Critical Review Keeping Bugs in Check: The Mucus Layer as a Critical Component in Maintaining Intestinal Homeostasis

Martin Faderl Mario Noti Nadia Corazza Christoph Mueller*

Division of Experimental Pathology, Institute of Pathology, University of Bern, Bern, Switzerland

Abstract In the mammalian gastrointestinal tract the close vicinity of abundant immune effector cells and trillions of commensal microbes requires sophisticated barrier and regulatory mechanisms to maintain vital host-microbial interactions and tissue homeostasis. During co-evolution of the host and its intestinal microbiota a protective multilayered barrier system was established to segregate the luminal microbes from the intestinal mucosa with its potent immune effector cells, limit bacterial translocation into host tissues to prevent tissue damage, while ensuring the vital functions of the intestinal mucosa and the luminal gut microbiota. In the present review we will focus on the different layers of protection in the intestinal tract that allow the successful mutualism between the microbiota and

the potent effector cells of the intestinal innate and adaptive immune system. In particular, we will review some of the recent findings on the vital functions of the mucus layer and its site-specific adaptations to the changing quantities and complexities of the microbiota along the (gastro-) intestinal tract. Understanding the regulatory pathways that control the establishment of the mucus layer, but also its degradation during intestinal inflammation may be critical for designing novel strategies aimed at maintaining local tissue homeostasis and supporting remission from relapsing intestinal inflammation in C 2015 IUBMB patients with inflammatory bowel diseases. V Life, 67(4):275–285, 2015

Keywords: disease models; genetic models

The inner body surfaces of the mammalian gastrointestinal tract are colonized at birth with an enormous number and diversity of microbes (1). In humans intestinal bacteria that populate the entire intestinal mucosa in a nonrandom manner exceed the number of body cells by an order of magnitude and represent an aggregate biomass of approximately 1.5 kg (1–3).

Abbreviations: AMP, antimicrobial peptides; DC, dendritic cells; DSS, dextran sodium sulfate; IEC, intestinal epithelial cells, IEL, intraepithelial lymphocytes; SEA, sperm protein, enterokinase and agrin domain; sIgA, secretory IgA C 2015 International Union of Biochemistry and Molecular Biology V Volume 67, Number 4, April 2015, Pages 275–285 *Address correspondence to: Christoph Mueller, Division of Experimental Pathology, Institute of Pathology, University of Bern, CH-3010 Bern, Switzerland. Tel.: 141-31-632-89-04. Fax: 141-31-381-87-64. E-mail: [email protected] Received 10 January 2015; Accepted 4 March 2015 DOI 10.1002/iub.1374 Published online 24 April 2015 in Wiley Online Library (wileyonlinelibrary.com)

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The density of the bacterial colonization increases in the intestinal tract from proximal to distal with the highest concentration of up to 1011 bacteria/gram feces in the colon (2). The intestinal microbiota is dominated by anaerobic bacteria and includes approximately 1,000 different species with a collective genome (“microbiome”), estimated to contain 100 times more genes than the entire human genome (2,4). Luminal bacteria provide relevant sources of energy to gut epithelial cells such as short chain fatty acids. Accordingly, mice maintained under germ-free conditions require 30% more dietary calories to maintain their body weight than their littermates housed under conventional conditions (5). Intestinal bacteria also produce essential vitamins and can actively modulate the local immune system via their metabolites (6). Besides providing vitamins and metabolites to the host, the commensal microbiota competes with potential pathogens for nutrients and niches and thus contributes to prevent colonization by potentially harmful microbes (“colonization resistance”) (7–9). This enormous and complex mass of—mostly—commensal bacteria is only separated by a mucus-covered, single layered epithelium that segregates the microbiota from the local innate

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and adaptive immune system with its numerous and potent effector cells. Hence, under homeostatic conditions, the intestinal mucosa represents an ideal model system to investigate the different strategies that evolved at the interface of two supersystems—the microbiota and the intestinal immune system—to prevent the induction of a potentially deleterious antimicrobial inflammatory response, triggered by luminal bacteria, while maintaining the vital functions of the intestinal mucosa, such as the absorption of nutrients and water and the exchange of electrolytes. Defective regulation of these complex host-microbiota interactions leads to potentially exacerbating inflammatory disorders. This is best illustrated in patients with inflammatory bowel disease (10) where an aberrant immune response to intestinal antigens, mostly of bacterial origin, leads to severe chronic intestinal inflammatory disease in genetically predisposed individuals which may affect the colon (ulcerative colitis) (11) or any part of the gastrointestinal tract (Crohn’s disease) (12). While the cellular and molecular adaptations of the innate and adaptive immune system to the antigen-rich microenvironment in the gastrointestinal tract have been discussed extensively elsewhere (9,13,14), this review will focus on the different layers of protection in the intestinal tract that allow the successful mutualism between the microbiota and the potent effector cells of the intestinal innate and adaptive immune system with a special emphasis on the mucus layer functioning as biochemical barrier to keep the microbiota in check.

