(425) Regulation of Neutrophil Heterogeneity and Effector Functions by the NLRP3 Inflammasome in Sepsis

Introduction/Rationale: 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.