Regulation of Neutrophil Heterogeneity and Effector Functions by the NLRP3 Inflammasome in Sepsis 2306743

Abstract Introduction Sepsis is a life-threatening condition marked by dysregulated inflammation and immunosuppression. Bacterial clearance relies on effector functions of neutrophils. Besides classic neutrophils (HDNs), inflammatory low-density neutrophils (LDNs) emerge in disease states, driving tissue damage and morbidity in sepsis. Although NLRP3 modulates neutrophil activity, its influence on neutrophil heterogeneity and effector functions remains elusive. Methods Sepsis model: Sepsis was induced in 8-12-week-old male C57BL/6 wild-type (WT) and Nlrp3-deficient (Nlrp3-/-) mice using the cecal ligation and puncture model. Functional Assays Neutrophil Extracellular Traps (NETosis) were quantified using SYTOX Green dye. Phagocytosis was measured using pHrodo-bacterial bioparticles., Single-cell RNA sequencing: Neutrophils were profiled using the 10X Genomics platform. Data were analyzed using CellRanger and Seurat. Data Analysis Results presented as Mean ± SEM. Data were analyzed using Student’s t-test or one-way ANOVA. Differences were considered statistically significant at p < 0.05. Results We found that Nlrp3 is not required for LDN/HDN recruitment in sepsis. However, the LDNs and HDNs from Nlrp3-deficient mice demonstrated reduced NETosis. In contrast, the Nlrp3-/- LDNs and Nlrp3-/- HDNs from the spleen and peritoneum, respectively, showed increased phagocytosis. Inhibition of NLRP3 in human LDNs and HDNs confirmed our mouse findings. scRNA-Seq analysis revealed that neutrophil subtype clustering is altered in Nlrp3-/- mice during sepsis. Conclusion NLRP3 is dispensable for LDN and HDN recruitment to tissues but essential for NETosis during sepsis. NLRP3 activation impairs bacterial phagocytosis. scRNA-Seq analysis revealed significant changes in LDN/HDN clusters in Nlrp3-/- mice, reflecting tremendous neutrophil heterogeneity and attenuation in pro-inflammatory genes. Human LDNs/HDNs analysis confirmed the findings from mouse neutrophils and suggests NLRP3 as a therapeutic target to improve sepsis outcomes. Funding Source R01AI157353 Topic Categories Innate Immune Responses and Host Defense: Cellular Mechanisms (INC)