Intestinal Epithelial Cells Regulators of Barrier Function and Immune Homeostasis

Specialized epithelial cells constitute barrier surfaces that separate mammalian hosts from the external environ-ment. The gastrointestinal tract is the largest of these barriers and is specially adapted to colonization by commensal bacteria that aid in digestion and markedly influence the development and function of the mucosal immune system. However, microbial colonization car-ries with it the risk of infection and inflammation if epi-thelial or immune cell homeostasis is disrupted. Key to the coexistence of commensal microbial communities and mucosal immune cells is the capacity to maintain the segregation between host and microorganism. The intes-tinal epithelium accomplishes this by forming a physical and biochemical barrier to commensal and pathogenic microorganisms. Furthermore, intestinal epithelial cells (IECs) can sense and respond to microbial stimuli to reinforce their barrier function and to participate in the coordination of appropriate immune responses, ranging from tolerance to anti-pathogen immunity. Thus, IECs maintain a fundamental immuno regulatory function that influences the development and homeostasis of mucosal immunecells.

The association between increased bacterial trans-location and risk of developing inflammatory bowel disease (IBD) suggests a central role for dysregulated epithelial barrier function in either the aetiology or the pathol-ogy of intestinal inflammation and IBD1. Increasing evidence also indicates that the loss of intestinal barrier

function contributes to systemic immune activation, which promotes the progression of chronic viral infec-tions, including infection with HIV and hepatitis virus2,3, and metabolic disease4,5. Furthermore, hostmicrobial interactions that occur at the IEC barrier contribute to a broad range of extra-intestinal autoimmune and inflam-matory diseases, including type1 diabetes, rheumatoid arthritis and multiple sclerosis69. Hence, a comprehen-sive understanding of the barrier and immunoregulatory properties of IECs could aid in the development of new strategies to prevent and treat multiple human infectious, inflammatory and metabolic diseases.

The topics of commensal bacterial diversity, microbial regulation of immune cell development and hostviral interactions in the intestine have been reviewed exten-sively elsewhere1014. Therefore, in this Review, we dis-cuss the role of IECs in promoting intestinal homeostasis through the segregation and regulation of commensal microorganisms and the host immune system. Recent advances in the understanding of the barrier, microbial-sensing and immunoregulatory functions of IECs are reviewed, with a particular focus on their relationship to intestinal health and disease. We discuss the barrier func-tion maintained by IEC-derived mucins and anti microbial proteins, the pathways through which IECs regulate innate and adaptive immune cells present at the intes-tinal barrier and the contribution of IEC recognition of microbial colonization to IEC function and homeostasis.

Intestinal epithelial cells: regulators of barrier function and immune homeostasisLance W.Peterson1 and David Artis1,2

Abstract | The abundance of innate and adaptive immune cells that reside together with trillions of beneficial commensal microorganisms in the mammalian gastrointestinal tract requires barrier and regulatory mechanisms that conserve hostmicrobial interactions and tissue homeostasis. This homeostasis depends on the diverse functions of intestinal epithelial cells (IECs), which include the physical segregation of commensal bacteria and the integration of microbial signals. Hence, IECs are crucial mediators of intestinal homeostasis that enable the establishment of an immunological environment permissive to colonization by commensal bacteria. In this Review, we provide a comprehensive overview of how IECs maintain hostcommensal microbial relationships and immune cell homeostasis in the intestine.

1Department of Microbiology and Institute for Immunology, Perelman School of Medicine, University of Pennsylvania.2Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.emails: [email protected]; [email protected]:10.1038/nri3608

Inflammatory bowel disease(IBD). A chronic condition of the intestine characterized by severe inflammation and mucosal destruction. The most common forms of IBD in humans are ulcerative colitis and Crohns disease, which have both distinct and overlapping pathological and clinical characteristics.

MucinsHeavily glycosylated proteins that are the major component of the mucus that coats epithelial barrier surfaces.

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CryptsTubular invaginations of the intestinal epithelium. Lining the base of the crypts are small intestinal Paneth cells, which produce numerous antimicrobial proteins, and stem cells, which continuously divide to give rise to the entire intestinal epithelium.

VilliProjections of the intestinal epithelium into the lumen ofthe small intestine that havean outer layer consistingof mature, absorptive enterocytes, mucus-secreting goblet cells and enteroendocrine cells.

Pluripotent intestinal epithelial stem cells(Pluripotent IESCs). Tissue-resident stem cells thatundergo continuous self-renewal and are responsible for regenerating all lineages of mature intestinal epithelial cells, including enterocytes, enteroendocrine cells, gobletcells and Paneth cells.

IEC regulation of barrier functionThe intestinal epithelium is the largest of the bodys mucosal surfaces, covering ~400 m2 of surface area with a single layer of cells organized into crypts and villi (FIG.1). This surface is continually renewed by pluripotent intestinal epithelial stem cells (pluripotent IESCs) that reside in the base of crypts, where the pro-liferation, differentiation and functional potential of epithelial cell progenitors is regulated by the local stem cell niche15,16 (BOX1). Although the majority of cells bordering the intestinal lumen are absorptive entero-cytes, which are adapted for metabolic and digestive function, the diversity of functions that the intestinal epithelium carries out is reflected by the presence of additional specialized IEC lineages.

Secretory IECs, including enteroendocrine cells, goblet cells and Paneth cells, are specialized for main-taining the digestive or barrier function of the epithe-lium. Enteroendocrine cells represent a link between the central and enteric neuroendocrine systems through the secretion of numerous hormone regulators of

digestive function. The luminal secretion of mucins and antimicrobial proteins (AMPs) by goblet cells and Paneth cells, respectively, establishes a physical and biochemical barrier to microbial contact with the epi-thelial surface and underlying immune cells17,18 (FIG.1). Collectively, the diverse functions of IECs result in a dynamic barrier to the environment, which protects the host from infection and continuous exposure to potentially inflammatory stimuli.

