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.
