Sweet-talk in the time of cholera: host mucin negotiates peace with Vibrio cholerae.

Vibrio cholerae, the causative agent of cholera, must first be converted to its toxigenic form and cross the sugar-rich mucus barrier before it can cause disease, but whether these hurdles are linked is unclear. In this issue, Wang et al (2022) provide new evidence that mucus O-glycans directly prevent toxigenic conversion and virulence factor expression in V. cholerae.

The gut mucus network: A dynamic liaison between microbes and the immune system.

A complex mucus network made up of large polymers of the mucin-family glycoprotein MUC2 exists between the large intestinal microbial mass and epithelial and immune cells. This has long been understood as an innate immune defense barrier against the microbiota and other luminal threats that reinforces the barrier function of the epithelium and limits microbiota contact with the tissues. However, past and recent studies have provided new evidence of how critical the mucus network is to act as a 'liaison' between host and microbe to mediate anti-inflammatory, mutualistic interactions with the microbiota and protection from pathogens. This review summarizes historical and recent insights into the formation of the gut mucus network, how the microbes and immune system influence mucus, and in turn, how the mucus influences immune responses to the microbiota.

Fecal-adherent mucus is a non-invasive source of primary human MUC2 for structural and functional characterization in health and disease.

The O-glycoprotein Mucin-2 (MUC2) forms the protective colon mucus layer. While animal models have demonstrated the importance of Muc2, few studies have explored human MUC2 in similar depth. Recent studies have revealed that secreted MUC2 is bound to human feces. We hypothesized human fecal MUC2 (HF-MUC2) was accessible for purification and downstream structural and functional characterization. We tested this via histologic and quantitative imaging on human fecal sections; extraction from feces for proteomic and O-glycomic characterization; and functional studies via growth and metabolic assays in vitro. Quantitative imaging of solid fecal sections showed a continuous mucus layer of varying thickness along human fecal sections with barrier functions intact. Lectin profiling showed HF-MUC2 bound several lectins but was weak to absent for Ulex europaeus 1 (α1,2 fucose-binding) and Sambucus nigra agglutinin (α2,6 sialic acid-binding), and did not have obvious b1/b2 barrier layers. HF-MUC2 separated by electrophoresis showed high molecular weight glycoprotein bands (∼1-2 MDa). Proteomics and Western analysis confirmed the enrichment of MUC2 and potential MUC2-associated proteins in HF-MUC2 extracts. MUC2 O-glycomics revealed diverse fucosylation, moderate sialylation, and little sulfation versus porcine colonic MUC2 and murine fecal Muc2. O-glycans were functional and supported the growth of Bacteroides thetaiotaomicron (B. theta) and short-chain fatty acid (SCFA) production in vitro. MUC2 could be similarly analyzed from inflammatory bowel disease stools, which displayed an altered glycomic profile and differential growth and SCFA production by B. theta versus healthy samples. These studies describe a new non-invasive platform for human MUC2 characterization in health and disease.

Control of intestinal homeostasis, colitis, and colitis-associated colorectal cancer by the inflammatory caspases.

Inflammatory caspases are essential effectors of inflammation and cell death. Here, we investigated their roles in colitis and colorectal cancer and report a bimodal regulation of intestinal homeostasis, inflammation and tumorigenesis by caspases-1 and -12. Casp1(-/-) mice exhibited defects in mucosal tissue repair and succumbed rapidly after dextran sulfate sodium administration. This phenotype was rescued by administration of exogenous interleukin-18 and was partially reproduced in mice deficient in the inflammasome adaptor ASC. Casp12(-/-) mice, in which the inflammasome is derepressed, were resistant to acute colitis and showed signs of enhanced repair. Together with their increased inflammatory response, the enhanced repair response of Casp12(-/-) mice rendered them more susceptible to colorectal cancer induced by azoxymethane (AOM)+DSS. Taken together, our results indicate that the inflammatory caspases are critical in the induction of inflammation in the gut after injury, which is necessary for tissue repair and maintenance of immune tolerance.