Keeping the bugs at bayIEC secretory defences. The secretion of highly glycosylated mucins into the intesti-nal lumen by goblet cells creates the first line of defence against microbial encroachment. The most abundant of these mucins, mucin2 (MUC2), plays an essential part in the organization of the intestinal mucous layers at the epithelial surface of the colon19. The importance of mucin production by goblet cells is emphasized by the spontaneous development of colitis and the predis-position to inflammation-induced colorectal cancers observed in MUC2-deficient mice20,21. Additional goblet

Figure 1 | The IEC barrier. Intestinal epithelial cells (IECs) form a biochemical and physical barrier that maintains segregation between luminal microbial communities and the mucosal immune system. The intestinal epithelial stem cell (IESC) niche, containing epithelial, stromal and haematopoietic cells, controls the continuous renewal of the epithelial cell layer by crypt-resident stem cells. Differentiated IECs with the exception of Paneth cells migrate up the cryptvillus axis, as indicated by the dashed arrows. Secretory goblet cells and Paneth cells secrete mucus and antimicrobial proteins (AMPs) to promote the exclusion of bacteria from the epithelial surface. The transcytosis and luminal release of secretory IgA (sIgA) further contribute to this barrier function. Microfold cells (Mcells) and goblet cells mediate transport of luminal antigens and live bacteria across the epithelial barrier to dendritic cells (DCs), and intestine-resident macrophages sample the lumen through transepithelial dendrites. TFF3, trefoil factor 3.

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AutophagyA cellular process by which cytoplasmic organelles and macromolecular complexes are engulfed by double membrane-bound vesicles for delivery to lysosomes and subsequent degradation. This process is involved in constitutive turnover of proteins and organelles and is central to cellular activities that maintain a balance between the synthesis and breakdown of various proteins.

Unfolded protein response(UPR). A response that increases the ability of the endoplasmic reticulum to fold and translocate proteins, decreases the synthesis of proteins, causes the arrest of the cell cycle and promotes apoptosis.

Plasma cellsTerminally differentiated cells of the Bcell lineage that secrete large amounts of antibodies.

Lamina propriaConnective tissue that underlies the epithelium of the mucosa and contains stromal and haematopoietic cells.

cell-derived products, such as trefoil factor 3 (TFF3) and resistin-like molecule- (RELM), further contribute to the regulation of a physical barrier in the intestine. TFF3 provides structural integrity to mucus through mucin crosslinking and acts as a signal that promotes epithe-lial repair, migration of IECs and resistance to apopto-sis22,23. RELM functions to promote MUC2 secretion, regulate macrophage and adaptive Tcell responses during inflammation and, in the setting of nematode infection, directly inhibit parasite chemotaxis24,25.

Intestinal barrier function is further reinforced by the secretion of AMPs by IECs. Enterocytes are capa-ble of producing some AMPs, such as the C-type lec-tin regenerating islet-derived protein III (REGIII), throughout the small intestine and colon. By contrast, Paneth cells are uniquely adapted for the secretion of many additional AMPs, including defensins (cryptdins in mice), cathelicidins and lysozyme, in the crypts of the small intestine18,26. These AMPs disrupt highly con-served and essential features of bacterial biology, such as surface membranes, which are targeted by pore-forming defensins and cathelicidins, and Gram-positive cell wall peptidoglycans, which are targeted by C-type lectins18,27. This strategy enables the broad regulation of both com-mensal and pathogenic bacteria and limits resistance of bacteria to antimicrobial responses. Regional vari-ation in AMP production exists along the longitudinal axis of the intestinal tract28. Although further analysis is required, this distribution may reflect anatomically restricted hostcommensal bacteria interactions that drive the differential regulation of IEC responses or serve to shape heterogeneity in the composition and localization of microbial communities.

Paneth cell- and enterocyte-derived REGIII has recently been described as a mediator of hostmicrobial segregation in the gut29. Similar to the function and regulation of MUC2 in the colon, REGIII acts to exclude bacteria from the epithelial surface of the small intestine, and its production is dependent on IEC-intrinsic recognition of commensal microbial sig-nals29. Interactions between AMPs, including REGIII, and mucins lead to concentrated antimicrobial activity at the epithelial surface30. Thus, the combined func-tions of secretory IECs seem to limit the quantity and diversity of live bacteria that can reach the epithe-lial surface or interact with the underlying mucosal immunesystem.

The importance of maintaining the health of secre-tory IECs is reflected in human IBD and models of murine intestinal inflammation, in which genetic defects in autophagy and the unfolded protein response (UPR) disrupt the function of Paneth and goblet cells and promote disease susceptibility 3137. Autophagy in IECs has been shown to act in an innate immune capacity to limit the dissemination of invasive bacteria passing through the epithelium38, but it also supports the packaging and exocytosis of Paneth cell granules33. When autophagy is disrupted in mice they become sus-ceptible to a form of experimental colitis33. Notably, this susceptibility is dependent on exposure to a common strain of an enteric virus (murine norovirus), providing an example of the compound genetic and environmen-tal interactions that contribute to disease pathogene-sis36. Disruption of UPR genes results in endoplasmic reticulum stress in secretory cells and spontaneous intestinal inflammation34. Notably, disruption of either autophagy or the UPR leads to the compensatory engagement of the other, supporting a model in which the two are interrelated39. Furthermore, the engage-ment of these pathways by Paneth cells is required for maintaining intestinal homeostasis in mice, and their combined absence leads to the development of a spon-taneous disease resembling human Crohns disease39. These findings, coupled with genetic evidence from patients with IBD for the role of autophagy and the UPR in disease pathogenesis31,32,34,37, support an impor-tant link between the disruption of Paneth cell function and the potential origins of intestinal inflammation.

Finally, IECs directly transport secretory immu-noglobulins across the epithelial barrier. Following their production by plasma cells in the lamina propria, dimeric IgA complexes are bound by the polymeric immunoglobulin receptor (pIgR) on the basolateral membrane of IECs and actively transcytosed into the intestinal lumen40. The collaboration between IgA-secreting Bcells and IECs provides an adaptive immune component to the epithelial barrier that reg-ulates commensal bacterial populations to maintain IEC and immune cell homeostasis4143. Future studies to better understand how mucus, AMP and secretory immunoglobulin dynamics can be regulated to sup-port barrier function will enable the development of therapeutic interventions for preserving intestinal homeostasis.

