Subgingival Plaque in Periodontal Health Antagonizes at Toll-Like Receptor 4 and Inhibits E-Selectin Expression on Endothelial Cells

Abstract

The ability of the subgingival microbial community to induce an inappropriate inflammatory response ultimately results in the destruction of bone and gingival tissue. In this study, subgingival plaque samples from both healthy and diseased sites in the same individual were obtained from adults with chronic periodontitis and screened for their ability to either activate Toll-like receptor 2 (TLR2) or TLR4 and to antagonize TLR4-specific activation by agonist, Fusobacterium nucleatum LPS. Subgingival plaque from diseased sites strongly activated TLR4, whereas matched plaque samples obtained from healthy sites were significantly more variable, with some samples displaying strong TLR4 antagonism, while others were strong TLR4 agonists when combined with F. nucleatum LPS. Similar results were observed when TLR4 dependent E-selectin expression by endothelial cells was determined. These results are the first to demonstrate TLR4 antagonism from human plaque samples and demonstrate that healthy but not diseased sites display a wide variation in TLR4 agonist and antagonist behavior. The results have identified a novel characteristic of clinically healthy sites and warrant further study on the contribution of TLR4 antagonism in the progression of a healthy periodontal site to a diseased one.

FIG 1

Bacterial load of matched healthy and diseased plaque samples. The copy number of 16S rRNA genes represent the total bacterial load for each sample. Differences were tested using a Wilcoxon matched-pair signed-rank test to determine the significance (*, P < 0.05). The results shown are means ± the standard deviations (SD) of three independent assays.

FIG 2

TLR2 (A) and TLR4 (B) activation by 100 μg ml⁻¹ subgingival plaque from matched healthy or diseased sites in chronic periodontitis patients. The TLR4- or TLR2-specific NF-κB activity was determined by calculating the ratio of NF-κB-dependent firefly luciferase activity to β-actin promoter-dependent Renilla luciferase activity (internal control). The results shown are means ± the standard deviations of three independent assays.

FIG 3

Relationships between TLR2- or TLR4-specific activation and clinical measurements of chronic periodontitis. TLR2 (A to D) and TLR4 (E to H) activation is represented by the NF-κB activity, and associations were determined with the corresponding clinical measurements: BOP status (A and E), plaque index (B and F), probing depth (C and G), and clinical attachment levels (D and H). The correlation was determined by calculating using the Spearman rank (r_s) to determine the significance (P < 0.05).

FIG 4

TLR4 antagonistic or stimulatory potential of matched diseased (A) and healthy (B) subgingival plaque. Each patient was designated a number and “D” or “H” label corresponds to diseased or healthy plaque, respectively. The percent stimulation of NF-κB above that of F. nucleatum LPS (FnLPS) activation alone was calculated by using the formula [(y − x)/x] × 100, where x is the NF-κB activity due to TLR4 stimulation with 100 ng/ml FnLPS alone, and y is activity of FnLPS combined with either plaque at 100 μg ml⁻¹ or Pg1435LPS control at 1 μg ml⁻¹. The results shown are means ± the standard deviations of triplicate wells determined in one of three independent assays. *, P < 0.05; **, P < 0.01.

FIG 5

Antagonism and stimulation of E-selectin expression of endothelial cells by matched diseased (A) and healthy (B) subgingival plaque. Each patient was designated a number and “D” or “H” label corresponds to diseased or healthy plaque, respectively. The percent stimulation of E-selectin expression above that of FnLPS activation alone was calculated as described for Fig. 4. The results shown are means ± the standard deviations of triplicate wells determined in one of three independent assays. *, P < 0.05; **, P < 0.01.