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  • Identification of novel microRNA signatures linked to human lupus disease activity and pathogenesis: miR-21 regulates aberrant T cell responses through regulation of PDCD4 expression

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Basic and translational research
Extended report
Identification of novel microRNA signatures linked to human lupus disease activity and pathogenesis: miR-21 regulates aberrant T cell responses through regulation of PDCD4 expression
  1. Elias Stagakis1,2,
  2. George Bertsias1,2,
  3. Panayotis Verginis1,3,
  4. Magdalene Nakou1,2,
  5. Maria Hatziapostolou4,5,
  6. Heraklis Kritikos1,†,
  7. Dimitrios Iliopoulos4,5,
  8. Dimitrios T Boumpas1,3
  1. 1Laboratory of Autoimmunity and Inflammation, University of Crete Medical School, Heraklion, Greece
  2. 2Graduate Program on Molecular Basis of Human Disease, School of Medicine, University of Crete, Heraklion, Greece
  3. 3Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology, Heraklion, Greece
  4. 4Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, Boston, Massachusetts, USA
  5. 5Department of Pathology, Harvard Medical School, Boston, Massachusetts, USA
  6. †Deceased
  1. Correspondence to Dimitrios T Boumpas, Laboratory of Autoimmunity and Inflammation, Department of Rheumatology, Clinical Immunology and Allergy, University of Crete, School of Medicine, PO Box 2208, Heraklion, Greece; boumpasd{at}med.uoc.gr

Abstract

Objective MicroRNAs (miRNAs) regulate the expression of genes involved in immune activation. A study was undertaken to characterise the miRNA signature and identify novel genes involved in the regulation of immune responses in systemic lupus erythematosus (SLE).

Methods The expression of 365 miRNAs in peripheral blood mononuclear cells of patients with SLE and healthy controls was analysed using TaqMan Low Density Arrays. The results were validated by quantitative real-time PCR and potential target genes were identified using prediction analysis software. The effect of miR-21 on T cell function was assessed by transfection with antago-miR-21 or pre-miR-21.

Results A 27-miRNA signature was identified in patients with SLE; 19 miRNAs correlated with disease activity. Eight miRNAs were deregulated specifically in T cells and four miRNAs in B cells. miR-21 was upregulated and strongly correlated with SLE disease activity (r2=0.92). Compared with controls, CD4 T lymphocytes from patients with SLE had higher basal and activation-induced miR-21 expression. Silencing of miR-21 reversed the activated phenotype of T cells from patients with SLE—namely, enhanced proliferation, interleukin 10 production, CD40L expression and their capacity to drive B cell maturation into Ig-secreting CD19+CD38hiIgD−(plasma cells. Overexpression of mMiR-21 in normal T cells led to acquisition of an activated phenotype. Investigation of putative gene- targets showed that PDCD4 (a selective protein translation inhibitor) was suppressed by miR-21 and its expression was decreased in active SLE.

Conclusions miRNAs represent potential biomarkers in SLE as their expression reflects underlying pathogenic processes and correlates with disease activity. Upregulated miR-21 affects PDCD4 expression and regulates aberrant T cell responses in human SLE.

https://doi.org/10.1136/ard.2010.139857

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    Introduction

    Systemic lupus erythematosus (SLE) is a prototypic autoimmune disease with multiple genetic and environmental factors contributing to its pathogenesis.1 2 Defects in T cell antigen receptor-mediated signalling in SLE have been shown to lead to breakdown of immunological tolerance. Aberrant costimulation and skewed cytokine production by CD4 T lymphocytes in SLE contribute to B cell hyper-responsiveness.3

    High-throughput techniques have been used to dissect disease mechanisms and identify novel molecular pathways involved in complex diseases including SLE.4 We and others have previously used cDNA microarrays to identify unique gene signatures in patients with SLE.5,–,7 Although these studies have provided insights into genetic pathways involved in disease pathogenesis, they offer limited information regarding regulatory mechanisms that control gene expression.

    MicroRNAs (miRNAs) are, in general, potent negative modulators of genes involved in several cellular processes.8 Compared with other gene regulatory mechanisms such as epigenetic and transcription factors, miRNA-mediated effects occur prior to protein synthesis, thus allowing for the fine tuning of gene expression. Deregulation of miRNA expression has been implicated in the pathogenesis of human diseases and miRNAs are thought to represent novel disease biomarkers and potential therapeutic targets.9

    miRNAs regulate the function of both the innate and the adaptive immune system and are involved in various immune pathways.8 10 In mice, deregulation of miRNAs leads to aberrant immune responses and development of systemic autoimmunity.11 12 Altered miRNA expression has been reported in human autoimmune diseases including SLE, rheumatoid arthritis and multiple sclerosis.13,–,16 However, the mechanisms by which these changes promote autoimmunity have not been thoroughly investigated.

