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

• 批准号: 10654752
• 负责人: CALVIN J KUO
• 金额: $ 39.35万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 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/10654752
• 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; Diarrhea; Economics; Epithelial Cells; Epithelium; Event; Fostering; Gluten; Health; 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; Maps; Medicine; Methods; Modeling; Myelogenous; Myeloid Cells; Natural Immunity; Nature; Nodal; Organ; Organoids; Pathogenesis; Pathology; Patients; Peripheral; Plasma; Predisposition; Public Health; 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; Type 2 Angiotensin II Receptor; Variant; Virus Diseases; Virus Replication; Vomiting; adaptive immune response; adaptive immunity; cell type; cross reactivity; cytokine release syndrome; data integration; enteric infection; global health; immune cell infiltrate; immunoregulation; in vitro Model; intestinal epithelium; lymph nodes; mesenteric lymph node; network models; novel; novel coronavirus; novel therapeutics; pharmacologic; reconstitution; response; single cell analysis; single-cell RNA sequencing; systemic inflammatory response; therapeutic development; therapeutic evaluation; tumor

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 由新型冠状病毒 SARS-CoV-2 引起的 COVID-19 大流行对公众健康构成严重威胁， 伴有肺部感染和呼吸衰竭。然而，肠道也是 SARS-CoV-2 的目标，因为许多 出现胃肠道症状且 SARS-CoV-2 受体血管紧张素转换酶 2 (ACE2) 的患者 肠上皮大量表达。 COVID-19 死亡率与系统性密切相关 “细胞因子风暴”中的炎症，促进了治疗性免疫调节的尝试，并提出了一个关键的问题 需要体外人体系统模拟 SARS-CoV-2 诱导的免疫细胞与肠道的相互作用 上皮。传统的 3D 类器官培养物（“肠类器官”）允许肠上皮 SARS-CoV-2 感染，但 不幸的是忽略了免疫细胞。在这里，我们生成了 SARS-CoV-2 感染的整体肠道体外模型 使用含有上皮细胞和浸润免疫细胞的气液界面（ALI）类器官，无需 人工重建。 ALI肠道类器官复杂的肠道免疫系统包含各种先天性的 和适应性免疫细胞、高度多样化的 T 细胞受体 (TCR) 和 B 细胞受体 (BCR) 库以及血浆 B 细胞衍生的抗体转录本。重要的是，这些 ALI 类器官中的免疫成分可以有效地做出反应 上皮损伤和 ALI 肠道类器官极易受到细菌和病毒感染。 在这里，我们利用这种独特的 ALI 类器官技术和集成免疫组件来 探索 SARS-CoV-2 感染的后遗症。目标 1 建立并优化 BSL3 SARS-CoV-2 感染 ALI 肠道类器官，利用一种新颖的外翻方法来重新定位表达 ACE2 的顶端 细胞外表面，从而可以调查小肠不同区域的 SARS-CoV-2 感染情况 冒号。组织驻留 SARS-CoV-2 诱导的肠上皮和肠道上皮之间串扰的时间过程 由于缺乏人体实验系统，免疫细胞尚不清楚。因此，目标 2 执行 scRNA- 对 ALI 类器官内 SARS-CoV-2 诱导的免疫反应进行 seq 和 CyTOF 研究，以 (1) 创建一个网络 免疫的时间传播以及上皮和免疫之间的双向通讯模型 细胞和（2）针对淋巴结脆弱性进行治疗测试，与临床状态（幼稚， 恢复期、免疫、交叉反应的冠状病毒）。目前的上皮类器官系统也不允许 适应性免疫的研究。因此，目标 3 通过共培养重现了 SARS-CoV-2 适应性免疫反应 ALI 肠道类器官与新开发的人类淋巴结类器官。在此类 SARS-CoV-2 感染者中 通过共培养，我们将适应性免疫反应与临床状态（包括先前的交叉反应）相关联 冠状病毒感染和 SARS-CoV-2 幼稚、恢复期和最终免疫状态。总的来说，我们 利用 Mark Davis（人类 LN 文化）以及 Catherine Blish 和 Scott Boyd（SARS-CoV-2）的合作 创建人体体外系统，模拟 SARS-CoV-2 诱导的先天性和适应性免疫，并具有相关性 用于发病机制研究和治疗测试。