology, 135, 529–538. Stelter, C., Ka¨ppeli, R., K€ onig, C., Krah, A., Hardt, W.-D., Stecher, B., et al. (2011). Salmonella-induced mucosal lectin RegIIIβ kills competing gut microbiota. PLoS One, 6, e20749. Sternini, C., Anselmi, L., & Rozengurt, E. (2008). Enteroendocrine cells: A site of “taste” in gastrointestinal chemosensing. Current Opinion in Endocrinology, Diabetes, and Obesity, 15, 73–78. Stowell, S. R., Arthur, C. M., Dias-Baruffi, M., Rodrigues, L. C., Gourdine, J.-P., Heimburg-Molinaro, J., et al. (2010). Innate immune lectins kill bacteria expressing blood group antigen. Nature Medicine, 16, 295–301. Sun, C. M., Hall, J. A., Blank, R. B., Bouladoux, N., Oukka, M., Mora, J. R., et al. (2007). Small intestine lamina propria dendritic cells promote de novo generation of Foxp3 T reg cells via retinoic acid. Journal of Experimental Medicine, 204, 1775–1785. Suzuki, K., Meek, B., Doi, Y., Muramatsu, M., Chiba, T., Honjo, T., et al. (2004). Aberrant expansion of segmented filamentous bacteria in IgA-deficient gut. Proceedings of the National Academy of Sciences of the United States of America, 101, 1981–1986. Swanson, P. A., Kumar, A., Samarin, S., Vijay-Kumar, M., Kundu, K., Murthy, N., et al. (2011). Enteric commensal bacteria potentiate epithelial restitution via reactive oxygen species-mediated inactivation of focal adhesion kinase phosphatases. Proceedings of the National Academy of Sciences of the United States of America, 108, 8803–8808. Swidsinski, A., Weber, J., Loening-Baucke, V., Hale, L. P., & Lochs, H. (2005). Spatial organization and composition of the mucosal flora in patients with inflammatory bowel disease. Journal of Clinical Microbiology, 43, 3380–3389. Tahoun, A., Mahajan, S., Paxton, E., Malterer, G., Donaldson, D. S., Wang, D., et al. (2012). Salmonella transforms follicle-associated epithelial cells into M cells to promote intestinal invasion. Cell Host & Microbe, 12, 645–656. Takeuchi, O., Sato, S., Horiuchi, T., Hoshino, K., Takeda, K., Dong, Z., et al. (2002). Cutting edge: Role of toll-like receptor 1 in mediating immune response to microbial lipoproteins. Journal of Immunology, 169, 10–14. Taylor, B. C., Zaph, C., Troy, A. E., Du, Y., Guild, K. J., Comeau, M. R., et al. (2009). TSLP regulates intestinal immunity and inflammation in mouse models of helminth infection and colitis. Journal of Experimental Medicine, 206, 655–667.


