title: Endosomal translocation of CpG-oligodeoxynucleotides inhibits DNA-PKcs-dependent IL-10 production in macrophages.
abstract: Synthetic oligodeoxynucleotides containing unmethylated CpG motifs (CpG-ODNs) function as powerful immune adjuvants by activating macrophages, dendritic cells, and B cells. However, the molecular recognition mechanism that initiates signaling in response to CpG-ODN has not fully been identified. We show in this study that peritoneal macrophages from SCID mice having mutations in the catalytic subunit of DNA-protein kinase (DNA-PKcs) were almost completely defective in the production of IL-10 and in ERK activation when treated with CpG-ODN. In contrast, IL-12 p70 production significantly increased. Furthermore, small interfering RNA (siRNA)-mediated knockdown of DNA-PKcs expression in the mouse monocyte/macrophage cell line RAW264.7 led to reduced IL-10 production and ERK activation by CpG-ODN. IL-10 and IL-12 p70 production, but not ERK activation, are blocked by chloroquine, an inhibitor of endosomal acidification. Endosomal translocation of CpG-ODN in a complex with cationic liposomes consisting of 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) (CpG-DOTAP-liposomes) decreased IL-10 production and ERK activation, whereas the endosomal escape of CpG-ODN in a complex with cationic liposomes consisting of DOTAP and dioleyl-phosphatidylethanolamine (DOPE) (CpG-DOTAP/DOPE-liposomes) increased. In contrast, IL-12 p70 production was increased by CpG-DOTAP-liposomes and decreased by CpG-DOTAP/DOPE-liposomes. IL-10 production induced by CpG-DOTAP/DOPE-liposomes was not observed in macrophages from SCID mice. Thus, our findings suggest that DNA-PKcs in the cytoplasm play an important role in CpG-ODN-induced production of IL-10 in macrophages. In addition, DNA-PKcs-mediated production of IL-10 and IL-12 p70 can be regulated by manipulating the intracellular trafficking of CpG-ODN in macrophages.
authors.affiliation: Tokyo University of Pharmacy and Life Science, Tokyo, Japan.
authors.forename: Satoshi
authors.initials: S
authors.lastname: Yotsumoto
authors.forename: Kazuharu
authors.initials: K
authors.lastname: Saegusa
authors.forename: Yukihiko
authors.initials: Y
authors.lastname: Aramaki
mesh_terms: D000818:Animals; D002460:Cell Line; D051747:DNA-Activated Protein Kinase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D004705:Endocytosis; D011992:Endosomes / Q000378:metabolism*; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D005229:Fatty Acids, Monounsaturated / Q000737:chemistry / Q000378:metabolism; D016753:Interleukin-10 / Q000096:biosynthesis*; D018664:Interleukin-12 / Q000378:metabolism; D008081:Liposomes / Q000737:chemistry; D017737:Macrophages, Peritoneal / Q000201:enzymology / Q000276:immunology*; D051379:Mice; D016513:Mice, SCID; D009687:Nuclear Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D009838:Oligodeoxyribonucleotides / Q000378:metabolism*; D000644:Quaternary Ammonium Compounds / Q000737:chemistry / Q000378:metabolism; D034741:RNA, Small Interfering / Q000494:pharmacology
chemical_list: C408982:CPG-oligonucleotide; D004268:DNA-Binding Proteins; D005229:Fatty Acids, Monounsaturated; D008081:Liposomes; D009687:Nuclear Proteins; D009838:Oligodeoxyribonucleotides; D000644:Quaternary Ammonium Compounds; D034741:RNA, Small Interfering; D016753:Interleukin-10; D018664:Interleukin-12; D051747:DNA-Activated Protein Kinase; C497264:Prkdc protein, mouse; D048049:Extracellular Signal-Regulated MAP Kinases; C070046:1,2-dioleoyloxy-3-(trimethylammonium)propane