Systemic joint destruction by chronic synovial inflammation is a major problem in rheumatoid arthritis (RA). Osteoclasts, the major bone resorptive cells, play a crucial role in joint destruction and are formed as multinucleated giant cells by the fusion of hematopoietic cells of the monocyte and macrophage lineage at or near the bone surface (1, 2). Macrophages in the RA synovium are capable of differentiating into osteoclasts by initiating a cascade of cellular signals, such as proinflammatory cytokines (3, 4). Prevention of osteoclast differentiation in inflamed joints is one of the best therapeutic strategies for RA patients (5).
MicroRNA (miRNA) are a class of noncoding RNA that regulate gene expression by binding the 3′-untranslated region (3′-UTR) of their target messenger RNA (mRNA), leading to translational repression or mRNA degradation (6–9). Several miRNA exhibit a tissue-specific or developmental stage–specific expression pattern and have been reported to be associated with human diseases, including RA (10–15). MicroRNA-146 (miR-146) has been shown to be strongly expressed in RA synovium and peripheral blood mononuclear cells (PBMCs) and to inhibit the expression of interleukin-1 receptor–associated kinase 1 (IRAK1) and tumor necrosis factor receptor–associated factor 6 (TRAF6) by binding to the 3′-UTR of their mRNA (13–16).
It has been reported that expression of miR-146a/b plays a role as a negative regulator of constitutive activity of NF-κB (16). NF-κB, which activates osteoclast precursor cells, is one of the most important molecules in osteoclastogenesis (17–19). TRAF6, which is one of the target genes of miR-146a, is one of the most important mediators of osteoclastogenesis, and therefore, there is a possibility that regulation of miR-146a could lead to suppression of osteoclastogenesis and subsequently to the prevention of joint destruction.
The aim of this study was to determine whether overexpression of miR-146a inhibits osteoclastogenesis. First, we confirmed that transfection of miR-146a into human PBMCs suppresses osteoclastogenesis induced by macrophage colony-stimulating factor (M-CSF) and either tumor necrosis factor α (TNFα) or RANKL in vitro. Second, we investigated the degree of efficacy of intravenous administration of double-stranded miR-146a for the prevention of joint destruction in vivo in mice with collagen-induced arthritis (CIA).
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Recently, miRNA has been attracting attention due to its crucial role in human disease and is shaping up to be a new therapeutic target. It has gradually become clear that miRNA participate in the pathogenesis of RA. Stanczyk et al reported that miR-146 and miR-155 are strongly expressed in RA synovial fibroblasts compared with osteoarthritis (OA) fibroblasts (13). We previously demonstrated that miR-146 is strongly expressed in RA synovial tissue compared with OA and normal synovial tissue. We also demonstrated that miR-146 is predominantly expressed in CD68+ macrophages but is also expressed in some CD3+ T cell subsets and CD79a+ B cells in RA synovial tissue (14). Pauley et al reported that PBMCs from RA patients exhibit high expression of miR-146a, miR-155, miR-132, and miR-16 compared with healthy individuals and control patients (15). This evidence, which proves that miRNA play a role in RA pathogenesis, may lead to a novel treatment strategy.
Several therapeutic trials to regulate miRNA in vivo have been undertaken. Tazawa et al demonstrated that local injection of double-stranded miR-34a into a tumor suppresses tumor growth in mice (22). We previously showed that local injection of miR-1, miR-133, and miR-206 accelerates muscle regeneration in the rat model of skeletal muscle injury (23). We have also shown that intraarticular injection of double-stranded miR-15a induces apoptosis in the synovium of arthritic mice (24). A novel therapy targeting miRNA might be developed for RA.
Taganov et al reported that the induction of miR-146a/b is regulated by NF-κB, and that miR-146a/b plays a role in fine-tuning innate immune responses by providing negative feedback with down-regulation of TRAF6 and IRAK1 genes (16). In RA synovium, the expression of miR-146a might be induced by NF-κB to regulate the expression of proinflammatory cytokines by negative feedback. Proinflammatory cytokines, especially TNFα, are one of the most important factors in osteoclastogenesis (25, 26).