Box 1 | The IESC niche

Along the cryptvillus axis of the epithelium, pluripotent intestinal epithelial stem cells (IESCs) residing in the base of crypts give rise to a transit-amplifying population of cells that undergo rapid proliferation and differentiation into the various intestinal epithelial cell (IEC) subsets. Terminally differentiated cells with the exception of Paneth cells migrate up the cryptvillus axis until they are lost from the epithelial layer. For this process to be maintained, epithelial stem cells must be able to undergo repeated rounds of replication and possess the capacity for continuous self-renewal16. Recent advances in stem cell biology have identified markers of IESCs that have contributed to the understanding of epithelial self-renewal and differentiation16,183185.

The patterning and distribution of proliferating crypt units in the intestine depend on paracrine signalling between the epithelium and the underlying mesenchyme. A balance between bone morphogenetic protein signals and antagonists, such as noggin and gremlin, provides a niche for proliferating stem cells while limiting ectopic crypt formation15. IESCs further rely on signalling through both the WNT-catenin and the Notch pathways for promoting self-renewal and directing differentiation towards secretory versus non-secretory lineage IEC fates16.

The responsiveness of epithelial progenitors to external regulation in settings of inflammation or infection remains less well understood. In particular, how immune system-mediated signalling integrates into the homeostatic pathways described above or acts through alternative pathways for altering stem cell function is poorly defined. However, several recent studies have given insight into the regulation of WNT-catenin signalling by the pro-inflammatory cytokines interferon- and tumour necrosis factor, offering an example of how immune signalling and homeostatic pathways for regulating the stem cell niche can converge186,187. Furthermore, cell-intrinsic mechanisms of integrating hostcommensal microorganism interactions into IEC homeostasis have been recently described188.

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Peyers patchesGroups of lymphoid aggregates located in the submucosa of the small intestine that contain many immune cells, including Bcells, Tcells and dendritic cells. They have a luminal barrier consisting of specialized epithelial cells, called microfold cells, which sample the lumen and transport antigens.

Pattern-recognition receptors(PRRs). Receptors that recognize structures shared by foreign microorganisms or endogenous molecules associated with pathogenesis. Signalling through these receptors promotes tissue-specific innate immune responses including the production of cytokines.

Toll-like receptor(TLR). An evolutionarily conserved pattern-recognition receptor located at the cell surface or at intracellular membranes. The natural ligands of TLRs are conserved molecular structures found in bacteria, viruses and fungi.

Sampling of luminal contents by IECs. Despite the bar-rier function supported by IECs (BOX 2), the intestinal epithelium includes specialized adaptations that conflict with the concept of complete segregation between host immune cells and microorganisms. Specialized IECs, called microfold cells (Mcells), mediate the sampling of luminal antigens and intact microorganisms for pres-entation to the underlying mucosal immune system44. These specialized IECs are concentrated in the follicle-associated epithelium overlaying the luminal surface of intestinal lymphoid structures, including Peyers patches and isolated lymphoid follicles44,45.

Although nonspecific uptake and transcytosis of anti-gens represents a well-established mechanism of sampling by Mcells, it has recently been demonstrated that more efficient mechanisms of receptor-mediated transport also exist. The surface glycoprotein GP2 acts as a receptor for the bacterial pilus protein FimH, and the Mcell-mediated transport of the pathogen Salmonella enterica across the epithelial barrier depends on GP2FimH interaction46. This suggests that Mcells are capable of both specific receptor-mediated microbial uptake and nonspecific antigen uptake from the intestinal lumen. Although the active transport of luminal contents across the epithelial barrier was thought to be a unique function of Mcells, it was recently shown that small-intestinal goblet cells also contribute to this process through the delivery of soluble luminal antigens to subepithelial dendritic cells (DCs)47. Although both Mcells and goblet cells seem to be capable of antigen delivery to the lamina propria, the functional importance and contribution of these two pathways to the development of anti-pathogen responses or to the maintenance of immune tolerance remains incompletely understood.

In addition to luminal antigen sampling by IECs, subepithelial mononuclear phagocytes, through inter-actions with IECs, sample luminal contents through transepithelial dendrites48,49 (discussed below). The adaptation of the epithelial barrier for the sampling of luminal contents accommodates limited and controlled bacterial and antigen translocation to direct appropriate tolerogenic or anti-pathogen responses. The influence of these transport pathways on the immune response is not well understood, but distinct pathways of acquiring antigens may influence the context in which immune cells interpret microbial signals50. Furthermore, trans-port through these pathways may alter bacteria and antigens to enable controlled transport of antigens to be differentiated from dysregulated bacterial transloca-tion50. Harnessing the functions of IECs in this sampling process holds promise for the development of mucosal vaccines and the regulation of intestinal inflammation.

IECs sentinels in intestinal homeostasisCentral to the capacity of IECs to maintain barrier and immunoregulatory functions is their ability to act as frontline sensors for microbial encounters and to integrate commensal bacteria-derived signals into anti-microbial and immunoregulatory responses (FIG.2). IECs express pattern-recognition receptors (PRRs) that enable them to act as dynamic sensors of the microbial envi-ronment and as active participants in the directing of mucosal immune cell responses (see Supplementary information S1 (table)). Members of the Toll-like recep-tor (TLR)51, NOD-like receptor (NLR)52,53 and RIG-I-like receptor (RLR) families54,55 provide distinct pathways for the recognition of microbial ligands or endogenous sig-nals associated with pathogenesis. Unlike sterile sites in the body where recognition of foreign microorganisms initiates highly inflammatory cascades, the abundance of symbiotic commensals in the intestine necessitates that IECs maintain a state of altered responsiveness (discussed below). Although the study of PRR pathways in haematopoietic cells has mostly focused on their pro-inflammatory properties in antigen-presenting and effector immune cell populations, their role in regulating tissue homeostasis and immune tolerance has emerged as a major component of their function in IECs (see Supplementary information S1 (table)).

