AB0226 NEUTROPHIL EXTRACELLULAR TRAPS ACTIVATE LUNG FIBROBLAST TO INDUCE MYOSITIS-RELATED INTERSTITIAL LUNG DISEASES IN MICE

Abstract

Background Excessive neutrophil extracellular traps (NETs) formation may contribute to myositis-associated interstitial lung diseases (ILD), but the underlying mechanism is not fully revealed. Objectives This research aimed to elucidate the mechanism by which NETs contribute to myositis-associated ILD. Methods Myositis mice model was established by injected with the rat skeletal muscle homogenate and pertussis toxin. Expressions of ACTA2, CCN2, ADAM12, miR-7 and Smad2 were detected by quantitative real-time polymerase chain reaction (qRT-PCR). Smad2, α-SMA, CCN2 and TLR9 signaling-related proteins were measured by western blot. Soluble collagen I-IV were detected by Sircol collagen assay kit. The proliferation and apoptosis of lung fibroblasts (LFs) were detected by MTT assay or flow cytometry analysis. The interaction between miR-7 and the Smad2 was confirmed by dual-luciferase reporter gene assay. Results NET accelerated the progression of ILD and promoted pulmonary fibrosis in vivo. miR-7 expression was downregulated in lung tissue of myositis group than control group, and NETs further decreased miR-7 expression. TLR9 and Smad2 were upregulated in lung tissue of myositis group than control group, and NETs further increased TLR9 and Smad2 expressions. In vitro experiments showed that PMA-treated NETs accelerated the proliferation of LF and their differentiation into myofibroblast (MF), whereas DNAse I decreased the promotion effect of NETs. NETs components myeloperoxidase (MPO) and histone 3 also promoted the proliferation and differentiation of LF. In addition, we demonstrated that TLR9 involved in the regulation of NETs on LF proliferation and differentiation, and confirmed the interaction between miR-7 and Smad2 in LF. Finally, miR-7-Smad2 pathway was confirmed to be involved in the regulation of TLR9 on LF proliferation and differentiation. Conclusion NETs promote myositis-related ILD, and TLR9-miR-7-Smad2 signaling pathway is involved in the proliferation of LFs and their differentiation into MFs.