ABSTRACT Embedded in the colonic mucus are cathelicidins, small cationic peptides secreted by colonic epithelial cells. Humans and mice have one cathelicidin-related antimicrobial peptide (CRAMP) each, LL-37/hCAP-18 and Cramp, respectively, with related structure and functions. Altered production of MUC2 mucin and antimicrobial peptides is characteristic of intestinal amebiasis. The interactions between MUC2 mucin and cathelicidins in conferring innate immunity against Entamoeba histolytica are not well characterized. In this study, we quantified whether MUC2 expression and release could regulate the expression and secretion of cathelicidin LL-37 in colonic epithelial cells and in the colon. The synthesis of LL-37 was enhanced with butyrate (a product of bacterial fermentation) and interleukin-1β (IL-1β) (a proinflammatory cytokine in colitis) in the presence of exogenously added purified MUC2. The LL-37 responses to butyrate and IL-1β were higher in high-MUC2-producing cells than in lentivirus short hairpin RNA (shRNA) MUC2-silenced cells. Activation of cyclic adenylyl cyclase (AMP) and mitogen-activated protein kinase (MAPK) signaling pathways was necessary for the simultaneous expression of MUC2 and cathelicidins. In Muc2 mucin-deficient ( Muc2 −/− ) mice, murine cathelicidin ( Cramp ) was significantly reduced compared to that in Muc2 +/− and Muc2 +/+ littermates. E. histolytica -induced acute inflammation in colonic loops stimulated high levels of cathelicidin in Muc2 +/+ but not in Muc2 −/− littermates. In dextran sodium sulfate (DSS)-induced colitis in Muc2 +/+ mice, which depletes the mucus barrier and goblet cell mucin, Cramp expression was significantly enhanced during restitution. These studies demonstrate regulatory mechanisms between MUC2 and cathelicidins in the colonic mucosa where an intact mucus barrier is essential for expression and secretion of cathelicidins in response to E. histolytica- and DSS-induced colitis.

MUC2 mucin is the major glycoprotein in colonic mucus that separates intestinal microbiota from underlying host cells and serves as a food source for some eubacteria. MUC2 deficiency results in impaired epithelial barrier function, imbalance in gut microbiota, and spontaneous colitis. Probiotics have been shown to have a protective effect against colitis. In this study we used Muc2 mucin-deficient (Muc2-/-) and Muc2+/+ littermates to test whether the probiotic mixture VSL#3 requires an intact mucin barrier to exert its beneficial effect. VSL#3 alone reduced basal colonic proinflammatory cytokine levels and improved epithelial barrier function in Muc2-/- animals. Similarly, in dextran sulfate sodium-induced colitis, VSL#3 dampened the proinflammatory chemokines KC, monocyte chemoattractant protein-1, and macrophage inflammatory protein-2 and upregulated the tissue regeneration growth factors transforming growth factor-β, fibroblast growth factor-1, and vascular endothelial growth factor-A, which accelerated resolution of colitis symptoms in Muc2-/- animals. Importantly, improved colonic health in VSL#3-treated animals was associated with attenuated reactive oxygen species production by peritoneal macrophages, restoration of antimicrobial peptide gene expression in the small intestine, and increased abundance of bacterial commensals in the gut. The beneficial effects of VSL#3 in Muc2-/- animals were mediated by acetate, an important short-chain fatty acid produced by gut bacteria. These studies provide evidence for the first time that VSL#3 can enhance epithelial barrier function by dampening the proinflammatory cytokine and chemokine response, accelerating restitution, and altering commensal microbiota in the absence of a functional mucus barrier. NEW & NOTEWORTHY:It is unclear whether probiotics require an intact mucin barrier to first colonize and/or exert their protective functions. In this study we used mucin-deficient (Muc2-/-) mice to interrogate if the multispecies probiotic mixture VSL#3 could enhance epithelial barrier function. In the absence of a mucus bilayer, VSL#3 dampened proinflammatory and chemokine production, accelerated restitution, and markedly improved gut permeability mediated by the short-chain fatty acid acetate in the colon.