Loss of mucin-type O-glycans impairs the integrity of the glomerular filtration barrier in the mouse kidney.

The kidney's filtration activity is essential for removing toxins and waste products from the body. The vascular endothelial cells of the glomerulus are fenestrated, flattened, and surrounded by podocytes, specialized cells that support glomerular endothelial cells. Mucin-type core 1-derived O-glycans (O-glycans) are highly expressed on both glomerular capillary endothelial cells and their supporting podocytes, but their biological role is unclear. Biosynthesis of core 1-derived O-glycans is catalyzed by the glycosyltransferase core 1 ß1,3-galactosyltransferase (C1galt1). Here we report that neonatal or adult mice with inducible deletion of C1galt1 (iC1galt1-/-) exhibit spontaneous proteinuria and rapidly progressing glomerulosclerosis. Ultrastructural analysis of the glomerular filtration barrier components revealed that loss of O-glycans results in altered podocyte foot processes. Further analysis indicated that O-glycan is essential for the normal signaling function of podocalyxin, a podocyte foot process-associated glycoprotein. Our results reveal a new function of O-glycosylation in the integrity of the glomerular filtration barrier.

Defective Intestinal Mucin-Type O-Glycosylation Causes Spontaneous Colitis-Associated Cancer in Mice.

BACKGROUND & AIMS: Core 1- and core 3-derived mucin-type O-linked oligosaccharides (O-glycans) are major components of the colonic mucus layer. Defective forms of colonic O-glycans, such as the Thomsen-nouveau (Tn) antigen, frequently are observed in patients with ulcerative colitis and colorectal cancer, but it is not clear if they contribute to their pathogenesis. We investigated whether and how impaired O-glycosylation contributes to the development of colitis-associated colorectal cancer using mice lacking intestinal core 1- and core 3-derived O-glycans. METHODS: We generated mice that lack core 1- and core 3-derived intestinal O-glycans (DKO mice) and analyzed them, along with mice that singly lack intestinal epithelial core 1 O-glycans (IEC C1galt1(-/-) mice) or core 3 O-glycans (C3Gnt(-/-) mice). Intestinal tissues were collected at different time points and analyzed for levels of mucin and Tn antigen, development of colitis, and tumor formation using imaging, quantitative polymerase chain reaction, immunoblot, and enzyme-linked immunosorbent assay techniques. We also used cellular and genetic approaches, as well as intestinal microbiota depletion, to identify inflammatory mediators and pathways that contribute to disease in DKO and wild-type littermates (controls). RESULTS: Intestinal tissues from DKO mice contained higher levels of Tn antigen and had more severe spontaneous chronic colitis than tissues from IEC C1galt1(-/-) mice, whereas spontaneous colitis was absent in C3GnT(-/-) and control mice. IEC C1galt1(-/-) mice and DKO mice developed spontaneous colorectal tumors, although the onset of tumors in the DKO mice occurred earlier (age, 8-9 months) than that in IEC C1galt1(-/-) mice (15 months old). Antibiotic depletion of the microbiota did not cause loss of Tn antigen but did reduce the development of colitis and cancer formation in DKO mice. Colon tissues from DKO mice, but not control mice, contained active forms of caspase 1 and increased caspase 11, which were reduced after antibiotic administration. Supernatants from colon tissues of DKO mice contained increased levels of interleukin-1ß and interleukin-18, compared with those from control mice. Disruption of the caspase 1 and caspase 11 genes in DKO mice (DKO/Casp1/11(-/-) mice) decreased the development of colitis and cancer, characterized by reduced colonic thickening, hyperplasia, inflammatory infiltrate, and tumors compared with DKO mice. CONCLUSIONS: Impaired expression of O-glycans causes colonic mucus barrier breach and subsequent microbiota-mediated activation of caspase 1-dependent inflammasomes in colonic epithelial cells of mice. These processes could contribute to colitis-associated colon cancer in humans.

