Dysregulation of U12-Type Splicing in Lupus Neutrophils

Abstract Objective Neutrophil dysfunction is a hallmark of systemic lupus erythematosus (SLE), but its molecular basis remains unclear. This study explores transcriptional and post-transcriptional changes in low-density granulocytes (LDGs), a proinflammatory neutrophil subset expanded in SLE, focusing on NADPH oxidase (Nox) function and minor intron splicing. Methods LDGs and normal-density neutrophils (NDGs) were isolated from SLE patients and healthy controls (HCs). CYBA (P22phox) expression was evaluated at transcript and protein levels. Nox activity was measured using luminol assays. Bulk RNA sequencing and rMATS software were used to assess alternative splicing, particularly of U12-type intron-containing genes. Results CYBA expression was reduced in SLE LDGs (n=11) compared to SLE NDGs and HCs (n=6), with levels resembling those in chronic granulomatous disease neutrophils. SLE LDGs exhibited impaired Nox activity (n=7 SLE, n=12 HC). CYBA is a U12 intron-containing gene, and transcriptomic analysis revealed broad downregulation of this gene class in SLE LDGs, suggesting minor spliceosome dysfunction. rMATS analysis showed increased U12-type intron retention and widespread splicing defects— including exon skipping and mutually exclusive exon use—in genes such as GBP5, MAEA and STX10 . These abnormalities were validated in an independent long-read RNA-seq dataset from SLE PBMCs. Importantly, splicing disruptions correlated with disease activity and autoantibody profiles. Conclusion Impaired U12-dependent splicing may contribute to neutrophil dysfunction in SLE, potentially via defective oxidative burst and altered immune regulation. These findings highlight the minor spliceosome as a novel player in lupus pathogenesis.