CpG DNA modulates interleukin 1beta-induced interleukin-8 expression in human bronchial epithelial (16HBE14o-) 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-1beta 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-1beta and CpG. TLR9 siRNA was used to silence TLR9 prior to treatment with IL-1beta and CpG. IkappaBalpha and p38 were assessed by Western blot, and EMSA's were performed to determine NF-kappaB activation. To investigate IL-8 mRNA stability, cells were treated with IL-1beta 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-1beta-induced IL-8 protein abundance, however treatment with CpG alone had no effect. CpC (a control ODN) had no effect on IL-1beta-induced IL-8 levels. In addition, CpG synergistically upregulated TNFalpha-induced IL-8 expression. Silencing TLR9 using siRNA or pretreatment of cells with chloroquine had little effect on IL-1beta-induced IL-8 levels, but abolished CpG-induced synergy. CpG ODN had no effect on NF-kappaB 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-1beta compared to treatment with IL-1beta alone.


CONCLUSIONS
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.
Parilla
N William
NW
Division of Critical Care Medicine, Cincinnati Children's Hospital Medical Center, Cincinnati, OH, USA. N.william_Parilla@URMC.Rochester.edu
Hughes
Valerie S
VS
Lierl
Kristin M
KM
Wong
Hector R
HR
Page
Kristen
K
D001980:Bronchi / Q000187:drug effects* / Q000378:metabolism; D002461:Cell Line, Transformed; D018899:CpG Islands / Q000235:genetics; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism; D005786:Gene Expression Regulation; D006801:Humans; D053583:Interleukin-1beta; D016209:Interleukin-8 / Q000235:genetics / Q000378:metabolism*; D009838:Oligodeoxyribonucleotides / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D051217:Toll-Like Receptor 9 / Q000235:genetics / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism*
C408982:CPG-oligonucleotide; D053583:Interleukin-1beta; D016209:Interleukin-8; D009838:Oligodeoxyribonucleotides; D012333:RNA, Messenger; D034741:RNA, Small Interfering; C495371:TLR9 protein, human; D051217:Toll-Like Receptor 9; D014409:Tumor Necrosis Factor-alpha; D048051:p38 Mitogen-Activated Protein Kinases