DLL4+ neutrophils induce alveolar macrophages to cause ADAM-17-mediated endothelial barrier disruption and the increase of ICAM1hiCXCR1lo neutrophils in sepsis.

Sepsis remains a leading cause of death. Reverse migrated neutrophils, characterized as ICAM1hiCXCR1lo, have been recognized as a key driver of systemic inflammation and organ injury in sepsis. We discovered a distinct delta-like ligand 4-positive (DLL4+) subset of neutrophils that accumulate in the lungs, contributing to lung injury; however, the underlying mechanism is less understood. In sepsis, ICAM1hiCXCR1lo neutrophils, being hyperactive, are detrimental. Here, we investigated how DLL4+ neutrophils activate alveolar macrophages (AMs) to cause endothelial cell barrier disruption and promote neutrophil reverse migration. AMs were treated with DLL4+ neutrophils or recombinant mouse DLL4, and a disintegrin and metalloprotease (ADAM17) generated by AMs was assessed at both mRNA and protein levels. Conditioned medium was subsequently applied to pulmonary vascular endothelial cells (pVECs); junctional adhesion molecule-C (JAM-C) protein was detected by Western blot assays; and ICAM1hiCXCR1lo neutrophils were detected by flow cytometry. We demonstrate that during sepsis induced by cecal ligation and puncture (CLP), DLL4+ neutrophils interact with AMs via the Notch1 pathway, leading to increase of ADAM17 expression. ADAM17 decreased JAM-C on pVECs, causing endothelial barrier disruption and ICAM1hiCXCR1lo neutrophil generation. A small-molecule inhibitor of ADAM17 effectively preserved pulmonary endothelial barrier integrity and reduced ICAM1hiCXCR1lo neutrophil accumulation. Importantly, we have developed a novel DLL4-Notch1 inhibitory peptide (NDI) that effectively suppresses ADAM17 expression, restores JAM-C, and reduces ICAM1hiCXCR1lo neutrophil accumulation in sepsis. These findings identify DLL4+ neutrophils as critical inflammatory mediators that exacerbate systemic inflammation and worsen sepsis, and highlight the DLL4-Notch1-ADAM17 axis as a promising therapeutic target.