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Effect of TLR9 methylation on its transcription in apical inflammation.

AIM
to explore the methylation pattern, its role on transcriptional regulation and potential modifiers of methylation of theTLR9 gene in chronic periapical inflammation.


METHODOLOGY
In this cross-sectional study, apical lesions of endodontic origin (ALEO, n=61) and healthy periodontal ligaments (HPL, n=15) were included. Products from bisulfited and PCR-amplified DNA were analyzed for their methylation profiles in the promoter region and at each CpG island. Additionally, TLR9 mRNA levels were quantified by qPCR and bivariate and multiple modelling were performed to better understand the influence of methylations on gene transcription.


RESULTS
TLR9 mRNA levels were upregulated in ALEO compared to HPL (p<0.001). TLR9 promoter CpG sites and CpG +2086 in the intragenic island 1 were demethylated in ALEO compared to HPL (p<0.05). Multivariate analysis, adjusted by smoking and gender, revealed that demethylation of TLR9 promoter sites enhanced transcriptional activity, specifically demethylated CpGs at positions -736 and -683, (p=0.02), which are close to CRE binding. Whereas ALEO reduced the global methylation of the gene-promoter and intragenic-island 2 (p<0.05) by -42.5 and -9.5 percentage points, respectively, age reduced the global methylation of intragenic-island 3 within the exon 2.


CONCLUSIONS
Demethylations of TLR9 promoter CpG sites, along with the intragenic DNA methylation status, were involved in higher transcription in ALEO. Hence, chronic periapical inflammation and aging modify the methylation status both in the gene promoter and in intragenic CpG islands.