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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Arthritis Rheum. Author manuscript; available in PMC 2010 April 1.
Published in final edited form as:
PMCID: PMC2792591
NIHMSID: NIHMS163040

Contrast in Aberrant MicroRNA Expression in Systemic Lupus Erythematosus and Rheumatoid Arthritis: Is MicroRNA-146 All We Need?

In the current issue of Arthritis & Rheumatism, the article by Tang et al (1) describes their initial experiments comparing RNA samples from isolated peripheral blood mononuclear cells (PBMCs) obtained from patients with systemic lupus erythematosus (SLE) and controls by microRNA (miRNA) profiling of 156 miRNA species. The majority of data presented follow up on the finding that microRNA-146 (miR-146) was underexpressed in SLE. The apparent interest in miR-146 is partly because it is a known negative regulator of the type I interferon (IFN) and Toll-like receptor 4 (TLR-4) signaling pathway. Thus, underexpression of miR-146 clearly may contribute to the disease activity of SLE because the elevated IFN is believed to correlate with disease activity. Since IFN plays a critical role in the development of lupus in animal models and since the development of lupus in non–rheumatic disease patients following treatment with IFN has been reported, miR-146 may also contribute to the development of disease.

The miRNA are ~22-nucleotide noncoding RNA molecules that function in the posttranscriptional regulation of ~30% of messenger RNAs (mRNA) by binding to their 3′-untranslated region (3′-UTR), thus targeting them for degradation or translational repression. Currently, miRNA are known to regulate cellular processes such as apoptosis, differentiation, cell cycle, and immune functions. To date, the miRNA sequence database lists over 800 predicted miRNA.

Defective miRNA function leads to autoimmune disease in mice

The role of certain miRNA in the regulation of the immune response and immune cell development is becoming evident. The involvement of miRNA in a new pathway regulating autoimmunity was recently demonstrated in T lymphocytes in the sanroque mouse by Yu et al (2). The sanroque mouse, originally selected from screening mutant mice derived from the chemical mutagen N-ethyl-N-nitrosourea, has a known mutation in the gene Roquin that encodes a ring-type ubiquitin ligase. In normal T cells, Roquin regulates the expression of inducible T cell costimulator (ICOS) by promoting the degradation of ICOS mRNA. In sanroque mice, however, the absence of this regulation leads to an accumulation of activated lymphocytes that is associated with a lupus-like autoimmune syndrome. In their article, Yu et al (2) reported that miR-101 is required for the Roquin-mediated degradation of ICOS mRNA. Introducing mutations into the miR-101 binding sites in the 3′-UTR of ICOS mRNA disrupted the repression of Roquin (2). These findings revealed an interesting miRNA-mediated regulatory pathway that prevents aberrantly activated lymphocyte accumulation and autoimmune disease.

Two recent studies reported the importance of miRNA regulation in safeguarding regulatory T (Treg) cell function in the prevention of autoimmune disease (3,4). In those studies, Treg cell development and function in the absence of functional miRNA was monitored in Dicer-deficient mice; Dicer is a key RNase III enzyme required for the maturation of all miRNA. Although thymic Treg cells developed normally in these miRNA-deficient mice, the cells exhibited altered differentiation and dysfunction in the periphery (4). Specifically, the Dicer-deficient Treg cells failed to remain stable and altered the expression of multiple genes and proteins associated with the Treg cell fingerprint, including FoxP3 (4). In addition to their instability, Dicer-deficient Treg cells lost suppressor activity in vivo, and the mice rapidly developed fatal systemic autoimmune disease (4).

Interestingly, Liston et al (3) found that in disease-free FoxP3Cre/wtDicerfl/fl mice, Dicer-deficient Treg cells retained some suppressive activity, albeit reduced in comparison with wild-type mice. However, in diseased FoxP3CreDicerfl/fl mice exhibiting inflammatory conditions, Dicer-deficient Treg cells were devoid of suppressor activity and instead showed a robust in vitro proliferative response that led to the progression of a fatal early-onset lymphoproliferative autoimmune syndrome indistinguishable from that observed in FoxP3-mutant mice devoid of Treg cells (3). These data suggest that Dicer and miRNA preserve stable Treg cell function under inflammatory conditions.

