Title: The intestinal epithelium as guardian of gut barrier integrity
Abstract: Cellular MicrobiologyVolume 17, Issue 11 p. 1561-1569 MicroreviewFree Access The intestinal epithelium as guardian of gut barrier integrity Kaiyi Zhang, Kaiyi Zhang Institute for Medical Microbiology, University Hospital RWTH Aachen, Aachen, GermanySearch for more papers by this authorMathias W. Hornef, Mathias W. Hornef Institute for Medical Microbiology, University Hospital RWTH Aachen, Aachen, GermanySearch for more papers by this authorAline Dupont, Corresponding Author Aline Dupont Institute for Medical Microbiology, University Hospital RWTH Aachen, Aachen, GermanyFor correspondence. E-mail [email protected]; Tel. (+49) 2418089511/510; Fax: (+49) 2418082483.Search for more papers by this author Kaiyi Zhang, Kaiyi Zhang Institute for Medical Microbiology, University Hospital RWTH Aachen, Aachen, GermanySearch for more papers by this authorMathias W. Hornef, Mathias W. Hornef Institute for Medical Microbiology, University Hospital RWTH Aachen, Aachen, GermanySearch for more papers by this authorAline Dupont, Corresponding Author Aline Dupont Institute for Medical Microbiology, University Hospital RWTH Aachen, Aachen, GermanyFor correspondence. E-mail [email protected]; Tel. (+49) 2418089511/510; Fax: (+49) 2418082483.Search for more papers by this author First published: 20 August 2015 https://doi.org/10.1111/cmi.12501Citations: 76AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Summary A single layer of epithelial cells separates the intestinal lumen from the underlying sterile tissue. It is exposed to a multitude of nutrients and a large number of commensal bacteria. Although the presence of commensal bacteria significantly contributes to nutrient digestion, vitamin synthesis and tissue maturation, their high number represents a permanent challenge to the integrity of the epithelial surface keeping the local immune system constantly on alert. In addition, the intestinal mucosa is challenged by a variety of enteropathogenic microorganisms. In both circumstances, the epithelium actively contributes to maintaining host–microbial homeostasis and antimicrobial host defence. It deploys a variety of mechanisms to restrict the presence of commensal bacteria to the intestinal lumen and to prevent translocation of commensal and pathogenic microorganisms to the underlying tissue. Enteropathogenic microorganisms in turn have learnt to evade the host's immune system and circumvent the antimicrobial host response. In the present article, we review recent advances that illustrate the intense and intimate host-microbial interaction at the epithelial level and improve our understanding of the mechanisms that maintain the integrity of the intestinal epithelial barrier. The intestinal epithelium — composed of different cell types overlaid by the mucus layer — forms the barrier that separates the microbe-filled and nutrient-filled gut lumen from the largely sterile underlying tissue. Whereas continuously proliferating stem cells and antimicrobial peptide-secreting Paneth cells remain situated at the base of the intestinal crypts, nutrient-absorbing enterocytes, mucus-producing goblet cells and neuroendocrine cells migrate along the crypt-villus axis and are eventually exfoliated from the constantly renewed epithelial surface. Here, we discuss recent discoveries on the role of the overlaying mucus layer, innate immune recognition, epithelial cell–cell junctions, cell autonomous immunity and enterocyte exfoliation and renewal in antimicrobial host defence and maintenance of host–microbial homeostasis. The mucus layer as a physico-chemical barrier The intestinal mucus represents the first barrier between the intestinal lumen and the mucosal tissue. It is mainly composed of the heavily modified glycoprotein mucin 2 (MUC2) assembled to a characteristic three-dimensional hydrophilic matrix. In the colon, it forms a thick layer (200 µm in humans, 50 µm in mice) made of a tight adherent inner part and a softer outer part that provides a nutrient source for surface-associated resident commensal bacteria. It is less pronounced in the small intestine, where it secures the crypt openings and covers the lower part of the protruding villi (Birchenough, Johansson, Gustafsson, Bergstrom and Hansson, 2015). Its functional importance is highlighted by the fact that mice with Muc2 deficiency or with intestinal epithelial cell-specific deficiency of core 1-derived O-glycans, one of the main components of mucins, spontaneously develop colitis (Van der Sluis et al., 2006, Fu et al., 2011). Of note, as the mucus layer collapses following formaldehyde fixation, mucus-preserving fixatives (e.g. Carnoy fixative) are required for its visualization. The tight inner layer of colonic mucus has previously been shown to be impermeable to particles the size of a bacterium rendering it devoid of