The homeostatic role of microbial recognition by IECs. Evidence of a role for PRRs in the protection against intestinal inflammation and repair of epithelial dam-age emerged from studies of mice deficient in TLRs and signalling adaptors or depleted of key commensal microorganisms56. Landmark work by Medzhitov and colleagues demonstrated, through the use of TLR- and MYD88-deficient and broad-spectrum antibiotic-treated mice, that commensal bacteria-derived signals contrib-ute to epithelial homeostasis and repair in a model of chemically induced colitis using dextran sodium sulphate (DSS)56. This and other studies defined beneficial roles of IEC-intrinsic TLR signalling that include the expression of cytoprotective heat-shock proteins, epidermal growth factor receptor ligands56,57, and TFF3 (REF.58), and the

Box 2 |IEC tight junctions and turnover

Below the mucous layers, intestinal epithelial cells (IECs) form a continuous physical barrier. Tight junctions connect adjacent IECs and are associated with cytoplasmic actin and myosin networks that regulate intestinal permeability. In the setting of inflammatory bowel disease (IBD), dysregulation of these interactions, mediated by tumour necrosis factor signalling and by myosin light chain kinase activity, leads to IEC cytoskeletal rearrangements that disrupt tight junctions and increase permeability189,190. These findings suggest that IEC tight junctions could be important targets for enhancing the integrity of the intestinal barrier in IBD.

As the IEC barrier is continuously renewed, the turnover of IECs provides an additional challenge to the maintenance of epithelial continuity. Recent studies have described pathways by which adjacent cells seal potential voids created during the extrusion of either apoptotic or live cells from the single-cell layer191,192. As dysregulated epithelial cell turnover and apoptosis are associated with intestinal inflammation, the contribution of these mechanisms to the limiting of barrier breaches and further inflammation is of relevance to our understanding of epithelial cells as an efficient physical barrier.

Although increased intestinal permeability has been correlated with IBD1,193,194, it remains unclear whether the loss of barrier function is a cause or a consequence of intestinal inflammation in human disease. Evidence from mouse models with genetic defects in tight-junction-associated proteins suggests that disruption of barrier function alone is not always sufficient to cause disease195,196. Notably, in mice with a deletion of the tight-junction protein junctional adhesion moleculeA, the secretion of commensal bacteria-specific IgA can compensate for the loss of barrier function and limit disease severity following chemically induced colitis195. Thus, compensatory immune mechanisms can act to protect against the development of colitis, even in the setting of barrier disruption, supporting a multi-hit model of disease susceptibility195.

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NOD-like receptor(NLR). A pattern-recognition receptor located in the cytosol. NLRs recognize a wide range of foreign structures and patterns associated with pathogenesis. Some members of this family form multiprotein complexes known as inflammasomes, which regulate the processing and secretion of pro-inflammatory cytokines.

RIG-I-like receptor(RLR). A pattern-recognition receptor located in the cytosol that responds to viral RNA.

Dextran sodium sulphate(DSS). A large polysaccharide that causes epithelial injury and inflammation in the intestinal tract and is commonly used in models of experimentally induced colitis for studying the response to intestinal injury.

enhanced integrity of apical tight-junction complexes59 (FIG.2). Furthermore, IEC-specific deletion of elements necessary for the activation of the transcription factor complex nuclear factor-B (NF-B) downstream of TLR signalling in mice, including the inhibitor of NF-B

(IB) kinase (IKK) complex or NF-B essential modula-tor (NEMO), results in enhanced DSS-induced or spon-taneous colitis60,61. These studies establish an essential role for TLRs, in addition to other NF-B signalling pathways, in epithelial homeostasis andrepair.

Figure 2 | Microbial recognition promotes IEC health and function. a|Pattern-recognition receptors (PRRs), including intestinal epithelial cell (IEC)-expressed Toll-like receptors (TLRs) and NOD-like receptors (NLRs), recognize conserved microbial-associated molecular motifs and pathogen-specific virulence properties. TLRs recruit the signalling adaptors MYD88 and TIR-domain-containing adaptor protein inducing interferon- (TRIF) on ligation to signal molecules via nuclear factor-B (NF-B), p50 and p65 subunit activation and the mitogen-activated protein kinase (MAPK) pathway (not shown). Nucleotide-binding oligomerization domain 1 (NOD1) and NOD2 signal through receptor-interacting protein 2 (RIP2) to activate NF-B and MAPKs, whereas other IEC-expressed NLRs, including NOD-, LRR- and pyrin domain-containing3 (NLRP3), NLRP6 and NOD-, LRR- and CARD-containing4 (NLRC4), form inflammasome complexes with pro-caspase1 for the cleavage and activation of interleukin-1 (IL-1) and IL-18. Polarized expression of PRRs by IECs at either the apical or basolateral membrane may contribute to the discrimination between commensal and pathogen microbial signals. For example, signalling through surface or endosomal TLR9 at the apical pole of IECs promotes the inhibition of NF-B signalling, whereas TLR signalling from the basolateral pole promotes NF-B activation. b|Microbial recognition is integrated by IECs. This promotes cell survival and repair (mediated by trefoil factor 3 (TFF3), heat-shock proteins and epidermal growth factor receptor (EGFR) ligand expression), barrier function (mediated by increased mucin and antimicrobial peptide (AMP) producton) and immunoregulatory responses (mediated by a proliferation-inducing ligand (APRIL), B cell-activating factor (BAFF), IL-25, retinoic acid, transforming growth factor- (TGF) and thymic stromal lymphopoietin (TSLP)), FRMPD2, FERM and PDZ domain-containing 2; IB, inhibitor of NF-B; IKK, IB kinase; ROS, reactive oxygen species; Ub, ubiquitin.


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Nuclear factor-B(NF-B). A family of transcription factors important for pro-inflammatory and anti-apoptotic responses that are activated by the ubiquitin-dependent degradation of their respective inhibitors, members of the inhibitor of NF-B (IB) family. This process is mediated by the kinases, IB kinase1 (IKK1) and IKK2.

InflammasomesMultiprotein complexes that contain a member of the NOD-like receptor family, adaptor proteins and the protease caspase 1. These complexes regulate the catalytic processing and secretion of pro-inflammatory cytokines, including interleukin-1 (IL-1) and IL-18.

Reactive oxygen species(ROS). Chemically reactive molecules containing oxygen that, when produced in large amounts, have pro-inflammatory and antimicrobial effects. Physiological levels of ROS have been shown to regulate cellular signalling pathways.

