MicroRNAs-novel regulators of systemic lupus erythematosus pathogenesis

reviews MicroRNAs—novel regulators of systemic lupus erythematosus pathogenesis Nan Shen, Dong Liang, YuanJia Tang, Niek de Vries and Paul-Peter Tak

s w e i v D e E R T e r C u E t R a N OR OF C O N R U P

Abstract | Dysregulation of gene expression can cause complex disease phenotypes. MicroRNAs (miRNAs) are well-known to fine tune cellular gene expression to control immune cell development and regulate adaptive and innate immune responses. Discoveries over the past decade have indicated that aberrant expression of miRNAs is associated with the pathogenesis of multiple immunological diseases, including systemic lupus erythematosus (SLE). Indeed, profiling miRNA expression in blood cells, body fluid and target tissues taken from patients with SLE has revealed unique miRNA signatures when compared with healthy individuals or those with other diseases. Moreover, dysregulation of these miRNAs has also been found to be associated with disease activity and major organ involvements. In our opinion, therefore, miRNAs have the potential to act as biomarkers for the diagnosis and assessment of patients with SLE. This Review provides an overview of the novel cellular and molecular mechanisms that seem to underlie the roles of miRNAs in SLE disease processes, as well as the future therapeutic potential of targeting miRNAs in the management of patients with SLE. Shen, N. et al. Nat. Rev. Rheumatol. advance online publication XX Month 2012; doi:10.10.38/nrrheum.2012.142

Introduction

MicroRNAs (miRNAs) are a novel class of endogenous, noncoding small RNAs of approximately 19–25 nucleotides in length. MiRNAs are ubiquitously expressed in a wide range of species including viruses, worms, flies, plants and animals1,2 and negatively regulate gene expression at the post-transcriptional level by targeting specific mRNAs for degradation or suppressing mRNA translation. The key role of miRNAs in multiple physio­logical and pathological processes, including stem cell develop­ ment, cell differentiation and organogenesis, proliferation and apoptosis, immune regulation and disease development, is now well established.3–5 Although the contribution of an individual miRNA in repression of a target gene is subtle (~50% reduction in expression), the 3' untranslated regions (UTR) of human mRNAs normally harbour multiple conserved miRNA binding sites that can be co-targeted by multiple miRNAs. Moreover, as one miRNA can regulate a set of genes functioning in the same pathway, this mechanism should ensure the final physio­ logical outcome. Furthermore, miRNAs, as novel regulatory factors, have now been shown to work co-operatively with transcription factors, contributing to the formation of a complex post-transcriptional regulatory network, which reduces the fluctuations in gene expression.6,7 The whole human genome is estimated to encode a minimum of 800 unique miRNAs,6 which are predicted to target one-third of human genes.9,10 Immune genes constitute an enriched source of miRNA targets. Indeed, a report from 20077 indicated that miRNAs preferentially target immune genes when compared with the whole Competing interests The authors declare no competing interests.

nature reviews | Rheumatology

genome, with more than 45% of immune genes predicted to contain 3' UTR miRNA binding sites. Systemic lupus erythematosus (SLE) is regarded as a prototypic systemic autoimmune disease,8 which can affect multiple organs and systems owing to its undefined aetiology, complex pathogenesis and a current lack of targeted treatment. Considering that miRNAs are known to function as regulators of several patho­physiological processes in the immune system, one can infer that they may also participate in the pathogenesis of SLE. In this Review, we summarize the current insights into miRNA bio­ genesis as well as novel functions of miRNA in regulation of immune responses and development of auto­immune diseases. Moreover, we discuss the roles of miRNAs in the pathogenesis of SLE and underscore the potential of using specific miRNAs as novel bio­markers and therapeutic targets for the future management of patients with SLE.

MiRNA biogenesis Genomic analyses of miRNA transcripts have revealed that a large proportion of miRNAs are located within introns of coding or noncoding regions, with a few in exons of long noncoding regions.9,10 Following the formation of a functional miRNA (Box 1), the target mRNA is then most commonly degraded,11 although miRNAs can also induce translational repression of target mRNAs.12–14 Of note, studies15,16 have also revealed that the ‘star’ form miRNA (that is, the non-dominant form derived from the mature miRNA), which was thought to be rapidly degraded during miRNA maturation, may also possess inhibitory activity and have a role in physio­logical processes similar to its miRNA partner.20,21 In some relatively rare cases, intronic miRNAs, termed mirtrons, can

Joint Molecular Rheumatology Laboratory of the Institute of Health Sciences and Shanghai Renji Hospital, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai Jiaotong University School of Medicine, 145 Shan Dong Middle Road, Shanghai 200001, China (N. Shen, Y. Tang). Division of Rheumatology and the Center for Autoimmune Genomics and Etiology, Cincinnati Children’s Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, OH 45229, USA (D. Liang). Division of Clinical Immunology and Rheumatology, Academic Medical Center, University of Amsterdam, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands (N. de Vries, P. P. Tack).