A Challenging Task: Maintaining Tissue Homeostasis in the Gastrointestinal Tract The abundance of innate and adaptive immune cells that reside together with trillions of commensal microbes in the mammalian gastrointestinal tract requires sophisticated barrier and regulatory mechanisms able to safeguard hostmicrobial interactions and tissue homeostasis. During coevolution of these two supersystems—the host and the microbiota—a protective multilayered barrier system depicted in Fig. 1 was established that aims to (i) segregate the luminal microbiota from the intestinal mucosa with its potent immune effector cells, (ii) limit bacterial invasion into host tissues, while (iii) being locally hypo-responsive to commensals to prevent tissue damage but to ensure the vital functions of the intestinal mucosa and the gut microbiota. The following sections will address how the host keeps the sheer number of microbes at bay by the formation of physical, immunological and biochemical barriers.

Intestinal Epithelial Cells: Key Regulators of Barrier Function and Local Immune Homeostasis The intestinal epithelium critically contributes to maintain tissue homeostasis by providing a physical barrier between the microbiota and the host. It consists of a single layer of

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columnar epithelial cells with several functionally specialized secretory epithelial cell subsets. Intestinal epithelial cells (IEC) are connected via tight junctions with their neighboring epithelial cells to form a semi-permeable physical barrier (15). In the small intestine the epithelium contains specialized microfold cells (M cells) which efficiently sample luminal antigens and microbes that upon transcytosis through the M cells are presented to dendritic cells (DCs) in the M-cell associated Peyer’s patches and solitary lymphoid follicles (16). IEC with secretory functions include hormone-secreting enteroendocrine cells which are involved in the endocrine regulation of digestion; Paneth cells, predominantly found in the crypts of the small intestine that are characterized by the secretion of anti-microbial proteins (AMP) including a2defensins (in mice: cryptdins), RegIIIb/c, and lysozymes. Goblet cells are the central cell type for the production of mucus that covers the gastrointestinal tract to form a protective mucous gel layer. In addition to mucus synthesis, goblet cells are also capable of secreting AMPs including RELMb that was shown to be induced by bacterial colonization (17). The defense strategies of the intestinal epithelium are directly influenced by the luminal microbiota. Epithelial cells express several receptors for pathogen-associated molecular patterns (PAMPs), such as TLR5 recognizing bacterial flagellin, NOD2 for intracellular recognition of muramyl dipeptide from gram-positive and negative bacteria, subsequently leading to NLRP3 inflammasome activation, and RIG-1, a sensor of viruses. The inflammasomes present in IEC, notably, NLRP3, NLRP6, NLRP12, and NLRC4 exert proinflammatory functions (e.g. induction of chemokine production) but also protective functions (e.g. during mucosal healing) as demonstrated by the phenotypes of the respective gene-deficient mouse lines (reviewed in ref. 18). Upon stimulation, IEC secrete cytokines at their basolateral side including IL-7 and IL-15, which regulate the proliferative capacity of innate lymphoid cells (ILC), intraepithelial lymphocytes (IEL) and lamina propria T cells; as well as IL-25, IL-33, and thymic stromal lymphopoietin (TSLP) known to promote type 2 helper T (TH2) cell-dependent immunity, inflammation, and tissue repair at barrier surfaces through the induction of multiple innate immune cell populations (19). Intraepithelial lymphocytes (IEL) represent a substantial fraction of the cells in the intestinal epithelium (20). These include antigen-experienced T cells expressing either the T cell receptor-cd (TCRcd) or TCRab T cells expressing either a CD8ab heterodimer (MHC class I restricted cytotoxic T cells) or a CD8aa homodimer named innate or unconventional T cells. The precise role of the unconventional IELs is not completely understood. Generally, unconventional IEL are believed to exert immunoregulatory functions (21,22) and thus may assist to preserve the integrity of the mucosal barrier by preventing microbial invasion in part by secretion of AMPs as shown for TCRcd IEL (23). Collectively, cells of the intestinal epithelium do not only provide a physical barrier to segregate luminal bacteria from

