Subversion of gastrointestinal host defenses by norovirus

描述（由申请人提供）：为了成功感染宿主，通过胃肠道进入的病原体必须克服肠道防御的多层系统，包括物理屏障（如粘液、紧密连接）和免疫反应（如分泌IgA）。微折叠细胞（Microfold cells， M）是肠上皮中吸收颗粒抗原或siga免疫复合物的特化细胞，在建立对肠道病原体的免疫应答中发挥重要作用。由于病毒在M细胞中可见，它们也可能为病原体进入肠道提供门户，但缺乏直接的实验证据证明M细胞是启动生产性感染所必需的。更好地了解病原体与肠道和相关淋巴细胞炎的相互作用，为早期干预提供了途径，但没有诺如病毒（NoVs）的信息。新病毒是肠道病毒，以特定物种的方式感染肠道，并通过粪-口途径传播。小鼠NoVs （mnv）是生物医学研究群体中最普遍的病毒，具有改变研究结果的潜力。与不可培养的人类NoVs （HuNoVs）相比，MNV在组织培养和小动物宿主中复制，并且可以进行基因修饰。本地鼠宿主也提供遗传易感性和广泛的可用资源。因此，MNV系统提供了一个多功能系统来研究肠道病毒与上皮屏障的相互作用，并能够解决肠道病毒发病机制中的一个基本问题：肠道病毒如何克服宿主的肠道屏障来建立生产感染？初步数据表明，在体外，在没有病毒复制或破坏上皮紧密连接的情况下，MNV通过细胞内转运穿过分化的小鼠肠上皮细胞单层。在M细胞诱导条件下或病毒粒子结合自然（非特异性）分泌IgA后，MNV转运增加。这些数据是中心假设的基础：MNV利用胃肠道宿主防御，利用自然分泌的IgA和M细胞穿过上皮屏障，进入允许的巨噬细胞和树突状细胞。这一假设将通过以下具体目标进行验证：1)确定MNV是否利用M细胞在其原生小鼠宿主中建立生产性感染；2)确定天然sIgA是否会增强MNV在小鼠胃肠道的感染。这些目标将通过利用体内（如M细胞缺陷，IgA-/-小鼠）和体外（如结扎回肠环）系统来实现。了解NoV如何突破肠上皮屏障以建立生产性感染将增加对肠道宿主-病原体相互作用基本原理的理解。本文揭示的原理可以稍后测试其对HuNoV和其他肠道病原体的适用性。这项研究具有创新性，因为它首次通过实验测试了肠道M细胞在生产性感染过程中是否需要，并且它可以首次证明病毒颠覆天然sIgA热防御途径以增强病毒感染。

DESCRIPTION (provided by applicant): To successfully infect their host, pathogens that enter via the gastrointestinal tract must overcome the multi- layered system of intestinal defenses including physical barriers (e.g., mucus, tight junctions) and immune responses (e.g., secretory IgA). Microfold (M) cells, specialized cells in the intestinal epithelium that take up particulate antigens or sIgA-immune complexes, play an important role in the establishment of immune responses to enteric pathogens. Since viruses were visualized in M cells, they may also provide portals for pathogen entry into the gut, but direct experimental evidence that M cells are required for initiating a productive infection is lacking. A better understanding of the interactio of pathogens with the intestine and associated lymphoid tis- sue offers avenues for early intervention, but no information is available for noroviruses (NoVs). NoVs are enteric viruses that infect the intestinal tract in a species-specific manner and are spread by the fecal-oral route. Murine NoVs (MNVs) are the most prevalent viruses in biomedical research colonies, with the potential to alter research outcomes. In contrast to the non-cultivatable human NoVs (HuNoVs), MNV replicates in tissue culture and a small animal host and can be genetically modified. The native murine host also offers genetic tractability and a wide array of available resources. Thus, the MNV system provides a versatile system to study the inter- action of an enteric virus with the epithelial barrier and enables addressing a fundamental question in enteric virus pathogenesis: how do enteric viruses overcome the intestinal barrier of the host to establish a productive infection? Preliminary data demonstrate that in vitro, MNV crosses a differentiated murine intestinal epithelial cell monolayer by intracellular transport in the absenc of viral replication or disruption of the epithelial tight junctions. MNV transport is increased uner M cell-inducing conditions or after virions bind to natural (non- specific) secretory IgA. These data are the foundation of the central hypothesis: MNV exploits gastrointestinal host defenses by using natural secretory IgA and M cells to cross the epithelial barrier and gain access to permissive macrophages and dendritic cells. This hypothesis will be tested by pursuing the fol- lowing specific aims: 1) Determine whether MNV exploits M cells to establish a productive infection in its native murine host, and 2) Determine whether natural sIgA enhances MNV infection of the murine gastrointestinal tract. These objectives will be achieved by exploiting bot in vivo (e.g. M cell deficient, IgA-/- mice) and ex vivo (i.e. ligated ileal loops) systems. Knowledge of how a NoV breaches the intestinal epithelial barrier to establish a productive infection will increase the understanding of fundamental principles during host-pathogen interac tion in the intestine. Principles uncovered herein can later be tested for their applicability to HuNoV and other enteric pathogens. The proposed research is innovative because it experimentally tests for the first time whether intestinal M cells are required during productive infections, and it could provide the first demonstration of viral subversion of the natural sIgA hot defense pathway to enhance viral infections.