Correspondence to: N. Shen nanshensibs@ gmail.com

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reviews Key points ■■ MicroRNAs (miRNAs) control immune cell differentiation, and regulate innate and adaptive responses; dysregulation of these immunologically important miRNAs can cause systemic autoimmunity and tissue inflammation ■■ Patients with systemic lupus erythematosus (SLE) and mouse models of SLE present abnormal miRNA expression, which are associated with disease activity and severity, when compared with healthy individuals or mice ■■ Differential expression of multiple miRNAs may contribute to SLE pathogenesis by regulating the type I interferon pathway, inflammatory cytokine expression, DNA methylation in T cells and local tissue inflammation ■■ MicroRNAs are potential novel biomarkers and could be future therapeutic targets for patients with SLE

pathogenesis of diverse autoimmune diseases such as multiple sclerosis,40 rheumatoid arthritis (RA),41,42 and SLE43–45 (Supplementary Table 1). The notion that miRNAs have a role in the control of immune responses has been firmly established by a series of in vitro and in vivo loss-of-function studies.39,46–49 The genetic ablation of dicer in mouse models severely impairs immune development and inflammatory responses to external stimuli, for example, parasitic [Au: OK?] infection,48 indicating a pivotal role for Dicer and the pathways that it signals through in normal immune system maintenance. Indeed, numerous miRNAs that have a role in the immune system have been unveiled, among which miR‑155 and miR‑146a have received extensive attention. Not only do these miRNAs target proinflammatory pathways that are closely associated with autoimmune diseases, but in vivo studies on these two miRNAs, have yielded the most comprehensive knowledge regarding the involvement of miRNA in autoimmune diseases. These studies suggest that miRNAs are promising therapeutic targets for patients with SLE.

s w e i v D e E R T e r C u E t R a N OR OF C O N R U P Box 1 | The formation of a functional microRNA

Generally, genes that encode microRNA (miRNA) are transcribed by RNA polymerase II to generate stem-loop primary miRNAs (pri-miRNA) composed of one or several miRNA hairpin structures.112 The primiRNAs are sequentially recognized by microprocessor complex subunit DGCR8, which forms a complex with ribonuclease 3 (also known as Drosha), to produce miRNA precursors (pre-miRNAs). After being actively transported to the cytoplasm via the Exportin‑5 pathway, the pre-miRNAs are further processed by endoribnuclease Dicer to yield miRNA duplex—the mature miRNA. One functional strand of this duplex is then recognized by the RNAi-induced silencing complex (RISC), an essential component of which is protein argonaute‑2, and loaded onto the target mRNA with imperfect complementarity.112

bypass Drosha processing, and be processed only by the endoribo­nuclease Dicer as pre-miRNAs.17–19 In addition, maturation of miR‑451 can be directly processed by protein argonaute‑2, without participation of Dicer.20 Interestingly, antibodies against autoantigen Su from patients with rheumatic diseases were reported to recognize argonaute 2 and Dicer,21 suggesting that these auto­ antibodies have the ability to interfere with the miRNA generating machinery. Their effects on miRNA biogenesis and the underlying mechanism, however, remain unclear. Evidence now indicates that protein factors involved in regulating the generation of miRNA can themselves be dynamically regulated under certain conditions to promote or suppress miRNA expression. For example, the far upstream element binding protein 2 promotes miR‑155 maturation through binding to the terminal loop of pre-miRNA in response to lipopolysaccharide (LPS) stimulation,22 whereas Dicer protein expression can be inhibited by multiple types of stresses including reactive oxygen species, phorbol esters and the Ras oncogenes [Au: OK?].23 Moreover, risk factors for SLE, such as Epstein– Barr virus (EBV) infection24,25 and sex hormones,26 can also trigger the dysregulation of miRNA expression.

MiRNAs in the immune system Since the discovery of miRNAs, their biological functions in both innate and adaptive immunity have been the focus of extensive study.27 A growing body of literature has revealed essential roles for miRNAs in the regulation of Toll-like receptor (TLR) signalling,28,29 immune cell differentiation and maturation3,30–39 as well as the 2  |  aDVANCE ONLINE PUBLICATION

MiR‑155 A role for miR‑155 in the immune system was first demon­strated in bic/miR‑155–/– mice. By specific regulation of T‑cell differentiation and germinal centre response, miR‑155 influences T‑cell-dependent antibody generation and controls the production of cytokines including tumour necrosis factor (TNF), lymphotoxin‑α, IL‑4, IL‑10 and interferon (IFN)‑γ.50 Despite normal lymphocyte development, these mice exhibited severe immune deficiencies in T cells and B cells, including altered T‑helper‑1 cell function, skewed T‑helper‑2 cell differentiation and defective B‑cell class switching.51 MiR‑155 has also been reported to negatively regulate toll-like receptor (TLR) signalling through targeted repression of multiple key components of the downstream pathway, 28 as well as contribute to the patho­ genesis of multiple inflammatory disorders such as atopic dermatitis,52 acute coronary syndrome53 and inflammatory arthritis54 (Supplementary Table 1). Furthermore, a report in 201015 indicated that miR‑155 and miR‑155* can co-operatively regulate type I IFN production in plasmacytoid dendritic cells, providing additional evidence for its potential role in SLE pathogenesis.