    In this study we have characterised the miRNA signature of human SLE, reporting a strong correlation of certain miRNAs with SLE disease activity. Importantly, the identified miRNAs are predicted to regulate genes and processes pertinent to the pathogenesis of SLE. Furthermore, in functional studies we show that miR-21, the miRNA with the highest correlation with disease activity, regulates key cellular functions that contribute to aberrant T cell phenotype in patients with SLE and affects PDCD4, a selective protein translation inhibitor of genes involved in immune responses.

    Methods

    Patients and healthy blood donors

    Patients with SLE fulfilling the American College of Rheumatology classification criteria17 were recruited from the Department of Rheumatology, University Hospital of Heraklion (Greece). The SLE Disease Activity Index (SLEDAI) was used to classify patients with active (SLEDAI ≥8) or inactive (SLEDAI <8) disease.18 Peripheral blood samples were obtained from 34 patients (18 with active and 16 with inactive disease) and 25 age- and sex-matched healthy blood donors. All subjects gave written informed consent prior to enrolment in the study, which was approved by the ethics committee of our hospital.

    Cell isolation

    Peripheral blood mononuclear cells (PBMCs) were freshly isolated by Ficoll-Histopaque (Sigma-Aldrich, St Louis, Missouri, USA) density gradient centrifugation of heparinised venous blood. Patients had not taken any medication for SLE during the 24 h prior to blood sampling. CD4 T and CD19 B lymphocytes were obtained by magnetic separation (Miltenyi Biotec, Miltenyi Biotec, Gladbach, Germany).

    MiRNA expression and miRNA target prediction analysis

    The expression of 365 miRNAs was analysed by TaqMan Low Density Arrays (TLDA human miRNA v1.0, Dana-Farber Molecular Diagnostics Facility, Harvard Medical School, Boston, Massachusetts, USA) using 2 μg total RNA. RNU48 was used to normalise microarray expression results. Potential miRNA gene targets were identified using the miRBase (http://microrna.sanger.ac.uk), PicTar (http://pictar.bio.nyu.edu) and TargetScan version 4.0 (http://www.targetscan.org/index.html) search engines. To optimise the accuracy of prediction, a potential gene target should be predicted by a minimum of two out of three programs and the targeted sequence should be conserved among species.

    Real-time PCR and western blot

    Microarray results were validated with the mirVana qRT-PCR miRNA Detection Kit and qRT-PCR Primer Sets (Ambion, Austin, Texas, USA). For western blot, cells were lysed with RIPA buffer containing a complete protease inhibitor cocktail. Proteins were separated by 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membrane. PDCD4 was detected using a monoclonal antibody (Cell Signaling Technology, Danvers, MA, USA) and protein loading was determined using a monoclonal antibody for β-actin (Santa Cruz, CA, USA). The secondary antibodies horseradish peroxidase (HRP)-conjugated antimouse IgG and antirabbit IgG were from Jackson Immunoresearch Laboratories (West Grove, PA, USA). Enhanced chemiluminescence was from Thermo Scientific (Vantaa, Finland).

    Transfection of pre-miRNAs or antisense-miRNAs

    The transfection agent siPORT NeoFX agent (Ambion) was used for silencing and overexpression of endogenous miRNAs. Freshly isolated T lymphocytes were seeded in 96-well tissue culture plates (Corning Incorporation, Corning, NY, USA) and mock-transfected or transfected with irrelevant (scramble) miR, antago-miR-21 or pre-miR-21 (all 75 nM) (Ambion).19 The efficiency and optimal dose of the transfection was determined by real-time PCR. Freshly isolated CD4 T lymphocytes were seeded in 96-well tissue culture plates and were transfected with 2 μg control (pEGFP-C1) vector or PDCD4-pEGFP-C1 expression vector (Cloneth Laboratories, USA). Culture supernatants were collected after 48 h to measure interleukin (IL)-10 (Ready-SET-Go, ELISA kit; eBioscience, San Diego, CA, USA).

    T cell assays and T/B lymphocytes co-cultures

    Full details are given in the online supplement.

