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Background Intra-articular glucocorticoid (GC) injections are widely used as a symptomatic treatment for osteoarthritis (OA). However, there are also concerns about their potentially harmful effects, and their detailed effects on chondrocyte phenotype remain poorly understood. Methods We studied the effects of dexamethasone on gene expression in OA chondrocytes with RNA-Seq. Chondrocytes were isolated from the cartilage from OA patients undergoing knee replacement surgery and cultured with or without dexamethasone for 24 h. Total RNA was isolated and sequenced, and functional analysis was performed against the Gene Ontology (GO) database. Results for selected genes were confirmed with RT-PCR. We also investigated genes linked to OA in recent genome-wide expression analysis (GWEA) studies. Results Dexamethasone increased the expression of 480 and reduced that of 755 genes with a fold change (FC) 2.0 or greater. Several genes associated with inflammation and cartilage anabolism/catabolism as well as lipid and carbohydrate metabolism were among the most strongly affected genes. In the GO analysis, genes involved in the extracellular matrix organization, cell proliferation and adhesion, inflammation, and collagen synthesis were enriched among the significantly affected genes. In network analysis, NGF, PI3KR1, and VCAM1 were identified as central genes among those most strongly affected by dexamethasone. Conclusions This is the first study investigating the genome-wide effects of GCs on the gene expression in OA chondrocytes. In addition to clear anti-inflammatory and anticatabolic effects, GCs affect lipid and glucose metabolism in chondrocytes, an observation that might be particularly important in the metabolic phenotype of OA.

Pinosylvin is a natural stilbenoid found particularly in Scots pine. Stilbenoids are a group of phenolic compounds identified as protective agents against pathogens for many plants. Stilbenoids also possess health-promoting properties in humans; for instance, they are anti-inflammatory through their suppressing action on proinflammatory M1-type macrophage activation. Macrophages respond to environmental changes by polarizing towards proinflammatory M1 phenotype in infection and inflammatory diseases, or towards anti-inflammatory M2 phenotype, mediating resolution of inflammation and repair. In the present study, we investigated the effects of pinosylvin on M2-type macrophage activation, aiming to test the hypothesis that pinosylvin could polarize macrophages from M1 to M2 phenotype to support resolution of inflammation. We used lipopolysaccharide (LPS) to induce M1 phenotype and interleukin-4 (IL-4) to induce M2 phenotype in J774 murine and U937 human macrophages, and we measured expression of M1 and M2-markers. Interestingly, along with inhibiting the expression of M1-type markers, pinosylvin had an enhancing effect on the M2-type activation, shown as an increased expression of arginase-1 (Arg-1) and mannose receptor C type 1 (MRC1) in murine macrophages, and C-C motif chemokine ligands 17 and 26 (CCL17 and CCL26) in human macrophages. In IL-4-treated macrophages, pinosylvin enhanced PPAR-γ expression but had no effect on STAT6 phosphorylation. The results show, for the first time, that pinosylvin shifts macrophage polarization from the pro-inflammatory M1 phenotype towards M2 phenotype, supporting resolution of inflammation and repair.

Interleukin-6 (IL-6) is involved in the pathogenesis of various inflammatory diseases, like rheumatoid arthritis (RA). In the present study, we investigated the role of IL-6 in osteoarthritis (OA) patients and the effects of the stilbenoids monomethyl pinosylvin and pinosylvin on the expression of the cartilage matrix components aggrecan and collagen II and the inflammatory cytokine IL-6 in human OA chondrocytes. Synovial fluid and plasma samples were obtained from 100 patients with severe OA [BMI 29.7 (8.3) kg/m2, age 72 (14) years, median (IQR); 62/38 females/males] undergoing total knee replacement surgery. IL-6 and matrix metalloproteinase (MMP) concentrations in synovial fluid and plasma were measured by immunoassay. The effects of pinosylvin on the expression of IL-6, aggrecan, and collagen II were studied in primary cultures of human OA chondrocytes. IL-6 levels in synovial fluid from OA patients [119.8 (193.5) pg/mL, median (IQR)] were significantly increased as compared to the plasma levels [3.1 (2.7) pg/mL, median (IQR)] and IL-6 levels in synovial fluid were associated with MMPs and radiographic disease severity. Natural stilbenoids monomethyl pinosylvin and pinosylvin increased aggrecan expression and suppressed IL-6 production in OA chondrocytes. The results propose that IL-6 is produced within OA joints and has an important role in the pathogenesis of OA. Stilbenoid compounds monomethyl pinosylvin and pinosylvin appeared to have disease-modifying potential in OA chondrocytes.