MiR‑146a MiR‑146a was originally identified in a search for ­endotoxin-responsive genes and was found to be regu­ lated by Nuclear factor‑κB (NF‑κB) in the THP‑1 human monocytic cell line.55 The elevated expression of miR‑146a in THP‑1 cells gave rise to endotoxin-induced tolerance56 and cross-tolerance57 through translational inhibition of TNF receptor-associated factor 6 (TRAF6) and IL‑1 receptor-associated kinase 1 (IRAK1). These findings were confirmed in vivo as miR‑146a conferred intestinal epithelial tolerance during the neonatal period.58 MiRNA‑146a has a repressive role in auto­immunity, as demonstrated in a study published in 2011 in which miR‑146a null mice displayed markedly increased LPS sensitivity and www.nature.com/nrrheum

reviews Table 1 | MiRNA expression profile in human SLE Dysregulated miRNAs

Source of sample

Platform

Study

miR‑31, miR‑95, miR‑99a, miR‑130b, miR‑10a, miR‑134, miR‑146a

More than sixfold lower in PBMCs

TaqMan miRNA Assay (156miRNAs)

Tang et al. 200963

miR‑1246, miR‑574-5p, miR‑1308, miR‑638, miR‑7, and miR‑126

Increased more than twofold in CD4+ T cells

miRNA array (873 miRNAs)

Zhao et al. 201170

miR‑142-5p, miR‑142-3p, miR‑31, miR‑186, and miR‑197

Decreased more than twofold in CD4+ T cells

miRNAarray (873 miRNAs)

Zhao et al. 201170

miR‑518c*, miR‑23a, miR‑638, miR‑198, miR‑583, miR‑200c, miR‑612, miR‑516-5p, miR‑142-5p, miR‑320, miR‑657, miR‑184, miR‑197, let-7e, miR‑134, miR‑494, miR‑513, miR‑575, let-7a, miR‑658, miR‑600, let-7a, miR‑433, miR‑185, miR‑324-5p, miR‑325, miR‑662, miR‑208, miR‑130b, miR‑30a-5p, miR‑601, miR‑622, miR‑608, miR‑195, miR‑124a, miR‑15b

Increased more than twofold in renal biopsy samples

miRNAarray (455 miRNAs)

Dai et al. 200965

miR‑296, miR‑150, miR‑365, miR‑324-3p, miR‑518b, miR‑346, miR‑637, miR‑133a, miR‑557, miR‑615, miR‑345, miR‑642, miR‑654, miR‑484, miR‑99a, miR‑223, miR‑611, miR‑30d, miR‑500, miR‑663, miR‑423, miR‑381, miR‑602, miR‑210, miR‑596, miR‑486, miR‑769-3p, miR‑629, miR‑92b, miR‑150

Decreased more than twofold in renal biopsy samples

miRNA array (455 miRNAs)

Dai et al. 200965

HMP-miR189, HMP-miR61, HMP-miR78, miR‑21, miR‑142-3p, miR‑342, miR‑299-3p

Increased more than twofold in PBMCs

miRNA array (331 miRNAs)

Dai et al. 200762

miR‑196a, miR‑17-5p, miR‑409-3p, HMP-miR141, miR‑383, HMP-miR112

Decreased more than twofold in PBMCs

miRNA array (331 miRNAs)

Dai et al. 200762

miR‑21, miR‑25, miR‑148a, miR‑148b, miR‑214, miR‑494, miR‑198, miR‑324-3P, miR‑342, miR‑373, miR‑106b, miR‑544, miR‑155

3.4–9.2-fold upregulated in PBMCs

TaqMan low density array (365 miRNAs)

Stagakis et al. 201166

miR‑296, miR‑196a, let‑17-5p, miR‑383, miR‑184, miR‑379, let‑15a, let‑16, miR‑150, miR-7a, miR-7d, miR-7g, miR‑98, miR‑532

2.2–18.0-fold downregulated in PBMCs

TaqMan low density array (365 miRNAs)

Stagakis et al. 201166

miR‑371-5P, miR‑423-5P, miR‑638, miR‑1224-3P, hsa-miR‑663

Dysregulated in EBVtransformed B cell lines

miRNA array (850 human, 584 mouse, 426 rat and 122 viral precursor miRNAs)

Te et al. 201064

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Abbreviations: EBV, Epstein–Barr virus; HMP, human predicted sequence; miRNA, microRNA; PBMCs, peripheral blood mononuclear cells; SLE, systemic lupus erythematosus.

overproduction of proinflammatory cytokines than did mice that expressed this miRNA.59 Moreover, miR‑146a was reported to be induced in T cells following T‑cell receptor stimulation leading to suppression of IL‑2 production.60 This finding highlights the involvement of miR‑146a in adaptive immunity.

