The mammalian target of rapamycin contributes to synovial fibroblast pathogenicity in rheumatoid arthritis.

Objectives: The mammalian target of Rapamycin (mTOR) is a metabolic master regulator of both innate and adaptive immunity; however, its exact role in stromal cell biology is unknown. In this study we explored the role of the mTOR pathway on Rheumatoid Arthritis synovial fibroblast (RASF) metabolism and activation and determined if crosstalk with the Hippo-YAP pathway mediates their effects. Methods: Primary RA synovial fibroblasts (RASF) were cultured with TNFα alone or in combination with the mTOR inhibitor Rapamycin or YAP inhibitor Verteporfin. Chemokine production, matrix metalloproteinase (MMP) production, and adhesion marker expression were quantified by real-time PCR, ELISA, and/or Flow Cytometry. Invasion assays were performed using Transwell invasion chambers, while wound repair assays were used to assess RASF migration. Cellular bioenergetics was assessed using the Seahorse XFe96 Analyzer. Key metabolic genes (GLUT-1, HK2, G6PD) were measured using real-time PCR. Reanalysis of RNA-Seq analysis was performed on RA (n = 151) and healthy control (HC) (n = 28) synovial tissue biopsies to detect differential gene and pathway expression. The expression of YAP was measured by Western Blot. Results: Transcriptomic analysis of healthy donor and RA synovial tissue revealed dysregulated expression of several key components of the mTOR pathway in RA. Moreover, the expression of phospho-ribosomal protein S6 (pS6), the major downstream target of mTOR is specifically increased in RA synovial fibroblasts compared to healthy tissue. In the presence of TNFα, RASF display heightened phosphorylation of S6 and are responsive to mTOR inhibition via Rapamycin. Rapamycin effectively alters RASF cellular bioenergetics by inhibiting glycolysis and the expression of rate limiting glycolytic enzymes. Furthermore, we demonstrate a key role for mTOR signaling in uniquely mediating RASF migratory and invasive mechanisms, which are significantly abrogated in the presence of Rapamycin. Finally, we report a significant upregulation in several genes involved in the Hippo-YAP pathway in RA synovial tissue, which are predicted to converge with the mTOR pathway. We demonstrate crosstalk between the mTOR and YAP pathways in mediating RASF invasive mechanism whereby Rapamycin significantly abrogates YAP expression and YAP inhibition significantly inhibits RASF invasiveness. Conclusion: mTOR drives pathogenic mechanisms in RASF an effect which is in part mediated via crosstalk with the Hippo-YAP pathway.

Metabolites as drivers and targets in rheumatoid arthritis.

Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by neovascularization, immune cell infiltration, and synovial hyperplasia, which leads to degradation of articular cartilage and bone, and subsequent functional disability. Dysregulated angiogenesis, synovial hypoxia, and immune cell infiltration result in a 'bioenergetic crisis' in the inflamed joint which further exacerbates synovial invasiveness. Several studies have examined this vicious cycle between metabolism, immunity, and inflammation and the role metabolites play in these interactions. To add to this complexity, the inflamed synovium is a multicellular tissue with many cellular subsets having different metabolic requirements. Metabolites can shape the inflammatory phenotype of immune cell subsets during disease and act as central signalling hubs. In the RA joint, the increased energy demand of stromal and immune cells leads to the accumulation of metabolites such as lactate, citrate, and succinate as well as adipocytokines which can regulate downstream signalling pathways. Transcription factors such as HIF1ɑ and mTOR can act as metabolic sensors to activate synovial cells and drive pro-inflammatory effector function, thus perpetuating chronic inflammation further. These metabolic intermediates may be potential therapeutic targets and so understanding the complex interplay between metabolites and synovial cells in RA may allow for identification of novel therapeutic strategies but also may provide significant insight into the underlying mechanisms of disease pathogenesis.