Caroline Ospelt

Altered Expression of MicroRNA-203 in Rheumatoid Arthritis Synovial Fibroblasts and Its Role in Fibroblast Activation

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.

Overexpression of toll-like receptors 3 and 4 in synovial tissue from patients with early rheumatoid arthritis: Toll-like receptor expression in early and longstanding arthritis†

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 alpha, interleukin-1beta (IL-1beta), 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.

Cells of the synovium in rheumatoid arthritis. Synovial fibroblasts.

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.

Prospective new biological therapies for rheumatoid arthritis.

Advances in the current knowledge of pathogenetic mechanisms of rheumatoid arthritis have contributed to the development of biological therapy, and translated research findings into clinical practice. TNF-alpha (infliximab, etanercept, adalimumab), IL-1 (anakinra) and IL-6 (tocilizumab) inhibitors, a B-cell depleting agent (rituximab) and a drug blocking T-cell costimulation (abatacept) have been approved for rheumatoid arthritis. The progress in manufacturing biotechnology has contributed to the development of several other prospective agents that may form the basis for the therapy of rheumatoid arthritis in the near future. New or modified TNF-alpha inhibitors (golimumab, certolizumab pegol), new monoclonal antibodies against other cytokines (e.g. IL-1, IL-6, IL-12, IL-15, IL-17, IL-23), and other agents targeting B-cell depletion (e.g. ocrelizumab, ofatumumab) are in various stages of development. Many pharmaceutical companies have focused on developing small molecule inhibitors with possible peroral administration, which are considered promising drugs for rheumatoid arthritis. In most cases, these small molecules inhibit cellular kinases (e.g. p38, JAK or Syk) that mediate the signaling and transcription of proinflammatory genes. In this review, we describe the cytokine inhibitors and modulators of the immune response currently in ongoing clinical trials, the results of which may further expand the spectrum of efficient therapies for chronic autoimmune diseases.

TLRs and chronic inflammation.

After the discovery of Toll-like receptors (TLRs), innate immune mechanisms came back in the focus of scientific research. With more and more mechanisms of TLR biology known, it has become clear that these and also other innate immune receptors are not only of crucial importance in the immune response to invading pathogens, but also play a role in the homeostasis of commensal flora and in the response to stress and danger signals. In this respect, increasing evidence is found that inappropriate quantity or quality of TLR ligands or aberrant response to TLR activation plays a role in a variety of chronic inflammatory diseases. In this review, an overview of the currently known TLRs and their signaling pathways is given and reports about their expression and activation in chronic inflammatory diseases are recapitulated.

Proteinases in the joint: clinical relevance of proteinases in joint destruction

Proteinases are involved in essential steps in cartilage and bone homeostasis. Consequently, efforts have been made to establish their potential role in the pathology of rheumatic conditions such as rheumatoid arthritis, osteoarthritis and spondyloarthritis. Matrix metalloproteinases (MMPs) are sensitive markers of disease severity and response to treatment, and therefore they have potential in the assessment of rheumatic diseases. Despite disappointing early results with synthetic inhibitors of MMPs, there is still much scope for developing effective and safe MMPs inhibitors, and consequently to deliver new options to inhibit joint destruction.

Down‐regulation of microRNA‐34a* in rheumatoid arthritis synovial fibroblasts promotes apoptosis resistance

OBJECTIVE To investigate the expression and effect of the microRNA-34 (miR-34) family on apoptosis in rheumatoid arthritis synovial fibroblasts (RASFs). METHODS Expression of the miR-34 family in synovial fibroblasts with or without stimulation with Toll-like receptor (TLR) ligands, tumor necrosis factor α (TNFα), interleukin-1β (IL-1β), hypoxia, or 5-azacytidine was analyzed by real-time polymerase chain reaction (PCR). Promoter methylation was studied by combined bisulfite restriction analysis. The effects of overexpression and silencing of miR-34a and miR-34a* on apoptosis were analyzed by annexin V/propidium iodide staining. Production of X-linked inhibitor of apoptosis protein (XIAP) was assessed by real-time PCR and immunohistochemistry analysis. Reporter gene assay was used to study the signaling pathways of miR-34a*. RESULTS Basal expression levels of miR-34a* were found to be reduced in synovial fibroblasts from RA patients compared to osteoarthritis patients, whereas levels of miR-34a, miR-34b/b*, and miR-34c/c* did not differ. Neither TNFα, IL-1β, TLR ligands, nor hypoxia altered miR-34a* expression. However, we demonstrated that the promoter of miR-34a/34a* was methylated and showed that transcription of the miR-34a duplex was induced upon treatment with demethylating agents. Enforced expression of miR-34a* led to an increased rate of FasL- and TRAIL-mediated apoptosis in RASFs. Moreover, levels of miR-34a* were highly correlated with expression of XIAP, which was found to be up-regulated in RA synovial cells. Finally, we identified XIAP as a direct target of miR-34a*. CONCLUSION Our data provide evidence of a methylation-specific down-regulation of proapoptotic miR-34a* in RASFs. Decreased expression of miR- 34a* results in up-regulation of its direct target XIAP, thereby contributing to resistance of RASFs to apoptosis.

