Decoding the Interferome by Mapping Genetic Interactions in Human Tissue

通过绘制人体组织中的遗传相互作用来解码干扰素

• 批准号: 10725446
• 负责人: Ryan Gilbert Gaudet
• 金额: $ 48.31万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-08-10 至 2028-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10725446
• 关键词: 3-Dimensional; Anatomy; Antibacterial Response; Bacterial Infections; Biological; Biological Assay; Bordetella pertussis; Buffers; CRISPR screen; Cell Communication; Cell Lineage; Cells; Chlamydophila pneumoniae; Chromosome Mapping; Clustered Regularly Interspaced Short Palindromic Repeats; Collaborations; Complex; Cytokine Signaling; Cytosol; Endothelium; Epithelium; Fibroblasts; Frustration; Future; Gene Combinations; Genes; Genetic; Genetic Screening; Genetic Transcription; Genomics; Goals; Health; Host Defense; Host resistance; Human; Human Genome; Image; Immune; Immune response; Immune signaling; Immune system; Immunity; Immunology; Individual; Infection; Interferon Type II; Interferons; Intestines; Link; Lung; Lymphocyte; Macrophage; Maps; Mediating; Mediator; Methods; Microbe; Modeling; Molecular; Network-based; Organ; Organ Donor; Organoids; Outcome; Pathologic; Pathology; Pathway interactions; Pattern; Phenotype; Postdoctoral Fellow; Predisposition; Proteins; Protozoan Infections; Role; Shapes; Shelter facility; Signal Transduction; Site; Structure; Structure of parenchyma of lung; System; Techniques; Tissues; Validation; Virus Diseases; Vision; Work; airway epithelium; arm; bactericide; cell type; cellular imaging; combat; combinatorial; cytokine; cytokine release syndrome; empowerment; functional outcomes; gene function; gene network; gene product; genomic locus; human model; human tissue; immunopathology; infancy; innovation; insight; intestinal epithelium; lung pathogen; mutant; novel; novel strategies; pathogen; programs; response; single-cell RNA sequencing; synergism; technology development; therapeutic target; virtual

PROJECT SUMMARY Non-immune cells greatly outnumber professional immune cells in the body, providing intracellular pathogens with many opportunities to shelter and take refuge. How does the immune system protect this huge landscape? My previous work described the ability of the immune cytokine interferon-g (IFN-g), classically considered a macrophage activating protein, to broadly activate non-immune cells and confer the ability to mount sterilizing cell-intrinsic responses through effectors encoded by Interferon-Stimulated Genes (ISGs), collectively termed the ‘interferome’. However, our understanding of how ISGs execute tissue localized responses is in its infancy, with remarkably few of these effectors being well characterized and virtually nothing known about how multiple ISGs functionally interact to achieve host defense. The principal barrier to understanding ISGs is that they rarely work in isolation; rather they are part of complex genetic networks that are buffered from the phenotypic effects of perturbation. I propose a radical new strategy that exploits this genetic complexity by mapping functional relationships between ISGs using combinatorial forward genetic screens. My central hypothesis is that systematically mapping genetic interactions will uncover the effectors of localized IFN-g signalling and enable hierarchical organization of ISG products into functional complexes and pathways (network) that execute specific protective and pathological responses. On a small scale with a single query gene in my postdoctoral work, this approach unveiled a pair of synergistic ISGs that execute unexpectedly potent bactericidal defense of the cytosol. Further development of this technology for use at a larger scale in my own lab will provide the framework to fully deconvolve the interferome into distinct host resistance pathways. I will develop an experimental pipeline that enlists a pooled CRISPR-based screening system for multi-locus gene perturbation paired with a novel single cell imaging and expression analysis platform. Focusing on the human airway epithelium, this innovative approach will be applied to dissect the ISG-encoded effectors that mediate cell-intrinsic control of three diverse pulmonary pathogens (C. pneumoniae, B. pertussis, and RSV), and those that mediate lung tissue damage downstream of cytokine storm. Newly identified ISGs and their connections will be validated for their protective or pathological activities using tissue explant systems from donated human lungs and models of human lung organoids. The ensuing genetic interaction network will provide unprecedented insight into the effectors that dictate infection outcome in the human lung during type 1 immune responses. These findings will reveal new local therapeutic targets and establish a paradigm for appreciating the full spectrum of immunity. The approaches pioneered here will also extend to the downstream effectors of other non-immune cells and tissues whose integrated study will define a new biological landscape of critical importance to human health.

项目总结 在体内，非免疫细胞的数量远远超过专业免疫细胞，提供了细胞内的病原体 有很多机会躲避和避难。免疫系统如何保护这片辽阔的土地？ 我之前的工作描述了免疫细胞因子干扰素-g(干扰素-g)的能力，经典的认为是 巨噬细胞激活蛋白，广泛激活非免疫细胞并赋予其杀菌能力 通过干扰素刺激基因(ISGs)编码的效应器的细胞本征反应，统称为 “干扰体”。然而，我们对ISGs如何执行组织局部反应的理解还处于初级阶段， 这些效应器中很少有被很好地刻画出来的，而且几乎不知道 ISGS在功能上进行交互以实现主机防御。理解ISG的主要障碍是它们很少 它们是孤立工作的；相反，它们是复杂遗传网络的一部分，这些遗传网络缓冲了表型效应 微扰的影响。我提出了一种激进的新策略，通过映射功能来利用这种遗传复杂性 使用组合正向遗传筛选的ISG之间的关系。我的中心假设是 系统地绘制遗传相互作用图将揭示局部干扰素-g信号的效应器并使 将ISG产品分层组织成执行特定任务的功能复合体和路径(网络) 保护性和病理性反应。在我的博士后工作中，在一个小范围内只有一个查询基因，这 Approach推出了一对协同的ISG，它们执行了出人意料的强大杀菌防御 胞浆。进一步开发这项技术，以便在我自己的实验室中更大规模地使用，将提供框架 以将干扰体完全解卷为不同的寄主抗性途径。我将开发一条试验性管道 这使得基于集合CRISPR的多基因座基因扰动筛查系统与一种新的 单细胞成像和表达分析平台。聚焦于人类呼吸道上皮，这一创新 该方法将被用来剖析ISG编码的效应器，这些效应器介导了三种不同的细胞内在控制 肺部病原体(肺炎衣原体、百日咳杆菌和呼吸道合胞病毒)以及那些介导肺组织损伤的病原体 在细胞因子风暴的下游。新确定的ISG及其连接将经过验证，以确保其保护 或使用来自捐赠的人肺和人肺模型的组织外植体系统的病理活动 有机化合物。随之而来的遗传相互作用网络将提供对影响因素的前所未有的洞察 在1型免疫反应中决定人类肺部的感染结果。这些发现将揭示新的 以局部治疗为靶点，并建立评价全谱免疫的范例。这个 这里首创的方法也将延伸到其他非免疫细胞和组织的下游效应器 其综合研究将确定对人类健康至关重要的新的生物景观。