MiRNAs in SLE

MiRNA expression signatures MiRNA expression profiles have been successfully utilized in classification of human cancers.61 Studies of miRNA expression profiles in patients with SLE or animal models of SLE are expected to reveal the biological and clinical relevance of miRNAs in SLE. Dai et al.62 identified seven downregulated and nine upregulated miRNAs in patients with SLE when compared with healthy individuals and patients with idio­pathic thrombocytopenic purpura (diseased controls) (Table 1). Furthermore, 42 miRNAs were found to be differentially expressed in samples of peripheral blood mononuclear cells (PBMCs) from patients with SLE when compared with those from healthy individuals (Table 1).63 Expression of seven of these miRNAs was more than sixfold lower in patients than in healthy controls; miR‑146a was the most substantially down­regulated. Using a miRNA microarray technique, another group investigated miRNA expression levels in EBV-transformed B-cell lines and frozen PBMCs obtained from patients with lupus nephritis and healthy controls in different racial nature reviews | Rheumatology

groups (African American and European American), and identified four upregulated miRNAs and one downregulated miRNA in patients. The upregulation of miR‑638 and miR‑663 is consistent with the data generated in a previous miRNA profiling study of kidney biopsy samples,64 in which miRNA micro­array chip analysis identified 66 miRNAs differentially expressed in patients with lupus nephritis (Table 1).65 TaqMan Low Density Arrays have been used to analyze the expression of 365 miRNAs in the PBMCs from 34 patients with SLE and 20 healthy controls. 14 miRNAs were identified to be considerably downregulated and 13 miRNAs upregulated in patients with active SLE.66 This study also showed that miR‑21, miR‑25 and miR‑106b are upregulated in both T cells and B cells from patients with SLE; eight miRNAs exhibited altered expression only in T cells from patients with SLE whereas four miRNAs had altered expression in B cells only. Another group also reported that 11 miRNAs are differen­tially expressed in CD4+ T cells from patients with SLE (Table 1).67 Using MRL/lpr mice at 5 and 16 weeks of age, Pan et al. 68 profiled miRNA expression in splenic CD4 + T cells, and identified two upregulated miRNAs—miR‑21 and miR‑148a. This observation was later confirmed in PBMCs from patients with SLE.71 Dai et al.69 also profiled miRNA expressions in splenic lymphocytes from three spontaneous genetically lupus-prone mouse models, and found a common set of upregulated miRNAs (miR‑31,

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Autoantigen

Pathogen Type I IFN

IFNAR1/2

by individual miRNA detection using real-time PCR or northern blot is preferable. Moreover, the application of new technologies, such as next-generation sequencing,72,73 in which parallel sequencing enables robust and sensitive detection of miRNA expression in a systematic manner, has the potential to greatly advance the progress of highthroughput miRNA profiling data capture and analysis.

Dysfunction of miRNAs

TLR7–TLR9 MyD88 IRAK1

miR-146a

STAT1

MiR‑146a–IFN hyperactivation The expression of miR‑146a is considerably decreased in patients with SLE74 when compared with healthy controls, but not in patients with other diseases such as BehÇet disease, (an autoinflammatory disease but not a classic autoimmune disease).63 Of note, the downregulation of miR‑146a was independent of the influence of medications (specifically steroids and secondary antirheumatic agents), indicating that miR‑146a is dysregulated in SLE. In contrast to the observations made in patients with SLE, miR‑146a was reported to be induced in patients with RA74 and Sjögren’s syndrome.75 This induction, however, failed to limit the production of proinflammatory cytokines such as, TNF, IL‑1b and IL‑18, probably owing to unknown mechanisms that lead to insufficient inhibition of TRAF6 and IRAK1—the two established mRNA targets of miR‑146a in THP‑1 cells.76 It is thus note­worthy that one may not expect that the same miRNA acts uniform­ly under different pathological conditions. The abnormal downregulation of miR‑146a in patients with SLE is strongly associated with clinical disease activity. Indeed, a lower level of miR‑146a expression is particularly observed in patients with active SLE and in those with concurrent presence of proteinuria.63 The expression level is also inversely correlated with SLEDAI score as well as renal SLEDAI score—two weighted and cumulative indexes of SLE activity. Moreover, the low expression of miR‑146a is associated with the activation of the type I IFN pathway—a pathway with a pivotal role in the pathogenesis of SLE. Our data showed that both IFN-inducible genes and, consequently, calculated IFN scores are inversely correlated with miR‑146a levels.63 Type I IFN activates signal transducer and activator of transcription (STAT) proteins and initiates transcription of IFN-inducible genes through stimulating IFNsensitive response elements (ISREs) in their promoter regions. MiR‑146a inhibits transcription of these genes by interfering with the function of the ISRE after stimulation with type I IFN.63 Moreover, the ability of miR‑146a to negatively regulate type I IFN production was further investigated in macrophages during viral infection77 and in THP‑1 cells in response to stimulation with LPS or viral mimics.78 MiR‑146a therefore seems to be a negative regulator of the type I IFN pathway. Basal expression and activation of STAT1 have been shown to be elevated in mouse models of SLE and patients with SLE and IRF5 is one of most well-­recognized SLE disease genes.79 Both IFN regulatory factor 5 (IRF5) and STAT1 were directly repressed by enforced miR‑146a expression in vitro.63 In addition, the inhibitory effect of miR‑146a on STAT1 was also confirmed in a study in

