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. 2016 Apr 28;139(6):1747–1761. doi: 10.1093/brain/aww084

MicroRNAs targeting TGFβ signalling underlie the regulatory T cell defect in multiple sclerosis

Mary E Severin 1,2, Priscilla W Lee 1,3, Yue Liu 1, Amanda J Selhorst 1, Matthew G Gormley 4, Wei Pei 5, Yuhong Yang 5, Mireia Guerau-de-Arellano 6,7, Michael K Racke 5,7, Amy E Lovett-Racke 1,7,✉
PMCID: PMC4892757  PMID: 27190026

Regulatory T cells (Tregs) are defective in multiple sclerosis. Severin et al. show that TGFβ signalling is reduced in T cells in multiple sclerosis owing to overexpression of miRNAs that negatively regulate the TGFβ pathway. This results in impaired Treg development, and implicates overexpression of specific miRNAs in multiple sclerosis susceptibility.

Keywords: multiple sclerosis, TGFbeta, microRNA, Tregs

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Regulatory T cells (Tregs) are defective in multiple sclerosis. Severin et al. show that TGFβ signalling is reduced in T cells in multiple sclerosis owing to overexpression of miRNAs that negatively regulate the TGFβ pathway. This results in impaired Treg development, and implicates overexpression of specific miRNAs in multiple sclerosis susceptibility.

Abstract

Transforming growth factor beta (TGFβ) signalling is critical for regulatory T cell development and function, and regulatory T cell dysregulation is a common observation in autoimmune diseases, including multiple sclerosis. In a comprehensive miRNA profiling study of patients with multiple sclerosis naïve CD4 T cells, 19 differentially expressed miRNAs predicted to target the TGFβ signalling pathway were identified, leading to the hypothesis that miRNAs may be responsible for the regulatory T cell defect observed in patients with multiple sclerosis. Patients with multiple sclerosis had reduced levels of TGFβ signalling components in their naïve CD4 T cells. The differentially expressed miRNAs negatively regulated the TGFβ pathway, resulting in a reduced capacity of naïve CD4 T cells to differentiate into regulatory T cells. Interestingly, the limited number of regulatory T cells, that did develop when these TGFβ-targeting miRNAs were overexpressed, were capable of suppressing effector T cells. As it has previously been demonstrated that compromising TGFβ signalling results in a reduced regulatory T cell repertoire insufficient to control autoimmunity, and patients with multiple sclerosis have a reduced regulatory T cell repertoire, these data indicate that the elevated expression of multiple TGFβ-targeting miRNAs in naïve CD4 T cells of patients with multiple sclerosis impairs TGFβ signalling, and dampens regulatory T cell development, thereby enhancing susceptibility to developing multiple sclerosis.

Introduction

Multiple sclerosis is a CNS demyelinating disease that is immune-mediated and postulated to be driven by myelin-specific pro-inflammatory T cells. Progress has been made towards understanding disease pathogenesis, but the cause of multiple sclerosis remains unknown. A tremendous need for defining susceptibility factors and new therapeutic targets remains. Epidemiological studies have implicated both environmental and genetic factors in multiple sclerosis susceptibility (Hafler et al., 2007; Ebers, 2008; Oksenberg et al., 2008; Baranzini et al., 2009a). While genetic studies have identified genes critical to the immune system as being potential risk factors (Baranzini et al., 2009b; De Jager et al., 2009; Beecham et al., 2013), many of the genetic contributors to this disease are unknown. An emerging area of interest in multiple sclerosis has been genetic regulation at the microRNA (miRNA) level (Du et al., 2009; Keller et al., 2009; Otaegui et al., 2009; De Santis et al., 2010; Guerau-de-Arellano et al., 2011; Noorbakhsh et al., 2011; Jr OeF et al., 2012; Smith et al., 2012; Gandhi et al., 2013; Ridolfi et al., 2013; Søndergaard et al., 2013). MiRNAs are small, non-coding RNA that regulate gene expression and, thus, modify cellular pathways and disease processes (Ambros, 2004; Bartel, 2004). In addition to being biological regulators, miRNAs are easily quantified from cells, tissue, and blood, making them attractive candidates as potential biomarkers of multiple sclerosis susceptibility.

Our lab previously performed a miRNA profiling study on naïve CD4 T cells of untreated patients with multiple sclerosis and healthy individuals, identifying 85 miRNAs as being differentially expressed in patients with multiple sclerosis (Guerau-de-Arellano et al., 2011). Pathway analysis predicted that the transforming growth factor-beta (TGFβ) signalling pathway was potentially altered by the differential expression of miRNAs in the naïve T cells of patients with multiple sclerosis. TGFβ is a cytokine known to be particularly important in the development and function of regulatory T cells (Tregs) (Yamagiwa et al., 2001; Zheng et al., 2002; Chen et al., 2003), protectors against autoimmune responses (Suri-Payer et al., 1998; Itoh et al., 1999; Sakaguchi, 2004). Tregs in patients with multiple sclerosis, while normal in number, demonstrate diminished suppressive effect on myelin-specific autoreactive T cells, low FOXP3 expression, and a less diverse T cell receptor (TCR) repertoire (Viglietta et al., 2004; Haas et al., 2005, 2007; Huan et al., 2005; Kumar et al., 2006; Venken et al., 2008). However, it was also shown that CD4+CD25hiCD127lo T cells of patients with multiple sclerosis had normal suppressive activity in polycloncal T cell activation assays (Michel et al., 2008), suggesting that the characterization of the Treg population, as well as the method to measure suppression, may influence the outcome. The mechanisms for Treg defects in patients with multiple sclerosis are unknown. Interestingly, adult mice deficient in TGFβ signalling exhibit a Treg phenotype with normal numbers, decreased suppressive function, and an incomplete TCR repertoire (Gorelik and Flavell, 2000; Marie et al., 2005, 2006; Liu et al., 2008; Ouyang et al., 2010), mirroring the observations seen in patients with multiple sclerosis. We hypothesized that dysregulated miRNAs in the naïve CD4 T cells of patients with multiple sclerosis modulate the TGFβ-signalling pathway, resulting in defective Tregs and enhanced susceptibility to developing multiple sclerosis.

