MicroRNA-146a contributes to abnormal activation of the type I interferon pathway in human lupus by targeting the key signaling proteins.код для вставкиСкачать
ARTHRITIS & RHEUMATISM Vol. 60, No. 4, April 2009, pp 1065–1075 DOI 10.1002/art.24436 © 2009, American College of Rheumatology MicroRNA-146a Contributes to Abnormal Activation of the Type I Interferon Pathway in Human Lupus by Targeting the Key Signaling Proteins Yuanjia Tang,1 Xiaobing Luo,1 Huijuan Cui,1 Xuming Ni,1 Min Yuan,1 Yanzhi Guo,1 Xinfang Huang,1 Haibo Zhou,1 Niek de Vries,2 Paul Peter Tak,2 Shunle Chen,1 and Nan Shen1 Objective. MicroRNA have recently been identified as regulators that modulate target gene expression and are involved in shaping the immune response. This study was undertaken to investigate the contribution of microRNA-146a (miR-146a), which was identified in the pilot expression profiling step, to the pathogenesis of systemic lupus erythematosus (SLE). Methods. TaqMan microRNA assays of peripheral blood leukocytes were used for comparison of expression levels of microRNA between SLE patients and controls. Transfection and stimulation of cultured cells were conducted to determine the biologic function of miR-146a. Bioinformatics prediction and validation by reporter gene assay and Western blotting were performed to identify miR-146a targets. Results. Profiling of 156 miRNA in SLE patients revealed the differential expression of multiple microRNA, including miR-146a, a negative regulator of innate immunity. Further analysis showed that underexpression of miR-146a negatively correlated with clinical disease activity and with interferon (IFN) scores in patients with SLE. Of note, overexpression of miR-146a reduced, while inhibition of endogenous miR-146a increased, the induction of type I IFNs in peripheral blood mononuclear cells (PBMCs). Furthermore, miR-146a directly repressed the transactivation downstream of type I IFN. At the molecular level, miR-146a could target IFN regulatory factor 5 and STAT-1. More importantly, introduction of miR-146a into the patients’ PBMCs alleviated the coordinate activation of the type I IFN pathway. Conclusion. The microRNA miR-146a is a negative regulator of the IFN pathway. Underexpression of miR-146a contributes to alterations in the type I IFN pathway in lupus patients by targeting the key signaling proteins. The findings provide potential novel strategies for therapeutic intervention. Supported in part by the National Basic Research Program of China (973 Program, grant 2007CB947900), the National High Technology Research and Development Program of China (863 Program, grant 2007AA02Z123), the National Natural Science Foundation of China (grant 30700734), the Key Basic Program of the Shanghai Commission of Science and Technology (grants 06JC14050 and 08JC1414700), and the Program of Shanghai Subject Chief Scientist (grant 07XD14021). 1 Yuanjia Tang, PhD, Xiaobing Luo, PhD, Huijuan Cui, MD, Xuming Ni, BS, Min Yuan, MD, Yanzhi Guo, MD, Xinfang Huang, MD, Haibo Zhou, PhD, Shunle Chen, MD, Nan Shen, MD: Joint Molecular Rheumatology Laboratory of the Institute of Health Sciences and Shanghai Renji Hospital, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, and Shanghai Jiaotong University School of Medicine, Shanghai, China; 2Niek de Vries, MD, PhD, Paul Peter Tak, MD, PhD: Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands. Drs. Tang, Luo, and Cui contributed equally to this work. Address correspondence and reprint requests to Nan Shen, MD, Department of Rheumatology, Renji Hospital, Shanghai Jiaotong University School of Medicine, 145 Shan Dong Road (Central), Shanghai 200001, China. E-mail: email@example.com. Submitted for publication March 17, 2008; accepted in revised form January 10, 2009. Systemic lupus erythematosus (SLE) is a complex autoimmune disease characterized by chronic immune activation and multiple immunologic phenotypes (for review, see ref. 1). The pathogenesis of SLE is poorly understood, and current therapies are based on nonspecific immunosuppression. Among numerous immunologic alterations present in lupus patients, the type I interferon (IFN) system is thought to play a pivotal role in pathogenesis (2–4). It is well known that many patients with SLE have elevated serum levels of IFN␣ (5) and that these increased levels correlate with disease activity and severity (6,7). Using peripheral blood samples from SLE patients, we and other groups of investigators have independently identified the expression patterns of IFN-inducible genes (8–11), which is referred to as the IFN signature. This signature is associated with severe manifestations of the disease, such as nephritis 1065 1066 TANG ET AL (12), and reflects coordinate activation of the type I IFN pathway in vivo (10). Recent recognition of the central role of type I IFN in SLE has led to a new understanding of the significant role of the innate immune system in the predisposition to, and amplification of, autoimmunity and tissue damage (4), especially the Toll-like receptor 7 (TLR-7) pathway, which has been shown to be the most important molecular