登革热病毒抗体依赖增强感染分子机制的体外模型研究

发布时间：2018-03-31 03:03

本文选题：登革病毒　切入点：抗体依赖性增强感染　出处：《北京协和医学院》2015年博士论文

【摘要】：登革热病毒(Dengue virus, DENV)是一种蚊媒病毒,在超过100多个国家中,尤其是亚洲和拉丁美洲,引起了巨大的公共卫生问题。据估计,每年有超过5千万至1亿人感染登革热病毒。随着其宿主埃及伊蚊和白纹伊蚊栖息地的扩张,全球面临越来越严重的登革感染风险。四型登革病毒中每一型单独感染均能导致一系列程度不一的临床症状,从自限性的登革热(DF)至危及生命的登革出血热(DHF)或登革休克综合症(DSS)等重症形式。基于临床观察,二次异型感染或登革患者胎儿随年龄增长均显著增加重症感染几率,抗体依赖增强假说(ADE)被提出以解释登革病毒重症感染病理机制。该理论认为,在二次异型登革病毒感染中预先存在的交叉抗体能够与病毒结合,通过抗体与靶细胞表面Fc受体的相互作用促进病毒对单核细胞,巨噬细胞,以及成熟DC细胞的感染。目前研究认为DENV-ADE能够通过抑制Ⅰ型干扰素产生及效应进而促进病毒增殖；其中抗炎症细胞因子,如IL-10等对干扰素途径的抑制作用在DENV-ADE感染中扮演的重要角色。然而有研究证明DENV-ADE感染的人初始单核细胞并不抑制干扰素或IL-10表达。这些研究显示DENV-ADE感染Fc受体阳性细胞存在一种更为普遍的机制,且不依赖于上调IL-10对一型干扰素抑制过程。在本研究中,我们使用Ⅰ型干扰素缺陷细胞K562建立了DENV3抗体依赖增感染体外模型,并体现出内部增强感染形式。通过检测抗病毒基因NOS2表达水平,发现DENV-ADE感染细胞中降低的NOS2表达及NO活性分子释放导致了病毒胞内复制增加。进一步分析NOS2基因上游调控途径,发现DENV-ADE感染中下调的NOS2基因表达源于固有免疫RIG-I/MDA-5—NF-κB—IRF-1信号途径抑制。过表达RIG-I和/或MDA-5能够协同促进NF-κB活化及NOS2表达。为验证免疫抑制因子IL-10在DENV-ADE固有免疫抑制中的作用,我们检测了IL-10/IL-6—SOCS3通路,发现DENV-ADE感染K562细胞未显著上调IL-10, IL-6, SOCS3表达。利用CRISP/CAS9技术获得IL-10敲出的K562细胞进行DENV-ADE模型表明,IL-10并不影响DENV-ADE感染。由于细胞自噬过程在DENV复制中扮演重要作用,我们比较了DENV直接感染与DENV-ADE感染中自噬程度差异。研究发现,DENV-ADE感染K562细胞诱导更高水平的自噬体形成及自噬相关蛋白ATG5, ATG12表达。通过药物促进或抑制细胞自噬进程能够剂量依赖性地促进或抑制胞内病毒复制,且完全敲除自噬相关蛋白ATG5显著抑制胞内病毒复制。结果表明,DENV-ADE感染通过诱导更强的自噬进而促进胞内病毒增殖。此外,过表达自噬蛋白ATG5能够通过抑制NF-κB活化及NOS2表达从而促进病毒增殖。本研究发现DENV-ADE感染中病毒复制增强是由RIG-I/MDA-5—NF-κB— IRF-1—NOS2固有免疫途径抑制导致的,且该抑制过程不依赖于IL-10—SOCS3对干扰素途径的抑制。此外DENV-ADE感染能够通过上调细胞自噬进程,抑制固有免疫途径,从而促进病毒增殖。该研究将有助于加深我们对DENV-ADE感染机制的理解,并为重症登革患者治疗提供了理论支持和潜在靶点,同时也表明自噬抑制剂在治疗DENV-ADE引起的重症登革热疾病,以及抑制病毒胞内复制方面具有潜在的应用价值。
[Abstract]:Dengue virus (Dengue virus DENV) is a mosquito borne virus, in more than more than 100 countries, especially in Asia and Latin America, causing a major public health problem. It is estimated that there are more than 50 million to 100 million people infected with dengue virus each year. With its host Egypt and Iraq mosquito Aedes albopictus habitat expansion the world, facing increasingly serious risk of dengue infections. Dengue virus type four in each type of single infection can cause a series of clinical symptoms in different degree, from self limited dengue fever (DF) to life-threatening dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS) in form. Based on clinical observation, two patients with dengue infection or abnormal fetal age were significantly increased in severe infection, antibody dependent enhancement hypothesis (ADE) is proposed to explain the dengue virus infection. The pathological mechanism of theory in the two different Dengue virus infection cross pre-existing antibodies can bind to the virus and antibody through interaction with the target cell surface Fc receptor on monocyte macrophage to promote viral infection, and, mature DC cells. The present study suggests that DENV-ADE can inhibit type I interferon production and effect and promote the proliferation of virus; anti inflammation cytokines, such as inhibition of IL-10 on interferon pathway in DENV-ADE infection plays important role. However, studies have shown that the initial DENV-ADE