Epigenetic reprogramming of epithelial cell TLR signaling by Porphyromonas gingivalis

Pathogen mediated gain of epigenetic reprogramming in humans is recently being implicated in cancer and immune mediated diseases.  Periodontitis is an immune mediated disease instigated by a biofilm pathogen Porphyromonas gingivalis.  The host innate immune system recognizes P. gingivalis through TLR2/4.  Nevertheless, P. gingivalis induced epigenetic reprogramming remains to be elucidated. Objective: We aimed to characterize P. gingivalis induced epigenetic reprogramming in human gingival epithelial cells (HGECs) because we hypothesized that these modifications may render periodontal disease susceptibility.  Method: We have isolated HGECs from patients and characterized on their inflammatory responses to P. gingivlalis as ‘normal’ (normal cytokine responders) ‘hypo-responsive’ (diminished cytokine responders).  DNA from these cells was subjected to methylation specific qPCR array for the TLR gene network. The ‘normal’ cells were stimulated with P. gingivalis (MOI:5) for 30 minutes at 0, 4, 8 and 16 h time intervals.  After 48 h of last stimulation, the cells were split and repeated stimulation cycle with or without 1 mM of 5-Aza-2’-deoxycytidine (decitabine).  After which, TLR2 promoter CpG methylation was determined by methylation specific qPCR assay.  DNA from healthy and periodontitis affected tissue was subjected bisulfite sequencing to determine CpG methylation. Time course P. gingivalis stimulation was done and protein was subjected to immunoblot against histone antibodies.   Results: We identified TLR2 promoter CpG hypermethylation in ‘hypo-responsive’ cells.  This methylation status was reversed by the use of decitabine restoring the TLR2 and pro-inflammatory cytokine expression. We observed the induction of TLR2 CpG methylation by P. gingivalis in HGECs. Moreover, the tissue from periodontal sites showed sporadic TLR2 CpG methylation compared to healthy sites as revealed by bisulfite sequencing. We also observed rapid histone modifications upon P. gingivalis stimulation in HGECs. Conclusion: Here we show previously unknown specific epigenetic reprogramming of epithelial chromatin by P. gingivalis that may contribute to periodontal disease susceptibility.