Single-cell RNA sequencing reveals the role of neutrophils in intestinal ischemia-reperfusion injury in mice.

Abstract

OBJECTIVE: Intestinal ischemia-reperfusion injury (IRI) severely compromises the mucosal barrier. While neutrophils are key responders, their specific roles and mechanisms remain unclear. This study aims to delineate the functional heterogeneity of neutrophils in intestinal IRI and identify the underlying molecular pathways. METHODS: A murine model of intestinal IRI was established by subjecting C57BL/6 mice to 45 min of ischemia and 4 h of reperfusion. Single-cell RNA sequencing (scRNA-seq) was performed on intestinal tissues. Neutrophil depletion, co-culture assays, and pharmacological modulation of endoplasmic reticulum stress (ERS) were employed for functional validation. RESULTS: scRNA-seq analysis revealed a significant expansion of neutrophils and inflammatory monocytes alongside a reduction in lymphocytes post-IRI. Notably, we identified a distinct pro-inflammatory neutrophil subcluster (C5), characterized by high expression of Atf4 and ERS-related genes. In vivo neutrophil depletion markedly alleviated intestinal damage, evidenced by improved histology, reduced serum DAO and IL-6 levels, and preserved Occludin expression. Mechanistically, neutrophils were shown to exacerbate injury via the ERS pathway. Pharmacological induction (tunicamycin) or inhibition (4-PBA) of ERS significantly augmented or mitigated IRI severity, respectively, with ATF4 playing a central role. CONCLUSION: This study identifies a pathogenic neutrophil subpopulation that drives intestinal IRI through the ATF4-mediated ERS pathway. These findings provide new insights into the immunopathology of IRI and highlight ATF4 or specific neutrophil subsets as potential therapeutic targets for mitigating intestinal damage.