IADR Abstract Archives
Inflammatory and TLR2-Methylation Profiles of PBMCs in Apical Periodontitis
Objectives: To determine the inflammatory profile, TLR2 gene methylation patterns, and expression in peripheral mononuclear blood cells (PBMCs) from individuals with apical periodontitis (AP) and controls.
Methods: Cross-sectional study. Otherwise healthy individuals with AP (n=27) and controls (n=30) who consulted at the Faculty of Dentistry, Universidad de Chile were included. PMBCs were isolated by Ficoll gradient, cultured for 24 hours, and both RNA and DNA were extracted. DNA was bisulfite-treated, and specific sites at the promoter region of the TLR2 gene were amplified by PCR and sequenced. mRNA expression of TLR2 was determined by qPCR. The levels of TNF-α, IL-6, IL-10, IL-6Rα, IL-1β and IL-12p70 were measured in the supernatants by Multiplex assay. The results were analyzed with the software STATA V.12 (p<0.05).
Results: PBMCs demonstrated a proinflammatory profile showing higher soluble levels of TNF-α, IL-6 and IL-1β in AP compared to controls (p<0.05). Higher TLR2 expression was also found in AP compared to controls (p<0.05), although the global methylation pattern of the promoter region of the gene showed no differences (p>0.05). The CpG individual sites at positions -69, -16 and -12 were 100% demethylated in controls, being significantly different compared to AP (p<0.05). However, only the unmethylated state of CpG -16 and -12 sites was associated with increased TLR2 mRNA expression in both groups (p<0.05).
Conclusions: PBMCs from AP individuals demonstrate higher cytokine and TLR2 expression in association with the methylation state of the gene promoter’s single sites. Consequently, PBMCs contribute to the sustained systemic inflammatory load in individuals with periapical endodontic diseases.
Methods: Cross-sectional study. Otherwise healthy individuals with AP (n=27) and controls (n=30) who consulted at the Faculty of Dentistry, Universidad de Chile were included. PMBCs were isolated by Ficoll gradient, cultured for 24 hours, and both RNA and DNA were extracted. DNA was bisulfite-treated, and specific sites at the promoter region of the TLR2 gene were amplified by PCR and sequenced. mRNA expression of TLR2 was determined by qPCR. The levels of TNF-α, IL-6, IL-10, IL-6Rα, IL-1β and IL-12p70 were measured in the supernatants by Multiplex assay. The results were analyzed with the software STATA V.12 (p<0.05).
Results: PBMCs demonstrated a proinflammatory profile showing higher soluble levels of TNF-α, IL-6 and IL-1β in AP compared to controls (p<0.05). Higher TLR2 expression was also found in AP compared to controls (p<0.05), although the global methylation pattern of the promoter region of the gene showed no differences (p>0.05). The CpG individual sites at positions -69, -16 and -12 were 100% demethylated in controls, being significantly different compared to AP (p<0.05). However, only the unmethylated state of CpG -16 and -12 sites was associated with increased TLR2 mRNA expression in both groups (p<0.05).
Conclusions: PBMCs from AP individuals demonstrate higher cytokine and TLR2 expression in association with the methylation state of the gene promoter’s single sites. Consequently, PBMCs contribute to the sustained systemic inflammatory load in individuals with periapical endodontic diseases.