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Interleukin-34 produced by human fibroblast-like synovial cells in rheumatoid arthritis supports osteoclastogenesis.

ABSTRACT: INTRODUCTION: Interleukin-34 (IL-34) is a recently defined cytokine, showing a functional overlap with macrophage colony stimulating factor (M-CSF). This study was undertaken to address the expression of IL-34 in rheumatoid arthritis (RA) patients and to investigate its regulation and pathogenic role in RA. METHODS: IL-34 levels were determined in the RA synovium, synovial fluid (SF) and fibroblast-like synovial cells (FLS) by immunohistochemistry, real-time PCR, enzyme-linked immunosorbent assay and immunoblotting. RA activity was assessed using Disease Activity Score 28 (DAS28) activity in the plasma collected at baseline and one year after treatment. Conditioned media (CM) were prepared from RA FLS culture with tumor necrosis factor alpha (TNF?) for 24 hours and used for functional assay. RESULTS: IL-34 was expressed in the synovium, SF, and FLS from RA patients. The production of IL-34 in FLS was up-regulated by TNF? in RA samples compared with osteoarthritis (OA) patients. Importantly, the preferential induction of IL-34 rather than M-CSF by TNF? in RAFLS was mediated by the transcription factor nuclear factor kappa B (NF-?B) and activation of c-Jun N-terminal kinase (JNK). IL-34 elevation in plasma from RA patients was decreased after the administration of disease-modifying anti-rheumatic drugs (DMARDs) in accordance with a decrease in DAS28. CM from RAFLS cultured with TNF? promoted chemotactic migration of human peripheral blood mononuclear cells (PBMCs) and subsequent osteoclast (OC) formation, effects that were attenuated by an anti-IL-34 antibody. CONCLUSIONS: These data provide novel information about the production of IL-34 in RA FLS and indicate that IL-34 is an additional osteoclastogenic factor regulated by TNF? in RA, suggesting a discrete role of IL-34 in inflammatory RA diseases.

SUBMITTER: Hwang SJ

PROVIDER: S-EPMC3392804 | biostudies-other | 2012

REPOSITORIES: biostudies-other

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Interleukin-34 produced by human fibroblast-like synovial cells in rheumatoid arthritis supports osteoclastogenesis.

Hwang Seung-Jun SJ Choi Bongkun B Kang Soon-Suk SS Chang Jae-Ho JH Kim Yong-Gil YG Chung Yeon-Ho YH Sohn Dong Hyun DH So Min Wook MW Lee Chang-Keun CK Robinson William H WH Chang Eun-Ju EJ

Arthritis research & therapy 20120120 1

<h4>Introduction</h4>Interleukin-34 (IL-34) is a recently defined cytokine, showing a functional overlap with macrophage colony stimulating factor (M-CSF). This study was undertaken to address the expression of IL-34 in rheumatoid arthritis (RA) patients and to investigate its regulation and pathogenic role in RA.<h4>Methods</h4>IL-34 levels were determined in the RA synovium, synovial fluid (SF) and fibroblast-like synovial cells (FLS) by immunohistochemistry, real-time PCR, enzyme-linked immun ...[more]

PMID: 22264405

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Project description:BackgroundRheumatoid arthritis (RA) is a chronic systemic inflammatory disease with persistent synovitis. In the present study, the impact of monocyte chemoattractant protein-1 (MCP-1) was explored to determine methods for the diagnosis and treatment of RA.MethodsFirst, fibroblast-like synoviocytes (FLSs) were obtained from a collagen-induced rat RA model. Next, MCP-1-overexpression plasmid and small interfering RNA were transfected into human and rat FLSs. Cell Counting Kit-8 (CCK-8), Transwell migration and flow cytometry assays were used to analyze cell proliferation, migration and apoptosis of FLSs following MCP-1 transfections, respectively. Furthermore, western blotting was used to analyze the expression levels of p-P38, p-PI3K, PI3K, CD31, VEGF, TNF-α and IL-β in FLSs following MCP-1 transfection. In addition, reverse transcription-quantitative PCR and ELISAs were used to analyze the expression levels of C-reactive protein (CRP), estrogen receptor, MCP-1 and pentraxin-3 in patients with clinical RA, followed by correlation analysis of clinical data. Finally, expression validation, diagnostic and protein-protein interaction (PPI) network analysis of MCP-1 were performed.ResultsMCP-1 promoted FLS proliferation and migration, and affected the apoptosis of FLSs. In addition, the expression levels of p-P38, p-PI3K, PI3K, CD31, VEGF, TNF-α and IL-β were also affected by MCP-1. In patients with clinical RA, the expression level of MCP-1 was increased. Moreover, CRP expression level was significantly up-regulated in RA. Clinically, MCP-1 was strongly correlated with tender joint count, swollen joint count, visual analog scale for general health and disease activity score 28 (DAS28)-MCP-1, and was moderately correlated with DAS28 and DAS28-CRP. PPI analysis showed that MCP-1 mainly interacted with other inflammatory cytokines.ConclusionIn conclusion, MCP-1 may play a significant regulatory role in RA, and could be used as a measurement index of clinical RA activity.

Project description:ObjectivesThe 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.MethodsPrimary 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.ResultsTranscriptomic 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.ConclusionmTOR drives pathogenic mechanisms in RASF an effect which is in part mediated via crosstalk with the Hippo-YAP pathway.

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Interleukin-34 produced by human fibroblast-like synovial cells in rheumatoid arthritis supports osteoclastogenesis.

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Interleukin-34 produced by human fibroblast-like synovial cells in rheumatoid arthritis supports osteoclastogenesis.

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