CpG motifs in bacterial DNA evokes peroxynitrite signaling in human neutrophils

Abstract

Bacterial DNA is emerging as an important regulator of functions of human neutrophil granulocytes. Bacterial DNA contains short sequences of nonmethylated CpG dinucleotides (CpG-DNA) that are recognized by TLR-9. Evidence suggests that peroxynitrite (ONOO-) may function as an intracellular signal for cytokine production in neutrophils. We investigated whether CpG-DNA evokes ONOO- signaling in neutrophils. High purity (>99.9%) human neutrophils express TLR-9 and respond to CpG-DNA (0.2-10 μg/ml), but not to thymus DNA, with secretion of IL-8 and, to a considerably lesser extent, IL-6. Methylation of cytosines in CpG-DNA resulted in a complete loss of activity. CpG-DNA-induced IL-8 mRNA expression and IL-8 release were blocked by inhibitors of endosomal acidification and L-NAME. CpG-DNA evoked concomitant formation of superoxide and NO, coinciding with ONOO- formation (assessed by dihydrorhodamine oxidation and nitrotyrosine formation). RT-PCR amplified endothelial NO synthase in neutrophils cultured with or without CpG-DNA. CpG-DNA evoked rapid mobilization of NF-kB and c-Fos to the nucleus that were attenuated by L-NAME parallel with inhibition of IL-8 mRNA expression. Pharmacological blockade of NF-kB activation attenuated ~75% of CpG-DNA-evoked IL-8 release. These results provide evidence that bacterial DNA and unmethylated CpG motifs in particular activate ONOO- signaling in neutrophils, underlying IL-8 gene and protein expression through activation of NF-kB and c-Fos. (Supported by CIHR MOP-64283).