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

使用 iPSC 衍生的肠道类器官剖析埃博拉病毒造成的肠上皮损伤机制

• 批准号: 10538716
• 负责人: Gustavo Mostoslavsky
• 金额: $ 27.38万
• 依托单位: BOSTON MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-07-05 至 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10538716
• 关键词: Abdominal Pain; Ablation; Affect; Africa; Animal Model; Area; Bacteremia; Bacteria; CDX2 gene; Case Fatality Rates; Case Study; Cell Culture System; Cell Lineage; Cell model; Cells; Cessation of life; Characteristics; Clustered Regularly Interspaced Short Palindromic Repeats; Complication; Defect; Development; Diarrhea; Disease; Disease Outbreaks; Disease Progression; Disease model; Distal; Ebola; Ebola Hemorrhagic Fever; Ebola virus; Emergency Situation; Endotoxins; Enterocytes; Epithelial; Epithelial Cells; Fever; Filoviridae Infections; Filovirus; Functional disorder; Genes; Genetic; Goals; Gold; Human; Hypovolemic Shock; Immune; Immune response; In Vitro; Individual; Infection; Inflammatory Response; Intercellular Junctions; Intestines; Ions; Liquid substance; Marburg Virus Disease; Marburgvirus; Mediating; Modeling; Molecular; Monoclonal Antibodies; Organ; Organoids; Pathogenesis; Pathogenicity; Pathology; Pathway interactions; Patients; Play; Positioning Attribute; Preparation; Proteins; Reporting; Role; Sampling; Septicemia; Symptoms; Testing; Therapeutic; Therapeutic Intervention; Therapeutic Monoclonal Antibodies; Tight Junctions; Tissues; Vaccines; Viral; Viral Pathogenesis; Virus; Virus Diseases; Vomiting; Zaire Ebola virus; Zoonoses; base; cell injury; common symptom; design; drug candidate; enteric infection; epithelial injury; gastrointestinal; gastrointestinal symptom; induced pluripotent stem cell; intestinal barrier; intestinal epithelium; macrophage; nonhuman primate; novel; regeneration model; self-renewal; stem cell differentiation; tissue regeneration; transcriptomics

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.

项目概要 丝状病毒，包括埃博拉病毒和马尔堡病毒，是人畜共患病毒的主要例子，可导致严重的 人类疾病。致病性最强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。许多死于该病的患者接受埃博拉治疗 当他们已经病情严重时。重要的是，没有可用于缓解晚期症状的治疗方法 埃博拉病毒病（EVD）。虽然埃博拉病毒发病机制的许多方面已在细胞中得到了广泛研究 尽管在培养系统和动物模型中，肠道在埃博拉病毒病中的参与尚不清楚 腹泻是最常见的症状之一（在某些案例研究中，腹泻为 78% 的患者），并且是最常见的症状之一 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）疾病的常见症状。目前没有可用的感染模型 可以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。我们将探索可能在病理生理学中发挥作用的两种潜在机制 丝状病毒感染引起的影响： i) 丝状病毒感染人类肠上皮导致屏障丧失 正直。 ii) 人类肠上皮的丝状病毒感染调节离子转运蛋白的功能。 确定丝状病毒感染引起严重腹泻的机制具有潜力 为缓解晚期严重肠道症状提供迫切需要的治疗方法 丝状病毒病。 人类诱导多能干细胞 (iPSC) 能够无限期地自我更新，并具有潜力 分化成任何组织特异性细胞谱系，包括人肠类器官（HIO）。准备中 在这个项目中，我们成功实现了 iPSC 衍生的 HIO 的强大 EBOV 和 MARV 感染。这 受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 已知在诱导腹泻中发挥作用的途径和一组离子转运蛋白。开始探索 肠道细胞对丝状病毒感染的内在宿主反应以及感染引起的细胞损伤的影响 关于屏障完整性，我们设计了以下具体目标： 具体目标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药物 作为肠上皮功能调节剂的候选者。