TBA (17A182)

JAK-STAT blockade alters synovial bioenergetics, mitochondrial function and pro-inflammatory mediators in Rheumatoid arthritis.

Author(s)

Trudy McGarry1, Carl Orr1, Sarah Wade2, Monika Biniecka1, Siobhan Wade2, Douglas Veale1, Ursula Fearon2 

Department(s)/Institutions

1Centre for Arthritis and Rheumatic Diseases, St Vincents University Hospital, University College Dublin, Dublin.

2Molecular Rheumatology, Trinity Biomedical Sciences Institute, Trinity College Dublin, Dublin.

 

Introduction

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. 

Aims/Background

This study examines the effect of JAK inhibitor tofacitinib on synovial cellular bioenergetics, mitochondrial function and subsequent pro-inflammatory mechanisms in RA.

Method

Ex-vivo RA synovial explants and primary RA synovial fibroblasts (RASFC) were cultured with tofacitinib (1µM)/DMSO control for 24-72hrs. RASFC were also cultured with Oncostatin M (OSM)(10ng/ml) in the presence and absence of tofacitinib (1µM) or DMSO control for 24 hrs. 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. RASFC mitochondrial mutagenesis was quantified using a mitochondrial random mutation capture assay (RMCA) and Lipid peroxidation (4HNE) by ELISA. Immunofluorescence was performed to demonstrate co-expression of pSTAT3 and mitochondrial markers. RASFC glycolysis and oxidative phosphorylation were assessed by the XF24-Flux-analyser, and key metabolic genes by real-time PCR. Western blotting was used to examine pSTAT3, PIAS3 and SOCS3 and ELISA used to quantify Using RA whole tissue synovial organotypic explant cultures, the effect of tofacitinib (1µM) on spontaneous release of pro-inflammatory mediators were quantified by ELISA/MSD multiplex assays and metabolic markers by real-time PCR. Finally, RASFC invasion and matrix degradation were assessed by Transwell invasion and ELISA.

Results

Tofacitinib differentially regulated key mitochondrial genes in RA synovial tissue, and pSTAT3 was co-localised with mitochondrial protein COX-IV. In parallel, tofacitinib significantly decreased MMP and MM and the production of ROS in RASFC. Tofacitinib significantly increased baseline oxidative phosphorylation, ATP production, maximal respiratory capacity and the respiratory reserve in RASFC, an effect paralleled by an and inhibition of key glycolytic genes, HK2, LDHA and HIF1α. OSM induced pSTAT3 and could also significantly decrease the OCR and increase ECAR, an effect which could be reversed in the presence of tofacitinib. In support of this data, tofacitinib inhibited the effect of OSM on IL-6, MCP-1 and RANTES promoting resolution of inflammation. Using RA whole tissue synovial organotypic explants, tofacitinib inhibited key metabolic genes Glut-1, PFK3B, PDK1, HK2, GSK3A, spontaneous secretion of pro-inflammatory mediators IL-6, IL-8, IL-1b, ICAM-1, VEGF, Tie2 and matrix degrading MMP-1 and RASFC invasion. 

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.