pathway responsible for induction of excessive type I IFN in both murine and human lupus (4). However, the molecular mechanisms by which the type I IFN pathway is activated in patients with SLE remains largely unknown. Scientists have only recently discovered that microRNA (miRNA), tiny fragments of single-stranded RNA, regulate gene expression via messenger RNA (mRNA) degradation and translational repression (13). It is estimated that miRNA target ⬃30% of the human transcriptome (14). A growing body of evidence indicates that miRNA have the ability to alter cellular pathways and events, such as development and differentiation. The role of miRNA in immunity is also beginning to be explored. A recent study that identified miR-146 as a key player in innate immunity was among the first to demonstrate the importance of miRNA in immune regulation (15). The role of miRNA in adaptive immunity has also been described, since miR-181a was found to modulate T cell sensitivity and selection (16). Investigations of the role of miRNA in immune-related diseases conducted by several groups of scientists have found altered expression of miRNA in rheumatoid arthritis (17–19). In this study, we examined the involvement of miRNA in SLE in humans. Using pilot expression profiling, we identified the underexpression of miR-146a in patients with SLE and further demonstrated an association between miR-146a levels and overactivation of the type I IFN pathway. We characterized the role of miR-146a as a negative regulator of the type I IFN pathway and explored the potential molecular mechanisms. The findings presented here reveal the relevance of miRNA genes in the biologic and clinical behavior of an autoimmune disease. PATIENTS AND METHODS Study subjects. We recruited 52 patients with SLE, 6 patients with Behçet’s disease, and 29 normal control subjects for the present study, after obtaining their informed consent. Participants with concurrent infection were excluded from the study. Patients with Behçet’s disease met the International study group criteria for the disease (20). All SLE patients Table 1. Clinical features of the 52 patients with SLE, by disease activity group* Characteristic Sex, no. male/female Age, mean ⫾ SD years Disease duration, mean ⫾ SD months SLEDAI, mean ⫾ SD score Anti-dsDNA, no. positive/negative† Lupus nephritis, no. positive/negative Proteinuria, no. positive/negative† Medications Steroids, no. taking‡ ⱕ10 mg/day 10–40 mg/day ⬎40 mg/day Secondary agents, no. taking/not taking§ Inactive SLE (n ⫽ 19) Active SLE (n ⫽ 33) 2/17 35 ⫾ 14 70.11 ⫾ 57.73 2/31 32 ⫾ 13 76.61 ⫾ 76.74 2⫾2 2/7 7/12 0/17 11 ⫾ 5 14/11 19/14 18/14 11 6 2 10/9 3 12 18 22/11 * Patients with systemic lupus erythematosus (SLE) were classified as having active disease if the Systemic Lupus Erythematosus Disease Activity Index (SLEDAI) score was ⬎4 and inactive disease if the SLEDAI score was ⱕ4. Anti-dsDNA ⫽ anti–double-stranded DNA. † Not all patients were evaluated. ‡ Prednisone or equivalent dosage of another steroid. Dosages of other steroids were converted to prednisone equivalents (e.g., 40 mg of methylprednisolone was considered to be equivalent to 50 mg of prednisone). § Some patients were receiving secondary antirheumatic agents, including chloroquine, cyclophosphamide, methotrexate, and azathioprine. fulfilled the American College of Rheumatology (ACR) classification criteria for SLE (21), and 26 of these patients met the ACR criteria for lupus nephritis (22). The Systemic Lupus Erythematosus Disease Activity Index (SLEDAI) score (23) was determined for each patient at the time of the blood draw. Patients were categorized as having active disease (scores ⬎4) or inactive disease (scores ⱕ4) based on the SLEDAI results. Renal SLEDAI scores were determined as described elsewhere (24). Additional clinical information on the SLE patients is given in Table 1. The study was approved by the Research Ethics Board of Renji Hospital. Sample handling and RNA processing. Peripheral blood samples obtained from each study subject were collected into tubes containing acid citrate dextrose Formula A. Erythrocytes were immediately lysed, and total RNA was extracted from the leukocytes using TRIzol (Invitrogen, San Diego, CA). Approximately 1 g of RNA was reverse transcribed into complementary DNA (cDNA) using Superscript II reverse transcriptase (Invitrogen) and oligo(dT) primers. The quantity of miRNA was determined by reverse transcription of 20 ng of RNA from each sample using a TaqMan Human MicroRNA Assays kit (Applied Biosystems, Foster City, CA). For the pilot expression profiling study, equal amounts of RNA from 8 healthy donors was mixed and divided into 2 control pools, each containing samples from 4 donors. Real-time polymerase chain reaction (PCR) analysis. For the pilot study, expression of the 156 miRNA included in the TaqMan MicroRNA Assays Human Panel–Early Access ABNORMAL ACTIVATION OF THE TYPE I IFN PATHWAY BY miR-146a IN SLE kit (Applied Biosystems) was examined according to the manufacturer’s protocol. In subsequent studies, the TaqMan kit specified for quantification of miR-146a