Goblet Cell Derived RELM-ß Recruits CD4+ T Cells during Infectious Colitis to Promote Protective Intestinal Epithelial Cell Proliferation.

Enterohemorrhagic Escherichia coli and related food and waterborne pathogens pose significant threats to human health. These attaching/effacing microbes infect the apical surface of intestinal epithelial cells (IEC), causing severe diarrheal disease. Colonizing the intestinal luminal surface helps segregate these microbes from most host inflammatory responses. Based on studies using Citrobacter rodentium, a related mouse pathogen, we speculate that hosts rely on immune-mediated changes in IEC, including goblet cells to defend against these pathogens. These changes include a CD4+ T cell-dependent increase in IEC proliferation to replace infected IEC, as well as altered production of the goblet cell-derived mucin Muc2. Another goblet cell mediator, REsistin-Like Molecule (RELM)-ß is strongly induced within goblet cells during C. rodentium infection, and was detected in the stool as well as serum. Despite its dramatic induction, RELM-ß's role in host defense is unclear. Thus, wildtype and RELM-ß gene deficient mice (Retnlb-/-) were orally infected with C. rodentium. While their C. rodentium burdens were only modestly elevated, infected Retnlb-/- mice suffered increased mortality and mucosal ulceration due to deep pathogen penetration of colonic crypts. Immunostaining for Ki67 and BrDU revealed Retnlb-/- mice were significantly impaired in infection-induced IEC hyper-proliferation. Interestingly, exposure to RELM-ß did not directly increase IEC proliferation, rather RELM-ß acted as a CD4+ T cell chemoattractant. Correspondingly, Retnlb-/- mice showed impaired CD4+ T cell recruitment to their infected colons, along with reduced production of interleukin (IL)-22, a multifunctional cytokine that directly increased IEC proliferation. Enema delivery of RELM-ß to Retnlb-/- mice restored CD4+ T cell recruitment, concurrently increasing IL-22 levels and IEC proliferation, while reducing mucosal pathology. These findings demonstrate that RELM-ß and goblet cells play an unexpected, yet critical role in recruiting CD4+ T cells to the colon to protect against an enteric pathogen, in part via the induction of increased IEC proliferation.

Loss of Core 1-derived O-Glycans Decreases Breast Cancer Development in Mice.

Mucin-type core 1-derived O-glycans, one of the major types of O-glycans, are highly expressed in mammary gland epithelium. Abnormal O-glycans such as Tn antigen are found in over 90% of breast cancers; however, the in vivo role of these aberrant O-glycans in the etiology of breast cancer is unclear. We generated mice with mammary epithelial specific deletion of core 1-derived O-glycans. By crossing with two spontaneous mouse breast cancer models, we determined that loss of core 1-derived O-glycans delays the onset and progression of breast cancer development. Deficiency of core 1 O-glycosylation impaired the localization of Muc1, a major O-glycoprotein, on the apical surfaces of mammary epithelium. Signaling mediated by Muc1, which is critical for breast cancer development, was also defective in the absence of core 1 O-glycans. This study reveals an unexpected role of core 1-derived O-glycans in breast cancer development in mice.

Loss of intestinal O-glycans promotes spontaneous duodenal tumors.

