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? Methods and Results The colon mucus layer of mice deficient in the Muc2 mucin, Core 1 O-glycans, Tlr5, IL10 and Slc9a3 (Nhe3) together with dextran sulfate (DSS) treated mice was immunostained for Muc2 and the bacterial localization in the mucus was analyzed. All murine colitis models revealed bacteria in contact with the epithelium. Additional analysis of the less inflamed IL10−/− mice revealed a thicker mucus layer than WT, 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 biopsies. In contrast, mucus on colon biopsies of ulcerative colitis (UC) patients with acute inflammation had a highly penetrable mucus. Most UC patients in remission had similar to controls an impenetrable mucus layer. Conclusions Normal human sigmoid colon has an inner mucus layer impenetrable to bacteria. The colon mucus in animal models that spontaneously develop colitis and in UC patients with active disease allows bacteria to penetrate and reach the epithelium. Thus colon mucus properties can be modulated and suggest a novel model of UC pathophysiology.

SUMMARY The intestinal mucus layer provides a barrier limiting bacterial contact with the underlying epithelium. Mucus structure is shaped by intestinal location and the microbiota. To understand how commensals modulate gut mucus, we examined mucus properties under germ-free (GF) conditions and during microbial colonization. Although the colon mucus structure of GF mice was similar to conventionally raised (Convr) mice, the GF inner mucus layer was penetrable to bacteria-sized beads. During colonization, in which GF mice were gavaged with Convr microbiota, the small intestine mucus required five weeks to be normally detached and colonic inner mucus six weeks to become impenetrable. The composition of the small intestinal microbiota during colonization was similar to Convr donors until three weeks when Bacteroides increased, Firmicutes decreased, and segmented filamentous bacteria became undetectable. These findings highlight the dynamics of mucus layer development and indicate that studies of mature microbe-mucus interactions should be conducted weeks after colonization.

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.

Intestinal cells are covered by mucus. In the small intestine, a single unattached mucus is present whereas the colon has both an inner attached mucus layer and an outer loose mucus. The attached mucus of the colon is impenetrable to bacteria while the loose mucus acts as a habitat for commensal bacteria. In germ-free (GF) mice, small intestinal mucus is attached to the epithelium and the inner colon mucus is penetrable. O-glycosylation plays an important role in the host-microbiota interactions as the commensal bacteria use glycans as nutrient sources and attachment sites. While mucus protein composition is relatively homogenous along the intestine, its main component the Muc2 mucin shows regiospecific O-glycan patterns. We have now analyzed the glycosyltransferase relative concentrations in the epithelial cells along the intestine in GF and conventionally raised mice and compared this with the O-glycans formed. As Muc2 is the main O-glycosylated product in mucus, we made the simplified assumption that most of the glycosyltransferases found in the epithelial cells are involved in Muc2 O-glycan biosynthesis. The O-glycosyltransferase abundances along the intestine correlated well with the Muc2 O-glycan patterns. Some of the glycosyltransferases involved in the O-glycan elongation were decreased in GF mice, something that is in concordance with the observed shorter Muc2 O-glycans.

The heavily O-glycosylated mucin MUC2 constitutes the major protein in the mucosal layer that acts as a physical barrier protecting the epithelial layer in the colon. In this study, Muc2 was purified from mucosal scrapings from the colon of wild-type (WT) mice, core 3 transferase knockout (C3Gnt(-/-)) mice and intestinal epithelial cell-specific core 1 knockout (IEC C1Galt1(-/-)) mice. The Muc2 O-glycans were released by reductive β-elimination and analyzed with liquid chromatography-mass spectrometry in the negative-ion mode. Muc2 from the distal colon of WT and C3Gnt(-/-) knockout mice carried a mixture of core 1- or core 2-type glycans, whereas Muc2 from IEC C1Galt1(-/-) mice carried highly sialylated core 3- and core 4-type glycans. A large portion of NeuAc in all mouse models was positioned on disialylated N-acetyllactosamine units, an epitope not reported on human colonic MUC2. Mass spectra and proton NMR spectroscopy revealed an abundant NeuAc linked to internally positioned N-acetylglucosamine on colonic murine Muc2, which also differs markedly from human MUC2. Our results highlight that murine colonic Muc2 O-glycosylation is substantially different from human MUC2, which could be one explanation for the different commensal microbiota of these two species.