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5-aza-2'-deoxycytidine may regulate the inflammatory response of human odontoblast-like cells through the NF-κB pathway.

AIM
To explore the role of DNA methylation in the innate immunity of dental pulp, this study investigated the effect of 5-aza-2'-deoxycytidine (AZA) on lipoteichoic acid (LTA)-induced cytokine production and related intracellular signaling pathways in human odontoblast-like cells (hOBs).


METHODOLOGY
hOBs were cultured and differentiated from human dental pulp tissue and the odontoblastic phenotype of the cells were detected using immunofluorescence, qRT-PCR and western blotting. hOBs were pretreated with AZA and then stimulated with 10 μg/mL LTA. The levels of 42 cytokines related to immunity and inflammation were examined using a cytokine antibody array and verified using qRT-PCR and ELISA. The effect of AZA on the LTA-induced NF-κB and MAPK signaling pathways was explored using western blotting. The cells were treated with the specific NF-κB inhibitor PDTC and MAPK inhibitors (the ERK inhibitor U0126, the p38 inhibitor SB203580, and the JNK inhibitor SP600125) to further confirm the role of the signaling pathways in LTA-treated hOBs. DNA immunoprecipitation-PCR was used to examine the dynamic methylation status of the gene promoters of myeloid differentiation primary response 88 (MyD88) and tumor necrosis factor receptor associated factor 6 (TRAF6) in the LTA-induced hOBs. Statistical analyses of the differences between two groups were performed using Student's t-test. One-way analysis of variance (ANOVA) or repeated-measures ANOVA with a post hoc Dunnett's test were used to assess the differences between multiple sets of data. P<0.05 was considered to be statistically significant.


RESULTS
The odontoblastic markers were significantly higher in hOBs than those in human dental pulp cells (hDPCs) (P<0.05). According to the cytokine antibody array results, hOBs pretreated with AZA had significantly increased production of several inflammatory cytokines (P<0.05), in which the expression levels of IL-6 and IL-8 were the most dramatically increased upon LTA stimulation (P<0.01). Furthermore, AZA resulted in the significant upregulation of p-IKKα/β, p-IκBα, p-p65, p-p38 and p-ERK in LTA-stimulated hOBs (P<0.01). Treatment with the NF-κB pathway inhibitor suppressed both IL-6 and IL-8 expression (P<0.05), whereas inhibitors of the MAPK pathway (SB203580 and SP600125) did not. In LTA-treated hOBs, AZA significantly increased the expression levels of TRAF6 and MyD88 (P<0.05). AZA induced MyD88 promoter hypomethylation but did not affect TRAF6 methylation.


CONCLUSION
AZA regulated the LTA-induced inflammatory response through NF-κB signal pathway in hOBs. This study highlights the important role of DNA methylation in the immunity defense of odontoblasts during the dental pulp immunity response to caries.