METHODS: THP-1 cells were stimulated with bDNA (80-100 ng/µl) from periodontal pathogens, including Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, Tannerella forsythia, and Fusobacterium nucleatum. Proinflammatory cytokine (IL-1beta) production was determined by ELISA (R&D). E.coli DNA (80ng/µl) and LPS (10 ng/ml) and calf thymus DNA (80-200ng/µl) were served as controls. DNA preparations were tested for LPS contamination using LAL assay (LONZA) and treated with polymyxin prior to stimulation. TLR9 expression on THP-1 cells was determined by real time PCR using TaqMan gene expression assays (Applied Biosystems). To elucidate the role of unmethylated CpG motifs and TLR9 signalling in bDNA-mediated cytokine induction, some experiments were carried out using methylated-bDNA while others were performed with THP-1 cells treated with chloroquine(10 µg/ml).
RESULTS: DNA from periodontal pathogens induced IL-1beta production from THP-1 cells in a time and dose-dependent manner. Methylation of bDNA resulted in significantly decreased cytokine production implicating that recognition of CpG motifs is involved in periodontal bDNA-mediated cytokine production. We also confirmed constitutive expression of TLR9 in THP1 cells and blocking TLR9 signalling with chloroquine significantly diminished cytokine production. Treatment of bDNA preparations with DNase completely abolished the cytokine response further providing evidence that the cytokine induction was the result of bDNA stimulation.
CONCLUSION: Periodontal bDNA has an ability to trigger IL-1beta production from human monocytic cells. In diseases involving multiple pathogens, such as periodontal diseases, elimination of whole bacteria may not be sufficient to stop inflammatory response. Therefore, alternative approaches targeted to eliminate bDNA might be beneficial to control inflammation and subsequently disease progression.(Supported by AD Williams Research Funds)