Intestinal organoid modeling of SARS-CoV-2-stimulated innate and adaptive immunity

SARS-CoV-2 刺激的先天性和适应性免疫的肠道类器官模型

基本信息

批准号：
10319858
负责人：
CALVIN J KUO
金额：
$ 39.36万
依托单位：
STANFORD UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-07-15 至 2024-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10319858
关键词：
2019-nCoV 3-Dimensional ACE2 Acute Air Antibodies Antiviral Response Apical B-Cell Antigen Receptor B-Lymphocytes Bacterial Infections Biological Models Biopsy COVID-19 mortality COVID-19 pandemic COVID-19 patient Cell Communication Cells Clinical Coculture Techniques Collaborations Colon Common Cold Communication Complex Convalescence Coronavirus Coronavirus Infections Data Diarrhea Epithelial Epithelial Cells Event Fostering Gluten Human Immune Immune checkpoint inhibitor Immune response Immune system Immunity Immunization Immunize Immunological Models In Vitro Incubators Infection Inflammation Innate Immune Response Intervention Intestines Investigation Liquid substance Lung Lung diseases Lung infections Malignant Neoplasms Medicine Methods Modeling Myelogenous Myeloid Cells Natural Immunity Nature Nodal Organ Organoids Pathogenesis Pathology Patients Peripheral Pharmacology Plasma Public Health Receptor Cell Reporting Respiratory Failure SARS-CoV-2 infection SARS-CoV-2 pathogenesis Serology Small Intestines Specificity Surface Surveys Suspension Culture Suspensions Symptoms System T-Cell Receptor T-Lymphocyte Technology Testing Therapeutic Time Tissue Model Tissue Preservation Tissues Tonsil Transcript Tumor-infiltrating immune cells Type 2 Angiotensin II Receptor Variant Virus Diseases Virus Replication Vomiting adaptive immune response adaptive immunity cell type co-infection cross reactivity cytokine release syndrome enteric infection global health health economics immunoregulation in vitro Model intestinal epithelium lymph nodes mesenteric lymph node network models novel novel coronavirus novel therapeutics reconstitution response single cell analysis single-cell RNA sequencing systemic inflammatory response therapeutic development therapeutic evaluation three dimensional cell culture tumor

项目摘要

ABSTRACT The COVID-19 pandemic, engendered by the novel coronavirus SARS-CoV-2, is a grave threat to public health, with lung infection and respiratory failure. However, the intestine is also targeted by SARS-CoV-2, as many patients present with GI symptoms and the SARS-CoV-2 receptor angiotensin-converting enzyme 2 (ACE2) is abundantly expressed by intestinal epithelium. COVID-19 mortality is strongly associated with systemic inflammation as in “cytokine storm”, fostering attempts at therapeutic immunomodulation, and raising a critical need for in vitro human systems modeling SARS-CoV-2-induced immune cell interactions with intestinal epithelium. Conventional 3D organoid cultures (“enteroids”) allow intestinal epithelial SARS-CoV-2 infection but unfortunately omit immune cells. Here we generate a holistic intestinal in vitro model of SARS-CoV-2 infection using air-liquid interface (ALI) organoids containing both epithelium and infiltrating immune cells en bloc without artificial reconstitution. The complex intestinal immune system of ALI intestinal organoids contains various innate and adaptive immune cells, highly diverse T cell receptor (TCR) and B cell receptor (BCR) repertoire, and plasma B cell-derived antibody transcripts. Importantly, immune components in these ALI organoids respond efficiently to epithelial damage and ALI intestinal organoids are highly susceptible to bacterial and viral infections. Here, we utilize this unique ALI organoid technology with integrated immune components to explore sequelae of SARS-CoV-2 infection. Aim 1 establishes and optimizes BSL3 SARS-CoV-2 infection of ALI intestinal organoids, exploiting a novel eversion method to relocate the apical aspect of ACE2-expressing cells to the external surface, allowing survey of SARS-CoV-2 infection of different regions of small intestine and colon. The time course of tissue-resident SARS-CoV-2-induced cross-talk between intestinal epithelium and immune cells is unknown, as hindered by lack of human experimental systems. Thus, Aim 2 performs a scRNA- seq and CyTOF study of SARS-CoV-2-induced immune responses within ALI organoids to (1) create a network model of the temporal propagation of immunity and bidirectional communication between epithelium and immune cells and (2) perform therapeutic testing against nodal vulnerabilities, correlating against clinical status (naïve, convalescent, immunized, cross-reactive coronavirus). Current epithelial organoid systems also do not allow study of adaptive immunity. Aim 3 thus recapitulates SARS-CoV-2 adaptive immune responses by co-culturing ALI intestinal organoids with newly developed human lymph node organoids. Within such SARS-CoV-2-infected co-cultures we correlate adaptive immune responses with clinical status including prior cross-reactive coronavirus infection and SARS-CoV-2 naïve, convalescent and ultimately immunized states. Overall, we leverage collaboration from Mark Davis (human LN culture) and Catherine Blish and Scott Boyd (SARS-CoV-2) to create human in vitro systems modeling SARS-CoV-2-induced innate and adaptive immunity, with relevance for pathogenesis investigations and therapeutics testing.

