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Neutrophil extracellular trap-associated carbamylation and histones trigger osteoclast formation in rheumatoid arthritis
  1. Liam J O'Neil1,2,
  2. Christopher B Oliveira1,
  3. Xinghao Wang1,
  4. Mario Navarrete2,
  5. Ana Barrera-Vargas3,
  6. Javier Merayo-Chalico3,
  7. Rwan Aljahdali1,
  8. Eduardo Aguirre-Aguilar3,
  9. Philip Carlucci1,
  10. Mariana J Kaplan1,
  11. Carmelo Carmona-Rivera1
  1. 1Systemic Autoimmunity Branch, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, Maryland, USA
  2. 2Manitoba Centre for Proteomics and Systems Biology, Department of Internal Medicine, University of Manitoba, Winnipeg, Manitoba, Canada
  3. 3Department of Immunology and Rheumatology, Instituto Nacional de Ciencias Medicas y de la Nutricion, Salvador Zubiran, Mexico City, Mexico
  1. Correspondence to Dr Carmelo Carmona-Rivera, National Institutes of Health, Bethesda, MD 20892, USA; carmelo.carmona-rivera{at}


Objective Neutrophil infiltration into the synovial joint is a hallmark of rheumatoid arthritis (RA), a disease characterised by progressive bone erosion. However, the mechanisms by which neutrophils participate in bone destruction remain unclear. Carbamylation is a posttranslational modification linked to increased bone erosion in RA and we previously showed that carbamylation is present in RA neutrophil extracellular traps (NETs). However, it remains unclear whether NETs and their carbamylated protein cargo directly promote bone destruction and alter osteoclast biology.

Methods NETs and carbamylated NETs (cNETs) were assessed for their capacity to induce osteoclast formation in CD14+ monocytes. Chemical inhibitors and neutralising antibodies were used to elucidate the pathway by which NETs induce osteoclastogenesis. HLA-DRB1*04:01 mice received intra-articular injection of cNETs for 4 weeks. Joints were isolated and assessed for osteoclast formation. Plasma and synovial fluid samples from patients with RA (n=32) were assessed for the presence of carbamylated histone, and correlations to disease specific outcomes were performed.

Results We found that NETs, when cNETs, instruct monocytes to undergo rapid osteoclast formation. NET-mediated osteoclastogenesis appears to depend on Toll-like receptor 4 signalling and NET-associated proteins including histones and neutrophil elastase. In vivo, we identified that the number of osteoclasts increased following immunisation with cNETs in HLA-DRB1*04:01 transgenic mice. Furthermore, carbamylated histones are increased in plasma and synovial fluid from patients with RA and correlate with active bone resorption and inflammatory markers.

Conclusions Our results suggest that NETs have a direct role in RA-associated bone erosion by promoting osteoclast formation.

  • Rheumatoid Arthritis
  • Inflammation
  • Autoimmune Diseases

Data availability statement

Data are available on reasonable request.

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Data availability statement

Data are available on reasonable request.

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  • Handling editor Josef S Smolen

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  • Correction notice This article has been corrected since it published Online First. The author affiliations have been updated.

  • Contributors LJO'N and CC-R designed and performed experiments and drafted the manuscript. AB-V, JM-C, EA-A collected patient samples and provided clinical information. CBO, RA, XW and MN performed experiments and contributed to statistical analysis. All authors contributed to the acquisition, analysis or interpretation of data for the work. MJK drafted the manuscript and supervised the work. PC and CC-R conceived, designed study and drafted the manuscript. CC-R is the guarantor.

  • Funding This work was supported by the NIAMS Intramural Research Programmes (ZIAAR041199).

  • Competing interests None declared.

  • Patient and public involvement Patients and/or the public were not involved in the design, or conduct, or reporting, or dissemination plans of this research.

  • 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.