was used, and the expression level of each sample was normalized to that of RNU66, a reference small nuclear RNA. To determine the quantity of mRNA, the cDNA was amplified by real-time PCR with SYBR Green (SYBR Premix Ex Taq RT-PCR kit; Takara, Shiga, Japan), and the expression of RPL13A was determined as the internal control. TaqMan and SYBR Green assays were performed in duplicate or in triplicate on a 7900HT real-time instrument (Applied Biosystems). Relative expression levels were calculated using the 2–⌬⌬Ct method. Primers used were as follows: for lymphocyte antigen 6 complex, locus E (Ly-6E), 5⬘-CTTACGGTCCAACATCAGAC (forward) and 5⬘-GCACACATCCCTACTGACAC (reverse); for 2⬘,5⬘-oligoadenylate synthetase 1, 40/46 kd (OAS-1), 5⬘-GA AGGCAGCTCACGAAAC (forward) and 5⬘-TTCTTAAAGC ATGGGTAATTC (reverse); for myxovirus resistance 1 (MX1), 5⬘-GGGTAGCCACTGGACTGA (forward) and 5⬘-AGG TGGAGCGATTCTGAG (reverse); for IFN-induced protein with tetratricopeptide repeats 3 (IFIT-3), 5⬘-AACTACGCCTGGGTCTACTATCACTT (forward) and 5⬘-GCCCTTTCATTTCTTCCACAC (reverse); for IFN␣, 5⬘-TCCATGAGATGATCCAGCAG (forward) and 5⬘-ATTTCTGCTCTGACAACCTCCC (reverse); for IFN␤, 5⬘-TCTAGCACTGGCTGGAATGAG (forward) and 5⬘-GTTTCGGAGGTAACCTGTAAG (reverse); and for ribosomal protein L13A (RPL13A), 5⬘-CCTGGAGGAGAAGAGGAAAGAGA (forward) and 5⬘-TTGAGGACCTCTGTGTATTTGTCAA (reverse). Calculation of IFN scores. Three representative genes (Ly-6E, OAS-1, and MX-1) were chosen to calculate IFN scores (8,10,12). First, the mean level of Ly-6E expression in the normal controls was subtracted from the level of Ly-6E expression in each lupus patient, and then the remainder was divided by the SD value for Ly-6E in normal controls to obtain the standardized expression level of the gene. The standardized expression levels of OAS-1 and MX-1 were calculated in the same manner. These 3 values were then summed to obtain the IFN score for each patient. The mean IFN score in SLE patients was 65.3 (range –0.45 to 412.60), and the mean IFN score in normal controls was 0 (range –1.88 to 6.55). Preparation of constructs. Overexpression of miR146a was accomplished by amplifying a genomic fragment of ⬃280 bp, corresponding to the miR-146a precursor, and inserting this fragment into the pSUPER.basic vector (OligoEngine, Seattle, WA), using the primer pair 5⬘GTGAGATCTGCATTGGATTTACC (forward) and 5⬘GACCTCGAGACTCTGCCTTCTGT (reverse). To create 3⬘untranslated region (3⬘-UTR) luciferase reporter constructs, fragments of 3⬘-UTR from the IFN regulatory factor 5 (IRF-5) or STAT-1 gene harboring the predicted miR-146a binding sites were cloned downstream of the firefly luciferase cassette in pMIR-REPORT vector (Ambion, Austin, TX). Primers used were as follows: for IRF-5 5⬘-GTCGAGCTCTCTTGTGTATATTC (forward) and 5⬘-GAGAAGCTTGGAGTGTGCAGAGAT (reverse); and for STAT-1 5⬘-GTGGAGCTCTTTACTGTTTGTTATGG (forward) and 5⬘-ACGAAGCTTAATAGACTAAATACCAC (reverse). All constructs were sequenced, and expression vectors were prepared with the use of an EndoFree Plasmid Maxi kit (Qiagen, 1067 Chatsworth, CA). Following transfection, overexpression of miR-146a was confirmed by quantitative PCR. Cell culture, transfection, and stimulation. For these studies, 293T and SMMC-7721 cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), and 293T/ISRE cells (i.e., 293T cells stably transfected with an IFN-stimulated response element [ISRE] reporter gene) were maintained in DMEM supplemented with 10% FBS and 200 g/ml of hygromycin B. These 3 cell lines were transfected using Lipofectamine 2000 (Invitrogen). Peripheral blood mononuclear cells (PBMCs) were isolated using a density gradient separation medium (Cedarlane, Burlington, NC) and were rested in RPMI 1640 supplemented with 10% FBS for 2 hours. For analysis of the induction of miR-146a, PBMCs from 5 healthy donors were incubated for 6 hours with each of the following stimuli separately: type I IFN (1,000 units/ml; PBL InterferonSource, Piscataway, NJ), ultrapure lipopolysaccharide (LPS) (10 g/ml, from Escherichia coli strain K12; InvivoGen, San Diego, CA), imiquimod R837 (5 g/ml; InvivoGen), and type A CpG-containing oligonucleotide (ODN) 2216 (5 M; InvivoGen). For transfection of PBMCs, 3 ⫻ 106 cells were electroporated with 1.5 g of an empty or miR-146a expression vector using a Nucleofector device (Amaxa, Cologne, Germany). Alternatively, PBMCs were transfected with 3 g of miRIDIAN (i.e., an hsa-miR-146a inhibitor) or with 3 g of scrambled oligonucleotides (all from Dharmacon, Lafayette, CO). In experiments using PBMCs from healthy donors, the medium was changed 24 hours after transfection, and cells were incubated in fresh medium alone or in the presence of type I IFN (1,000 units/ml) for 6 hours or in the presence of imiquimod R837 (5 g/ml) for 2 hours. Reporter gene assay. SMMC-7721 cells were seeded in the wells of a 96-well plate and then transfected with a mixture of 20 ng of 3⬘-UTR luciferase reporter vector and 10 ng of pRL-TK vector, along with 270 ng of either an empty vector or the miR-146a expression plasmid. After 24 hours, cells were lysed, and luciferase activity was measured with a