Functional characterization of SARS-CoV-2 myeloid cell receptors as an immunopathogenic mechanisms of COVID-19

项目摘要 SARS-CoV-2的快速传播和由此产生的冠状病毒病2019(新冠肺炎)构成了全球 大流行。SARS-CoV-2及其家族成员有一些令人费解的独特病理特征，大多数 值得注意的是急性呼吸窘迫综合征(ARDS)、细胞因子释放综合征(CRS)和淋巴细胞减少症， 尽管有过度的以髓系细胞为主的炎症，这与新冠肺炎的严重程度有关。多么 该病毒参与并失调免疫系统目前尚不清楚；尽管血管紧张素转换 酶2(ACE2)是典型的SARS-CoV-2受体，免疫细胞，特别是髓系细胞，表达很少， 如果有的话，ACE2的水平，尽管有证据表明有直接的病毒接触。 基于我们在免疫调节受体配体的发现和表征方面的丰富经验 相互作用，我们试图解决SARS-CoV-2如何与髓系细胞相互作用，这在 病毒的先天感知和宿主免疫的调节，通过发展一种髓系细胞受体 异位表达屏幕。在正在进行的实验中，我们已经确定了几个新的依赖于糖的宿主 SARS-CoV-2 Spike(S)蛋白的相互作用伙伴，包括几个C型凝集素和Twety家族 成员2(TTYH2)。新冠肺炎患者肺组织单细胞核酸序列分析 表明这些受体以髓系细胞为主表达，而不是ACE2。在初步研究中， 我们已经证明这些分子主要通过ACE2受体结合区以外的区域相互作用。 结构域(RBD)，这表明它们可能在病毒入侵之外提供了一种新的功能。尽管这些受体 不支持主动复制正宗的SARS-CoV-2，由病毒与髓系细胞直接接触诱导 一个强大的促炎反应，它被受体诱骗蛋白和微克分子亲和力抗亲和力所阻断 也通过ACE2阻止病毒感染的钉状双特异性纳米体。总而言之，我们的发现提供了第一个 SARS-CoV-2直接免疫调节的证据，可能针对治疗益处。 鉴于这些发现，我们假设在髓系细胞中新的SARS-CoV-2病毒与受体的相互作用 构成新冠肺炎的致病途径，作为信号受体直接驱动髓系细胞 监管失调。这些新的病毒-免疫直接相互作用也可能具有迄今未被探索的功能 间接影响其他细胞。这些假设将在以下具体目标内得到解决：(1) 确定每个髓样细胞受体在诱导促炎反应中的功能贡献 在SARS-CoV-2接触时，以及(2)确定这些相互作用在调节失调中的作用 ACE2受体介导的获得性免疫与SARS-CoV-2的跨性感染我们对新型病毒髓系的研究 控制异常免疫反应的细胞受体将极大地有助于我们对COVID- 19病机，揭示了针对新冠肺炎的新的治疗靶点，造福于全人类。

Project Summary The rapid spread of SARS-CoV-2 and the resulting Coronavirus Disease 2019 (COVID-19) pose a global pandemic. SARS-CoV-2 and its family members share some puzzling, unique pathological features, most notably acute respiratory distress syndrome (ARDS), cytokine release syndrome (CRS), and lymphopenia, despite excessive myeloid cell–dominant inflammation, which has been correlated with COVID-19 severity. How the virus engages and dysregulates the immune system is currently unknown; although angiotensin-converting enzyme 2 (ACE2) is the canonical SARS-CoV-2 receptor, immune cells, particularly myeloid cells, express little, if any, levels of ACE2 despite evidence for direct viral engagement. Based on our extensive experience in the discovery and characterization of immune-modulatory receptor-ligand interactions, we sought to address how SARS-CoV-2 interacts with myeloid cells, which play essential roles in both virus innate sensing and the modulation of host immunity, by developing a myeloid cell receptor-focused ectopic expression screen. In ongoing experiments, we have identified several novel glycan-dependent host interaction partners for SARS-CoV-2 Spike (S) protein, including several C-type lectins and Tweety Family Member 2 (TTYH2). Pulmonary single-cell RNA sequencing (scRNA-seq) analysis in COVID-19 patients indicates a myeloid cell-dominant expression of these receptors as opposed to ACE2. In the preliminary studies, we have shown that these molecules interact mostly through regions outside of the ACE2 receptor-binding domain (RBD), suggesting that they may provide a novel function outside of virus entry. Although these receptors do not support the active replication of authentic SARS-CoV-2, the direct virus-myeloid cell engagement induces a robust pro-inflammatory response, which is blocked by receptor-decoy proteins and a picomolar-affinity anti- spike bispecific nanobody that also blocks virus infection through ACE2. Together, our findings provide the first evidence for direct immune modulation by SARS-CoV-2, potentially targeted for therapeutic benefit. Given these findings, we hypothesize that novel SARS-CoV-2 virus-receptor interactions in myeloid cells constitute a pathogenic pathway for COVID-19, serving as signaling receptors that directly drive myeloid cell dysregulation. These new direct virus-immune interactions may also have heretofore unexplored functions to affect other cells indirectly. These hypotheses will be addressed within the following Specific Aims: (1) to determine the functional contribution of each myeloid cell receptor in the induction of pro-inflammatory responses upon SARS-CoV-2 engagement, and (2) to determine the role of these interactions in the dysregulation of adaptive immunity and SARS-CoV-2 trans-infection through ACE2 receptor. Our study on novel virus myeloid cell receptors that govern aberrant immune responses would greatly contribute to our understanding of COVID- 19 pathogenesis, revealing new therapeutic targets against COVID-19 for the benefit of humanity at large.