Single-Cell RNA-Sequencing of Peripheral Blood Reveals Neutrophil Heterogeneity in Childhood ANCA-Associated Vasculitis

Background: The intolerance of the body to neutrophil antigens drives the pathogenesis of childhood anti-neutrophil cytoplasmic autoantibody-associated vasculitis (AAV), making neutrophils a central focus of research on the disease’s underlying mechanisms. Methods: We performed single-cell RNA sequencing on 18 peripheral blood samples (including three healthy human samples, twelve samples from patients with different types of AAV, and three samples from patients after AAV treatment) to investigate neutrophil heterogeneity in AAV. These bioinformatics findings were subsequently validated through comprehensive experimental approaches, including in vitro cellular assays, in vivo animal studies, clinical sample analyses, and correlation with patient clinical data. Results: In AAV, neutrophils were reclassified into seven distinct clusters, Neutrophils2 (N2, CD10 + CD11B ++ ) exhibited significant expansion (P< 0.01), while Neutrophils5 (N5, CD10 − CD11B ++ ) showed a trend toward increase (P> 0.05). This phenomenon has been confirmed by the experimental autoimmune vasculitis model (CD10 + CD11B ++ , P< 0.001; CD10 − CD11B ++ , P< 0.05). The bioinformatics results indicated that both CD10 + CD11B ++ neutrophils and CD10 − CD11B ++ neutrophils exhibited dominant pro-inflammatory activities, including neutrophil activation, reactive oxygen species production, neutrophil extracellular trap formation, and degranulation. Pseudotime trajectory and RNA velocity analyses indicated direct differentiation of CD10 + CD11B ++ neutrophils from CD10 − CD11B ++ neutrophils. Experiments using HL-60 cells demonstrated that the peripheral blood microenvironment of AAV promotes the generation of CD11B ++ neutrophils (P< 0.001). Functionally relevant metabolic profiling revealed a substantial reduction in glutamine metabolism in the CD10 + CD11B ++ neutrophils of AAV patients compared to controls (P< 0.001), which was restored following treatment (P< 0.001). Experimental results demonstrated that increasing glutamine concentration in the microenvironment of HL-60 cells can decrease the production of CD11B ++ neutrophils (P< 0.001). Clinical data demonstrate that peripheral blood GGT concentrations increase in patients following treatment (P< 0.01). Conclusion: These findings identify two novel pathogenic neutrophil subsets and suggest that modulation of glutamine metabolism is a promising avenue for further investigation as a potential therapeutic strategy in childhood AAV. Keywords: single-cell RNA sequencing, neutrophil, AAV, heterogeneity, metabolic reprogramming