ObjectiveRetinol binding protein 4 (RBP4) is a member of the lipocalin family and a vitamin A carrier in the blood. More recently, RBP4 has been described as an adipokine that is involved in insulin resistance and metabolic syndrome (MetS). As obesity, MetS and some adipokines contribute to the pathogenesis of osteoarthritis (OA), we investigated RBP4 in patients with OA.Materials and methodsCartilage, synovial fluid and blood samples were collected from 100 OA patients undergoing total knee replacement surgery. Primary chondrocytes and cartilage tissue were cultured to measure the RBP4 expression. The concentrations of RBP4, other adipokines (adipsin, adiponectin, leptin and resistin) and biomarkers of OA (COMP, MMP-1, MMP-3 and YKL-40) were measured by immunoassay, and gene expression was measured by next-generation RNA sequencing.ResultsThe OA cartilage samples released RBP4 into the culture medium, and the levels correlated positively with the expression of the adipokines adipsin, adiponectin, leptin and resistin. RBP4 was the most prominently expressed of these adipokines in the OA chondrocytes, and the expression of the RBP4 receptors STRA6 (stimulated by retinoic acid gene homologue 6) and TLR4 (Toll-like receptor 4) was also detected. Within the cartilage culture medium, RBP4 showed a positive correlation with MMP-1, MMP-3 and YKL-40. RBP4 was also present in the synovial fluid from the OA patients and correlated positively with the concentrations of RBP4 found in the plasma and the cartilage culture medium. Plasma RBP4 concentrations also showed a positive correlation with MMP-3 and adipsin.ConclusionsWe show here, for the first time, that RBP4 is produced within OA joints and that it is associated with increased levels of adipokines and MMPs. The results suggest a role for RBP4 in the pathogenesis of OA and as a possible target for the disease-modifying drugs for the treatment of OA.

Background Previous studies provide evidence that adipokine leptin increases production of catabolic and proinflammatory factors in chondrocytes and serves as a link between obesity and osteoarthritis (OA). However, the magnitude of the response to leptin treatment varies greatly between chondrocytes from different donor patients. In the present study, we investigated the regulatory role of suppressor of cytokine signaling-3 (SOCS-3) in the leptin-induced responses in OA cartilage. Methods Cartilage and synovial fluid samples from 97 patients with OA undergoing knee replacement surgery were collected. Cartilage samples were cultured with leptin (10 μg/ml), and the levels of proinflammatory and catabolic factors in synovial fluid and in the cartilage culture media, and SOCS-3 expression in the cartilage were measured. The role of SOCS-3 in leptin signaling was further studied in H4 murine chondrocytes by downregulating SOCS-3 with siRNA. Results Leptin-induced expression of matrix metalloproteinases MMP-1, MMP-3, MMP-13, interleukin-6 (IL-6), inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) were higher in the cartilage samples with low SOCS-3 expression. Accordingly, downregulation of SOCS-3 by siRNA in H4 chondrocytes led to enhanced leptin-induced expression of MMP-3, MMP-13, IL-6 and iNOS. Synovial fluid leptin was associated positively, and cartilage SOCS-3 negatively with synovial fluid levels of MMPs in a multivariate model in obese (body mass index (BMI) >30 kg/m 2 ) but not in non-obese (BMI <30 kg/m 2 ) patients. Conclusions Our results show, for the first time, that SOCS-3 regulates leptin-induced responses in cartilage, and could thus be a future drug target in the treatment or prevention of OA, especially in obese patients.