Overexpression of miR-146 in rheumatoid arthritis (RA)

It is becoming increasingly clear that proper miRNA regulation is critical for the prevention of autoimmunity and normal immune functions. However, it is not yet well understood whether miRNA dysregulation could play a role in the pathogenesis of autoimmune disease in humans. Three recent studies have discovered altered miRNA expression in RA patients as compared with controls (5-7). Two of these studies examined miRNA expression in RA synovial tissue and fibroblasts. Stanczyk et al (7) reported increased miR-146 and miR-155 expression in RA synovial fibroblasts compared with osteoarthritis (OA) synovial fibroblasts. Furthermore, miR-155 expression was higher in RA synovial tissue than in OA synovial tissue, and miR-155 expression was higher in RA synovial fluid monocytes than in RA peripheral blood monocytes (7). Transfection of miR-155 in RA synovial fibroblasts revealed matrix metalloproteinase 3 as a potential target of miR-155, suggesting that miR-155 might modulate downstream tissue damage (7). Nakasa et al (5) reported that miR-146 was highly expressed in RA synovial tissue compared with OA and normal synovial tissue. In situ hybridization studies revealed that miR-146 expression could be detected in RA synovial tissue primarily in CD68+ macrophages, but also in some CD3+ T cell subsets and CD79a+ B cells (5).

The third study, which was reported by our group (6), demonstrated differential expression of miRNA in RA PBMCs, with between 1.8-fold and 2.6-fold increases in miR-16, miR-132, miR-146, and miR-155 expression, whereas miRNA let-7a expression was not significantly different, as compared with healthy controls (6). Interestingly, increased miR-16 and miR-146 expression correlated with active disease in RA patients; however, there was no correlation between the observed increase in miRNA expression and the patients' age, race, or medications. Two known targets of miR-146, tumor necrosis factor receptor–associated factor 6 (TRAF6) and interleukin-1 receptor–associated kinase 1 (IRAK1), were examined, and despite increased miR-146 expression in RA patients, there was no significant difference in mRNA or protein levels of TRAF6 or IRAK1 between RA patients and healthy controls (6). In vitro studies revealed that repression of TRAF6 and/or IRAK1 in THP-1 human monocytes resulted in up to an 86% reduction in tumor necrosis factor α (TNFα) production, indicating that normal miR-146 function could be critical for the regulation of TNFα production (6). Given that prolonged TNFα production is known to play a role in RA pathogenesis, these data suggest a possible mechanism contributing to RA pathogenesis, where miR-146 is up-regulated but unable to properly regulate TRAF6/IRAK1, leading to prolonged TNFα production in RA patients.

MicroRNA in SLE

In 2007, Dai et al (8) reported the findings of their microarray expression analysis of miRNA in PBMCs from 23 SLE patients and 10 healthy controls. In these SLE patients, 7 miRNA (miR-196a, miR-17-5p, miR-409-3p, miR-141, miR-383, miR-112, and miR-184) were down-regulated and 9 miRNA (miR-189, miR-61, miR-78, miR-21, miR-142-3p, miR-342, miR-299-3p, miR-198, and miR-298) were up-regulated as compared with healthy controls. Recently, Dai et al (9) reported the miRNA profile of kidney biopsy samples from lupus nephritis patients and healthy controls and found 66 differentially expressed miRNA (36 up-regulated and 30 down-regulated) in patients with lupus nephritis. These data suggest a possible role for miRNA as diagnostic markers and as factors involved in the pathogenesis of SLE. However, those investigators did not provide further data to show how these changes in miRNA may play a role in SLE disease pathogenesis. Furthermore, they did not detect changes in miR-146 using their custom chip-based microarray methodology, which is technically different from the real-time polymerase chain reaction analysis used by Tang et al (1).

Antigens of the miRNA pathway are targets of autoantibodies

A few of the key components of the miRNA pathway are known autoantibody targets. Ago2 has been shown to cleave mRNA targeted by small interfering RNA (siRNA) and is known as the catalytic enzyme of RNA interference (RNAi). In addition to Ago proteins, many other proteins are required for miRNA functioning, including GW182 and Ge-1, and these proteins all localize in discrete cytoplasmic foci known as GW bodies (GWB). In mammals, these foci were discovered in 2002, using an autoimmune serum from a patient with motor and sensory neuropathy, and most subsequent anti-GWB–positive sera have been identified in patients with neurologic symptoms (33%), Sjögren's syndrome (31%), and various other autoimmune disorders, including SLE (12%), RA (7%), and primary biliary cirrhosis (10%) (10,11). About the same time period, similar foci were discovered in yeast and referred to as processing bodies (P-bodies) or Dcp-containing foci.

