Malin E. V. Johansson

The inner of the two Muc2 mucin-dependent mucus layers in colon is devoid of bacteria

We normally live in symbiosis with ∼1013 bacteria present in the colon. Among the several mechanisms maintaining the bacteria/host balance, there is limited understanding of the structure, function, and properties of intestinal mucus. We now demonstrate that the mouse colonic mucus consists of two layers extending 150 μm above the epithelial cells. Proteomics revealed that both of these layers have similar protein composition, with the large gel-forming mucin Muc2 as the major structural component. The inner layer is densely packed, firmly attached to the epithelium, and devoid of bacteria. In contrast, the outer layer is movable, has an expanded volume due to proteolytic cleavages of the Muc2 mucin, and is colonized by bacteria. Muc2−/− mice have bacteria in direct contact with the epithelial cells and far down in the crypts, explaining the inflammation and cancer development observed in these animals. These findings show that the Muc2 mucin can build a mucus barrier that separates bacteria from the colon epithelia and suggest that defects in this mucus can cause colon inflammation.

The two mucus layers of colon are organized by the MUC2 mucin, whereas the outer layer is a legislator of host–microbial interactions

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.

The gastrointestinal mucus system in health and disease

Mucins—large, highly glycosylated proteins—are important for the luminal protection of the gastrointestinal tract. Enterocytes have their apical surface covered by transmembrane mucins and goblet cells produce the secreted gel-forming mucins that form mucus. The small intestine has a single unattached mucus layer, which in cystic fibrosis becomes attached, accounting for the intestinal manifestations of this disease. The stomach and colon have two layers of mucus; the inner layer is attached and the outer layer is less dense and unattached. In the colon, the outer mucus layer is the habitat for commensal bacteria. The inner mucus layer is impervious to bacteria and is renewed every hour by surface goblet cells. The crypt goblet cells have the ability to restitute the mucus layer by secretion, for example after an ischaemic challenge. Proteases of certain parasites and some bacteria can cleave mucins and dissolve the mucus as part of their pathogenicity. The inner mucus layer can, however, also become penetrable to bacteria by several other mechanisms, including aberrations in the immune system. When bacteria reach the epithelial surface, the immune system is activated and inflammation is triggered. This mechanism might occur in some types of ulcerative colitis.

Bacteria penetrate the normally impenetrable inner colon mucus layer in both murine colitis models and patients with ulcerative colitis

Objective The inner mucus layer in mouse colon normally separates bacteria from the epithelium. Do humans have a similar inner mucus layer and are defects in this mucus layer a common denominator for spontaneous colitis in mice models and ulcerative colitis (UC)? Methods and results The colon mucus layer from mice deficient in Muc2 mucin, Core 1 O-glycans, Tlr5, interleukin 10 (IL-10) and Slc9a3 (Nhe3) together with that from dextran sodium sulfate-treated mice was immunostained for Muc2, and bacterial localisation in the mucus was analysed. All murine colitis models revealed bacteria in contact with the epithelium. Additional analysis of the less inflamed IL-10−/− mice revealed a thicker mucus layer than wild-type, but the properties were different, as the inner mucus layer could be penetrated both by bacteria in vivo and by fluorescent beads the size of bacteria ex vivo. Clear separation between bacteria or fluorescent beads and the epithelium mediated by the inner mucus layer was also evident in normal human sigmoid colon biopsy samples. In contrast, mucus on colon biopsy specimens from patients with UC with acute inflammation was highly penetrable. Most patients with UC in remission had an impenetrable mucus layer similar to that of controls. Conclusions Normal human sigmoid colon has an inner mucus layer that is impenetrable to bacteria. The colon mucus in animal models that spontaneously develop colitis and in patients with active UC allows bacteria to penetrate and reach the epithelium. Thus colon mucus properties can be modulated, and this suggests a novel model of UC pathophysiology.

The mucus and mucins of the goblet cells and enterocytes provide the first defense line of the gastrointestinal tract and interact with the immune system

The gastrointestinal tract is covered by mucus that has different properties in the stomach, small intestine, and colon. The large highly glycosylated gel‐forming mucins MUC2 and MUC5AC are the major components of the mucus in the intestine and stomach, respectively. In the small intestine, mucus limits the number of bacteria that can reach the epithelium and the Peyers patches. In the large intestine, the inner mucus layer separates the commensal bacteria from the host epithelium. The outer colonic mucus layer is the natural habitat for the commensal bacteria. The intestinal goblet cells secrete not only the MUC2 mucin but also a number of typical mucus components: CLCA1, FCGBP, AGR2, ZG16, and TFF3. The goblet cells have recently been shown to have a novel gate‐keeping role for the presentation of oral antigens to the immune system. Goblet cells deliver small intestinal luminal material to the lamina propria dendritic cells of the tolerogenic CD103+ type. In addition to the gel‐forming mucins, the transmembrane mucins MUC3, MUC12, and MUC17 form the enterocyte glycocalyx that can reach about a micrometer out from the brush border. The MUC17 mucin can shuttle from a surface to an intracellular vesicle localization, suggesting that enterocytes might control and report epithelial microbial challenge. There is communication not only from the epithelial cells to the immune system but also in the opposite direction. One example of this is IL10 that can affect and improve the properties of the inner colonic mucus layer. The mucus and epithelial cells of the gastrointestinal tract are the primary gate keepers and controllers of bacterial interactions with the host immune system, but our understanding of this relationship is still in its infancy.

