A modified cholera toxin B subunit containing an ER retention motif enhances colon epithelial repair via an unfolded protein response

Abstract

Cholera toxin B subunit (CTB) exhibits broad-spectrum biologic activity upon mucosal administration. Here, we found that a recombinant CTB containing an endoplasmic reticulum (ER) retention motif (CTB-KDEL) induces colon epithelial wound healing in colitis via the activation of an unfolded protein response (UPR) in colon epithelial cells. In a Caco2 cell wound healing model, CTB-KDEL, but not CTB or CTB-KDE, facilitated cell migration via interaction with the KDEL receptor, localization in the ER, UPR activation, and subsequent TGF-b signaling. Inhibition of the inositol-requiring enzyme 1/X-box binding protein 1 arm of UPR abolished the cell migration effect of CTB-KDEL, indicating that the pathway is indispensable for the activity. CTB-KDEL's capacity to induce UPR and epithelial restitution or wound healing was corroborated in a dextran sodium sulfate-induced acute colitis mouse model. Furthermore, CTB-KDEL induced a UPR, up-regulated wound healing pathways, and maintained viable crypts in colon explants from patients with inflammatory bowel disease (IBD). In summary, CTB-KDEL exhibits unique wound healing effects in the colon that are mediated by its localization to the ER and subsequent activation of UPR in epithelial cells. The results provide implications for a novel therapeutic approach for mucosal healing, a significant unmet need in IBD treatment.-Royal,

Repeated Oral Administration of a KDEL-Tagged Recombinant Cholera Toxin B Subunit Effectively Mitigates DSS Colitis despite a Robust Immunogenic Response

“…The principal virulence factor of Vibrio cholerae is cholera toxin (CT), which is comprised of a toxic A subunit and a non-toxic homopentameric B subunit (CTB). Recently, we have shown that a recombinant variant of CTB containing a KDEL endoplasmic reticulum (ER) retention motif (CTB-KDEL) has the ability to induce mucosal healing, facilitate colon epithelial wound repair, and enhance recovery from an acute dextran sulfate sodium (DSS) colitis model in mice [1,2]. These effects were attributed to the addition of the C-terminal ER retention motif, KDEL, to CTB.…”

A recombinant cholera toxin b subunit variant (CTB-KDEL) exhibits unique colon mucosal healing effects that have therapeutic implications for inflammatory bowel disease.

“…Upon ER localization, CTB-KDEL induced an unfolded protein response (UPR) and subsequent TGFβ signaling. In particular, the inositol-requiring enzyme 1 (IRE1)/X-box binding protein 1 (XBP1) arm of the UPR was indispensable for wound healing activity[147]. These findings were corroborated in primary mouse colon epithelial cells, where CTB-KDEL induced an UPR, while CTB and the non-GM1 binding mutant G33D-CTB-KDEL failed to exhibit such an effect.…”

“…These findings were corroborated in primary mouse colon epithelial cells, where CTB-KDEL induced an UPR, while CTB and the non-GM1 binding mutant G33D-CTB-KDEL failed to exhibit such an effect. Moreover, in an ex vivo experiment using colectomy tissue from inflammatory bowel disease (IBD) patients, CTB-KDEL, but not CTB, induced an UPR, upregulated wound healing pathways, and maintained viable crypts[147]. These findings provide implications for the potential use of CTB-KDEL in the treatment of IBD.…”

“…In conclusion, the data herein reveal that CTB-KDEL exhibits unique colon mucosal would healing effects that are mediated by its localization in the ER via KDELR and subsequent activation of a UPR and TGFβ signaling in colon epithelial cells. Further investigation of the role of the UPR in TGFβ activation in colon epithelial cells may shed light on the mechanistic link to epithelial restitution and repair, facilitating the development of a new agent for the treatment of inflammatory diseases of the mucosa.CHAPTER 4: REPEATED ORAL ADMINISTRATION OF A KDEL-TAGGED RECOMBINANT CHOLERA TOXIN B SUBUNIT EFFECTIVELY MITIGATES DSS COLITIS DESPITE A ROBUST IMMUNOGENIC RESPONSE 44.1 IntroductionThe principal virulence factor of Vibrio cholerae is cholera toxin (CT), which is comprised of a toxic A subunit and a non-toxic homopentameric B subunit (CTB).Recently, we have shown that a recombinant variant of CTB containing a KDEL endoplasmic reticulum (ER) retention motif (CTB-KDEL) has the ability to induce mucosal healing, facilitate colon epithelial wound repair, and enhance recovery from an acute dextran sulfate sodium (DSS) colitis model in mice[50,147]. These effects were attributed to the addition of the C-terminal ER retention motif, KDEL, to CTB.…”

“…Thus, to address this immunogenicity question, specifically, the induction of ADAs, we used a mouse model of acute DSS colitis in which mice were orally administered with 30 µg of CTB-KDEL twice prior to 3% DSS exposure to induce an immunogenic response to the protein (the aforementioned vaccine dosing regimen[89]). At the end of DSS exposure, mice were orally administered 3 µg of CTB-KDEL; this single therapeutic dose has proven to be efficacious in C57BLl/6J mice[89,147]. The dosing regimens and groups are depicted inFigure 14.…”