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 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). METHODOLOGY: 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 10 µg mL-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. 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/ß, 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 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.

MyD88 hypermethylation mediated by DNMT1 is associated with LTA-induced inflammatory response in human odontoblast-like cells.

Dental caries is a chronic, infectious, and destructive disease that allows bacteria to break into the dental pulp tissue. As caries-related bacteria invade the human dentinal tubules, odontoblasts are the first line of dental pulp that trigger the initial inflammatory and immune responses. DNA methylation is a key epigenetic modification that plays a fundamental role in gene transcription, and its role in inflammation-related diseases has recently attracted attention. However, whether DNA methylation regulates the inflammatory response of human odontoblasts is still unknown. In the present study, we investigated the expression of DNA methyltransferase (DNMT)-1 in lipoteichoic acid (LTA)-stimulated human odontoblast-like cells (hOBs) and found that DNMT1 expression showed a decline that is contrary to the transcription of inflammatory cytokines. Knockdown of the DNMT1 gene increased the expression of several cytokines, including IL-6 and IL-8, in the LTA-induced inflammatory response. DNMT1 knockdown increased the phosphorylation of IKKα/ß, IκBα, and p65 in the NF-κB pathway and the phosphorylation of p38 and ERK in the MAPK pathway; however, only the NF-κB pathway inhibitor PDTC suppressed both IL-6 and IL-8 expression, whereas inhibitors of the MAPK pathway (U0126, SB2035580, and SP600125) did not. Furthermore, DNMT1 knockdown upregulated the expression of MyD88 and TRAF6 but only attenuated the MyD88 gene promoter methylation in LTA-treated hOBs. Taken together, these results demonstrated that DNMT1 depletion caused hypomethylation and upregulation of MyD88, which resulted in activation of the NF-κB pathway and the subsequent release of LTA-induced inflammatory cytokines in hOBs. This study emphasizes the critical role of DNA methylation in the immune defense of odontoblasts when dental pulp reacted to caries.

Expression and distribution of three transient receptor potential vanilloid(TRPV) channel proteins in human odontoblast-like cells.

Odontoblasts have been suggested to contribute to nociceptive sensation in the tooth via expression of the transient receptor potential (TRP) channels. The TRP channels as a family of nonselective cation permeable channels play an important role in sensory transduction of human. In this study, we examined the expression of transient receptor potential vanilloid-1 (TRPV1), transient receptor potential vanilloid-2 (TRPV2) and transient receptor potential vanilloid-3 (TRPV3) channels in native human odontoblasts (HODs) and long-term cultured human dental pulp cells with odontoblast phenotyoe (LHOPs) obtained from healthy wisdom teeth with the use of immunohistochemistry (IHC), immunofluorescence (IF), quantitative real-time polymerase chain reaction (qRT-PCR),western blotting (WB) and immunoelectron microscopy (IEM) assay. LHOPs samples were made into ultrathin sections, mounted on nickel grids, floated of three TRPV antibodies conjugated with 10 nm colloidal gold particles and observed under IEM at 60,000 magnifications. The relative intracellular distributions of these three channels were analyzed quantitatively on IEM images using a robust sampling, stereological estimation and statistical evaluation method. The results of IHC and IF convinced that TRPV1, TRPV2 and TRPV3 channels were expressed in native HODs and (LHOPs). The result of qRT-PCR and WB confirmed that the gene and protein expression of TRPV1, TRPV2, and TRPV3 channels and TRPV1 mRNA are more abundantly expressed than TRPV2 and TRPV3 in HODs (P < 0.05). Quantitative analysis of IEM images showed that the relative intracellular distributions of these three channels are similar, and TRPV1, TRPV2 and TRPV3 proteins were preferential labeled in human odontoblast processes, mitochondria, and endoplasmic reticulum. Thus, HODs could play an important role in mediating pulp thermo-sensation due to the expression of these three TRPV channels. The difference of relative intracellular distributions of three channels suggests that special structures such as processes may have an important role to sensing of the outer stimuli first.