| Multiple sclerosis(MS) and its animal model, experimental autoimmune encephalomyelitis(EAE), are autoimmune diseases of the central nervous system(CNS) which mediated by both Th1 and Th17 cells. It is thought that, in the initial stage of MS, myelin-reactive CD4+ T cells activated in the periphery infiltrate into the CNS, followed closely by a broad range of other immune cells, including T cells, macrophages, dendritic cells(DCs), B cells, and neutrophils. Once in the CNS, myelin-reactive T cells become reactivated and produce pro-inflammatory mediators, which induce chemokine secretion by CNS resident cells, thus recruiting more inflammatory cells from the periphery and triggering an immunologic cascade that results in myelin damage. While it appears that both IFN-γ-producing Th1 and IL-17-producing Th17 cells play important roles in EAE pathogenesis, the mechanisms by which autoreactive helper T cells initiate the disease process remain only partially understood. Although the pathophysiology of MS/EAE remains unclear, many immune regulators or immune suppressors have been identified for MS treatment, of which, IFN-β is the first FDA-approved medicine and has been widely used for MS treatment. However, where endogenous IFN-β comes from and how it involved in MS treatment remains unknown.Central nervous system in situ immune surrounding, immune regulation and the responsiveness of CNS cells to inflammation play important roles in disease development. Thus, identifying how lesion tissues responsive to neuroimmunology and neuroinflammation are key steps for uncovering pathophysiology mechanisms and will help to identifying novel therapeutic targets. Previously, based on the specific characters of mi RNAs—multiple targets and the numerous reports about theirs important roles in development and many diseases, we focused on the CNS lesion enriched mi RNAs and tried to explore the role of such mi RNAs in MS/EAE. In this work, the expression pattern of mi RNAs in EAE lesion was characterized. Further, in the view of epigenetics, we try to identify the role of CNS lesion enriched mi RNA-mi R-146 a in MS/EAE. The results are as follows:(1) In the 200 μg MOG35-55 immunized EAE model, the mice were sacrificed and perfused at peak disease(d 20 after immunization), the spinal cord lesions were cut and used for mouse mi RNAs expression PCR array analysis. The results showed that the expression of mi R-146a、mi R-147、mi R-155、mi R-223、mi R-509-3p were greatly up-regulated, while the expression of mi R-124, mi R-132 and mi R-338 were significantly decreased during EAE. We focused on mi R-146 a, a greatly up-regulated mi RNA in the lesion of EAE mouse, and its expression was confirmed by mi RNA in situ hybridization analysis and got the similar results. Further, the primary astrocytes and microglia cells were purified and treated with proinflammatory cytokines IL-17, TNF-α, and IL-17 plus TNF-α, the results showed that proinlammatory cytokines stimulation induced the expression of mi R-146 a by both astrocytes and microglia cells and IL-17 and TNF-α have the synergistic effects in mi R-146 a induction.(2) A lentiviral vector harboring mi R-146 a sponge were constructed to inhibit the expression of mi R-146 a and named LV-mi R-146 a inhibitor. The virus were packaged and tittered. The virus were then concentrated and diluted as 5×108 IU/ml by PBS for future use. The EAE model was constructed by immunized with 200 μg MOG35-55 in 8-12 week-old C57BL/6 female mice. The 20 μl LV-mi R-16 a inhibitor lentivirus and the control virus were separately i.c.v. injected into the EAE mice at disease onset(d 14 after immunization) or at the peak disease(d 20 after immunization). The clinical score was record daily after MOG immunization and the results showed that inhibiting the expression of mi R-146 a decreased the disease severity in the groups either the mi R-146 a inhibitor was given at disease onset or at peak disease. In addition, the mi R-146 a inhibitor administration decreased CNS inflammatory infiltration and demyelination as assayed by H&E staining, Fast blue staining. Further, immunohistochemical staining showed that inhibited the expression of mi R-146 a I CNS decreased the number of white blood cells, macrophages, microglia cells, and neutrophils in the CNS lesions, while i.c.v. administration of mi R-146 a did not affect the peripheral immune system as reflected by Th1/Th17 population, MOG specific T cells proliferation and MOG induced cytokines secretion by splenic mononuclear cells. Also mi R-146 a administration decreased astrogliosis n EAE mice.(3) We found at different stages of EAE, the lesion expression of mi R-146 a was negative correlated with TRAF6 and IFN-β. Bioinformatics analysis showed that the 3’-UTR of TRAF6 gene had two mi R-146 a binding sites(localized at 447-468 and 506-532). In the in vitro cultured astrocytes, mi R-146 a transfection significantly inhibited the expression of TRAF6 in a dose-dependent manner. Dual luciferase reporter experiment showed that in the CNS lesion of EAE, TRAF6 was the direct target of mi R-146 a. Further in the in vivo experiment, inhibiting mi R-146 a plus blocking the expression of TRAF6 did not affect disease severity and solely overexpresson of TRAF6 in CNS decreased the disease severity to some extent. The results showed that in the CNS lesion of EAE, mi R-146 a functions at least to some extent through regulating the expression of TRAF6 and its downstream type 1 interferon signaling.The above results showed that CNS lesion restricted mi R-146 a involves in the pathogenesis of EAE through regulating the expression of TRAF6 and it downsream type 1 interferon signaling. Inhibiting CNS enriched mi R-146 a could significantly relief disease severity of EAE. These works will help to uncover the pathophysiological mechanisms of MS/EAE and identify novel therapeutic target for disease treatment. |
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