Single-cell transcriptomics identifies NF-κB/STAT3-associated neutrophil inflammatory reprogramming in rheumatoid arthritis synovium

To elucidate neutrophil heterogeneity in rheumatoid arthritis (RA) synovium, we performed single-cell RNA sequencing on 8 RA patients (66,539 cells), stratified by neutrophil infiltration status. Infiltrating neutrophils predominantly displayed inflammatory C1 (Chemokine + ) and C3 (S100A8/A9 + ) phenotypes. Co-expression analysis (hdWGCNA) identified two functional modules (M3/M4) enriched in NF-κB and type I interferon signaling. Furthermore, these neutrophils actively reshaped the synovial microenvironment via CXCL, IL1, and MIF communication pathways. Transcriptional inference and comparative in silico knockouts revealed the NF-κB/STAT3 axis as a candidate regulatory node, with NFKB1 uniquely perturbing S100A8/A9 effector genes. Key findings, including M4 module activation and its correlation with systemic inflammatory markers (ESR/CRP), were validated in an independent bulk RNA-seq cohort ( n = 19). This study comprehensively characterizes RA neutrophil inflammatory reprogramming and nominates the NF-κB/STAT3 axis for future functional validation. • Inflammatory C1/C3 neutrophil subsets dominate infiltrated RA synovium. • hdWGCNA identifies NF-κB/IFN-driven functional modules in these neutrophils. • Neutrophils actively reshape the synovial niche via CXCL, IL1, and MIF pathways. • In silico knockout reveals NFKB1 uniquely regulates S100A8/A9 effector genes. • Independent cohort links the M4 module to systemic inflammatory markers in RA.