Objectives Recent studies demonstrate that extracellular-released aminoacyl-tRNA synthetases (aaRSs) play unique roles in immune responses and diseases. This study aimed to understand the role of extracellular aaRSs in the pathogenesis of rheumatoid arthritis (RA).
Methods Primary macrophages and fibroblast-like synoviocytes were cultured with aaRSs. aaRS-induced cytokine production including IL-6 and TNF-α was detected by ELISA. Transcriptomic features of aaRS-stimulated macrophages were examined using RNA-sequencing. Serum and synovial fluid (SF) aaRS levels in patients with RA were assessed using ELISA. Peptidyl arginine deiminase (PAD) 4 release from macrophages stimulated with aaRSs was detected by ELISA. Citrullination of aaRSs by themselves was examined by immunoprecipitation and western blotting. Furthermore, aaRS inhibitory peptides were used for inhibition of arthritis in two mouse RA models, collagen-induced arthritis and collagen antibody-induced arthritis.
Results All 20 aaRSs functioned as alarmin; they induced pro-inflammatory cytokines through the CD14-MD2-TLR4 axis. Stimulation of macrophages with aaRSs displayed persistent innate inflammatory responses. Serum and SF levels of many aaRSs increased in patients with RA compared with control subjects. Furthermore, aaRSs released PAD4 from living macrophages, leading to their citrullination. We demonstrate that aaRS inhibitory peptides suppress cytokine production and PAD4 release by aaRSs and alleviate arthritic symptoms in a mouse RA model.
Conclusions Our findings uncovered the significant role of aaRSs as a novel alarmin in RA pathogenesis, indicating that their blocking agents are potent antirheumatic drugs.
- Arthritis, Rheumatoid
Data availability statement
Data are available on reasonable request.
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Handling editor Josef S Smolen
Contributors AK, HH and HS designed the study. AK and HS wrote the manuscript. AK performed experiments and analyzed the data. TTa, TTh, SSa and TIt performed affinity-beads screening. IS, MO, HA, SSe and TN participated in performing experiments. HI, TU, MM and KW generated recombinant aaRS proteins. KM shared MD2 KO mice. NI and NO provided serum samples from COVID-19 patients. HY, HK, HT and KF provided serum and synovial fluid samples from RA and OA patients. TIg performed protein-protein docking simulation. TN and HT participated in data discussion and interpretation. HH provided affinity-beads screening expertise. HS supervised the project and accepts final responsibility for this work as guarantor.
Funding This work was supported by Grant-in-Aid for Scientific Research (C) from the Japan Society for the Promotion of Science (JSPS; grant 21K07088); grants from National Center for Global Health and Medicine (30-shi-2008 and 20-A-1001); grants from National Center for Global Health and Medicine (20A1003, 20A2005D, 29A1036); Grant-in-Aid for Scientific Research (B) from the Japan Society for the Promotion of Science (JSPS; grant 19H03492).
Competing interests None declared.
Patient and public involvement Patients and/or the public were involved in the design, or conduct, or reporting, or dissemination plans of this research. Refer to the Methods section for further details.
Provenance and peer review Not commissioned; externally peer reviewed.
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