Article Text

THU0540 Toll like Receptor 2 (TLR2) Induces Mitochondrial Dysfunction and Activation of the NLRP3 Inflammasome in Rheumatoid Arthritis
  1. M. Connolly,
  2. M. Biniecka,
  3. J. McCormick,
  4. T. McGarry,
  5. D.J. Veale,
  6. U. Fearon
  1. St. Vincents University Hospital, Dublin, Ireland


Background Activation of the NLRP3 inflammasome by microbial and environmental stimuli enables the caspase -1-dependent processing and secretion of IL-1β. While the inflammasome has traditionally been thought to regulate infection and inflammation, impaired mitochondrial metabolism may mediate this process.

Objectives In this study we examined the interplay between the inflammasome and mitochondrial function following TLR2 activation.

Methods Mitochondrial mutagenesis was assessed by Random Mutation Capture Assay. Primary RASFC were stimulated with a TLR2 agonist, PAMCSK, in the presence of absence of N-Acetyl Cysteine (NAC). IL-1β and IL-18 expression was examined by PCR. Nlrp3 expression was assessed by real-time PCR and Western blot. In parallel the effect of an TLR endogenous ligand, A-SAA, on RASFC function was examined in the presence of a specific neutralising anti-TLR2 mAb (1μg/ml) and matched IgG isotype control Ab (1μg/ml). RASFC proliferation, adhesion, chemokine expression, migration and invasion were assessed by proliferation assays, flow cytometry, Taqman PCR, ELISA, wound repair and transwell assays. Finally to establish whether A-SAA activates TLR2 Human embryonic kidney (HEK) -TLR2 or -TLR4 cells were cultured in the presence of A-SAA and NFκB luciferase activity was examined.

Results PAM3CYSK4 and A-SAA significantly induced mitochondrial mutagenesis in RA synovial explants cultures (p<0.05). PAM3CYSK4 and A-SAA significantly induced NLRP3 mRNA and protein expression. TLR2-induced IL-6, IL-8, IL-1, IL-18 expression were significantly inhibited in presence of NAC and ROS inhibitor (all p<0.05). In parallel, A-SAA-induced RASFC proliferation, ICAM-1 expression, invasion and migration was significantly inhibited in the presence of anti-TLR2 (all p<0.05), suggesting its role as an endogenous TLR2 ligand. Finally we demonstrated that A-SAA induced TLR2 mediated NF-κB lucifearse activity (p<0.05), with no effect observed for NFκB in HEK-TLR4 cells.

Conclusions This is the first study to demonstrate that mitochondrial dysfunction may be involved in regulation of downstream inflammasome activation. Furthermore we suggest that TLR2 induced pro-inflammatory effects in RA may in part be activated by endogenous secretion of A-SAA from the synovium at the site of inflammation.

Disclosure of Interest : M. Connolly: None declared, M. Biniecka: None declared, J. McCormick: None declared, T. McGarry: None declared, D. Veale Grant/research support: AbbVie, Pfizer, MSD, Roche, Consultant for: Pfizer, Roche, Speakers bureau: Pfizer, Roche, MSD, Abbott, U. Fearon: None declared

DOI 10.1136/annrheumdis-2014-eular.5351

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