s w e i v D e E R T e r C u E t R a N OR OF C O N R U P TRAF6

IRF5

Pre-miR-146a

Gene variants Loss of ETS1 binding

IFN-inducible genes

Pri-miR-146a

Proinflammatory cytokines

Figure 1 | Roles of miRNA in type I IFN abnormal activation in SLE. In physiological processes, activation of TLR receptors (for example, TLR7–TLR9) triggers sequential signalling and leads to the production of type I IFN, which in turn bind to their receptors and induce downstream activation. In this scenario, various negative regulators, including miR‑146, are simultaneously induced. The mature miR‑146 uses inhibitory machinery to reduce expression of its target genes, including those that encode IRAK1, TRAF6, IRF5, and STAT1, thereby attenuating positive signalling. In SLE, the binding site of the transcription factor ETS1 in the miR‑146a promoter region is disrupted by the presence of the risk allele of the SNP (rs57095329) resulting in a reduction in its expression. The consequent aberrant accumulation of miR‑146a targeted proteins (TRAF6, IRAK1, IRF5 and STAT1) leads to cascade signal amplification, contributing to the altered activation of the IFN pathway. Abbreviations: IFN, interferon; IRAK1, IL-1 receptor-associated kinase 1; IRF5, interferon regulatory factor 5; miRNA, microRNA; SLE, systemic lupus erythematosus; STAT1, signal transducer and activator of transcription 1; TLR, Toll-like receptor; TRAF6, tumour necrosis factor receptor-associated factor 6.

miR‑155 and miR‑182‑96‑183 cluster). This result, how­ ever, is partially inconsistent with data generated from patients: upregulation of miR‑155 was confirmed in PBMCs and CD19+ B cells,66 but miR‑31 was found to be downregulated in PBMCs63 and CD4+ T cells.70 The dysregulation of miR‑182‑96‑183 cluster was not reported in any miRNA expression profile studies in patients with SLE. Although numerous dysregulated miRNAs have been identified in patients with SLE and mouse models of SLE, relatively limited overlap is observed between the ‘positive’ miRNAs generated in these studies (Table 1). This observation was especially apparent in the early stage of miRNA profiling studies, when precise normalization methods and optimized technical procedures were not well established. The lack of overlap in the miRNAs identified from early miRNA profiling studies was also observed in the profiling of multiple sclerosis.71 Furthermore, miRNA profiling is affected by the diversity in disease severity, medical history and race of patients with SLE, as well as differences in cell types and sample species. In this case, miRNA profiling data from either miRNA microarray or real-time PCR miRNA array should be used with caution and viewed only as a guide, and further confirmation

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reviews 201080 using a miR‑146a null mouse model, in which the inhibition of miR‑146a resulted in an elevated amount of both total and phosphorylated stat1 in T‑regulatory cells and non‑T-regulatory cells. Collectively, miR‑146a seems to regulate immune activation by targeting key signalling proteins in the type I IFN pathway. A previous study 37 indicated that miRNAs inhibit expression of most targets at the post-trancriptional level in a relatively mild manner. Using mass spectroscopy and microarray technique, Selbach et al.81 and Baek et al.82 also reported that miRNAs negatively regulate hundreds of targets only to a modest degree. Despite the relatively subtle inhibitory effects, miR‑146a is capable of exerting a long-lasting and accumulative influence by targeting multiple key components of type I IFN signalling.55,63 The aberrant accumulation of these targeted proteins (STAT1, IRF5, TRAF6 and IRAK1) results in cascade signal amplification, contributing ultimately to the hyperactivation of the IFN pathway (Figure 1). As the expression of miR‑146a can be robustly induced by various stimuli (such as lipopolysaccharide, imiquimod‑R837, CpG‑A/ODN2216, and type I IFN) in PBMCs from healthy individuals, miR‑146a could be an inducible negative regulators that controls the innate immune response.55 This observation also raises a question regarding whether the dysregulation of miR‑146a is an etiologic factor in SLE development. Splenic mononuclear cells from the NZB/W mice, but not MRL/lpr lupus-prone mice, at the pre-autoimmune stage were reported to have increased expression of many known IFN-regulated genes. 83 We therefore compared the expression of miR‑146a in CD19+ and CD14+ cells isolated from 8‑week-old BALB/c and NZB/NZW mice and found a similar reduction in the expression of miR‑146a in both subsets (unpublished data), implying that the miR‑146a reduction in SLE represents a causal factor, rather than the consequence, of SLE. Of note, miR‑146a was identified to be downregulated by oestrogen,26 suggesting an association between miRNA dysregulation and female dominance in the prevalence of SLE. As miR‑146a fine-tunes gene expression but strongly inhibits downstream signalling of the IFN pathway, it could serve as a therapeutic target for SLE. Indeed, exo­ genous introduction of miR‑146a into PBMCs from patients with SLE alleviates the coordinate activation of the type I IFN pathway, as indicated by a substantial reduction (~75%) in mRNA levels of three selected IFNinducible genes: IFN-induced protein with tetratrico­ peptide repeats 3 (IFIT3), myxoviurs resistance 1 (MX1), and 2',5'-­oligoadenylate synthetase 1 (OAS1).63 It is thus noteworthy that, in addition to traditional immuno­ suppressive treatment, manipulation of miR‑146a expression may bring a benefit to patients with SLE [Au: Sentence OK?].