bacteria under homeostatic conditions (Johansson et al., 2008, Ermund et al., 2013). Two recent studies further clarified the functional importance of the mucus layer in the host–microbe interaction. Jakobsson et al. discovered that the structural filter function of the colonic mucus is microbiota-dependent and that even slight variations in the microbiota composition alter the permeability of the inner colonic mucus layer (2015). Ermund et al. analysed the function of the softer, bacteria-permeable mucus layer in the small intestine. They found that the continuous shedding of mucus into the lumen hinders bacteria from coming in close proximity to the epithelium (2013). A newly discovered mechanism may reinforce the more permeable mucus matrix in the small intestine. Chu et al. described that Paneth cell-secreted human α-defensin (HD) 6 forms self-assembled nanonet structures and entangles luminal bacteria (2012). HD6 thus enhances the physical barrier function of the mucus layer in the small intestine upon secretion. Recently, a number of antimicrobial peptides, including HD6, were noted to gain activity under low pO2 conditions similar to the one found in the intestinal lumen (Albenberg et al., 2014, Schroeder et al., 2015). In addition to HD6, the mucus layer is enriched in many other antimicrobial peptides and proteins produced by crypt-based Paneth cells and enterocytes, conferring local antibacterial activity and contributing to the bacteria-free zone between the epithelial surface and intestinal lumen (Meyer-Hoffert et al., 2008, Vaishnava et al., 2011, Antoni et al., 2013, Loonen et al., 2014, Dupont et al., 2015). Besides their antibacterial activity, antimicrobial peptides exert potent anti-inflammatory properties inhibiting, for example the immunostimulatory activity of bacterial endotoxin (Dupont et al., 2015). Similarly, intestinal alkaline phosphatase – a phosphatase secreted by intestinal epithelial cells and associated with the mucus matrix – detoxifies bacterial endotoxin and reduces its local and systemic immunostimulatory potential (Kaliannan et al., 2013, Zarepour et al., 2013). The mucus matrix thus represents a physico-chemical barrier with potent antibacterial and anti-inflammatory properties. Secretory IgA (SIgA) molecules represent yet another important constituent of the intestinal mucosal surface. SIgA are translocated by the epithelial polymeric immunoglobulin receptor and bind bacteria-derived and food-derived antigens. SIgA thereby helps to retain toxins or bacteria within the lumen. In accordance, bacterial strains with colitogenic potential are preferentially coated with SIgA, and mice with low fecal SIgA levels exhibit a significantly enhanced susceptibility to colonic inflammation (Palm et al., 2014, Moon et al., 2015). Interestingly, the Stappenbeck laboratory demonstrated that low immunoglobulin levels could be the result of the SIgA degrading activity of unidentified anaerobic bacteria. These bacterial species might thus confer enhanced susceptibility to inflammation in a transferrable manner (Moon et al., 2015). IgG can as well be transported across the epithelium. Kamada et al. recently proposed that IgG bound to virulent Citrobacter rodentium within the intestinal lumen mediates phagocytosis and elimination by polymorphonuclear cells that transmigrated from the lamina propria (Kamada et al., 2015). Similarly, the group of Yasmine Belkaid has shown that polymorphonuclears emigrate into the gut lumen during inflammation and contain luminal dysbiotic commensal bacteria by keeping them from reaching the epithelial surface (Molloy et al., 2013). Epithelial innate immune recognition Pathogen-associated molecular patterns (PAMPs) represent evolutionary conserved structural motifs produced by both commensal and pathogenic microorganisms. Pattern recognition receptors (PRRs) recognize PAMPs initiating transcriptional and non-transcriptional cellular responses. Intestinal epithelial cells express a variety of PRRs and are able to sense the presence of commensal and pathogenic microorganisms (Pott & Hornef, 2012). Toll-like receptor 1/2, 2/6, 3, 4, 5 and 9, nucleotide-binding oligomerization domain 1 and 2, the RNA sensors retinoic acid-inducible gene (Rig)-I-like helicases and melanoma differentiation-associated protein-5, as well as the DNA sensors interferon-γ inducible protein (IFI)16 and cyclic GMP-AMP synthase (cGAS), facilitate transcriptional epithelial responses. PRR-induced transcriptional responses are mediated by NF-κB and the mitogen activated protein (MAP) kinases p38, JNK and ERK as well as IRF3 leading to secretion of types I and III interferon (IFN). Strong IFN expression was observed after stimulation of the most recently discovered cytosolic DNA sensor, cGAS, upon invasive infection with bacterial pathogens such as Listeria monocytogenes, Chlamydia trachomatis or Mycobacterium