Screening for regulators of SARS CoV-2 infection and inflammation

先天免疫系统的细胞，如树突状细胞和巨噬细胞，不断巡逻宿主粘膜表面和外周组织，寻找感染或损伤的迹象。由于许多感染SARS-CoV-2的人在没有出现症状的情况下清除了病毒，先天免疫反应的各个方面可能是击败这种病毒的关键。不利的细胞因子风暴的可能性取决于感染部位不同细胞类型的相互作用。这突出了需要研究在上皮细胞（感染的主要目标）和先天免疫细胞（如巨噬细胞）（炎症的关键驱动因素）的组合环境中对SARS-CoV-2的免疫反应。 由于该项目刚刚开始，我们将主要总结未来的研究计划。我们建议首先建立一种共培养测定法，该测定法可以测量Calu 3人上皮细胞（一种已建立的CoV-2允许系）中的病毒复制和THP 1人巨噬细胞的IL-6表达。由于两种测定读数都是基于FACS的，这将允许使用合并的CRISPR筛选方法，这在全基因组范围内劳动密集度较低，并且在NIAID BSL-3实验室的可用设备下更可行。 感染后，我们将从CD 324+（E-钙粘蛋白）Calu 3细胞中分选CD 32 + THP 1细胞，并测定细胞的病毒水平和IL-6表达。 将使用我们从UTMB收到的最近开发的mNeonGreen荧光报告病毒测定病毒水平，而IL-6将通过细胞内细胞因子染色的成熟实验室方案测定。我们的期望是Calu 3细胞将成为CoV-2感染的主要靶标，而THP 1将成为主要的IL-6产生者。 出于这个原因，我们最初试图确定THP 1细胞中IL-6的调节因子，同时将SARS-CoV-2感染性的宿主因素的研究限制在Calu 3细胞中。一旦主校区的BSL-3实验室可供我们进行SARS-CoV-2研究，我们将建立病毒感染和IL-6表达测定，作为基于FACS的读数，并建立筛选测定的可重复性，阳性对照基因由特定的CRISPR/Cas9 gRNA靶向。我们期望确认的CoV-2的人受体ACE 2是病毒感染的适当阳性对照，也可能是IL-6应答的适当阳性对照。我们将优化病毒感染时间、感染复数（MOI）、高尔基体阻断孵育期、细胞固定、IL-6染色条件和荧光病毒感染水平和IL-6表达的FACS分选。 一旦建立了初步测定，我们建议使用NCATS提供的基于CRISPR的混合形式的慢病毒文库（Cellecta）。THP 1细胞和Calu 3细胞将分别用慢病毒感染，并在BSL-2条件下进行药物选择。为了确保每个细胞平均1个gRNA，我们将使用0.3的MOI进行慢病毒感染。将保留慢病毒感染的THP 1和Calu 3细胞的对照池用于后续测序步骤，以鉴定由于对细胞活力的影响而从文库池中丢失的gRNA。 SARS-CoV-2的制备和感染将在BSL-3实验室进行。如前所述，我们将通过以1：1的比例混合CRISPR文库感染的细胞，在共培养系统中筛选Calu 3细胞和分化的THP 1细胞。 在用SARS-CoV-2感染后，细胞将用高尔基体块处理，用4%PFA固定，并按照先前优化的方案对IL-6、CD 32和CD 324进行染色。我们将遵循BSL-3 CoV-2工作的标准操作规程，以允许在BSL-2条件下对固定细胞进行FACS分选。 假设Calu 3细胞是主要的病毒靶标，THP 1是主要的IL-6产生者，我们将分选六个细胞群：最高和最低20%的THP 1细胞被染色用于IL-6表达，中间60%的IL-6染色细胞作为对照，最高和最低20%的Calu 3细胞被分析用于病毒荧光，中间60%的细胞也作为对照。将分离基因组DNA，通过PCR扩增gRNA序列，并使用深度测序鉴定与中间60%相比在高/低20%组中富集的gRNA。 我们将一式三份重复筛选以鉴定在高/低20%群体中一致出现的gRNA。由这些gRNA靶向的基因将作为候选命中进行跟踪。 于2020财政年度，我们已在BSL-2条件下对人类冠状病毒OC 43进行中试检测。我们已经证实，我们可以在共培养和病毒感染后有效地FACS分离上皮细胞和巨噬细胞，目前正在评估该系统是否驱动强的巨噬细胞IL-6反应。

Cells of the innate immune system, such as dendritic cells and macrophages, constantly patrol host mucosal surfaces and peripheral tissues for signs of infection or injury. Since many people infected with SARS-CoV-2 clear the virus without developing symptoms, aspects of the innate immune response may hold the key to defeating this virus. The potential for an adverse cytokine storm depends on the interaction of different cell types at the site of infection. This highlights the need to study the immune response to SARS-CoV-2 in a combined environment of both epithelial cells, which are the primary target of infection, and innate immune cells such as macrophages, which are crucial drivers of inflammation. Since this project has only recently started, we will primarily summarize the future research plan. We propose to first establish a co-culture assay which can measure both viral replication in Calu3 human epithelial cells, an established CoV-2-permissive line, and IL-6 expression from THP1 human macrophages. As both assay readouts will be FACS-based, this will permit used of a pooled CRISPR screening approach, which will be less labor intensive on a genome-wide scale and more feasible with available equipment in the NIAID BSL-3 lab. Post-infection, we will sort CD32+ THP1 cells from CD324+ (E-cadherin) Calu3 cells, and assay the cells for viral levels and IL-6 expression. Viral levels will be assayed using a recently developed mNeonGreen fluorescent reporter virus which we have received