Induction of immunological paralysis by CpG DNA

描述（由申请人提供）：如果不受控制，由CpG DNA诱导的促炎性免疫应答可能导致慢性炎性疾病和脓毒性休克样综合征。我们已经发现，巨噬细胞预先暴露于CpG DNA变得对随后的挑战与细菌产品的低反应。CpG DNA诱导巨噬细胞低反应性的机制尚未深入研究。本研究的长期目标是阐明CpG DNA诱导巨噬细胞低反应性的生化机制。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诱导的巨噬细胞低反应性中的生物学作用，并了解CpG DNA抑制IRAK 1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IRAK 1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IRAK 1表达的生化机制。特别是，我们将表征IRAK 1启动子区域，并确定CpG DNA是否通过内体pH敏感性TLR 9/MyD 88依赖性途径抑制IRAK 1表达。我们将使用小干扰RNA技术制备表达显性负性形式的信号调节剂的各种突变体RAW264.7细胞和特异性基因敲低的RAW264.7细胞。这些突变体RAW264.7细胞和特定基因敲除稳定转染子将与野生型和各种基因敲除小鼠的骨髓来源巨噬细胞一起用于研究特定信号调节剂在CpG DNA诱导的巨噬细胞低反应性和IRAK 1抑制中的作用。将通过ELISA、电泳迁移率变动试验、荧光素酶试验、实时PCR、体外激酶试验和蛋白质印迹试验分析细胞的选定细胞因子产生、各种基因表达以及转录因子和信号传导调节剂的活化。我们还将分析CpG DNA预防小鼠感染性休克的能力。通过增强我们对CpG DNA诱导的巨噬细胞低反应性机制的理解，本研究可能提供有用的信息，以预防感染性休克和应对感染性休克幸存者的免疫瘫痪。

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.