Patient with SLE RasGRP

MEKK1

miR-21 miR-148a miR-126

DNMTs ? DNA hypomethylation

Epigenetic alterations

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MiRNAs and DNA hypomethylation CD4+ T cells from patients with SLE patients display aberrant DNA hypomethylation. The mechanism that leads to the epigenetic aberrance remains unknown. Studies in cancer 84,85 have linked multiple miRNAs with gene nature reviews | Rheumatology

Methylation-sensitive gene CD11a, CD70, CD40L

Figure 2 | Roles of miRNA in SLE hypomethylation. Upregulation of miR‑21 indirectly inhibits DNMT1 by targeting RasGRP. MiR‑148 and miR‑126 can directly inhibit DNMT1. This inhibition in turn reduces the CpG methylation level and causes upregulation of autoimmune-associated genes in SLE, such as those that encode CD70, CD11a and CD40L. Abbreviations: DNMT1, DNA methyltransferase 1; miRNA, microRNA; SLE, systemic lupus erythematosus.

hypomethylation. Our group previously identified a total of 125 miRNAs differentially expressed in CD4+ T cells of MRL/lpr lupus mice.68 MiR‑21 and miR‑148a are among the 24 miRNAs that are more than fourfold higher in CD4+ T cells from MRL/lpr mice when compared with controls. MiR‑21 was also previously found to be upregulated in PBMCs from patients with SLE and highly correlated with SLE disease activity in studies by two other groups.62,66 DNA methyltransferase 1 (DNMT1) is one of the major epigenetic regulators and has also been linked with DNA hypomethylation in T cells from patients with SLE.86 DNMT1 protein level was considerably decreased by overexpression of miR‑21 and miR‑21 reduced Ras–MAPK– ERK signalling, a major upstream signalling pathway of DNMT1 in T cells.63 Bioinformatics tools identified the potential targets of miR‑21, and indicated that miR‑21 base pairs with sequences in the 3' UTR of RAS guanyl releasing protein 1 (RASGRP1), an important autoimmune gene that activates the Erk–MAP kinase cascade and regulates T‑cell development and homeostasis.87 MiR‑148a directly downregulated DNMT1 expression by targeting the protein coding region of its transcript.63 These data provide evidence that elevated expression of miR‑21 and miR‑148 contribute to the development of SLE through their effects on inhibiting DNA methylation in T cells. In another study, Zhao et al. 67 reported that DNA methyla­tion can also be modulated in SLE CD4+ T cells by highly expressed levels of miR‑126. DNMT1 was identified to be directly targeted by miR‑126, which led to translational suppression of this protein and ultimately resulted in T cell DNA hypomethylation in SLE. Multiple miRNAs may, therefore, contribute actively to mechanisms that underlie the low DNA methylation level in SLE (Figure 2). Taken together, these results demonstrate a crucial functional link between miRNAs and the aberrant DNA hypomethylation in lupus CD4+ T cells and could help to develop new therapeutic approaches.

