Interferon-mediated control mechanisms in human cells

人类细胞中干扰素介导的控制机制

• 批准号: 10041166
• 负责人: L. David Sibley
• 金额: $ 23.63万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-06-16 至 2022-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10041166
• 关键词: Adult; Animals; Anti-Bacterial Agents; Antibiotics; Antiviral Agents; Autophagocytosis; Back; Binding Proteins; Binding Sites; CRISPR screen; CRISPR/Cas technology; Candidate Disease Gene; Cell Line; Cells; Chronic; Complex; Cyst; DNA Binding; Development; Disease; GBP1 gene; Genes; Growth; Hematopoietic; Human; Immune response; Immune signaling; Immune system; Immunity; Immunocompromised Host; Individual; Infection; Infection Control; Interferon Type II; Interferon-beta; Interferons; Intervention; Knock-out; Knowledge; Lead; Libraries; Life; Lymphoid Cell; Maps; Mediating; Mediator of activation protein; Molecular; Monitor; Mus; Natural Immunity; Nitrogen; Nutrient; Nutrient Depletion; Outcome Study; Oxygen; Parasite Control; Parasites; Pathway interactions; Process; Production; Proteins; Resistance; Risk; Role; Rupture; System; T-Lymphocyte; Testing; Tissues; Toxoplasma; Toxoplasma gondii; Toxoplasmosis; Up-Regulation; Vaccines; Vacuole; Zoonoses; acute infection; adaptive immunity; antigen-specific T cells; antimicrobial; base; cell type; chronic infection; effective intervention; genome wide screen; genome-wide; guanylate; high throughput analysis; immune clearance; improved; in vitro Assay; macrophage; monocyte; novel; obligate intracellular parasite; overexpression; pathogen; response; transcription factor

Abstract Toxoplasma gondii is a widespread parasite of animals that causes zoonotic infection in humans. Although acute infections in healthy adults are normally resolved without serious consequences, infection of immunocompromised individuals can lead to serious complications. Toxoplasma gondii is an obligate intracellular parasite, capable of infecting all nucleated cells in the body, including monocytes and macrophages. Control of infection relies on production of IFN-γ, which is essential to upregulate antimicrobial pathways in both hematopoietic and non-hematopoietic cells. Interferons activate STAT transcription factors to upregulate interferon stimulated genes (ISGs), many of which participate in antimicrobial defenses. Compared to studies in the murine system, our knowledge of how these pathways control T. gondii replication in human cells is relatively limited. Treatment of human cells with type I (IFN-β) or type II (IFN-γ) interferon leads to control of T. gondii replication. A variety of different control pathways have been implicated including nutrient limitation, guanylate binding proteins, autophagy, and production of reactive oxygen or nitrogen species. However prior studies have only focused on a few of the components that operate in each pathway, limiting our knowledge of their relative importance. To provide a comprehensive analysis of ISGs that are required for IFN- mediated control of T. gondii, we have developed a FACS-based screen to monitor parasite replication in different human cell types. Genome wide CRISPR/Cas9 screens will be employed to identify genes whose loss results in an inability to control T. gondii replication in IFN-treated cells. In parallel, we will use a lentiviral over- expression strategy to screen libraries of type I and type II ISGs to identify effectors that induce control of parasite replication. Collectively these approaches should define the repertoire of human genes that are necessary and sufficient to control intracellular parasites. Understanding such mechanisms may be important in overcoming the resistance to clearance seen in chronic toxoplasmosis and may also be important in control of other intracellular pathogens.

抽象的 弓形虫是一种广泛传播的动物寄生虫，可引起人类人畜共患感染。 健康成人的急性感染通常会得到解决，不会产生严重后果， 免疫功能低下的个体可能会导致严重的弓形虫并发症。 细胞内寄生虫，能够感染体内所有有核细胞，包括单核细胞和 巨噬细胞的感染控制依赖于 IFN-γ 的产生，这对于上调抗菌药物至关重要。 干扰素激活造血细胞和非造血细胞中的 STAT 转录因子。 上调干扰素刺激基因 (ISG)，其中许多参与抗菌防御。 通过对小鼠系统的研究，我们了解这些途径如何控制弓形虫在人类中的复制 使用 I 型 (IFN-β) 或 II 型 (IFN-γ) 干扰素治疗人类细胞的效果相对有限。 弓形虫复制的控制涉及多种不同的控制途径，包括营养物质。 限制、鸟苷酸结合蛋白、自噬以及活性氧或氮的产生。 然而，之前的研究只关注在每个途径中起作用的几个组件，限制了我们的研究 了解它们的相对重要性，对 IFN-所需的 ISG 进行全面分析。 为了介导弓形虫的控制，我们开发了一种基于 FACS 的屏幕来监测寄生虫在体内的复制 不同的人类细胞类型将采用全基因组 CRISPR/Cas9 筛选来识别丢失的基因。 导致无法控制弓形虫在 IFN 处理的细胞中的复制。同时，我们将使用慢病毒过度治疗。 筛选 I 型和 II 型 ISG 文库的表达策略，以确定诱导控制的效应子 总的来说，这些方法应该定义人类基因的全部内容。 了解这些机制对于控制细胞内寄生虫来说是必要且充分的。 克服慢性弓形虫病中出现的清除阻力，并且对于控制也可能很重要 其他细胞内病原体。