Sensors and signaling of the interferon stimulatory DNA (ISD) pathway

干扰素刺激 DNA (ISD) 通路的传感器和信号传导

• 批准号: 8894192
• 负责人: Daniel B Stetson
• 金额: $ 42.92万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-08-01 至 2015-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8894192
• 关键词: Address; Antiviral Response; Autoimmune Diseases; Bacteria; Binding; Cell Death; Cell Line; Cells; Chronic; Clustered Regularly Interspaced Short Palindromic Repeats; Cyclic GMP; DNA; DNA Binding; DNA Viruses; DNA receptor; Detection; Dinucleoside Phosphates; Evaluation; Family; Gene Targeting; Genes; Genome engineering; Health; Host Defense; Human; Human Cell Line; Immune; Immune response; Immunity; Infection; Interferon Activation; Interferon Type I; Interferons; Knock-out; Ligands; Mediating; Mus; Pathway interactions; Play; Production; Receptor Gene; Regulation; Relative (related person); Retroviridae; Role; Signal Pathway; Signal Transduction; Subfamily lentivirinae; Testing; Tropism; Vaccination; Virus Diseases; adapter protein; base; cytokine; in vivo; monocyte; mouse model; novel; nucleic acid detection; plasmid DNA; receptor; response; sensor; tool

DESCRIPTION (provided by applicant): Innate immune detection of nucleic acids is essential for protective immunity against viral infection, but can cause autoimmune disease if dysregulated. This application is based on the study of an antiviral response triggered by detection of intracellular DNA called the interferon stimulatory DNA (ISD) pathway. Despite the established importance of DNA sensing in immunity and certain human autoimmune diseases, the identities of the DNA receptors that activate these responses remains controversial. In particular, AIM2-like receptors (ALRs) and cyclic GMP-AMP synthase (cGAS) have emerged as attractive candidate DNA sensors, but the relative contributions of these receptors to immunity, alone and together, remain to be established. We developed novel tools to explore the functions of ALRs, cGAS, and other candidate DNA sensors: mice that lack all thirteen ALR genes, and knockout human cell lines lacking candidate DNA sensors. We will use these exciting new tools to address the following specific aims. Aim 1: What are the relative roles of murine ALRs and cGAS in intracellular DNA sensing? Using cells from mice that lack all ALRs, cGAS, or both ALRs and cGAS, we will perform a comprehensive evaluation of DNA-activated innate immune responses to pure DNA ligands, as well as to infections with DNA viruses and intracellular bacteria. Aim 2: What are the in vivo roles of candidate DNA sensors in host defense and autoimmune disease? We will establish the relative contributions of ALRs and cGAS to activation of immunity in vivo using a plasmid DNA-based vaccination approach. We will then determine the roles of ALRs and cGAS in a mouse model of autoimmune disease caused by chronic activation of the ISD pathway. Aim 3: What are the functions of candidate DNA sensors in human cells? We have used CRISPR-mediated genome engineering to achieve biallelic disruption of genes in the relevant human monocyte cell line THP-1. We will establish panels of gene-targeted THP-1 cells for a rigorous analysis of the roles of candidate DNA sensors in activation of the ISD pathway and the inflammasome in response to pure ligands, and during infection with DNA viruses and lentiviruses that have exclusive tropism for human cells. Together, these proposed studies will enable us to explore in detail the mechanisms of intracellular DNA sensing in both mouse and human cells.

描述（由申请人提供）：核酸的先天免疫检测对于防止病毒感染的保护性免疫至关重要，但如果失调，可能会引起自身免疫性疾病。该应用基于对检测到称为干扰素刺激DNA（ISD）途径的细胞内DNA触发的抗病毒反应的研究。尽管DNA感应在免疫和某些人类自身免疫性疾病中具有确定的重要性，但激活这些反应的DNA受体的身份仍然存在争议。特别是，AIM2样受体（ALR）和环状GMP-AMP合酶（CGA）已成为有吸引力的候选DNA传感器，但是这些受体对免疫的相对贡献（单独及其共同）仍然待确定。我们开发了新的工具来探索ALR，CGA和其他候选DNA传感器的功能：缺乏所有13个ALR基因的小鼠以及缺乏候选DNA传感器的敲除人类细胞系。我们将使用这些激动人心的新工具来解决以下特定目标。 AIM 1：鼠ALR和CGA在细胞内DNA传感中的相对作用是什么？使用缺乏所有ALR，CGA或ALRS和CGA的小鼠的细胞，我们将对DNA激活的先天免疫反应对纯DNA配体以及DNA病毒和细胞内细菌感染进行全面评估。目标2：候选DNA传感器在宿主防御和自身免疫性疾病中的体内角色是什么？我们将使用基于质粒DNA的疫苗接种方法来建立ALR和CGA对体内免疫激活的相对贡献。然后，我们将确定由ISD途径长期激活引起的ALR和CGA在自身免疫性疾病的小鼠模型中的作用。 AIM 3：人类细胞中候选DNA传感器的功能是什么？我们已经使用CRISPR介导的基因组工程来实现相关人类单核细胞系THP-1中基因的双重破坏。我们将建立一组基因靶向的THP-1细胞，以严格分析候选DNA传感器在ISD途径激活中的作用和响应于纯配体的炎症体，以及在感染DNA病毒和andiveruse期间，具有独家恐怖症对于人类细胞。这些提出的研究一起将使我们能够详细探讨小鼠和人类细胞中细胞内DNA感应的机制。