Induction of immunological paralysis by CpG DNA

描述（由申请人提供）：如果不受控制，CpG DNA诱导的促炎免疫反应可能导致慢性炎症性疾病和脓毒性休克样综合征。我们发现，预先暴露于CpG DNA的巨噬细胞对随后的细菌产物挑战反应迟钝。CpG DNA诱导巨噬细胞低反应性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，并将确定CpG DNA是否通过内体ph敏感的TLR9/ myd88依赖途径抑制IRAK1表达。我们将利用小干扰RNA技术制造各种表达显性负性信号调制剂形式的RAW264.7突变细胞和特异性基因敲除RAW264.7细胞。与野生型和各种敲除小鼠的骨髓源性巨噬细胞一起，这些突变RAW264.7细胞和特异性基因敲除稳定的转染物将被用来研究特异性信号调节剂在CpG dna诱导的巨噬细胞低反应性和IRAK1抑制中的作用。将通过ELISA、电泳迁移转移试验、荧光素酶试验、实时PCR、体外激酶试验和western blot试验分析细胞产生选定的细胞因子、各种基因的表达、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诱导的巨噬细胞低反应性机制的理解，本研究可能为感染性休克幸存者预防感染性休克和应对免疫瘫痪提供有用的信息。

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.