infection of monocytes did not inhibit interferon or IL-10 expression. These studies showed the presence of a common infection mechanism of DENV-ADE Fc receptor positive cells, and does not depend on the up regulation of IL-10 inhibit the process of a type of interferon. In this study, we use the type I interferon deficient cells K562 established DENV3 antibody dependent enhancement of infection. Outside the model, and reflect the internal form of infection. Enhanced expression levels by detection of antiviral gene NOS2, found that the virus has led to increased release of intracellular replication molecule NOS2 expression cells decreased and the activity of NO DENV-ADE infection. Further analysis of NOS2 gene upstream regulatory pathways, found that DENV-ADE infection in down-regulation of NOS2 gene expression in innate immune RIG-I/MDA-5 NF- B - kappa IRF-1 signaling pathway inhibition. Overexpression of RIG-I and / or MDA-5 can synergistically promote NOS2 activation and expression of NF- kappa B. In order to verify the immunosuppressive factor IL-10 in innate immune suppression in DENV-ADE, we examined the IL-10/IL-6 SOCS3 pathway, found DENV-ADE infected K562 cells did not significantly up-regulated the expression of IL-10, IL-6, SOCS3 expression get out of IL-10. By using the technology of CRISP/CAS9 K562 cell DENV-ADE model showed that IL-10 did not affect DENV-ADE infection due to autophagy in DENV. Play an important role in replication, we compared the autophagy of DENV direct infection and DENV-ADE infection in difference. The study found that autophagy induced higher levels of DENV-ADE infected K562 cells and the formation of autophagy related protein ATG5, ATG12 expression. The drugs promote or inhibit autophagy process could dose dependently promote or inhibit virus replication in cells completely, and knockdown of autophagy related protein ATG5 significantly inhibited the virus replication in cells. The results showed that DENV-ADE infection induced by stronger autophagy and promote the proliferation of virus in the cells. Moreover, overexpression of autophagy protein ATG5 can inhibit NF- B activation and NOS2 expression to promote proliferation of virus. This study found that DENV-ADE infection enhanced virus replication is caused by RIG-I/MDA-5 - NF- - IRF-1 - NOS2 - B innate immune pathways inhibition, and the inhibition process does not depend on the IL-10 - SOCS3 of interferon The way to inhibit DENV-ADE infection. In addition to the upregulation of cell autophagy process, inhibition of innate immune pathways, thereby promoting proliferation of virus. The study will help to deepen our understanding of the mechanism of DENV-ADE infection, and to provide theoretical support and a potential target for treatment of patients with severe leather also shows that, in the treatment of DENV-ADE induced autophagy inhibitors the severe dengue disease has potential application value and inhibit viral intracellular replication.

【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R392

【共引文献】