Materials and methods

Human subjects

Information on patients with multiple sclerosis and control subjects used in the miRNA profiling study, as well as the methods and data from this miRNA profiling study can be found in Guerau-de-Arellano et al. (2011). The data shown in Fig. 1 were obtained from these samples. The healthy control peripheral blood mononuclear cells (PBMCs) for the current study were obtained from the Red Cross.

Figure 1.

Figure 1

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Differentially expressed miRNAs in the naïve CD4 T cells of patients with multiple sclerosis predicted to target the TGFβ signalling pathway. (A) miRNA profiling was performed on the naïve CD4 T cells from healthy controls (HC; n = 16) and patients with multiple sclerosis (MS; n = 22) using a TaqMan® real-time PCR array. Of the 85 differentially expressed miRNAs, 19 (>3-fold expression change) were predicted to target the TGFβ signalling pathway. (B) Real-time quantitative PCR was used to measure the expression of TGFBR1, TGFBR2, SMAD2, and SMAD4 in the naïve CD4 T cells of healthy controls (n = 7) and untreated patients with multiple sclerosis (n = 22). Fold-change was calculated using healthy control subject median Ct, and Mann-Whitney analyses were performed. TGFBR1 and SMAD4 were found to be significantly decreased (P-value < 0.05) in patients with multiple sclerosis. (C) For the miRNAs predicted to target TGFBR1 and SMAD4, miRNA fold-changes were calculated relative to healthy control subject geometric mean Ct. Mean fold-change values are represented by a line. Statminer’s Limma test P-values for the healthy control subject (n = 16) to multiple sclerosis (n = 22) groups’ comparisons are shown.

Real-time polymerase chain reaction

Complementary DNA was transcribed from the RNA of the profiled samples using random primers. TaqMan® real-time polymerase chain reactions were performed using hTGFβR1, hTGFβR2, hSMAD2, hSMAD4, and hHPRT primer sets (Applied Biosystems). Results were analysed using the comparative Ct method. Relative fold-change expression was calculated relative to the median Ct of the healthy controls group.

Luciferase assay

For human TGFBR1 (NM_004612), two segments of the 3’UTR were individually cloned into PGL3 vectors (Promega). TGFBR1 3’UTR base pairs 1563–2752 contain binding sites for miR-27b and miR-128, and TGFBR1 3’UTR base pairs 3275–4038 contain binding sites for miR-141, miR-500a, and let-7 (Fig. 2A). For human SMAD4 (NM_00539), four segments of the 3’UTR were made and cloned into PGL3 vectors (Fig. 2B). SMAD4 3’UTR base pairs 1–787 contain binding sites for miR-708 and miR-212. SMAD4 3’UTR base pairs 1525–2275 contain binding sites for miR-500a, miR-27b, and miR-128. SMAD4 3’UTR base pairs 3927–4582 contain binding sites for miR-128, miR-628-3p, miR-141, and miR-27b. SMAD4 3’UTR base pairs 4570–5563 contain binding sites for miR-103a, miR-141, and miR-18a. Cos-7 cells were transfected with one of the constructs containing a 3’UTR segment and a single miRNA using Lipofectamine® 2000 (Life Technologies). miR-NS was used as the negative control. The transfected cells were lysed and processed using the Luciferase Assay System (Promega). A luminometer measured relative light units (RLU). RLU was normalized to the protein concentration of the samples and per cent RLU was calculated. Significance (P > 0.05) was calculated comparing per cent RLU between nonsense (miR-NS) and test miRNA groups.

Figure 2.

Figure 2

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Differentially expressed miRNAs bind and regulate TGFBR1 and SMAD4. Luciferase assays were conducted using PGL3 vector constructs containing segments of the 3’UTRs of TGFBR1 (A) and SMAD4 (B). Significance was calculated using an unpaired t-test comparing the relative luciferase units (RLU) of the miR-NS control and individual miRNA groups (*P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001).

TransIT-TKO® (Mirus) was used to transfect PBMCs with 0.05 µM of single miRNA mimics (Dharmacon; miR-27b, miR-103a, miR-128, miR-141, miR-212, miR-500a, miR-628-3p, miR-708, let-7a, and let-7b) or miRNAs in combination (miR-103/212/708; miR-141/500a/let-7b; miR-128/628-3p/let-7ab). A nonsense miRNA (miR-NS) was used as a negative control for miRNA transfection. Cells were transfected for 48 h at 37 °C according to manufacturer’s protocol prior to analysis or differentiation.

