Intestinal epithelial pH-sensing receptor GPR65 maintains mucosal homeostasis via regulating antimicrobial defense and restrains gut inflammation in inflammatory bowel disease

Intestinal epithelial cell (IEC) regulation of barrier function and mucosal homeostasis enables the establishment of a harmonious gut microenvironment. However, host-derived regulatory networks that modulate intestinal antimicrobial defenses have not been fully defined in intestinal homeostasis and inflammation. We generated mice with IEC-specific deletion of Gpr65 and investigated the role of epithelial GPR65 using DSS- and C. rodentium-induced murine colitis models. RNA sequencing analysis was conducted on colonic IECs from mice with IEC-specific deletion of Gpr65 and control littermates. Colonoids and colonic epithelial cell lines were used to evaluate the pH-sensing effect of GPR65. The expression of GPR65 was determined in IECs from IBD patients and DSS colitis mice by qRT-PCR, Western blot, and immunohistochemistry. We observed that the absence of GPR65 in IECs abrogated homeostatic antimicrobial programs, including the production of antimicrobial peptides (AMPs) and defense response-associated proteins. Mice lacking epithelial GPR65 displayed dysbiosis of the gut microbiota and increased susceptibility to DSS- and C. rodentium-induced colitis, as characterized by significantly disrupted epithelial antimicrobial responses, pathogen invasion, and increased inflammatory infiltrates in the inflamed colon. RNA sequencing analysis revealed that deletion of GPR65 in IECs provoked dramatic transcriptome changes with respect to the downregulation of immune and defense responses to bacteria. Forced AMP induction assays conducted in vivo or in ex vivo colonoids revealed that IEC-intrinsic GPR65 signaling drove antimicrobial defense. Mechanistically, GPR65 signaling promotes STAT3 phosphorylation to optimize mucosal defense responses. Epithelial cell line and colonoid assays further confirmed that epithelial GPR65 sensing pH synergizes with IL-22 to facilitate antimicrobial responses. Finally, the expression of GPR65 was markedly decreased in the inflamed epithelia of IBD patients and DSS colitis mice. Our findings define an important role for epithelial GPR65 in regulating intestinal homeostasis and mucosal inflammatory response and point towards a potential therapeutic approach by targeting GPR65 in the treatment of IBD.