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

Abstract

To explore the role of DNA methylation in the innate immunity of the dental pulp, this study investigated the effect of 5-aza-2'-deoxycytidine (AZA) on lipoteichoic acid (LTA)-induced cytokine production and related intracellular signalling pathways in human odontoblast-like cells (hOBs). hOBs were cultured and differentiated from human dental pulp tissue, and the odontoblastic phenotype of the cells was detected using immunofluorescence, qRT-PCR and Western blotting. hOBs were pretreated with AZA and then stimulated with 10μgmL-1 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 signalling 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 signalling 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 tumour 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 was used to assess the differences between multiple sets of data. P<0.05 was considered to be statistically significant. 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. AZA regulated the LTA-induced inflammatory response through the NF-κB signal pathway in hOBs. This study highlights the important role of DNA methylation in the immunity defence of odontoblasts during the dental pulp immunity response to caries.