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

了解导致严重COVID-19的免疫反应对于识别可能发生以下疾病的患者至关重要： 成为危重病，并辨别哪些途径的治疗干预的目标。炎性小体是 这是一种由许多病毒激活的抗病毒和促炎途径，但其在COVID-19中的作用尚未被证实。 完全定义。炎性小体激活导致称为焦亡的炎性类型的细胞死亡， 释放促炎细胞因子，包括白细胞介素（IL）-18。炎性细胞因子是 病毒控制，但过度或长期激活可增强许多呼吸道病毒的发病机制 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，我们测量了IL-18和36种其他细胞因子， 新冠肺炎患者血浆中的趋化因子。虽然大多数人在COVID-19中没有显著差异， IL-18和IL-1受体拮抗剂（RA）水平在COVID-19插管患者与非COVID-19插管患者中升高。 插管COVID-19和住院流感患者。人巨噬细胞与SARS-CoV-2的孵育 在体外产生IL-18、IL-1 β、IL-RA、IL-6和IL-8。我们使用了一种人巨噬细胞系， 炎性体基因被破坏，表明caspase-1和NLRP 3是SARS-CoV-2激活 炎性小体我们将建立SARS-CoV-2激活炎性小体的机制， 使用一组内吞作用抑制剂来确定该途径的抑制如何改变先天免疫信号传导， 具有特异性炎性小体和其他先天传感基因被破坏的巨噬细胞系，以及特异性 在原代人巨噬细胞中的先天感应抑制剂。早期研究表明，抗体（Abs） 调节SARS-CoV-2的先天感应。为了测试COVID-19期间产生的抗体是否会改变先天性 我们将SARS-CoV-2与单克隆抗体或患者血清孵育， 巨噬细胞，并测量上清液细胞因子。为了研究细胞功能，我们开发了一种流动 基于细胞仪的平台，能够对传统细胞表面标记物进行单细胞分析， 参与代谢程序的蛋白质的细胞内染色。利用这个平台，我们确定了骨髓 衍生的抑制细胞（MDSC），具有与严重COVID-19相关的独特代谢特征。 长期炎症诱导癌症、肥胖和慢性感染中的MDSC。我们将使用单细胞RNA 测序以表征这些新型MDSC并评估COVID-19中产生的细胞因子如何调节 MDSC代谢编程。总体目标是确定SARS-CoV-2激活的机制 炎症途径，抗体调节，MDSC的作用，以及它们如何交叉介导SARS-CoV-2 免疫控制和病理学。这将确定治疗干预的靶点， 病理学而不损害抗病毒免疫作为新的临床试验和疾病标志物的基础 这一进展允许将资源针对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.