Composition and functional role of the mucus layers in the intestine

In discussions on intestinal protection, the protective capacity of mucus has not been very much considered. The progress in the last years in understanding the molecular nature of mucins, the main building blocks of mucus, has, however, changed this. The intestinal enterocytes have their apical surfaces covered by transmembrane mucins and the whole intestinal surface is further covered by mucus, built around the gel-forming mucin MUC2. The mucus of the small intestine has only one layer, whereas the large intestine has a two-layered mucus where the inner, attached layer has a protective function for the intestine, as it is impermeable to the luminal bacteria.

Bacteria penetrate the inner mucus layer before inflammation in the dextran sulfate colitis model.

Background Protection of the large intestine with its enormous amount of commensal bacteria is a challenge that became easier to understand when we recently could describe that colon has an inner attached mucus layer devoid of bacteria (Johansson et al. (2008) Proc. Natl. Acad. Sci. USA 105, 15064–15069). The bacteria are thus kept at a distance from the epithelial cells and lack of this layer, as in Muc2-null mice, allow bacteria to contact the epithelium. This causes colitis and later on colon cancer, similar to the human disease Ulcerative Colitis, a disease that still lacks a pathogenetic explanation. Dextran Sulfate (DSS) in the drinking water is the most widely used animal model for experimental colitis. In this model, the inflammation is observed after 3–5 days, but early events explaining why DSS causes this has not been described. Principal Findings When mucus formed on top of colon explant cultures were exposed to 3% DSS, the thickness of the inner mucus layer decreased and became permeable to 2 µm fluorescent beads after 15 min. Both DSS and Dextran readily penetrated the mucus, but Dextran had no effect on thickness or permeability. When DSS was given in the drinking water to mice and the colon was stained for bacteria and the Muc2 mucin, bacteria were shown to penetrate the inner mucus layer and reach the epithelial cells already within 12 hours, long before any infiltration of inflammatory cells. Conclusion DSS thus causes quick alterations in the inner colon mucus layer that makes it permeable to bacteria. The bacteria that reach the epithelial cells probably trigger an inflammatory reaction. These observations suggest that altered properties or lack of the inner colon mucus layer may be an initial event in the development of colitis.

Transient Inability to Manage Proteobacteria Promotes Chronic Gut Inflammation in TLR5-Deficient Mice

Colitis results from breakdown of homeostasis between intestinal microbiota and the mucosal immune system, with both environmental and genetic influencing factors. Flagellin receptor TLR5-deficient mice (T5KO) display elevated intestinal proinflammatory gene expression and colitis with incomplete penetrance, providing a genetically sensitized system to study the contribution of microbiota to driving colitis. Both colitic and noncolitic T5KO exhibited transiently unstable microbiotas, with lasting differences in colitic T5KO, while their noncolitic siblings stabilized their microbiotas to resemble wild-type mice. Transient high levels of proteobacteria, especially enterobacteria species including E. coli, observed in close proximity to the gut epithelium were a striking feature of colitic microbiota. A Crohns disease-associated E. coli strain induced chronic colitis in T5KO, which persisted well after the exogenously introduced bacterial species had been eliminated. Thus, an innate immune deficiency can result in unstable gut microbiota associated with low-grade inflammation, and harboring proteobacteria can drive and/or instigate chronic colitis.

Loss of intestinal core 1-derived O-glycans causes spontaneous colitis in mice

Mucin-type O-linked oligosaccharides (O-glycans) are primary components of the intestinal mucins that form the mucus gel layer overlying the gut epithelium. Impaired expression of intestinal O-glycans has been observed in patients with ulcerative colitis (UC), but its role in the etiology of this disease is unknown. Here, we report that mice with intestinal epithelial cell-specific deficiency of core 1-derived O-glycans, the predominant form of O-glycans, developed spontaneous colitis that resembled human UC, including massive myeloid infiltrates and crypt abscesses. The colitis manifested in these mice was also characterized by TNF-producing myeloid infiltrates in colon mucosa in the absence of lymphocytes, supporting an essential role for myeloid cells in colitis initiation. Furthermore, induced deletion of intestinal core 1-derived O-glycans caused spontaneous colitis in adult mice. These data indicate a causal role for the loss of core 1-derived O-glycans in colitis. Finally, we detected a biosynthetic intermediate typically exposed in the absence of core 1 O-glycan, Tn antigen, in the colon epithelium of a subset of UC patients. Somatic mutations in the X-linked gene that encodes core 1 β1,3-galactosyltransferase-specific chaperone 1 (C1GALT1C1, also known as Cosmc), which is essential for core 1 O-glycosylation, were found in Tn-positive epithelia. These data suggest what we believe to be a new molecular mechanism for the pathogenesis of UC.

Bicarbonate and functional CFTR channel are required for proper mucin secretion and link cystic fibrosis with its mucus phenotype

Ileal mucus in CftrΔ508 mice is more adherent, denser, and less penetrable than that of WT mice, but addition of bicarbonate normalizes the properties of CftrΔ508 mucus.