Flow cytometry

After 48 h of transfection with miRNAs, PBMCs were analysed using a FACSCanto™ II (BD Biosciences) for expression of TGFβR1 and SMAD4. Cells were first stained with antibodies for the human surface markers CD4 (BD Biosciences) and CD45RA (BioLegend). The FOXP3/Transcription Factor Staining Buffer Set (eBioscience) was used to fix and permeabilize the cells. The cells were subsequently stained with antibodies for intracellular components of human TGFβR1 V 1-22 (Santa Cruz Biotechnology) and SMAD4 (R&D Systems). The TGFβR1 detection required an additional staining with FITC conjugated goat anti-rabbit secondary antibody (Abcam). For Treg analysis, the flow cytometry was performed at 72 h post anti-CD3/anti-CD28 activation, and cells were initially stained with antibodies for CD4 (BD Biosciences) and, CD45RA (BioLegend), and CD25 (BD Biosciences), followed by intracellular staining for FOXP3 (eBioscience). The data were analysed using FlowJo software (Tree Star). The cells were initially gated on CD4+CD45RA+ naïve T cells, and this population was further analysed for CD25 and FOXP3 expression so that only the Tregs that differentiated from the naïve CD4 T cell population were quantitated.

In vitro human Treg induction

For Figs 5, 6A–C, and Supplementary Figs 3 and 4, PBMCs were cultured on 48-well plates coated with 1 µg/ml of anti-human CD3/CD28 in the presence of 1 U/ml IL-2, 0.5 ng/ml TGFβ1, and 2.5 nM all trans retinoic acid for 72 h at 37 °C. For Fig. 6D–F and Supplementary Fig. 2, PBMCs were cultured on 48-well plates coated with 1 µg/ml of anti-human CD3/CD28 in the presence of 500 U/ml IL-2, 5 ng/ml TGFβ1, and 10 nM all trans retinoic acid for 96 h at 37 °C. This method was used to enhance Treg numbers in the miRNA-transfected cells so that the number of Tregs was sufficient for suppression assays.

Figure 5.

Figure 5

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Overexpression of TGFβ-targeting miRNAs decreases inducible Treg induction. PBMCs from healthy controls were transfected with each miRNA and cultured in inducible Treg inducing conditions. The cells were analysed using flow cytometry and inducible Tregs (CD4+CD45RA+CD25+ FOXP3+) generated from naïve CD4 T cells were quantified (red). (A) Flow plots are representative data from a single individual. (B) Each line represents a unique PBMC sample (n = 13–19) with the red line representing the data in A. A Wilcoxon matched pairs test comparing miR-NS and individual miRNA was used to calculate significance (P < 0.05). (C) Inducible Treg flow plots for the miRNA combinations from the same individual in A are shown (left; miR-NS in A is the control) and the highly significant changes in inducible Tregs in the PBMC samples for the miRNA combinations are shown on the right.

Figure 6.

Figure 6

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Inducible Tregs generated from cells overexpressing TGFβ-targeting miRNAs retain suppressive function. (A) PBMCs from a healthy control subject were transfected with 0.05 μM of miRNA and cultured in inducible Treg inducing conditions. Inducible Tregs were analysed using flow cytometry. (B) IL-10 was measured in the supernatants of the inducible Treg cultures at 72 h. (C) A non-parametric Pearson correlation test analysed the degree of relatedness between the levels of IL-10 and percent of inducible Tregs generated. Data in A–C are representative of four independent experiments. (D) PBMCs from a Healthy donor were transfected with miRNAs (miR-NS; miR-103,212,708; and miR-141,500, let-7b) and cultured in Treg-inducing conditions. The per cent of inducible Tregs (CD4 + CD45RA + CD25 + FOXP3+) generated for each condition was determined using flow cytometry. (E) Inducible Tregs were co-cultured at various ratios with CFSE-labelled PBMCs. Gates for five generations of proliferation were set using flow cytometry. The amount of cells per generation of proliferation indicated in E was determined. (F) Using the per cent of cells in each generation, proliferation patterns of Teff cells alone (bottom) and Teff cells co-cultured at a 2:1 ratio with Tregs (top) from the three miRNA conditions were compared. Data in D–F are representative of six PBMCs samples from two independent experiments.

IL-10 ELISA

Supernatants were collected from the inducible Treg cultures. ELISA was performed using purified rat anti-human detection antibodies and biotinylated anti-human/viral IL-10 detection antibodies (BD Biosciences). IL-10 concentrations were calculated from known standards of recombinant IL-10 protein (R&D Systems) and analysed via SoftMax® Pro Software (Molecular Devices).

Human CSFE suppression assay

PBMCs were isolated from healthy controls. The PBMCs were subsequently transfected with control miRNA (miR-NS) or miRNAs in combinations (miR-103, 212, 708; miR-141, 500, let-7b) as described above. Inducible Tregs were generated from the transfected PBMCs by culturing the cells on 48-well plates coated with 1 µg/ml of anti-human CD3/CD28 in the presence of 500 U/ml IL-2, 5 ng/ml TGFβ1, and 10 nM all trans retinoic acid for 96 h at 37 °C. Effector T cells (Teffs) were generated by culturing additional autologous PBMCs in the presence of 1 U/ml IL-2 at 37 °C for the duration of the transfection and Treg induction steps (6 days). The Teff cells were subsequently labelled using the CellTrace™ CFSE Cell Proliferation Kit Protocol (ThermoFisher Scientific). The inducible Treg cultured cells were washed and mixed with the CSFE-labelled Teff cells at varying ratios (Teff:Treg; 1:0, 1:1, 2:1, and 4:1) on 48-well plates coated with 1 µg/ml anti-human CD3/28 for 96 h at 37 °C. After 96 h, the CFSE-labelled CD4+Teff cells were evaluated for proliferation using flow cytometry. Analysis was performed using the FlowJo Proliferation Platform (Tree Star).

