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Master Thesis

Identification, quantification et caractérisation de l'ADN mitochondrial au cours de la polyarthrite rhumatoïde

Abstract : Introduction: Mitochondrial DNA (mtDNA) is very similar to bacterial DNA. Recent works suggest that this mtDNA could act like an alarmine, especially in inflammatory situations. Objectives: To identify and quantify this mtDNA in synovial fluids (SF) and plasma of patients suffering from rheumatoid arthritis (RA), and to search proinflammatory characteristics. To study the putative role of mtDNA on the RANK/RANK-L system. Material and Methods: The plasma and SF of patients suffering from osteoarthritis (OA) were collected from the Rheumatology department of the Teaching Hospital of Bordeaux, during current medical acts. The samples were first purified on special kits, before searching and quantifying extracellular mtDNA through quantitative PCR (qPCR). The polymorphonuclears (PMNs) were also isolated from the same samples and purified. We searched through Western-Blotting of the SF the mitochondrial transcription factor A (TFAM), well known for its association to mtDNA and its proinflammatory role. We searched an excessive oxidation of mtDNA in RA DNA samples compared to controls through ELISA quantification of proinflammatory 8-oxo-2'-deoxyguanosine (8-oxo-dG) motives. PMNs were incubated with mtDNA extracted from JRT3 cells, and were then analysed through flow-cytometry to check whether PMNs stimulation by mtDNA could up-regulate RANK-L expression on their membranes. Results: 1) RA SF (n=27) contain statistically more mtDNA copies than OA SF (n=12): mean 749147 copies/mL +/- SD 310776 versus 5460 copies/mL +/- 4507, p=0,0007, Kruskal-Wallis test. The higher the disease activity was, the higher this mean extracellular mtDNA level was. 2) The levels were statistically higher in RA plasma (n=40) when compared to healthy donors plasma (n=15): respectively mean of 124989 copies/mL +/- 25952 versus 24210 copies/mL +/- 11067, p=0,0237, Kruskal-Wallis test. 3) In patients with RA, the intracellular mt DNA content, evaluated through mt DNA/ nuclear mean ration, was 4,59 times higher inside the blood PMN than inside the articular PMNs. 4) TFAM was inconstantly found in RA SF (6 out of 14), but never in OA SF. 5) There was no increased level of 8-oxo-dG in RA DNA when compared to healthy subjects. 6) The incubation of PMNs with mtDNA up-regulates RANK-L expression on the cellular membrane of these cells. Conclusion: RA SF have higher levels of extracellular mt DNA compared to OA SF. On the contrary, SF PMNs have a decreased intracellular level of mtDNA, suggesting that those cells could be a source of mt DNA liberation in the joint. Mitochondrial DNA stimulates RANK-L expression on the cellular membrane of PMNs. Thus, mtDNA could directly contribute to RA physiopathology.
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Master Thesis
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https://dumas.ccsd.cnrs.fr/dumas-01165082
Contributor : Bu Carreire Université de Bordeaux <>
Submitted on : Thursday, June 18, 2015 - 12:20:34 PM
Last modification on : Wednesday, August 23, 2017 - 4:30:14 PM
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Stéphane Mitrovic. Identification, quantification et caractérisation de l'ADN mitochondrial au cours de la polyarthrite rhumatoïde. Médecine humaine et pathologie. 2015. ⟨dumas-01165082⟩

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