Analysis of Subgingival Plaque Ability to Stimulate Toll-Like Receptor 2 and 4.

BACKGROUND: It has been shown that toll-like receptor (TLR) 2- and TLR4-stimulating abilities of supragingival plaque (SPP) are associated with periodontal conditions. It is hypothesized that SPP might affect the periodontium through its influence on subgingival plaque (SBP). This study investigates relationships between TLR2- and TLR4-stimulating abilities of SBP and periodontal conditions. METHODS: One hundred thirteen SBP samples were collected from the deepest pockets in patients with chronic periodontitis. TLR2- and TLR4-stimulating abilities were measured using genetically engineered nuclear factor-kappa B reporter cells. Numbers of Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans in each plaque sample were determined by real-time polymerase chain reaction. Peripheral blood mononuclear cells (PBMCs) were stimulated with SBP samples in presence or absence of TLR4 or TLR2 inhibitor. Production of tumor necrosis factor (TNF)-α and interleukin (IL)-8 was analyzed by enzyme-linked immunosorbent assay. RESULTS: TLR4-stimulating ability of SBP was associated with plaque index (PI), but not with other clinical parameters at sampling sites. TLR2-stimulating ability of SBP was associated with none of the parameters. Number of P. gingivalis and A. actinomycetemcomitans in each plaque sample was not associated with TLR2- or TLR4-stimulating ability of SBP. PBMCs stimulated with SBP samples produced TNF-α and IL-8, which was inhibited by TLR4 but not by TLR2 inhibitor. CONCLUSION: TLR4- but not TLR2-stimulating ability of SBP is associated with PI. Enhanced TLR4-stimulating ability at sites with accumulated plaque may mediate gingival inflammation.

Recombinant Porphyromonas gingivalis FimA preproprotein expressed in Escherichia coli is lipidated and the mature or processed recombinant FimA protein forms a short filament in vitro.

The gram-negative anaerobic bacterium Porphyromonas gingivalis is an etiologically important pathogen for chronic periodontal diseases in adults. Our previous study suggested that the major structural components of both Fim and Mfa fimbriae in this organism are secreted through their lipidated precursors. In this study, we constructed Escherichia coli strains expressing various fimA genes with or without the 5'-terminal DNA region encoding the signal peptide, and we determined whether lipidation of recombinant FimA proteins occurred in E. coli. Lipidation occurred for a recombinant protein from the fimA gene with the 5'-terminal DNA region encoding the signal peptide but not for a recombinant protein from the fimA gene without the signal-peptide-encoding region, as revealed by [3H]palmitic acid labeling experiments. A TLR2-dependent signaling response was induced by the recombinant protein from the fimA gene with the signal-peptide-encoding region but not by a recombinant protein from the fimA gene with the signal-peptide-encoding region that had a base substitution causing an amino acid substitution (C19A). Electron microscopic analysis revealed that recombinant FimA (A-47 - W-383) protein was autopolymerized to form filamentous structures of about 80 nm in length in vitro. The results suggest that FimA protein, a major subunit of Fim fimbriae, is transported to the outer membrane by the lipoprotein sorting system, and a mature or processed FimA protein on the outer membrane is autopolymerized to form Fim fimbriae.

Ability of supragingival plaque to induce toll-like receptor 4-mediated stimulation is associated with cytokine production by peripheral blood mononuclear cells.

BACKGROUND: In our previous study, we found that the ability of supragingival plaque to induce Toll-like receptor (TLR)4-mediated stimulation was positively associated with plaque score and bleeding on probing (BOP) at the sampled sites and that the ability to induce TLR2-mediated stimulation was negatively associated with probing depth (PD) and clinical attachment level (CAL). Because signaling from TLR leads to the induction of pro- and anti-inflammatory cytokines, we further analyzed the influence of the ability of supragingival plaque to induce TLR2-/TLR4-mediated stimulation of cytokine production by peripheral blood mononuclear cells (PBMCs). METHODS: The abilities of 125 plaque samples to induce TLR2- or TLR4-mediated stimulation were determined using genetically engineered Chinese hamster ovary reporter cells that express a reporter molecule upon activation of nuclear factor-kappa B through TLR2 or TLR4. PBMCs were stimulated with each plaque sample, and the production of proinflammatory cytokines (tumor necrosis factor-alpha and interleukin [IL]-6 and -8) and an anti-inflammatory cytokine (IL-10) was analyzed by enzyme-linked immunosorbent assay. RESULTS: The levels of the cytokines produced by PBMCs all correlated with the ability of supragingival plaque to induce TLR4-mediated stimulation but not with its ability to induce TLR2-mediated stimulation. Cytokine production was inhibited by an anti-TLR4 monoclonal antibody and a TLR4 antagonist, compound 406. The levels of cytokines were associated with plaque index, BOP, PD, and CAL at the sampled sites. CONCLUSIONS: The production of pro-/anti-inflammatory cytokines by PBMCs was associated with the ability of supragingival plaque to induce TLR4-mediated stimulation. The cytokines induced by supragingival plaque via TLR4 might modulate periodontal status.

Analysis of the activity to induce toll-like receptor (TLR)2- and TLR4-mediated stimulation of supragingival plaque.

BACKGROUND: The deleterious effects of the accumulation of supragingival plaque are well known, but the role of the proinflammatory property of supragingival plaque in periodontal diseases has not been completely elucidated. The aim of this study was to determine the relevance of Toll-like receptor (TLR)2- and TLR4-stimulating activity of supragingival plaque to periodontal parameters. METHODS: We isolated 144 supragingival plaque samples and analyzed TLR2- and TLR4-stimulating activity using genetically engineered Chinese hamster ovary reporter cells that express a reporter molecule upon activation of nuclear factor-kappa B through TLR2 or TLR4. The numbers of Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans (previously Actinobacillus actinomycetemcomitans), and Streptococcus mutans cells in each plaque sample were determined by real-time polymerase chain reaction. RESULTS: The activity to induce TLR4-mediated stimulation, but not TLR2-mediated stimulation, was positively associated with the plaque score and bleeding on probing score of the teeth from which the plaque samples were taken. The activity to induce TLR2-mediated stimulation, but not TLR4-mediated stimulation, was negatively associated with probing depth and clinical attachment level. The ratio of TLR4-/TLR2-mediated stimulation was positively associated with all of those parameters. The number of P. gingivalis cells in each plaque sample was associated with the plaque score and clinical attachment level, but no strong association was observed between the ratio of examined bacteria in each plaque sample and the activity to induce TLR2- or TLR4-mediated stimulation, except for a weak correlation between the ratio of A. actinomycetemcomitans cells and the activity to induce TLR4-mediated stimulation. CONCLUSION: The TLR2- and TLR4-stimulating activity of supragingival plaque is associated with clinical parameters for gingivitis and periodontitis.