Objective: MicroRNA (miRNA) are recognized as important regulators of a variety of fundamental biologic processes. Previously, we described increased expression of miR‐155 and miR‐146a in rheumatoid arthritis (RA) and showed a repressive effect of miR‐155 on matrix metalloproteinase (MMP) expression in RA synovial fibroblasts (RASFs). The present study was undertaken to examine alterations in expression of miR‐203 in RASFs and analyze its role in fibroblast activation.Methods: Differentially expressed miRNA in RASFs versus osteoarthritis synovial fibroblasts (OASFs) were identified by real‐time polymerase chain reaction (PCR)–based screening of 260 individual miRNA. Transfection of miR‐203 precursor was used to analyze the function of miR‐203 in RASFs. Levels of interleukin‐6 (IL‐6) and MMPs were measured by real‐time PCR and enzyme‐linked immunosorbent assay. RASFs were stimulated with IL‐1β, tumor necrosis factor α (TNFα), lipopolysaccharide (LPS), and 5‐azacytidine (5‐azaC). Activity of IκB kinase 2 was inhibited with SC‐514.Results: Expression of miR‐203 was higher in RASFs than in OASFs or fibroblasts from healthy donors. Levels of miR‐203 did not change upon stimulation with IL‐1β, TNFα, or LPS; however, DNA demethylation with 5‐azaC increased the expression of miR‐203. Enforced expression of miR‐203 led to significantly increased levels of MMP‐1 and IL‐6. Induction of IL‐6 by miR‐203 overexpression was inhibited by blocking of the NF‐κB pathway. Basal expression levels of IL‐6 correlated with basal expression levels of miR‐203.Conclusion: The current results demonstrate methylation‐dependent regulation of miR‐203 expression in RASFs. Importantly, they also show that elevated levels of miR‐203 lead to increased secretion of MMP‐1 and IL‐6 via the NF‐κB pathway and thereby contribute to the activated phenotype of synovial fibroblasts in RA.

Objective: To analyze the expression, regulation, and biologic relevance of Toll‐like receptors (TLRs) 1–10 in synovial and skin fibroblasts and to determine the expression levels of TLRs 2, 3, and 4 in synovial tissues from patients with early rheumatoid arthritis (RA), longstanding RA, and osteoarthritis (OA).Methods: Expression of TLRs 1–10 in RA synovial fibroblasts (RASFs), OASFs, and skin fibroblasts was analyzed by real‐time polymerase chain reaction (PCR). Fibroblasts were stimulated with tumor necrosis factor α, interleukin‐1β (IL‐1β), bacterial lipopeptide, poly(I‐C), lipopolysaccharide, and flagellin. Production of IL‐6 was determined by enzyme‐linked immunosorbent assay and induction of TLRs 2–5, matrix metalloproteinases (MMPs) 3 and 13 messenger RNA by real‐time PCR. Expression of TLRs 2–4 in synovial tissues was analyzed by immunohistochemistry.Results: Synovial fibroblasts expressed TLRs 1–6, but not TLRs 7–10. Among the expressed TLRs, TLR‐3 and TLR‐4 were the most abundant in synovial fibroblasts, and stimulation of synovial fibroblasts with the TLR‐3 ligand poly(I‐C) led to the most pronounced increase in IL‐6, MMP‐3, and MMP‐13. In contrast, skin fibroblasts did not up‐regulate MMP‐3 or MMP‐13 after stimulation with any of the tested stimuli. In synovial tissues from patients with early RA, TLR‐3 and TLR‐4 were highly expressed and were comparable to the levels of patients with longstanding RA. These expression levels were elevated as compared with those in OA.Conclusion: Our findings of high expression of TLRs, particularly TLRs 3 and 4, at an early stage of RA and the reactivity of synovial fibroblasts in vitro to TLR ligands suggest that TLR signaling pathways resulting in persistent inflammation and joint destruction are activated early in the disease process.

