Date on Master's Thesis/Doctoral Dissertation

5-2018

Document Type

Master's Thesis

Degree Name

M.S.

Department

Pharmacology and Toxicology

Degree Program

Pharmacology and Toxicology, MS

Committee Chair

Matoba, Nobuyuki

Committee Co-Chair (if applicable)

Ceresa, Brian

Committee Member

Ceresa, Brian

Committee Member

Siskind, Leah

Committee Member

Yaddanapudi, Kavitha

Committee Member

Li, Chi

Author's Keywords

cholera toxin b; CTB; wound healing; ulcerative colitis; plant-made pharmaceutical

Abstract

Cholera toxin B subunit (CTB) is a mucosal immunomodulatory protein that induces robust mucosal and systemic antibody responses. This well-known biological activity has been exploited in cholera prevention (as a component of Dukoral® vaccine) and vaccine development for decades. On the other hand, several studies have investigated CTB’s immunotherapeutic potential in the treatment of inflammatory diseases such as Crohn’s disease and asthma. Furthermore, we recently found that a plant-made variant of CTB (CTBp) could induce colon epithelial wound healing in mouse colitis models. In this thesis, it is revealed that the wound healing effects are unique to the plant-made variant, as it has an ER retention signal KDEL sequence that provides the protein with new functions. This was determined by investigating how the C-terminal KDEL sequence contributes to the protein’s wound healing activity in vivo, in vitro, and ex vivo. In a mouse model of dextran sodium sulfate (DSS)-induced colitis, CTBp, but not CTB, mitigated colitis as characterized by lower disease activity index and inflammation scores, colon shrinkage protection, blunted escalation of blood leukocyte levels, and observable histological epithelial restitution. In vitro, a Caco2 cell wound healing model revealed CTBp’s epithelial healing activity, intracellular retention, and unique signaling pathways that were reliant on the protein’s KDEL sequence. It was determined that, upon internalization of CTBp, the KDEL sequence enables ER colocalization and retention of the protein, leading to the activation of the inositol-requiring enzyme 1 (IRE1)/X-box binding protein 1 (XBP1) arm of unfolded protein response and subsequent TGFβ signaling. Lastly, using a ulcerative colitis (UC) patient colon explants, CTBp’s therapeutic potential was evaluated, which demonstrated efficacy as manifested by the induction of TGFB gene expression, upregulation of wound healing pathways and presence of viable crypts in the mucosa. In summary, CTBp exhibits unique colon mucosal would healing effects that are mediated by its colocalization to the ER and subsequent activation of IRE1/XBP1 signaling in colon epithelial cells. Furthermore, the results presented herein provide implications for the unique therapeutic potential of CTBp that may address a significant unmet need in UC treatment.

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