Transient receptor potential ankyrin 1 (TRPA1) is a membrane-bound ion channel found in neurons, where it mediates nociception and neurogenic inflammation. Recently, we have discovered that TRPA1 is also expressed in human osteoarthritic (OA) chondrocytes and downregulated by the anti-inflammatory drugs aurothiomalate and dexamethasone. We have also shown TRPA1 to mediate inflammation, pain, and cartilage degeneration in experimental osteoarthritis. In this study, we investigated the role of TRPA1 in joint inflammation, focusing on the pro-inflammatory cytokine interleukin-6 (IL-6). We utilized cartilage/chondrocytes from wild-type (WT) and TRPA1 knockout (KO) mice, along with primary chondrocytes from OA patients. The results show that TRPA1 regulates the synthesis of the OA-driving inflammatory cytokine IL-6 in chondrocytes. IL-6 was highly expressed in WT chondrocytes, and its expression, along with the expression of IL-6 family cytokines leukemia inhibitory factor (LIF) and IL-11, were significantly downregulated by TRPA1 deficiency. Furthermore, treatment with the TRPA1 antagonist significantly downregulated the expression of IL-6 in chondrocytes from WT mice and OA patients. The results suggest that TRPA1 is involved in the upregulation of IL-6 production in chondrocytes. These findings together with previous results on the expression and functions of TRPA1 in cellular and animal models point to the role of TRPA1 as a potential mediator and novel drug target in osteoarthritis.

Background Transient receptor potential ankyrin 1 (TRPA1) is a membrane-associated cation channel, widely expressed in neuronal cells and involved in nociception and neurogenic inflammation. We showed recently that TRPA1 mediates cartilage degradation and joint pain in the MIA-model of osteoarthritis (OA) suggesting a hitherto unknown role for TRPA1 in OA. Therefore, we aimed to investigate whether TRPA1 is expressed and functional in human OA chondrocytes. Methods Expression of TRPA1 in primary human OA chondrocytes was assessed by qRT-PCR and Western blot. The functionality of the TRPA1 channel was assessed by Ca 2+ -influx measurements. Production of MMP-1, MMP-3, MMP-13, IL-6, and PGE 2 subsequent to TRPA1 activation was measured by immunoassay. Results We show here for the first time that TRPA1 is expressed in primary human OA chondrocytes and its expression is increased following stimulation with inflammatory factors IL-1β, IL-17, LPS, and resistin. Further, the TRPA1 channel was found to be functional, as stimulation with the TRPA1 agonist AITC caused an increase in Ca 2+ influx, which was attenuated by the TRPA1 antagonist HC-030031. Genetic depletion and pharmacological inhibition of TRPA1 downregulated the production of MMP-1, MMP-3, MMP-13, IL-6, and PGE 2 in osteoarthritic chondrocytes and murine cartilage, respectively. Conclusions The TRPA1 cation channel was found to be functionally expressed in primary human OA chondrocytes, which is an original finding. The presence and inflammatory and catabolic effects of TRPA1 in human OA chondrocytes propose a highly intriguing role for TRPA1 as a pathogenic factor and drug target in OA.

