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The normal intestinal microbiota inhabits the colon mucus without triggering an inflammatory response. The reason for this and how the intestinal mucus of the colon is organized have begun to be unraveled. The mucus is organized in two layers: an inner, stratified mucus layer that is firmly adherent to the epithelial cells and approximately 50 μm thick; and an outer, nonattached layer that is usually approximately 100 μm thick as measured in mouse. These mucus layers are organized around the highly glycosylated MUC2 mucin, forming a large, net-like polymer that is secreted by the goblet cells. The inner mucus layer is dense and does not allow bacteria to penetrate, thus keeping the epithelial cell surface free from bacteria. The inner mucus layer is converted into the outer layer, which is the habitat of the commensal flora. The outer mucus layer has an expanded volume due to proteolytic activities provided by the host but probably also caused by commensal bacterial proteases and glycosidases. The numerous O-glycans on the MUC2 mucin not only serve as nutrients for the bacteria but also as attachment sites and, as such, probably contribute to the selection of the species-specific colon flora. This observation that normal human individuals carry a uniform MUC2 mucin glycan array in colon may indicate such a specific selection.

MUC2, the major colonic mucin, forms large polymers by N-terminal trimerization and C-terminal dimerization. Although the assembly process for MUC2 is established, it is not known how MUC2 is packed in the regulated secretory granulae of the goblet cell. When the N-terminal VWD1-D2-D'D3 domains (MUC2-N) were expressed in a goblet-like cell line, the protein was stored together with full-length MUC2. By mimicking the pH and calcium conditions of the secretory pathway we analyzed purified MUC2-N by gel filtration, density gradient centrifugation, and transmission electron microscopy. At pH 7.4 the MUC2-N trimer eluted as a single peak by gel filtration. At pH 6.2 with Ca2+ it formed large aggregates that did not enter the gel filtration column but were made visible after density gradient centrifugation. Electron microscopy studies revealed that the aggregates were composed of rings also observed in secretory granulae of colon tissue sections. The MUC2-N aggregates were dissolved by removing Ca2+ and raising pH. After release from goblet cells, the unfolded full-length MUC2 formed stratified layers. These findings suggest a model for mucin packing in the granulae and the mechanism for mucin release, unfolding, and expansion.

In the development of inflammatory bowel disease (IBD), peritoneal macrophages contribute to the resident intestinal macrophage pool. Previous studies have demonstrated that oral administration of L-fucose exerts an immunomodulatory effect and repolarizes the peritoneal macrophages in vivo in mice. In this study, we analyzed the phenotype and metabolic profile of the peritoneal macrophages from Muc2−/− mice, as well as the effect of L-fucose on the metabolic and morphological characteristics of these macrophages in vitro. The investigation utilized flow cytometry, quantitative PCR (qPCR), measurement of the intracellular ATP and Ca2+ concentrations, an analysis of mitochondrial respiration and membrane potential, and transmission electron microscopy (TEM) for ultrastructural evaluations. The Muc2−/− mice exhibited lower intracellular ATP and Ca2+ levels in their peritoneal macrophages, a higher percentage of stellate macrophages, and an increased oxygen consumption rate (OCR), combined with a higher percentage of mitochondria displaying an abnormal ultrastructure. Additionally, there was a five-fold increase in condensed mitochondria compared to their level in C57BL/6 mice. The number of CD209+ peritoneal macrophages was reduced three-fold, while the number of M1-like cells increased two-fold in the Muc2−/− mice. L-fucose treatment enhanced ATP production and reduced the expression of the Parp1, Mt-Nd2, and Mt-Nd6 genes, which may suggest a reduction in pro-inflammatory factor production and a shift in the differentiation of peritoneal macrophages towards the M2 phenotype.

Chronic stress is known to promote inflammatory bowel disease (IBD), but the underlying mechanism remains largely unresolved. Here, we found chronic stress to sensitize mice to dextran sulfate sodium (DSS)-induced colitis; to increase the infiltration of B cells, neutrophils, and proinflammatory ly6Chi macrophages in colonic lamina propria; and to present with decreased thymus and mesenteric lymph node (MLN) coefficients. Circulating total white blood cells were significantly increased after stress, and the proportion of MLN-associated immune cells were largely changed. Results showed a marked activation of IL-6/STAT3 signaling by stress. The detrimental action of stress was not terminated in IL-6-/- mice. Interestingly, the composition of gut microbiota was dramatically changed after stress, with expansion of inflammation-promoting bacteria. Furthermore, results showed stress-induced deficient expression of mucin-2 and lysozyme, which may contribute to the disorder of gut microbiota. Of note is that, in the case of cohousing, the stress-induced immune reaction and decreased body weight were abrogated, and transferred gut microbiota from stressed mice to control mice was sufficient to facilitate DSS-induced colitis. The important role of gut microbiota was further reinforced by broad-spectrum antibiotic treatment. Taken together, our results reveal that chronic stress disturbs gut microbiota, triggering immune system response and facilitating DSS-induced colitis.

