Topical application of Porphyromonas gingivalis into the gingival pocket in mice leads to chronic‑active infection, periodontitis and systemic inflammation

Abstract

Porphyromonas gingivalis (Pg), one of the 'red‑complex' perio‑pathogens known to play a critical role in the development of periodontitis, has been used in various animal models to mimic human bacteria‑induced periodontitis. In order to achieve a more realistic animal model of human Pg infection, the present study investigated whether repeated small‑volume topical applications of Pg directly into the gingival pocket can induce local infection, including periodontitis and systemic vascular inflammation in wild‑type mice. Freshly cultured Pg was topically applied directly into the gingival pocket of the second molars for 5 weeks (3 times/week). After the final application, the mice were left in cages for 4 or 8 weeks and sacrificed. The status of Pg colony formation in the pocket, gingival inflammation, alveolar bone loss, the expression levels of pro‑inflammatory cytokines in the serum and aorta, the presence of anti‑Pg lipopolysaccharide (LPS) and gingipain (Kpg and RgpB) antibodies in the serum, as well as the accumulation of Pg LPS and gingipain aggregates in the gingiva and arterial wall were evaluated. The topical application of Pg into the gingival pocket induced the following local and systemic pathohistological changes in mice when examined at 4 or 8 weeks after the final topical Pg application: Pg colonization in the majority of gingival pockets; increased gingival pocket depths; gingival inflammation indicated by the increased expression of TNF‑α, IL‑6 and IL‑1β; significant loss of alveolar bone at the sites of topical Pg application; and increased levels of pro‑inflammatory cytokines, such as TNF‑α, IL‑1β, IL‑17, IL‑13, KC and IFN‑γ in the serum in comparison to those from mice receiving PBS. In addition, the Pg application/colonization model induced anti‑Pg LPS and gingipain antibodies in serum, as well as the accumulation of Pg LPS and gingipain aggregates in the gingivae and arterial walls. To the best of our knowledge, this mouse model represents the first example of creating a more sustained local infection in the gingival tissues of wild‑type mice and may prove to be useful for the investigation of the more natural and complete pathogenesis of the bacteria in the development of local oral and systemic diseases, such as atherosclerosis. It may also be useful for the determination of a treatment/prevention/efficacy model associated with Pg‑induced colonization periodontitis in mice.

Keywords: Porphyromonas gingivalis; gingipains; intrapocket infection; periodontitis; systemic inflammation.

Figure 1

Research design and electrophoretic images of amplified Pg DNA from the gingival pocket swabs of mice collected at 4 and 8 weeks after the final PBS or Pg inoculation for 5 weeks. (A) Schematic diagram of the research design. (B) Electrophoretic image of amplified Pg DNA from mice sacrificed 4 weeks after completing the last inoculation (inoculation duration, 5 weeks). (C) Electrophoretic image of amplified Pg DNA from mice sacrificed 8 weeks after completing the last inoculation (inoculation duration, 5 weeks). The lanes indicate the following: +, positive control (1,000 CFU of purified Pg DNA); -, negative control; 1–10, samples taken from the gingival pocket. Pg, Porphyromonas gingivalis.

Figure 2

Topical Pg application directly into the gingival pocket induces clinical epithelial attachment loss and alveolar bone loss. (A) Representative images of hematoxylin and eosin staining of the maxillary second molar and periodontal tissue. Scale bar, 100 μm. White arrows indicate the CEJ. (B) Depth of gingival pocket histologically measured at the mesiobuccal pocket of the maxillary second molars from CEJ to the base of pocket. ^****P<0.001 (n=5). Results represent the mean ± SEM. (C) Representative two or three dimensional μCT images of mice maxillae. The mice were sacrificed at 4 (right) or 8 (left) weeks after the final PBS or Pg inoculation. Scale bar, 1 mm. The average distance from the palatal and mesiobuccal CEJ to the ABC of (D) upper first molar, (E) second molar, and (F) third molar. Results represent the means ± SEM performed in ten samples. ^****P<0.001. ns, not significant (P>0.05); Pg, Porphyromonas gingivalis; CEJ, cement-enamel junction; ABC, alveolar bone crest.

Figure 3

Topical Pg application directly into the gingival pocket increases the expression of pro-inflammatory cytokines in gingival tissues. Reverse transcription-quantitative PCR of pro-inflammatory cytokines (A) IL-1β, (B) IL-6, (C) TNF-α, (D) iNOS, and (E) IL-17 from the gingival tissue of second molars of mice receiving ligature placement for 1 week followed by PBS or Pg inoculation into the gingival pocket. The cytokine levels were determined from the tissues obtained at 4 or 8 weeks after the final Pg inoculation. GAPDH served as the loading control. ^*P<0.05 and ^**P<0.01. Results represent the mean ± SEM performed in triplicate; n=8–12 mice per group. Each dot represents a result from 1 mouse. Pg, Porphyromonas gingivalis; IL, interleukin; TNF-α, tumor necrosis factor α; iNOS, inducible nitric oxide synthase.

