Phosphatidylinositol 3-kinase-mediated regulation of IL-10 and IL-12 production in macrophages stimulated with CpG oligodeoxynucleotide.

The aim of this study was to clarify the role of phosphatidylinositol 3-kinase (PI3K) on the production of interleukine-10 (IL-10) and IL-12 in mouse peritoneal macrophages stimulated with CpG-ODN. CpG-ODN-induced IL-10 mRNA expression and protein production decreased following the treatment of macrophages with wortmannin and LY294002, specific inhibitors for PI3K. In contrast, IL-12 p40 mRNA expression and p70 protein production increased. Neutralizing anti-IL-10 monoclonal antibody (anti-IL-10 mAb) exactly mimicked the effects of PI3K inhibitors to enhance IL-12 p70 production. The enhancement effect of PI3K inhibitors on IL-12 p70 production almost completely disappeared by the treatment with anti-IL-10 mAb. PI3K inhibitors suppressed the activation of extracellular-regulated kinase (ERK), a member of the mitogen-activated protein kinases, by CpG-ODN. A specific ERK inhibitor, U0126, as well as PI3K inhibitors, differentially regulated IL-10 and IL-12 p70 productions. These results suggest that PI3K positively and negatively regulates the production of CpG-ODN-induced IL-10 and IL-12 p70, respectively, and negatively regulates IL-12 p70 production in macrophages through ERK-mediated IL-10 induction.

Endosomal translocation of CpG-oligodeoxynucleotides inhibits DNA-PKcs-dependent IL-10 production in macrophages.

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.