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THU0049 Involvement of Mitochondrial DNA in Rheumatoid Arthritis Pathogenesis Through Up-Regulation of RANK-L Expression by Neutrophils
  1. S. Mitrovic1,
  2. A. Contis2,
  3. B. Faustin3,
  4. R. Rossignol4,
  5. C. Goizet4,
  6. M.-E. Truchetet1,
  7. E. Lazaro2,
  8. C. Contin-Bordes3,
  9. P. Duffau2,
  10. C. Richez1
  1. 1Rheumatology
  2. 2Internal Medicine, CHU de Bordeaux
  3. 3UMR CNRS 5164
  4. 4EA4576 MRGM, Université de Bordeaux, Bordeaux, France

Abstract

Background Mitochondrial DNA (mtDNA) is a highly potent inflammatory trigger. It is found as a circulating factor in the blood of various inflammatory diseases. Among the cells implicated in the pathology of rheumatoid arthritis (RA), neutrophils possess the greatest cytotoxic potential, notably owing to their ability to release neutrophils extracellular traps (NETs). We hypothesized that neutrophils could also release metabolism-related products including mtDNA, and that this alarmin could induce, through a paracrine loop, the expression of RANK-L on neutrophils surface.

Objectives To identify and quantify mtDNA in synovial fluids (SF) and plasma of patients with rheumatoid arthritis (RA). To identify the source of this mtDNA. To study the putative role of mtDNA on the RANK/RANK-L system.

Methods The plasma and SF of patients were collected in the Rheumatology department. Samples were purified on appropriated kits, before quantifying extracellular mtDNA through quantitative PCR (qPCR). Neutrophils were isolated from SF and plasma and intracellular mtDNA was quantified by qPCR and normalized using intracellular nuclear DNA level. Mitochondrial transcription factor A (TFAM) presence was searched by Western-Blot. Neutrophils were incubated with mtDNA extracted from JRT3 cells, and expression of RANK-L was analyzed by flow-cytometry.

Results Extracellular mtDNA copies were found in SF at higher levels in RA patients compared to osteoarthritis (OA) patients (p=0.0007), especially in RA patients with high disease activity (figure 1 enclosed). Furthermore, extracellular mtDNA levels were statistically higher in RA plasma than in healthy donors plasma (p=0,02). Interestingly, we found a direct correlation between SF extracellular mtDNA levels and the number of neutrophils present in SF suggesting that neutrophils may be the source of mtDNA. Indeed, extracellular mtDNA is likely to be released by neutrophils since SF neutrophils from RA patients were characterized by a decrease of intracellular mtDNA level compared to blood neutrophils from healthy donors and RA patients. Moreover, TFAM was found in RA SF (6 out of 14), but never in OA SF. Finally incubation of neutrophils with mtDNA extracted from JRT3 cells up-regulated RANK-L expression by neutrophils at the cell surface.

Conclusions Our findings identify extracellular mtDNA, potentially released by SF neutrophils, as a potent autocrine amplifying loop mechanism leading to RANK-L up-regulation, and therefore to structural damages in RA patients.

Acknowledgements This work was supported by a grant from Société Française de Rhumatologie and SFR Transbiomed.

Disclosure of Interest None declared

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