luminometer (TR717; Applied Biosystems) by using a Dual-Luciferase Reporter Assay system (Promega, Madison, WI). The ratio of firefly luciferase to Renilla luciferase was obtained for each well. In addition, 293T/ISRE cells were similarly seeded and transfected with 300 ng of expression plasmids. After 18 hours, cells were stimulated with type I IFN (1,000 units/ml) for 6 hours. The cells were then lysed, and the firefly luciferase activity was measured. Western blotting. In a 6-well plate, 293T cells were seeded and transfected with 3 g of miR-146a expression vector per well. Twenty-four hours after transfection, cells were lysed, and proteins were extracted. Supernatants were then subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis, blotted with the indicated antibodies, and detected with Luminol/Enhancer Solution (Pierce, Rockford, IL). IRF-5 and GAPDH antibodies were obtained from Abcam (Cambridge, UK) and Chemicon (Temecula, CA), respectively. STAT-1 and horseradish peroxidase–conjugated secondary antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Relative expression levels were quantified using Quantity One software, version 4.52 (Bio-Rad, Richmond, CA). 1068 TANG ET AL Figure 1. Underexpression of microRNA-146a (miR-146a) in patients with systemic lupus erythematosus (SLE) as compared with patients with Behçet’s disease (BD) and normal control (NC) subjects, and its correlation with SLE disease activity. A, Expression of miR-146a in 47 patients with SLE, 6 patients with Behçet’s disease, and 21 normal controls. Values are the mean and SEM. B, Expression of miR-146a in 29 patients with active SLE, 18 patients with inactive SLE, and 21 normal controls. Horizontal bars show the mean. C, Association of miR-146a expression with the concurrent presence of proteinuria in SLE. Horizontal bars show the mean. D, Correlation between miR-146a levels and activation of the type I interferon (IFN) pathway. IFN scores were calculated by integrating the expression of 3 representative IFN-inducible genes (see Patients and Methods for details) to reflect the coordinate activation of the type I IFN pathway. Statistical analysis. Data were analyzed using Prism 4 software, version 4.03 (GraphPad Software, San Diego, CA). The nonparametric Mann-Whitney U test was used to draw comparisons between groups, with the exception that an unpaired t-test was used to compare reporter gene activity. Spearman’s test was used for correlation studies. P values (2-tailed) less than 0.05 were considered statistically significant. RESULTS Decreased expression of miR-146a in patients with SLE and correlation between miR-146a levels and disease activity. In an initial effort to identify differentially expressed miRNA in lupus patients, we profiled the expression of 156 miRNA by using a TaqMan microRNA Assay Human Panel–Early Access kit from Applied Biosystems. This method, which involves stemloop reverse transcription followed by TaqMan real-time PCR analysis, amplifies only mature miRNA and can discriminate among similar miRNA that differ by as few as 1 nucleotide. The high sensitivity and specificity of this method have been well established (25–27). RNA samples were obtained from 5 patients and 2 control pools, each of which was a composite of RNAs from 4 healthy donors. The study revealed differential expression of 42 miRNA in patients with SLE as compared with normal controls. Seven miRNA (miR-31, miR-95, miR-99a, miR-130b, miR-10a, miR-134, and miR-146a) were more than 6-fold lower in patients versus controls (data not shown). Among these miRNA, miR-146a in particular has been reported to negatively regulate the innate immune response by targeting interleukin-1 receptor– associated kinase 1 (IRAK1) and tumor necrosis factor receptor–associated factor 6 (TRAF6) (15). Because defects in the negative regulation system can cause unabated immune activation, even autoimmune diseases ABNORMAL ACTIVATION OF THE TYPE I IFN PATHWAY BY miR-146a IN SLE 1069 Figure 2. Effects of microRNA-146a (miR-146a) on the activation of the type I interferon (IFN) pathway. A, Overexpression of miR-146a represses the production of type I IFNs. Peripheral blood mononuclear cells (PBMCs) from 3 normal donors were electroporated with an expression plasmid for miR-146a, followed by stimulation with imiquimod R837 for 2 hours. Shown are the relative mRNA levels of IFN␣ and IFN␤. The IFN␣ primers were designed to recognize most IFN␣ subtypes. Values are the mean and SEM. B, Silencing of miR-146a augments the production of type I IFNs. PBMCs from two donors (#1 and #2) were transfected with an inhibitor against miR-146a or scrambled oligonucleotides, followed by stimulation with imiquimod R837 for 2 hours. Shown are the relative mRNA levels of IFN␣ and IFN␤. C, MicroRNA-146a inhibits the activation downstream of type I IFN in vitro. To examine this, 293T/ISRE cells (i.e., 293T cells stably transfected with an IFN-stimulated response element [ISRE] reporter gene) were transfected to express miR-146a, followed by incubation with type I IFN for 6 hours. The activity of the ISRE luciferase reporter gene