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，是引起严重感染的人畜共患病毒的主要例子。 人类的疾病。最致病的埃博拉病毒物种是埃博拉病毒（扎伊尔埃博拉病毒; EBOV）， 死亡率从40%到90%不等。许多死于该疾病的患者入院接受埃博拉治疗 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减轻晚期 埃博拉病毒病（EVD）。虽然EBOV发病机制的许多方面已经在细胞中进行了广泛的研究， 尽管在培养系统和动物模型中，肠在EVD中的参与完全不被理解， 腹泻是最常见的症状之一（在一些病例研究中占患者的78%）， 死亡的主要原因。胃肠道表现，包括呕吐、腹痛和腹泻， 也是马尔堡病毒（MARV）病的常见症状。目前没有可用的感染模型 从而研究肠道丝状病毒感染的后果。为了填补这一空白，我们建议设立 人类肠道感染平台，以剖析丝状病毒诱导损伤的分子机制 肠道器官。我们将探讨可能在病理生理学中发挥作用的两种潜在机制。 由丝状病毒感染诱导的作用：i）丝状病毒感染人肠上皮导致屏障丧失 完整ii）人肠上皮的丝状病毒感染调节离子转运蛋白的功能。 确定丝状病毒感染中导致严重腹泻的机制具有潜在的 为缓解晚期严重肠道症状提供急需的治疗方法 丝状病毒病 人类诱导多能干细胞（iPSC）能够无限自我更新，并具有向人类分化的潜力。 分化成任何组织特异性细胞谱系，包括人类肠类器官（HIO）。为筹备 在该项目中，我们成功实现了iPSC衍生HIO的EBOV和MARV强有力感染。的 感染的细胞表现出细胞损伤的迹象，转录组学分析表明细胞连接的调节 途径和一组已知在诱导腹泻中起作用的离子转运蛋白。为了开始探索 肠道细胞对丝状病毒感染的内在宿主反应和感染诱导的细胞损伤的影响 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学影响， 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药物 作为肠上皮功能调节剂的候选物。

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.