Objective: To study possible mechanisms that mediate induction of the recently described adipocytokine pre–B cell colony‐enhancing factor (PBEF) in joints of patients with rheumatoid arthritis (RA), and to analyze whether levels of PBEF correlate with disease severity and whether PBEF itself has the potential to act as a proinflammatory and destructive mediator in RA.Methods: RA synovial fibroblasts (RASFs) and monocytes were stimulated with Toll‐like receptor (TLR) ligands, cytokines, and recombinant human PBEF or were transfected with PBEF expression constructs or with PBEF‐specific small interfering RNA. Production of interleukin‐6 (IL‐6), IL‐8, and tumor necrosis factor α (TNFα) was measured by enzyme‐linked immunosorbent assay, and expression of matrix metalloproteinases (MMPs) was assessed by real‐time polymerase chain reaction. PBEF expression in synovial tissue, synovial fluid, serum, and SFs was assessed by immunohistochemistry, in situ hybridization, Western blotting, and enzyme immunoassays.Results: In RASFs, PBEF was up‐regulated by TLR ligands and cytokines that are characteristically present in the joints of patients with RA. In synovial tissue, RASFs were the major PBEF‐expressing cells. A predominance of PBEF was found in the synovial lining layer and at sites of invasion into cartilage. Levels of PBEF in serum and synovial fluid correlated with the degree of inflammation and clinical disease activity. Moreover, PBEF itself activated the transcription factors NF‐kB and activator protein 1 and induced IL‐6, IL‐8, MMP‐1, and MMP‐3 in RASFs as well as IL‐6 and TNFα in monocytes. PBEF knockdown in RASFs significantly inhibited basal and TLR ligand–induced production of IL‐6, IL‐8, MMP‐1, and MMP‐3.Conclusion: Our findings establish PBEF as a proinflammatory and destructive mediator of joint inflammation in RA and identify PBEF as a potential therapeutic target.

For some time synovial fibroblasts have been regarded simply as innocent synovial cells, mainly responsible for synovial homeostasis. During the past decade, however, a body of evidence has accumulated illustrating that rheumatoid arthritis synovial fibroblasts (RASFs) are active drivers of joint destruction in rheumatoid arthritis. Details regarding the intracellular signalling cascades that result in long-term activation and synthesis of proinflammatory molecules and matrix-degrading enzymes by RASFs have been analyzed. Molecular, cellular and animal studies have identified various interactions with other synovial and inflammatory cells. This expanded knowledge of the distinct role played by RASFs in the pathophysiology of rheumatoid arthritis has moved these fascinating cells to the fore, and work to identify targeted therapies to inhibit their joint destructive potential is underway.

Background Identification of parameters for early diagnosis and treatment response would be beneficial for patients with early rheumatoid arthritis (ERA) to prevent ongoing joint damage. miRNAs have features of potential biomarkers, and an altered expression of miRNAs was shown in established rheumatoid arthritis (RA). Objective To analyse RA associated miRNAs in the sera of patients with ERA to find markers of early disease, clinical activity or predictors of disease outcome. Methods Total RNA was isolated from whole sera in ERA patients (prior to and after 3 and 12 months of therapy with disease modifying antirheumatic drugs), in patients with established RA and in healthy controls (HC) using phenol–chloroform extraction. Expression of miR-146a, miR-155, miR-223, miR-16, miR-203, miR-132 and miR-124a was analysed by TaqMan Real Time PCR. Results From all analysed miRNAs, levels of miR-146a, miR-155 and miR-16 were decreased in the sera of ERA patients in comparison with established RA. A change in circulating miR-16 in the first 3 months of therapy was associated with a decrease in DAS28 in long term follow-up in ERA (p=0.002). Levels of circulating miR-223 in treatment naïve ERA correlated with C reactive protein (p=0.008), DAS28 (p=0.031) and change in DAS28 after 3 months (p=0.003) and 12 months (p=0.011) of follow-up. However, neither miR-16 nor miR-223 could distinguish ERA from HC. Conclusions Differential expression of circulating miR-146a, miR-155 and miR-16 in the sera of ERA patients may characterise an early stage of the disease. We suggest miR-223 as a marker of disease activity and miR-16 and miR-223 as possible predictors for disease outcome in ERA.