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FIG 1

A multilayered barrier system segregates the intestinal microbiota from its host. Goblet cells of the intestinal epithelium secrete mucins that form the mucus layer present in the colon as a dense, bacteria-free inner layer, and a loose outer mucus layer colonized by bacteria. The mucins are cross-linked by goblet cell-derived proteins like trefoil factors (TFF). Antimicrobial proteins (AMPs) like a-defensins or lysozymes are secreted by Paneth cells (located in the small intestine), epithelial cells, and intraepithelial cells (IEL) (e.g. RegIIIc) into the mucus layer to further contribute to the formation of a biochemical barrier and to enhance the segregation of bacteria from the host. Via tight junctions (TJ), epithelial cells form not only a physical barrier, but also actively contribute to the regulation of immunological barrier integrity via the production and secretion of cytokines at their basolateral side. Notably, epithelial-derived IL-7, IL-15, IL-25, IL-33, and TSLP have various key functional properties in the regulation and expansion of innate and adaptive immune cell populations. Further, intestinal epithelial cells (IEC) are able to secrete the TNF family members APRIL and BAFF that support T-cell independent plasma cell differentiation in lymphoid follicles. Secreted IgA is transported as dimerized secretory IgA (sIgA) through the epithelial cells and released where it is integrated in the mucus and prevents the direct access of luminal bacteria to the epithelial surface. Luminal bacteria that breached the epithelial barrier are efficiently taken up and degraded by subepithelial macrophages (Mh) while upon endocytosis of bacteria, lamina propria dendritic cells (DCs) can migrate to the draining lymph node where they may initiate a potent adaptive immune reaction.

the intestinal lamina propria, they also integrate environmental cues, that is, mostly microbiota-derived signals to regulate the production of immunoregulatory mediators and antimicrobial peptides (24).

Immunological Barrier: Immune Responses to Invading Pathogenic Bacteria Despite the presence of different physical and biochemical layers of protection, occasionally luminal microbes can breach the epithelium and get access to the underlying intestinal lamina propria where they are facing the next layer of host defense, that is, the immunological barrier. Hereby, innate immune cells especially mononuclear phagocytes, that is, macrophages, DCs, but also granulocytes play an important role in the front line defense. Subepithelial macrophages form a dense network of highly phagocytic scavenger cells that rapidly phagocytose and degrade luminal bacteria and antigens (25). In contrast to blood monocytes or macrophages in the periphery, resident intestinal macrophages lack several PAMPs or

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DAMPs receptors including TLR4, CD14 or TREM-1 (25). These functional adaptations allow the local up-take and degradation of bacteria without the induction of pro-inflammatory responses that otherwise may harm the integrity of the epithelial barrier. One of the key features of the local humoral immune response is the copious production of antigen-specific IgA in the intestinal lamina propria and associated lymphoid follicles (26). This potent humoral immune response is supported by the local secretion of the epithelial cell-derived TNF family members BAFF and APRIL, which enhance the differentiation of B cells into IgA plasma cells even in the absence of T cells (27). Upon dimerization and addition of a secretory component, IgA is taken up by a polymeric Ig receptor-mediated endocytosis at the basolateral side of IEC and released at the apical side into the gut lumen (28) where secretory IgA (sIgA) is integrated into the mucus layer and exerts critical functions

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in trapping luminal bacteria to prevent unrestricted access of microbes to the epithelial surface.

Biochemical Barrier: Minimizing Bacteria-Epithelial Cell Contact in the Intestine The intestinal epithelium is coated by mucus, that is, a viscous gel layer that serves as lubricant for the movement of luminal contents, protects the non-stratified intestinal epithelium from physical friction due to peristalsis, chemical digestion or bacterial infection (29). Goblet cells crucially contribute to the formation and maintenance of the intestinal mucus layer. They constantly secrete mucin glycoproteins to form a mucus layer that prevents the physical contact of bacteria to the intestinal epithelium or even their translocation into the underlying lamina propria. In addition to providing a physical barrier, the mucus layer acts as a biochemical wall by trapping digestive enzymes, AMPs and sIgA. The formation and function of this physico-biochemical mucus layer will be the focus of the following chapter.

Mucus Biosynthesis, Secretion, and Gel Formation The mucus layer is an integral component of the gastrointestinal tract serving as biochemical defense barrier, lubricant of the epithelium and transport system between the luminal contents and the epithelial lining. The viscoelastic, polymer-like structure of the mucus is derived from large highly glycosylated glycoproteins with protein backbone structures rich in serine and threonine linked to a wide variety of O-linked oligosaccharide side chains. Up to 20 different mucin genes have been identified so far in humans and mice, which are expressed in a tissue and cell type-specific manner (for detailed review see refs. 29 and 30) and are broadly classified into two types, secretory and transmembrane mucins. Among the gel-forming secretory mucins (MUC2, MUC5AC, MUC5B and MUC6), MUC2 is the major secretory mucin synthesized and secreted by goblet cells of the small- and large intestine and will be discussed in more detail below. Transmembrane mucins derived from goblet cells or absorptive cells include MUC1, MUC3, MUC4, MUC13, and MUC17 (29,30). The following section will discuss (i) the distinct structural features and biosynthetic pathways of secretory and membrane-bound mucins and (ii) the composition and functional roles of the intestinal mucus layer.

