Background Rheumatoid arthritis (RA) is a chronic joint disease, characterised by synovial inflammation and a shift in the metabolic profile of cells to a more destructive phenotype. The JAK-STAT signalling pathway is implicated in the pathogenesis of RA.
Objectives To examine the effect of tofacitinib, a selective JAK inhibitor, on synovial cellular bioenergetics, mitochondrial function and subsequent pro-inflammatory mechanisms in RA.
Methods Ex-vivo RA whole tissue synovial explants and primary RA synovial fibroblasts (RASFC) and were cultured with tofacitinib (1μM) for 24–72hrs. RASFC metabolism was assessed by the XF24-Flux-analyser and mitochondrial mutagenesis was quantified using a mitochondrial random mutation capture assay. 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. Mitochondrial structural morphology was assessed by transmission electron microscopy. Lipid peroxidation (4HNE) was measured by specific ELISA. Dual staining of pSTAT3 and mitochondrial marker Cox-IV was demonstrated by confocal microscopy. The effect of tofacitinib (1μM) in RA synovial explant on markers of cellular bioenergetics and pro-inflammatory mediators, including cytokines and growth factors were quantified by ELISA, MSD multiplex assays and Real-time PCR.
Results An initial screen demonstrated alterations in 18 key genes involved in mitochondrial function in RA synovial tissue in response to tofacitinib. Supporting this, tofacitinib inhibited ROS production, decreased the MMP and MM (all p<0.05), coupled with altered mitochondrial morphology. No effect observed for mtDNA mutations or 4HNE levels. Tofacitinib significantly inhibited the expression of glycolytic genes HIF1α, HK2, LDHA, GSK3A and PDK1 (all p<0.05) suggesting altered energy metabolism. This was paralleled by inhibition of baseline ECAR (glycolysis) with a concomitant increase in baseline OCR (oxidative phosphorylation), ATP production, maximal respiratory capacity and in the respiratory reserve in RASFC, confirming a bioenergetic switch in synovial cells in response to tofacitinib. Furthermore, we demonstrated co-localisation of pSTAT3 with Cox-IV in RASFC, suggested that in addition to nuclear transcription, pSTAT3 may also act as a mitoTF, regulating mitochondrial function directly. Finally, in RA whole tissue explants, tofacitinib significantly inhibited glycolytic genes HK2, GSK3A and PDK1 which was paralleled by a significant decrease in the spontaneous secretion of inflammatory mediators IL-6, IL-8, IL-1β, ICAM-1, VEGF, Tie2 and MMP1 (all p<0.05).
Conclusions In this study, we describe a potential mechanism of action for tofacitinib, through reversing mitochondrial dysfunction and subsequent switch in cellular bioenergetics, in favour of a less glycolytic microenvironment leading to the reduction of inflammatory mediators. Thus, we have demonstrated that pathological cellular metabolism may be reversed by therapeutic treatment with tofacitinib.
Disclosure of Interest None declared