Using the GoFlowChip to understand SARS-CoV-2 infection of the gastrointestinal mucosa of humans and bats

摘要 新出现的严重急性呼吸综合征冠状病毒2(SARS-CoV-2)是一种高度传染性的 人类呼吸道病原体，有能力感染许多不同的器官系统，包括 胃肠道(GI)。这个项目的总体目标是阐明胃肠道粘膜在SARS中的作用。 冠状病毒2型的感染和传播。具体地说，我们试图确定涉及到的粘膜免疫机制 新冠肺炎致病机制和比较人类和蝙蝠肠道对SARS-CoV-2的上皮反应。 研究蝙蝠细胞很重要，因为蝙蝠被认为是SARS-CoV-2的原始宿主，但不是 一旦感染就会发展为显性疾病。我们最近开发的GOFlowChip平台，集成了3- 微流控芯片上的三维GI有机物和免疫细胞，具有管腔和基底侧向流动能力，是 非常适合于探讨SARS-CoV-2感染肠道粘膜的机制。在这里，我们 建议利用GOFlowChip阐明病毒感染、复制和传播的机制 在胃肠道的SARS-CoV-2感染。具体地说，我们试图(1)定义SARS-CoV-2感染是如何 胃肠道上皮有助于病毒的传播。(2)确定胃肠道黏膜免疫机制的程度 调节SARS-CoV-2在胃肠道的感染和传播。(3)比较胃肠道上皮对SARS的反应- 蝙蝠和人类之间的CoV-2病毒。针对具体目标1，我们将优化GOFlowChip以在BSL-3下使用 实验室条件，随后进行SARS-CoV-2感染实验，以阐明易感性和病毒 不同肠段的复制动力学。特殊目标2将利用我们的有机单核 巨噬细胞(MNP)共培养系统阐明MNP依赖的转运机制是否影响病毒 胃肠道粘膜的侵袭，我们将筛选SARS-CoV-2反应性患者血清的调节能力 肠道感染。为了达到特定的目的3，我们将建立蝙蝠胃肠道组织的类器系，以便进行比较 用RNA测序了解蝙蝠和人对SARS-CoV-2的上皮细胞应答 人类和蝙蝠之间病毒致病性的差异。建议的工作直接与我们的 现有项目，因为我们将使用GOFlowChip来研究SARS-CoV-2如何与上皮细胞相互作用 细胞和免疫系统组件穿过胃肠道屏障。我们的研究在技术上具有创新性， 因为我们在完全包含的组织芯片设计中使用肠道器官-免疫细胞共培养来研究 BSL-3级病原菌。我们的项目在概念上是创新的，因为它是第一个比较肠道有机化合物的项目 从牙买加果蝠到人类有机化合物，它们抵御SARS-CoV-2感染的能力和 细胞抗病毒反应。这项拟议的研究意义重大，因为它将产生关于 胃肠道粘膜在新冠肺炎发病机制中的作用，可能为未来识别病毒携带者的策略提供参考。 防止病毒传播，设计新的抗病毒治疗和疫苗。

Summary The emerging severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a highly infectious human respiratory tract pathogen that has the capacity to infect many different organ systems, including the gastrointestinal (GI) tract. The overall goal of this project is to elucidate the role of the GI mucosa in SARS- CoV-2 infection and transmission. Specifically, we seek to identify mucosal immune mechanisms involved in COVID-19 pathogenesis and compare epithelial responses to SARS-CoV-2 in the gut of humans and bats. Investigating bat cells is important, because bats are considered the original hosts for SARS-CoV-2, but do not develop overt disease upon infection. Our recently developed GOFlowChip platform, which integrates 3- dimensional GI organoids and immune cells on a millifluidic chip with luminal and basolateral flow capacity, is ideally suited to probe the mechanisms involved in SARS-CoV-2 infection of the gut mucosa. Here, we propose to utilize the GOFlowChip to elucidate mechanisms of viral infection, replication and spread involved in SARS-CoV-2 infection of the GI tract. Specifically, we seek to (1) Define how SARS-CoV-2 infection of the GI epithelium contributes to viral spread. (2) Determine to what extent GI mucosal immune mechanisms regulate SARS-CoV-2 infection and spreading in the GI tract. (3) Compare GI epithelial responses to SARS- CoV-2 between bats and humans. For Specific Aim 1, we will optimize the GOFlowChip for use under BSL-3 laboratory conditions, followed by SARS-CoV-2 infection experiments that will elucidate susceptibility and viral replication dynamics in different gut compartments. Specific Aim 2 will leverage our organoid-mononuclear phagocyte (MNP) co-culture system to elucidate whether MNP-dependent transport mechanisms impact viral invasion of the GI mucosa, and we will screen SARS-CoV-2 reactive patient sera for their ability to modulate gut infection. For Specific Aim 3, we will establish organoid lines from bat GI tissues in order to compare epithelial responses to SARS-CoV-2 in bats and humans using RNA sequencing in order to understand differences in viral pathogenicity between humans and bats. The proposed work is directly integrated with our existing project, because we will use out GOFlowChip to investigate how SARS-CoV-2 interacts with epithelial cells and immune system components to cross the GI barrier. Our research is technologically innovative, because we are using gut organoid-immune cell co-cultures in a fully contained tissue chip design to study a BSL-3 level pathogen. Our project is conceptually innovative, because it is the first to compare gut organoids from Jamaican fruit bats to human organoids for their ability to sustain SARS-CoV-2 infection and to mount cellular antiviral responses. The proposed research is significant because it will yield crucial information on the role of the GI mucosa in COVID-19 pathogenesis that may inform future strategies to identify viral carriers, prevent viral transmission, and design novel antiviral treatments and vaccines.