CpG DNA modulates interleukin 1β-induced interleukin-8 expression in human bronchial epithelial (16HBEI4o-) cells

Background: Recognition of repeat unmethylated CpG motifs from bacterial DNA through Toll-like receptor (TLR-9) has been shown to induce interleukin (IL)-8 expression in immune cells. We sought to investigate the role of CpG oligodeoxynucleotides (ODN) on a human bronchial epithelial cells. Methods: RT-PCR and Western blot analysis were used to determine expression of TLR-9 in human bronchial epithelial cells (16HBE14o-). Cells were treated with CpG ODN in the presence or absence of IL-1 beta and IL-8 protein was determined using ELISA. In some cases cells were pretreated with chloroquine, an inhibitor of TLR-9 signaling, or SB202190, an inhibitor of the mitogen activated protein kinase p38, prior to treatment with IL-1 beta and CpG. TLR9 siRNA was used to silence TLR9 prior to treatment with IL-1 beta and CpG. I kappa B alpha and p38 were assessed by Western blot, and EMSA's were performed to determine NF-kappa B activation. To investigate IL-8 mRNA stability, cells were treated with IL-1 beta in the absence or presence of CpG for 2 h and actinomycin D was added to induce transcriptional arrest. Cells were harvested at 15 min intervals and Northern blot analysis was performed. Results: TLR-9 is expressed in 16HBE14o- cells. CpG synergistically increased IL-1 beta-induced IL-8 protein abundance, however treatment with CpG alone had no effect. CpC ( a control ODN) had no effect on IL-1 beta-induced IL-8 levels. In addition, CpG synergistically upregulated TNF alpha-induced IL-8 expression. Silencing TLR9 using siRNA or pretreatment of cells with chloroquine had little effect on IL-1 beta-induced IL-8 levels, but abolished CpG-induced synergy. CpG ODN had no effect on NF-kappa B translocation or DNA binding in 16HBE14o- cells. Treatment with CpG increased phosphorylation of p38 and pretreatment with the p38 inhibitor SB202190 attenuated the synergistic increase in IL-8 protein levels. Analysis of the half-life of IL-8 mRNA revealed that IL-8 mRNA had a longer half-life following the co-treatment of CpG and IL-1 beta compared to treatment with IL-1 beta alone. Conclusion: Together, these data demonstrate that CpG modulates IL-8 synthesis in the presence of a pro-inflammatory mediator utilizing TLR9 and post-transcriptional mechanisms involving the activation of p38 and stabilization of IL-8 mRNA.