Phenotypic profiling of neutrophils in acute Clostridioides difficile infection identifies a TNF-induced activation signature associated with epithelial damage

Neutrophils are key first responders in the host response to Clostridioides difficile infection (CDI). Although a high number of tissue and blood neutrophils clearly correlates with adverse outcomes in CDI patients, their functional role remains poorly defined. Using murine (CMT-93) and human (Caco-2) intestinal epithelial cell (IEC) lines co-cultured with neutrophils and a pre-clinical mouse model of CDI, we show that activated neutrophils exacerbate C. difficile-induced IEC injury. To identify neutrophil subtypes and biological pathways that contribute to CDI-associated IEC damage, we utilized single-cell RNAseq (scRNAseq) to generate the first transcriptomic atlas of murine bone marrow, blood, and colonic neutrophils in acute CDI. Our analyses identified a novel neutrophil population in the colonic lamina propria, "cNeu3," which exhibited high expression of genes associated with neutrophil activation and degranulation. Informed by cNeu3 signature genes from the scRNAseq dataset, we validated the presence of these neutrophils in mouse colon using flow cytometry (CD11bhiCD63hiIL-1βhiMIP-1αhiMALT1hi cells). We found that their abundance correlated with increased epithelial damage. Tumor Necrosis Factor (TNF) was sufficient to polarize mouse bone marrow neutrophils to a cNeu3 phenotype in vitro. Finally, TNF-primed human neutrophils worsened C. difficile toxin-induced IEC damage, which was improved when neutrophil degranulation was blocked. Collectively, our data provide novel insights into neutrophil-mediated pathology during CDI.