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b

KLF13

miR-125a

CCL5

Epigenetic alterations or gene variants

P13K

? PDCD4

miR-21

patients with SLE, which presents a positive correlation with disease activity.66 Inhibition of miR‑21 expression in SLE CD4+ T cells increases expression level of its target programmed cell death 4, a tumour suppressor, which results in decreased T-cell proliferation and impaired production of IL‑10 and CD40L66 (Figure 3b). The IL‑2 production deficiency in patients with SLE and in mouse models of SLE has long been recognized as one of the pathogenic factors that causes T-cell dysfunction and defective immune response.92 Thanks to many years of concerted efforts invested on understanding of the underlying molecular basis, we now know that multiple transcriptional regulators (including cAMP responsive element modulator [CREM] and cAMP response-­binding protein [CREB]) and transcription factors (NF-κB, AP‑1 and nuclear factor of activated T cells [NFAT]) may cooperatively regulate transcription of IL‑2 in T cells in patients with SLE.93 Current working models, however, do not fully account for all of the phenotypes observed. In 2012, Fan et al.94 reported that the downregulation of miR‑31 in SLE T cells may contribute to impaired IL‑2 production. RhoA, a negative regulator of NFAT transcription activity, was previously identified as a direct target of miR‑31 in the MDA-MB‑231 breast cancer cell line.95 Moreover, the overexpression of miR‑31 reduced both mRNA and protein expression levels of RhoA, and the expression levels of miR‑31 and RhoA are inversely correlated in patients with SLE. These lines of evidence suggest a direct relationship between miR‑31 and its target RhoA in SLE. However, RhoA is most probably not the only target, considering that miRNAs are capable of targeting a set of genes in a shared pathway.96 This observation also explains an increased nuclear expression of NFAT upon miR‑31 overexpression in stimulated T cells, an unexpected result in response to RhoA inhibition.97 MiR‑31 expression was also positively correlated with IL‑2 production, and a restoration of normal IL‑2 production was achieved through ectopic miR‑31 expression in activated T cells from patients with SLE. Overall, this study provides new mechanistic insights into IL‑2 defect in SLE and suggests a promising therapeutic utility of miR‑31.

s w e i v D e E R T e r C u E t R a N OR OF C O N R U P Cell proliferation Cell survival IL-10 production CD40L

Figure 3 | Role of miR‑125a and miR‑21 in regulation of cytokine/chemokine production in T cells in SLE. a | A regulatory feedback loop involves expression of miR‑125a, KLF13, and CCL5 in activated T cells. CCL5 induced after stimulation requires the binding of KLF13 to its promoter. This schematic representation shows in patients with SLE, miR‑125a acts as a negative regulator that reduces CCL5 expression by targeting KLF13. In SLE T cells, decreased expression of miR‑125a leads to upregulation of the transcription factor KLF13, which, in turn, contributes to the elevation of the level of the inflammatory chemokine CCL5. b | MiR‑21 was identified to be upregulated in SLE peripheral mononuclear blood cells. In patients with SLE, miR‑21 directly targets PDCD4, a selective protein translation inhibitor, and consequently, leads to increased T-cell proliferation and survival along with enhanced IL‑10 production. Abbreviations: CCL5, CC chemokine ligand 5 (also known as RANTES); SLE, systemic lupus erythematosus; TCR, T cell receptor.

Abnormal cytokine secretion Altered expression of cytokines such as IL‑6, IL‑10, CC chemokine ligand 5 (CCL5, also known as RANTES) and IL‑2 have crucial roles in SLE development. For example, the inflammatory cytokine CCL5 is abnormally over­ expressed in blood sera from patients with SLE, whereas the expression level of IL‑2 is dramatically lower in T cells from patients with SLE. CCL5 has a fundamental role in regulation of immune cell movement from bloodstream into tissues and contributes to tissue damage such as glomerulo­nephritides.88 The development of a molecular complex containing Krueppel-like factor 13 (KLF13) is required for CCL5 expression in activated T cells.88 IL‑2 is a multifunctional cytokine that has a vital role in T‑cell activation, proliferation and contraction.89 MiR‑125a is significantly downregulated in PBMCs from patients with SLE and is selectively expressed in T cells and B cells, with T cells expressing the highest level, suggesting a role of miR‑125a in T‑cell function.90 Two evolutionarily conserved sequences in 3' UTR of KLF13, an important in vivo regulator of CCL5 expression,91 are potential binding sites of miR‑125a. Given that CCL5 expression level is considerably higher in patients with SLE than in healthy individuals, we proposed that the decreased miR‑125a results in upregulation of KLF13 and contributes to elevated CCL5 production in T cells in patients with SLE. Indeed, miR‑125a markedly downregulates KLF13 and CCL5 expression at both the mRNA and protein level.90 Further investigation revealed that miR‑125a inhibits the T‑cell-mediated secretion of CCL5 via direct targeting of the 3' UTR in KLF1390 (Figure 3a). In addition, upregulation of miR‑21 was reported in