Murine CSFE suppression assay

The protocol used for these experiments received prior approval by the OSU Institutional Animal Care and Use Committee and were conducted in accordance with the United States Public Health Service’s Policy on Humane Care and Use of Laboratory Animals. The MBP Ac1-11-specific TCR transgenic mice are on a B10.Pl background and were bred at the OSU animal facility (Governman et al., 1993). The ovalbumin 323-339-specific TCR transgenic mice are on a C57Bl/6 and were purchased from Jackson Laboratories. Splenocytes were isolated from MBP Ac1-11-specific TCR transgenic mice, as well as ovalbumin 323-339-specific TCR transgenic mice. The splenocytes were subsequently transfected with control miRNA (miR-NS) or miRNAs in combinations (miR-103, 212, 708; miR-141, 500, let-7b) as described above. Inducible Tregs were generated from the transfected splenocytes by culturing the cells on a 24-well plate in the presence of 10 µg/ml MBP Ac1-11 peptide (or ovalbumin 323-339), 500 U/ml IL-2, 5 ng/ml TGFβ, and 10 nM retinoic acid for 72 h at 37 °C. Teff cells were generated from fresh splenocytes labelled using the CellTrace™ CFSE Cell Proliferation Kit Protocol (ThermoFisher Scientific). The inducible Treg cultured cells were washed and mixed with the CSFE-labelled Teff cells at varying ratios (Teff:Treg; 1:0, 1:1, 2:1, and 4:1) in the presence of 10 µg/ml MBP Ac1-11 peptide for 96 h at 37 °C. After 96 h, the CFSE-labelled CD4+Teff cells were evaluated for proliferation using flow cytometry. Analysis was performed using the FlowJo Proliferation Platform (Tree Star).

Results

TGFβ-associated genes are decreased in patients with multiple sclerosis

In our previous comprehensive miRNA profiling study of patients with multiple sclerosis CD4 T cells (Guerau-de-Arellano et al., 2011), 19 of 85 differentially expressed miRNAs in naïve CD4 T cells were predicted to target the TGFβ signalling pathway (Fig. 1A). To determine if the TGFβ signalling pathway is defective in patients with multiple sclerosis, real-time quantitative PCR was performed on the naïve CD4+CD45RA+ T Cells of 22 untreated patients with multiple sclerosis and seven healthy control samples. The samples used were the same RNA isolates utilized in the initial miRNA profiling array. This strategy allowed us to evaluate whether the patients with multiple sclerosis whose differentially expressed miRNAs were predicted to target the TGFβ signalling pathway also had lower expression of TGFβ-associated genes (TGFBR1, TGFBR2, SMAD2, and SMAD4). Of the four predicted targets analysed, TGFBR1 and SMAD4 were significantly decreased in patients with multiple sclerosis (Fig. 1B). The differential expression of miRNAs predicted to target TGFBR1 and SMAD4 in the miRNA profiling (n = 16 healthy control subjects and n = 22 multiple sclerosis) are shown in Fig. 1C and are the focus of this study. These data suggested that overexpression of miRNAs in patients with multiple sclerosis may limit TGFβ signalling and may be the mechanism responsible for the Treg defect in patients with multiple sclerosis. Interestingly, these miRNAs had not previously been identified as playing a role in Treg development.

Dysregulated miRNAs directly bind and regulate TGFBR1 and SMAD4

To identify which miRNAs directly bind and potentially regulate their predicted target genes, TGFBR1 and SMAD4, luciferase assays were performed. Segments of the 3’UTRs of human TGFBR1 (Fig. 2A) and SMAD4 (Fig. 2B), which contain miRNA binding sites, were inserted into luciferase vectors and co-transfected into cos-7 cells with the appropriate individual miRNA. A reduction in luciferase activity would be indicative of miRNA regulation. Nonsense miRNA (miR-NS) was used as a control to indicate the normal levels of luciferase activity. All of the miRNAs predicted to target TGFBR1 were shown to cause a significant reduction in the RLU when co-transfected with the TGFBR1 constructs (Fig. 2A). All the miRNAs predicted to target SMAD4, except miR-18a, had a significant reduction in RLU (Fig. 2B). These data indicate that the miRNAs can bind their predicted target genes, suggesting that these miRNAs could modulate TGFβ signalling through their regulation of TGFBR1 and SMAD4.