was then measured. Values are the mean and SEM. D, MicroRNA-146a inhibits the activation downstream of type I IFN in primary immune cells. PBMCs from 3 donors were transfected to express miR-146a, followed by incubation with type I IFN for 6 hours. The mRNA levels of 3 IFN-inducible genes (lymphocyte antigen 6 complex, locus E [LY6E], 2⬘,5⬘-oligoadenylate synthetase 1, 40/46 kd [OAS1], and myxovirus resistance 1 [MX1]) were quantified. Values are the mean and SEM. (28–30), we further explored the role of miR-146a in SLE. We used the TaqMan PCR method described above to subsequently examine miR-146a expression in a larger group of samples: 47 patients with SLE, 6 patients with Behçet’s disease, and 21 normal controls. As shown in Figure 1A, the expression of miR-146a was significantly lower in lupus patients compared with normal controls (P ⬍ 0.0001), while the level of expression in patients with Behçet’s disease appeared normal. Since our patients were receiving various medications, we conducted the following analysis to evaluate the influence of drugs on the expression of miR-146a. We selected 29 patients with active SLE who were receiving a relatively high dose of steroids. Higher doses of steroids were not associated with altered expression of miR-146a (P ⫽ 0.1422) (data not shown). Moreover, when all 47 SLE patients were divided into 2 groups according to whether they were receiving a secondary antirheumatic agent apart from steroids, no difference was detected (P ⫽ 0.7149) (data not shown). Taken together, the results suggested that drugs do not affect the expression of miR-146a and confirm that this miRNA is intrinsically underexpressed in lupus patients. We next performed an analysis to determine whether there was any correlation between miR-146a levels and clinical features. As shown in Figure 1B, the expression of miR-146a was lower in patients with 1070 TANG ET AL Figure 3. Identification of interferon regulatory factor 5 (IRF-5) and STAT-1 as targets of microRNA-146a (miR-146a). A, Schematic presentation of the potential miR-146a binding sites in the 3⬘-untranslated regions (3⬘-UTRs) of IRF-5 and STAT-1. B, Relative activity of the luciferase reporter gene fused to the fragment of 3⬘-UTRs of IRF-5 or STAT-1. Shown is a comparison between cotransfection in SMMC-7721 cells with an empty control vector and with a miR-146a expression plasmid. Values are the mean and SEM of quadruplicate assays, each of which was performed 3 times. ⴱⴱ ⫽ P ⬍ 0.01. C, Effect of miR-146a on IRF-5 (left) and STAT-1 (right) in 293T cells. MicroRNA-146a was overexpressed in the cells, and cell lysates were used for Western blotting. The ratios of IRF-5 to GAPDH and of STAT-1 to GAPDH ratio in vector-transfected cells were arbitrarily set at 1. inactive SLE, and even lower in those with active SLE, compared with the expression in normal controls. SLE patients with concurrent proteinuria had much lower levels of miR-146a expression than did those without proteinuria (Figure 1C). Moreover, a direct inverse correlation was observed between the miR-146a levels and the SLEDAI scores (r ⫽ –0.2882, P ⫽ 0.0247) (data not shown), as well as between the miR-146a levels and the renal SLEDAI scores (r ⫽ –0.3815, P ⫽ 0.0081) (data not shown). Thus, we concluded that miR-146a expression levels correlate negatively with SLE disease activity. It has been established that type I IFN plays a key etiologic role in SLE and that it can be induced by various stimuli. Since miR-146a deficiencies might reflect defects in negative regulation of the immune response, we explored whether sustained underexpression of miR-146a affected, or was associated with, activation of the type I IFN pathway in lupus patients. We examined mRNA expression of 3 representative IFNinducible genes and calculated IFN scores accordingly (8,10,12) to determine the coordinate activation of the type I IFN pathway in our cohorts. The associations were then analyzed. It is interesting, although not surprising, that a negative correlation was found between miR-146a levels and IFN scores (r ⫽ –0.3073, P ⫽ 0.0378) (Figure 1D). Negative regulation of the type I IFN pathway by miR-146a. To explore the association between miR-146a levels and activation of the type I IFN pathway, we first sought to determine whether miR-146a could intrinsically modulate the onset and activation of the pathway. The effects of miR-146a on type I IFN production were initially explored in primary immune cells. Normal PBMCs were electroporated with a miR-146a expression vector; 24 hours later, cells were stimulated with imiquimod R837, the TLR-7 ligand, to induce production of IFNs. As shown in Figure 2A, overexpression of miR146a greatly reduced the induction of IFN␣ and IFN␤. Furthermore, silencing the endogenous miR-146a via transfection with inhibitory oligonucleotides increased IFN production (Figure 2B). Thus, the results suggested that miR-146a negatively regulates the production of type I IFN. ABNORMAL ACTIVATION OF THE TYPE I IFN PATHWAY BY miR-146a IN SLE A common response to type I IFN is the activation of STAT proteins. Activated STAT-1 