RNA sequencing of primary Sjögren's syndrome neutrophils

Objective Neutrophils and aberrant NETosis have been implicated in the pathogenesis of diverse autoimmune diseases, however, their roles in primary Sjögren's syndrome (pSS) remain unclear. We aimed to reveal the potential roles of neutrophils and neutrophil extracellular traps (NETs) in this study. Methods pSS patients were enrolled according to the corresponding diagnostic criteria. NETosis markers were measured in plasma and small salivary gland using ELISA and immunofluorescence. The gene signatures of neutrophils were assessed by RNA-Seq and RT-PCR. Reactive oxygen species (ROS), mitochondrial ROS (mitoROS) production and JC-1 was measured by flow cytometry. Results NETosis markers including cell free-DNA (cf-DNA), myeloperoxidase (MPO) in plasma and small salivary gland from pSS patients were significantly higher than healthy controls (HCs) and were associated with disease activity. RNA sequencing and RT-qPCR revealed activated Type I IFN signaling pathway and higher expression of type I interferon related genes in pSS neutrophils. Further stimulating with IFN-a 2a in vitro significantly induced ROS production and JC-1 monomer percentage in pSS neutrophils. Conclusions Our data suggest the involvement of neutrophils and enhanced NETosis in pSS patients. Further mechanism study in vitro revealed that type I IFN activation in pSS neutrophils led to mitochondrial damage and related ROS production which finally result in the generation of NETs. Overall design: RNA sequencing of 7 pSS patients and 6 healthy controls