Overexpression of multiple sclerosis-associated miRNAs decrease TGFβR1 and SMAD4 levels

To determine if the TGFβ-targeting miRNAs could decrease TGFβR1 and SMAD4 in naïve CD4 T cells, miRNAs were overexpressed in human PBMCs and analysed via flow cytometry. PBMCs from healthy individuals, not patients with multiple sclerosis since they already have altered miRNA levels, were transfected with single miRNAs or miRNAs in combination, and flow cytometry was used to analyse the TGFβR1 and SMAD4 levels in naïve CD4+CD45RA+ T cells. TGFβR1 and SMAD4 are constitutively expressed in T cells and therefore, to detect changes in protein levels on a per cell basis, mean fluorescence intensity (MFI) was analysed. Figure 3A illustrates the changes in TGFβR1 levels following miRNA transfection for one individual’s naïve CD4+CD45RA+ T cells sample. The quantification for this representative sample is shown in red in Fig. 3B, as well as the changes for each miRNA in 15 human samples. For TGFβR1, six of seven miRNAs significantly decreased the MFI. To determine if having multiple miRNAs overexpressed, as was typically observed in the patients with multiple sclerosis, augments changes in TGFβR1 levels, a combination of miR-141, miR-500a and let-7b was analysed, demonstrating a more significant decrease in TGFβR1 levels. Similarly, five of eight miRNAs predicted to target SMAD4 significantly decreased the MFI of SMAD4 (Fig. 4). The observed shifts in MFI indicate that there is less TGFβR1 and SMAD4 on a per cell basis when these miRNAs are elevated in naïve CD4 T cells. Given that TGFβ signalling is required for the development of Tregs (Yamagiwa et al., 2001; Zheng et al., 2002; Chen et al., 2003), the elevated expression of these miRNAs in patients with multiple sclerosis’ naïve CD4 T cells suggests that these miRNAs play a role in the Treg defect observed in patients with multiple sclerosis.

Figure 3.

Figure 3

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Overexpression of TGFβ-targeting miRNAs reduces TGFβR1 expression. Individual or combinations of miRNAs predicted to target TGFBR1 were transfected into PMBCs from healthy controls. Flow cytometry was used to analyse changes in TGFβR1 levels. (A) Flow plots representative of data from a single individual. (B) Each line represents a unique PBMC sample (n = 15) with the red line representing the data in A. The geometric mean florescence intensity (MFI) was measured and compared between miR-NS and individual or combined miRNA groups. A Wilcoxon matched pairs test calculated significance (P < 0.05).

Figure 4.

Figure 4

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Overexpression of TGFβ-targeting miRNAs reduces SMAD4 expression. Individual or combinations of miRNAs predicted to target SMAD4 were transfected into PMBCs from healthy controls. Flow cytometry was used to analyse changes in SMAD4 levels. (A) Flow plots representative of data from a single individual. (B) Each line represents a unique PBMC sample (n = 15) with the red line representing the data in A. The geometric mean florescence intensity (MFI) was measured and compared between miR-NS and individual or combined miRNA groups. A Wilcoxon matched pairs test calculated significance (P < 0.05).

Overexpression of TGFβ-associated miRNAs decrease Treg development

To determine if overexpression of these TGFβ-targeting miRNAs could decrease the differentiation of naïve CD4+CD45RA+ T cells into inducible Tregs, miRNAs were transfected into PBMCs from healthy individuals and flow cytometry was used to analyse the percentage of naïve CD4 T cells that were able to differentiate into CD25+ FOXP3+ inducible Tregs. The mean number of endogenous CD25+ FOXP3+ Tregs present in the CD4+CD45RA+ population prior to differentiation was 1.6% ± 0.75%. Given that the number of endogenous Tregs was very low in the human PBMCs and the fact that each PBMC sample transfected with a nonsense miRNA served as its own control, this small population of endogenous Tregs was controlled in each experiment. Figure 5A illustrates the percentage of inducible Tregs generated by differentiation with anti-CD3/anti-CD28 in the presence of TGFβ, IL-2 and trans retinoic acid from naïve CD4+CD45RA+ T cells in one PBMC sample when the miRNAs are overexpressed individually. Analysis of a panel of PBMCs found that miR-27b, 103a, 128, 628-3p, and 708 significantly decreased inducible Treg development from naïve CD4 T cells (Fig. 5B, the sample shown in Fig. 5A is indicated by red line). To ensure that the transfection process and our Treg induction protocol induce functional Tregs, a CFSE-based suppression assay was performed using miR-NS transfected inducible Tregs, demonstrating that these Tregs suppress effector CD4 T cells (Supplementary Fig. 1). In addition, to confirm that the inducible Treg induction protocol was directly effecting the differentiation of naïve CD4 T cells into inducible Tregs and not simply affecting the small population of endogenous Tregs that are also CD4+ CD25RA+, we tracked CSFE-labelled endogenous Tregs during the inducible Treg differentiation protocol. To perform this experiment, endogenous Tregs were isolated from the PBMCs, these Tregs were labelled with CFSE, and added back to the PMBCs at either 2% or 5% of the total PBMCs concentration. These PBMCs with the labelled Tregs were then subjected to the inducible Treg differentiation protocol. After 72 h of inducible Treg differentiation, the endogenous Tregs had only increased to 2.7% and 6.7% of the total PBMC sample for the 2% and 5% CFSE-labelled cells, respectively (Supplementary Fig. 2A), indicating that the endogenous Tregs had minimal proliferation during the inducible Treg differentiation. The endogenous CFSE-labelled Tregs and the non-labelled inducible Tregs were analysed in the naïve CD4 T cell population within the PBMCs. The total number of Tregs in the CD4+CD45RA+ exceeded 60%, with only 3.1% and 6.9% of the Tregs in this population labelled with CFSE, corresponding to the 2% and 5% CSFE-labelled endogenous Tregs (Supplementary Fig. 2B). This indicates that the changes in Treg percentages in the miRNA-transfected PBMCs are due to changes in the differentiation of inducible Tregs, and not significantly affected by the small population of endogenous Tregs present in the PBMCs. One additional experiment was performed to confirm that the endogenous Tregs were not a major factor in the miRNA-mediated inhibition of inducible Treg development. Endogenous Tregs were partially depleted or enriched in PBMCs by isolating endogenous Tregs from PBMCs and adding the isolated Tregs back to half the PBMCs, and comparing the PBMCs with partial deletion of endogenous Tregs to the PBMCs with elevated numbers of endogenous Tregs. Both PMBC samples were subjected to miRNA transfection and inducible Treg differentiation. Overall, there was no significant difference in the ability of the naïve CD4+CD45RA+ T cells to differentiate into inducible Tregs in either condition (Supplementary Fig. 3), indicating that the miRNA were affecting the naïve CD4 T cells differentiate into inducible Tregs, with minimal influence by the small population of endogenous Tregs in the PBMC samples.