The mucus that covers and protects the epithelium of the intestine is built around its major structural component, the gel-forming MUC2 mucin. The gel-forming mucins have traditionally been assumed to be secreted as nonattached. The colon has a two-layered mucus system where the inner mucus is attached to the epithelium, whereas the small intestine normally has a nonattached mucus. However, the mucus of the small intestine of meprin β-deficient mice was now found to be attached. Meprin β is an endogenous zinc-dependent metalloprotease now shown to cleave the N-terminal region of the MUC2 mucin at two specific sites. When recombinant meprin β was added to the attached mucus of meprin β-deficient mice, the mucus was detached from the epithelium. Similar to meprin β-deficient mice, germ-free mice have attached mucus as they did not shed the membrane-anchored meprin β into the luminal mucus. The ileal mucus of cystic fibrosis (CF) mice with a nonfunctional cystic fibrosis transmembrane conductance regulator (CFTR) channel was recently shown to be attached to the epithelium. Addition of recombinant meprin β to CF mucus did not release the mucus, but further addition of bicarbonate rendered the CF mucus normal, suggesting that MUC2 unfolding exposed the meprin β cleavage sites. Mucus is thus secreted attached to the goblet cells and requires an enzyme, meprin β in the small intestine, to be detached and released into the intestinal lumen. This process regulates mucus properties, can be triggered by bacterial contact, and is nonfunctional in CF due to poor mucin unfolding.

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.

A dense mucus layer in the large intestine prevents inflammation by shielding the underlying epithelium from luminal bacteria and food antigens. This mucus barrier is organized around the hyperglycosylated mucin MUC2. Here we show that the small intestine has a porous mucus layer, which permitted the uptake of MUC2 by antigen-sampling dendritic cells (DCs). Glycans associated with MUC2 imprinted DCs with anti-inflammatory properties by assembling a galectin-S-Dectin-1-FcyRIIB receptor complex that activated β-catenin. This transcription factor interfered with DC expression of inflammatory but not tolerogenic cytokines by inhibiting gene transcription through nuclear factor κB. MUC2 induced additional conditioning signals in intestinal epithelial cells. Thus, mucus does not merely form a nonspecific physical barrier, but also constrains the immunogenicity of gut antigens by delivering tolerogenic signals.

Enterotoxigenic Escherichia coli (ETEC) infections are a leading cause of diarrheal illness, responsible for an estimated 100,000 deaths annually. ETEC pathogenesis is driven by various virulence factors, including toxins, adhesins, and noncanonical factors such as the protease EatA. The first line of host defense against intestinal pathogenic bacterial infections is the protective intestinal mucus layer. Here, we demonstrate the mechanism by which EatA degrades the core mucus component MUC2, thereby facilitating access to the epithelial cell surface and promoting infection. We identify the specific cleavage site region localized at the C-terminal of MUC2. EatA’s protease activity depends on the interaction between two distinct, uniquely spaced domains in human MUC2, which defines species specificity. We confirm this using a novel transgenic mouse model exclusively expressing human MUC2, which allows us to study the role of the mucus layer in the infection by human intestinal pathogens. These findings highlight how ETEC is adapted to specifically degrade the mucus layer of its human host. In this work, authors show how the enterotoxigenic Escherichia coli (ETEC) protease EatA cleaves the human mucus protein MUC2 at a C-terminal site, allowing bacteria to cross the intestinal mucus, reach epithelial cells, and promote infection, as demonstrated using a human MUC2 transgenic mouse model.

An intact mucus layer is important in managing inflammatory bowel disease (IBD). Dairy Propionibacterium freudenreichii has probiotic potential, produces propionic acid and is known to promote health. The aim of this study was to evaluate the effects of P. freudenreichii on the improvement of colitis. LS 174T goblet cells and a dextran sodium sulfate (DSS)-induced colitis rat model were used to investigate the P. freudenreichii -induced stimulation of mucin production in vitro and in vivo , respectively. The mRNA and protein expression levels of MUC2, a main component of intestinal mucus, increased in the supernatant of P. freudenreichii culture (SPFC)-treated LS 174 cells. The SPFC and live P. freudenreichii (LPF) reduced the disease activity index (DAI) in the rats with DSS-induced colitis. After treatment with SPFC or LPF, the mRNA levels of typical pro-inflammatory cytokines decreased and the inflammatory state was histologically improved in the rats with DSS-induced colitis. The SPFC and LPF treatments increased the gene and protein expression levels of MUC2 in the rats with DSS-induced colitis compared with the expression levels in the negative control rats, and immunohistochemistry (IHC) showed an increase of the intestinal MUC2 level. In addition, SPFC and LPF augmented the level of propionate in the faeces of the rats with DSS-induced colitis. In conclusion, P. freudenreichii might improve acute colitis by restoring goblet cell number and stimulating the expression of MUC2 in intestinal goblet cells.

Intestinal goblet cells are potentially key players in controlling susceptibility to ulcerative colitis (UC). Although impaired mucin (Muc2) production by goblet cells increases microbial stimulation of the colonic mucosa, goblet cells secrete other mediators that may influence or promote UC development. Correspondingly, Muc2-deficient (−/−) mice develop spontaneous colitis, concurrent with the dramatic upregulation of the goblet cell mediator, resistin-like molecule-beta (RELM-β). Testing RELM-β's role, we generated Muc2−/−/Retnlb−/− mice, finding that RELM-β deficiency significantly attenuated colitis development and symptoms compared with Muc2−/− mice. RELM-β expression in Muc2−/− mice strongly induced the production/secretion of the antimicrobial lectin RegIIIβ, that exerted its microbicidal effect predominantly on Gram-positive Lactobacillus species. Compared with Muc2−/−/Retnlb−/− mice, this worsened intestinal microbial dysbiosis with a selective loss of colonic Lactobacilli spp. in Muc2−/− mice. Orally replenishing Muc2−/− mice with murine Lactobacillus spp., but not with a probiotic formulation containing several human Lactobacillus spp. (VSL#3), ameliorated their spontaneous colitis in concert with increased production of short-chain fatty acids. These studies demonstrate that the goblet cell mediator RELM-β drives colitis in Muc2−/− mice by depleting protective commensal microbes. The ability of selective commensal microbial replacement to ameliorate colitis suggests that personalized bacterial therapy may prove beneficial for treatment of UC.