Kumar M, Kissoon-Singh V, Coria AL, Moreau F, Chadee K. Probiotic mixture VSL#3 reduces colonic inflammation and improves intestinal barrier function in Muc2 mucin-deficient mice. Am J Physiol Gastrointest Liver Physiol 312: G34–G45, 2017. First published November 17, 2016; doi:10.1152/ajpgi.00298.2016.—MUC2 mucin is the major glycoprotein in colonic mucus that separates intestinal microbiota from underlying host cells and serves as a food source for some eubacteria. MUC2 deficiency results in impaired epithelial barrier function, imbalance in gut microbiota, and spontaneous colitis. Probiotics have been shown to have a protective effect against colitis. In this study we used Muc2 mucin-deficient (Muc2 / ) and Muc2 / littermates to test whether the probiotic mixture VSL#3 requires an intact mucin barrier to exert its beneficial effect. VSL#3 alone reduced basal colonic proinflammatory cytokine levels and improved epithelial barrier function in Muc2 / animals. Similarly, in dextran sulfate sodium-induced colitis, VSL#3 dampened the proinflammatory chemokines KC, monocyte chemoattractant protein-1, and macrophage inflammatory protein-2 and upregulated the tissue regeneration growth factors transforming growth factor, fibroblast growth factor-1, and vascular endothelial growth factor-A, which accelerated resolution of colitis symptoms in Muc2 / animals. Importantly, improved colonic health in VSL#3-treated animals was associated with attenuated reactive oxygen species production by peritoneal macrophages, restoration of antimicrobial peptide gene expression in the small intestine, and increased abundance of bacterial commensals in the gut. The beneficial effects of VSL#3 in Muc2 / animals were mediated by acetate, an important short-chain fatty acid produced by gut bacteria. These studies provide evidence for the first time that VSL#3 can enhance epithelial barrier function by dampening the proinflammatory cytokine and chemokine response, accelerating restitution, and altering commensal microbiota in the absence of a functional mucus barrier.

The MUC2 mucus layer is the first line of host defense in the gut. How MUC2 interacts with antimicrobial peptides in host defense is not well understood. In this study we investigated the interactions between MUC2 and β‐defensin. Of the colonic cells tested, β‐ defensin 2 expressions was the highest in MUC2 producing human LS 174T and the lowest in Caco‐2 cells. Similarly, expression of murine β‐defensin 4 (orthologous to human β‐defensin 2) was low in the colon of Muc2−/−mice as compared to Wt. Purified MUC2 attenuated the stimulation of β‐defensin as IL‐1β and sodium butyrate induced the expression of β‐defensin in Caco‐2, but not LS 174T cells. The antimicrobial activity of β‐defensin 2 differed among enteric bacteria; Escherichia coli were more susceptible than Bacteroides vulgatus and Clostridium difficile in killing assays. Our studies revealed that MUC2 served as a food source for enteric bacteria, which explains why Muc2−/− harbored less commensal Bacteroides and Firmicutes spp. Mechanistically, MUC2 N‐ and O‐linked oligosaccharides bound bacteria and protected them against β‐defensin 2 antimicrobial activities. MUC2 protected the microbiota by serving as a food source and by inhibiting the antimicrobial activity of β‐defensin. When the mucus barrier is compromised, β‐defensin kills susceptible bacteria whereas β‐defensin resistant bacteria may colonize and cause disease.

Human mucin-2 (MUC-2) is the first line of innate host defense in preventing pathogen-induced epithelial injury. Entamoeba histolytica (Eh) colonizes the mucus layer by binding of the parasite's surface galactose lectin to galactose and N-acetyl-D-galactosamine residues on colonic MUC-2, preventing parasite contact-dependent cytolysis of epithelial cells. We quantified early innate responses to Eh in wild-type and MUC-2-deficient mice (Muc2(-/-)) using closed colonic loops. Eh infection in wildtype but not Muc2(-/-) mice induced a time-dependent increase in H-3-labeled mucin and nonmucin glycoprotein secretions. Immunohistochemical staining revealed intense MUC-2 secretion, which formed a thick, protective mucus plug overlying the surface epithelium, entrapping Eh. In Muc2(-/-) mice, Eh induced a pronounced time-dependent secretory exudate with increased gross pathology scores and serum albumin leakage. Colonic pathology, secretory responses, and increased proinflammatory cytokine secretions of TNF-alpha, IFN-gamma, and IL-13 correlated with altered expression of the tightjunction proteins claudin-2, occludin, and Z0-1. We identified the putative Eh virulence factor that elicits the proinflammatory responses and alters tight junction permeability as Eh cysteine protease A5 (EhCP-A5). The present findings demonstrate that colonic mucins confer both luminal and epithelial barrier functions and that, in the absence of MUC-2, mice are more susceptible to Eh-induced secretory and proinflammatory responses mediated by EhCP-A5. (Am 3 Pathol 2013, 182: 852-865; http:// dx.doi.org/10.1016/jajpath.2012.11.035)