    Statistical analysis

    The GraphPad Prism software was used for statistical analysis and calculation of mean±SEM values. Comparisons were performed using the Mann–Whitney U test (independent samples) and the paired t test (paired samples). miRNA expression was plotted against SLEDAI scores of individual patients and r2 values (coefficient of determination) were obtained from polynomial trend lines. A two-tailed p value <0.05 was considered statistically significant.

    Results

    Microarray analysis reveals a distinct pattern of miRNA expression in PBMCs of patients with SLE

    We analysed the expression of 365 miRNAs in the PBMCs of 34 patients with SLE and 20 healthy individuals. The microarray analysis identified 14 miRNAs significantly downregulated (2.2–18.0-fold) and 13 miRNAs significantly upregulated (3.4–9.2-fold) in patients with active SLE compared with controls (figure 1A). miR-21, miR-25, miR-148b and miR-155 showed the highest induction (6.3–9.2-fold) whereas miR-196a, miR-150 and hsa-let-7a were the most downregulated miRNAs. The microarray results were validated by quantitative real-time PCR (figure 1B). Several of the identified miRNAs are predicted to regulate genes implicated in biological processes pertinent to the pathogenesis of SLE (figure 1C). Four miRNAs (miR-15a, miR-16, miR-21, miR-25) are predicted to regulate genes involved in apoptosis, a process known to be impaired in SLE.20 Upregulated miR-148a and miR-148b regulate DNA methyltransferase 3b (DNMT3b), which may be involved in epigenetic modifications in SLE.21 miR-150 has been identified as a major regulator of B cell development by targeting the transcription factor c-myb,22 and hsa-let-7a regulates Stat3 expression.23 In accordance, an inverse correlation between miR-150 and c-myb, as well as between hsa-let-7a and Stat3, was documented in our SLE cohort (figure 1D). Gene ontology analysis identified miRNA gene networks significantly enriched in patients with SLE involving anti-apoptotic mechanisms, immune responses, DNA methylation and insulin receptor signalling (see table 1 in online supplement). Together these data suggest that miRNAs may regulate major pathogenic pathways in SLE.

    Figure 1
    Figure 1

    Microarray analysis reveals a distinct pattern of microRNA (miRNA) expression in peripheral blood mononuclear cells (PBMCs) of patients with systemic lupus erythematosus (SLE), with predicted gene targets implicated in immune processes pertinent to the pathogenesis of SLE. (A) Heat map representation of the miRNA microarray analysis of RNA from healthy controls (CT1→10), patients with active SLE (SLE1A→11A) and those with inactive SLE (SLE1I→6I). Red represents higher miRNA expression and green represents lower miRNA expression in healthy controls compared with patients with active SLE (or vice versa). The metric scale (top right) represents the log10-fold change in miRNA expression using the group (healthy controls or active SLE) with the lower expression as denominator. Fourteen miRNAs were significantly downregulated (2.2–18.0-fold) and 13 miRNAs were significantly upregulated (3.4–9.2-fold) in patients with active SLE compared with controls. (B) Validation of miRNA microarrays with quantitative real-time PCR. Representative data of hsa-miR-25, miR-21, miR-150 and let-7a expression in healthy controls, patients with inactive SLE and those with active SLE are shown. miR-150 and let-7a were downregulated whereas miR-25 and miR-21 was upregulated in active SLE. (C) Gene targets of downregulated (red) and upregulated (green) miRNAs in patients with SLE as predicted by at least two out of three bioinformatic algorithms. (D) Experimental validation of predicted miRNA gene targets. Inverse correlation of miR-150/c-myb (left) and let-7a/STAT3 (right) mRNA levels in total PBMCs of patients with SLE. Expression levels of STAT3 mRNA were normalised to GAPDH and let-7a microRNA levels were normalised to RNU48.

    miRNA expression correlates with disease activity in patients with SLE

    We next analysed miRNA expression according to SLE disease activity. Nine miRNAs were significantly downregulated and 10 miRNAs were significantly upregulated in active SLE compared with inactive SLE (figure 2A). Expression of miR-21, miR-25, miR-106b and miR-148b showed a significant positive correlation with disease activity and variation in miR expression could explain 84–92% of the SLEDAI variation (r2=0.84–0.92, figure 2B). Notably, miR-196a and miR-379, both downregulated in active SLE, showed a significant inverse association with SLEDAI (r2=0.89 and 0.90, respectively; figure 2C). To characterise the subsets of PBMCs that account for these differences, we repeated the analysis in purified CD4 T and CD19 B cells from a subset of cases and controls. Five miRNAs were significantly downregulated and six miRNAs were significantly upregulated in patients with SLE compared with controls (figure 2D). In B cells, three miRNAs were downregulated and four miRNAs were upregulated in patients with SLE compared with controls (figure 2E). miR-21, miR-25 and miR-106b were upregulated in both T and B lymphocytes from patients with SLE compared with healthy controls; eight miRNAs (let-7a, let-7d, let-7g, miR-148a, miR-148b, miR-324-3p, miR-296, miR-196a) showed altered expression only in T cells from patients with SLE and four miRNAs (miR-15a, miR-16, miR-150, miR-155) only in B cells from patients with SLE (figure 2F).

