Rheumatoid arthritis (RA) is a systemic inflammatory autoimmune disease affecting ∼1% of the population and is one of the most prevalent causes of disability worldwide (1). Its pathogenesis, however, although studied intensively, has not yet been clarified. Evidence from animal models and from therapeutic trials in patients with RA has suggested an involvement of T cells, B cells, osteoclasts, and several cytokines in the induction and/or perpetuation of the disease (2–8). However, because a substantial number of RA patients still do not respond sufficiently to current treatment options, the development of new therapeutic targets is needed (9). Evidence has emerged to indicate that microRNAs (miRNA) play a crucial role as posttranscriptional regulators of gene expression (10–12). MicroRNA-mediated gene regulation normally results in a reduction of the total amount of target protein that is produced. However, the fate of most messenger RNAs (mRNA) that are targeted by miRNA remains unclear.
The miRNA have been shown to repress expression of some of their target genes at the translational level, with mRNA levels remaining constant and the level of the encoded protein declining, whereas in other cases, miRNA repress target gene expression by triggering the degradation of target mRNA (13, 14). In the first years after the discovery of miRNA, they were thought to be involved mostly in controlling proliferation, cell differentiation, or apoptosis (15–17). More recently, it has become clear that they also exert powerful regulatory functions in cancer development and the immune system, including regulation of T cells, B cells, and dendritic cells (DCs) (11, 14, 18–22).
The miRNA miR-155 has been detected in the joints and synovial fibroblasts of patients with RA and has been found to be involved in the regulation of matrix metalloproteinase 3 (23). RA is a disease characterized by chronic, destructive arthritis resulting from complex alterations of not only the adaptive immune system, but also the innate immune system. Whereas the adaptive immune system is thought to play an important role mainly in the initial phase of RA, disturbances of the innate immune system become more important at later stages of the disease (24). We therefore investigated the role of miR-155 in the pathogenesis of autoimmune-mediated arthritis, by analyzing 2 different animal models, each of them representing a main immune system pathway in RA. The collagen-induced arthritis (CIA) model depends on adaptive immune responses, whereas the K/BxN serum-transfer arthritis model depends on alterations of the innate immune response.
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The contribution of miRNA to the pathogenesis of complex systemic autoimmune diseases such as RA is not known. This study is the first to demonstrate that the lack of a single miRNA, miR-155, is sufficient to interfere with 2 major pathways of destructive arthritis, namely, the adaptive and innate immune systems. It has been previously reported that miR-155 is involved in antibody formation after immunization with KLH or tetanus toxoid (20, 29, 30). Indeed, miR-155 deficiency completely prevents the development of CIA, which is dependent on adaptive immune-regulatory mechanisms, by reducing the levels of pathogenic anticollagen autoantibodies. This reduction is apparent in all classes of IgG analyzed, namely, IgG1, IgG2a, and IgG2c. However, anticollagen IgM levels, B cell numbers, and the expression of costimulatory molecules on these cells are not altered by the absence of miR-155.
In addition, proliferation of antigen-specific T cells after restimulation with collagen is reduced in miR-155−/− mice compared to WT animals. These results demonstrate that miR-155 also plays an important role in regulating autoreactive T cell responses in our model. Furthermore, after stimulation with an anti-CD3 antibody, miR-155−/− mouse T cells showed reduced production of the Th17 cytokines IL-17 and IL-22, both of which have been shown to be important in the development of CIA (31, 32). Therefore, our experiments demonstrate that after immunization with collagen, miR-155−/− mice show a selective defect in Th17 polarization.
In vitro assays have previously shown that miR-155−/− mouse T cells preferentially differentiate into Th2 cells, and in vivo immunization experiments using tetanus immunization have revealed decreased production of IFNγ and IL-2 in miR-155−/− mouse T cells, indicating that Th1 polarization is reduced (20, 29). Interestingly, in our study, after immunization with collagen, the production of IFNγ and IL-4 by miR-155−/− mouse T cells was not affected after nonspecific stimulation with an anti-CD3 antibody.
The miRNA miR-155 also plays an important role in regulating innate effector mechanisms of destructive arthritis. In the K/BxN serum-transfer arthritis model, we found similar amounts of synovial inflammation in WT and miR-155−/− mice. This was somewhat surprising, since previous studies have shown an important involvement of miR-155 in innate inflammation pathways (18, 33). However, we noticed an uncoupling of inflammation and bone destruction, since osteoclast-mediated bone erosions were significantly reduced in miR-155−/− mice compared to WT mice. In line with our in vivo findings, miR-155 deficiency also decreased RANKL-induced osteoclastogenesis in vitro.
Taken together, these findings show that miR-155 has a dual effect on controlling autoimmune-triggered destructive arthritis. On the one hand, miR-155 deficiency inhibits the generation of pathogenic self-reactive T and B cell responses. On the other hand, miR-155 controls the development of local bone destruction. These data identify miR-155 as a possible novel target in the treatment of autoimmune arthritis.
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- MATERIALS AND METHODS
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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. Redlich 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. Blüml, Bonelli, Hayer, Koenders, Smolen, Redlich.
Acquisition of data. Blüml, Bonelli, Niederreiter, Puchner, Mayr.
Analysis and interpretation of data. Blüml, Bonelli, Hayer, Koenders, van den Berg, Smolen, Redlich.