Synthesis of Secreted and Membrane-associated Mucins Goblet cells distributed throughout the gastrointestinal tract are key for the synthesis and secretion of mucins. Their cell morphology is mainly shaped by voluminous theca containing mucin granules, located underneath the apical membrane, which are secreted into the lumen by either constitutive secretion or compound exocytosis in response to a variety of bioactive factors (31). The syntheses of the secretory, and trans-

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FIG 2

Biosynthesis and secretion of mucins in the intestine. Mucins can be classified into two major types: transmembrane and secretory mucins. Transmembrane mucins are the major components of the epithelial glycocalyx that covers the brush border of the IEC and serve as anchor for the network of the secretory mucins. Upon translation, transmembrane mucins undergo autocatalytic cleavage in the ER at the SEA domain positioned close to the transmembrane domain at the extracellular part of the mucin. Prior to transport to the Golgi apparatus, the mucin monomer is N-glycosylated close to the autocatalytic cleavage site of the extracellular domain. Upon Oglycosylation in the Golgi network, membrane-bound mucins are transported to the cell surface for incorporation into the cell membrane. Secretory mucins are formed as C-terminally linked homodimers in the ER, to become O-glycosylated in the Golgi apparatus. Upon N-terminal oligomerization they are packed into the compact mucin granules stored in the goblet cell thecae. Upon their exocytosis, secreted mucins form a complex net of mucin multimers serving as diffusion barrier against bacteria and a scaffold for antimicrobial peptides. In the colon, the mucus layer is divided into a dense inner mucus layer devoid of bacteria and anchored to the colonic epithelium by cell-surface mucins, and an outer loose mucus layer, which is a preferred habitat for commensal bacteria.

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membrane mucins are graphically depicted in Fig. 2. Upon their translation in the goblet cells’ endoplasmatic reticulum (ER), secretory mucins like MUC2 form homodimers through covalent dimerization at the C-terminal cysteine knot domain (CK) via disulfide bonds. Correctly assembled MUC2 dimers then migrate to the Golgi apparatus for O-glycosylation of the two centrally localized PTS domains, (i.e. domains rich in proline, threonine, and serine). Starting by adding N-acetyl galactosamine (GalNAc) sugars to these residues, complex oligosaccharides are assembled, named mucin domains that form rod like structures. The terminally glycosylated MUC2 dimer increases to a size of about 5 kD. In the trans-Golgi network MUC2 is then sorted to pass to the regulated secretory pathway. To eventually form a mucin network at the epithelial surface, the mucin dimers are N-terminally trimerized through the formation of disulfide bonds at low pH and high calcium concentration. These polymeric macromolecules are then packed into secretory goblet cell vesicles, stored in the theca in a concentrated and highly organized system which allows the massive expansion in volume after exocytosis (31,32). Upon mucin granule exocytosis the densely packed mucus granules are hydrated and expand massively (up to 3,000-fold) to form stacked planar networks of the colonic inner mucus layer (33) with a total thickness of about 50 lm in the mouse, and approximately 200 to 300 lm in the human colon (34). The transmembrane members of the mucin family (e.g. MUC1, MUC3, MUC4, MUC12, MUC13, and MUC17) do not form gel-like structures but rather serve as anchor for the network of the secretory mucins (mostly Muc2). Transmembrane mucins are the major components of the epithelial glycocalyx that covers the brush border of IEC. Characterized by a single transmembrane domain, the cytoplasmic tail of transmembrane mucins interacts with the cytoskeleton while the extracellular part is highly glycosylated at PTS domains and is thought to form a filamentous structure (35,36). Transmembrane mucins are single proteins that upon translation undergo autocatalytic cleavage in the ER at their SEA domain positioned close to the transmembrane domain at the extracellular part of the mucin. The extracellular mucin domain still binds to the transmembrane fragment by strong protein interactions. This allows for subsequent release of the glycosylated extracellular domain from the surface, most likely to prevent damage of the apical membrane by mechanical stress (31,32). Prior to transport to the Golgi apparatus, the mucin monomer is N-glycosylated close to the autocatalytic cleavage site of the extracellular domain. Upon O-glycosylation in the Golgi network, transmembrane mucins are transported to the cell surface for incorporation into the cell membrane. These membrane-bound mucins can be shed from the cell surface in two ways: either by the separation of two subunits at the SEA domain, or by alternative splicing (35–37). Beyond their role in providing anchor function to secreted mucins to form the mucus layer, transmembrane mucins such as murine Muc3 (38) and human MUC17 (39) inhibit epithelial cell apoptosis and enhance epithelial cell aggregation and migration as

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demonstrated by the endogenous inhibition of MUC17 (39) and Muc3 expression (38) in epithelial cell lines in vitro. Administration of a recombinant MUC17 fragment consisting of its extracellular portion further accelerated mucosal healing in acetic acid-, and DSS-induced experimental colitis in mice (39). These results thus indicate a relevant contribution of these transmembrane mucins in epithelial restitution and mucosal healing. Obviously, these transmembrane mucinmediated effects described in experimental settings in vitro and in vivo need to be corroborated also in patients to directly assess their therapeutic potential for attenuating epithelial erosion and ulceration during chronic intestinal inflammation.