As additional families of TLRs, such as the NLRs and RLRs, have been ascribed roles in regulating inflamma-tory immune cell responses, they have also been shown to be important in IECs for the regulation of intestinal homeostasis5255. The identificationof nucleotide-bind-ing oligomerization domain 2 (NOD2), an NLR family member that recognizes bacterial muramyl dipeptide (MDP), as the first genetic susceptibility locus for Crohns disease has fuelled interest in the role of this PRR and the related protein NOD1 in both immune cells and the intestinal epithelium37,62,63. Moreover, inflammasomes formed by caspase1 and NLRs, includ-ing IEC-expressed NOD-, LRR- and pyrin domain- containing3 (NLRP3), NLRP6, NLRP12 and NOD-, LRR- and CARD-containing4 (NLRC4), have a complex influence over inflammation and epithelial repair, as dem-onstrated by both pathological and protective roles in con-stitutive knockout mouse models52,53,64 (see Supplementary informationS1 (table)). As NLRs are expressed by sev-eral cell populations in the intestine, conditional knock-out models will be required to elucidate the precise haematopoietic, epithelial and stromal contributions of these PRRs during inflammation and repair.

Finally, reactive oxygen species (ROS) produced in response to commensal or pathogenic bacteria have a role in IEC-intrinsic signalling that acts to promote epithelial repair, independently of their microbicidal effects65,66. Through the inactivation of cellular redox-sensitive tyrosine phosphatases, ROS promote the form-ation by IECs of focal adhesions, which are necessary for cell migration and wound healing65,66. Strikingly, these findings show remarkable symmetry with studies in Drosophila melanogaster, in which ROS also promote epithelial homeostasis, suggesting an evolutionarily conserved role for ROS in mediating protective effects of commensal microorganism-dependent cellular responses6769.

The protective effects of microbial recognition by IECs may come at a cost. Although commensal micro-bial signals are protective in settings of tissue damage or infection, they can drive tumorigenesis and cancer when homeostatic responses become dysregulated60,70. Epithelial cell-intrinsic TLR, MYD88 and NF-B signalling have all been implicated in promoting tumour development and progression in multiple genetic7073 and inflammation-induced60,72,74,75 mouse models of colorectal cancer. The convergence between PRR signalling and pro-oncogenic signalling pathways could partly explain the tumorigenic effects of microbial stimulation. The stabilization of key oncogenic proteins, such as MYC, has been shown to be promoted by MYD88 signalling71. Furthermore, NF-B can enhance WNT signalling in terminally differentiated IECs to promote their dedifferentiation into stem cell-like tumour initiators73.

Paradoxically, some NLR signalling pathways protect against tumorigenesis, partly through the regulation of cell death and proliferation in damaged or transformed IECs7680 and through the regulation of tissue repair responses mediated by interleukin-18 (IL-18) signal-ling53,81,82. The complexity of the multiple roles of micro-bial recognition by IECs serves to further highlight the

delicate nature of the balance that exists between homeo-stasis and inflammation and its importance in maintain-ing healthy hostmicroorganism symbiosis.

Specialized regulation of PRR pathways in IECs. The proximity of IECs to an abundance of luminal microbial signals necessitates specialized mechanisms for maintain-ing altered or hyporesponsive PRR signalling in response to commensal bacteria-dependent stimuli83,84. In support of this, IECs express negative regulators of PRR-dependent pro-inflammatory signalling75,83,85,86 (see Supplementary informationS1 (table)). The disruption of these regula-tory pathways or constitutive activation of NF-B pre-dispose mice to dysregulated epithelial homeostasis and exaggerated inflammation72,75,85,87. Furthermore, it has been appreciated that commensal bacteria-dependent production of ROS by IECs can attenuate the activation of NF-B, broadly tolerizing IECs to microbial stimula-tion through PRR signalling88,89. Although additional mechanisms exist for the negative regulation of PRR sig-nalling pathways90, in most cases the extent to which they are active in IECs and their contributions to intestinal homeostasis remain to be determined.

In addition to maintaining the hyporesponsiveness of IECs, innate immune pathways must differentiate between signals derived from commensal and patho-genic microorganisms for the scaling of an appropriate inflammatory response91. The polarized nature of the intestinal epithelium allows for the anatomical segrega-tion of PRRs (FIG.2). Invitro and invivo models demon-strate differential responsiveness of IECs to apical versus basolateral stimulation with multiple TLR ligands9294. For example, although basolateral exposure of IECs to TLR9 ligands results in canonical activation and nuclear translocation of NF-B, apical exposure results in a net inhibitory effect through the stabilization of IB94. This apical signal induces tolerance to subsequent TLR stimulation, demonstrating a unique adaptation for the cross-tolerance of microbial recognition pathways and a differential response to microbial signals based on anatomical location94 (FIG.2).

This concept of subcellular segregation and polarized distribution of TLRs has been translated to the regula-tion of additional PRR pathways95,96. Through a series of elegant genetic screens, FERM and PDZ domain-containing2 (FRMPD2) which is a positive regula-tor of NOD2-mediated NF-B activation in response to MDP recognition was recently identified to act as a scaffold protein that promotes basolateral membrane localization and selective basolateral activation through interactions with the leucine-rich repeat (LRR) domain of NOD2 (REF. 96) (FIG.2). Common Crohns disease-associated variants of NOD2 contain mutations in this LRR domain. These NOD2-mutant proteins were shown invitro to lack the ability to interact with FRMPD2, to colocalize at the basolateral membrane of epithelial cells and to respond to stimulation with NOD2 ligands62,63,96. These studies give insight into the mechanism of NOD2 dysfunction associated with IBD and how IECs may spatially regulate the activation of PRR signals at the intestinal barrier.

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Viability-associated PAMPs(Vita-PAMPs). Members of a special class of pathogen- associated molecular patterns recognized by the innate immune system to signify microbial life. These patterns differentiate dead and living microorganisms to allow for scaling of appropriate immune responses based on the level of threat the microbial signals represents.

Finally, mechanisms by which IECs may break their relative tolerance to microbial signals in settings of path-ogen infection are poorly defined. In contrast to sterile sites in the body, control of inflammation in the intes-tine may be more adapted to relying on the recognition of danger signals associated with pathogenesis, rather than on the presence of microbial signals alone97. The recognition of danger has been proposed to be medi-ated through the detection of properties associated with microbial viability, termed viability-associated PAMPs (vita-PAMPs), that distinguish living pathogens from inert microbial debris, as well as through the detection of conserved virulence factors of pathogens, such as bac-terial secretion systems and toxins that penetrate into the cellular cytosol91,98. Although these mechanisms for scal-ing microbial threats have been studied and identified in phagocytes and antigen-presenting cells, their function and relevance in IECs are less well understood.

