Odontoblasts are involved in innate immunity against invading microorganisms. However, the mechanisms of host inflammatory responses to bacterial DNA in odontoblasts are not fully understood. The purpose of this study was to investigate whether microbial cytidine-phosphate-guanosine (CpG) DNA influences interleukin-8 (IL-8) expression in odontoblasts and the signaling pathways involved.
The effect of CpG oligonucleotide (CpG ODN) on IL-8 mRNA and protein expression levels in the mouse odontoblast-like cell line MDPC-23 was investigated by real-time polymerase chain reaction (PCR) analysis and enzyme-linked immunosorbent assay (ELISA). Whether Toll-like receptor 9 (TLR9), myeloid differentiation marker 88 (MyD88), nuclear factor kappa B (NF-κB), or mitogen-activated protein kinase (MAPK) pathways were involved in the CpG ODN–induced IL-8 expression was determined by examined real-time PCR, ELISA, and luciferase activity assay. Extracellular signal-regulated kinase (ERK) activation and TLR9 protein expression were measured by Western blot analysis.
Exposure to CpG ODN induced significant up-regulation of IL-8 mRNA and protein in MDPC-23 cells. CpG ODN–induced IL-8 up-regulation was attenuated by TLR9 inhibitor (chloroquine) and MyD88 inhibitory peptide. CpG ODN also increased the expression of TLR9 mRNA and protein in MDPC-23 cells. Treatment of MDPC-23 cells with NF-κB inhibitors (pyrrolidine dithiocarbamate), IκBα phosphorylation inhibitors (Bay 117082), or IκB protease inhibitor (L-1-tosylamido-2-phenylethyl chloromethyl ketone) decreased CpG ODN–induced IL-8 expression. Furthermore, stimulation of cells with CpG ODN enhanced κB-luciferase activity, and the activity was diminished by the overexpression of dominant negative mutants of MyD88 and IκBα. In addition, CpG ODN–induced IL-8 expression was markedly suppressed by U0126, but not by SB203580 and SP600125. Moreover, CpG ODN activated ERK phosphorylation in a time-dependent manner.
These data demonstrate that CpG ODN–induced IL-8 expression was mediated through TLR9, MyD88, NF-κB, and ERK pathways in MDPC-23 cells and suggest a possible role for the CpG DNA–mediated immune response in odontoblasts with involvement of TLR9, MyD88, and ERK pathways in this process.
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Published online: April 06, 2012
Wenxi He and Yaqing Zhang contributed equally to this work.
Supported by grant 30872869 and grant 81070831 from the Chinese National Natural Science Foundation.
© 2012 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.