Composition and Functional Roles of the Intestinal Mucus Layer The organization of the protective mucus layer varies markedly along the mammalian digestive tract. The evolution of different structural characteristics of the mucus layer in small and large intestine may be attributed to changing rates of microbial colonization along the gastrointestinal tract. This is reflected by the distinct spatial variation and frequency of mucin secreting goblet cells with high frequencies at sites of dense microbial colonization, that is, in the distal colon. To maintain tissue homeostasis, the gastrointestinal tract established a sophisticated tissue-specific mucus layer system to assure the different needs of tissues by allowing digestion and absorption of ingested food while protecting from uncontrolled microbial colonization. While the small intestinal epithelium is only covered with a single, nonanchored loose layer of mucus that enables absorption of nutrients, the colon handles the dense microbial colonization with a stratified two-layered mucus system. While in the colon the inner dense mucus layer, anchored to the IEC glycocalyx remains sterile under homeostatic conditions, the outer loose colonic mucus layer as well as the small intestinal mucus layer provide a preferred habitat for different mucin-derived glycan foraging microbes (40). Despite the formal distinction into an inner and outer mucus layer, these layers should not be regarded as two clearly segregated, distinct mucus compartments, but likely represent two dynamic compartments where the outer layer is degraded by the luminal microbiota and may also become transported distally together with the intestinal content, while it is constantly replenished by mucus from the inner layer. The netlike, viscous, gel-forming structure of the mucus layer provides an important host defense against endogenous and exogenous irritants, but also against microbial invasion (29). Importantly, the dense gel forming structure of the mucus layer acts as a trap for mucus stabilizing proteins, AMPs and sIgA antibodies. In addition to secretion of mucins, goblet cells produce a number of proteins that contribute to crosslinking and stabilization of the MUC2 mucin network including trefoil factors (TFF), Fcgamma binding proteins (FCG-BP), the Anterior Gradient 2 (AGR2), Chloride Channel Accessory 1 (CLCA1), and the Zymogen Granule Protein 16 (ZG16). The trefoil factors 1, 2, and 3

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FIG 3

Dynamics of the colonic mucus layer and its effects on the segregation of luminal bacteria from the intestinal mucosa during homeostasis, inflammation and resolution. (A) Under homeostatic conditions the stratified colonic mucus layer with its dense inner layer (stained with anti Muc-2, green) separates the luminal bacteria from the single layer of epithelial cells (containing numerous mucus-filled goblet cell) (blue: DAPI stained cell nuclei). (B) During inflammatory conditions the mucus layer is disrupted and luminal bacteria can get access to the epithelium and breach the epithelial barrier. The ensuing recruitment of inflammatory cells (e.g. neutrophils and inflammatory macrophages) to the lamina propria and the secretion of proinflammatory mediators, such as TNF-a may further damage the epithelial barrier. During active inflammation the number of mucus filled goblet cells is drastically reduced due to the accelerated release of mucin-containing granules. (C) During the resolution of the inflammatory response, the mucus layer is restored and the numbers of mucus-filled goblet cells are increasing although increased cellularity in the lamina propria and increased epithelial cell proliferation may still be present during the early stages of remission from inflammation.

(TFF1-3) are a family of peptides that plays important roles in the protection and repair of epithelial surfaces. Previous studies suggested an important role for goblet cell-derived TFF3 in the enhancement of mucus barrier function (41–43). Similarly, FCG-BP containing cysteine rich vWD domains have been demonstrated to stabilize the MUC2 network by covalently crosslinking several MUC2 mucins (31). The Anterior Gradient 2 (AGR2) protein is highly abundant in the intestinal mucus layer and absence of Agr2 in mice has been associated with a poorly formed inner colonic mucus layer and an increased susceptibility to develop colitis (44). Another component of the MUC2 network is the highly abundant Chloride Channel Accessory 1 (CLCA1) protein that in addition to controlling Ca21 secretion may have mucin inducing properties. However, its exact modes of action are still under investigation (45,46). While the above-mentioned proteins contribute to the stabilization of the mucus layer, Paneth cells, enterocytes and IEL can secrete AMPs such as a-defensins and cathelicidins in response to microbial stimulation. This results in the formation of a gradi-

