The role of tuft cells in norovirus pathogenesis

项目摘要 人类诺沃克病毒是全球急性胃肠炎的主要原因，每年造成多达20万人死亡 年。然而，目前还没有抗病毒药物或疫苗。我们使用的是亲缘关系密切的小鼠或小鼠诺如病毒 (MNV)作为了解人类诺沃克病毒如何导致感染和疾病的模式系统。更广泛地说，我们 也使用MNV作为工具来揭示病毒如何与宿主免疫相互作用的新的和基本的方面 系统和其他肠道微生物，包括细菌和蠕虫。 我们最近发现CD300lf是MNV的受体。我们利用这一发现将簇状细胞鉴定为 MNV在小鼠肠道中的靶细胞。簇状细胞是一种罕见的感觉和启动免疫的上皮细胞 对肠道蠕虫的反应。丛生细胞在调节肠道炎症、创伤等方面也很重要 康复，和几个粘膜癌。 在初步数据中，我们开发了新的簇状细胞缺陷小鼠和CD300lf条件性基因敲除 和敲入小鼠，研究丛状细胞在急性和慢性诺沃克病毒感染中的作用。W我们也 开发了一种MNV簇状细胞体外培养系统，使我们能够剖析MNV簇状细胞之间复杂的相互作用 诺如病毒、丛生细胞和免疫系统。这项提案的目标是确定 丛状细胞在诺如病毒感染中的作用，以确定丛状细胞感染的免疫学后果，以及 发现感染的簇状细胞如何逃避CD8+T细胞的杀伤。 在目标1中，我们将确定病毒感染是否损害簇状细胞功能。我们将确定Tuft的角色 急性和持续性MNV感染中的细胞。我们将确定MNV在慢性感染期间是如何杀死丛生细胞的， 病毒感染是如何干扰丛生细胞转录组的，以及病毒感染是否会损害丛生细胞的转录能力 保护宿主免受蠕虫感染的细胞。 在目标2中，我们将确定丛生细胞作为免疫缺陷小生境的机制。 为MNV。在慢性感染期间，MNV特异性CD8+T细胞忽略感染的簇状细胞，但它们仍具有功能 并且能够在体外检测抗原。我们将通过以下方式确定簇状细胞趋向性如何使人对CD8+T细胞产生抵抗 结合新的小鼠品系、病毒遗传学、簇状细胞和T细胞共培养以及过继转移两种簇状细胞 MNV特异性CD8+T细胞。特别是，我们将确定静止的长寿命簇状细胞，一种簇状细胞亚型， 是一种治疗慢性感染的新型免疫保护剂。 我们期望这项工作将产生对细胞嗜性调节诺沃克病毒的详细了解。 传播，病毒感染在II型免疫中的作用，以及诺沃克病毒逃避免疫的机制 CD8+T细胞。这对我们破译人类诺沃克病毒机制的长期目标至关重要。 诺如病毒的致病机制和研制成功的人用诺如病毒疫苗。

Project Summary Human norovirus is the leading cause of acute gastroenteritis globally causing up to 200,000 deaths per year. However, there are no antiviral drugs or vaccines. We use the closely related mouse or murine norovirus (MNV) as a model system to understand how human norovirus causes infection and disease. More broadly, we also use MNV as a tool to uncover novel and fundamental aspects of how viruses interact with the host immune system and other intestinal microbes including bacteria and worms. We recently discovered CD300lf as a receptor of MNV. We leveraged this finding to identify tuft cells as a target cell of MNV in the mouse intestines. Tuft cells are rare epithelial cells that sense and initiate an immune response against intestinal worms. Tuft cells are also important in regulating intestinal inflammation, wound recovery, and several mucosal cancers. In preliminary data, we developed novel tuft cell deficient mice and both CD300lf conditional knockout and knock-in mice to study the role of tuft cells during acute and chronic norovirus infection. W We also developed an in vitro MNV tuft cell culture system that will enable us to dissect the complex interactions between norovirus, tuft cells, and the immune system. The objectives of this proposal are to determine the role of tuft cells in norovirus infection, to determine the immunological consequences of tuft cell infection, and to discover how infected tuft cells evade CD8+ T cell killing. In Aim 1, we will determine if viral infection impairs tuft cell function. We will identify the role of tuft cells in acute and persistent MNV infection. We will determine how MNV kills tuft cells during chronic infection, how infection perturbs the tuft cell transcriptome, and whether virus infection of tuft cells impairs the ability of tuft cells to protect the host against worm infection. In Aim 2, we will determine the mechanism by which tuft cells act as an immunoprivileged niche for MNV. MNV-specific CD8+ T cells ignore infected tuft cells during chronic infection yet they remain functional and able to detect antigen ex vivo. We will identify how tuft cell tropism enables resistance to CD8+ T cells by combining novel mouse lines, viral genetics, tuft cell and T cell co-culture, and adoptive transfer of both tuft cell and MNV-specific CD8+ T cells. In particular, we will determine if quiescent long-lived tuft cells, a tuft cell subtype, are a novel immunoprivileged reservoir for chronic infection. We anticipate this work will generate a detailed understanding about the cell tropism regulating norovirus transmission, the role of viral infection in type II immunity, and the mechanism by which norovirus evades immune CD8+ T cells. This is critical to our long-term goal of deciphering mechanisms of human norovirus pathogenesis and developing a successful human norovirus vaccine.