tuberculosis (Hansen et al., 2014, Zhang et al., 2014b, Dey et al., 2015, Watson et al., 2015). Soluble IFN via the IFN receptors subsequently provides antiviral protection and may also contribute to antibacterial host defence. Interestingly, antiviral protection of intestinal epithelial cells is mediated specifically by type III IFN (Pott et al., 2011). Additional members of the nucleotide-binding oligomerization domain-like receptor family such as Nlrp3, Nlrp6, Nlrc4 and the DNA sensors absent in melanoma 2 initiate assembly of the inflammasome leading to caspase 1-mediated IL-1β and IL-18 release as well as pyroptosis, a sort of inflammatory cell death. Thereby, inflammasome stimulation can confer protection. For example, early epithelial activation of Nlrp3 improves the outcome after C. rodentium infection (Song-Zhao et al., 2014). Also, Nlrp6 stimulation regulates mucus secretion by goblet cells and may reinforce the mucus layer (Wlodarska et al., 2014). In contrast, Nlrp12 seems to negatively regulate mucosal inflammation, and Salmonella enterica serovar Typhimurium was shown to exploit this receptor to suppress antimicrobial host responses (Zaki et al., 2014). A new non-canonical inflammasome complex composed of murine caspase 11 (caspase 4 in humans) that senses endotoxin from cytosolic gram-negative bacteria and leads to IL-18 cleavage and epithelial cell exfoliation has been described (Hagar et al., 2013, Knodler et al., 2014a, Shi et al., 2014). Of note, microbial ligands have not yet been identified for all receptors. Beside structural motifs, alterations in the cell homeostasis such as the activation of small Rho GTPases by translocated effector proteins from S. Typhimurium might be sensed (Keestra et al., 2013). Also, innate immune stimulation of one epithelial cell can be propagated to neighbouring cells via spread of the cGAS product 2′–3′-cyclic GMP-AMP through gap junctions or via release of nitric oxide, facilitating a coordinated epithelial response to a local challenge (Dolowschiak et al., 2010, Kasper et al., 2010, Ablasser et al., 2013). In contrast to most other cell types, intestinal epithelial cells are permanently exposed to PAMPs, explaining their need for tight controls in order to avoid inappropriate immune stimulation and inflammation (Pott & Hornef, 2012). Nevertheless, homeostatic innate immune signalling helps to maintain epithelial barrier function both in the small and large intestine (Sodhi et al., 2012, Stockinger et al., 2014). Enhanced epithelial innate signalling during S. Typhimurium infection via the toll-like receptor adaptor molecule MyD88 promotes an antibacterial host response and protects from bacterial translocation (Bhinder et al., 2014). However, this induced antimicrobial activity also represses the enteric microbiota, creating a niche for pathogen colonization (Behnsen et al., 2014). Strikingly, some pathogenic bacteria even exploit the host inflammatory immune response. Salmonella Typhimurium was shown to specifically seek the inflammatory host response to boost its luminal growth by metabolizing the inflammatory byproducts (Rivera-Chavez et al., 2013). In line with this, S. Typhimurium promotes colitis by decreasing epithelial expression of the negative regulatory peroxisome proliferator-activated receptor-γ (Kundu et al., 2014). Other pathogens such as non-invasive enteropathogenic Escherichia coli (EPEC) pursue another strategy: they dampen the epithelial JNK and NF-κB signalling pathways via type 3 secretion system (T3SS)-translocated effector proteins to reduce the antimicrobial host response (Nadler et al., 2010, Baruch et al., 2011). Finally, several reports recently highlighted the importance of epithelial protein fucosylation in antimicrobial host defence. Epithelial expression of the fucosyltransferase Fut2 is enhanced by innate lymphoid cell-derived IL-22 and leads to the release of fucosylated proteins into the lumen. Fucosylated proteins are used as energy source by commensal bacteria that support the host–microbiota mutualism and protect from invading pathogens such as S. Typhimurium or C. rodentium (Goto et al., 2014, Pham et al., 2014, Pickard et al., 2014). Barrier formation and transepithelial transport mechanisms Adherence and tight junction complexes composed of E-cadherin, claudins, junctional adhesion molecules and occludin link epithelial cells to form a non-penetrable barrier and define their polarized phenotype. Under homeostatic conditions, they provide a tight barrier to macromolecules and bacteria. Their structural organization, however, is highly dynamic, and inflammatory mediators such as TNF-α as well as innate immune stimuli influence their permeability. Also, enteric pathogens manipulate tight junction integrity. The S. Typhimurium pathogenicity island-1 encoded effector molecules SopB, SopE/E2 and SipA are able to stimulate Rho family GTPases and