Mucin-type O-glycans, primarily core 1- and core 3-derived O-glycans, are the major mucus barrier components throughout the gastrointestinal tract. Previous reports identified the biological role of O-glycans in the stomach and colon. However, the biological function of O-glycans in the small intestine remains unknown. Using mice lacking intestinal core 1- and core 3-derived O-glycans [intestinal epithelial cell C1galt1(-/-);C3GnT(-/-) or double knockout (DKO)], we found that loss of O-glycans predisposes DKO mice to spontaneous duodenal tumorigenesis by ∼1 yr of age. Tumor incidence did not increase with age; however, tumors advanced in aggressiveness by 20 mo. O-glycan deficiency was associated with reduced luminal mucus in DKO mice before tumor development. Altered intestinal epithelial homeostasis with enhanced baseline crypt proliferation characterizes these phenotypes as assayed by Ki67 staining. In addition, fluorescence in situ hybridization analysis reveals a significantly lower bacterial burden in the duodenum compared with the large intestine. This phenotype is not reduced with antibiotic treatment, implying O-glycosylation defects, rather than bacterial-induced inflammation, which causes spontaneous duodenal tumorigenesis. Moreover, inflammatory responses in DKO duodenal mucosa are mild as assayed with histology, quantitative PCR for inflammation-associated cytokines, and immunostaining for immune cells. Importantly, inducible deletion of intestinal O-glycans in adult mice leads to analogous spontaneous duodenal tumors, although with higher incidence and heightened severity compared with mice with O-glycans constitutive deletion. In conclusion, these studies reveal O-glycans within the small intestine are critical determinants of duodenal cancer risk. Future studies will provide insights into the pathogenesis in the general population and those at risk for this rare but deadly cancer.

Resistin-like molecule α promotes pathogenic Th17 cell responses and bacterial-induced intestinal inflammation.

Resistin-like molecule (RELM)α belongs to a family of secreted mammalian proteins that have putative immunomodulatory functions. Recent studies have identified a pathogenic role for RELMα in chemically induced colitis through effects on innate cell populations. However, whether RELMα regulates intestinal adaptive immunity to enteric pathogens is unknown. In this study, we employed Citrobacter rodentium as a physiologic model of pathogenic Escherichia coli-induced diarrheal disease, colitis, and Th17 cell responses. In response to Citrobacter, RELMα expression was induced in intestinal epithelial cells, infiltrating macrophages, and eosinophils of the infected colons. Citrobacter-infected RELMα(-/-) mice exhibited reduced infection-induced intestinal inflammation, characterized by decreased leukocyte recruitment to the colons and reduced immune cell activation compared with wild-type (WT) mice. Interestingly, Citrobacter colonization and clearance were unaffected in RELMα(-/-) mice, suggesting that the immune stimulatory effects of RELMα following Citrobacter infection were pathologic rather than host-protective. Furthermore, infected RELMα(-/-) mice exhibited decreased CD4(+) T cell expression of the proinflammatory cytokine IL-17A. To directly test whether RELMα promoted Citrobacter-induced intestinal inflammation via IL-17A, infected WT and IL-17A(-/-) mice were treated with rRELMα. RELMα treatment of Citrobacter-infected WT mice exacerbated intestinal inflammation and IL-17A expression whereas IL-17A(-/-) mice were protected from RELMα-induced intestinal inflammation. Finally, infected RELMα(-/-) mice exhibited reduced levels of serum IL-23p19 compared with WT mice, and RELMα(-/-) peritoneal macrophages showed deficient IL-23p19 induction. Taken together, these data identify a proinflammatory role for RELMα in bacterial-induced colitis and suggest that the IL-23/Th17 axis is a critical mediator of RELMα-induced inflammation.

CD4+ T cells drive goblet cell depletion during Citrobacter rodentium infection.

