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CPG DNA引起的促炎性免疫反应不受控制，可能会导致慢性炎症性疾病和败血性休克样综合征。我们发现，预先暴露于CpG DNA的巨噬细胞对随后对细菌产物的挑战产生了反应。尚未深入研究CpG DNA诱导巨噬细胞低调的机制。我们研究的长期目标是阐明生化机制，通过这种机制，CpG DNA诱导了巨噬细胞的不足。为了实现这一长期目标，拟议的研究的目标是了解IRAK家族蛋白在CpG DNA诱导的巨噬细胞低调的生物学作用，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，我们将通过下调IRAK1表达和上调IRAK-M表达来确定CPG DNA是否在体内和体外诱导巨噬细胞的低温性。其次，我们将确定一种生化机制，通过该机制，CpG DNA降低了IRAK1表达。特别是，我们将表征IRAK1启动子区域，并将确定CPG DNA是否通过内体pH敏感的TLR9/MYD88依赖性途径抑制IRAK1表达。我们将使用小型干扰RNA技术制作各种突变的RAW264.7细胞，表达信号调节剂的主要负形式和特定基因敲低RAW264.7细胞。这些突变的RAW264.7细胞和特定基因敲低稳定转染剂将与野生型和各种基因敲除小鼠衍生的巨噬细胞一起，用于研究特定信号调节剂在CPG DNA诱导的巨噬细胞中的特定信号调节剂的作用。将通过ELISA，电泳迁移率转移测定法，荧光素酶测定，实时PCR，体外激酶测定法和Western Blot测定法对细胞的产生，各种基因的表达以及转录因子的激活以及转录因子的激活和信号调节剂的激活分析。我们还将分析CpG DNA预防小鼠败血性休克的能力。通过增强我们对CpG DNA诱导的巨噬细胞低调的机制的理解，这项研究可能会为防止败血性休克和应对败血性休克幸存者的免疫瘫痪提供有用的信息。