    Figure 2
    Figure 2

    Correlation of microRNA (miRNA) expression with disease activity in patients with systemic lupus erythematosus (SLE). (A) Specific miRNA signatures in total peripheral blood mononuclear cells (PBMCs) distinguish patients with SLE (n=19) according to disease activity. Nine miRNAs were downregulated and 10 were upregulated in active SLE compared with inactive SLE (see legend to figure 1 for more details). (B) miR-21, miR-25, miR-148b and miR-106b mRNA levels correlate significantly with SLEDAI (r2=0.85–0.92, polynomial regression). The deltaCT values of the miRNAs were calculated and samples with the maximum SLE Disease Activity Index (SLEDAI) score were considered to have miRNA expression equal to 1. (C) miR-196a and miR-379, both downregulated in patients with SLE, are inversely correlated with SLEDAI score (r2=0.89–0.90). (D) Expression of miRNAs in purified CD4 T lymphocytes; five miRNAs were significantly downregulated and six were significantly upregulated in patients with SLE (n=16) versus controls (n=10). Eight miRNAs showed altered expression between active and inactive SLE. (E) Expression of miRNAs in purified CD19 B lymphocytes; three miRNAs were downregulated and four were upregulated in patients with SLE (n=5) versus healthy controls (n=3). (F) Venn diagram with overlapping signatures in T and B cell-expressed miRNAs that are differentially expressed (upregulated or downregulated) in patients with SLE compared with healthy controls. miR-21, miR-25 and miR-106b were upregulated in both SLE T and B lymphocytes compared with healthy controls; eight miRNAs showed altered expression only in SLE T cells and five miRNAs only in SLE B cells.

    miR-21 is upregulated and controls aberrant T cell responses in active SLE

    Among the upregulated miRNAs, miR-21 exhibited the strongest correlation with disease activity (r2=0.92), being upregulated in both T and B lymphocytes of patients with active SLE (figure 2). miR-21 is upregulated in murine-activated effector and memory T cells and exerts significant cellular proliferative effects.24 25 T cells from patients with SLE show enhanced TCR/CD28-mediated proliferative responses and also provide ‘help’ to B cells for autoantibody production. miR-21 mRNA was 2.3-fold upregulated in freshly isolated CD4 T cells from patients with SLE compared with healthy controls (figure 3A). Anti-CD3/anti-CD28 stimulation induced miR-21 to levels that were significantly higher in patients with SLE than controls (mean 4.0-fold vs 1.6-fold, respectively).

