MicroRNA-181 Variants Regulate T Cell Phenotype in the Context of Autoimmune Neuroinflammation.

BACKGROUND: Recent studies have revealed that multiple sclerosis (MS) lesions have distinct microRNA (miRNA) expression profiles. miR-181 family members show altered expression in MS tissues although their participation in MS pathogenesis remains uncertain. Herein, we investigated the involvement of miR-181a and miR-181b in the pathogenesis of MS and its animal model, experimental autoimmune encephalomyelitis (EAE). METHODS: miR-181a and -b levels were measured in the central nervous system (CNS) of patients with MS and mice with EAE as well as relevant leukocyte cultures by real-time RT-PCR. To examine the role of the miRNAs in leukocyte differentiation and function, miR-181a and -b mimic sequences were transfected into cultured primary macrophages and purified CD4+ T cells which were then analyzed by RT-PCR and flow cytometry. Luciferase reporter assays were performed to investigate the interaction of miR-181a and -b with the 3'-UTR of potential target transcripts, and the expression of target genes was measured in the CNS of EAE mice, activated lymphocytes, and macrophages. RESULTS: Expression analyses revealed a significant decrease in miR-181a and -b levels in brain white matter from MS patients as well as in spinal cords of EAE mice during the acute and chronic phases of disease. Suppression of miR-181a was observed following antigen-specific or polyclonal activation of lymphocytes as well as in macrophages following LPS treatment. Overexpression of miR-181a and -b mimic sequences reduced proinflammatory gene expression in macrophages and polarization toward M1 phenotype. miR-181a and -b mimic sequences inhibited Th1 generation in CD4+ T cells and miR-181a mimic sequences also promoted Treg differentiation. Luciferase assays revealed Suppressor of mothers against decapentaplegic 7 (Smad7), as a direct target of miR-181a and -b. CONCLUSION: Our data highlight the anti-inflammatory actions of miR-181a and -b in the context of autoimmune neuroinflammation. miR-181a and -b influence differentiation of T helper cell and activation of macrophages, providing potential therapeutic options for controlling inflammation in MS.

miR-181 interacts with signaling adaptor molecule DENN/MADD and enhances TNF-induced cell death.

MicroRNAs are small noncoding RNAs, which regulate the expression of protein coding transcripts through mRNA degradation or translational inhibition. Numerous reports have highlighted the role of miRNAs in regulating cell death pathways including the expression of genes involved in the induction of apoptosis. Tumor necrosis factor alpha (TNF-α) is a proinflammatory cytokine which can send pro-death signals through its receptor TNFR1. Diverse adaptor molecules including DENN/MADD adaptor protein have been shown to modulate TNF-α pro-death signaling via recruitment of MAP kinases to TNFR1 and activation of pro-survival NFκB signaling. Herein, we investigated the role of microRNA-181 (miR-181) in regulating DENN/MADD expression levels and its subsequent effects on TNF-α-induced cell death. Using bioinformatics analyses followed by luciferase reporter assays we showed that miR-181 interacts with the 3' UTR of DENN/MADD transcripts. miR-181 overexpression also led to decreased endogenous DENN/MADD mRNA levels in L929 murine fibroblasts. Flow cytometric analysis of miR-181 transfected cells showed this miRNA accentuates mitochondrial membrane potential loss caused by TNF-α. These findings were associated with enhanced apoptosis of L929 cells following TNF-α treatment. Overall, these data point to the potential role of miR-181 in regulating TNF-α pro-death signaling, which could be of importance from pathogenesis and therapeutic perspectives in inflammatory disorders associated with tissue degeneration and cell death.

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

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.