171

Turer, E. E., Tavares, R. M., Mortier, E., Hitotsumatsu, O., Advincula, R., Lee, B., et al. (2008). Homeostatic MyD88-dependent signals cause lethal inflammation in the absence of A20. Journal of Experimental Medicine, 205, 451–464. Turnbaugh, P. J., Ley, R. E., Mahowald, M. A., Magrini, V., Mardis, E. R., & Gordon, J. I. (2006). An obesity-associated gut microbiome with increased capacity for energy harvest. Nature, 444, 1027–1131. Uematsu, S., Fujimoto, K., Jang, M. H., Yang, B.-G., Jung, Y.-J., Nishiyama, M., et al. (2008). Regulation of humoral and cellular gut immunity by lamina propria dendritic cells expressing toll-like receptor 5. Nature Immunology, 9, 769–776. Vaishnava, S., Behrendt, C. L., Ismail, A. S., Eckmann, L., & Hooper, L. V. (2008). Paneth cells directly sense gut commensals and maintain homeostasis at the intestinal hostmicrobial interface. Proceedings of the National Academy of Sciences of the United States of America, 105, 20858–20863. Vaishnava, S., & Hooper, L. V. (2007). Alkaline phosphatase: Keeping the peace at the gut epithelial surface. Cell Host & Microbe, 2, 365–367. Vaishnava, S., Yamamoto, M., Severson, K. M., Ruhn, K. A., Yu, X., Koren, O., et al. (2011). The antibacterial lectin RegIIIgamma promotes the spatial segregation of microbiota and host in the intestine. Science, 334, 255–258. van Es, J. H., Jay, P., Gregorieff, A., van Gijn, M. E., Jonkheer, S., Hatzis, P., et al. (2005). Wnt signalling induces maturation of Paneth cells in intestinal crypts. Nature Cell Biology, 7, 381–386. Virgin, H. W. (2014). The virome in mammalian physiology and disease. Cell, 157, 142–150. Wehkamp, J., Fellermann, K., Herrlinger, K. R., Baxmann, S., Schmidt, K., Schwind, B., et al. (2002). Human beta-defensin 2 but not beta-defensin 1 is expressed preferentially in colonic mucosa of inflammatory bowel disease. European Journal of Gastroenterology & Hepatology, 14, 745–752. Wehkamp, J., Salzman, N. H., Porter, E., Nuding, S., Weichenthal, M., Petras, R. E., et al. (2005). Reduced Paneth cell α-defensins in ileal Crohn’s disease. Proceedings of the National Academy of Sciences of the United States of America, 102, 18129–18134. Wellcome Trust Case Control Consortium (2007). Genome-wide association study of 14,000 cases of seven common diseases and 3,000 shared controls. Nature, 447, 661–678. Wertz, I. E., O’Rourke, K. M., Zhou, H., Eby, M., Aravind, L., Seshagiri, S., et al. (2004). De-ubiquitination and ubiquitin ligase domains of A20 downregulate NF-kappaB signalling. Nature, 430, 694–699. Wilson, C. L., Ouellette, A. J., Satchell, D. P., Ayabe, T., Lo´pez-Boado, Y. S., Stratman, J. L., et al. (1999). Regulation of intestinal α-defensin activation by the metalloproteinase matrilysin in innate host defense. Science, 286, 113–117. Wingender, G., & Kronenberg, M. (2008). Role of NKT cells in the digestive system. IV. The role of canonical natural killer T cells in mucosal immunity and inflammation. American Journal of Physiology Gastrointestinal and Liver Physiology, 294, G1–G8. Wlodarska, M., Thaiss, C. A., Nowarski, R., Henao-Mejia, J., Zhang, J.-P., Brown, E. M., et al. (2014). NLRP6 inflammasome orchestrates the colonic host-microbial interface by regulating goblet cell mucus secretion. Cell, 156, 1045–1059. Wolk, K., Kunz, S., Witte, E., Friedrich, M., Asadullah, K., & Sabat, R. (2004). IL-22 increases the innate immunity of tissues. Immunity, 21, 241–254. Wostmann, B. S., Larkin, C., Moriarty, A., & Bruckner-Kardoss, E. (1983). Dietary intake, energy metabolism, and excretory losses of adult male germfree Wistar rats. Laboratory Animal Science, 33, 46–50. Xu, J., Bjursell, M. K., Himrod, J., Deng, S., Carmichael, L. K., Chiang, H. C., et al. (2003). A genomic view of the human-Bacteroides thetaiotaomicron symbiosis. Science, 299, 2074–2076.

