Identification and validation of hub genes associated with neutrophil immunosuppression during Sepsis

Sepsis involves a transition from hyperinflammation to immunosuppression, with neutrophils playing a central role in both phases. However, the molecular basis of neutrophil-driven immunosuppression remains unclear. This study aimed to identify and validate hub genes regulating neutrophil immunosuppression in sepsis to uncover potential therapeutic targets.

Neutrophil transcriptomic datasets (GSE64457, GSE180387) from the GEO database were analyzed using WGCNA to identify modules correlated with sepsis progression, followed by GO/KEGG enrichment and PPI network construction. Candidate hub genes were validated through single-cell RNA sequencing of human peripheral blood neutrophils. A cecal ligation and puncture (CLP) murine sepsis model was further used to evaluate neutrophil-mediated immunosuppression via CD4⁺ T cell co-culture assays, with hub gene expression confirmed by Western blot analysis and qPCR.

WGCNA identified immune-related modules strongly associated with sepsis progression, enriched in cytokine signaling, immune receptor activity, and neutrophil degranulation. Intersection analysis revealed 34 common genes, and PPI networks prioritized three hub genes: NFKBIZ, TNFAIP2, and CTSS. Single-cell analysis demonstrated distinct expression of these hub genes in immunosuppressive versus non-immunosuppressive neutrophils. In the CLP model, late-stage neutrophils (day 5) significantly suppressed CD4⁺ T cell proliferation compared to early-stage neutrophils (day 1, p < 0.01). Protein validation confirmed upregulation of NFKBIZ and downregulation of TNFAIP2 and CTSS in late-stage sepsis.

Integrated bioinformatics and experimental validation identified NFKBIZ, TNFAIP2, and CTSS as potential regulators of neutrophil immunosuppression in sepsis. Their dynamic expression highlights key mechanistic features associated with immune dysfunction and suggests new targets for reversing sepsis-induced immunoparalysis.