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周围组织，寻找感染或损伤的迹象。由于许多感染SARS-CoV-2病毒的人清除了病毒而没有出现症状，因此先天免疫反应方面可能是击败这种病毒及其变种的关键。一场不利的细胞因子风暴的可能性取决于感染部位不同类型细胞的相互作用。这凸显了在两种环境下研究SARS-CoV-2免疫反应的必要性，上皮细胞是感染的主要目标，天然免疫细胞如巨噬细胞是炎症的关键驱动因素。 在2022财年，我们获得了NIAID SARS病毒学核心实验室(SVC)的访问权限，此后我们建立了与人上皮细胞、已建立的CoV-2允许细胞和人巨噬细胞的联合培养试验。使用针对SARS-CoV-2刺突蛋白的抗体，我们已经能够测量上皮细胞的病毒感染以及巨噬细胞状态如何调节病毒感染。 在一个相关的项目中，我们研究了生物活性小分子如何在免疫细胞中传递调制效应，在某些情况下，提供延长的长期记忆。在对具有生物活性的小分子进行肿瘤坏死因子诱导调节的筛选中，我们鉴定了几种具有诱导人巨噬细胞训练作用的化合物。在这些化合物中，Syk激酶抑制剂(SYKi IV)的筛选可促进对内毒素的增强反应，与先前报道的β-葡聚糖类似。经SYKI IV培养的巨噬细胞对甲型流感显示出高度的抵抗力。 为了将这些研究扩展到冠状病毒，我们首先确定用SYKI IV训练的巨噬细胞显示出对OC43季节性冠状病毒的感染减少。然后，我们研究了上皮/巨噬细胞共培养试验中对SARS CoV-2的影响，发现SYKi IV巨噬细胞训练显著减少了上皮细胞的感染。我们在广泛的SYK激酶抑制剂小组中发现了类似的结果，突出了SYK靶向作为SARS-CoV-2易感性调节剂的潜在应用。这项工作最近被接受发表在《细胞报告》上。 我们继续评估在先天免疫调节的背景下，进行大规模筛查以识别可能影响SARS-CoV-2感染的基因扰动或小分子的后勤工作。 我们还在开发额外的输出分析来评估宿主细胞对感染的反应，例如基于多重高含量显微镜的单细胞基因诱导分析。这样的检测可以在固定的细胞中进行，因此，可以在病毒感染步骤之后在BSL-3实验室之外进行。

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 and its variants. 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. In FY2022, we obtained access to the NIAID SARS Virology Core lab (SVC), and we have since established a co-culture assay with human epithelial cells, an established CoV-2-permissive cell, and human macrophages. Using an antibody specific to the SARS-CoV-2 spike protein, we have been able to measure viral infection of the epithelial cells and how this can be modulated by the macrophage state. In a related project, we have investigated how biologically active small molecules can impart modulatory effects in immune cells, in some cases, providing extended long-term memory. In a screen of biologically active small molecules for regulators of TNF induction, we identified several compounds with the ability to induce training effects on human macrophages. Among these compounds, a Syk kinase inhibitor (SYKi IV) screen hit promoted an enhanced response to LPS similar to that previously reported for beta-glucan. Macrophages trained with SYKi IV showed a high degree of resistance to influenza A. To extend these studies to coronavirus, we first determined that macrophages trained with SYKi IV showed reduced infection with the OC43 seasonal coronavirus. We then investigated effects in the epithelial/macrophage co-culture assays for SARS CoV-2, and found that SYKi IV macrophage training led to dramatically reduced epithelial cell infection. We find similar results with a broad panel of SYK kinase inhibitors, highlighting a potential application of SYK targeting as a modulator of SARS-CoV-2 susceptibility. This work was recently accepted for publication in Cell Reports. We continue to evaluate the logistics of running large scale screen to identify gene perturbations or small molecules that can influence SARS-CoV-2 infection in the context of innate immune modulation. We are also developing additional output assays to evaluate host cell responses to infection, such as multiplex high content microscopy-based single cell analysis of gene induction. Such assays can be conducted in fixed cells and as such, could be run outside the BSL-3 lab after the viral infection step.