disrupt tight junctions, and this may promote bacterial invasion (Boyle et al., 2006). Recently, the effector molecules EspG1/G2 produced by EPEC were shown to perturb tight junction assembly and cause delayed epithelial barrier repair, while NleD counteracts the tight junction supporting effect of type I IFN (Glotfelty et al., 2014, Long et al., 2014). Despite these barrier perturbations, EPEC usually remains within the intestinal lumen during infection. Several mechanisms facilitate the delivery of luminal antigen to intestinal lymphoid organs, prerequisite to an active contribution of the adaptive immune system to mucosal homeostasis. Microfold (M) cells are specialized enterocytes that belong to the follicle-associated epithelium overlaying Peyer's patches. They deliver luminal antigen as well as material up to the size of a bacterium to the underlying immune cells. The translocation is facilitated by the binding of the M cell surface glycoprotein2 to the type 1 pilus protein FimH of gram-negative bacteria (Hase et al., 2009). Thereby, non-invasive bacteria such as Alcaligenes spp. may reach the mucosal lymphoid tissue, and a number of important enteric pathogens such as S. Typhimurium, Yersinia enterocolitica and Shigella spp. have been shown to hijack M cells to reach the lamina propria (Jones et al., 1994, Hase et al., 2009, Chiba et al., 2011, Sonnenberg et al., 2012, Tahoun et al., 2012, Ponnusamy et al., 2013). Interestingly, S. Typhimurium, via its translocated effector molecule SopB, promotes M cell differentiation and thereby enhances its own translocation (Tahoun et al., 2012). Somehow surprisingly, also goblet cells were shown to actively internalize low molecular weight antigen from the lumen and forward it to underlying CD103+ dendritic cells (McDole et al., 2012). CD103+ dendritic cells as well as CX3CR1hi intestinal phagocytes also directly sample antigen via extending cell protrusions in the gut lumen (Farache et al., 2013). Salmonella Typhimurium exploits this mechanism for its own benefit to reach the lamina propria and spread to systemic organs (Muller et al., 2012). In contrast, the uptake of C. rodentium by CX3CR1hi intestinal phagocytes leads to the secretion of protective IL-22 and antibacterial protection (Manta et al., 2013). Cell autonomous immunity Cell autonomous immunity describes evolutionary highly conserved mechanisms of the cell-intrinsic anti-infectious host defence. The mechanisms involved act against both viruses and bacteria and are found in many different cell types including intestinal epithelial cells. Recognition of microbial structures or infection-associated cellular damage induces antimicrobial effector mechanisms such as nutrient deprivation, containment by de novo membrane synthesis and killing by the generation of oxygen and nitrogen radicals. Despite its housekeeping function, its critical importance in vivo has only recently been appreciated (Randow et al., 2013). The discovery of specific 'eat-me' signals such as polyubiquitination or galectin-8 on cytosolic bacteria or their vesicle remnants in combination with the identification of autophagy cargo receptors like p62, NDP52 or optineurin explained the molecular mechanisms of this potent defence mechanism (Cemma et al., 2011). Potential targets are pathogens that reach the cytosol as part of their life cycle such as L. monocytogenes, Shigella spp. or Francisella tularensis. Consistent with the recent observation that S. Typhimurium is able to lyse the endosomal membrane in a fraction of epithelial cells leading to rapid proliferation within the cytosol, two independent studies have illustrated the importance of autophagosomal degradation of S. Typhimurium in intestinal epithelial cells in vivo (Benjamin et al., 2013, Conway et al., 2013, Knodler et al., 2014b). Not surprisingly, bacterial pathogens have found ways to escape autophagy. For example, S. Typhimurium secretes SseL that deubiquitinates p62 (Mesquita et al., 2012). Also, Shigella IcsB competitively prevents binding of ATG5, and L. monocytogenes ActA recruits the host Arp2/3 actin nucleation complex preventing ubiquitination and autophagic detection (Perrin et al., 2004, Ogawa et al., 2005, Yoshikawa et al., 2009). Surface receptor-mediated phagocytosis of immunoglobulin-coated microorganisms has been known for a long time. In addition, the cytosolic tripartite motif-containing protein (TRIM) 21 recognizes IgG or IgM bound to bacterial or viral particles, induces NF-κB, MAPK and interferon signalling and directs the microorganism to proteasomal degradation (Mallery et al., 2010). Now, TRIM21 has been shown to be also activated by cytosolic SIgA and to trigger protection from viral infection in an adenoviral infection model (Bidgood et al., 2014). TRIM21 might thus play a significant role at mucosal surfaces where SIgA concentrations are high and serve a critical function