Osteoclastogenesis induced by TNFα occurs independently of RANK signaling. Biologic agents such as TNFα blockers have been shown to successfully decrease joint destruction. In the present study, overexpression of miR-146a inhibited osteoclastogenesis induced by both TNFα and RANKL, although TNF induced osteoclastogenesis independently of TRAF6, which is recognized as the target gene of miR-146a. However, the mechanism by which miR-146 inhibits osteoclastogenesis remains unclear. Bhaumik et al demonstrated that expression of miR-146a/b in the metastatic human breast cancer cell line functions to negatively regulate NF-κB activity (27). When miR-146a is overexpressed in PBMCs in the osteoclastogenesis culture system induced by TNFα, miR-146a has the potential to inhibit osteoclastogenesis via negative regulation of NF-κB activity independently of the TRAF6 pathway. It is possible that miR-146a also inhibits the expression of TRAF6, although TRAF6 is recognized to be the critical signaling molecule in RANKL-mediated osteoclastogenesis (28–30).
The number of target genes of miRNA is estimated to range between one and several hundred, based on target predictions using the bioinformatics approach (31). Several factors other than TRAF6 and IRAK1 might contribute to the inhibition of osteoclastogenesis by miR-146a. Moreover, the mechanism of action of osteoclastogenesis has not been elucidated.
In this study, since osteoclastogenesis was successfully inhibited by transfection of double-stranded miR-146a in vitro, double-stranded miR-146a was administered to mice in vivo via intravenous injection. Administration of double-stranded miRNA-146a prevented joint destruction in mice. However, it did not completely ameliorate joint inflammation. Although bone and cartilage destruction were prevented in mice by administration of miRNA-146a, histologic analysis showed that synovitis was not completely abrogated. However, there was no significant difference in the arthritis score between the mice that received nonspecific double-stranded RNA and those that received miR-146a.
Immunofluorescence analysis showed that administration of miR-146a to mice did not completely suppress the expression of TNFα, IL-1β, or IL-6. First administration of double-stranded miR-146a was conducted after the onset of distinct arthritis. MicroRNA suppressed the production of protein such as TNFα by targeting mRNA, but miRNA did not suppress the protein that had already been secreted.
Administering double-stranded miR-146a by intravenous injection might not have a great enough antiinflammatory effect; this may explain the lack of a significant difference in arthritis score between mice that received nonspecific double-stranded RNA and those that received miR-146a. The combination of a biologic agent and disease-modifying antirheumatic drugs might be more effective. Khoury et al reported that an intravenous injection of a cocktail of 3 siRNA for IL-1, IL-6, and IL-18 ameliorated joint inflammation and degradation (32). The combination of several miRNA, which play a crucial role in RA pathogenesis, has greater potential to effectively reduce inflammation.
Moreover, the methods of administration of synthetic miRNA, including dosage and delivery system, should be examined to determine which methods ameliorate arthritis most efficiently. We examined the distribution of double-stranded miR-146a 24 hours after injection using real-time qRT-PCR, as previously described (24). The results revealed that miR-146a was up-regulated in the liver, spleen, and kidney. However, there was no notable difference in the hind paws between mice that received an injection of miR-146a and mice that received no injection (data not shown). There is a possibility that miR-146a affects circulating cells that play a role in arthritis, including bone destruction; however, the functional mechanism of double-stranded miR-146a could not be elucidated. The tissue distribution and cell type that takes up the administered double-stranded miRNA should be examined in the future.
This is the first study to evaluate the systemic administration of double-stranded miRNA for arthritis. Although this preliminary study might provide a useful optional tool for RA treatment, further studies are needed to determine more comprehensive therapeutic strategies.
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All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Dr. Nakasa had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.
Study conception and design. Nakasa, Ochi.
Acquisition of data. Nakasa, Shibuya, Nagata.
Analysis and interpretation of data. Nakasa, Shibuya, Nagata, Niimoto.