Characteristics of peripheral blood neutrophil subsets in patients with primary Sjogren’s syndrome based on single-cell RNA sequencing

Background Neutrophils, which are polymorphonuclear leukocytes of the phagocytic system, serve as critical mediators of inflammation-induced injury and are implicated in various diseases. However, the role of neutrophils in primary Sjögren’s syndrome (pSS) remains underexplored. Objectives Single-cell RNA sequencing (scRNA-seq) was used to construct a map of neutrophil subsets and cell interactions in pSS, and to explore the roles and mechanisms of each subset in the development and progression of pSS, thereby providing new theoretical support for the pathogenesis of pSS and clinical drug development. Methods Include newly diagnosed patients with pSS who presented to the Rheumatology and Immunology Department of Ningxia Medical University General Hospital between December 2022 and January 2025. Peripheral blood neutrophils were obtained from pSS and healthy control (HC) subjects. Following quality control, dimensionality reduction, clustering, and cell annotation, we analyzed compositional and distributional differences in neutrophil subsets between the pSS and HC groups. Significantly differentially expressed genes (DEGs) in neutrophils underwent Gene Ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis, along with molecular interaction and pseudo-time analysis between cells. The DEGs LY6E and CLEC12A were further verified by RT-qPCR. Statistical analysis was performed using the non-paired two-tailed Wilcoxon rank-sum test, Mann-Whitney U test, and non-paired two-tailed Student’s t-test. Visualization was conducted using Monocle and CellphoneDB software. Results Neutrophil counts were significantly lower in the pSS group compared to the HC group. Neutrophils were further clustered into six subsets (Neutrophil 1, Neutrophil 2, Neutrophil 3, Neutrophil 4, Neutrophil 5, and Neutrophil 6), with Neutrophil 2 exhibiting distinct characteristics. The DEGs in neutrophils were primarily involved in biological processes such as viral response and defense, type I interferon signaling pathways, and were enriched in signaling pathways including interferon-α response and inflammatory response. Neutrophils interact with natural killer (NK) cells and plasmacytoid dendritic cells (pDCs) via CCL5-CCR1, LGALS9-HAVCR2, FTH1-SCARA5, and FTL-SCARA5. Pseudo-time analysis revealed that neutrophils first differentiate into the Neutrophil 2 subset and ultimately into the Neutrophil 4 subset. This progression is characterized by progressively enhanced cellular signaling and behavioral regulation capabilities, while concurrently diminishing immune defense responses and neutrophil-mediated immune functions. Conclusion Each subset of neutrophils is involved in the occurrence and development of pSS disease at different stages. In particular, LY6E and CLEC12A among the DEGs specifically upregulated by pSS neutrophils may be biomarkers and potential therapeutic targets for pSS.