    Figure 3
    Figure 3
    Figure 3

    The microRNA miR-21 is upregulated in patients with active systemic lupus erythematosus (SLE) and contributes to the aberrant phenotype of T lymphocytes. (A) miR-21 mRNA was 2.3-fold upregulated in freshly isolated CD4 T cells from patients with SLE (n=3) compared with healthy controls (n=3). Stimulation of CD4 T cells with anti-CD3/anti-CD28 monoclonal antibody (mAb) resulted in induction of miR-21 that was significantly higher in patients with SLE than controls (relative expression 9.1±2.1 vs 1.6±0.3). Error bars represent mean±SEM expression; **p<0.01, Mann–Whitney test for comparison between patients with SLE and controls. (B) Purified peripheral blood CD4 T cells from healthy controls were stimulated with anti-CD3/anti-CD28 mAb and simultaneously transfected with mock (75 nM), antago-miR-21 (50, 75 nM) or pre-miR-21 (75 nM) as described in the Methods section. Cells were harvested after 3 and 6 days and miR-21 mRNA levels were measured. Expression values are presented relative to mock-transfected cells (results from 2–3 independent experiments). (C) CFSE-labelled CD4 T cells from patients with SLE were stimulated with anti-CD3/anti-CD28 mAb and transfected with 75 nM of mock, irrelevant (scramble) miR, or antago-miR-21 Antago-miR-21-transfected T cells had reduced proliferation based on CFSE dilution on day 6 (left panel). The mean±SEM percentage of undivided T cells was 48±12% in mock-transfected cells vs 66±9% in antago-miR-21-transfected cells; p<0.05, paired t test, 4 independent experiments (right panel). (D) Purified CD4 T cells from patients with SLE were either unstimulated or were stimulated with anti-CD3/anti-CD28 mAb and transfected with mock or antago-miR-21 as previously described. After 48 h culture supernatants were collected and interleukin 10 (IL-10) was measured by ELISA. Antago-miR-21 transfection significantly reduced IL-10 production (612±137 ng/ml in mock-transfected cells vs 347±101 ng/ml in antago-miR-21-transfected cells; p<0.01, paired t test, 5 independent experiments). (E) CD4 T cells from patients with SLE were treated as previously described. After 18 h the cells were examined for surface CD40L expression by flow cytometry. Silencing of miR-21 suppressed the activation-induced expression of CD40L by SLE T cells (left panel: representative flow cytometry histogram of CD40L expression in unstimulated and stimulated T cells from one patient with SLE). The mean±SEM percentage of CD40L CD4 T cells was 16.8±1.9% in mock-transfected cells vs 10.1±1.8% in antago-miR-21-transfected cells; p<0.01, paired t test, 5 independent experiments) (right panel). (F) CD4 T cells from patients with SLE were transfected with mock or antago-miR-21 and were co-cultured with autologous CD19 B cells at 1:1 ratio (105 cells/well) in the presence of anti-CD3 mAb (100 ng/ml). After 8 days the cells were harvested and assessed for plasma cell differentiation by flow cytometry, defined as CD19+CD38hiIgD− cells. Antago-miR-21-transfected T cells from patients with SLE had reduced capacity to drive B cell differentiation into plasma cells. (G) Dot plot analysis of plasma cell generation in cultures with mock- versus antago-miR-21-transfected T cells from patients with SLE. The mean±SEM percentage of plasma cells was 16.9±2.5% in mock-transfected cells vs 10.5±2.3% in antago-miR-21-transfected cells (p<0.01, paired t test, 4 independent experiments). (H) In the previous experiments, day 8 culture supernatants were assayed for total IgG by ELISA. Dot plot analysis of IgG concentration in cultures with mock- or antago-miR-21-transfected T cells from patients with SLE. The mean±SEM IgG concentration was 362±63 ng/ml in mock-transfected cells vs 252±72 ng/ml in antago-miR-21-transfected cells (p<0.05, paired t test, 4 independent experiments).

    To explore whether aberrant miR-21 expression contributes to T cell hyperactivity in SLE, we stimulated CD4 T cells from patients with SLE with anti-CD3/anti-CD28 monoclonal antibodies and transfected them with antago-miR-21 to silence miR-21 expression (figure 3B). By using CFSE-labelled T cells, we found that silencing of miR-21 reduced T cell proliferation (mean±SEM percentage of undivided T cells on day 6: 48±12% in mock-transfected cells vs 66±9% in antago-miR-21-transfected cells; p<0.05, figure 3C). We also studied T cell IL-10 production and membrane CD40L expression in SLE, both implicated in B cell hyperactivity of SLE. Antago-miR-21 transfection significantly reduced IL-10 production by stimulated CD4 T cells from patients with SLE (612±137 ng/ml in mock-transfected cells vs 347±101 ng/ml in antago-miR-21-transfected cells; p<0.01, figure 3D). miR-21 silencing also suppressed the activation-induced upregulation of membrane CD40L (16.8±1.9% in mock-transfected cells vs 10.1±1.8% in antago-miR-21-transfected cells; p<0.01, figure 3E).

    Autoantibody production by B cells in SLE is a T cell-driven response mediated by soluble factors and cell surface interactions. We performed autologous co-cultures of T/B cells from patients with SLE to study the effect of T cell miR-21 silencing in plasma cell generation. In the presence of suboptimal anti-CD3 monoclonal antibody (100 ng/ml), T cells from patients with SLE induced the differentiation of B cells into CD19+CD38hiIgD− plasma cells (figure 3F). Antago-miR-21 transfection significantly reduced the proportion of plasma cells (16.9±2.5% in mock-transfected cells vs 10.5±2.3% in antago-miR-21-transfected cells; figure 3G). Accordingly, the total IgG concentration was reduced in cultures containing antago-miR-21-transfected T cells from patients with SLE (252±72 ng/ml vs 362±63 ng/ml in mock-transfected cells; p<0.05, figure 3H). Together, miR-21 upregulation contributes to the activated T cell phenotype of SLE and the exaggerated T cell-driven B cell differentiation of SLE into Ig-secreting plasma cells.