and STAT-2, together with IRF-9, form the IFN-stimulated transcription factor 3 (ISGF-3) transcriptional complex to initiate transcription of IFN-inducible genes whose promoters harbor ISREs (31). Thus, we evaluated the direct effects of miR-146a on downstream transactivation of type I IFN by performing ISRE reporter gene assays. Transient miR-146a expression in 293T/ISRE cells substantially inhibited ISRE reporter gene activity after stimulation with type I IFN (Figure 2C), indicating that miR-146a directly regulates the activation downstream of IFN receptors. We further clarified the regulatory role of miR146a by monitoring its effect on the expression of IFN-inducible genes. Normal PBMCs were similarly transfected and stimulated with type I IFN. Overexpression of miR-146a consistently reduced the expression of the selected IFN-inducible genes (Figure 2D). Taken together, the results indicate that miR-146a could also effectively regulate the coordinate activation downstream of type I IFN. To further investigate the involvement of miR146a in the type I IFN pathway, we used PBMCs from healthy donors to determine whether triggers of the type I IFN pathway could induce the expression of miR-146a. We found that mature miR-146a can be induced by imiquimod R837 (TLR-7 ligand), type A CpG (TLR-9 ligand), and type I IFN, in addition to LPS (TLR-4 ligand) (see discussion of Figure 5A below). Regulation of the type I IFN pathway by miR146a by targeting multiple key components. Our findings thus far have revealed that miR-146a is a negative regulator of the type I IFN pathway. To gain insight into the molecular mechanism of this regulator, we used bioinformatics tools to identify its potential targets. Previous computational analyses have revealed that miRNA predominantly target positive regulatory motifs, highly connected scaffolds, and downstream network components to facilitate robust transitions of cellular responses to extracellular signals and maintain cellular homeostasis (32). Hence, we compiled a list of all key components related to IFN signaling and searched each of these proteins for potential miR-146 binding sites. Using algorithms from miRBase (available at http://microrna.sanger.ac.uk/targets/v5/) and TargetScan (available at http://www.targetscan.org/), we discovered that miR-146a base-pairs with sequences in the 3⬘-UTR of IRF-5 and STAT-1 (Figure 3A), in addition to the 2 1071 Figure 4. Potential alleviation of the overactivation of the type I interferon (IFN) pathway by microRNA-146a (miR-146a) in patients with systemic lupus erythematosus (SLE). Peripheral blood mononuclear cells from 5 patients with SLE who had high IFN scores were transfected with a miR-146a expression plasmid for 24 hours, and then mRNA levels of the selected IFN-inducible genes (IFN-induced protein with tetratricopeptide repeats 3 [IFIT3], myxovirus resistance 1 [MX1], and 2⬘,5⬘-oligoadenylate synthetase 1, 40/46 kd [OAS1]) were quantified. Values are the mean and SEM percentage reduction of the expression levels by transfection with miR-146a as compared with transfection with an empty vector (designated as 100%). established miR-146 targets, IRAK1 and TRAF6 (15). All of the proteins were key components in the signaling cascade of the type I IFN pathway and were essential either for the production of type I IFN (33–36) or for the transactivation downstream of IFN (31). More intriguingly, a haplotype that elevates the expression of multiple isoforms of IRF-5 has been shown to confer lupus susceptibility across multiple ethnic backgrounds (37), and basal expression and activation of STAT-1 is known to be elevated in lupus patients and murine models (38–40). We thus performed a biologic validation to confirm our new predictions. The 3⬘-UTR fragments of IRF-5 and STAT-1 were cloned downstream of the luciferase reporter gene. The constructs were then transiently transfected into SMMC-7721 cells, together with either an empty control vector or an expression plasmid for miR-146a. As expected, overexpression of miR-146a effectively attenuated luciferase activity (Figure 3B), while 3 irrelevant miRNA (miR-98, miR-202, and miR-224) had no effect on those reporter constructs (data not shown), implying the inhibitory effect of miR-146a via the predicted binding sites. We then introduced miR-146a into 293T cells and analyzed the expression of IRF-5 and STAT-1 by Western blotting. Overexpression of miR-146a consistently reduced the expression of IRF-5 and STAT-1 at the protein level (Figure 3C). Thus, miR-146a appears to regulate the immune activation by targeting key signaling proteins in the type I IFN pathway. 