Study On The Mechanism Of Central Nervous System Cell - Specific IFN - γ Signaling Pathway In Experimental Autoimmune Encephalomyelitis

Multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE), are autoimmune diseases of the central nervous system (CNS) which mediated by both Thl and Th17 cells. This disease usually affects young adults and it is one of the key reasons for neurological disability of young adults. IFN-y, the hallmark cytokine of the Thl cells, plays an important role in EAE. Thus far, the role of IFN-y in EAE has been largely studied through its effects on immune cells and whereas much less is known about its effects on CNS cells, especially in vivo. Thus in this study, we specifically block IFN-y signaling in astrocytes or microglia cells to test the function and the mechanisms of cell specific IFN-y signal pathway in the MOG35-55 immunization induced EAE model. Our study will help to define novel mechanisms of action of IFN-y in EAE pathogenesis, and also highlight an opportunity for the development of multiple sclerosis therapies directed at CNS cells. The main results are as follows:1. Immunofluorescent double staining showed that IFN-yRl is expressed in astrocytes, microglia and oligodendrocytes, but not in neurons;2. Immunofluorescent staining and real-time quantitative RT-PCR experiments showed that the expression of IFN-yRl was induced both in brain and spinal cord during EAE development, suggested that the CNS restricted IFN-y signaling may involved in EAE pathogenesis;3. We constructed miR-30 based shRNA cassette targeting IFN-yRl which driven by cell-type specific promoter GFAP or CD11b. Both in vitro and in vivo experiments showed that the lentiviral vector could effectively knock down the expression of IFN-yRl in astrocytes or microglia cells;4. IFN-y signaling in astrocytes and microglia has opposite roles in EAE development as assayed by clinical evaluation. Blocking IFN-y signaling in astrocytes ameliorated EAE, while silencing the signal pathway in microglia aggravated the disease;5. H&E staining, the spinal cord purified mononuclear cell (MNC) counting and FACS staining analysis showed that locking IFN-y signaling in astrocytes inhibited CNS inflammatory cells infiltration and reduced demyelination;6. In the ex vivo experiments, silencing IFN-γR1 in astrocytes decreased the expression of proinflammatory cytokine IL-6 and IL-17A, meanwhile, the expression of the chemokines in spinal cord including CXCL2, CCL20, CXCL12, MMP9 were decreased as well. In the in vitro cultured primary astrocytes, knocking down the expression of IFN-γR1 decreased the expression of IL-17 responsive chemokines including CXCL1, CXCL2 and CCL20 as stimulated by IL-17 or IL-17 plus TNF, and it also suppressed the expression of IFN-y-responsive chemokines, such as CXCL9, CXCL10 and CXCL11, as stimulated by IFN-y or IFN-y plus TNF. The chemoattract experiment showed that IFN-γR1I deficient astrocytes attracted less inflammatory cells when stimulated by IFN-y plus TNF;7. Blocking IFN-y signaling in micorglia cells promoted CNS inflammatory cells infiltration and expanded the microglia/macrophages numbers in the spinal cord. The H&E staining, the spinal cord purified mononuclear cell (MNC) counting and FACS staining analysis showed that microglia specific silencing IFN-y signaling enhanced immune cells infiltration in brain stem and spinal cord. Real-time quantitative RT-PCR analysis showed that interfering IFN-y signaling in microglia had little effect on chemokines expression in CNS tissues, but enhanced the expression of IFN-y while decreased IL-10 expression. Immunofluorescent staining and FACS analysis showed that blocking IFN-y signaling in microglia cells expanded microglia/macrophages numbers in CNS;8. The in vitro experiment showed that IFN-y or IFN-y plus TNF treatment significantly restricted microglia cells numbers and this effect was not observed in IFN-yRI deficient microglia cells. The real-time quantitative RT-PCR analysis exhibited IFN-y treatment could induce the expression of inflammation cytokines and chemokines, but the most striking role IFN-y signaling is restricting microglia cell numbers. Further analysis showed that IFN-y treatment could increase the expression of p21 and decrease the expression of c-myc in microglia, while IFN-y has no effect on the expression of these two genes in astrocytes, suggesting IFN-y signaling in microglia may control the cells numbers through affecting cell cycles. The caspase staining and LDH analysis also supported that this restricted cell number was not duing to IFN-y induced cell apoptosis or cell death;9. Silencing IFN-y signaling in astrocytes both at disease onset or at the peak of disease ameliorated EAE. Further, blocking IFN-y signaling in astrocytes alleviated both Th1- and Thl7-mediated adoptive EAE, indicating an important role for IFN-y signaling in astrocytes in autoimmune CNS inflammation.The above results indicated CNS cell type-specific inflammatory signaling may have different roles in CNS inflammatory diseases. Blocking IFN-y signaling in astrocytes alleviates EAE by decreasing chemokine expression, whereas silencing IFN-y signaling in microglia, and likely in some infiltrating macrophages, promotes disease severity via enhanced proliferation of these cells. The results obtained in this study will not only help us understand the complex role of IFN-y in the development of EAE, but will also provide a promising CNS-specific therapeutic approach for MS/EAE treatment by blocking the encephalitogenicity of both myelin-reactive Thl and Th17 cells, the two major pathogenic mechanisms in CNS autoimmunity.