    Normal CD4 T cells transfected with pre-miR-21 acquire an activated phenotype

    We next addressed whether miR-21 overexpression could render normal T cells hyperactive. Purified CD4 T cells from healthy controls were stimulated with anti-CD3/anti-CD28 monoclonal antibody and transfected with pre-miR-21 to induce miR-21 expression (figure 3B). Compared with mock-transfected or irrelevant miR-transfected cells, pre-miR-21-transfected T cells had enhanced proliferation (mean±SEM percentage of divided T cells on day 6: 31±3% in scramble-transfected vs 46±1% in pre-miR-21-transfected cells; p<0.05, figure 4A). In accordance with previous studies,26 normal T cells expressed low levels of membrane CD40L; pre-miR-21 transfection caused a small but consistent increase in CD40L expression (figure 4B,C). Moreover, pre-miR-21 transfection caused a significant induction in activation-induced IL-10 secretion (567±0.4 ng/ml vs 255±4 ng/ml in scramble-transfected cells; p<0.001, figure 4D). Finally, miR-21 overexpression in normal CD4 T cells enhanced their capacity to promote autologous CD19 B cell differentiation into CD19 CD38hi IgD− plasma cells (figure 4E), and this effect was associated with increased IgG production (figure 4F). Together, these results suggest that miR-21 overexpression can induce normal T cells to acquire an activated ‘lupus-like’ phenotype.

    Figure 4
    Figure 4
    Figure 4

    Normal CD4 T cells transfected with pre-miR-21 acquire activated phenotype. (A) CFSE-labelled CD4 T cells from healthy controls were stimulated with anti-CD3/anti-CD28 monoclonal antibody (mAb) and transfected with 75 nM of mock, irrelevant (scramble) miR or pre-miR-21. Pre-miR-21-transfected T cells showed increased proliferation. The mean±SEM percentage of divided (CFSElow) T cells on day 6 was 31±3% in scramble-transfected cells vs 46±1% in pre-miR-21-transfected cells (p<0.05, paired t test, 3 independent experiments). (B) CD4 T cells from healthy controls were either unstimulated or stimulated as previously described. After 18 h the cells were examined for surface CD40L expression by flow cytometry. Stimulated T cells showed upregulated CD40L expression (albeit to lesser degree than in patients with active SLE) that was increased following pre-miR-21 transfection. Representative flow cytometry dot plot of CD40L expression in unstimulated and stimulated T cells from one healthy control. (C) The mean±SEM percentage of CD40L CD4 T cells was 5.8±0.6% in scramble-transfected cells vs 7.3±0.2% in pre-miR-21-transfected cells (p=0.085, paired t test, 3 independent experiments). (D) CD4 T cells from healthy controls were either unstimulated or stimulated with anti-CD3/anti-CD28 mAb and transfected with mock, scramble miR or pre-miR-21 as previously described. After 48 h the culture supernatants were collected and interleukin 10 (IL-10) was measured by ELISA. Pre-miR-21 transfection significantly increased IL-10 production (567±0.4 ng/ml vs 255±4 ng/ml in scramble-transfected cells and 262±12 ng/ml in mock-transfected cells; p<0.001, 3 independent experiments). (E) CD4 T cells were transfected with mock, scramble (irrelevant) or pre-miR-21 and co-cultured with autologous CD19 B cells at a ratio of 1:1 (105 cells/well) in the presence of anti-CD3 mAb (100 ng/ml). After 8 days the cells were harvested and assessed for plasma cell differentiation by flow cytometry (CD19+CD38hiIgD−). Pre-miR-21-transfected T cells showed enhanced capacity to drive normal B cell differentiation into plasma cells (mean±SEM 13.5±3.1% vs 7.2±1.3% in scramble miR-transfected cells, p=0.120, paired t test, 4 independent experiments). (F) In the previous assay, day 8 culture supernatants were assayed for total IgG by ELISA. Dot plot analysis of IgG concentration in cultures with mock-, scramble- or pre-miR-21-transfected T cells. The mean±SEM IgG concentration was 288±199 ng/ml in mock-transfected cells, 398±163 ng/ml in scramble-miR-transfected cells and 998±4 ng/ml in pre-miR-21-transfected cells (p<0.05, paired t test between scramble and pre-miR-21, 3 independent experiments).