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ent of anti-microbial peptides trapped in the mucus matrix that is highest in the dense inner mucus layer, free of microbes. AMPs are small cysteine-rich cationic proteins that can limit bacterial growth by disruption of their anionic cell membranes. Interestingly, intestinal microbes are also confronted with an oxygen gradient as oxygen is continuously released from the blood towards the mucus layer. This is of particular interest as aerobic conditions increase the killing efficiency of several AMPs (47). Importantly, alterations in AMP expression and or biologic activity, as seen in matrilysindeficient mice with a combined defect of all cryptdins resulted in increased survival and virulence of orally administered bacteria (48). Further, loss of the antimicrobial lectin RegIIIc has been associated with increased bacterial colonization of the intestinal epithelial surface and enhanced activation of intestinal adaptive immune responses by the microbiota (49). In addition to AMPs, sIgA that is transported across the intestinal epithelium and trapped in the outer mucus may provide an additional line of defense. By binding luminal bacteria in an

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antigen-specific manner thus limiting uncontrolled migration of potential harmful microbes to the intestinal epithelium while maintaining a mutual relationship with members of the commensal flora (50). Studies in mice deficient in activationinduced cytidine deaminase (AID) that are unable to class switch from IgM to IgA suggested that defective class switching is associated with profound increases in the number of anaerobic bacteria in the small intestine. While IgA-deficient mice have no phenotype under normal laboratory housing conditions, secretory IgM may have compensatory activities in IgAdeficient mice. These observations in mice can be compared to human IgA deficiency in which a maturation defect in B cells is commonly observed. IgA-deficient patients are generally asymptomatic but do exhibit a tendency to develop airway infections and gastrointestinal disorders.

The Mucus Layer In Homeostasis, Inflammation, and Remission The mucus layer provides a physico-biochemical barrier that function as protective spacer fronting the epithelium. The dense network of glycoproteins provides a molecular scaffold for IEC- and immune cell-derived factors that exert stabilizing or host-protective functions by the formation of an antimicrobial gradient limiting bacterial penetration through the mucus layer. In this last part of the review we will focus on the dynamic nature of the intestinal mucus layer during homeostasis, intestinal inflammation and tissue damage and resolution of disease (Fig. 3). In adaptation to the specific main functions of the distinct segments of the intestinal tract the mucus layer shows quantitative and qualitative differences in the small, and large intestine. The small intestinal epithelium is covered by a loose, non-epithelial anchored mucus layer that is continuously transported together with luminal contents in a distal direction via peristalsis. In the densely colonized colonic lumen, a loose outer mucus layer colonized by luminal bacteria is stratified by an adjacent dense inner mucus layer (34,51). This inner colonic mucus layer is generally impermeable for luminal bacteria. For an appropriate segregation of colonic microbiota and epithelium, the inner mucus layer needs to be appropriately formed since mice with a defect in mucus layer formation are predisposed to spontaneous intestinal inflammation (51). As described in Chapter 2, MUC2 represents the core structure of the inner mucus layer. This highly glycosylated protein, which polymerizes to form large net like structures becomes highly hydrated upon release from mucin-secreting cells resulting in the viscous consistency of the mucus. The inner mucus layer is firmly anchored to the colonic epithelial cells, but is subject to a high turnover rate: under homeostatic conditions the inner mucus layer is rebuilt within 1 to 2 h by a continuous release of sheets of MUC2 from goblet cell-derived, mucin-containing granules.

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The precise molecular mechanisms responsible for the conversion of the inner to the loose outer mucus layer are not completely defined yet. Since germ-free mice have a stratified colonic mucus bilayer with a dense inner and a loose outer layer, microbial colonization may have a limited effect on the formation of a stratified colonic mucus layer under homeostatic conditions, although the microbial colonization is likely to greatly affect the biochemical composition of the mucus (e.g. higher sIgA, AMPs concentrations in colonized mice), and hence, also the anti-microbial barrier functions of the mucus layer. Host-derived proteases are likely candidates involved in dynamically converting the inner to the outer mucus layer which is accessible for colonization by luminal microbes (34). Recently, the metalloproteinase meprin-b has been identified as a host-derived protease that can contribute to the cleavage of mucins, in particular of Muc2 in the small intestine, leading to the detachment of mucus and its release into the small intestinal lumen. Together with the released mucus, trapped bacteria are transported with the fecal stream out of the intestinal tract. Intriguingly, the release of membrane associated meprin-b which is essential for the cleavage of Muc2 appears to be dependent on the presence of commensal bacteria (52). The enzymatic cleavage of the outer mucus layer into smaller fragments leads to a larger pore size, making this layer more accessible to luminal bacteria that can further degrade the mucus-derived glycans by bacteria-derived exoglycosidases. These glycans are a major source of energy for microbes and commensal species-specific glycan-degrading properties may represent a selective advantage for commensals over pathogens, thus contributing to colonization resistance (40). Whether luminal bacteria-derived proteases also contribute to mucus degradation has not been comprehensively addressed so far. Porphyromonas gingivalis, a bacterium that can colonize the colon also contains proteases able to cleave MUC2, yet, only once the O-glycans are enzymatically removed from this heavily glycosylated protein (53). Intriguingly, in intestinal biopsies obtained from patients with inflammatory bowel diseases mucin glycan-degrading bacterial species such as Ruminococcus gnavus and R. torques are overrepresented when compared to biopsies obtained from non-IBD patients (54). These mucolytic bacteria efficiently deglycosylate MUC2 in vitro, and may thus make mucins more susceptible to a rapid proteolytic degradation by host derived proteases (53,54). Despite these findings a causative role for mucus-degrading bacteria in the pathogenesis of IBD remains to be demonstrated. The complexity of the factors that regulate the mucus layer in the colon is further illustrated by a recent analysis of two wild type C57BL/6 mouse colonies maintained in the same animal facility. The two colonies were found to differ in their intestinal microbial composition, and most intriguingly, also in the quality of their inner mucus layer: while in one mouse colony the inner colonic mucus layer was sterile, the inner colonic mucus layer was colonized by commensal bacteria in the other colony (55). These distinct