during homeostasis and infection. Epithelial cell proliferation, migration and exfoliation Intestinal epithelial cells constantly grow from the pool of crypt-based stem cells. Upon differentiation, they migrate along the crypt-villus axis to finally exfoliate into the gut lumen. This process allows the complete renewal of the epithelium every 3–5 days and represents an important defence strategy against pathogens (Chang et al., 2013). It is further enhanced during infection. Enhanced epithelial proliferation mediated by the Wnt/β catenin signalling pathway was observed, for example after infection with C. rodentium (Sellin et al., 2009). Enhanced cell proliferation has, however, to be complemented by concomitant cell differentiation as the generation of undifferentiated dysfunctional epithelial cells in R-spondin 2 overexpressing mice increases the susceptibility to colonic infection (Papapietro et al., 2013). Cell renewal facilitates exfoliation of infected enterocytes into the gut lumen, a protective mechanism used during S. Typhimurium infection (Sellin et al., 2014). This process requires Naip proteins and Nlrc4 that facilitate recognition of the bacterial flagellum and T3SS (Zhao and Shao, 2015). Interestingly, the rapid epithelial cell turnover only starts after the postnatal period in mice (Harper et al., 2011, Muncan et al., 2011). Oral infection of neonate animals therefore facilitates S. Typhimurium invasion and the formation of large intraepithelial microcolonies (Zhang et al., 2014a). The balance between epithelial cell proliferation and exfoliation is tightly regulated to maintain surface integrity and overcome infection-induced epithelial cell loss. Various cell death pathways have been described in intestinal epithelial cells, namely anoikis, apoptosis, necrosis and necroptosis (Gunther et al., 2013). The critical importance of cell death regulation even under homeostatic conditions is illustrated by the severe intestinal damage observed in animals deficient in cell death regulatory molecules (Dannappel et al., 2014, Takahashi et al., 2014). Pathogenic microorganisms have developed strategies to manipulate epithelial cell proliferation and turnover interfering with cell death regulating processes. For example, NleB, an EPEC-derived T3SS effector, blocks the host cell death pathway by binding to Fas-associated protein with death domain, TNFRSF1A-associated via death domain and reporter-interacting protein kinase 1. It thereby promotes the epithelial lifespan and improves attachment of EPEC to the epithelial surface (Pearson et al., 2013). Another EPEC T3SS effector, the cell cycle inhibitor factor, delays cell death by inducing G2 and G1 cell cycle arrest (Samba-Louaka et al., 2009, Morikawa et al., 2010). Similarly, the Shigella effector molecule IpaB causes cell cycle arrest during the G2/M phase and decreases the intestinal epithelial cell renewal process (Iwai et al., 2007). Shigella additionally produces OspE that enhances epithelial cell adherence to the basement membrane by interacting with integrin-linked kinase (Kim et al., 2010). Conclusive remarks During the last years, epithelial innate immune recognition, signal transduction and antimicrobial responses have attracted increasing attention. Exciting discoveries have been made, and it has become clear that epithelial cells play a non-redundant, dynamic and critical role and intimately interact with other cells of the mucosal tissue to facilitate a coordinated response to microbial challenge (Figure 1). With their ability to sense the presence of commensal and pathogenic microorganisms and their manifold ways to maintain and reinforce the epithelial barrier, they significantly contribute to mucosal homeostasis and anti-infectious host responses. Figure 1Open in figure viewerPowerPoint Epithelial barrier mechanisms. A, B. The epithelial surface of the small (A) and large (B) intestine. The mucus layer and the presence of Paneth cell-derived and enterocyte-derived antimicrobial peptides as well as SIgA generate a physico-chemical barrier overlaying the epithelium. Continuous epithelial proliferation, migration and exfoliation facilitate cell renewal and shedding of infected cells. Delivery of luminal antigen (red arrows) is facilitated by directed transport through M cells and goblet cells as well as phagocytes and dendritic cells extending protrusions into the gut lumen. C. PRRs sense the presence of microorganisms at the epithelial surface as well as within endosomal compartments and the cell cytosol, stimulating a transcriptional cell response. In addition, they mediate IL-1β/IL-18 cleavage and pyroptosis. 'Eat-me' signals such as polyubiquitination or galectin 8 binding induce autophagosomal degradation of cytosolic bacteria. Conflict of interest The authors declare that there is no conflict of interest. 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