Subversion of gastrointestinal host defenses by norovirus

描述（由申请人提供）：为了成功感染其宿主，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（例如，粘液，紧密连接）和免疫应答（例如，分泌型伊加）。微折叠（M）细胞是肠上皮中的特化细胞，其摄取颗粒抗原或sIgA-免疫复合物，在建立对肠道病原体的免疫应答中起重要作用。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表明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组织的相互作用为早期干预提供了途径，但没有关于诺如病毒（NoV）的信息。NoV是以物种特异性方式感染肠道并通过粪-口途径传播的肠道病毒。小鼠NoV（MNV）是生物医学研究群体中最常见的病毒，具有改变研究结果的潜力。与不可培养的人NoV（HuNoV）相反，MNV在组织培养物和小动物宿主中复制，并且可以被遗传修饰。天然鼠宿主还提供遗传易处理性和广泛的可用资源。因此，MNV系统提供了研究肠道病毒与上皮屏障的相互作用的通用系统，并且能够解决肠道病毒发病机制中的基本问题：肠道病毒如何克服宿主的肠道屏障以建立生产性感染。初步数据表明，在体外，MNV通过细胞内运输穿过分化的小鼠肠上皮细胞单层，在没有病毒复制或上皮紧密连接破坏的情况下。在M细胞诱导条件下或在病毒粒子与天然（非特异性）分泌伊加结合后，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，通过使用天然分泌型伊加和M细胞穿过上皮屏障，并获得进入许可的巨噬细胞和树突状细胞。将通过追求以下具体目标来检验该假设：1）确定MNV是否利用M细胞在其天然鼠宿主中建立生产性感染，和2）确定天然sIgA是否增强鼠胃肠道的MNV感染。这些目的将通过利用体内（例如M细胞缺陷型、伊加-/-小鼠）和离体（即连接的回肠环）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中宿主-病原体相互作用期间的基本原理的理解。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在生产性感染期间是否需要肠道M细胞，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.