1072 TANG ET AL Potential alleviation of overactivation of the type I IFN pathway by miR-146a in patients with SLE. The results described thus far demonstrate that miR-146a regulates the type I IFN pathway by targeting critical proteins in the signaling cascade. Thus, we performed an exploratory examination of whether manipulation of miR-146a levels could alleviate overactivation of the type I IFN pathway in lupus patients. PBMCs were obtained from 5 patients who had high IFN scores (i.e., ⬎2 SD above the mean IFN score in normal controls) and were electroporated for expression of miR-146a. As expected, miR-146a notably reduced the expression of selected IFN-inducible genes (Figure 4). This indicates that manipulation of miR-146a levels could potentially provide a therapeutic benefit to lupus patients. DISCUSSION Ever since the discovery of miRNA, tremendous effort has been devoted to determining their biologic functions and their relevance to diseases. Dysregulation of miRNA has been associated with certain human diseases, such as leukemia and heart disease (15,41). By elucidating the presence of an miRNA signature and its contribution to alterations of the type I IFN pathway in patients with SLE, the present study now extends the role of miRNA in the pathogenesis of autoimmune diseases. The strong association between the miR-146a level and clinical disease activity indicates that it may serve as a new disease biomarker. Dai et al (42) recently identified several differentially expressed miRNA in lupus patients by microarray analysis; however, dysregulation of miR-146a was not reported. This is likely due to the difference between the quantification methods and patient cohorts used in their study as compared with ours. There is now considerable evidence of the contribution of TLRs to autoimmunity through their role in the recognition of endogenous ligands (43,44). Immune complexes of autoantibodies to RNA binding nucleoproteins and self DNA from lupus patients are known to induce IFN␣ in plasmacytoid dendritic cells through TLR-7 or TLR-9 (45–47). These responses require sequential activation of signaling adaptors and transcription factors, including myeloid differentiation factor 88, IRAK1, TRAF6, IRF-5, and IRF-7 (33–35). Recently reported data underscore the critical role of IRF-5 in the induction of type I IFN (36). Once induced, type I IFN binds to its receptors to initiate downstream signaling by activation of STAT proteins, which ultimately leads to the transcription of target genes (“IFN-inducible genes”) (31). Physiologically, cells of the immune system spontaneously use various mechanisms to negatively regulate TLR signaling so as to avoid abnormal activation and to maintain the immunologic balance. Such mechanisms involve intracellular negative regulators, which are present constitutively or are up-regulated by TLR signaling in a feedback loop (28). Defects in negative regulators could lead to overactivation of positive signaling and result in diseases (28). This notion is supported by genetic studies in patients with asthma, in whom inactive lesions in the IRAK-M gene are found (29), and in mice deficient in suppressor of cytokine signaling protein 1, in which systemic autoimmune phenotypes occur (30). Recent studies also add miRNA to the list of negative regulators of innate immunity. It has been reported that miR-146 regulates TLR-4 signaling through a feedback loop in THP-1 cells (15). We used PBMCs from healthy donors and found that mature miR-146a can also be induced upon TLR-7, TLR-9, and type I IFN ligation in addition to LPS (Figure 5A). This indicates that miR-146a is indeed involved in the much more complex regulatory network of innate immunity. In the present study, we showed that miR-146a negatively regulates type I IFN induction by TLR-7 signaling and directly represses activation downstream of type I IFN. During the preparation of our manuscript, a group studying Epstein-Barr virus determined via array analysis that miR-146a modulates the expression of plenty of IFN-inducible genes (48). These findings further support our data. To explore the molecular mechanisms of the regulation by miR-146a, we used bioinformatics tools to search for its potential targets among the key components involved in signaling upstream and downstream of IFN. This led to the identification of 2 novel targets of miR-146a (i.e., IRF-5 and STAT-1), in addition to the 2 previously established miR-146 targets (i.e., IRAK1 and TRAF6). All of these proteins are important mediators of IFN signaling, and are critical for the induction of type I IFN or for transactivation downstream of type I IFN. Thus, miR-146a is licensed to target multiple components in the signaling cascade of the type I IFN pathway (Figure 5B). Given the essential characteristics of these proteins, the negative regulation by miR-146a on the activation of the pathway could be very effective, which is an overall result of the integration of its function at multiple targets with quantitatively graded modulation, al- ABNORMAL ACTIVATION OF THE TYPE I IFN PATHWAY BY miR-146a IN SLE 1073 Figure 5. Induction of microRNA-146a (miR-146a) to target multiple key components in the interferon (IFN) signaling pathway in a feedback loop. A, Induction of miR-146a in peripheral blood mononuclear cells (PBMCs) by various stimuli. Shown is the fold change in expression of miR-146a in PBMCs from 5 healthy donors after 6 hours of incubation with lipopolysaccharide (LPS), imiquimod R837, type A CpG, and type I IFN. Values are the mean and SEM. B, Overview of the negative