Overexpression of a single TGFβ-targeting miRNA was not unusual in the healthy control population, as seen in Fig. 1C, but patients with multiple sclerosis typically had multiple miRNAs overexpressed. Therefore, combinations of miRNAs found to be overexpressed in the multiple sclerosis patients’ naïve CD4 T cells were tested to determine if there may be a synergistic effect of the TGFβ-targeting miRNAs. Three miRNA combinations observed in patients with multiple sclerosis were transfected into PBMCs of healthy controls and evaluated for inducible Treg development. All three combinations demonstrated highly significant reductions in inducible Tregs in naïve CD4 T cells (Fig. 5C). In fact, miR-141/500/let-7b, which had no significant effect individually on inducible Treg development, showed a significant decrease in combination, indicative of the synergistic effect. Similarly, the per cent decrease in the other two miRNA combinations were more significant than the individual miRNAs, suggesting that the elevated expression of multiple TGFβ-targeting miRNAs in naïve CD4 T cells of patients with multiple sclerosis diminishes their capacity to generate inducible Tregs.

TGFβ-associated miRNAs do not alter Treg function

To address the functional status of the Tregs generated from naïve T cells overexpressing these miRNAs, miRNAs were transfected into PBMCs of healthy individuals and inducible Treg numbers were compared to IL-10 production, an anti-inflammatory cytokine often expressed by Tregs. Overall, IL-10 levels were decreased (Fig. 6B), as were the inducible Treg percentages (Fig. 6A). There was a positive correlation between the numbers of inducible Tregs and levels of IL-10, indicating that the Tregs that did develop were able to produce normal levels of IL-10 (Fig. 6C). These data suggest that these miRNAs limit Treg development, but not their ability to produce the anti-inflammatory cytokine IL-10.

Previous studies have shown that patients with multiple sclerosis have Tregs with reduced suppressive function and a retracted TCR repertoire (Viglietta et al., 2004; Haas et al., 2005, 2007; Huan et al., 2005; Kumar et al., 2006; Venken et al., 2008). Interestingly, similar observations of reduced Treg function and TCR diversity has been reported in mice deficient in TGFβ signalling (Gorelik and Flavell, 2000; Marie et al., 2005, 2006; Liu et al., 2008; Ouyang et al., 2010). Currently, it is unclear whether the observed defect is a result of disrupted Treg machinery or a lack of Treg clonal diversity. To determine if the suppressive capacity of the human inducible Tregs is altered due to the TGFβ-targeting miRNAs, inducible Tregs generated following transfection with miRNA combinations miR-103,212,708 and miR-141,500, let-7b were assayed for their ability to suppress polyclonal T cell activation. Human inducible Tregs were generated from miRNA-transfected PBMCs (Fig. 6D) and then co-cultured with CFSE-labelled PBMCs, Teff cells, from the same donor on anti-CD3/CD28-coated plates. While the CD4+ Teff cells proliferated robustly in the absence of inducible Tregs (Fig. 6E), the addition of inducible Tregs generated from miR-NS or the miRNA combinations resulted in similar suppression of proliferation (Fig. 6E). To compare patterns of proliferation between the three miRNA conditions, the per cent of CD4 T cells in each generation was calculated using the gates indicated in Fig. 6E. The co-culture proliferation profiles were similar among the three groups containing inducible Tregs generated from miRNA transfected cells (Fig. 6F). The cells from the inducible Treg cultures suppressed Teff proliferation similarly despite reduced Treg induction in the miRNA combination groups, indicating that the inducible Tregs that do develop from TGFβ-targeting miRNA transfection have the capacity to function normally with polyclonal activation.

To determine if inducible Tregs generated from naïve T cells overexpressing TGFβ-targeting miRNAs have reduced ability to suppress antigen-specific Teff cells, TCR transgenic T cells were used as the source of inducible Tregs and Teff cells. By overexpressing the TGFβ-targeting miRNAs in naïve T cells with the same TCR, an antigen-driven system was used to eliminate effects due to TCR repertoire. Naïve myelin basic protein (MBP) Ac1-11 peptide-specific TCR splenocytes were transfected with two miRNA combinations and the T cells were stimulated with MBP Ac1-11 under optimal inducible Treg conditions to minimize differences in inducible Treg numbers due to the miRNAs (Fig. 7A). The inducible Tregs were co-cultured with naïve MBP Ac1-11-specific TCR splenocytes, the Teff cells, in the presence of MBP Ac1-11. When cultured alone, Teff cells underwent nine detectable rounds of proliferation (Fig. 7B). However, when co-cultured at a 2:1 (Teff:Treg) ratio, inducible Tregs from all three miRNA conditions (miR-NS; miR-103,212,708; and miR-141, 500, let-7b) were capable of suppressing Teff cell proliferation (Fig. 7B and C). Similar findings were observed using ovalbumin-specific TCR transgenic mice to generate Teff and inducible Tregs specific for ovalbumin 323-339 (data not shown). These data indicate that when TCR repertoire is intact inducible Tregs generated from cells overexpressing TGFβ-targeting miRNAs possess the machinery necessary for suppressing Teff cell proliferation. This indicates that the TGFβ-targeting miRNAs suppress the generation of Tregs, but that the Tregs that do develop function normally.

