RNAi Screening in Hematopoietic Cells

造血细胞中的 RNAi 筛选

• 批准号: 10692140
• 负责人: Iain Fraser
• 金额: $ 102.24万
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10692140
• 关键词: Address; Bioinformatics; Biological; Biological Assay; Blood - brain barrier anatomy; CRISPR screen; CRISPR/Cas technology; Candidate Disease Gene; Cell Line; Cell Lineage; Cell model; Cells; Childhood; Collaborations; Computer Analysis; Computer software; Dangerousness; Data; Defect; Development; Diphosphates; Disease; Encephalitis; Endotoxic Shock; Family; Genes; Genetic Screening; Genetic Transcription; Genome; Goals; Gram-Negative Bacterial Infections; Hematopoietic; Human; Image; Immune; Immune response; Infection; Inflammasome; Inflammatory; Inflammatory Response Pathway; Integration Host Factors; Interleukin-1; Interleukin-1 alpha; Investigation; Knock-out; Ligands; Mammalian Cell; Metabolism; Methodology; Mitochondria; Modeling; Molecular; Mus; National Center for Advancing Translational Sciences; National Institute of Allergy and Infectious Disease; Nucleic Acids; Nucleotides; Only Child; Orthobunyavirus; Outcome; Pathogenicity; Pathway Analysis; Pathway interactions; Patients; Pattern; Phosphotransferases; Physiological; Predisposition; Process; Protein Isoforms; Protocols documentation; RNA Interference; RNA Splicing; RNA analysis; RNA interference screen; Reporter; Research; Research Personnel; Resistance; Role; Screening Result; Septic Shock; Signal Transduction; Small Interfering RNA; Software Tools; Spliceosomes; Statistical Data Interpretation; Stimulus; TLR4 gene; Technology; Transcript; United States National Institutes of Health; Validation; age related; analytical method; base; cytokine; differential expression; gene function; genome editing; genome wide screen; genome-wide; immune activation; information processing; innate immune mechanisms; insight; macrophage; mitochondrial fitness; novel; novel therapeutics; nucleic acid delivery; pathogen; pathogenic bacteria; pathogenic virus; programs; response; screening; the sun; therapeutic target; tool

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.

RNAi和CRISPR/CAS9遗传筛查技术的发现和开发为研究人员提供了宝贵的工具，用于广泛和快速的遗传筛查。我们的研究计划的一个主题是开发有效应用这些筛查技术在免疫细胞谱系中的方法，并在人和小鼠造血细胞中实施筛查，以询问免疫细胞对病原刺激的机械基础。我们的努力集中在巨噬细胞上，因为它们构成了针对众多细菌和病毒病原体的第一道防线，并且这些初始相遇的表征对于LISB的协作努力是产生宿主 - 病原体相互作用的集成模型的核心。 遗传筛查数据易于多种实验偏差，其中一些可以通过计算分析来减轻我们以前已经开发出复杂的软件工具（例如信号（通过迭代途径组选择）和网络分析循环：https://signal.niaid.nih.gov）。我们已将信号分析应用于先前描述的人类和小鼠巨噬细胞对LPS屏幕的筛选（Sun等人（2017）Sci。Data。4：170007; Li等人（2017）Sci。Data。4：170008）。对普遍富集途径的分析确定了先天免疫过程的预期富集，但对剪接体成分的强烈富集。在2022财年，我们使用长阅读（PACBIO）和深入（Illumina）测序的组合对LPS挑战的巨噬细胞中的RNA剪接进行了深入分析。这揭示了一个额外的调节层，该层延伸超出了LPS引起的众所周知的基因转录变化，即使在骨料基因转录水平没有变化的基因中，也具有强烈的差异同工型使用模式。与MDS患者相比，我们目前正在比较健康患者巨噬细胞中这些LPS诱导的剪接模式，以确定该PRR驱动的剪接反应中的缺陷是否可以解释该疾病中观察到的一些先天免疫失调。 除了我们对TLR4途径对细菌LP的响应的持续研究外，我们还扩展了研究以询问最近发现的胞质LPS传感途径，该途径激活了非典型的炎性体反应并释放IL-1家族炎性细胞因子。最近的研究表明，这是对LP的更广泛生理反应的关键组成部分，并且是革兰氏阴性细菌感染中败血性休克结果的主要贡献者。我们已经与NIH-NCATS筛查设施合作，以完成IL-1 Alpha对胞质LPS响应的基因组规模屏幕，我们正在积极研究该项目中出现的基因命中。在2022财年，我们继续研究线粒体和细胞代谢在炎性体激活中的重要作用。在从筛网中出现的基因中，我们已经鉴定出三种核苷酸双磷酸激酶，并且我们还进一步研究了NME4基因在炎性体激活中的作用。这项研究揭示了NME4依赖性线粒体适应性检查点，该检查点支持对感染和细胞因子释放的巨噬细胞反应。与该模型一致，我们发现NME4缺陷型小鼠由于炎症细胞因子反应减少而抵抗LPS诱导的内毒性休克。我们正在继续研究此屏幕上的进一步基因，这些基因可能会发现非典型炎症体的新型调节剂。 在2022财年，我们还与落基山实验室的Karin Petersons Niaid Lab进行了合作，以筛选宿主因素，以调节对La Crosse Bunyavirus（LACV）的易感性。 LACV是小儿脑炎的主要原因，因为只有儿童通过血液脑屏障妥协表现出对这种病原体的敏感性。我们正在使用深入的转录分析和靶向siRNA筛选的组合来识别候选基因，其年龄相关的差异表达可能是小儿对LACV的敏感性的基础。我们正在进行的研究有可能确定可以构成这种危险儿童疾病的新药物治疗基础的治疗靶标。