Commensal microorganism-dependent regulation of barrier function. In addition to the homeostatic role of microbial recognition by IECs, the intestinal epithelium acts as an essential integrator of environmental signals for the regulation of microbial colonization, barrier function and mucosal immune responses. As previously discussed, the production of an apical mucous layer, the secretion of broadly targeted AMPs and the transcytosis of secre-tory IgA contribute to epithelial barrier function. Reduced mucous layer thickness in germ-free mice can be reversed by treatment with TLR ligands, indicating that commen-sal bacteria-dependent signals regulate mucus produc-tion by goblet cells17. Similarly, the expression of many epithelial cell-derived AMPs is enhanced by, or dependent on, the presence of commensal microbial signals18,29,99101. As cells with specialized antimicrobial function, Paneth cells play a particularly important part in the regulation of AMP production through cell-intrinsic expression of MYD88 and NOD2 (REFS100,101).

The transport of IgA across the epithelial barrier is regulated, in part, by the expression of pIgR on the basolateral membrane of IECs, which is promoted by MYD88- and NF-B-dependent signalling in response to commensal microbial signals40,102. Finally, the integ-rity of tight junctions and transepithelial permeability are regulated by commensal microbial signals, including TLR2-dependent redistribution of the tight-junction pro-teins to apical cellcell contacts59. Thus, the ability of IECs to sense their microbial surroundings has an integral role in regulating their barrier function.

Regulation of immune cells by IECsIECs produce numerous immunoregulatory signals that are necessary for tolerizing immune cells, limiting steady-state inflammation and directing appropriate innate and adaptive immune cell responses against pathogens and commensal bacteria. Many of these responses depend on the translation of commensal bacteria-derived sig-nals by IECs to mucosal immune cells. The produc-tion of the cytokines thymic stromal lymphopoietin (TSLP)103105, transforming growth factor- (TGF)104,106 and IL-25 (REF.107) and the Bcell-stimulating factors

a proliferation-inducing ligand (APRIL; also known as TNFSF13) and Bcell-activating factor (BAFF; also known as TNFSF13B)108,109 by IECs is promoted by com-mensal bacteria via PRR signalling (FIG.2). We discuss below the immunoregulatory functions of IECs, describ-ing their contribution to the priming of adaptive immune cell responses, regulation of innate effector responses and homeostasis of adaptive immune cell function in the intestinal environment.

Mononuclear phagocytes and antigen presentation. IECs exert their influence over the priming of both cellular and humoral adaptive immune responses via a continuous dialogue with antigen-presenting mononuclear phago-cytes (FIG.3). IEC-derived TSLP, TGF and retinoic acid, produced in response to commensal bacteria-derived sig-nals, promote the development of DCs and macrophages with tolerogenic properties, including the production of IL-10 and retinoic acid103,104,110. Considerable hetero-geneity exists among intestinal mononuclear phagocytes, the classification of which has been previously compli-cated by conflicting nomenclature, as well as phenotypical and functional plasticity in settings of inflammation111. However, two distinct populations that have been characterized are the pre-DC-derived CD11c+CD103+ DCs and monocyte-derived CD11clowF4/80+CX3CR1

hi intestine-resident macrophages112115.

CD103+ DCs act as migratory antigen-presenting cells and upon activation traffic to secondary lymphoid tis-sues, including the mesenteric lymph nodes and Peyers patches, carrying with them antigenic material and live bacteria for presentation to adaptive immune cells116,117. Influenced by their previous interactions with IECs at the intestinal barrier, these migratory DCs promote immune tolerance through the differentiation of forkhead boxP3 (FOXP3+) regulatory Tcells by a TGF- and retinoic acid-dependent mechanism116,118,119. Furthermore, the production of retinoic acid by IEC-conditioned CD103+ DCs is responsible for the imprinting of gut-homing properties on Tcells, allowing for the targeting of recircu-lating mature cells to the original site of antigen encoun-ter in the intestinal lamina propria120124. Thus, in addition to promoting naive Tcell maturation based on antigen specificity, CD103+ DCs relay the original context of antigenic encounter at the intestinal epithelial barrier.

In contrast to CD103+ DCs, CX3CR1hi intestine-

resident macrophages lack migratory properties in the steady state and instead persist in close physical contact with IECs, where they act as avid phagocytes to medi-ate clearance of pathogens and commensal bacteria that traverse the epithelial barrier116,125. Their expres-sion of tight-junction proteins allows the formation of trans epithelial dendrites that penetrate into the lumen of the intestine for sampling of exogenous antigens48,125. Reflecting the functional dependence of these cells on the epithelium, the extension of these trans epithelial dendrites is initiated by TLR signalling, not in myeloid cells themselves, but in IECs49. The CX3CR1

hi intestine- resident macrophage population has also been impli-cated in the maintenance of mucosal tolerance, as they have been shown to promote the survival and local

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Innate lymphoid cells(ILCs). A group of innate immune cells that are lymphoid in morphology and developmental origin, but lack properties of adaptive B cells and T cells such as recombined antigen-specific receptors. They function in the regulation of immunity, tissue homeostasis and inflammation in response to cytokine stimulation.

expansion of previously primed regulatory Tcells126. CX3CR1

hi macrophages promote tolerance in the intes-tinal lamina propria through the production of IL-10, which leads to suppression of inflammatory cytokine production by colitogenic Tcells and promotion of regulatory Tcell function127,128. IECs maintain this tolerogenic function through their production of solu-ble factors, such as TSLP, TGF and retinoic acid103,104,110, as well as through contact-dependent interactions involving IEC expression of the integrin ligand sema-phorin 7A, which induces IL-10 expression by CX3CR1

hi macrophages and promotes intestinal homeostasis129.

IECs also play an important part in the induction of T helper 2 (TH2) cell responses during helminth infec-tion. In this setting, the IEC-derived cytokines TSLP and IL-25 promote the expansion and differentiation of haematopoietic progenitor cells towards mononuclear

and myeloid cell phenotypes that promote the develop-ment of type2 cytokine responses at mucosal sites130133. These cells include a distinct population of basophil pro-genitors and a population of multipotent progenitor cells, which undergo extramedullary haematopoiesis and rep-resent an innate link between IEC-derived signals and the polarization of TH2 cell immune responses to helminths and allergens132,133.

