Inhibition of glycolytic pathways induce resolution of Inflammation
Rochelle Dowding1, Sharon Ansboro1, Clare Cunningham1, Gao W2, Biniecka M2, Canavan M1,2, Candice Low2, Douglas Veale2, Ursula Fearon1,2.
Molecular Rheumatology, Trinity Biomedical Sciences Institute, Trinity College Dublin Eular Centre for Arthritis and Rheumatic Diseases, St Vincents University Hospital, University College Dublin.
Inflammatory cells require energy to maintain their activation state. Recent evidence suggests that altered cellular metabolism plays a key role in driving synovial infiltration and invasive mechanisms in RA.
This study examines the role of cellular-metabolism in driving inflammatory pathways in RA and in at-risk of RA subjects (arthralgia).
Synovial-tissue and PBMC were obtained from RA, OA, HC and arthralgia (ACPA+/RF+, normal-CRP, no-synovitis). Primary RA synovial fibroblasts (RASFC) RASFC were also obtained. Expression of metabolic-mediators Glut-1,-PFKFB3,-PKM2, pro-inflammatory cytokines (IL-6,-IL-8,-MCP-1) and transcription-factor pSTAT3 and HIF1a were quantified by RT-PCR, western-blot analysis and immunohistology. Synovial-tissue bioenergetics was assessed by real-time Seahorse-XF24-Flux-Analyzer. The effect of metabolic-inhibitors on proinflammatory mediators and transcriptional regulation in RA synovial-explants and RASFC was quantified by RT-PCR, ELISA western-blot and invasion/migration assays.
PFKFB3, Glut-1, IL-6, IL-8 and pSTAT3 mRNA expression were significantly increased in synovial-tissue and PBMC from RA vs OA/-HC. Interestingly, increased expression of PFKFB3, IL-6,-IL-8,-MCP-1 were also observed in arthralgia vs OA/-HC. PFKFB3 and Glut-1 were localised in the synovial vascular and lining-layer regions, levels increasing in a stepwise-progression from OA to arthralgia to RA. This was paralleled by real-time analysis of synovial-tissue explants demonstrating a shift in the bioenergetic-profile where an increase in ATP-consumption and the glycolytic/oxidative-phosphorylation ratio was demonstrated in RA vs arthralgia vs OA. Addition of glycolytic-inhibitors to RASFC and RA synovial-explant cultures significantly decreased transcriptional activation of pSTAT3 and HIF1a, secretion of pro-inflammatory cytokines (IL-6,-IL-8,-MCP-1), paralleled by inhibition of synovial fibroblast invasive and migrative mechanisms.
This study demonstrates a switch in the metabolic-profile in favour of glycolysis in RA synovial-tissue and cells, an effect also observed in arthralgia subjects suggesting that cells may already be primed pre-onset of RA. Glycolytic-inhibitors promoted resolution of inflammation suggesting potential new therapeutic strategies.