    PDCD4 levels correlate inversely with miR-21 and are decreased in patients with active SLE

    MicroRNAs inhibit target gene expression usually at the post-transcriptional level. Previous studies have identified PDCD4, a protein translation inhibitor involved in immune responses,27 as a target gene of miR-21. We found diminished PDCD4 mRNA and protein expression in patients with active SLE compared with controls (figure 5A). To further examine the inverse relationship between miR-21 and PDCD4, we prospectively studied two patients with active SLE until they reached remission. Upon remission, miR-21 mRNA levels were decreased whereas PDCD4 protein levels were significantly increased (figure 5B). Moreover, anti-CD3/anti-CD28 stimulation of T cells resulted in significant suppression of PDCD4 expression (figure 5C). To directly demonstrate that miR-21 negatively regulates PDCD4, normal T cells were transfected with mock or pre-miR-21 to induce miR-21 levels, and PDCD4 protein levels were assessed after 48 h. Pre-miR-21-transfected T cells had reduced PDCD4 compared with mock-transfected cells (figure 5D). In accordance, PDCD4 overexpression significantly reduced IL-10 production (495±23 ng/ml in empty vector vs 312±9 ng/ml in mock-transfected cells, p<0.001) by anti-CD3/anti-CD28-stimulated normal CD4 T cells (figure 5E). Collectively, these results corroborate previous studies showing that miR-21 negatively regulates PDCD4 and imply that the effects of miR-21 in T cells of patients with active SLE may, at least in part, be due to diminished PDCD4 expression.

    Figure 5
    Figure 5

    PDCD4 is decreased in patients with active systemic lupus erythematosus (SLE) and is inversely correlated with miR-21 expression levels. (A) Reduced levels of PDCD4 mRNA (left panel) and protein (right panel) in CD4 T cells from patients with active SLE compared with healthy controls assessed by quantitative real-time PCR and western blot analysis, respectively. mRNA levels were assessed in 15 healthy controls, 8 patients with inactive SLE and 12 patients with active SLE; protein levels were assessed in 5 healthy controls and 5 patients with active SLE. In humans, two transcripts encoding different isoforms of PDCD4 have been identified. Our blot picture is consistent with this finding. (B) miR-21 mRNA levels are downregulated (right panel) and PDCD4 protein levels are upregulated (left panel) and in CD4 T cells from two patients with SLE who entered clinical remission after successful therapy. (C) Purified CD4 T cells from patients with SLE downregulate PDCD4 protein levels upon activation with anti-CD3/anti-CD28 monoclonal antibody (mAb) for 48 h. (D) CD4 T cells from healthy controls were mock-transfected or pre-miR-21-transfected (75 nM) and protein extracts were collected after 48 h for western blot analysis. PDCD4 protein levels were downregulated in pre-miR-21-transfected T cells compared with mock-transfected T cells. (E) Purified CD4 T cells from healthy controls were stimulated with anti-CD3/anti-CD28 mAb and transfected with PDCD4 expression vector as described in the Methods section. After 48 h the culture supernatants were collected and interleukin 10 (IL-10) was measured by ELISA.

    Discussion

    In this study we characterised the miRNA signature of human SLE in PBMCs and in isolated T and B lymphocytes and found a strong correlation of certain miRNAs with SLE disease activity. The identified miRNAs are predicted to regulate genes and processes pertinent to the pathogenesis of SLE, such as DNA methylation, apoptosis and proliferation. miR-21, the miRNA with the highest correlation with disease activity, was found to regulate key cellular functions that contribute to aberrant T cell phenotype in SLE. Finally, the paper reports an inverse correlation between miR-21 and its putative gene target, PDCD4, a selective protein translation inhibitor of genes involved in immune responses.

    Our analysis revealed several differentially expressed miRNAs in active versus inactive SLE. Among these genes, miR-21, miR-25, miR-106b (expressed by both T and B lymphocytes) and miR-148b (expressed by T cells) correlated strongly with SLEDAI. The longitudinal analysis of two patients who entered remission showed a significant decrease in miR-21 (figure 5B), suggesting the potential use of miRNAs as disease biomarkers. To this end, miRNAs are attractive as potential biomarkers in SLE because their expression pattern reflects the underlying pathophysiological processes correlating with disease activity, and they can be detected in a variety of tissues.28 Unsupervised clustering of additional cases and controls and longitudinal studies in large patient cohorts would be required to define the use of miRNAs as disease biomarkers.

