Date of Award

Spring 3-31-2026

Embargo Period

4-16-2028

Document Type

Thesis - MUSC Only

Degree Name

Master of Biomedical Science

Department

Regenerative Medicine and Cell Biology

College

College of Graduate Studies

First Advisor

Melinda Engevik

Abstract

Background: Clostridioides difficile (C. difficile) is a toxin-producing enteric pathogen that causes severe diarrhea and inflammation. During infection, intestinal mucins play an essential role in epithelial protection and wound repair. Previous studies have shown that the cytokine IL-22 regulates adherent mucin expression during Citrobacter rodentium infection, but the relationship between IL-22 and adherent mucins, such as MUC13, during C. difficile infection remains unclear. We hypothesized that C. difficile infection stimulates IL-22 production, promoting transcription of the adherent mucin MUC13 in the colon. Methods and Results: To model infection, antibiotic-treated mice were gavaged with PBS (vehicle control) or C. difficile, and disease progression was monitored for 10 days. RNA sequencing identified differentially expressed genes at days 3, 4, 5, 8, and 10 post-infection, while immunostaining and histological analyses were performed at day 3. Infected mice exhibited significant weight loss, peaking at day 3 post-infection. At this time point, infected mice showed increased colonic IL-22 expression and elevated serum IL-22 protein compared to controls. Correspondingly, MUC13 expression and protein levels were significantly increased in the colon, as demonstrated by RNAseq and immunostaining. To determine whether IL-22 directly regulates MUC13, human and murine colonic cells and organoids were treated with IL-22. IL-22 treatment consistently increased MUC13 expression across all models, indicating that IL-22 is sufficient to induce MUC13 production. Since IL-22 is primarily produced by type 3 innate lymphoid cells (ILC3s), we next tested whether C. difficile toxins directly stimulate IL-22 production. Treatment of MNK3 (ILC3) cells with toxins failed to induce IL-22 secretion. However, macrophage-derived IL-1β is known to stimulate IL-22 production. Peritoneal macrophages exposed to C. difficile toxin B (TcdB) generated IL-1β, and conditioned macrophage supernatant or recombinant IL-1β induced robust IL-22 production in MNK3 cells. These findings suggest that C. difficile toxins indirectly promote IL-22 production through macrophage-derived IL-1β signaling. Finally, to evaluate therapeutic potential, mice were gavaged with engineered Limosilactobacillus reuteri designed to release IL-22. Treatment significantly reduced intestinal damage, enhanced mucus barrier integrity, and decreased inflammation. Conclusions: Together, these results indicate that IL-22-mediated upregulation of MUC13 represents a potential therapeutic strategy to limit tissue damage during C. difficile infection.

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Copyright is held by the author. All rights reserved.

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Available for download on Sunday, April 16, 2028

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