Differential Metabotypes in Synovial Fibroblasts and Synovial Fluid in Hip Osteoarthritis Patients Support Inflammatory Responses

Abstract

Changes in cellular metabolism have been implicated in mediating the activated fibroblast phenotype in a number of chronic inflammatory disorders, including pulmonary fibrosis, renal disease and rheumatoid arthritis. The aim of this study was therefore to characterise the metabolic profile of synovial joint fluid and synovial fibroblasts under both basal and inflammatory conditions in a cohort of obese and normal-weight hip OA patients. Furthermore, we sought to ascertain whether modulation of a metabolic pathway in OA synovial fibroblasts could alter their inflammatory activity. Synovium and synovial fluid was obtained from hip OA patients, who were either of normal-weight or obese and were undergoing elective joint replacement surgery. The synovial fluid metabolome was determined by 1H NMR spectroscopy. The metabolic profile of isolated synovial fibroblasts in vitro was characterised by lactate secretion, oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) using the Seahorse XF Analyser. The effects of a small molecule pharmacological inhibitor and siRNA targeted at glutaminase-1 (GLS1) were assessed to probe the role of glutamine metabolism in OA synovial fibroblast function. Obese OA patient synovial fluid (n = 5) exhibited a different metabotype, compared to normal-weight patient fluid (n = 6), with significantly increased levels of 1, 3-dimethylurate, N-Nitrosodimethylamine, succinate, tyrosine, pyruvate, glucose, glycine and lactate, and enrichment of the glutamine-glutamate metabolic pathway, which correlated with increasing adiposity. In vitro, isolated obese OA fibroblasts exhibited greater basal lactate secretion and aerobic glycolysis, and increased mitochondrial respiration when stimulated with pro-inflammatory cytokine TNFα, compared to fibroblasts from normal-weight patients. Inhibition of GLS1 attenuated the TNFα-induced expression and secretion of IL-6 in OA synovial fibroblasts. These findings suggest that altered cellular metabolism underpins the inflammatory phenotype of OA fibroblasts, and that targeted inhibition of glutamine-glutamate metabolism may provide a route to reducing the pathological effects of joint inflammation in OA patients who are obese.

Keywords: IL6; glutamine; inflammation; metabolism; osteoarthritis; synovial fibroblast.

Figure 1

NMR spectroscopy reveals different metabotypes between obese and normal-weight OA patient synovial fluid (A) Concentration (μM) of selected metabolites that was found to be significantly different in synovial fluid from obese (OB; n = 5) and normal-weight (NW; n = 6) OA patient synovial fluids. * = p < 0.05; (B) top metabolites ranked on Variable Importance in Projection (VIP) score between obese (OB; n = 5) and normal-weight (NW; n = 6) OA synovial fluid; (C) enrichment analysis using MetaboAnalyst, of metabolites with VIP scores ≥1 between obese and normal-weight synovial fluids, showing enrichment ratio and associated p-values for the most relevant metabolic pathways.

Figure 2

Lactate secretion is greater in obese OA synovial fibroblasts and is induced during the inflammatory response. The effect of TNFα (10 ng/mL) stimulation of synovial fibroblasts from normal-weight (NW) or obese OA patients (n = 3) on (A) the expression of IL-6 and (B) secretion of lactate after 4 h and 24 h stimulation. Data expressed as mean ± SEM. ** = p < 0.01, *** = p < 0.001, significantly different from time 0. ‡ = p < 0.01, significantly different between synovial fibroblasts from patients with different BMI.

Figure 3

Obese and normal-weight OA synovial fibroblasts exhibit differential mitochondrial respiration. (A–C) OA synovial fibroblasts from obese patients (n = 6 patients, with 5 biological replicates per patient) stimulated with TNFα at 10 ng/mL for 24 h show increased mitochondrial respiration, compared to fibroblasts from normal weight patients. (D) Non-mitochondrial oxygen consumption does not increase in OA synovial fibroblasts from obese or normal weight patients with TNFα stimulation. (E) basal respiration; (F) maximal respiration; and (G) ATP production from mitochondrial respiration significantly increase in obese OA synovial fibroblasts when stimulated with TNFα at 10 ng/mL for 24 h. All data presented as mean ± SEM. * = p < 0.05, ** = p < 0.01.

Figure 4

Obese and normal-weight OA synovial fibroblasts exhibit differential glycolytic metabolism. (A–C) OA synovial fibroblasts from obese patients (n = 6 patients) stimulated with TNFα at 10 ng/mL for 24 h show elevated aerobic glycolysis, compared to stimulated and unstimulated OA synovial fibroblasts from normal weight (n = 6 patients, with 5 biological replicates per patient). (D) Glycolysis levels are significantly increased in obese OA synovial fibroblasts when stimulated with TNFα at 10 ng/mL for 24 h. (E) Glycolytic capacity of normal weight OA synovial fibroblasts is elevated when challenged with TNFα at 10 ng/mL for 24 h. (F) Glycolytic reserve is increased in unstimulated obese OA synovial fibroblasts, compared to unstimulated normal weight OA synovial fibroblasts. All data presented as mean ± SEM. * = p < 0.05.

Figure 5

Inhibition of glutaminase-1 attenuates the IL-6 inflammatory response in OA synovial fibroblasts. (A) GLS1 inhibitor, BPTES (20 µM), attenuates TNFα induced IL-6 secretion in synovial fibroblasts from normal weight and obese hip OA patients (n = 3 patients). Bars represent mean IL-6 secretion (pg/mL). (B) % knockdown of GLS1 expression in OA synovial fibroblasts after 24 h post-transfection with a GLS1 targeting siRNA (5 nM or 100 nM), compared to a non-targeting control (NTC) siRNA (n = 4 patients). (C) Confirmation of siRNA-mediated GLS1 protein knockdown at 24 h and 48 h post-transfection using 100 nM siRNA. (D) Effect of sRNA-mediated knockdown of GLS1 on IL-6 expression in OA synovial fibroblasts at 24 h post-transfection (n = 4 patients). Data are presented as mean ± SEM. * = p < 0.05, ** = p < 0.01, *** = p < 0.001.