P-162 Core 1- and Core 3-Derived Mucin-Type O-Glycans Collectively Maintain the Colonic Mucus Barrier and Protect Against Spontaneous Colitis in Mice

Abstract

Background: The colon is covered with a thick mucus layer, which is at the interface between gut bacteria and mucosal epithelial and immune cells. Mucus consists mainly of O-linked oligosaccharides (O-glycans), and defects in O-glycosylation have been reported in ulcerative colitis (UC) patients with unclear etiology. Core 1- and core 3-derived mucin-type O-glycans are primary components of the mucus layer and are important for mucus function. However, how both types of O-glycans maintain mucus barrier function to preserve hostmicrobe homeostasis throughout the colon is unclear. Methods: We analyzed mRNA expression of C1GalT1 and C3GnT, which respectively control biosynthesis of core 1 or 3 O-glycans, in the colon. We compared mucus barrier function, structure, thickness, glycosylation status, and colitis susceptibility between WT mice and mice lacking core 3 (C3GnT-/-), intestinal core 1 (IEC C1galt1-/-), and both intestinal core 1- and 3-derived O-glycans (DKO). This was performed using various histologic approaches, immunostaining for Muc2 and Tn-antigen, lectin staining, and fluorescent in situ hybridization (FISH) for bacteria. Mucins from WT and DKO mice were additionally isolated and analyzed by composite agarose-polyacrylamide gel electrophoresis to measure mucin integrity in the presence or absence of bacteria and bacterial-derived proteases. Results: C1GalT1 expression was high and ubiquitous throughout the colon, whereas C3GnT was highly expressed in the proximal colon. Consistent with this differential expression, IEC C1galt1-/- mice develop spontaneous colitis primarily in the distal colon, whereas DKO mice developed spontaneous colitis in both distal and proximal colon. In neonates (7-21 day-old), DKO mice showed an early onset and more severe colitis than IEC C1galt1-/- mice, which correlated with a more rapid dissolution of the distal colon mucus layer and increased contact of bacteria with the mucosa. Further, mice with inducible deletion of O-glycans in adult ages (8-12 weeks) showed impaired mucus layer prior to spontaneous colitis. These results indicate that an impaired mucus barrier due to O-glycosylation defects alone plays a causative role in colitis. Antibiotic treatment led to rescue of the inner mucus layer in DKO mice prior to colitis resolution, and DKO mucins were more susceptible to proteolytic attack versus WT mucins. These data support that the mucus layer defect in DKO mice is due to its increased susceptibility to microbiota-mediated mucolytic activity but not inflammation. Finally, microbiota-dependent mucus breakdown of O-glycan-deficient mucus drives spontaneous colitis since antibiotic treatment also ameliorated colitis in DKO mice. Conclusions: Our results indicate that both core 1- and 3-derived O-glycans collectively contribute to mucus barrier function by protecting it from degradation by bacterial-derived proteases. Moreover, mice lacking intestinal core-1 and 3-derived O-glycans represent a robust, highly reproducible model of severe spontaneous UC-like colitis to investigate both causes of, and novel therapeutic strategies for, IBD.