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02.33 Altered bioenergetics, mitochondrial function and pro-inflammatory pathways in ra synovium in response to tofacitinib
  1. Carl Orr1,
  2. Trudy McGarry2,
  3. Sarah Wade2,
  4. Monika Biniecka1,
  5. Siobhan Wade2,
  6. Douglas Veale1,
  7. Ursula Fearon2
  1. 1Centre for Arthritis and Rheumatic Diseases, St Vincents University Hospital, University College Dublin, Dublin
  2. 2Molecular Rheumatology, Trinity Biomedical Sciences Institute, Trinity College Dublin, Dublin

Abstract

Background/purpose Rheumatoid arthritis (RA) is a chronic joint disease, characterised by synovial inflammation and destruction of articular cartilage/bone. The Janus-Kinase and Signal Transducer and Activator of Transcription (JAK-STAT) signalling pathway is implicated in the pathogenesis of RA. The objective of our research was to examine the effect of tofacitinib, a selective JAK inhibitor, on metabolic activity, mitochondrial function and pro-inflammatory mechanisms in RA.

Methods Primary RA synovial fibroblasts (RASFC) and ex-vivo RA synovial explants were cultured with tofacitinib (1 µM) for 24–48 hours. RASFC metabolism was assessed by the XF24-Flux-analyser and RASFC mitochondrial mutagenesis was quantified using a mitochondrial random mutation capture assay (RMCA). Mitochondrial function was assessed for reactive oxygen species (ROS), mitochondrial membrane potential (MMP) and mitochondrial mass (MM) using the specific cell fluorescent probes and differential gene expression by mitochondrial gene arrays or RT-PCR. Lipid peroxidation (4HNE) was measured by specific ELISA. The effect of tofacitinib (1 µM) on RA pro-inflammatory mediators, including cytokines and growth factors were quantified by ELISA and MSD multiplex assays.

Results During an initial screen, we observed alteration of 18 key genes involved in mitochondrial function in RA synovial tissue in response to tofacitinib. Supporting this, tofacitinib inhibited the production of ROS (p<0.05), MMP (p<0.06) and MM (p<0.05) in RASFC with no effect observed for mitochondrial DNA-mutations or 4HNE. Tofacitinib significantly inhibited the expression of glycolytic genes HIF1α, HK2, GSK3A and PDK1 (all p<0.05) suggesting altered energy metabolism. This was paralleled by a significant decreased baseline ECAR (glycolysis)(p<0.05), with an observed simultaneous increase in baseline OCR (oxidative phosphorylation)(p<0.05). In addition, tofacitinib increased ATP production, the maximal respiratory capacity and the respiratory reserve in RASFC (all p<0.05). In RA whole tissue synovial explants, which maintain the architecture and cell-cell contact of the joint thus closely reflecting the in-vivo environment, tofacitinib significantly inhibited spontaneous secretion of IL-6, IL-8, IL-1b, ICAM-1, VEGF, Tie2 and MMP1 (all p<0.05), confirming a bioenergetic switch in favour of resolution of inflammation.

Conclusion This study demonstrates that tofacitinib inhibition of pro-inflammatory mechanisms is paralleled by metabolic reprogramming, and further supports JAK-STAT inhibition as a therapeutic target for the treatment of RA.

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