Both idiopathic and infectious forms of colitis disrupt normal intestinal epithelial cell (IEC) proliferation and differentiation, although the mechanisms involved remain unclear. Recently, we demonstrated that infection by the attaching and effacing murine pathogen Citrobacter rodentium leads to a significant reduction in colonic goblet cell numbers (goblet cell depletion). This pathology depends on T and/or B cells, as Rag1(-/-) mice do not suffer this depletion during infection, instead suffering high mortality rates. To address the immune mechanisms involved, we reconstituted Rag(-/-) mice with either CD4(+) or CD8(+) T cells. Both T cell subsets increased Rag1(-/-) mouse survival during infection, with mice that received CD8(+) T cells developing colonic ulcers but not goblet cell depletion. In contrast, mice that received CD4(+) T cells showed goblet cell depletion in concert with exaggerated IEC proliferation. To define the possible involvement of T cell-derived cytokines, we infected gamma interferon receptor gene knockout (IFN-γR(-/-)) mice and wild-type mice given interleukin 17A (IL-17A) neutralizing antibodies and found that IFN-γ signaling was required for both goblet cell depletion and increased IEC proliferation. Immunostaining revealed that C. rodentium cells preferentially localized to nonhyperplastic crypts containing numerous goblet cells, whereas hyperplastic, goblet cell-depleted crypts appeared protected from infection. To address whether goblet cell depletion benefits the C. rodentium-infected host, we increased goblet cell numbers using the γ-secretase inhibitor dibenzazepine (DBZ), which resulted in greatly increased pathogen burdens and mortality rates. These results demonstrate that goblet cell depletion reflects host immunomodulation of IEC homeostasis and reflects a novel host defense mechanism against mucosal-adherent pathogens.

Mucin-type O-glycans and their roles in intestinal homeostasis.

Mucin-type O-glycans are the primary constituents of mucins that are expressed on various mucosal sites of the body, especially the bacteria-laden intestinal tract. Mucins are the main components of mucus, which is secreted by goblet cells and forms a protective homeostatic barrier between the resident microbiota and the underlying immune cells in the colon. However, the specific role of mucin-type O-glycans in mucus barrier function has been uncertain. Recent studies utilizing mice deficient in key glycosyltransferases involved in O-glycan biosynthesis on intestinal mucins have underscored the importance of mucin-type O-glycosylation in mucus barrier function. This review will highlight recent advances in our understanding of mucin-type O-glycan function in the mucus barrier and how they promote mutualism with our resident microbiota.

Innate host responses to enteric bacterial pathogens: a balancing act between resistance and tolerance.

Infection by enteric bacterial pathogens activates pathogen recognition receptors, leading to innate responses that promote host defence. While responses that promote host 'resistance' to infection, through the release of antimicrobial mediators, or the recruitment of inflammatory cells aimed at clearing the infection are best known, recent studies have begun to identify additional innate driven responses that instead promote intestinal tissue repair and host survival. Described as infection 'tolerance' responses, we and others have primarily studied these responses in the Citrobacter rodentium infection model. In this review we discuss the impact of innate resistance mechanisms on host defence, and describe how 'tolerance' responses act primarily on the intestinal epithelium, triggering epithelial cell proliferation, repair or promoting barrier function. Resistance and tolerance responses appear to work together, with tolerance repairing the tissue injury caused by resistance driven inflammation. Tolerance responses fit a pattern where innate immunity and inflammation are tightly regulated in the gastrointestinal tract. Moreover, tolerance may have developed due to the successful subversion and avoidance of host resistance by enteric bacterial pathogens. Further studies are needed to clarify the contribution of different pathogen recognition receptors to tolerance and resistance responses against bacterial pathogens, in the gut or in other host tissues.

Requirement of epithelial integrin-linked kinase for facilitation of Citrobacter rodentium-induced colitis.

