Olfactomedin-4 ⁺ neutrophils exacerbate intestinal epithelial damage in <i>Clostridioides difficile</i> infection

Neutrophils are dominant cells during acute immune response to <i>Clostridioides difficile</i> infection (CDI). A higher number of infiltrating colonic neutrophils is clearly linked to greater tissue damage and severe CDI (3, 4). However, the mechanism(s) by which neutrophils exacerbate tissue damage in CDI remain unknown. We investigated the role of a neutrophil subset marked by Olfactomedin-4 expression (OLFM4⁺ neutrophils) during CDI. Single-cell transcriptomics reveal that <i>Olfm4</i> is increased in blood neutrophils of infected mice, and these cells exhibit gene signatures characterized by high expression of degranulation genes. In <i>C. difficile</i>-infected mice, OLFM4⁺ neutrophils aggregate to areas of severe intestinal epithelial cell (IEC) damage, and plasma OLFM4 was significantly increased in both <i>C. difficile</i>-infected mice and patients. <i>In vitro</i>, OLFM4⁺ neutrophils and recombinant OLFM4 protein exacerbated <i>C. difficile</i> toxin-induced IEC damage. In sum, our studies provide novel insights into neutrophil-mediated pathology and highlight the role of OLFM4⁺ neutrophils in worsening CDI-induced IEC damage.