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phenotypes of the inner colonic mucus layer in inbred wildtype mice harboring distinct commensal communities were transmissible to germ-free mice upon fecal transplantation suggesting that some bacterial communities may have the ability to either shape host derived factors that influence the formation of the mucus layer or have direct mucolytic properties that facilitate penetration of the mucus layer. Hence the current data suggest that mucus degradation of the stratified colonic mucus layer may be regulated in a bidirectional manner, that is, from the outer layer by the commensal bacteria that forage (mostly) host O-glycans, thereby enlarging the pore size of the outer layer and allowing further bacterial colonization, while proteases released from the colonic mucosa may lead to a rapid proteolytic degradation and detachment of the mucins at the inner and outer layer. This process is enhanced and accelerated during colonic inflammation, possibly triggered by bacteria that may enhance the production and release of these proteases from the colonic mucosa. This may contribute, as described below, to the observed rapid loss of the colonic mucus layer during local inflammation.

Dynamics of Mucus Layer Formation During Inflammatory Conditions The importance of an intact mucus layer in providing protection from microbial invasion and associated intestinal inflammation and tissue damage has been best illustrated in mice lacking key components of the mucus layer. Muc2-deficient mice lack an intestinal mucus layer and are predisposed to parasitic infections and, when maintained under conventional conditions, develop spontaneous colitis with bloody diarrhea, weight loss, and rectal prolapses (56–59). A recent comprehensive study by Johansson et al. demonstrated that in both, animal models of intestinal inflammation and patients with ulcerative colitis, development of intestinal inflammation strictly correlated with degradation of the mucus layer and the presence of bacteria in direct contact with the colonic epithelium, particularly, in the crypts, whereas in unaffected wild type mice the inner colonic mucus layer remained sterile and provided an efficient spatial segregation of the luminal microbes from the epithelium (51). Intriguingly, some of the key features of colitis induction in Muc2-deficient mice lacking a functional mucus layer closely reflect clinical and cellular features of active ulcerative colitis in patients (51,56,60). A hallmark of intestinal inflammation is the drastic and rapid reduction in the number of mucus-filled goblet cells that can be visualized by immunostaining for Muc2 (Fig. 3B) or by Periodic Acid–Schiff (PAS) staining (51).This observation was first attributed to a complete loss of goblet cells as a consequence of the longstanding inflammation and the presence of pro-inflammatory cytokines such as IFNc and particularly TNF-a, known to induce apoptosis and detachment of intestinal epithelial cells (61). However, recent findings suggest that the apparent absence of goblet cells during intestinal inflammation is mainly the result of an exhaustion and a dysbalance between mucus production and exocytosis of the mucin-

