EVALUATING THE ROLE OF GOBLET CELL ASSOCIATED ANTIGEN PASSAGES (GAPS) IN THE DEVELOPMENT OF MUCOSAL IMMUNE TOLERANCE IN THE CFTR KO INTESTINE

Abstract

Abstract Greater than 70,000 individuals worldwide are living with the monogenetic disease cystic fibrosis (CF). The development of chronic intestinal inflammation, with clinical signs resembling inflammatory bowel disease-like conditions, is a common yet poorly understood occurrence in CF patients. This inflammation is typically neutrophilic in human and animal models with a heightened basal pro-inflammatory cytokine release. Prior research utilizing intestinal organoids (enteroids) cultured from Cftr knockout mice has shown that goblet cells in the CF mouse intestine demonstrate defective clearance of mucin granules and abnormal mucus retention. Goblet cell-associated antigen passages (GAPs), located in the small intestine and colon, deliver intraluminal antigens to antigen-presenting dendritic cells in the submucosa. This mechanism serves as an important step in the development and maintenance of tolerogenic dendritic cell populations expressing receptors to luminal antigens with involvement of regulatory T cell activation and release of IL-10. We hypothesized that mucus plugging of goblet cells in the CF intestine leads to defective GAP formation and a consequent decrease in the expansion of tolerogenic dendritic cells. To test this hypothesis, Cftrtm1Unc (Cftr KO) and wild type (WT) sex-matched littermate pairs (n=2) maintained on a commercially available liquid diet (Peptamen®) were anesthetized with ketamine/xylazine for a laparotomy to inject a luminal fluorescent 10kD dextran dye into the mid-jejunum. After 30 min, the mice were euthanized with CO2, and the intestine was collected for immunofluorescent staining to evaluate GAP formation. In the WT intestine, the dextran dye was observed within goblet cells outlined by CK18 immunofluorescence, a goblet cell marker. Punctate dextran dye was observed in the submucosa, suggestive of dendritic cell uptake. In contrast, the Cftr KO mice demonstrated defective GAP formation, i.e., without dye penetration of goblet cells, and the lack of punctate dextran fluorescence in the submucosa. To evaluate the population of tolerogenic dendritic cells, small intestinal segments from Cftr KO-WT sex-matched littermate pairs (3-female and 2-male pairs) were collected for FACS sorting of submucosal CD103+ (tolerogenic) and CD103- (pro-inflammatory) dendritic cells. The WT mice had a significantly higher population of CD103+ tolerogenic dendritic cells compared to the CF mice (WT: 20.5+/-2, CF: 9.2+/-3, P < 0.006). A trend towards an increase in CD103- dendritic cells was seen in the CF intestine. In summary, the CF mice were found to have defective intraluminal antigen transfer through the GAP pathway and a significant decrease in tolerogenic dendritic cells in the intestine.