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

摘要 由新型冠状病毒SARS-CoV-2引起的COVID-19大流行对公共卫生构成严重威胁， 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 患者出现胃肠道症状，SARS-CoV-2受体血管紧张素转换酶2（ACE2） 在肠上皮中大量表达。COVID-19死亡率与系统性 炎症，如"细胞因子风暴"，促进治疗性免疫调节的尝试，并提出关键的 需要体外人体系统模拟SARS-CoV-2诱导的免疫细胞与肠道相互作用 上皮常规的3D类器官培养物（"类肠"）允许肠上皮SARS-CoV-2感染， 不幸的是忽略了免疫细胞。在这里，我们建立了一个完整的肠道SARS-CoV-2感染的体外模型 使用含有上皮细胞和浸润免疫细胞的气液界面（ALI）类器官， 人工重建ALI肠道类器官的复杂肠道免疫系统包含各种先天性免疫系统， 和适应性免疫细胞、高度多样的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物。重要的是，这些ALI类器官中的免疫组分有效地响应 上皮损伤和ALI肠类器官对细菌和病毒感染高度敏感。 在这里，我们利用这种独特的ALI类器官技术与集成的免疫组件， 探索SARS-CoV-2感染的后遗症。目的1建立并优化BSL 3 SARS-CoV-2感染 ALI肠类器官，利用一种新的外翻方法重新定位ACE2表达的顶端方面 细胞的外表面，允许调查SARS-CoV-2感染的不同区域的小肠， 结肠SARS-CoV-2诱导的肠上皮细胞和大肠杆菌细胞间的相互作用的时间过程 免疫细胞是未知的，因为缺乏人类实验系统。因此，Aim 2执行scRNA- 在ALI类器官内SARS-CoV-2诱导的免疫应答的seq和CyTOF研究，以（1）创建网络 免疫的时间传播模型和上皮与免疫之间的双向通信 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， 康复期、免疫、交叉反应性冠状病毒）。目前的上皮类器官系统也不允许 适应性免疫研究因此，目的3通过共培养来重现SARS-CoV-2适应性免疫应答 ALI肠类器官与新开发的人淋巴结类器官。在这种SARS-CoV-2感染者中， 我们将适应性免疫应答与临床状态相关联，包括先前的交叉反应， 冠状病毒感染和SARS-CoV-2初治、恢复期和最终免疫状态。总的来说，我们 利用Mark Davis（人类LN培养）和Catherine Blish和Scott Boyd（SARS-CoV-2）的合作 建立模拟SARS-CoV-2诱导的先天性和适应性免疫的人类体外系统， 用于发病机理研究和治疗试验。

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.