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containing granules rather than a selective loss of goblet cells (62). This notion is in agreement with the observed rapid reappearance of mucus-filled goblet cells early after induction of remission from colitis. In accordance to this idea the absence of mucus-filled granules in goblet cells is likely not a consequence of their damage. This concept is further supported by the fact that in response to intestinal insult the release of mucin granules stored in goblet cell thecae is accelerated at the top of the villi as an attempt to overcome pathogenic invasion and to restore its barrier function, a process which has previously been described as compound exocytosis (63,64). A defect in Muc2 formation and/or exocytosis by goblet cells has also been associated with increased susceptibility to intestinal inflammation. For instance autophagy, a catabolic cellular process degrading cellular components to maintain energy homeostasis (65) has been shown to play an essential role for goblet cell exocytosis (66). In the absence of a functional mucus layer, the local gradient of AMPs, but also of sIgA trapping luminal bacteria that get access to the mucus layer cannot be maintained and these potent antimicrobial defense systems are transported with the fecal stream out of the intestine. The severe phenotypes seen in patients with hypomorphic mutations for genes involved directly, or indirectly in regulating goblet cell functions and mucin-production, but also in the production of AMPs by Paneth cells (e.g. autophagy related genes ATG16L1 (67), ATG5, ATG7 (66)), the intracellular sensor of bacterial muramyl dipeptides NOD2, and the XBP1 gene involved in ER stress (68) highlight the importance of this dynamic defense system for controlling the mutualistic interactions between the microbiota and the immune system. As a direct consequence of an impaired mucus layer luminal bacteria can gain direct access to the epithelium where they activate extracellular and intracellular innate immune sensors (e.g. NLRP6, TLRs, NOD) to trigger the recruitment and activation of proinflammatory leukocyte subsets, notably neutrophils, inflammatory macrophages, but also pro-inflammatory CD4 T-cell subsets and may even translocate to the intestinal mucosa leading to local and/or systemic inflammatory immune responses (Fig. 3B). While our understanding of the events leading to inflammation-induced mucus degradation is gradually emerging, insight on the molecular and cellular events that lead to a resolution of a chronic intestinal inflammation and how this will affect the dynamics of mucus layer formation are poorly understood. In an initial attempt to follow the dynamics of mucus layer formation in response to mucosal healing, we recently established an animal model system in which remission from active intestinal inflammation and associated tissue damage can be induced in a time-specific manner (unpublished data). Interestingly, upon experimental induction of remission from colitis, the colonic mucus layer was reestablished in mice with established disease within 3 to 5 days resulting in the separation of luminal microbes and the gut epithelium (Fig. 3C). Thus, the regeneration of the colonic

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mucus layer precedes the complete histopathological recovery by several days. The precise mechanisms that are involved in the rapid re-establishment of the colonic mucus structure are not known yet, but several mechanisms may be involved: glycosylated Muc2, which may gain access during severe intestinal inflammation to the lamina propria can bind to the cell surface of DCs and thus reduce the production of inflammatory cytokines by DCs exposed to bacteria. Moreover, Muc2 can induce an increased expression of IL-10 and other immunosuppressive factors by DCs (69). Other cytokines that reportedly enhance mucus synthesis and/or exocytosis include IL-4, IL-13 or TNF-a that act via NF-jB signaling to enhance mucin synthesis. Furthermore, short-chain fatty acids produced by the luminal microbiota are a substantial source of energy for gut epithelial cells, but also induce the expression of Muc2 gene expression in cell lines (70). In conclusion, in the context of intestinal protection the mucus layer with its capacity to form highly dense stratified glycoprotein networks is a growing field of research. Until recently, the dynamic nature of the intestinal mucus layer and its complex regulatory interactions with both the intestinal microbiota and the local immune system were poorly described. However, in the past decade important findings on the different structure of the mucus layer in distinct intestinal compartments, its dynamic nature during onset of inflammation, but also the pleiotropic functions of mucins, including their immunomodulatory effects on antigen-presenting cells in the lamina propria, emerged that led to new concepts on how this biochemical barrier is positioned not only as a stand-alone component, but closely interacts with the physical and immunological barrier of the intestinal mucosa. While the defensive nature of the mucus layer lies in part in its capacity to entrap microbes, bacterial adhesion to specific mucin epitopes may also facilitates mucus colonization by commensal bacteria. In these niches, microbes are protected from rapid expulsion via the hydrokinetic properties of the mucin glycoproteins and therefore contribute to health by providing colonization resistance against potential pathogenic organisms. Growth advantages of commensal mucus residing bacteria over harmful microorganisms may be overcome by pathogens with high mucolytic properties resulting in the degradation of the mucus barrier and associated unrestricted access of bacteria to the intestinal epithelium. Alternatively, while pathogen-induced acute immune responses may have a direct stimulatory effect on goblet cell-regulated mucus synthesis, chronic inflammatory conditions could result in exhaustion of goblet cell-derived mucogenesis. Thus, one future challenging task will be to define the taxonomy as well as the temporal and spatial distribution of mucus residing bacteria, their role in the dynamic regulation of the intestinal mucus layer and their interaction with the local immune system. Such studies will be key to gain deeper insight into the mutualistic interactions between the intestinal microbiota and the complex regulatory network that controls the synthesis, release and appropriate formation of the components of the

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mucus layer in health and disease. Understanding these regulatory mechanisms controlling mucus synthesis could provide broad biomedical applications in the treatment of gastrointestinal disorders associated with a compromised mucus barrier system.

Acknowledgements The authors apologize to our colleagues whose work could not be cited in this review due to space restrictions. Experimental work of the authors is supported by the Swiss National Science Foundation.

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Keeping bugs in check: The mucus layer as a critical component in maintaining intestinal homeostasis.

In the mammalian gastrointestinal tract the close vicinity of abundant immune effector cells and trillions of commensal microbes requires sophisticate...
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