EPIGENETIC AND TRANSCRIPTIONAL DYNAMIC IN PERIODONTAL DISEASE

Abstract

Objectives: Several studies have shown the involvement of epigenetics with periodontal disease. Since functional dissociation of Paracellular permeability is expected during bacterial infection, we hypothesize that the methylation of host oral epithelial DNA represents an important element in the disruption of barrier function and pathogenesis of periodontal diseases. With this In vitro study, we aimed to assess whether there is altered epithelial permeability measuring trans epithelial resistance after Porphyromonas gingivalis (P. gingivalis), Campylobacter rectus (C. rectus) and Fusobacterium nucleatum (F. nucleatum) infection. Plakophilin2 (PKP2) methylation status and expression levels were also investigated. In addition, investigate the potential effects of DNA methyltransferase (DNMT) inhibitors on epithelial barrier function in response to infection with periodontal pathogen in human gingival epithelial cells. Methods: Primary human gingival epithelial cells (HGEPs) were stimulated with P. gingivalis, strain, C. rectus and F. nucleatum (MOI 50) either in the presence or absence of DNMT inhibitors (10 μM of RG108 or EGCG). CellTiter-Blue® Cell Viability Assay (Promega) was used to determine an optimum cell density and maximum inhibitor concentration at which cell viability is maintained. Transepithelial electrical resistance (TER) at various time points were performed using an EVOM® electrical resistance system. DNA methylation was quantified by PCR using EpiTect Methyl II PCR Primer Assays for PKP2. Immunofluorescence analysis was performed using PKP2 antibody and analysis performed using Zeiss710 confocal microscope. Results: Exposure of HGEPs to P. gingivalis resulted in decreased TER (P=<0.001) associated with increased cell permeability. Methylation assays showed increased methylation levels of the PKP2 in comparison to non-infected controls (P=<0.001) and an associated PKP2 down-regulation (P=<0.005). For infected cells treated with DNMT inhibitors, PKP2 mRNA expression was increased (P=<0.001) and TER values similar to non-infected cells. Comparatively, immunofluorescent staining of the PKP2 protein showed reduced protein expression in infected cells not treated with DNMT inhibitors. Conclusion: DNA methylation levels of PKP2 can affect epithelial barrier function potentially conferring increased susceptibility to infection. DNMT inhibitors can affect cell adhesion dissociation in response to infection minimizing the disturbance to the barrier function.