MicroRNA-142 regulates inflammation and T cell differentiation in an animal model of multiple sclerosis

Abstract

Background: MicroRNAs have emerged as an important class of modulators of gene expression. These molecules influence protein synthesis through translational repression or degradation of mRNA transcripts. Herein, we investigated the potential role of miR-142a isoforms, miR-142a-3p and miR-142a-5p, in the context of autoimmune neuroinflammation.

Methods: The expression levels of two mature isoforms of miR-142 were measured in the brains of patients with multiple sclerosis (MS) and the CNS tissues from mice with experimental autoimmune encephalomyelitis (EAE), an animal model of MS. Expression analyses were also performed in mitogen and antigen-stimulated splenocytes, as well as macrophages and astrocytes using real-time RT-PCR. The role of the mature miRNAs was then investigated in T cell differentiation by transfection of CD4⁺ T cells, followed by flow cytometric analysis of intracellular cytokines. Luciferase assays using vectors containing the 3'UTR of predicted targets were performed to confirm the interaction of miRNA sequences with transcripts. Expression of targets were then analyzed in activated splenocytes and MS/EAE tissues.

Results: Expression of miR-142-5p was significantly increased in the frontal white matter from MS patients compared with white matter from non-MS controls. Likewise, expression levels of miR-142a-5p and miR-142a-3p showed significant upregulation in the spinal cords of EAE mice at days 15 and 25 post disease induction. Splenocytes stimulated with myelin oligodendrocyte glycoprotein (MOG) peptide or anti-CD3/anti-CD28 antibodies showed upregulation of miR-142a-5p and miR-142a-3p isoforms, whereas stimulated bone marrow-derived macrophages and primary astrocytes did not show any significant changes in miRNA expression levels. miR-142a-5p overexpression in activated lymphocytes shifted the pattern of T cell differentiation towards Th1 cells. Luciferase assays revealed SOCS1 and TGFBR1 as direct targets of miR-142a-5p and miR-142a-3p, respectively, and overexpression of miRNA mimic sequences suppressed the expression of these target transcripts in lymphocytes. SOCS1 levels were also diminished in MS white matter and EAE spinal cords.

Conclusions: Our findings suggest that increased expression of miR-142 isoforms might be involved in the pathogenesis of autoimmune neuroinflammation by influencing T cell differentiation, and this effect could be mediated by interaction of miR-142 isoforms with SOCS1 and TGFBR-1 transcripts.

Keywords: Experimental autoimmune encephalomyelitis; MicroRNA; Multiple sclerosis; Neuroinflammation; T cell differentiation.

Fig. 1

miR-142-3p and miR-142-5p levels in human brain tissue samples and EAE spinal cords. Expression of microRNAs was measured in CNS tissues by real-time RT-PCR. The level of miR-142-5p was significantly increased in human MS samples compared with non-MS controls (a) (n = 6, Mann–Whitney U test, *p ≤ 0.05). EAE was induced in C57BL6 mice and spinal cord tissues were extracted at three time points after disease induction (b). Expression levels of miR-142a-3p and miR-142a-5p were significantly increased in spinal cord during the peak of disease and post peak phases of EAE (c). Data are shown as mean ± SEM. Number of mice in each group = 10, *p < 0.05, **p < 0.01, Kruskal–Wallis tests

Fig. 2

miR-142a-3p and miR-142a-5p expression levels in splenocytes, macrophages, and astrocytes. Expression levels of miR-142a-3p and miR-142a-5p were analyzed in MOG-treated splenocytes from immunized animals (a, b) or splenocytes stimulated with anti-CD3 and anti-CD28 for different time points (c, d). Bone marrow-derived macrophages (e) and primary astrocytes (f) were stimulated with two concentrations of LPS for 12 h, before expression analysis by RT-PCR. Data are shown as mean ± SEM, n = 3. Experiment was repeated twice. *p < 0.05, **p < 0.01, Kruskal–Wallis test

Fig. 3

Overexpression of miR-142a-5p affects the differentiation of CD4⁺ T cells. Naïve CD4⁺ T cells were isolated from mouse splenocytes to a purity of approximately 95% (a). miR-142a-3p, miR-142a-5p, and negative control sequences were transfected into CD4⁺ T cells which were activated and polarized under relevant cytokine regimens. The frequencies of Th1, Th17, and Treg cells in CD4⁺ T cells were determined by intracellular staining and flow cytometry after 4 days. Representative dot plots and the percentages of IFN-γ, IL-17, and FoxP3 immunopositive cells within the CD4⁺ T cells (b). Average cell frequencies (c). Data are shown as mean ± SEM, n = 3. Data are from a single experiment representative of three independent experiments.(*p < 0.05, one-way ANOVA)

Fig. 4

Overexpression of miRNA sequences affects target gene expression in stimulated splenocytes. The expression of potential target genes was examined in cells transfected with miRNA sequences by real-time RT-PCR. TGFBR1 and ADCY9 transcripts were significantly suppressed in cells overexpressing miR-142a-3p compared with miRNA negative control transfected cells (a). SOCS1 and TGFBR2 transcripts were also significantly suppressed in cells overexpressing miR-142a-5p compared with miRNA negative control overexpressing cells (b). Data are shown as mean ± SEM, n = 3. Experiment was repeated twice. **p < 0.01, *p < 0.05, Student’s t test

Fig. 5

miR-142a-3p and miR-142a-5p predicted target genes expression in activated splenocytes. Expression of miR-142 isoforms target genes in stimulated splenocytes was determined by real-time RT-PCR. The levels of TGFBR1 and ADCY9 genes in 24, 48, and 72 h (a, b). TGFBR2 in 24 and 48 h after stimulation were significantly decreased in stimulated splenocytes compared with unstimulated cells (c). SOCS1 levels were initially upregulated followed by a significant reduction (d). Data are shown as mean ± SEM, n = 3. Experiment was repeated twice. *p < 0.05, one-way ANOVA)

Fig. 6

Luciferase-3′-untranslated region reporter assay. Interaction of miR-142a-3p with TGFBR1 and miR-142a-5p with TGFBR2 and SOCS1 transcripts were assessed using luciferase assay system. Co-transfection of HEK293T cells containing luciferase-3′UTR construct from TGFBR1 together with miR-142a-3p mimics showed significant suppression of luciferase activity in comparison with cells transfected with a control miRNA sequence (a). miR-142a-5p transfection did not lead to significant suppression of luciferase activity in cells co-transfected with TGFBR2 3′UTR constructs (b). Similar experiments with luciferase-3′UTR from SOCS1 and miR-142a-5p mimics showed suppression of luciferase activity compared with cells transfected a control sequence (c). Firefly luciferase levels were normalized against renilla luciferase, expressed as an internal control in the vector. Data are shown as mean ± SEM, n = 5. Data are from a single experiment representative of three independent experiments. *p < 0.05, **p < 0.01, Student’s t test

Fig. 7

miR-142a-3p and miR-142a-5p target gene expression in EAE tissue and human MS brain samples. miR-142a-3p target gene, TGFBR1, was significantly increased in peak of disease and post peak phase of EAE in spinal cord (a). SOCS1 and TGFBR2 were significantly decreased in peak of disease and post peak phases of EAE in comparison with pre-onset phase (b). In human autopsy samples, SOCS1 showed significant reduction in MS samples but the levels of TGFBR1 and TGFBR2 did not differ between MS and control tissues (c). Data are shown as mean ± SEM, **p < 0.01, *p < 0.05, one-way ANOVA