In 1994, Satoh et al (12) characterized autoantigens of 100/102 and 200 kd recognized by anti-Su autoantibodies. Autoantibodies that immunoprecipitated the 100/102 and 200–kd proteins were detected in sera from up to 20% of patients with SLE, scleroderma, and overlap syndromes (12). In 2006, Jakymiw et al (13) reported that anti-Su autoantibodies from human patients and from the pristane-induced murine lupus model of autoimmunity recognize Ago2, the catalytic enzyme in the RNAi/miRNA pathways, as well as Ago1, Ago3, Ago4, and Dicer. Additionally, the anti-Su autoantibodies were shown by immunofluorescence analysis to recognize GWBs (13).

A recent followup study of the clinical and serologic features of patients with autoantibodies to GWBs revealed that the most common clinical presentations of these patients were neurologic symptoms, Sjögren's syndrome, SLE, RA, and primary biliary cirrhosis (10). The most common autoantigens targeted by sera from these patients were Ge-1/Hedls (58%), GW182 (40%), and Ago2 (16%), and 18% of GWB-reactive sera did not react to any of the antigens analyzed, indicating that there are other target autoantigens yet to be discovered (10).

These data demonstrate an autoimmune response to key components of the RNAi/miRNA pathways, which could indicate the involvement of the miRNA pathway in the induction and production of autoantibodies in these systemic rheumatic diseases. It is interesting to speculate that aberrant expression of certain miRNA may change the dynamic of the macro-molecular complex, or an up-regulation of GWB by miRNA may lead to a loss of tolerance and to the production of autoantibodies to these self proteins. To date, there is no evidence of autoantibodies directed toward miRNA as a group or toward individual miRNA.

MicroRNA-146 as a master gene regulator

Two recent articles (14,15) have changed our concept concerning how much a single miRNA can affect global protein expression. Using state of the art quantitative mass spectrometry, the investigators in these 2 studies analyzed changes in protein levels from the increased expression of a single miRNA or the knockdown of the same miRNA in cultured cells. Their results are consistent with each other and demonstrate that changes in the level of a single miRNA may have a significant impact on the levels of hundreds or even thousands of proteins. However, the changes in protein levels may be variable and many may be relatively mild, indicating that for most interactions, miRNA act as “rheostats to make fine-scale adjustments to protein output” (14).

These important studies are the first to show the impact of miRNA on global protein output; however, miR-146 was not included in these studies, and the magnitude of its effect on protein expression has yet to be determined. Tang et al (1) have shown that miR-146 regulates the level of at least TRAF6, IRAK1, STAT-1, and IFN regulatory factor 5 (IRF-5), all of which are important for the IFN pathway. The reported reduction of miR-146 in PBMCs from SLE patients (1) will likely affect the levels of these factors significantly and contribute to overexpression of type I IFN and, thus, disease activity.

It is intriguing that independent studies have demonstrated an increased level of miR-146 in RA patients, but a decreased level in SLE patients, as compared with healthy controls. Given that RA and SLE are both systemic rheumatic diseases, one may be surprised by the finding that miR-146 levels are contradictory in these diseases, and yet, it should not be surprising, since this may simply be reflecting a difference in the overall cytokine profiles between the two diseases, with type I IFN playing a dominant role in SLE, whereas TNFα, interleukin-1 (IL-1), and IL-6 are the principle cytokines in RA. In the coming years, one can expect research reports on miRNA expression in many other autoimmune diseases, as well as more-complete profiling data, with disease activity correlations or a lack thereof.

Whether this novel finding will translate to a promising RNAi-based therapeutics remains to be determined. A recent report on using a T cell–specific siRNA delivery system to suppress human immunodeficiency virus type 1 infection in humanized mice has shown some promising data (16). Briefly, a single-chain Fv antibody to CD7 was used to deliver siRNA, apparently successfully, in vitro as well as in the humanized mouse model. An analogous strategy can potentially be developed for other cell types to “correct” the level of miRNA. The hypothesis that many miRNA are master regulators of gene expression remains attractive for those planning to select interesting target miRNA for the development of new therapeutics.

Acknowledgments

Supported in part by the NIH (grant AI-47859) and the Andrew J. Semesco Foundation, Ocala, Florida. Dr. Pauley's work was supported by the NIH (grant DE-007200).

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