RNAi Screening in Hematopoietic Cells

RNAi和CRISPR/Cas9基因筛查技术的发现和发展为研究人员提供了大规模和快速基因筛查的宝贵工具。我们研究计划的一个主题是开发方法学，以便在免疫细胞谱系中有效地应用这些筛选技术，并在人和小鼠的造血细胞中实施筛选，以询问免疫细胞对致病刺激反应的机制基础。我们的工作重点是巨噬细胞，因为它们形成了抵御众多细菌和病毒病原体的第一道防线，而这些初始接触的特征是LISB合作努力的核心，以生成宿主-病原体相互作用的集成模型。 遗传筛选数据容易受到无数实验偏差的影响，其中一些可以通过计算分析来缓解，我们之前已经为其开发了复杂的软件工具，如Signal(由迭代路径小组和网络分析循环选择：https://signal.niaid.nih.gov).我们已经将信号分析应用于我们先前描述的人和小鼠巨噬细胞对LPS屏幕的反应屏幕(Sun等人(2017)科学。数据。4：170007；李等人(2017年)科学。数据。4：170008)。对常见的富集化途径的分析确定了预期的先天免疫过程的富集性，但也对剪接体成分进行了强烈的富集性。在2022财年，我们使用长读测序(PacBio)和深度测序(Illumina)相结合的方法对内毒素攻击的巨噬细胞中的RNA剪接进行了深入分析。这揭示了一个额外的调节层，它超出了众所周知的由内毒素诱导的基因转录变化，即使在基因转录水平不变的基因中也出现了明显的差异异构体使用模式。目前，我们正在比较内毒素诱导的健康患者巨噬细胞和MDS患者的这些剪接模式，以确定这种PRR驱动的剪接反应的缺陷是否可以解释这种疾病中观察到的一些先天免疫失调。 除了我们继续研究细菌内毒素对TLR4通路的反应外，我们还在扩大我们的研究范围，以询问最近发现的胞浆内毒素感应通路，该通路激活非典型性炎症体反应和IL-1家族炎症细胞因子的释放。最近的研究表明，这是内毒素更广泛的生理反应的关键组成部分，也是革兰氏阴性细菌感染中败血症休克结果的主要因素。我们已经与NIH-NCATS筛选设施合作，完成了对胞浆内毒素对IL-1α反应的基因组规模筛选，我们正在积极研究这一项目产生的基因命中。在2022财年，我们继续研究线粒体和细胞代谢在炎症小体激活中的重要作用。在我们筛选出的基因中，我们已经鉴定了三个核苷酸二磷酸激酶，并进一步研究了Nme4基因在炎症体激活中的作用。这项研究揭示了Nme4依赖的线粒体适合性检查点，它支持巨噬细胞对感染和细胞因子释放的反应。与这一模型一致，我们发现Nme4基因缺陷的小鼠由于炎性细胞因子反应减弱而抵抗内毒素休克。我们正在继续从这一屏幕上进一步研究基因，可能会发现非典型性炎症体的新调节因子。 在2022财年，我们还继续与落基山实验室的Karin Petersons NIAID实验室合作，筛选调节La Crosse BunyaVirus(LACV)易感性的宿主因素。LACV是儿童脑炎的主要原因，因为只有儿童通过血脑屏障损害表现出对这种病原体的易感性。我们正在结合深入的转录谱分析和靶向siRNA筛选来确定候选基因，这些基因的年龄相关差异表达可能是儿童对LACV易感性的基础。我们的研究正在进行中，有可能确定治疗靶点，这些靶点可能构成这种危险的儿童疾病的新药治疗的基础。

The discovery and development of RNAi and CRISPR/Cas9 genetic screening technologies have provided researchers with invaluable tools for wide-scale and rapid genetic screening. A theme of our research program has been to develop methodology for efficient application of these screening technologies in immune cell lineages, and to implement screens in both human and mouse hematopoietic cells to interrogate the mechanistic basis of immune cell responses to pathogenic stimuli. Our efforts are focused on macrophages as they form the first line of defense against numerous bacterial and viral pathogens and characterization of these initial encounters are central to collaborative efforts in the LISB to generate integrated models of host-pathogen interactions. Genetic screen data are susceptible to a myriad of experimental biases, some of which can be mitigated by computational analysis for which we have previously developed sophisticated software tools such as SIGNAL (Selection by Iterative pathway Group and Network Analysis Looping: https://signal.niaid.nih.gov). We have applied the SIGNAL analysis to our previously described screens of the human and mouse macrophage responses to LPS screens (Sun et al (2017) Sci. Data. 4:170007; Li et al (2017) Sci. Data. 4:170008). Analysis of commonly enriched pathways identified the expected enrichment of innate immune processes, but also a strong enrichment for spliceosome components. In FY2022, we have conducted in-depth analysis of RNA splicing in LPS-challenged macrophages using a combination of long-read (PacBio) and in-depth (Illumina) sequencing. This has revealed an additional regulatory layer that extends beyond the well-known gene transcription changes that are induced by LPS, with a strong pattern of differential isoform usage emerging even among genes whose aggregate gene transcription levels do not change. We are currently comparing these LPS-induced splicing patterns in healthy patient macrophages compared to MDS patients to determine whether defects in this PRR-driven splicing response might explain some of the innate immune dysregulation observed in this disease. Beyond our continued study of the TLR4 pathway response to bacterial LPS, we are also extending our studies to interrogate the more recently discovered cytosolic LPS sensing pathway, which activates the non-canonical inflammasome response and the release of IL-1 family inflammatory cytokines. Recent studies have shown this to be a critical component of the broader physiological response to LPS and a major contributor to septic shock outcomes in Gram-negative bacterial infections. We have collaborated with the NIH-NCATS screening facility to complete a genome-scale screen of the IL-1 alpha response to cytosolic LPS and we are actively studying the gene hits that have emerged from this project. In FY 2022, we have continued investigation of an important role for the mitochondria and cellular metabolism in inflammasome activation. Among genes emerging from our screen, we have identified three nucleotide diphosphate kinases, and we have further investigated the role of the Nme4 gene in inflammasome activation. This study reveals an Nme4-dependent mitochondrial fitness checkpoint which supports the macrophage response to infection and cytokine release. Consistent with this model, we find that Nme4-deficient mice resist LPS-induced endotoxic shock, due to a diminished inflammatory cytokine response. We are continuing our investigation of further genes from this screen that may uncover novel regulators of the non-canonical inflammasome. In FY2022 we have also continued a collaboration with Karin Petersons NIAID lab at the Rocky Mountain Labs to screen for host factors that regulate susceptibility to the La Crosse bunyavirus (LACV). LACV is a leading cause of pediatric encephalitis as only children show susceptibility to this pathogen through blood brain barrier compromise. We are using a combination of in-depth transcript profiling and targeted siRNA screening to identify candidate genes whose age-related differential expression might underlie the pediatric susceptibility to LACV. Our studies, which are ongoing, have the potential to identify therapeutic targets that could form the basis of new drug treatments for this dangerous childhood disease.