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2019年冠状病毒病（COVID-19）构成了全球性的威胁 流行病SARS-CoV-2及其家族成员具有一些令人困惑的独特病理特征， 特别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， 尽管过度的髓系细胞为主的炎症，这与COVID-19的严重程度相关。如何 目前尚不清楚该病毒是否参与免疫系统并使免疫系统失调;尽管血管紧张素转化 酶2（ACE 2）是典型的SARS-CoV-2受体，免疫细胞，特别是骨髓细胞，表达很少， 如果有的话，尽管有直接病毒参与的证据，ACE 2的水平。 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， 我们试图解决SARS-CoV-2如何与骨髓细胞相互作用，骨髓细胞在 病毒的先天感应和宿主免疫的调节，通过开发一个髓样细胞受体集中， 异位表达筛选。在正在进行的实验中，我们已经确定了几个新的聚糖依赖性宿主 SARS-CoV-2 Spike（S）蛋白的相互作用伴侣，包括几种C型凝集素和Tweety家族 成员2（TTYH 2）。COVID-19患者的肺部单细胞RNA测序（scRNA-seq）分析 表明与ACE 2相反，这些受体的髓样细胞显性表达。在初步研究中， 我们已经证明，这些分子主要通过ACE 2受体结合区以外的区域相互作用， 结构域（RBD），这表明它们可能提供病毒进入之外的新功能。虽然这些受体 不支持真正的SARS-CoV-2的主动复制，直接的病毒-骨髓细胞接合诱导 一个强大的促炎反应，这是阻断受体诱饵蛋白和皮摩尔亲和抗- 刺突双特异性纳米抗体，其也通过ACE 2阻断病毒感染。总之，我们的发现提供了第一个 SARS-CoV-2直接免疫调节的证据，可能具有治疗益处。 鉴于这些发现，我们假设新的SARS-CoV-2病毒-受体相互作用在骨髓细胞， 构成COVID-19的致病途径，作为信号受体直接驱动骨髓细胞 失调这些新的直接病毒免疫相互作用也可能具有迄今为止尚未探索的功能， 间接影响其他细胞。这些假设将在以下具体目标范围内得到解决：（1） 确定每种髓样细胞受体在诱导促炎反应中的功能贡献 在SARS-CoV-2参与，和（2）确定这些相互作用的作用，在失调的 获得性免疫和SARS-CoV-2通过ACE 2受体的反式感染。新型髓系病毒的研究 控制异常免疫反应的细胞受体将极大地有助于我们对COVID的理解， 19发病机制，揭示了新的治疗靶点，以对抗COVID-19，造福全人类。

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.