The intestinal MUC2 mucus layer is the first line of innate host defense against pathogens. Mice deficient in Muc2 spontaneously develop colitis and are more susceptible to DSS induced injury. The colonic parasite Entamoeba histolytica (Eh) exploit the mucus layer by binding of the parasite Gal/GalNAc lectin to mucin oligosaccharides to facilitate colonization leading to infectious disease in humans. In this study we investigated the innate host defenses in Muc2−/− mice challenged with virulent Eh in a colonic loop model of infection. Eh induced a time dependent robust secretory response in Muc2−/− mice associated with increased pathology and leakage of serum albumin (p<0.01). The acute inflammation was dominated by elevated IFN‐γ and TNF‐α protein secretion. Surprisingly, Eh caused an increased expression of the pore forming tight junction protein claudin‐2 and a corresponding decreased expression of claudin‐4 and occludin (p<0.05). In the absence of Muc2, Eh elicits a robust acute inflammatory response and expression of TJ proteins leading to increased intestinal permeability emphasizing the role of Muc2 in luminal and epithelial barrier functions.

The intestinal epithelium is layered by a barrier composed of MUC2 mucin produced by goblet cells and β‐defensin 2, an inducible peptide produced by epithelial cells that exhibits antimicrobial and chemotactic activity. Bacteria infections and Ulcerative colitis and Crohn's diseases are associated with less MUC2 and altered defensin expression. In this study, we quantified the interaction between MUC2 mucin and β‐defensins. Human colonic mucin co‐incubated with β‐defensin 2 impaired defensin antimicrobial activities against Escherichia coli. However, addition of mucin 10–30 minutes following bacterial incubation with β‐defensin 2 did not alter antimicrobial activity. Mucin protective function on bacteria was partially abrogated by digesting the mucin terminal sialic acid residues with neuraminidases and oxidizing carbohydrate residues with sodium metaperiodate. Expression of murine β‐defensin 4 mRNA, an orthologue of human β‐defensin 2, was constitutively increased in the colons of mice deficient in MUC2 (Muc2−/−) and in dextran sodium sulfate induced colitis. These studies suggest that MUC2 abrogates the antimicrobial effect of β‐defensin in the intestinal mucosa by binding bacteria and therefore, mucin may shape the normal microflora by limiting β‐defensin antimicrobial activity. In contrast, β‐defensins are up regulated during mucosal damage when the mucus barrier is breached.

MUC2 mucin (murine Muc2) produced by goblet cells forms the mucus layer as the first line of host defence against enteric pathogens. E. histolytica (Eh) binds Gal/GalNAc residues of MUC2 and prevents Eh from damaging the underlying epithelia. As Eh do not invade the mouse colon, we tested the host responses towards Eh in the colon of wild type (Wt) and Muc2−/− mice (no mucus barrier) challenged with 106 virulent Eh trophozoites in colonic loop studies. In Muc2−/− colons, Eh evoked a robust time‐dependent (1–6h) secretory response concomitant with increased leakage of serum albumin, IgA and IgG as compared to Wt controls. In Wt mice, there was a significant increase in the secretion of high molecular weight mucin, non‐mucus glycoproteins and IgA. Moreover, although Eh induced robust pro‐inflammatory and protein expression (TNF‐α, IFN‐γ, COX‐2) in Wt animals, the responses in Muc2−/− mice was dampened. Neither Wt nor Muc2−/− animals showed increased gene expression of the goblet cell mediators TFF‐3 or RELM‐β. These data demonstrate that Muc2 mucin plays a critical role in protection against paracellular secretory responses toward Eh in the gut. Curiously, undefined compensatory mechanisms in Muc2−/− mice abrogated an excessive intestinal pro‐inflammatory response. This work was funded by CIHR.

