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Resistin upregulates chemokine production by fibroblast-like synoviocytes from patients with rheumatoid arthritis.

ABSTRACT: Adipokines are bioactive hormones secreted by adipose tissues. Resistin, an adipokine, plays important roles in the regulation of insulin resistance and inflammation. Resistin levels are known to be increased in the serum and synovial fluid of rheumatoid arthritis (RA) patients. However, the pathogenic role of resistin in RA has not yet been elucidated.The expression of resistin and adenylate cyclase-associated protein 1 (CAP1), a receptor for resistin, was examined immunohistochemically in synovial tissue. CAP1 expression in in vitro cultured fibroblast-like synoviocytes (FLSs) was assessed with a reverse transcription-polymerase chain reaction (PCR) and western blotting. The gene expression of resistin-stimulated FLSs was evaluated by RNA sequencing (RNA-Seq) and quantitative real-time PCR. Concentrations of chemokine (C-X-C motif) ligand (CXCL) 8, chemokine (C-C motif) ligand (CCL) 2, interleukin (IL)-1?, IL-6 and IL-32 in culture supernatants were measured by enzyme-linked immunosorbent assay. Small interfering RNA (siRNA) for CAP1 was transfected into FLSs in order to examine inhibitory effects.The expression of resistin and CAP1 in synovial tissue was stronger in RA than in osteoarthritis (OA). Resistin was expressed by macrophages in the RA synovium, while CAP1 was expressed by macrophages, FLSs and endothelial cells. In vitro cultured RA FLSs also expressed CAP1. RNA-Seq revealed that the expression levels of 18 molecules were more than twofold higher in resistin-stimulated FLSs than in unstimulated FLSs. Seven chemokines, CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL8, and CCL2, were included among the 18 molecules. Increases induced in the expression of CXCL1, CXCL8, and CCL2 by the resistin stimulation were confirmed by real-time PCR. The stimulation with resistin increased the protein levels of CXCL8 and CCL2 produced by RA FLSs, and the upregulated expression of CXCL8 was inhibited by the abrogation of CAP1 by siRNA for CAP1. Production of IL-6 by FLSs was also increased by resistin. Expression of IL-1? and IL-32 was not detected by ELISA.Resistin contributes to the pathogenesis of RA by increasing chemokine production by FLSs via CAP1 in synovial tissue.

SUBMITTER: Sato H

PROVIDER: S-EPMC5709830 | biostudies-literature | 2017 Dec

REPOSITORIES: biostudies-literature

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Resistin upregulates chemokine production by fibroblast-like synoviocytes from patients with rheumatoid arthritis.

Sato Hiroshi H Muraoka Sei S Kusunoki Natsuko N Masuoka Shotaro S Yamada Soichi S Ogasawara Hideaki H Imai Toshio T Akasaka Yoshikiyo Y Tochigi Naobumi N Takahashi Hiroshi H Tsuchiya Kazuaki K Kawai Shinichi S Nanki Toshihiro T

Arthritis research & therapy 20171201 1

<h4>Background</h4>Adipokines are bioactive hormones secreted by adipose tissues. Resistin, an adipokine, plays important roles in the regulation of insulin resistance and inflammation. Resistin levels are known to be increased in the serum and synovial fluid of rheumatoid arthritis (RA) patients. However, the pathogenic role of resistin in RA has not yet been elucidated.<h4>Methods</h4>The expression of resistin and adenylate cyclase-associated protein 1 (CAP1), a receptor for resistin, was exa ...[more]

