Dissecting the mechanisms of intestinal epithelial injury by Ebola virus using iPSC-derived intestinal organoids

项目摘要 丝状病毒，包括埃博拉病毒和马尔堡病毒，是人畜共患病病毒的主要例子，导致严重的 人类的疾病。最具致病性的埃博拉病毒种类是埃博拉病毒(扎伊尔埃博拉病毒；EBOV)，病例 死亡率从40%到90%不等。许多死于埃博拉病毒的患者被送往接受埃博拉治疗 当他们已经病得很重的时候。重要的是，没有可用的治疗方法来缓解晚期 埃博拉病毒病(EVD)。虽然EBOV致病的许多方面已经在细胞中得到了广泛的研究 培养系统和动物模型，肠道在EVD中的参与还完全不清楚，尽管 腹泻是最常见的症状之一(在一些案例研究中，78%的患者)，并且是 死亡的主要原因。胃肠道症状，包括呕吐、腹痛和腹泻， 也是马尔堡病毒(Marv)病的常见症状。目前还没有可用的感染模型 这使得研究肠道丝状病毒感染的后果成为可能。为了填补这一空白，我们建议建立 人类肠道感染平台剖析丝状病毒引起损伤的分子机制 肠道器官。我们将探索两种可能在病理生理学中发挥作用的潜在机制。 丝状病毒感染引起的效应：I)丝状病毒感染人肠上皮细胞导致屏障丧失 正直。2)人肠道上皮细胞的丝状病毒感染可调节离子转运体的功能。 在丝状病毒感染中识别导致严重腹泻的机制是有可能的 告知迫切需要的治疗方法以缓解晚期严重的肠道症状 丝状病毒病。 人类诱导多能干细胞(IPSCs)能够无限期自我更新，并有可能 分化为任何组织特异性细胞系，包括人类肠道器官(HIO)。为…做准备 在这个项目中，我们成功地实现了IPSC来源的HIO对EBOV和MARV的强大感染。这个 被感染的细胞显示出细胞损伤的迹象，转录组学分析表明细胞连接的调节 途径和一组已知的离子转运体在腹泻的诱导中发挥作用。开始探索 肠道细胞对丝病毒感染的固有宿主反应及其对细胞损伤的影响 在屏障完整性方面，我们制定了以下具体目标： 具体目的1：研究EBOV和MARV感染对肠道的病理生理影响 上皮完整性。 具体目标2：通过基因消融验证单个基因的作用并测试潜在的药物 作为肠上皮功能调节剂的候选药物。

Project Summary Filoviruses, including ebola- and marburgviruses, are prime examples of zoonotic viruses that cause severe disease in humans. The most pathogenic ebolavirus species is Ebola virus (Zaire ebolavirus; EBOV) with case fatality rates ranging from 40 to 90%. Many patients who succumb to the disease are admitted to Ebola treatment units when they are already severely ill. Importantly, there are no therapeutics available to mitigate late-stage Ebola Virus Disease (EVD). While many aspects of EBOV pathogenesis have been extensively studied in cell culture systems and animal models, the involvement of the intestine in EVD is not at all understood, despite diarrhea being among the most frequent symptoms (78% of patients in some case studies) and being one of the main causes for demise. Gastrointestinal manifestations, including vomiting, abdominal pain, and diarrhea, are also common symptoms of Marburg virus (MARV) disease. There are currently no infection models available that allow to study the consequences of filovirus infection of the gut. To fill this gap, we propose to establish human intestinal infection platforms to dissect the molecular mechanisms underlying filovirus-induced damage of the intestinal organs. We will explore two potential mechanisms that might play a role in the pathophysiological effects induced by filovirus infection: i) Filovirus infection of the human intestinal epithelium leads to loss of barrier integrity. ii) Filovirus infection of the human intestinal epithelium modulates the function of ion transporters. Identifying mechanisms that contribute to the induction of severe diarrhea in filovirus infection has the potential to inform urgently needed therapeutic approaches to mitigate the severe intestinal symptoms in late-stage filovirus disease. Human induced pluripotent stem cells (iPSCs) are capable of indefinite self-renewal and have the potential to differentiate into any tissue-specific cell lineage, including human intestinal organoids (HIOs). In preparation for this project, we have successfully achieved robust EBOV and MARV infections of iPSC-derived HIOs. The infected cells showed signs of cell damage, and transcriptomics analysis indicated the modulation of cell junction pathways and a set of ion transporters known to play a role in the induction of diarrhea. To begin to explore the intrinsic host response of intestinal cells to filovirus infection and the impact of infection-induced cellular damage on barrier integrity, we have designed the following specific aims: Specific Aim 1: To study the pathophysiological effects of EBOV and MARV infection on intestinal epithelial integrity. Specific Aim 2: To validate the role of individual genes by genetic ablation and test potential drug candidates as modulators of intestinal epithelial function.