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MiRNAs in local tissue inflammation Autoimmune diseases including SLE share a common feature of local tissue lesion caused by inflammation. IL‑17 is one of the pathogenic proinflammtory cytokines that is abnormally elevated and responsible for exacerbated inflammatory damage in patients with RA, multiple sclerosis or SLE. MiRNAs in peripheral blood cells can regulate inflammatory response, contributing to autoimmune pathogenesis. However, the role of miRNAs in local resident cells, such as fibroblast-like synoviocytes (FLSs) and kidney cells, in autoimmune pathogenesis is unclear. Zhu et al.98 identified three commonly dysregulated miRNAs (miR‑23b, miR‑30a and miR‑214) in affected tissues of patients with RA or SLE as well as in three mouse models of SLE, RA and multiple sclerosis, by comprehensively profiling miRNAs in local inflammatory lesions using human or mouse miRNA microarrays. The function of miR‑23b was chosen for further investigation as it was the most www.nature.com/nrrheum

reviews substantially downregulated miRNA identified. MiR‑23b can target transforming growth factor-β activated kinase 1/MAP3K7-(TAK)-binding protein 2 (TAB2), TAB3 and IkB kinase‑α (IKK‑α), key molecules acting in signalling pathways regulated [Au: OK?] by proinflammatory cytokines such as IL‑17, TNF and IL‑1β. Interestingly, the expression of miR‑23b is also negatively regulated following IL‑17 stimulation. Overall, these findings for the first time highlight the role of miRNAs in local tissue-resident cells in SLE pathogenesis via targeting major proinflammatory cytokine-mediated pathways.

caused by the risk allele, resulted in elevated SPI1 mRNA level contributing to SLE development. Of note, a computational target prediction revealed that all the tested 72 lupus susceptibility genes in humans or mice can be potentially targeted by miRNAs, most of them possessing multiple binding sites for over 140 conserved miRNAs.108 Whether these miRNA binding sites can be impaired by SNPs requires further experimental evidence.

Conclusions As miRNAs fine tune gene expression and have the potential to suppress multiple targets109 they could affect different stages of a signalling pathway. Using miRNA as a therapeutic target theoretically provides advantages over current drug design strategies which are focused on single gene targeting. A study in 2011 revealed that systemic delivery of a tiny seed-targeting locked nuclei acid efficiently silenced miR‑21 expression in vivo, and reversed splenomegaly, one of the cardinal manifestations of autoimmunity in B6.Sle123 mice.110 Besides, owing to the remarkable stability of miRNAs in body fluids as well as ease and reliability of detection, miRNAs isolated from blood samples or urine samples of patients also hold promise as novel noninvasive clinical biomarkers,111 especially for early diagnosis. In spite of many intriguing observations and considerable progress made in past decades, numerous fundamental questions remain unanswered. Potential links between SLE risk factors, such as genetic variation, EBV infection and oestrogen, and dysregulation of miRNAs await deep and careful exploration.108 Provocative ideas on the miRNA and its role in pathogenesis of diverse autoimmune diseases including SLE, have been put forward, and need to be further investigated, especially in vivo. The identification and characterization of candidate miRNAs that target molecules implicated in SLE pathogenesis should shed light on novel molecular mechanisms for SLE and pave the way for potential therapies in the future.

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SLE risk alleles SLE is an autoimmune disease with strong genetic disposition. Genome-wide association studies have identified multiple susceptibility loci associated with SLE.99–102 Studies on the roles of miRNA in cancer indicated that either altered miRNA expression, or polymorphisms in the sequence of miRNA or miRNA target sites can provide the intrinsic link between miRNA and disease mechanism.103 Using a candidate gene approach, Luo et al.104 identified, in multiple Asian cohorts, a novel genetic variant (rs57095329) in the promoter region of miRNA‑146a to be highly associated with SLE susceptibility. The individuals carrying the risk-associated G allele exhibited significantly reduced expression level of miR‑146a relative to those carrying the protective C allele. Further exploration showed an allelic difference of rs57095329 in miR‑146a promoter activity as revealed by altered binding affinity of ETS1, a transcription factor strongly associated with SLE susceptibility.100 Of note, within a region close to the gene encoding miR‑146a, another SLE-risk SNP (rs2431697) was identified in a European cohort of patients with SLE, confirming the genetic association of miR‑146a with SLE.105 The potential for variation in miRNA target sites is great. Some disease-related SNPs, located either in 3'UTR or even in coding sequence region can regulate gene expression through introducing or abolishing miRNA binding sites. Brest et al.106 reported that the risk allele of a synonymous SNP (rs10065172) in the gene encoding the immunity related GTPase family M protein (IRGM) can render higher susceptibility to Crohn’s disease through altering the binding site for miR‑196. In line with this finding, Hikami et al.107 also demonstrated that a functional polymorphism (rs1057233) in 3' UTR of SPI1 gene was in strong linkage disequilibrium with SLE. The disruption of miR‑569 binding site, 1.

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Acknowledgements This work was partially supported by the National Natural Science Foundation of China (No.30971632, No.81025016) and the Program of the Shanghai Commission of Science and Technology (No.10JC1409300). Author contributions N. Shen and Y. Tang researched data for the article. N. Shen, D. Liang, N. de Vries and P. P. Tack substantially contributed to the discussion of content, wrote the article and edited the article prior to submission. D. Liang also contributed to the writing of the article. Supplementary information Supplementary information is linked to the online version of the paper at www.nature.com/nrrheum

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