PMID: 29191223

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Project description:In recent years target based drug discovery has expanded our therapeutic armamentarium in the treatment of inflammatory and autoimmune diseases. Despite these advances and adverse effects, glucocorticoids remain reliable agents that are used in many of these diseases. The anti-inflammatory mechanisms of glucocorticoids include the suppression of transcription factor activity like nuclear factor kappa B (NF-κB). By reanalyzing data from two prior high throughput screens (HTS) that utilized a NF-κB reporter construct in THP-1 cells, we identified 1824 small molecule synthetic compounds that demonstrated NF-κB suppressive activities similar to the glucocorticoids included in the original >134,000 compound libraries. These 1824 compounds were then rescreened for attenuating NF-κB activity at 5 and 16 h after LPS stimuli in the NF-κB THP-1 reporter cells. After a "Top X" selection approach 122 hit compounds were further tested for toxicity and suppression of LPS induced CXCL8 release in THP-1 cells. Excluding cytotoxic compounds, the remaining active compounds were grouped into chemotype families using Tanimoto based clustering. Promising representatives from clustered chemotype groups were commercially purchased for further testing. Amongst these index compounds a lead chemotype: 1H-pyrazolo [3,4 d] pyrimidin-4-amine, effectively suppressed CXCL8, and TNF production by THP-1 cells when stimulated with LPS, TNF or IL-1ß. Extending these studies to primary cells, these lead compounds also reduced IL-6 and CXCL8 production by TNF stimulated fibroblast-like synoviocytes (FLS) from rheumatoid arthritis (RA) patients. Importantly a lead 1H-pyrazolo [3,4 d] pyrimidin-4-amine compound demonstrated synergistic effects with dexamethasone when co-administered to TNF stimulated THP-1 cells and RA FLS in suppressing chemokine production. In summary, a cell based HTS approach identified lead compounds that reduced NF-κB activity and chemokine secretion induced by potent immunologic stimuli, and one lead compound that acted synergistically with dexamethasone as an anti-inflammatory agent showing a dose-sparing effect.

Project description:ObjectiveUp-regulation of glucose metabolism has been implicated not only in tumor cell growth but also in immune cells upon activation. However, little is known about the metabolite profile in rheumatoid arthritis (RA), particularly in fibroblast-like synoviocytes (FLS). This study was undertaken to evaluate whether changes in glucose metabolism in RA FLS could play a role in inflammation and joint damage.MethodsSynovium and FLS were obtained from patients with RA and patients with osteoarthritis (OA). The rate of glycolysis after stimulation of FLS with lipopolysaccharide and platelet-derived growth factor BB was measured using glycolysis stress test technology. FLS function was evaluated using a glycolysis inhibitor, 2-deoxy-d-glucose (2-DG). After stimulation of the FLS, a migration scratch assay, MTT assay, and enzyme-linked immunosorbent assay were performed to measure the effect of 2-DG on FLS migration, viability of the FLS, and cytokine secretion, respectively. IRDye 800CW 2-DG was used to assess glucose uptake in the arthritic joints and stromal cells of mice after K/BxN mouse serum transfer. The mice were injected daily, intraperitoneally, with 3-bromopyruvate (BrPa; 5 mg/kg) to assess the effect of inhibition of glycolysis in vivo.ResultsCompared to human OA FLS, the balance between glycolysis and oxidative phosphorylation was shifted toward glycolysis in RA FLS. Glucose transporter 1 (GLUT1) messenger RNA (mRNA) expression correlated with baseline functions of the RA FLS. Glucose deprivation or incubation of the FLS with glycolytic inhibitors impaired cytokine secretion and decreased the rate of proliferation and migration of the cells. In a mouse model of inflammatory arthritis, GLUT1 mRNA expression in the synovial lining cells was observed, and increased levels of glucose uptake and glycolytic gene expression were detected in the stromal compartment of the arthritic mouse joints. Inhibition of glycolysis by BrPa, administered in vivo, significantly decreased the severity of arthritis in this mouse model.ConclusionTargeting metabolic pathways is a novel approach to understanding the mechanisms of disease. Inhibition of glycolysis may directly modulate synoviocyte-mediated inflammatory functions and could be an effective treatment strategy for arthritis.

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Resistin upregulates chemokine production by fibroblast-like synoviocytes from patients with rheumatoid arthritis.

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Resistin upregulates chemokine production by fibroblast-like synoviocytes from patients with rheumatoid arthritis.

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