    To account for any potential influence of drugs on miRNA expression, all medication for SLE was held for 24 h prior to blood sampling. This time interval is 3–4 times greater than the half-life of most drugs (including glucocorticoids); however, the possibility of a drug effect cannot be ruled out completely. Nevertheless, the strong correlation between the expression of certain miRNAs and disease activity suggests that aberrant miRNA expression is predominantly disease-driven.

    Our findings agree with those of Pan et al29 who found increased miR-21 and miR-148b levels in T cells from patients with active SLE. In contrast, we did not observe the previously reported downregulation of miR-146a in PBMCs from patients with SLE, which correlates with activation of the type I interferon (IFN) pathway.16 This is in agreement with previous work from our laboratory showing no activation of the type I IFN pathway in cDNA microarrays from our SLE cohort,7 and highlights the effect of genetic background in the miRNA profile and SLE phenotype.

    Having defined the miRNA signature of human SLE, we next sought to identify novel molecules involved in the pathogenesis of SLE. We selected miR-21 as this was one of the most upregulated miRNAs and displayed the strongest correlation with disease activity. Previous work has reported that miR-21 is overexpressed in several types of malignancies and may contribute to carcinogenesis by promoting cell proliferation.24 30 miR-21 is also upregulated upon stimulation with lipopolysaccharide27 and within inflammatory milieu,31 32 indicating that this miRNA may represent an inflammatory marker.

    T cells from patients with SLE had increased miR-21 expression and silencing of miR-21 reversed their aberrant phenotype, including enhanced activation-induced proliferation, IL-10 production and surface CD40L expression. This is consistent with the observation that miR-21 is upregulated in murine effector/memory versus naive T cells.25 In SLE, T cells promote B cell maturation, isotype switch and production of high-affinity pathogenic autoantibodies, and this effect is mediated by both cell contact interactions and soluble factors.33 Inhibition of miR-21 in T cells from patients with SLE significantly reduced their capacity to drive differentiation of B cells from patients with SLE into immunoglobulin-secreting plasma cells. Together, miR-21 may regulate multiple pathways that contribute to T and B cell abnormalities in SLE with important therapeutic implications. Of note, miR-21 was also found to be upregulated in B cells from patients with SLE, and this is currently under investigation.

    miR-21 regulates multiple gene targets including PDCD4,34 35 a selective protein translation inhibitor of genes involved in immune responses.27 PDCD4 levels were significantly decreased in active SLE and were restored upon disease remission. Accordingly, activated T cells from patients with SLE upregulated miR-21 and suppressed PDCD4. PDCD4 has been shown to regulate IL-10 production, indicating that the effect of miR-21 on IL-10 production by T cells in SLE may be through inhibition of PDCD4.27 36 Moreover, PDCD4 inhibits AP-1,37 which is an important transcription factor for CD40L,38 also shown to be regulated by miR-21. These findings suggest that the effects of miR-21 on T cells in SLE may, at least in part, be mediated through inhibition of PDCD4. In support of this, we found that PDCD4 overexpression significantly reduced IL-10 production by normal T cells. The beneficial effect of translation-inhibiting drugs such as rapamycin in murine and human SLE T cells39 makes PDCD4 a plausible target for treatment.

    In summary, we have provided evidence for altered expression of miRNAs in patients with active SLE, underscoring the importance of this novel class of genes in regulation of immune responses and pathogenesis of autoimmunity. miR-21 correlated strongly with SLE activity and regulated several functions that contribute to the aberrant phenotype of T cells in SLE. By fine-tuning gene regulation, miRNAs can maintain the balance between immune activation and tolerance, and could be exploited as novel therapeutic targets for SLE.

    Acknowledgments

    The authors acknowledge the help of Eva Choustoulaki, Prodromos Sidiropoulos, Amalia Raptopoulou, Aryro Repa and Eleni Kteniadaki in sample collection, and Christianna Choulaki, Irene Kyrmizi, Eleni Krasoudaki, Melina Kavoussanaki and Marriana Ioannou for technical assistance. The authors also wish to thank Themis Allisafi, Elena Kontaki and Dimitra Vyrla.

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    Supplementary materials

    Footnotes

    • ES, GB, AI and DTB contributed equally

    • Funding This work is supported by the Hellenic Society of Rheumatology, the Pancretan Health Association, the Hellenic Ministry of Education, Hellenic Republic and the European Union (EPEAEK Fund and Sixth Framework Programme AutoCure program).

    • Competing interests None.

    • Patient consent Obtained.

    • Ethics approval This study was conducted with the approval of the University Hospital of Heraklion, Crete, Greece.

    • Provenance and peer review Not commissioned; externally peer reviewed.