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

摘要 由新型冠状病毒SARS-CoV-2引发的新冠肺炎疫情对公众健康构成严重威胁， 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 出现胃肠道症状和SARS-CoV-2受体血管紧张素转换酶2(ACE2)的患者 肠上皮细胞大量表达。新冠肺炎死亡率与系统性疾病密切相关 炎症，如“细胞因子风暴”，促进了治疗性免疫调节的尝试，并引发了一个关键的 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的必要性 上皮组织。传统的3D器官培养(肠样培养)允许肠道上皮细胞感染SARS-CoV-2，但 不幸的是，省略了免疫细胞。在这里，我们建立了一个SARS-CoV-2感染的整体肠道体外模型 使用气液界面(ALI)有机物同时包含上皮细胞和浸润性免疫细胞，而不是 人工重建。ALI肠道脏器复杂的免疫系统含有多种先天免疫成分 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)谱系，以及血浆 B细胞来源的抗体转录本。重要的是，这些ALI类有机物中的免疫成分可以有效地 对上皮损伤和ALI肠道脏器高度敏感，易受细菌和病毒感染。 在这里，我们利用这一独特的ALI有机体技术和集成的免疫成分来 探索SARS-CoV-2感染的后遗症。目的1建立和优化BSL3 SARS-CoV-2感染小鼠 ALI肠道器官，利用一种新的外翻方法重新定位ACE2表达的顶端 细胞外表面，允许调查SARS-CoV-2感染在不同区域的小肠和 冒号。组织内滞留SARS-CoV-2诱导的肠上皮和肠上皮细胞间相互作用的时间进程 免疫细胞是未知的，因为缺乏人类实验系统而受到阻碍。因此，AIM 2执行scRNA- 用SEQ和CyTOF研究SARS-CoV-2诱导的ALI器官内免疫反应(1)形成网络 免疫的时间传播模型及上皮细胞与免疫细胞的双向通讯 细胞和(2)执行针对结节脆弱性的治疗性测试，与临床状态相关(幼稚， 恢复期、免疫、交叉反应冠状病毒)。目前的上皮器官系统也不允许 适应性免疫的研究。因此，目标3通过共培养来概括SARS-CoV-2获得性免疫反应 ALI肠器官与新近开发的人淋巴组织器官。在这些感染了SARS-CoV-2的人中 共培养我们将获得性免疫反应与临床状态相关联，包括先前的交叉反应 冠状病毒感染和SARS-CoV-2幼稚、康复和最终免疫状态。总体而言，我们 利用Mark Davis(人类LN文化)和Catherine Blish和Scott Boyd(SARS-CoV-2)的合作 建立模拟SARS-CoV-2诱导的先天免疫和获得性免疫的人体体外系统 用于发病机制研究和治疗试验。

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.