Figure 7.

Figure 7

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Inducible Tregs generated from cells overexpressing TGFβ-targeting miRNAs retain suppressive function. (A) Splenocytes from MBP TCR-Tg mice were transfected with miRNAs (miR-NS; miR-103,212,708; and miR-141,500, let-7b) and cultured in Treg inducing conditions. The per cent of inducible Tregs (CD4 + CD25 + FOXP3+) generated for each condition was determined using flow cytometry. (B) Inducible Tregs were co-cultured at various ratios with CFSE-labelled Teff cells. Gates for nine generations of proliferation were set using flow cytometry. The amount of cells per generation of proliferation indicated in B was determined. (C) Using the per cent of cells in each generation, proliferation patterns of Teff cells alone (bottom) and Teff cells co-cultured at a 2:1 ratio with Tregs (top) from the three miRNA conditions were compared.

Discussion

Together, these data indicate that enhanced levels of TGFβ-targeting miRNAs observed in naïve CD4 T cells of patients with multiple sclerosis result in a decreased capacity to generate Tregs and may be a risk factor for the development of multiple sclerosis. Although there has been some controversy as to whether patients with multiple sclerosis have a reduced number of Tregs, the majority of studies suggest that the defect is not in number but in function (Viglietta et al., 2004; Haas et al., 2005, 2007; Huan et al., 2005; Kumar et al., 2006; Venken et al., 2008). In mice with deficiencies in TGFβ signalling, the number of Tregs in adult mice is normal; however, the number of Tregs postnatally is significantly reduced (Marie et al., 2005; Liu et al., 2008; Ouyang et al., 2010). This limited pool of Tregs, which are CD25+, proliferate in response to IL-2 resulting in normal numbers of Tregs. This expanded pool of Tregs has a diminished capacity to suppress effector T cells as evidenced by the uncontrolled autoimmunity observed in mice with deficiencies in TGFβ signalling (Gorelik and Flavell, 2000; Marie et al., 2006; Liu et al., 2008). The inability of Tregs from TGFβ signalling-impaired mice to suppress autoimmunity has been attributed to decreased FOXP3 expression and a decreased repertoire of Tregs. Interestingly, patients with multiple sclerosis have also been found to have a decreased TCR repertoire in their Tregs (Haas et al., 2007), indicating that they may have insufficient diversity to respond to self-antigens and prevent autoimmunity. In addition, thymic output of Tregs was shown to be reduced in paediatric and adult patients with multiple sclerosis, and differences in suppressive capacity of Tregs in patients with multiple sclerosis and controls were dependent on the number of new thymic emigrant Tregs, not total number of Tregs (Haas et al., 2007; Balint et al., 2013). The miRNAs identified in this study may limit natural Treg development in the thymus and inducible Treg differentiation in the periphery since both are derived from naïve CD4 T cells, resulting in the generation of a less diverse population of Tregs causing diminished suppressive capacity and increased risk of autoimmunity. As such, these miRNAs may be differentially expressed in other immune-mediated diseases and have broader implications on CD4 T cell development and differentiation. It is well established that miRNAs play key roles in the development and function of Tregs (Gao et al., 2012; Josefowicz et al., 2012; Dooley et al., 2013), and differential expression of miRNAs have been found in patients with multiple sclerosis (Du et al., 2009; Keller et al., 2009; Otaegui et al., 2009; De Santis et al., 2010; Guerau-de-Arellano et al., 2011; Noorbakhsh et al., 2011; Jr OeF et al., 2012; Smith et al., 2012; Gandhi et al., 2013; Ridolfi et al., 2013; Søndergaard et al., 2013). To our knowledge none of the miRNAs identified in this study had been previously defined to play a role in Tregs. However, miR-141 and Treg numbers were shown to fluctuate in relapsing and remitting phases of multiple sclerosis (Naghavian et al., 2015). In addition, miRNAs, such has miR-155, which regulate normal Treg development and function (Lu et al., 2009; Gao et al., 2012; Josefowicz et al., 2012; Dooley et al., 2013; Kohlhaas et al., 2009), were not found to be altered in the naïve CD4 T cells of patients with multiple sclerosis (Guerau-de-Arellano et al., 2011). Thus, it is important to determine if these TGFβ-targeting miRNAs have roles in normal Treg development that are dysregulated in patients with multiple sclerosis.