Innate lymphocyte function. In addition to the myeloid cell and granulocyte populations, a recently identified innate immune cell population of innate lymphoid cells (ILCs) plays a crucial part in intestinal immune homeo-stasis. ILCs lack properties of adaptive lymphocytes, such as recombined antigen-specific receptors134. They are found at barrier surfaces, including mouse and human lung135, skin136 and intestine137, where they function

Figure 3 | IECs regulate innate and adaptive immunity. Intestinal epithelial cell (IEC)-derived cytokines interleukin-25 (IL-25) and thymic stromal lymphopoietin (TSLP) elicit the expansion and differentiation of basophil progenitors and multipotent progenitor type2 (type2 MPP) cells, respectively. IL-25, IL-33 and TSLP stimulate group 2 innate lymphoid cells (ILC2s), whereas IL-25 suppresses innate lymphoid cell subset 1 (ILC1) and ILC3 function by limiting macrophage production of pro-inflammatory cytokines IL-1, IL-12 and IL-23. IECs condition dendritic cells (DCs) and macrophages towards a tolerogenic phenotype through the production of TSLP, transforming growth factor- (TGF) and retinoic acid (RA). TheseDCs promote the differentiation of naive CD4+ Tcells into regulatory T (T

Reg) cells and the maturation of Bcells

into IgA-secreting plasma cells. Mucosal cell-derived DCs also imprint a gut-homing phenotype on primed Bcells and Tcells through the production of RA. After trafficking to the intestine, T

Reg cells are expanded in number by macrophages

that are conditioned to produce IL-10 by TSLP-mediated stimulation and through contact-dependent interactions with IEC-expressed semaphorin 7A (SEMA7A). The production of a proliferation-inducing ligand (APRIL) and Bcell-activating factor (BAFF) by IECs and by TSLP-stimulated macrophages and DCs promotes class-switch recombination and the production of IgA by Bcells in the intestinal lamina propria. IEL, intra-epithelial lymphocyte; IFN, interferon-; sIgA, secretory IgA; TCR, Tcell receptor; TLA, thymus leukaemia antigen; TNF, tumour necrosis factor.


ILC1

Lamina propria

Peyers patchor mesentericlymph node

ILC3ILC2

DC

IEL

TLACommensalbacterium

IL-25

Innate immune regulation Adaptive immune regulation

IL-12

IFN,TNF

IL-1,IL-23IL-17,

IL-22

IL-25,IL-33,TSLP

IL-13,amphiregulin

IL-10,RA,TGF

TSLP,TGF,RA

IL-7,IL-15

APRIL,BAFF

TSLP

SEMA7AsIgA

IL-10

TSLP

IL-25

Type 2MPP

Basophil

Basophil

Macrophage Monocyte

Mast cell B cell

Naive T cell

MHCTCR

TReg cell

TReg cell

IgA+ plasma cell

Basophilprogenitor

RA,TGF

Direct IEC eect

Indirect IEC eect

Immune response

Dierentiation

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Natural killer cells(NK cells). A subset of innate lymphoid cells originally defined on the basis of their cytolytic activity against tumour targets but now recognized to serve a broader role in host defence and inflammation through the production of cytokines.

as regulators of tissue homeostasis, inflammation and early innate response to infection. ILCs are regulated, in part, by epithelial cell-derived immunoregulatory signals (FIG.3). ILCs display phenotypical and functional heterogeneity, which has been reviewed extensively elsewhere134,138140. ILCs are characterized by their devel-opmental requirements and differential cytokine expres-sion into group 1, group 2 and group 3 ILCs, which share functional similarities with the adaptive CD4+ TH1, TH2 and TH17 cell populations, respectively.

Group 1 ILCs include classical natural killer cells (NK cells) and innate lymphoid cell subset 1 (ILC1) cells, and are characterized by the production of the TH1 cell-associated cytokines interferon- (IFN) and tumour necrosis factor (TNF) in response to IL-12 and/or IL-15 (REF.140). Although NK cells can directly kill target cells through cytotoxic activity, other ILC1s are limited to cytokine production in response to stimula-tion. Although these ILC1s have a less well-understood function than NK cells, several recent reports suggest a possible role in mediating intestinal inflammation in murine colitis models and human IBD141,142.

Group 2 ILCs (collectively termed ILC2s) produce the TH2 cell-associated cytokines IL-5 and IL-13 (REF.140). These factors contribute to an early innate response to intestinal helminth infection and invoke a protective epithelial response, including goblet cell hyperplasia and enhanced mucus secretion143145. Furthermore, ILC2s pre-sent in the lung promote airway hyperresponsiveness or tissue repair in mouse models of allergy and influenza virus infection146149. This suggests that ILC2s may have analogous functions in the intestine, perhaps during food allergy or wound repair; however, evidence for these roles has yet to be described. The proliferation and activation of ILC2s is supported by the predominantly epithelial cell-derived cytokines IL-25, IL-33 and TSLP143145,150. The contribution of microbial stimulation to these sig-nals reinforces the idea of the epithelium as an integrator of environmental signals for the regulation of immune cell function103,104,107.

Finally, group 3 ILCs produce TH17 and TH22 cell-associated cytokines, including IL-17A and IL-22, in response to stimulation by IL-23 (REF.140). This group includes ILC3s, as well as lymphoid tissue inducer (LTi) cells, which have a well-established role in secondary lymphoid tissue organogenesis, mediated by interactions with stromal cells during embryonic development151. IL-22 has an important role in protecting the intestinal epithelium following injury or infection by bacterial pathogens152,153. In addition, ILC3-derived IL-22 sup-ports the anatomical containment of gut-associated lymphoid tissue-resident commensal bacteria and the protection of IESCs in models of graft-versus-host dis-ease154156. These tissue-protective functions of IL-22 are balanced by detrimental effects in certain inflammatory settings and in the initiation of inflammation-induced cancer82,156,157. Collectively, these studies demonstrate the context-dependent nature of IL-22 function. By con-trast, ILC3-derived IL-17 is thought to have a primarily pro-inflammatory effect in the intestine and has been implicated in both mouse colitis and human IBD158160.

