Autophagy in Antiviral Immunity

描述(申请人提供)：自噬是一种古老的进化保守的途径，旨在通过降解细胞质中的长寿命蛋白质和细胞器来维持细胞内的动态平衡。它也被用作饥饿条件下的一种生存机制。最近的研究表明，自噬被先天和获得性免疫系统的细胞用来对抗病毒感染。对病毒的先天识别通过两条不同的途径发生。在专业的病毒传感器，浆细胞样树突状细胞(PDC)中，病毒的识别是通过Toll样受体(TLR)7和9在内体中进行的。我们最近的工作表明，自噬在通过TLR7识别PDCs中病毒感染的特征方面发挥了关键作用。与pDC不同的是，大多数身体其他类型的细胞通过RIG-I样受体(RLR)家族利用细胞内病毒复制的传感器。参与自噬的分子已被证明可以阻断RLR信号。此外，最近的报道表明，自噬将内源性病毒抗原运送到MHC II类负荷室，允许激活CD4T细胞。然而，这些途径在体内病毒感染过程中的相关性尚不清楚。在这个应用中，我们提供了初步的数据，揭示了ATG5，一个形成自噬小体所需的关键分子，在通过TLR9传递信号的过程中，导致在单纯疱疹病毒(HSV；TLR9激动剂)感染时pDC中I型干扰素基因的激活。此外，我们还发现，在水泡性口炎病毒感染(VSV；RIG-I激动剂)时，自噬负向调节非浆细胞样树突状细胞中的RLR通路。最后，我们在体内证明了自噬在处理和呈递HSV-1感染时树突状细胞中MHC II类不同形式的抗原中的关键作用。在这些初步研究的基础上，我们建议使用各种分子和细胞生物学技术并使用已建立的病毒感染小鼠模型来检查自噬在先天和获得性免疫反应中的重要性。在第一个目标中，我们将利用分子和细胞生物学技术，确定ATG5和/或自噬通过TLR9在pDC中介导HSV感染的信号转导机制。在第二个目标中，我们建议通过蛋白质组学和生化方法来确定自噬如何调节VSV感染时RNA传感器的激活。在最终目的中，我们将询问树突状细胞如何利用自噬在体外和体内处理和呈递细胞外病毒抗原，在树突状细胞种群内选择性地缺乏自噬的小鼠中。通过提供对自噬如何协调产生针对病毒感染的先天和获得性免疫的基本了解，这些研究将有助于建立重要的基础，以用于设计针对各种病毒病原体的疫苗和抗感染措施。 与公共卫生相关：虽然自噬是一种古老的进化保守的途径，旨在通过降解细胞质中长寿命的蛋白质和细胞器来维持细胞的动态平衡，但我们团队和其他人最近的研究揭示了自噬在免疫系统中的作用。在这一应用中，我们建议使用成熟的遗传、生化和细胞生物学工具以及RNA和DNA病毒感染的活体动物模型来检验自噬在先天和获得性抗病毒免疫反应中的重要性。从拟议的研究中获得的理解不仅将为免疫系统如何利用自噬提供科学进展，而且还将有助于为设计免疫干预措施和预防各种病毒疾病的措施奠定关键基础。

DESCRIPTION (provided by applicant): Autophagy is an ancient evolutionarily conserved pathway designed to maintain cellular homeostasis by degrading long-lived proteins and organelles in the cytosol. It is also used as a survival mechanism under starvation conditions. Recent studies demonstrated that autophagy is utilized by the cells of the innate and adaptive immune systems to combat viral infections. Innate recognition of viruses occurs via two distinct pathways. In professional viral sensors, the plasmacytoid dendritic cells (pDC), recognition of viruses occurs in the endosomes via Toll-like receptors (TLR) 7 and 9. Our recent work has demonstrated that autophagy plays a key role in recognizing signatures of viral infection in pDCs through TLR7. In contrast to pDCs, most other cell types of the body utilize cytosolic sensors of viral replication via RIG-I like receptor (RLR) family. Molecules involved in autophagy have been shown to block RLR signaling. In addition, recent reports indicate that autophagy delivers endogenous viral antigens to the MHC class II loading compartment, allowing activation of CD4 T cells. However, the relevance of such pathways during in vivo virus infection is unknown. In this application, we present preliminary data that reveal the requirement for Atg5, a key molecule required for formation of autophagosomes, in the transduction of signaling through TLR9 leading to the activation of type I IFN genes in pDCs upon herpes simplex virus (HSV; TLR9 agonist) infection. In addition, we show that autophagy negatively regulates RLR pathway in non-plasmacytoid dendritic cells upon vesicular stomatitis virus infection (VSV; RIG-I agonist). Finally, we demonstrate a key in vivo role for autophagy in the processing and presentation of various forms of antigens on MHC class II in dendritic cells upon HSV-1 infection. Building on these preliminary studies, we propose to examine the importance of autophagy in both innate and adaptive immune responses using a variety of molecular and cell biological techniques and using established mouse models of virus infection. In the first Aim, we will determine the mechanism by which Atg5 and/or autophagy mediates signaling through TLR9 upon HSV infection in pDCs through the use of molecular and cellular biological techniques. In the second Aim, we propose to determine how autophagy regulates RNA sensor activation upon VSV infection through proteomics and biochemical approaches. In the final Aim, we will interrogate how dendritic cells utilize autophagy for processing and presentation of extracellular viral antigens in vitro and in vivo in mice selectively deficient in autophagy within the dendritic cell populations. By providing basic understanding of how autophagy orchestrates the generation of innate and adaptive immunity against virus infections, these studies will help to establish important foundation with which to design vaccines and anti-infective measures against a variety of viral pathogens PUBLIC HEALTH RELEVANCE: While autophagy is an ancient evolutionarily conserved pathway designed to maintain cellular homeostasis by degrading long-lived proteins and organelles in the cytosol, recent studies from our group and others have revealed the role of autophagy in the immune system. In this application, we propose to examine the importance of autophagy in both innate and adaptive antiviral immune responses using well-established genetic, biochemical and cell biological tools as well as in vivo animal models of both RNA and DNA virus infections. The understanding gained from the proposed studies will not only provide scientific advances in how autophagy is utilized by the immune system, but also to help establish critical foundation with which to design immunological interventions and preventative measures against a wide variety of viral diseases.