regulation of the type I IFN pathway by miR-146a. Activation of Toll-like receptors (TLRs) (e.g., TLRs 7–9) triggers sequential signaling and leads to the production of type I IFNs, which in turn, bind to their receptors and induce downstream activation. In this scenario, various negative regulators, including miR-146a, are simultaneously induced. The mature miR-146a uses inhibitory machinery to reduce expression of its target genes, including interleukin-1 receptor–associated kinase 1 (IRAK1), tumor necrosis factor receptor–associated factor 6 (TRAF6), IFN regulatory factor 5 (IRF-5), and STAT-1, thereby attenuating the positive signaling. MyD88 ⫽ myeloid differentiation factor 88; IFNAR1 ⫽ interferon-␣/␤/ receptor 1. though the effect on a single target might be modest or subtle. This is similar to the role of miR-181a in T cells: by bringing about modest reductions in multiple phosphatases, miR-181a can single-handedly tune the excitation threshold of a T cell, whereas the robust repression of individual targets via siRNA is insufficient to achieve this regulation (16). Deficiency of miR-146a could thus be substantially deleterious; it would lead to accumulation of its target proteins, albeit with varied concentrations. This, in turn, could result in sustained overproduction of type I IFNs and downstream activation. Therefore, it appears that miR-146a deficiency is one of the causal factors in the abnormal activation of the type I IFN pathway in SLE. The correlation we observed between miR-146a levels and activation of the type I IFN pathway in lupus patients echoes this idea and implicates defective miRNA regulation in the pathogenesis of autoimmune conditions. On the other hand, some patients with decreased miR-146a expression do not have overactivation of the type I IFN pathway (Figure 1D). This difference may reflect the heterogeneous nature of lupus, and it indicates that miR-146a may have other etiologic roles. Previous studies have shown that miR-146 expression is up-regulated in Th1 cells and down-regulated in Th2 cells relative to its expression in naive T cells, thus suggesting that miR-146 might be involved in cell fate determination (49). It is known that T cells from lupus patients display numerous signaling abnormalities that contribute to the pathogenesis of SLE (50). Therefore, it would be interesting to determine the function of miR146 in T cells and explore whether miR-146a deficiencies contribute to the skewed expression of cytokines in SLE, thereby expanding the role of miR-146a to alterations in adaptive immunity. To explore possible reasons for the underexpression of miR-146a in lupus patients, we performed a bioinformatic analysis and identified a potential CpG island in the promoter of miR-146a. This island corresponded exactly to the position of a fragment harboring the putative STAT-1 binding site and one of the validated NF-B binding sites (15,48) (data not shown). Previous studies have shown the epigenetic regulation of several miRNA by IL-6 in malignant human cholangiocytes (51). Interestingly, a number of studies have shown an increase in serum IL-6 levels in SLE patients (for review, see ref. 1). It would be interesting to explore whether DNA methylation changes caused by IL-6 account for the lowered expression of miR-146a in lupus patients. 1074 TANG ET AL The type I IFN pathway has emerged as a significant contributor to the pathogenesis of SLE. Thus, antagonists for TLRs or IFNs appear to be promising strategies for the treatment of this disease. However, these strategies should be applied with considerable caution because of the potential for life-threatening immune deficiencies (52). Since miRNA provide quantitative regulation of genes, rather than on/off signals, they can be thought of as molecules that fine-tune a cell’s responses to external influences (53). Consequently, manipulation of miRNA levels could lead to novel therapeutic strategies to combat SLE. Interestingly, when miR-146a was introduced in PBMCs from the SLE patients, the coordinate activation of the type I IFN pathway was notably reduced, as revealed by downregulation of several IFN-inducible genes. These results suggest that miR-146a levels could be manipulated to provide useful therapeutic interventions for SLE. Studies in knockout and transgenic animal models would further identify the role of miR-146a in autoimmune diseases. In conclusion, our results demonstrate that underexpression of miR-146a in lupus patients is relevant to the biologic and clinical behavior of SLE. Our findings suggest that miRNA could serve as therapeutic targets for the treatment of SLE via regulation of the type I IFN pathway. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. AUTHOR CONTRIBUTIONS Dr. Shen 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 design. Tang, Chen, Shen. Acquisition of data. Luo, Cui, Ni, Yuan, Guo, Huang, Zhou. Analysis and interpretation of data. Tang, Luo, Cui, de Vries, Tak, Shen. Manuscript preparation. Tang, Luo, de Vries, Tak, Shen. Statistical analysis. Tang, Luo, Cui, Ni. 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