NETosis-Dependent Generation of Immunodeficient Low-Density Neutrophils Exacerbates Sepsis-Induced Acute Lung Injury

The mechanisms underlying the generation of low-density neutrophils (LDNs), along with their phenotypic characteristics and role in organ injury during sepsis, remain poorly understood. This study utilized lipopolysaccharide (LPS) stimulation to mimic the septic microenvironment. LDNs and high-density neutrophils (HDNs) were isolated via density gradient centrifugation. Single-cell RNA sequencing, in vitro functional assays, and a cecal ligation and puncture (CLP) murine sepsis model were employed, alongside techniques including immunohistochemistry and flow cytometry, to investigate LDN heterogeneity and their role in sepsis-associated acute lung injury (ALI). Results demonstrated that LPS stimulation significantly increased the LDN proportion. Single-cell transcriptomics revealed substantial heterogeneity within LDNs, which exhibited a hyperactivated yet immunodeficient phenotype characterized by delayed apoptosis, impaired migration and phagocytosis, and a heightened capacity to suppress T-cell proliferation. In vivo, the NETosis inhibitor GSK484 reduced LDN generation and alleviated sepsis-associated ALI. In conclusion, sepsis induces the generation of immunodeficient LDNs via a NETosis-dependent pathway, which exacerbates lung injury. Targeting this pathway may represent a novel therapeutic strategy for sepsis.