DLL4+ Neutrophils Induce Alveolar Macrophages to Cause ADAM-17-mediated Endothelial Barrier Disruption and the Increase of ICAM1hiCXCR1lo Neutrophils in Sepsis

Abstract Sepsis remains a leading cause of death. Reverse migrated (RM) neutrophils, characterized as ICAM1hiCXCR1lo, have been recognized as a key driver of systemic inflammation and organ injury in sepsis. We have recently discovered a distinct 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, were shown to be 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 (rmDLL4), 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 neutrophils generation. Small-molecule inhibitor of ADAM17 effectively preserved pulmonary endothelial barrier integrity, and reduced ICAM1hiCXCR1lo neutrophils accumulation. Importantly, we have developed a novel DLL4-Notch1 inhibitory peptide (NDI) that effectively suppresses ADAM17 expression, restores JAM-C, and reduces ICAM1hiCXCR1lo neutrophils accumulation in sepsis. These findings identify DLL4+ neutrophils as a critical inflammatory mediator that exacerbate systemic inflammation and worsen sepsis, highlight the DLL4-Notch1-ADAM17 axis as a promising therapeutic target.