IL-33 Expands M2 Macrophages population and Restrains Inflammatory Monocyte Recruitment During Clostridioides difficile Infection 2332432

Abstract

Abstract Introduction 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. Funding Source R01 AI152477 Topic Categories Innate Immune Responses and Host Defense: Cellular Mechanisms (INC)