Figure 4

Topical Pg application directly into the gingival pocket induces systemic inflammation. ELISA measuring the levels (pg/ml) of (A) TNF-α, (B) IL-13, (C) IL-1β, (D) IL-17, (E) KC, and (F) IFN-γ from mouse sera at 4 and 8 weeks after the final PBS or Pg inoculation. ^*P<0.05, ^**P<0.01, ^***P<0.005 and ^****P<0.001 determined using one-way ANOVA. Results represent the mean ± SEM; n=8–12 mice per group. Each dot represents a result from 1 mouse. Pg, Porphyromonas gingivalis; IL, interleukin; TNF-α, tumor necrosis factor α; KC, keratinocyte chemoattractant; IFN-γ, interferon γ.

Figure 5

Anti-Pg gingipain and anti-Pg LPS antibodies are detected in the sera of mice receiving Pg inoculation in the gingival pocket. (A) ELISA was used to measure the serum levels of anti-Kgp antibody. Mouse sera examined 4 weeks after the final PBS inoculation (PBS) exhibited only background levels of Kgp antibody. Significance was analyzed using an unpaired t-test. Tested sera were diluted 1:800. Error bars indicate the mean ± SEM. (B) ELISA was used to measure the serum level of anti-RgpB antibody. Mouse sera examined 4 weeks after the final PBS inoculation (PBS) exhibited only background levels of RgpB antibody. Significance was analyzed using an unpaired t-test. Tested sera were diluted 1:800. Error bars indicate the mean ± SEM. ^*P<0.05. (C) ELISA was used to measure the serum level of anti-Pg LPS antibody. Anti-Pg-LPS IgG levels in mouse sera were measured using a Mouse Anti-Pg-LPS antibody assay kit according to the manufacturer's protocol. Tested sera were diluted 1:800. Absorbance was read at 450 nm using a microplate reader. Significance was analyzed using an unpaired t-test. Error bars indicate the mean ± SEM. ^*P<0.05 and ^****P<0.001, determined using one-way ANOVA. ns, not significant (P>0.05). Results represent the mean ± SEM. Pg, Porphyromonas gingivalis; LPS, lipopolysaccharide; Kgp, lysine-gingipain; RgpB, arginine-gingipain B.

Figure 6

Pg gingipains and LPS aggregates are present in the gingival tissues of mice receiving the topical Pg inoculation into the gingival pocket. (A) IF analysis using Kgp. PBS and Pg were from the mice sacrificed at 4 or 8 weeks after the final PBS or Pg inoculation. Kgp IF-2 is the higher-magnified image of blue-dotted square in Kgp IF. Kgp (bright green color dots, arrows) was present only in the gingival tissue of mouse receiving topical Pg not in those receiving PBS. (B) IF analysis using RgpB. PBS and Pg were from the mice sacrificed at 4 or 8 weeks after the final PBS or Pg inoculation. RgpB IF-2 is the higher-magnified image of blue-dotted square in RgpB IF. The RgpB (bright green color dots, arrows) was present only in the gingival tissue of mouse receiving topical Pg not in those receiving PBS. (C) IF analysis using Pg LPS. PBS and Pg were from the mice sacrificed at 4 or 8 weeks after the final PBS or Pg inoculation. Pg LPS -2 is the higher-magnified image of blue-dotted square in Pg LPS IF. The Pg LPS (bright green color dots, arrows) was present only in the gingival tissue of mouse receiving topical Pg not in those receiving PBS. DAPI was heavily stained in pulpal and gingival tissues. Scale bars, 100 μm. M, upper second molar; G, gingival tissue; AB, alveolar bone; P, pulpal tissue; Pg, Porphyromonas gingivalis; IF, immunofluorescence; LPS, lipopolysaccharide; Kgp, lysine-gingipain; RgpB, arginine-gingipain B.

Figure 7

Pg gingipain and Pg LPS aggregates are found in the aortic roots of mice receiving Pg inoculation into the gingival pocket. (A) IF analysis using Kgp. PBS and Pg were from the mice sacrificed at 4 or 8 weeks after the final PBS or Pg inoculation. Kgp IF-2 is the higher-magnified image of blue-dotted square in Kgp IF. The RgpB (bright green color dots, arrows) was present only in the aortic leaflet and aortic wall of mouse receiving topical Pg, not in those receiving PBS. (B) IF analysis using RgpB. PBS and Pg were from the mice sacrificed at 4 or 8 weeks after the final PBS or Pg inoculation. RgpB IF-2 is the higher-magnified image of blue-dotted square in RgpB IF. The RgpB (bright green color dots, arrows) was present only in the aortic leaflet and aortic wall of mouse receiving topical Pg, not in those receiving PBS. (C) IF analysis using Pg LPS. PBS and Pg were from the mice sacrificed at 4 or 8 weeks after the final PBS or Pg inoculation. Pg LPS -2 is the higher-magnified image of blue-dotted square in Pg LPS IF. The LPS (bright green color dots, arrows) was present only in the aortic leaflet and aortic wall of mouse receiving topical Pg, not in those receiving PBS. Nuclei of cells were localized with DAPI staining. Scale bars, 100 μm. L, lumen; AW, aortic wall; AL, aortic leaflet; Pg, Porphyromonas gingivalis; IF, immunofluorescence; LPS, lipopolysaccharide; Kgp, lysine-gingipain; RgpB, arginine-gingipain B.