BACKGROUND: Integrin-linked kinase (ILK) is a serine-threonine kinase that transduces extracellular matrix-related cues into intracellular signals, with fundamental roles in cell motility, development and cancer. Recently ILK been shown to have an important role in bacterial epithelial cell attachment, through ILK-bacterial OspE binding. Here we report on the role of epithelial derived ILK in response to Citrobacter rodentium infection. METHODS: C. rodentium was administered to both control and intestinal epithelial cell ILK knockout mice. Histological inflammatory scores were assessed, and cytokines measured by ELISA as well as RT-PCR, in mouse colons. Bacterial colonization was determined by plating homogenates onto MacConkey agar, and immunofluorescence microscopy performed using anti-LPS and anti-Tir antibodies. RESULTS: ILK-ko mice exhibited reduced weight loss at 15 days post-infection (p < 0.01) and demonstrated reduced histological inflammatory scores (p < 0.01), reduced CCL2 and pro-inflammatory cytokines. This was not due to reduced colonization, but was associated with an altered pattern of C. rodentium bacterial migration. Attenuated fibronectin expression was found in the ILK-ko mice. C. rodentium exposure was shown to increase ILK expression in cell lines, and in murine epithelium in vivo. In ILK-ko mice reduced activation of ser473Akt and reduced crypt proliferation, together with reduced cyclin D1 expression were observed. CONCLUSIONS: ILK influences the host response to C. rodentium -induced infection, independently of reduced colonization in the ILK knockout mice. The reduced inflammation and dramatically attenuated hyperplastic cryptal response to infection in this group, are at least in part the result of, the reduction in CCL2 and cyclin D1 expression respectively.

Extraction and Verification of Mouse and Human Mucins from Tissue and Fecal Material.

Gel-forming mucins are highly O-glycosylated polymeric glycoproteins which have critical roles in tissue protection from environmental insult. To understand their biochemical properties, these samples must be extracted and enriched from biological samples. Here we describe how to extract and semi-purify human and murine mucins from intestinal scrapings or fecal material. As mucins have high molecular weights, traditional gel electrophoresis methods are unable to effectively separate these glycoproteins for analysis. We describe the procedure for making composite sodium dodecyl sulfate urea agarose-polyacrylamide (SDS-UAgPAGE) gels, which allows for accurate verification and band separation of extracted mucins.

Role of mucin glycosylation in the gut microbiota-brain axis of core 3 O-glycan deficient mice.

Alterations in intestinal mucin glycosylation have been associated with increased intestinal permeability and sensitivity to inflammation and infection. Here, we used mice lacking core 3-derived O-glycans (C3GnT-/-) to investigate the effect of impaired mucin glycosylation in the gut-brain axis. C3GnT-/- mice showed altered microbial metabolites in the caecum associated with brain function such as dimethylglycine and N-acetyl-L-tyrosine profiles as compared to C3GnT+/+ littermates. In the brain, polysialylated-neural cell adhesion molecule (PSA-NCAM)-positive granule cells showed an aberrant phenotype in the dentate gyrus of C3GnT-/- mice. This was accompanied by a trend towards decreased expression levels of PSA as well as ZO-1 and occludin as compared to C3GnT+/+. Behavioural studies showed a decrease in the recognition memory of C3GnT-/- mice as compared to C3GnT+/+ mice. Combined, these results support the role of mucin O-glycosylation in the gut in potentially influencing brain function which may be facilitated by the passage of microbial metabolites through an impaired gut barrier.

MyD88 signaling promotes both mucosal homeostatic and fibrotic responses during Salmonella-induced colitis.