IECs play an indirect part in the regulation of ILC3s in response to commensal bacteria-derived signals. For example, IEC-derived IL-25 leads to the suppression of IL-23 production by macrophages and decreased IL-22 production by ILC3s161. By contrast, commensal bacteria-dependent signals have also been shown to stimulate the production of IL-7 by IECs162, which supports the pro-duction of IL-22 by ILC3s through the stabilization of the transcription factor retinoid-related orphan receptor-t (RORt; encoded by RORC)162,163. These seemingly con-flicting roles for IECs in regulating ILC3 function in response to commensal bacterial stimulation may be explained by heterogeneity among intestinal ILCs and by differential targeting of cell types capable of producing pro-inflammatory versus tissue-protective cytokines139.

Although the function of ILCs has been appreciated in numerous mouse models, the importance and relative contribution of these cells to inflammation in settings of human disease remain incompletely defined. Future work in this field will be required to further characterize the heterogeneity and tissue-specific functions of these cells, elaborate our understanding of their contribu-tions to human disease and develop means of clinically targeting their protective or detrimental functions.

Tissue-resident Tcells. Following priming by intestine-derived antigen-presenting cells in secondary lymphoid tissues, conventional effector Tcells recirculate through the body before settling in the intestine, where they exert their tolerogenic or inflammatory effect on the local environment (FIG.3). Here, mature Tcells are subject to the direct influence of IECs for their functional main-tenance and survival in the lamina propria. Specialized cells known as intraepithelial lymphocytes (IELs) exist in intimate contact with the IEC layer, and bidirectional interactions between IELs and IECs maintain immune homeostasis at the intestinal barrier164166. IELs display an activated phenotype and include conventional Tcells, as well as subsets of cells expressing a restricted repertoire of Tcell receptor specificities and specialized properties, including Tcells and NKTcells165,167. Recent studies have advanced the understanding of the developmental origin of these cells and the functions that they have at the intestinal barrier165. These include the demonstration that committed CD4+ Tcells can undergo transcriptional reprogramming when they become IELs to develop a distinct phenotype resembling that of CD8+ cytotoxic Tcells168. Although the influence of the local environ-ment in promoting this developmental change has not been explored, the intimate interactions that these cells have with IECs suggest that epithelial cell-derived signals may promote their maintenance and function.

Tissue-resident conventional Tcells primed to act as rapidly responsive effectors are important during on going inflammation and infection, as well as for the protection of the mucosal barrier against future chal-lenge. This is thought to be particularly important in CD8+Tcell-dependent memory responses169. As such, CD8+ Tcells with a tissue-resident memory phenotype are uniquely enriched among Tcells present in the intestinal IEL compartment of mice and humans169,170.

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Class-switch recombination(CSR). The process by which proliferating Bcells rearrange their DNA to switch from expressing IgM (or another class of immunoglobulin) to expressing a different immunoglobulin heavy-chain constant region, thereby producing antibody with different effector functions.

These tissue-resident memory T (TRM) cells interact with IECs through CD103 (also known as E7 integ-rin), which binds the adhesion molecule E-cadherin on IECs171,172. This may promote retention of these and other cells at the intestinal epithelium.

Mouse IECs were recently demonstrated to contrib-ute to the refinement of the CD8+ TRM cell pool in favour of high-affinity precursors that allow for a more efficient memory response to secondary mucosal challenge173. This occurs through the contact-dependent selective expansion and survival of high-affinity or high-avidity CD8+ Tcell populations expressing homodimers of the co-receptor subunit CD8 (known as CD8+ IELs), which interact with the IEC-expressed MHC classI-like molecule, thymus leukaemia antigen (TLA)173. Understanding how such memory cell populations are maintained is of particular interest in the design of effi-cient vaccines against pathogens that invade mucosal surfaces. Strategies have been explored for generating CD8+ TRM cells with protective effects at extra-intestinal sites174,175. Through an improved understanding of how IELs are maintained within the intestinal epithelium, we can hope to improve vaccine strategies for prevent-ing infections with pathogens such as HIV and enteric viruses176.

IgA-secreting plasma cells. The maturation of naive Bcells into mature IgA-secreting plasma cells through heavy chain class-switch recombination (CSR) depends on priming by mucosal DCs carrying antigen and live bacteria from the intestinal epithelium117,177. Similar to the priming of a Tcell mucosal phenotype, these DCs are conditioned by IEC-derived signals to promote IgA class switching and a gut-homing phenotype through the production of nitric oxide (NO), IL-10 and retinoic acid, in conjunction with TGF signalling117,124,178 (FIG.3).

In the presence of a cognate CD4+ Tcell response, Tcell expression of CD40L acts as a necessary signal for Bcell CSR. In the absence of help from Tcells, CSR can occur through the stimulation of Bcells by APRIL and BAFF, and signalling through transmembrane activator and CAML interactor (TACI) and BAFF receptor (BAFFR; also known as TNFRSF13C)108,109,179,180. This process is directly supported by IECs through the production of APRIL and BAFF in response to commensal bacteria-induced NF-B signalling108,109. Moreover, IECs induce APRIL and BAFF production by mucosal DCs through TSLP signalling, which acts to amplify the effect on Bcell stimulation108,109. This pathway is of clinical relevance to the most prevalent human primary immunodeficiencies, common variable immunodeficiency and IgA deficiency, in which a subset of patients have mutations in the gene encoding the TACI receptor that lead to defects in CSR and IgA production181,182.

Concluding remarksCollectively, the studies highlighted in this Review dem-onstrate the diverse and multifaceted roles that IECs have in the continuous maintenance of intestinal home-ostasis. Through secretory epithelial cell responses and the maintenance of a continuous cell layer, IECs effec-tively sustain a physical and biochemical barrier between hosts and their environment. As cells forming a uniquely adapted barrier surface, IECs actively respond to their local environment through regulatory mechanisms that earn IECs recognition as central mediators of microbial and immune homeostasis in the intestine. As much of what is understood of IEC function has been derived from studies using mouse models, a future challenge lies in the translation of this understanding into human systems and the development of novel therapeutics for targeting the pathways that contribute to human health.

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Intestinal Epithelial Cells Regulators of Barrier Function and Immune Homeostasis

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Intestinal Epithelial Cells Regulators of Barrier Function and Immune Homeostasis