Project 1: Mechanisms of innate sensing and pathogenesis of SARS-CoV-2

了解导致严重新冠肺炎的免疫反应对于识别可能发生 病情危重，辨别哪些路径是治疗干预的目标。发炎的是 一种可被多种病毒激活的抗病毒和促炎途径，但其在新冠肺炎中的作用尚未得到证实 完全定义。炎性小体激活会导致一种称为下垂的炎症性细胞死亡，以及 包括白介素18在内的促炎细胞因子的释放。炎症体细胞因子是 病毒控制，但过度或长时间的激活增强了许多呼吸道病毒的致病作用 感染，包括禽流感和SARS-CoV-1。广泛研究了炎性小体的作用 细胞因子在多种其他病毒感染的发病机制中的作用，我们检测了IL-18和其他36种细胞因子 新冠肺炎患者血浆中的趋化因子。虽然大多数在COVID-19中没有显著差异， 新冠肺炎患者气管插管后IL-18和IL-1受体拮抗剂(RA)水平升高 为新冠肺炎患者插管和住院流感患者。人巨噬细胞与SARS-CoV-2的孵育 体外产生IL-18、IL-1、IL-RA、IL-6和IL-8。我们使用了一种人巨噬细胞系， 炎症体基因被打乱表明caspase-1和NLRP3是SARS-CoV-2激活所必需的 发炎的人。我们将建立SARS-CoV-2激活炎症小体和 利用一组内吞作用抑制剂，确定抑制这一途径如何改变先天免疫信号， 巨噬细胞系具有特定的炎症体和其他固有感觉基因的干扰，并特异性 原代人巨噬细胞固有感觉的抑制物。早期研究表明，抗体(Abs) 调节SARS-CoV-2的先天感知。测试新冠肺炎中产生的抗体是否会改变先天 信号，我们将SARS-CoV-2与单抗或患者血清孵化，接种原代或永生化 巨噬细胞，并测定上清液中的细胞因子。为了研究细胞功能，我们开发了一种流 基于细胞学的平台，使传统细胞表面标记的单细胞分析与 参与代谢编程的蛋白质的细胞内染色。利用这个平台，我们识别了髓系 具有不同代谢特征的衍生抑制细胞(MDSCs)与严重新冠肺炎相关。 长期的炎症会导致MDSCs出现在癌症、肥胖和慢性感染中。我们将使用单细胞RNA 测序鉴定这些新的MDSCs并评估新冠肺炎产生的细胞因子如何调节 MDSC代谢规划。总体目标是确定SARS-CoV-2激活的机制 炎症途径、抗体调节、MDSCs的作用及其如何交叉介导SARS-CoV-2 免疫控制和病理学。这将确定治疗干预的目标，将炎症降至最低 不损害抗病毒免疫的病理学作为新的临床试验和疾病标记物的基础 进展允许将资源定向到最有可能经历严重疾病的患者。

Understanding immune responses that contribute to severe COVID-19 is essential to identify patients likely to become critically ill and to discern which pathways to target for therapeutic intervention. The inflammasome is an antiviral and proinflammatory pathway activated by many viruses, but its role in COVID-19 has not been defined fully. Inflammasome activation results in an inflammatory type of cell death called pyroptosis as well as the release of proinflammatory cytokines that include interleukin (IL)-18. Inflammasome cytokines are central to viral control, but excessive or prolonged activation enhances pathogenesis of numerous respiratory virus infections, including avian influenza and SARS-CoV-1. Having extensively studied the role of inflammasome cytokines in the pathogenesis of multiple other viral infections, we measured IL-18 and 36 other cytokines and chemokines in plasma from patients with COVID-19. While most were not significantly different in COVID-19, IL-18 and IL-1 receptor antagonist (RA) levels are elevated in intubated patients with COVID-19 versus non- intubated COVID-19 and hospitalized influenza patients. Incubation of human macrophages with SARS-CoV-2 in vitro produced IL-18, IL-1, IL-RA, IL-6, and IL-8. We used a human macrophage cell-line with various inflammasome genes disrupted to show that caspase-1 and NLRP3 are required for SARS-CoV-2 activation of the inflammasome. We will establish the mechanism by which SARS-CoV-2 activates the inflammasome and determine how inhibition of this pathway alters innate immune signaling using a panel of endocytosis inhibitors, macrophage cell lines with specific inflammasome and other innate sensing genes disrupted, and specific inhibitors of innate sensing in primary human macrophages. Early investigations suggest that antibodies (Abs) modulate innate sensing of SARS-CoV-2. To test whether Abs produced during COVID-19 alter innate signaling, we will incubate SARS-CoV-2 with monoclonal Abs or patient sera, inoculate primary or immortalized macrophages, and measure supernatant cytokines. To investigate cellular function, we developed a flow cytometry-based platform that enables single cell analysis of traditional cell surface markers combined with intracellular staining for proteins involved in metabolic programming. Using this platform, we identified myeloid derived suppressor cells (MDSCs) with distinct metabolic profiles that correlated with severe COVID-19. Prolonged inflammation induces MDSCs in cancer, obesity, and chronic infections. We will use single cell RNA sequencing to characterize these novel MDSCs and assess how cytokines produced in COVID-19 regulate MDSC metabolic programming. The overall goal is to define the mechanism by which SARS-CoV-2 activates inflammatory pathways, Ab modulation, the role of MDSCs, and how they intersect to mediate SARS-CoV-2 immune control and pathology. This will identify targets for therapeutic intervention that minimize inflammatory pathology without impairing antiviral immunity as the foundation of novel clinical trials and markers of disease progression that allow targeting resources to patients most likely to experience severe disease.