中枢神经系统细胞特异性IFN-γ信号通路在实验性自身免疫性脑脊髓炎中的作用机制研究

【作者】 丁小丽；

【导师】 黄原； 张光先；

【作者基本信息】 陕西师范大学 ， 动物学， 2015， 博士

【摘要】 多发性硬化症(Multiple sclerosis, MS)是一种由1型辅助T细胞(Thl)和17型辅助T细胞(Thl7)介导的中枢神经系统自身免疫性炎性脱髓鞘疾病。该病主要累及青壮年,是造成青壮年神经系统功能性残障的最主要因素之一。实验性自身免疫性脑脊髓炎(Experimental autoimmune encephalomyelitis, EAE)是为了研究多发性硬化症(Multiple sclerosis, MS)而建立的动物模型,对该动物模型的研究表明,Thl细胞的特征性细胞因子IFN-y通过作用于免疫细胞在EAE的疾病发生发展中起着重要的作用,但是其如何作用于中枢神经系统(central nervous system; CNS)的细胞,尤其是在体内情况下其作用效果研究的较少。因此,本研究利用髓鞘少突胶质糖蛋白(Myelin oligodendrocyte glycoprotein, MOG)免疫诱导的EAE动物模型,通过侧脑室注射特异性敲低星形胶质细胞(Astrocyte)或小胶质细胞(microglia)IFN-γ受体(IFN-γR1)表达的方法阻断不同CNS细胞IFN-y信号通路,研究CNS不同细胞特异性IFN-y信号通路在EAE中的作用及其作用机制,以期为更好的理解MS/EAE的病程机制并寻找新的细胞特异性靶点奠定基础。所取得的主要研究结果如下：1.利用免疫荧光双染技术发现,在小鼠脊髓切片中,IFN-γR1在星形胶质细胞、小胶质细胞以及少突胶质细胞中均有表达,神经元中不表达IFN-yr1;2.利用免疫荧光染色及实时荧光定量PCR技术发现在MOG免疫诱导的EAE模型的脊髓和脑组织中,相较于健康小鼠,IFN-γR1呈诱导表达,提示CNS的IFN-γ信号通路可能参与了EAE的病程发生;3.构建基于miR-30的星型胶质细胞或小胶质细胞／单核巨噬细胞特异性IFN-γR1敲低的慢病毒载体,体内体外实验验证,该慢病毒可以有效的敲低IFN-γR1的表达；4.EAE临床神经功能评分提示星形胶质细胞或小胶质细胞特异性沉默IFN-γ信号通路在EAE诱导发病过程中具有截然相反的作用,星形胶质细胞特异性阻断IFN-γ信号通路会减轻疾病的严重程度,然而,小胶质细胞特异性阻断该信号通路则会加重疾病病情；5.通过H&E染色,脊髓单个核细胞计数以及流式细胞分析发现,星型胶质细胞特异性阻断IFN-γ信号通路减少了中枢神经系统的炎性细胞浸润,从而使得脱髓鞘程度减轻；6.体内实验发现,阻断星形胶质细胞降低了脊髓中致炎性细胞因子IL-6和IL-17A的表达,同时,趋化因子CXCL2、CXCL12、CCL20和MMP9等的表达显著降低。体外星形胶质细胞原代培养实验发现,敲低IFN-γR1之后,星型胶质细胞在IL-17以及IL-17和TNF刺激下,相应IL-17信号通路的趋化因子CXCL1和CXCL2以及CCL20的表达显著降低,而在IFN-γ或IFN-γ和TNF的刺激下,相应IFN-γ信号通路的化学趋化因子CXCL9、CXCL10以及CXCL11的表达显著下降。化学趋化实验证实,IFN-γR1缺失的星形胶质细胞招募或趋化单个核细胞的能力显著下降；7.阻断小胶质细胞的IFN-γ信号通路促进了中枢的炎性浸润和小胶质细胞的增殖。H&E染色、脊髓单个核细胞计数以及流式细胞分析发现小胶质细胞特异性阻断IFN-γ信号通路增加了脑干和脊髓的炎性细胞浸润。实时荧光定量PCR显示,小胶质细胞特异性阻断IFN-γ信号通路对CNS趋化因子表达的影响不明显,IFN-γ的表达量显著降低,而抗炎性细胞因子IL-10的表达量显著降低。免疫荧光染色以及流式细胞分析显示小胶质细胞特异性阻断IFN-γ信号显著增加了CNS小胶质细胞／单核巨噬细胞的数量。8.利用体外培养的原代小胶质细胞发现,IFN-γ处理显著降低了小胶质细胞的数量,而IFN-γ处理对IFN-γR1缺失的小胶质细胞的数量则没有影响。实时荧光定量PCR实验发现,IFN-γ能够促进小胶质细胞趋化因子以及致炎性细胞因子的表达,但其更显著的作用表现在小胶质细胞IFN-γ信号通路能显著控制或抑制该类细胞的数量。进一步分析发现相较于星形胶质细胞,IFN-γ或IFN-γ结合TNF的处理可以显著提高p21的表达并抑制cmyc的表达,提示小胶质细胞上的IFN-γ信号通路通过影响细胞周期来控制细胞的数量。同时caspase免疫荧光染色以及LDH活性测定表明IFN-γ信号通路影响小胶质细胞的数量不是通过促进细胞凋亡或坏死来实现的；9.在发病初期或发病高峰期特异性阻断星形胶质细胞IFN-γ信号通路可以有效缓解Thl细胞和Th17细胞介导的EAE疾病的严重程度。上述结果表明中枢神经系统不同细胞同一信号通路可能在疾病中起着不同的病理作用。星形胶质细胞的IFN-γ信号通路通过影响趋化因子的表达来影响疾病发展,而小胶质细胞的该信号通路则通过影响控制小胶质细胞的数量来影响疾病进程。该研究结果对于进一步理解MS/EAE的病程机制具有重要意义,也有利于发现新的细胞特异性的治疗靶点。更多还原

【Abstract】 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.更多还原