(469) IL-33 Expands M2 Macrophages population and Restrains Inflammatory Monocyte Recruitment During Clostridioides difficile Infection

Introduction/Rationale: Antibiotic exposure predisposes to Clostridioides difficile infection (CDI) by disrupting intestinal immune homeostasis. In the colon, TRMs are defined as CD11b⁺ Ly6C⁻ MHCII⁺ CD4⁺ TIM4⁺ macrophages and represent a long-lived population adapted to the intestinal microenvironment. We previously showed that IL-33 promotes a protective type-2 immune program during CDI by activating colonic ILC2s that limit epithelial damage and mortality. However, how antibiotics and IL-33 signaling influence TRMs and related macrophage programs during CDI remains incompletely understood.

Methods: Using spectral flow cytometry, we analyzed colonic immune populations at day 2 post-CDI. Uninfected mice with or without antibiotic treatment served as controls.

Results: CDI significantly reduced TRMs and was associated with decreased CX3CR1 expression across the CD11b⁺ CD64+ Ly6C⁻ MHCII⁺ macrophage compartment, indicating disruption of resident macrophage identity. Within this macrophage pool, IL-33 selectively expanded CD4⁺ TIM4⁻ macrophages, an intermediate population with both tissue-resident and monocyte-derived features. IL-33 also enhanced CD206 expression, consistent with a reparative and regulatory macrophage program, and significantly increased F4/80⁺ macrophages compared to PBS-treated infected controls, suggesting preservation of mature, tissue-adapted macrophages. In contrast, CDI induced a marked accumulation of Ly6Chi inflammatory monocytes in PBS-treated mice, which was significantly reduced following IL-33 administration.

Conclusion: Collectively, these findings demonstrate that antibiotics and CDI disrupt defined TRM populations, while IL-33 reshapes macrophage composition toward immune-regulatory and tissue-repair phenotypes, promoting restoration of intestinal immune balance during CDI.