from UTMB, while IL-6 will be assayed by well-established lab protocols for intracellular cytokine staining. Our expectation is that the Calu3 cells will be the primary target for CoV-2 infection, and THP1 the primary IL-6 producers. For this reason, we initially seek to identify regulators of IL-6 in THP1 cells, while limiting the study of host factors of SARS-CoV-2 infectivity to Calu3 cells. As soon as the BSL-3 lab in the main campus is available to us for SARS-CoV-2 research, we will establish the viral infection and IL-6 expression assays as FACS-based readouts, and establish the reproducibility of the screen assays with positive control genes targeted by specific CRISPR/Cas9 gRNAs. We expect the confirmed human receptor for CoV-2, ACE2, to be an appropriate positive control for viral infection and possibly also for the IL-6 response. We will optimize the viral infection time, multiplicity of infection (MOI), incubation period for golgi block, cell fixation, IL-6 staining conditions and FACS-sorting for both fluorescent virus infection levels and IL-6 expression. Once the primary assay is established, we propose to use an NCATS-provided CRISPR-based lentiviral library in pooled format (Cellecta). THP1 cells and Calu3 cells will be separately infected with lentivirus and drug-selected under BSL-2 conditions. To ensure an average of 1 gRNA per cell, we will use an MOI of 0.3 for the lentiviral infection. Control pools of lentivirus-infected THP1 and Calu3 cells will be retained for the later sequencing step to identify gRNAs that are lost from the library pool due to effects on cell viability. SARS-CoV-2 preparation and infection will be performed in the BSL-3 lab. As described earlier, we will screen Calu3 cells and differentiated THP1 cells in a co-culture system by mixing the CRISPR library-infected cells in a 1:1 ratio. After infection with SARS-CoV-2, cells will be treated with golgi block, fixed with 4% PFA and stained for IL-6, CD32 and CD324 following the previously optimized protocols. We will follow standard operating procedures for BSL-3 CoV-2 work to permit the fixed cells to be FACS-sorted under BSL-2 conditions. Assuming that Calu3 cells are the primary viral target and THP1 the primary IL-6 producers, we will sort six populations of cells: Highest and lowest 20% of THP1 cells stained for IL-6 expression and intermediate 60% of IL-6 stained cells as control, and highest and lowest 20% of Calu3 cells analyzed for viral fluorescence also with intermediate 60% of cells as control. Genomic DNA will be isolated, gRNA sequences amplified by PCR and deep sequencing will be used to identify gRNAs that are enriched in the high/low 20% sets compared to the intermediate 60%. We will repeat the screen in triplicate to identify gRNAs that appear consistently in the high/low 20% populations. Genes targeted by these gRNAs will be followed up as candidate hits. In FY2020, we have conducted pilot assays under BSL-2 conditions with the human coronavirus OC43. We have confirmed we can effectively FACS-separate the epithelial cells and macrophages after co-culture and viral infection, and are currently evaluating whether this system drives a strong macrophage IL-6 response.