Siglec-F+ neutrophils are pro-inflammatory and exacerbate C. difficile colitis 2257588

Abstract Introduction Neutrophils exhibit a dual role in C. difficile infection (CDI), contributing to tissue homeostasis by containing pathogens while potentially causing tissue damage through excessive inflammatory mediator release. We identified a unique population of neutrophils expressing the eosinophil marker Siglec-F that rapidly accumulates in the colon during CDI. While Siglec-F+ neutrophils have been observed in other disease contexts, their specific role in CDI pathogenesis remains unclear. Methods We used CDI mouse models to characterize colon-infiltrating neutrophils. Flow cytometry and staining confirmed Siglec-F expression on mature neutrophils, distinguishing them from SiglecFhigh eosinophils. Functional depletion employed anti-Siglec-F antibodies in eosinophil-deficient mice. RNA sequencing compared gene expression profiles to identify differences. Further metabolic studies were done by using a Flow cytometry-based assay, SCENITH. Results We verified that Siglec-F+ cells are bona fide mature neutrophils distinct from eosinophils and other myeloid populations. These neutrophils demonstrated significant metabolic alterations (Glycolytic capacity, 5.153776494 %, 66.72 %) compared to conventional neutrophils. Depletion of Siglec-F+ neutrophils using anti-Siglec-F antibodies reduced CDI (p = 0.049) in eosinophil-deficient mice. RNA sequencing revealed that Siglec-F+ neutrophils exhibit enhanced phagocytic activity, increased extracellular trap formation, and elevated production of proinflammatory TH1-associated genes. Conclusion Siglec-F+ neutrophils represent a metabolically distinct neutrophil subset that contributes to CDI pathogenesis through enhanced inflammatory functions. Their depletion ameliorates disease severity, suggesting these cells play a detrimental role in CDI. This population may serve as a therapeutic target for managing severe CDI, as their unique metabolic profile and enhanced inflammatory capacity distinguish them from protective conventional neutrophils in the infected colon. Funding Source n/a Topic Categories Innate Immune Responses and Host Defense: Cellular Mechanisms (INC)