A10 DEFINING A ROLE FOR GOBLET CELL MUCUS-ASSOCIATED PROTEINS IN INNATE HOST DEFENSE

MUC2 mucin secreted by goblet cells is first line of innate host defense separating the single layer of epithelial cells that lines the gut. Mucus serves as a substrate and food source for bacteria and creates a viscous physical barrier between the lumen and the underlying epithelial cells. Even though MUC2 mucin is important in health and disease, there are numerous other mucus-associated proteins (MAPs) that are uncharacterized includingcalcium-activated chloride channel regulator 1 (CLCA1), Fcfragment of IgG binding protein (FCGBP) and kallikrein 1 (KLK1). The functions of these MAPs are not known but are believed to stabilized MUC2 and/or aid in their protective functions. The goal of this study is to functionally characterize MAPs to define their role in mucosal homeostasis and host defense. Hypothesis: MUC2 mucin and MAPs play important roles in innate host defense. The specific aims are: 1. To determine if MUC2 mucin regulates the expression and production of MAPs 2. To quantify a role for MAPs in colitis To determine if high MUC2 mucin production regulates the expression of MAPs, human HT29/LS174T goblet and CRISPR/Cas9 MUC2 KO cells were used. QPCR and Western blotting were used to determine changes at the mRNA and protein level, respectively. HT29 WT and CRISPR/Cas9 MUC2 KO cells were mechanically wounded in vitroto model epithelial injury. Muc2+/+and Muc2-/-littermates were used to basally characterize the expression of MAPs and in response to DSS-induced colitis. The temporal expression ofMUC2, FCGBP, CLCA1and KLK1in WT and CRISPR/Cas9 HT29/LS174T MUC2 KO cells peaked at day 5 and remained high up to day 9. Similar basal mRNA expressions were noted from healthy human colonic biopsy specimens that were significantly decreased in active UC but restored to basal levels in UC in remission. In wounded HT29 CRISPR/Cas9 MUC2 KO cells as early as day 2, FCGBP, CLCA1 andKLK1were upregulated as compared to WT cells. In Muc2+/+and Muc2-/-littermates and organoids derived from Muc2+/+and Muc2-/-colons, MAPs were basally expressed at similar levels. However in DSS colitis, MAPs mRNA transcripts and protein levels significantly decreased steadily up to day 15 in Muc2+/+but not Muc2-/-littermates. At the height of colitis (days 8–12) Clca1expression disappeared and Fcgbp, Clca1 and Klk1were not restored during restitution (day 15). This study demonstrates that the presence of MUC2 mucin and MAPs are not mucus-associated or developmentally regulated but highly susceptible to DSS colitis and active UC. CIHR

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