Salmonella enterica serovar Typhimurium is a clinically important gram-negative, enteric bacterial pathogen that activates several Toll-like receptors (TLRs). While TLR signaling through the adaptor protein MyD88 has been shown to promote inflammation and host defense against the systemic spread of S. Typhimurium, curiously, its role in the host response against S. Typhimurium within the mammalian gastrointestinal (GI) tract is less clear. We therefore used the recently described Salmonella-induced enterocolitis and fibrosis model: wild-type (WT) and MyD88-deficient (MyD88(-/-)) mice pretreated with streptomycin and then orally infected with the ΔaroA vaccine strain of S. Typhimurium. Tissues were analyzed for bacterial colonization, inflammation, and epithelial damage, while fibrosis was assessed by collagen quantification and Masson's trichrome staining. WT and MyD88(-/-) mice carried similar intestinal pathogen burdens to postinfection day 21. Infection of WT mice led to acute mucosal and submucosal inflammation and edema, as well as significant intestinal epithelial damage and proliferation, leading to widespread goblet cell depletion. Impressive collagen deposition in the WT intestine was also evident in the submucosa at postinfection days 7 and 21, with fibrotic regions rich in fibroblasts and collagen. While infected MyD88(-/-) mice showed levels of submucosal inflammation and edema similar to WT mice, they were impaired in the development of mucosal inflammation, along with infection-induced epithelial damage, proliferation, and goblet cell depletion. MyD88(-/-) mouse tissues also had fewer submucosal fibroblasts and 60% less collagen. We noted that cyclooxygenase (Cox)-2 expression was MyD88-dependent, with numerous Cox-2-positive cells identified in fibrotic regions of WT mice at postinfection day 7, but not in MyD88(-/-) mice. Treatment of WT mice with the Cox-2 inhibitor rofecoxib (20 mg/kg) significantly reduced fibroblast numbers and collagen levels without altering colitis severity. In conclusion, MyD88 and Cox-2 signaling play roles in intestinal fibrosis during Salmonella-induced enterocolitis.

Muc2 protects against lethal infectious colitis by disassociating pathogenic and commensal bacteria from the colonic mucosa.

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.

The single IgG IL-1-related receptor controls TLR responses in differentiated human intestinal epithelial cells.

Intestinal epithelial cells (IECs) are constantly exposed to enteric microbes. Although IECs express TLRs that recognize bacterial products, the activation of these TLRs is strictly controlled through poorly understood mechanisms, producing a state of hyporesponsiveness and preventing unwanted inflammation. The single IgG IL-1-related receptor (Sigirr) is a negative regulator of TLRs that is expressed by IECs and was recently shown to inhibit experimental colitis. However, the importance of Sigirr in IEC hyporesponsiveness and its distribution within the human colon is unknown. In this study, we investigated the role of Sigirr in regulating epithelial-specific TLR responses and characterized its expression in colonic biopsy specimens. Transformed and nontransformed human IECs were cultured as monolayers. Transient gene silencing and stable overexpression of Sigirr was performed to assess innate IEC responses. Sigirr expression in human colonic biopsy specimens was examined by immunohistochemistry. Bacterial infection of IECs and exposure to flagellin transiently decreased Sigirr protein expression, concurrent with secretion of the neutrophil chemokine IL-8. Sigirr gene silencing augmented chemokine responses to bacterial flagellin, Pam3Cys, and the cytokine IL-1beta. Conversely, stable overexpression of Sigirr diminished NF-kappaB-mediated IL-8 responses to TLR ligands. We also found that Sigirr expression increased as IECs differentiated in culture. This observation was confirmed in biopsy sections, in which Sigirr expression within colonic crypts was prominent in IECs at the apex and diminished at the base. Our findings show that Sigirr broadly regulates innate responses in differentiated human IECs; therefore, it may modulate epithelial involvement in infectious and inflammatory bowel diseases.

The barrier and beyond: Roles of intestinal mucus and mucin-type O-glycosylation in resistance and tolerance defense strategies guiding host-microbe symbiosis.

Over the past two decades, our appreciation of the gut mucus has moved from a static lubricant to a dynamic and essential component of the gut ecosystem that not only mediates the interface between host tissues and vast microbiota, but regulates how this ecosystem functions to promote mutualistic symbioses and protect from microbe-driven diseases. By delving into the complex chemistry and biology of the mucus, combined with innovative in vivo and ex vivo approaches, recent studies have revealed novel insights into the formation and function of the mucus system, the O-glycans that make up this system, and how they mediate two major host-defense strategies - resistance and tolerance - to reduce damage caused by indigenous microbes and opportunistic pathogens. This current review summarizes these findings by highlighting the emerging roles of mucus and mucin-type O-glycans in influencing host and microbial physiology with an emphasis on host defense strategies against bacteria in the gastrointestinal tract.