Running a marathon causes strenuous joint loading and increased energy expenditure. Adipokines regulate energy metabolism, but recent studies have indicated that they also exert a role in cartilage degradation in arthritis. Our aim was to investigate the effects of running a marathon on the levels of adipokines and indices of cartilage metabolism. Blood samples were obtained from 46 male marathoners before and after a marathon run. We measured levels of matrix metalloproteinase-3 (MMP-3), cartilage oligomeric protein (COMP) and chitinase 3-like protein 1 (YKL-40) as biomarkers of cartilage turnover and/or damage and plasma concentrations of adipokines adiponectin, leptin and resistin. Mean marathon time was 3:30:46±0:02:46 (h:min:sec). The exertion more than doubled MMP-3 levels and this change correlated negatively with the marathon time (r = -0.448, p = 0.002). YKL-40 levels increased by 56% and the effect on COMP release was variable. Running a marathon increased the levels of resistin and adiponectin, while leptin levels remained unchanged. The marathon-induced changes in resistin levels were positively associated with the changes in MMP-3 (r = 0.382, p = 0.009) and YKL-40 (r = 0.588, p<0.001) and the pre-marathon resistin levels correlated positively with the marathon induced change in YKL-40 (r = 0.386, p = 0.008). The present results show the impact of running a marathon, and possible load frequency, on cartilage metabolism: the faster the marathon was run, the greater was the increase in MMP-3 levels. Further, the results introduce pro-inflammatory adipocytokine resistin as a novel factor, which enhances during marathon race and associates with markers of cartilage degradation.

YKL-40, a chitinase-like glycoprotein associated with inflammation and tissue remodeling, is produced by joint tissues and recognized as a candidate auto-antigen in rheumatoid arthritis (RA). In the present study, we investigated YKL-40 as a potential biomarker of disease activity in patients with early RA at baseline and during intensive treatment aiming for early remission. Ninety-nine patients with early DMARD-naïve RA participated in the NEO-RACo study. For the first four weeks, the patients were treated with the combination of sulphasalazine, methotrexate, hydroxychloroquine and low dose prednisolone (FIN-RACo DMARD combination), and subsequently randomized to receive placebo or infliximab added on the treatment for further 22 weeks. Disease activity was evaluated using the 28-joint disease activity score and plasma YKL-40 concentrations were measured by immunoassay. At the baseline, plasma YKL-40 concentration was 57 ± 37 (mean ± SD) ng/ml. YKL-40 was significantly associated with the disease activity score, interleukin-6 and erythrocyte sedimentation rate both at the baseline and during the 26 weeks' treatment. The csDMARD combination decreased YKL-40 levels already during the first four weeks of treatment, and there was no further reduction when the tumour necrosis factor-α antagonist infliximab was added on the combination treatment. High YKL-40 levels were found to be associated with disease activity in early DMARD-naïve RA and during intensive treat-to-target therapy. The present results suggest YKL-40 as a useful biomarker of disease activity in RA to be used to steer treatment towards remission.

YKL-40 is associated with tissue injury and inflammation, and consequently to diseases in which these mechanisms lead to tissue degradation, for example, asthma and rheumatoid arthritis. The purpose of the present study was to investigate if YKL-40 is also a significant factor in osteoarthritis (OA) by assessing associations of YKL-40 with mediators related to the pathogenesis of OA: cartilage destructing matrix metalloproteinases (MMPs) and proinflammatory cytokines interleukin-6 (IL-6) and interleukin-17 (IL-17). Cartilage, synovial fluid (SF), and plasma samples were obtained from 100 OA patients undergoing total knee replacement surgery. SF levels of YKL-40 (1027.9 ± 78.3 ng/mL) were considerably higher than plasma levels (67.2 ± 4.5 ng/mL) and correlated with YKL-40 released from cartilage samples obtained from the same patients (r=0.37, P=0.010), indicating that YKL-40 is produced by OA cartilage. Interestingly, YKL-40 concentrations in OA SF correlated positively with MMP-1 (r=0.36, P=0.014), MMP-3 (r=0.46, P=0.001), IL-6 (r=0.57, P<0.001), and IL-17 (r=0.52, P=0.010) levels. Moreover, IL-6 and IL-17 enhanced YKL-40 production in human primary chondrocyte cultures. The present study introduces YKL-40 as a cartilage-derived factor associated with mediators of inflammation and cartilage destruction involved in the pathogenesis of OA.