TGFβ is a pleiotropic cytokine that has potent immunological effects other than promoting Treg development and function. TGFβ has been shown to promote the development of Th17 cells in the presence of IL-6 in mice (Bettelli et al., 2006; Mangan et al., 2006; Veldhoen et al., 2006), suggesting that diminished TGFβ signalling may negatively modulate Th17 cells. However, in the case of encephalitogenic T cells, we and others have found that myelin-specific Th17 cells differentiated with IL-6 and TGFβ are not encephalitogenic (Yang et al., 2009; Ghoreschi et al., 2010; Lee et al., 2015). In fact, TGFβ is a negative regulator of T-bet, a key transcription factor in encephalitogenic T cells, irrespective of whether it has a Th1 or Th17 phenotype (Gocke et al., 2007; Yang et al., 2009; Lee et al., 2015). Thus, suppression of the TGFβ pathway enhances T-bet expression, as well as promotes the differentiation of pathogenic Th17 cells, suggesting that miRNAs that suppress TGFβ signalling may promote the development of autoreactive effector T cells. Furthermore, differentiation of human Th17 occurs independently of TGFβ (Acosta-Rodriguez et al., 2007; Wilson et al., 2007), and TGFβ induces IL-10 expression in autoreactive Th1 cells, promoting a self-regulation mechanism (Huss et al., 2010). IL-10 production has been shown to be impaired in patients with multiple sclerosis (Cao et al., 2015), but positively associated with therapeutic benefit (Du Pasquier et al., 2014). Thus, diminished TGFβ signalling would potentially allow encephalitogenic Th1 cells to mediate pathology more robustly due to a lack of inherent regulation. As TGFβ signalling not only promotes Treg development, but also influences the differentiation of naïve T cells into effector T cells and suppresses the function of effector/memory T cells (Huss et al., 2010), the TGFβ-targeting miRNAs may have the potential to influence naïve T cell activation in our inducible Treg differentiation assays. However, the percentage of naïve CD4 T cells that remain unactivated increases when inducible Treg differentiation is suppressed in miRNA-transfected naïve CD4 T cells, suggesting that an increase in naïve T cell activation in miRNA-transfected PBMCs is not skewing the percentage of inducible Tregs observed.

One question that is not readily answered in patients with multiple sclerosis is whether the miRNA dysregulation in naïve CD4 T cells is inherent or a result of the disease. To address this question, miRNA profiling was performed on naïve CD4 T cells from mice with experimental autoimmune encephalomyelitis (EAE) and their non-EAE littermates (data not shown). There were very few differences in miRNA levels between the EAE and non-EAE groups, and no differences were observed in miRNAs found differentially expressed in the multiple sclerosis patients’ naïve CD4 T cells. Therefore, it is unlikely that inflammation is causing the miRNA changes in naïve CD4 T cells in patients with multiple sclerosis, and this supports our hypothesis that overexpression of these TGFβ-targeting miRNAs are susceptibility factors for multiple sclerosis.

TGFβ was used as a successful therapy to ameliorate EAE (Racke et al., 1991), a model system in which the TGFβ signalling pathway is normal. However, a clinical trial using TGFβ as a therapy for multiple sclerosis failed (Calabresi et al., 1998). The reduced TGFβR1 and SMAD4 in CD4 T cells of patients with multiple sclerosis (Fig. 1B) may make patients less responsive to TGFβ therapy and thus, may be partially responsible for the lack of therapeutic benefit. Therapeutically targeting miRNAs may be problematic, given that these miRNAs have the capacity to regulate critical genes outside of the TGFβ signalling pathway and may cause unforeseen side effects. As previously demonstrated, systemic administration of small RNAs can have therapeutic benefits in CNS autoimmunity (Lovett-Racke et al., 2004; Gocke et al., 2007; Yang et al., 2010). The miRNA profiling demonstrated that there was heterogeneity in both the control and multiple sclerosis patient populations with respect to the level of expression of a specific miRNA (Fig. 1C). However, the patients with multiple sclerosis typically had several miRNAs overexpressed that targeted TGFβ signalling, suggesting that these miRNAs may serve as biomarkers for multiple sclerosis susceptibility.

In summary, patients with multiple sclerosis had reduced levels of TGFβ signalling components in their naïve CD4 T cells. The differentially expressed miRNAs negatively regulated the TGFβ pathway, resulting in a reduced capacity of naïve CD4 T cells to differentiate into Tregs, yet the function of the limited repertoire of Tregs appeared normal. The observation that Treg numbers appear normal in patients with multiple sclerosis in most studies is probably due to the fact that high expression of CD25 allows the Tregs that do develop to proliferate and appear normal in number, as observed in mice with defected TGFβ signalling (Liu et al., 2008). As compromising the TGFβ signalling pathway reduces the Treg repertoire, which results in an inability to control autoimmunity and patients with multiple sclerosis have a reduced Treg repertoire, these TGFβ-targeting miRNAs impair TGFβ signalling, dampen Treg development, enhancing susceptibility to developing multiple sclerosis.

Funding

This study was supported by grants from the National Multiple Sclerosis Society [RG 5241-A-5 (A.E.L.R) and RG 4742-A-14 (M.K.R.)], the National Institutes of Health (R21NS078390 – A.E.L.R.), and a NIH funded Clinical Translational Science Award fellowship for P.W.L. (TL1TR00091-05).

Supplementary material

Supplementary material is available at Brain online.

Supplementary Material

Supplementary Data
Click here for additional data file. (926.2KB, zip)

Glossary

Abbreviations

PBMC =

peripheral blood mononuclear cells

TCR =

T cell receptor

Teff =

effector T cell

TGFβ =

transforming growth factor beta

Treg =

regulatory T cell

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