10 A VARIANT OF CHOLERA TOXIN B SUBUNIT ENHANCES COLON EPITHELIAL WOUND HEALING VIA AN UNFOLDED PROTEIN RESPONSE

Abstract

Cholera toxin B subunit (CTB) exhibits multifaceted biological activity in the mucosa via binding to GM1 ganglioside on intestinal epithelial cells. We recently found that a recombinant variant of CTB, which contains an artificial KDEL endoplasmic reticulum (ER) retention signal (CTB-KDEL), induces mucosal healing in a mouse dextran sodium sulfate (DSS) acute colitis model. The objectives of this study are to investigate CTB-KDEL’s epithelial wound repair mechanism and characterize its efficacy in preclinical models. We hypothesize that CTB-KDEL’s wound healing effect is mediated through ER retention within epithelial cells, thereby facilitating mucosal healing in IBD. The effects of CTB-KDEL, CTB and G33D-CTB-KDEL (a non-GM1-binding variant) were analyzed for cell migration, subcellular localization, an unfolded protein response (UPR) and TGFβ expression in Caco2 cells. In a DSS acute colitis model, C57BL/6J mice were gavaged with a single dose of CTB-KDEL, CTB or PBS at the end of 7-day DSS exposure. An optimal dosing regimen of CTB-KDEL was evaluated in mice exposed to repeated DSS cycles. Efficacy was measured on the basis of colon length, Disease Activity Index, histopathological scoring, E-cadherin immunohistochemistry and wound healing gene/cytokine expression in the colon. For ex vivohuman colonic explant experiments, colon tissues were obtained from consenting IBD patients who underwent total colectomy, which were cultured with CTB-KDEL, CTB or a PBS for 24 hours. Caco2 cell migration was enhanced by CTB-KDEL at concentrations of 0.3 – 3 μM, while CTB and G33D-CTB-KDEL failed to show such an effect. CTB-KDEL showed longer residence in the ER than CTB, leading to the activation of UPRs, particularly the inositol-requiring enzyme 1 (IRE1)/X-box binding protein 1 (XBP1) arm, and subsequent TGFβ signaling in Caco2 cells. In the acute colitis model, CTB-KDEL, but not CTB, facilitated recovery from colonic damage as evident in macroscopic and histological evaluations, with significant upregulation of wound healing genes and E-cadherin positive staining in the colon (Fig. 1). Weekly oral administration of 3 μg CTB-KDEL showed significant efficacy against established colitis in the chronic DSS model. In human IBD colon explants, CTB-KDEL at 1 μM induced significant upregulation of the IRE1/Xbp1 pathway as well as wound healing genes including TGFB1and the E-cadherin gene CDH1, along with the retention of viable crypts in the colon. The results demonstrated CTB-KDEL’s unique colon epithelial wound healing activity via a UPR, providing implications for new IBD treatment strategies.