Study On The Regulatory Mechanism Of MicrorRNA MiR-20b In Th17Cell Differentiation And Experimental Autoimmune Encephalomyelitis Pathogenesis

Interleukin17(IL-17)-producing T helper (Th17) cells have been recognized as a third subset of effector CD4+T cells, which characterized critical pathological roles in multiple sclerosis (MS). Experimental autoimmune encephalomyelitis (EAE), the most widely used animal model for human MS, is a CD4+T cell-mediated inflammation of the central nervous system associated with demyelination and infiltration of inflammatory Thl and Th17cells. Recently, miRNAs have been shown to play an important role in the context of Th17-driven autoimmunity, such as human MS and EAE. In two studies, miR-20b has been found as a down-regulated miRNA in blood cells of MS patients. However, the role of miR-20b on Th17differentiation and its regulatory function on EAE have not been explored.In current study, miR-20b was found to be significantly down-regulated in CD4+T cells from EAE mice, and in the in vitro differentiation model, Th17cells had lower expression of miR-20b than did Thl, Th2or inducible Treg cells. Based on these results, we predicted that miR-20b might function to ongoing inflammation in EAE and Th17cell differentiation. Because miRNAs function by targeting gene regulation, we then searched for potential targets of miR-20b relative to Th17differentiation.We used TargetScanMouse to predict the candidate target genes of miR-20b. RORyt and STAT3were identified and confirmed as the potential target of miR-20b by Luciferase assay, GFP repression experiments and immunoblot analysis. We also found that miR-20b down-regulate the target proteins through different mechanisms, by mRNA degradation to STAT3and by inhibition of translation to RORyt.Considering that RORyt and STAT3participate in Th17differentiation, we then sought to determine if miR-20b directly regulated the differentiation program of Th17cells. We then found IL-17A were significantly down-regulated in those from mimics-transfected cells. In contrast, IL-17A was slight higher in those from inhibitor-transfected cells. Consistently, the expression of Th17-lineage marker genes (IL-17A and IL-17F) and IL-17A in culture supernatants were also significantly down-regulated by miR-20b mimics. We further used miR-20b NC or mimics labeled with FAM fluorescence to confirmed the above results. Our results also demonstrate that miR-20b negatively regulates Th17differentiation in vitro without influencing the T cells proliferation or apoptosis.To further investigate our hypothesis that miR-20b may function to ongoing inflammation in EAE, we constructed lentiviral vectors for miR-20b manipulation in vivo. On day7post viruses delivery, the mice were immunized with MOG peptide35-55, LV-miR-20b-infection led to milder EAE. Histological analysis of spinal cord sections also showed that amounts of infiltration and demyelination were lower in the LV-miR-20b-infected mice. And the mice were also injected through the tail vein with LV-miR-20b or LV-ctrl at day14post immunization (disease onset), the data shown that LV-miR-20b treatment at the time point of diseae onset ameliorated the disease severity of EAE. These results suggest that miR-20b plays a suppressive role in the development of EAE.We further tested the impact of miR-20b on the generation of Th17cells in EAE. We found fewer CD4+T cells accumulated in the CNS of LV-miR-20b-infected mice. Intracellular cytokine staining showed that the frequency of Th17in LV-miR-20b-infected mice was notably lower in the CD4+population. We also found fewer Th17cells in DLNs of LV-miR-20b mice, and the Th17responses in DLNs cells restimulated with MOG peptide35-55in vitro from LV-miR-20b-infected mice were lower. Our results indicate that miR-20b suppresses the in vivo generation of Th17cells during EAE.Finally, we found RORyt and STAT3protein was down-regulated in LV-miR-20b-infected mice. We further confirmed that miR-20b suppressed EAE development and Th17cell generation in vivo by targeting RORyt and STAT3.Taken together, our findings contribute to a growing body of data that show the critical role of miRNAs in Th17differentiation and the pathogenesis of multiple sclerosis and EAE. Perhaps miR-20b will be applied as a prognostic biomarker or a therapeutic biotarget in the future.