自噬通过抑制Ⅰ型干扰素产生负向调控抗病毒固有免疫反应
发布时间：2018-11-19 21:32
【摘要】：固有免疫(innate immunity)是机体抵抗病毒的第一道防线。当病毒入侵机体后,即被模式识别受体(pattern-recognition receptors,PRRs)识别,快速诱导固有免疫应答,产生大量的Ⅰ型干扰素(interferon,IFN)和促炎因子。Ⅰ型干扰素可以与胞膜上相应的受体结合,诱导一系列抗病毒蛋白高表达,进而抑制病毒的复制和感染,发挥强有力的抗病毒效应[1,2]。因此,Ⅰ型干扰素的表徶调控一直是抗病毒免疫研究领域的热点。自噬是一种发生在真核细胞内并且高度保守的自我稳态机制,对细胞抵抗外部刺激有非常重要的意义。病毒感染可以引起自噬的产生,但是目前关于自噬在病毒感染过程中所起的作用的研究很少,机制尚不明确。所以我们对自噬在抗病毒固有免疫应答中的作用机制进行了探究。我们构建了自噬相关基因7(Autophagy related gene 7,ATG7)在髓系细胞中特异性敲除的小鼠(Atg7f/f Lysm-Cre)。用VSV-GFP病毒感染小鼠的腹腔巨噬细胞,发现自噬缺陷之后小鼠的腹腔巨噬细胞对病毒的清除率增强,具有更强的抗感染能力。随后,我们给小鼠腹腔注射VSV病毒观察小鼠的存活率,得到了和体外实验一致的结果,自噬缺陷组小鼠的存活率明显升高,肺部组织炎性浸润增强,巨噬细胞、中性粒细胞富集明显增多,病毒复制减少,小鼠抗病毒能力明显增强。因为Ⅰ型干扰素在抗病毒固有免疫中发挥着重要作用,所以我们接着探究了自噬缺陷引起的这种抗病毒效应是否是因为影响了Ⅰ型干扰素的产生。所以我们用病毒感染小鼠的腹腔巨噬细胞,检测了对照组和自噬敲除组小鼠腹腔巨噬细胞Ⅰ型干扰素mRNA的表徶情况,发现病毒感染之后自噬缺陷组的Ⅰ型干扰素mRNA表达明显增强。证实了自噬缺陷确实影响了Ⅰ型干扰素的合成,从而增强了抗病毒效应。VSV病毒是一种dsRNA病毒,主要通过活化TLR3信号通路和RIG-Ⅰ信号通路来引起Ⅰ型干扰素的产生,随后我们对RNA病毒活化的信号通路进行了筛选和验证。我们用免疫印迹实验对不同信号通路主要的抗病毒蛋白的降解情况进行了检测,发现自噬缺陷之后TBK1的磷酸化水平增加,IRF3总蛋白和磷酸化水平都增加,而NF-κB的降解没有差异。同时还检测了 IRF3 mRNA水平,结果显示自噬缺陷后IRF3的合成亦增多。综上所述,我们发现自噬缺陷可以引起IRF3活化形式增多,促进Ⅰ型干扰素的产生,进而增强机体抗病毒能力。本研究揭示了自噬在抗病毒固有免疫中重要的负调控作用,为抗病毒感染免疫的调控机制提供新的观点,为病毒感染的临床治疗方法提供指导,为药物新靶点的设计提供了思路。
[Abstract]:Innate immune (innate immunity) is the body's first line of defense against the virus. When the virus invades the body, it is recognized by the pattern recognition receptor (pattern-recognition receptors,PRRs), which can quickly induce the innate immune response and produce a large number of interferon type I (interferon,). IFN) and pro-inflammatory factor.Interferon I can bind to the corresponding receptors on the cell membrane, induce a series of high expression of antiviral proteins, and then inhibit the replication and infection of the virus, and play a powerful antiviral effect [1t2]. Therefore, epidermal regulation of interferon type I has been a hot topic in the field of antiviral immunity. Autophagy is a highly conserved self-homeostasis mechanism occurring in eukaryotic cells, which plays an important role in the resistance of cells to external stimuli. Virus infection can induce autophagy, but there is little research on the role of autophagy in the process of virus infection, and the mechanism is unclear. So we explored the mechanism of autophagy in anti-viral innate immune response. We constructed a mouse (Atg7f/f Lysm-Cre) with specific knockout of autophagy associated gene 7 (Autophagy related gene 7 (ATG7) in myeloid cells. The peritoneal macrophages of mice infected with VSV-GFP virus were found to have stronger anti-infection ability and the clearance rate of peritoneal macrophages was enhanced after autophagy deficiency. Then, we injected VSV virus intraperitoneally into mice to observe the survival rate of mice. The results were consistent with in vitro experiment. The survival rate of mice in autophagy deficiency group was significantly increased, the inflammatory infiltration of lung tissue was enhanced, and macrophages were also observed. The concentration of neutrophil increased, the replication of virus decreased, and the anti-viral ability of mice increased obviously. Because interferon type I plays an important role in the innate antiviral immunity, we then explore whether the antiviral effect of autophagy deficiency is due to the influence of interferon type I production. So we used the virus to infect the peritoneal macrophages of mice, and we detected the expression of interferon type I mRNA in the peritoneal macrophages of the control group and the mice in the autophagy knockout group. It was found that the expression of interferon type I mRNA in autophagy deficiency group was significantly increased after viral infection. It is confirmed that autophagy deficiency does affect the synthesis of interferon type 鈪，
本文编号：2343482
[Abstract]:Innate immune (innate immunity) is the body's first line of defense against the virus. When the virus invades the body, it is recognized by the pattern recognition receptor (pattern-recognition receptors,PRRs), which can quickly induce the innate immune response and produce a large number of interferon type I (interferon,). IFN) and pro-inflammatory factor.Interferon I can bind to the corresponding receptors on the cell membrane, induce a series of high expression of antiviral proteins, and then inhibit the replication and infection of the virus, and play a powerful antiviral effect [1t2]. Therefore, epidermal regulation of interferon type I has been a hot topic in the field of antiviral immunity. Autophagy is a highly conserved self-homeostasis mechanism occurring in eukaryotic cells, which plays an important role in the resistance of cells to external stimuli. Virus infection can induce autophagy, but there is little research on the role of autophagy in the process of virus infection, and the mechanism is unclear. So we explored the mechanism of autophagy in anti-viral innate immune response. We constructed a mouse (Atg7f/f Lysm-Cre) with specific knockout of autophagy associated gene 7 (Autophagy related gene 7 (ATG7) in myeloid cells. The peritoneal macrophages of mice infected with VSV-GFP virus were found to have stronger anti-infection ability and the clearance rate of peritoneal macrophages was enhanced after autophagy deficiency. Then, we injected VSV virus intraperitoneally into mice to observe the survival rate of mice. The results were consistent with in vitro experiment. The survival rate of mice in autophagy deficiency group was significantly increased, the inflammatory infiltration of lung tissue was enhanced, and macrophages were also observed. The concentration of neutrophil increased, the replication of virus decreased, and the anti-viral ability of mice increased obviously. Because interferon type I plays an important role in the innate antiviral immunity, we then explore whether the antiviral effect of autophagy deficiency is due to the influence of interferon type I production. So we used the virus to infect the peritoneal macrophages of mice, and we detected the expression of interferon type I mRNA in the peritoneal macrophages of the control group and the mice in the autophagy knockout group. It was found that the expression of interferon type I mRNA in autophagy deficiency group was significantly increased after viral infection. It is confirmed that autophagy deficiency does affect the synthesis of interferon type 鈪，
本文编号：2343482
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