Induction of immunological paralysis by CpG DNA

CpG DNA 诱导免疫麻痹

• 批准号: 6819585
• 负责人: AE-KYUNG YI
• 金额: $ 29.2万
• 依托单位: UNIVERSITY OF TENNESSEE HEALTH SCI CTR
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-05-01 至 2009-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6819585
• 关键词: CpG islands; DNA; RNA interference; bacterial DNA; biochemistry; biological signal transduction; cytokine receptors; enzyme linked immunosorbent assay; gel mobility shift assay; gene expression; genetically modified animals; immune response; immunoregulation; inflammation; laboratory mouse; macrophage; pathologic process; protein structure function; septic shock; septicemia; small interfering RNA; toll like receptor; western blottings

DESCRIPTION (provided by applicant): If uncontrolled, pro-inflammatory immune response induced by CpG DNA may result in chronic inflammatory diseases and a septic shock-like syndrome. We have found that macrophages pre-exposed to CpG DNA become hyporesponsive to subsequent challenge with bacterial products. The mechanisms by which CpG DNA induces macrophage hyporesponsiveness have not been intensively studied. The long-term objective of our study is to the elucidate biochemical mechanisms by which CpG DNA induces hyporesponsiveness of macrophages. To accomplish this long-term objective, the goals of the proposed study are to understand the biological role of IRAK family proteins in CpG DNA-induced macrophage hyporesponsiveness and to understand the biochemical mechanisms by which CpG DNA represses IRAK1. We propose to pursue these goals with the following two specific aims. First, we will determine whether CpG DNA induces macrophage hyporesponsiveness in vivo and in vitro by down-regulating IRAK1 expression and up-regulating IRAK-M expression. Second, we will determine a biochemical mechanism by which CpG DNA down-regulates IRAK1 expression. In particular, we will characterize the IRAK1 promoter region and will determine whether CpG DNA suppresses IRAK1 expression through an endosomal pH-sensitive TLR9/MyD88-dependent pathway. We will make various mutant RAW264.7 cells expressing a dominant negative form of signaling modulators and specific gene knockdown RAW264.7 cells using the small interfering RNA technique. Together with bone marrow derived macrophages from wild type and various knockout mice, these mutant RAW264.7 cells and specific gene knockdown stable transfectants will be used to investigate the roles of specific signaling modulators in the CpG DNA-induced macrophage hyporesponsiveness and IRAK1 inhibition. The cells will be analyzed for production of selected cytokines, expression of various genes, and activation of transcription factors and signaling modulators by ELISA, electrophoretic mobility shift assay, luciferase assay, real-time PCR, in vitro kinase assay, and western blot assay. We will also analyze the ability of CpG DNA to prevent septic shock in mice. By enhancing our understanding of the mechanisms of CpG DNA-induced macrophage hyporesponsiveness, this study may provide information useful to preventing septic shock and coping with immune paralysis in septic shock survivors.

描述（由申请人提供）：如果不加控制，CpG DNA 诱导的促炎性免疫反应可能会导致慢性炎症性疾病和感染性休克样综合征。我们发现，预先暴露于 CpG DNA 的巨噬细胞对随后的细菌产物攻击反应迟钝。 CpG DNA 诱导巨噬细胞反应低下的机制尚未得到深入研究。我们研究的长期目标是阐明 CpG DNA 诱导巨噬细胞反应性低下的生化机制。为了实现这一长期目标，本研究的目标是了解 IRAK 家族蛋白在 CpG DNA 诱导的巨噬细胞反应低下中的生物学作用，并了解 CpG DNA 抑制 IRAK1 的生化机制。我们建议通过以下两个具体目标来实现这些目标。首先，我们将确定 CpG DNA 是否通过下调 IRAK1 表达和上调 IRAK-M 表达在体内和体外诱导巨噬细胞低反应性。其次，我们将确定 CpG DNA 下调 IRAK1 表达的生化机制。特别是，我们将表征 IRAK1 启动子区域，并确定 CpG DNA 是否通过内体 pH 敏感的 TLR9/MyD88 依赖性途径抑制 IRAK1 表达。我们将使用小干扰RNA技术制备表达显性失活形式的信号调节剂的各种突变RAW264.7细胞和特定基因敲低的RAW264.7细胞。与来自野生型和各种敲除小鼠的骨髓来源的巨噬细胞一起，这些突变体 RAW264.7 细胞和特定基因敲低的稳定转染子将用于研究特定信号调节剂在 CpG DNA 诱导的巨噬细胞反应低下和 IRAK1 抑制中的作用。将通过 ELISA、电泳迁移率变动测定、荧光素酶测定、实时 PCR、体外激酶测定和蛋白质印迹测定来分析细胞的选定细胞因子的产生、各种基因的表达以及转录因子和信号调节剂的激活。我们还将分析 CpG DNA 预防小鼠感染性休克的能力。通过加深我们对 CpG DNA 诱导的巨噬细胞反应低下机制的理解，这项研究可能为预防感染性休克和应对感染性休克幸存者的免疫麻痹提供有用的信息。