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  • Extracellular aaRSs drive autoimmune and inflammatory responses in rheumatoid arthritis via the release of cytokines and PAD4

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Rheumatoid arthritis
Extracellular aaRSs drive autoimmune and inflammatory responses in rheumatoid arthritis via the release of cytokines and PAD4
  1. Akihiro Kimura1,
  2. Takeshi Takagi2,
  3. Thiprampai Thamamongood3,
  4. Satoshi Sakamoto2,
  5. Takumi Ito4,
  6. Iwao Seki5,
  7. Masahiro Okamoto5,
  8. Hiroyuki Aono5,
  9. Satoshi Serada6,
  10. Tetsuji Naka6,
  11. Hiroaki Imataka7,
  12. Kensuke Miyake8,
  13. Takuya Ueda9,
  14. Miki Miyanokoshi10,
  15. Keisuke Wakasugi10,
  16. Noriko Iwamoto11,
  17. Norio Ohmagari11,
  18. Takahiro Iguchi12,
  19. Takeshi Nitta12,
  20. Hiroshi Takayanagi12,
  21. Hiroyuki Yamashita13,
  22. Hiroshi Kaneko13,
  23. Haruka Tsuchiya14,
  24. Keishi Fujio14,
  25. Hiroshi Handa4,
  26. Harumi Suzuki1
  1. 1 Dep of Immunology and Pathology, Research Center for Hepatitis and Immunology, Research Institute, National Center for Global Health and Medicine, Ichikawa-shi, Chiba, Japan
  2. 2 School of Life Science and Technology, Tokyo Institute of Technology, Yokohama, Kanagawa, Japan
  3. 3 National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Pathumthani, Thailand
  4. 4 Center for Future Medical Research, Tokyo Medical University, Shinjuku-ku, Tokyo, Japan
  5. 5 Research and Development Department, AYUMI Pharmaceutical Corporation, Chuo-ku, Tokyo, Japan
  6. 6 Institute for Biomedical Sciences Molecular Pathophysiology, Iwate Medical University, Morioka, Iwate, Japan
  7. 7 Department of Applied Chemistry, Graduate School of Engineering, University of Hyogo, Himeji, Hyogo, Japan
  8. 8 Division of Innate Immunity, The Institute of Medical Science, The University of Tokyo, Minato-ku, Tokyo, Japan
  9. 9 Department of Integrative Bioscience and Biomedical Engineering, Graduate School of Science and Engineering, Waseda University, Shinjuku-ku, Tokyo, Japan
  10. 10 Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo, Meguro-ku, Tokyo, Japan
  11. 11 Disease Control and Prevention Center, National Center for Global Health and Medicine, Shinjuku-ku, Tokyo, Japan
  12. 12 Department of Immunology, Graduate School of Medicine and Faculty of Medicine, The University of Tokyo, Bunkyo-ku, Tokyo, Japan
  13. 13 Division of Rheumatic Diseases, National Center for Global Health and Medicine, Shinjuku-ku, Tokyo, Japan
  14. 14 Department of Allergy and Rheumatology, Graduate School of Medicine, The University of Tokyo, Bunkyo-ku, Tokyo, Japan
  1. Correspondence to Dr Harumi Suzuki, Dep of Immunology and Pathology, NCGM, Ichikawa-shi, Chiba-ken, Japan; hsuzuki{at}ri.ncgm.go.jp; Dr Hiroshi Handa, Center for Future Medical Research, Tokyo Medical University, Shinjyuku-ku, Tokyo, Japan; hhanda{at}tokyo-med.ac.jp

Abstract

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
  • Cytokines
  • Inflammation
  • Autoimmunity

Data availability statement

Data are available on reasonable request.

https://doi.org/10.1136/ard-2023-224055

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    Data availability statement

    Data are available on reasonable request.

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    Footnotes

    • Handling editor Josef S Smolen

    • Twitter @Bluebeans02

    • 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.

    • Supplemental material This content has been supplied by the author(s). It has not been vetted by BMJ Publishing Group Limited (BMJ) and may not have been peer-reviewed. Any opinions or recommendations discussed are solely those of the author(s) and are not endorsed by BMJ. BMJ disclaims all liability and responsibility arising from any reliance placed on the content. Where the content includes any translated material, BMJ does not warrant the accuracy and reliability of the translations (including but not limited to local regulations, clinical guidelines, terminology, drug names and drug dosages), and is not responsible for any error and/or omissions arising from translation and adaptation or otherwise.