Screening for regulators of SARS CoV-2 infection and inflammation

点击翻译按钮获取中文摘要

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.