Cysteine proteases of the protozoan parasite Entamoeba histolytica are key virulence factors involved in overcoming host defences. These proteases are cathepsin-like enzymes with a cathepsin-L like structure, but cathepsin-B substrate specificity. In the host intestine, amoeba cysteine proteases cleave colonic mucins and degrade secretory immunoglobulin (Ig) A and IgG rendering them ineffective. They also act on epithelial tight junctions and degrade the extracellular matrix to promote cell death. They are involved in the destruction of red blood cells and the evasion of neutrophils and macrophages and they activate pro-inflammatory cytokines IL-1 beta and IL-18. In short, amoeba cysteine proteases manipulate and destroy host defences to facilitate nutrient acquisition, parasite colonization and/or invasion. Strategies to inhibit the activity of amoeba cysteine proteases could contribute significantly to host protection against E. histolytica.

Cysteine proteases of the protozoan parasite Entamoeba histolytica are key virulence factors involved in overcoming host defences. These proteases are cathepsin-like enzymes with a cathepsin-L like structure, but cathepsin-B substrate specificity. In the host intestine, amoeba cysteine proteases cleave colonic mucins and degrade secretory immunoglobulin (Ig) A and IgG rendering them ineffective. They also act on epithelial tight junctions and degrade the extracellular matrix to promote Cell death. They are involved in the destruction of red blood cells and the evasion of neutrophils and macrophages and they activate pro-inflammatory cytokines IL- 1β and IL-18. In short, amoeba cysteine proteases manipulate and destroy host defences to facilitate nutrient acquisition, parasite colonization and/or invasion. Strategies to inhibit the activity of amoeba cysteine proteases could contribute significantly to host protection against E. histolytica.

Despite recent advances in our understanding of the pathogenesis of attaching and effacing (A/E) Escherichia coli infections, the mechanisms by which the host defends against these microbes are unclear. The goal of this study was to determine the role of goblet cell-derived Muc2, the major intestinal secretory mucin and primary component of the mucus layer, in host protection against A/E pathogens. To assess the role of Muc2 during A/E bacterial infections, we inoculated Muc2 deficient (Muc2(-/-)) mice with Citrobacter rodentium, a murine A/E pathogen related to diarrheagenic A/E E. coli. Unlike wildtype (WT) mice, infected Muc2(-/-) mice exhibited rapid weight loss and suffered up to 90% mortality. Stool plating demonstrated 10-100 fold greater C. rodentium burdens in Muc2(-/-) vs. WT mice, most of which were found to be loosely adherent to the colonic mucosa. Histology of Muc2(-/-) mice revealed ulceration in the colon amid focal bacterial microcolonies. Metabolic labeling of secreted mucins in the large intestine demonstrated that mucin secretion was markedly increased in WT mice during infection compared to uninfected controls, suggesting that the host uses increased mucin release to flush pathogens from the mucosal surface. Muc2 also impacted host-commensal interactions during infection, as FISH analysis revealed C. rodentium microcolonies contained numerous commensal microbes, which was not observed in WT mice. Orally administered FITC-Dextran and FISH staining showed significantly worsened intestinal barrier disruption in Muc2(-/-) vs. WT mice, with overt pathogen and commensal translocation into the Muc2(-/-) colonic mucosa. Interestingly, commensal depletion enhanced C. rodentium colonization of Muc2(-/-) mice, although colonic pathology was not significantly altered. In conclusion, Muc2 production is critical for host protection during A/E bacterial infections, by limiting overall pathogen and commensal numbers associated with the colonic mucosal surface. Such actions limit tissue damage and translocation of pathogenic and commensal bacteria across the epithelium.