Trichostatin A sensitises rheumatoid arthritis synovial fibroblasts for TRAIL-induced apoptosis

Background: Histone acetylation/deacetylation has a critical role in the regulation of transcription by altering the chromatin structure. Objective: To analyse the effect of trichostatin A (TSA), a streptomyces metabolite which specifically inhibits mammalian histone deacetylases, on TRAIL-induced apoptosis of rheumatoid arthritis synovial fibroblasts (RASF). Methods: Apoptotic cells were detected after co-treatment of RASF with TRAIL (200 ng/ml) and TSA (0.5, 1, and 2 μmol/l) by flow cytometry using propidium iodide/annexin-V-FITC staining. Cell proliferation was assessed using the MTS proliferation test. Induction of the cell cycle inhibitor p21Waf/Cip1 by TSA was analysed by western blot. Expression of the TRAIL receptor-2 (DR5) on the cell surface of RASF was analysed by flow cytometry. Levels of soluble TRAIL were measured in synovial fluid of patients with RA and osteoarthritis (OA) by ELISA. Results: Co-treatment of the cells with TSA and TRAIL induced cell death in a synergistic and dose dependent manner, whereas TRAIL and TSA alone had no effect or only a modest effect. RASF express DR5 (TRAIL receptor 2), but treatment of the cells with TSA for 24 hours did not change the expression level of DR5, as it is shown for cancer cells. TSA induced cell cycle arrest in RASF through up regulation of p21Waf1/Cip1. Levels of soluble TRAIL were significantly higher in RA than in OA synovial fluids. Conclusion: Because TSA sensitises RASF for TRAIL-induced apoptosis, it is concluded that TSA discloses sensitive sites in the cascade of TRAIL signalling and may represent a new principle for the treatment of RA.

Association of circulating miR-223 and miR-16 with disease activity in patients with early rheumatoid arthritis

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.

Abundant expression of the interleukin (IL)23 subunit p19, but low levels of bioactive IL23 in the rheumatoid synovium: differential expression and Toll-like receptor-(TLR) dependent regulation of the IL23 subunits, p19 and p40, in rheumatoid arthritis

Objective: Interleukin (IL)23, composed of a p19 and a p40 subunit, is suggested to play key roles in rheumatoid arthritis (RA), dependent on the promotion and proliferation of IL17-producing T helper (Th)17 cells. However, previous studies on IL23 expression in human tissues were based on the p19 subunit only. We aimed to study the expression and regulation of IL23 subunits p19 and p40 in RA compared to patients with osteoarthritis (OA). Methods: The expression of p19 and p40 in synovial tissues was analysed by in situ hybridisation and immunohistochemistry. IL23 in RA and OA synovial fluids and sera was determined by ELISA. Toll-like receptor (TLR)-dependent induction of p19, p40 and bioactive IL23 was determined in RA synovial fibroblasts (RASF), monocytes and monocyte-derived dendritic cells (MDDCs) by real-time PCR and reverse transcriptase (RT)-PCR, Western blot and functional assays. Results: The p19 subunit was abundantly expressed in RA but not in OA synovial tissues. p19 was most prominently expressed by RASF in the synovial lining layer and at the site of invasion, but no heterodimeric IL23 was detected at these sites. Correspondingly, soluble IL23 was not detectable or found at very low levels in synovial fluids and sera of patients with RA. By in vitro experiments, we confirmed that TLR-activated RASF expressed p19 but not p40, in contrast to monocytes, which produced IL23 following TLR stimulation. Conclusion: The TLR-dependent induction of p19 but not p40 in RASF and the abundant expression of p19 along with the low or undetectable levels of IL23 in patients with RA provides strong evidence that p19 does not necessarily indicate the presence of IL23, as has been proposed to date.