Role of DLL4 in Neutrophil Aging and Impaired Clearance in Sepsis

Sepsis remains a major cause of mortality, largely due to acute lung injury (ALI) driven by excessive neutrophil mediated inflammation. We recently identified a pathogenic subset of DLL4⁺ neutrophils that expands in inflamed lungs and promotes ALI, yet the underlying mechanisms remain unclear. Here, we investigated how DLL4⁺ neutrophils modulate alveolar macrophage (AM) function to exacerbate lung injury. AMs were treated with DLL4⁺ neutrophils or recombinant DLL4 (rmDLL4), and macrophage polarization was assessed by flow cytometry. Conditioned medium was subsequently applied to bone marrow-derived neutrophils (BMDNs) to evaluate neutrophil aging and CD47 expression (don't eat me signal on neutrophils). We showed that DLL4⁺ neutrophils activate the Notch1 signaling pathway in AMs, promoting toward a proinflammatory M1 phenotype. M1 cells markedly upregulate leukotriene B4 (LTB4) production through 5-lipoxygenase activation (5-LOX), inducing the accumulation of aged neutrophils (CXCR4^hiCD62L^low). These aged neutrophils markedly increase CD47 expression, leading to impaired macrophage mediated phagocytosis and amplified lung inflammation. Importantly, we developed a novel DLL4-Notch1 inhibitory peptide (NDI) that reprograms AMs toward an anti-inflammatory M2 phenotype, reduces LTB4 release and aged neutrophils, lowers CD47 expression in a murine model of sepsis. Together, we identify DLL4⁺ neutrophils as key orchestrators of macrophage dysregulation in sepsis-induced ALI and demonstrate NDI may represent a potential therapeutic candidate in sepsis-induced ALI.