Induction of immunological paralysis by CpG DNA

描述(申请人提供)：如果不加以控制，CpG DNA诱导的促炎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品挑战反应迟钝。CpG DNA诱导巨噬细胞低反应性的机制尚未得到深入研究。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的表达和上调IRAK-M的表达而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，我们将确定IRAK1启动子区域的特征，并确定CpG DNA是否通过内体pH敏感的TLR9/MyD88依赖的途径抑制IRAK1的表达。我们将利用小干扰RNA技术使各种突变的RAW264.7细胞表达显性的负性信号调节剂和特定基因敲除的RAW264.7细胞。结合野生型和不同基因敲除小鼠骨髓来源的巨噬细胞，这些突变的RAW264.7细胞和特异性基因敲除稳定的转染体将被用来研究特定的信号调节因子在CpG DNA诱导的巨噬细胞低反应和IRAK1抑制中的作用。通过酶联免疫吸附试验、凝胶迁移率改变分析、荧光素酶分析、实时定量聚合酶链式反应、体外激酶分析和蛋白质印迹分析，分析细胞产生选定的细胞因子，各种基因的表达，以及转录因子和信号调节因子的激活。我们还将分析CpG DNA预防小鼠感染性休克的能力。通过加深对CpG DNA诱导的巨噬细胞低反应机制的了解，本研究可能为感染性休克的预防和应对感染性休克幸存者的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.