A pro-inflammatory neutrophil subpopulation drives intestinal ischemia-reperfusion injury via the ATF4-mediated endoplasmic reticulum stress pathway.

This study investigated the role of neutrophils in intestinal ischemia–reperfusion injury (IRI) in mice. We combined single-cell RNA sequencing (scRNA-seq) with in vivo and in vitro functional assays to characterize cellular dynamics. Single-cell RNA sequencing of IRI model tissue revealed a significant increase in neutrophils and inflammatory monocytes, alongside a decrease in T cells, B cells, and NK cells. In vivo neutrophil depletion markedly alleviated intestinal damage, as indicated by reduced serum diamine oxidase (DAO) and IL-6 levels, improved histopathological scores, and preserved Occludin protein integrity. Mechanistically, scRNA-seq identified a pro-inflammatory neutrophil subcluster (C5) characterized by enrichment of endoplasmic reticulum stress (ERS) markers, particularly the transcription factor ATF4. In vitro and in vivo studies confirmed that neutrophils exacerbate IRI severity by inducing ERS via the ATF4 pathway. Pharmacological inhibition of ERS or genetic ablation of ATF4 significantly attenuated neutrophil-driven inflammation and mucosal injury. These findings demonstrate that a specific neutrophil subpopulation aggravates intestinal IRI through the intrinsic ERS/ATF4 pathway, providing a novel perspective on IRI pathophysiology and highlighting a potential therapeutic target for mitigating intestinal damage. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1038/s41598-026-36938-9.