抽象的由新颖的冠状病毒SARS-COV-2引起的Covid-19-19大流行是对公共卫生的严重威胁，肺部感染和呼吸衰竭。但是，肠子也受到SARS-COV-2的瞄准，因为许多出现胃肠道符号和SARS-COV-2受体血管紧张素转换酶2（ACE2）的患者为几乎用肠上皮表达。 COVID-19死亡率与全身性密切相关炎症如“细胞因子风暴”，促进治疗性免疫调节的尝试并提高关键需要在体外人体系统建模SARS-COV-2诱导的免疫细胞与肠道的相互作用上皮。常规的3D器官培养物（“肠动物”）允许肠上皮SARS-COV-2感染，但不幸的是，省略了免疫细胞。在这里，我们生成了SARS-COV-2感染的整体体外肠道模型使用含有上皮和浸润的免疫细胞的空气界面（ALI）类器官，无需人造重构。 Ali肠道器官的复杂肠道免疫系统包含各种先天和自适应免疫电池，高度潜水T细胞受体（TCR）和B细胞受体（BCR）曲目以及等离子体 B细胞衍生的抗体转录本。重要的是，这些ALI器官中的免疫成分有效地反应对上皮损伤和Ali肠道类器官非常容易受到细菌和病毒感染的影响。在这里，我们利用这种独特的Ali类器官技术，具有集成的免疫组件探索SARS-COV-2感染的后遗症。 AIM 1建立并优化了BSL3 SARS-COV-2感染 Ali肠道器官，利用一种新型的Eversation方法来重新放置ACE2表达的顶端细胞到外表面，允许对小肠不同区域的SARS-COV-2感染进行调查冒号。肠上皮和由于缺乏人类实验系统的阻碍，免疫细胞是未知的。那是AIM 2执行scrna- SARS-COV-2-2诱导的ALI器官中SARS-COV-2诱导的免疫反应的SEQ和CYTOF研究（1）创建网络上皮和免疫之间免疫和双向通信的暂时传播模型细胞和（2）针对淋巴结漏洞进行治疗测试，与临床状况相关（幼稚，康复，免疫抑制，交叉反应性冠状病毒）。当前的上皮器官系统也不允许适应性免疫学研究。 AIM 3因此，通过共培养来概括SARS-COV-2自适应免疫学反应带有新发育的人淋巴结器官的Ali肠道器官。在此类SARS-COV-2感染中共培养我们将适应性免疫复杂与临床状态相关联，包括先前的交叉反应冠状病毒感染和SARS-COV-2幼稚，疗养和最终免疫抑制状态。总体而言，我们利用马克·戴维斯（Mark Davis）（人类LN文化）和凯瑟琳·布莱什（Catherine Blish）和斯科特·博伊德（Scott Boyd）（SARS-COV-2）的合作建立人类的体外系统建模SARS-COV-2诱导的先天和适应性免疫学，并具有相关性用于发病机理研究和治疗测试。