172

Lora V. Hooper

Xu, W., He, B., Chiu, A., Chadburn, A., Shan, M., Buldys, M., et al. (2007). Epithelial cells trigger frontline immunoglobulin class switching through a pathway regulated by the inhibitor SLPI. Nature Immunology, 8, 294–303. Yan, N., & Chen, Z. J. (2012). Intrinsic antiviral immunity. Nature Immunology, 13, 214–222. Yarovinsky, F., Zhang, D., Andersen, J. F., Bannenberg, G. L., Serhan, C. N., Hayden, M. S., et al. (2005). TLR11 activation of dendritic cells by a protozoan profilin-like protein. Science, 308, 1626–1629. Yatsunenko, T., Rey, F. E., Manary, M. J., Trehan, I., Dominguez-Bello, M. G., Contreras, M., et al. (2012). Human gut microbiome viewed across age and geography. Nature, 486, 222–227. Zaph, C., Troy, A. E., Taylor, B. C., Berman-Booty, L. D., Guild, K. J., Du, Y., et al. (2007). Epithelial-cell-intrinsic IKK-β expression regulates intestinal immune homeostasis. Nature, 446, 552–556. Zasloff, M. (2002). Antimicrobial peptides of multicellular organisms. Nature, 415, 389–395. Zeuthen, L. H., Fink, L. N., & Frokiaer, H. (2008). Epithelial cells prime the immune response to an array of gut-derived commensals towards a tolerogenic phenotype through distinct actions of thymic stromal lymphopoietin and transforming growth factor-beta. Immunology, 123, 197–208. Zhao, Z., Fux, B., Goodwin, M., Dunay, I. R., Strong, D., Miller, B. C., et al. (2008). Autophagosome-independent essential function for the autophagy protein Atg5 in cellular immunity to intracellular pathogens. Cell Host & Microbe, 4, 458–469. Ziegler, S. F., & Artis, D. (2010). Sensing the outside world: TSLP regulates barrier immunity. Nature Immunology, 11(4), 289–293.

Intestinal Epithelial Cell-Intrinsic Deletion of Setd7 Identifies Role for Developmental Pathways in Immunity to Helminth Infection.

Intestinal epithelial tuft cells initiate type 2 mucosal immunity to helminth parasites.

Epigenetics in Intestinal Epithelial Cell Renewal.

Epithelial Histone Deacetylase 3 Instructs Intestinal Immunity by Coordinating Local Lymphocyte Activation.

The Microbiome Activates CD4 T-cell-mediated Immunity to Compensate for Increased Intestinal Permeability.

The intestinal epithelial cell cycle: uncovering its 'cryptic' nature.

Compartmentalizing intestinal epithelial cell toll-like receptors for immune surveillance.

Interleukin-22 promotes intestinal-stem-cell-mediated epithelial regeneration.

Intestinal epithelial cell protein phosphorylation in enteropathogenic Escherichia coli diarrhoea.

Enterotoxigenic Escherichia coli infection induces intestinal epithelial cell autophagy.

DUSP10 regulates intestinal epithelial cell growth and colorectal tumorigenesis.

[Epithelial cell in intestinal homeostasis and inflammatory bowel diseases].

Calcium transport by intestinal epithelial cell basolateral membrane.

Autophagy, viruses, and intestinal immunity.

Molecular cloning, sequence analysis, and function of the intestinal epithelial stem cell marker Bmi1 in pig intestinal epithelial cells.

Gentamicin sulphate permeation through porcine intestinal epithelial cell monolayer.

Microblebs of intestinal epithelial cell microvilli of Citellus tridecemlineatus.

Epithelial cell differentiation in normal and transgenic mouse intestinal isografts.

6 mice.

Characterization of a vasoactive intestinal peptide-sensitive adenylate cyclase in rat intestinal epithelial cell membranes.

Intestinal Epithelial Cell Tyrosine Kinase 2 Transduces IL-22 Signals To Protect from Acute Colitis.

GATA4 regulates epithelial cell proliferation to control intestinal growth and development in mice.

Th17 Cell Induction by Adhesion of Microbes to Intestinal Epithelial Cells.

Basement membrane influences intestinal epithelial cell growth and presents a barrier to the movement of macromolecules.