Innate immune recognition of microbe-associated molecular patterns (MAMPs) by multiple families of pattern-recognition molecules (PRMs) is key for the initiation of the first-line host and adaptive immune system to cope with pathogen intrusion.Recent observations have demonstrated that innate immune sensing of peptidoglycan (PGN) by the Nod-like receptor (NLR) family member NLRC1/Nod1 contributes to the priming of pathogen-specific T and B cell immunity as well as to the intestinal lymphoid tissue genesis induced by commensals.To further elucidate the underlying cellular and molecular mechanisms, we studied the first-line host immune response upon peripheral NLRC1/Nod1 stimulation and analyzed if NLRC1/ Nod1-mediated PGN recognition of commensals impacts on the immune cell composition of the lamina propria.Peripheral stimulation by its specific agonist revealed a differential role of NLRC1/Nod1 in stromal and hematopoietic cells for the regulation of the first-line host responses.In contrast, wild type and NLRC1/Nod1-deficient animals harbor equal ratios of lamina propria resident CD4+ and CD8+ T cells, B1 and B2 cells, IgA+ plasma cells, suggesting a NLRC1 alone is not solely responsible for commensal driven intestinal homeostasis.

Gastrointestinal mucins produced by goblet cells comprise the main structural components of the mucus layer. Mucins play a critical role in the maintenance of mucosal homeostasis and are responsible for the differential effector and regulatory responses against a plethora of microorganisms, including commensals and pathogens. In this review, we present a comprehensive overview on mucin biology, its properties, classification and gene assembly. We also consider the structure of the mucin gene, its proteins and its role in innate host defenses. We compare the various mucin secretagogues and the differential regulatory pathways involved in mucin biosynthesis and secretion during normal and diverse pathogenic conditions. Finally, we summarize the putative uncharted aspects of mucin-derived innate host defenses, whose exploration will help drug developers to identify factors that can strengthen mucosal integrity and will facilitate basic science research into curative treatments for gastrointestinal diseases.

Introduction The mucosal surface of the gastrointestinal tract is a complex organization of epithelium, immune cells and resident microbiota [1]. The intestinal epithelium is covered by specialized mucin-producing goblet cells, which together with other substances such as antimicrobial peptides, lysozymes and resident microbiota collectively constitute 'innate immunity' and form the front line of defense against pathogenic microorganisms [2]. The lu-minal surface, which is made up of epithelial cells and mucins, acts as a protective barrier that separates the contents of the harsh luminal environment from the layers of tissue comprising the internal milieu. Goblet cells, found in the columnar epithelium, secrete high molecular weight glycoproteins called mucins. These mucins have a high negative surface charge and a large hydration capacity. Mucins act as the main structural component of the mucus layer, giving rise to its polymer-ic, viscoelastic and protective properties. Mucins are mainly found at the periphery of epithelial cells and their extracellular environment or covering epithelial cells. The surface of the gastrointestinal epithelium is continually exposed to numerous macromolecules and microorganisms including chemical irritants, digested foods, tox-ins, resident bacteria, intestinal pathogens and their prod-Abstract Gastrointestinal mucins produced by goblet cells comprise the main structural components of the mucus layer. Mucins play a critical role in the maintenance of mucosal homeo-stasis and are responsible for the differential effector and regulatory responses against a plethora of microorganisms, including commensals and pathogens. In this review, we present a comprehensive overview on mucin biology, its properties, classification and gene assembly. We also consider the structure of the mucin gene, its proteins and its role in innate host defenses. We compare the various mucin se-cretagogues and the differential regulatory pathways involved in mucin biosynthesis and secretion during normal and diverse pathogenic conditions. Finally, we summarize the putative uncharted aspects of mucin-derived innate host defenses, whose exploration will help drug developers to identify factors that can strengthen mucosal integrity and will facilitate basic science research into curative treatments for gastrointestinal diseases. 124 ucts [3]. The mesh-like structure of the mucin gel not only impedes the diffusion of offending macromolecules but also serves numerous other functions. These include lubrication for the passage of particulate matter, maintenance of a hydrated layer over the epithelium, forming a barrier to noxious substances and creating a permeable gel layer for the exchange of gases and nutrients with the underlying epithelium [4]. The best-established function is the prevention of the entry …