Divergence of the systemic immune response following oral infection with distinct strains of Porphyromonas gingivalis

Abstract

Periodontitis is a polymicrobial oral infection characterized by the destruction of tooth-supporting structures that can be linked to systemic diseases such as cardiovascular disease, diabetes or rheumatoid arthritis. Porphyromonas gingivalis, a bacterium implicated in the etiology of periodontitis, has shown variation in inducing T-cell responses among different strains. Therefore, in this study we investigated the strain-specific immune response using a murine experimental model of periodontitis. Periodontitis was induced by P. gingivalis strains A7A1-28, W83 and W50, and later confirmed by the presence of P. gingivalis in the oral microflora and by alveolar bone resorption. Splenocytes were evaluated for gene expression, cellular proteins and cytokine expression. Dendritic cells were stimulated in vitro for T helper cell-cytokine profiling. Results showed that P. gingivalis had the ability to alter the systemic immune response after bacterial exposure. Strains W50 and W83 were shown to induce alveolar bone loss, whereas the A7A1-28 strain did not significantly promote bone resorption in mice. Splenocytes derived from mice infected with strains W50 and W83 induced expression of high levels of interleukin-4 (IL-4) but A7A1-28 stimulated increased IL-10. Stimulation of dendritic cells in vitro showed a similar pattern of cytokine expression of IL-12p40, IL-6 and transforming growth factor-β among strains. A distinct systemic response in vivo was observed among different strains of P. gingivalis, with IL-10 associated with the least amount of alveolar bone loss. Evaluation of pathogen-driven systemic immune responses associated with periodontal disease pathogenesis may assist in defining how periodontitis may impact other diseases.

Figure 1

Schematic diagram illustrating the study experimental design. This timeline displays the in vivo experiment, including mice acclimation, antibiotic treatment, Porphyromonas gingivalis (Pg) infection period and periodontitis developmental (PD) phase, oral microflora sampling, spleen harvest and sacrifice.

Figure 2

Polymerase chain reaction (PCR) analysis of Porphyromonas gingivalis oral infection determined by Arg-gingipain (201 bp). (A) Confirmation of P. gingivalis colonies of strains W83, W50 and A7A1-28 before gavage. (B) Porphyromonas gingivalis colony-forming units (CFU) at 0, 10², 10³ and 10⁴ were added to a sample collected from the oral microflora of mice to determine the detection limit via PCR. Oral microflora analysis: samples of mice infected with P. gingivalis at baseline (upper panel) and at 42 days after the first gavage (lower panel). Numbers 1 to 8 represent individual mice gavaged with (C) vehicle alone, (D) A7A1-28, (E) W83 and (F) W50.

Figure 3

Alveolar bone loss measured by micro-computed tomography. (A) Representative images of the alveolar bone loss found in mice at 42 days after gavage was performed with the three different strains A7A1-28, W83 and W50. (B) Analysis performed for tissue mineral content, bone mineral content, bone volume and bone volume fraction. Error bars indicate standard error. Asterisks denote differences found among groups by Tukey test (P < 0.05).

Figure 4

Histomorphometric image analysis performed in maxillae sections of mice at 42 days after gavage; the distance between the alveolar bone crest to the cementum–enamel junction in M1 was measured in micrometers (depicted with black arrows) (hematoxylin & eosin, 20×)

Figure 5

Flow cytometric analysis of splenocytes derived from mice at 42 days after gavage with Porphyromonas gingivalis for regulatory markers. (A) CD3⁺ CD4⁺ Foxp3⁺ cell percentage, (B) CD3⁺ CD4⁺ IL-10⁺ cell percentage, (C) mRNA FasL relative expression of non-activated splenocytes (towards GAPDH), and (D) CD5⁺ B220⁺ cell percentage. Error bars indicate standard error. Asterisks denote differences found among groups by Tukey test (*P < 0.05, **P < 0.001).

Figure 6

Cytokine expression of 42 days post-gavage murine splenocytes treated with phorbol 12-myristate 13-acetate (PMA)/ionomycin for 48 h in vitro. (A) Differences were found among groups for the expression of interleukin-10 (IL-10; P = 0.0006) and no differences for (B) transforming growth factor- β (TGF-β); (C) interferon-γ (IFN-γ) expression was higher in A7A1-28 strain, and (D) IL-4 expression was higher in W83 strain. No differences were found in expression of (E) IL-17 or (F) IL-6. Experiments were performed in duplicates. Error bars indicate standard error. Asterisks denote differences found among groups by Tukey test (*P < 0.05, **P < 0.001).

Figure 7

Dendritic cells stimulated with Porphyromonas gingivalis strains A7A1-28, W83 and W50 at multiplicities of infection 1:10, 1:1, 1 : 0.1 showed an expression of (A) interleukin-12 (IL-12) p40, (B) IL-6 and (C) transforming growth factor-β (TGF-β) in a dose-dependent manner. Experiments were performed in duplicates. Error bars indicate standard error.