Figure 1.
P. gingivalis exacerbation of CIA in DBA/1 mice. Mice (n = 7) were inoculated with 1×108 P. gingivalis strain W83 cells and subsequently immunized with CII.
(A) Kaplan-Meier plots showing differences in the clinical onset of arthritis (log-rank test, p<0.001) (n = 7). (B) Mean severity of arthritis in mice followed for 5–6 weeks after immunization. Two-way ANOVA with Bonferroni's post-test was used for the statistical evaluation. (n = 7). (C) H&E staining of the representative inflamed tarsal joints from CII- immunized DBA/1, and (F) mice infected with wild-type P. gingivalis strain W83 (D) Representative picture of the inflamed lower paw in CIA and (E) CIA after infection with wild type P. gingivalis. (G) Scatter dot plot showing MPO activity in 1 g of joint tissue extract prepared at day 45 after CII immunization. Horizontal bar and error bars represent the mean and SEM, respectively. Differences between groups were analyzed using Mann-Whitney U test (n = 20). *** denotes p<0.001.
Figure 2.
Aggravation of collagen-induced arthritis was specific to exposure to viable P. gingivalis.
(A) Development of CIA at 5–6 weeks in DBA/1 mice (n = 7) were compared after exposure to viable P. gingivalis and heat-killed P. gingivalis; (B) Mice were immunized with P. gingivalis cell-envelope fractions (n = 10) 3 weeks prior to CII challenge and the development CIA were monitored at the 5–6 weeks period. (C) Mice were challenged with the periodontal pathogen P. intermedia strain 17 as compared to wild-type P. gingivalis. Points represent mean ± SEM. Two-way ANOVA with Bonferroni's post-test was used for the statistical evaluation (*** p<0.001, **** p<0.0001).
Figure 3.
Aggravation of collagen-induced arthritis in DBA/1 mice was dependent on the production of P. gingivalis peptidylarginine deiminase (PPAD).
Mice (n = 7) were inoculated with 1×108 CFU P. gingivalis wild-type strain W83 or PPAD-knockout strain (dPAD) and subsequently immunized with CII. (A) Kaplan-Meier plots displaying the clinical onset of arthritis (log-rank test, p = 0.002). (B) Mean severity of arthritis in mice followed for 5–6 weeks after immunization. (C) Histological signs of synovitis and (D) erosion on day 45 after CII immunization. (E) Scatter dot plot showing MPO activity in 1 g of joint tissue extract at day 45 after CII immunization. H&E staining of the representative inflamed tarsal joints from (F) CII- immunized DBA/1, (G) mice infected with wild-type P. gingivalis strain W83, and (H) isogenic dPAD mutant. Horizontal bar and error bars represent the mean and SEM, respectively. Statistical evaluation using two-way ANOVA with Bonferroni's post-test were employed for data in (B) and statistical differences between groups were analyzed using Mann-Whitney U test (C) or one way ANOVA (D). ** denotes p<0.01; ***, p<0.001; ****, p<0.0001.
Figure 4.
Aggravation of collagen-induced arthritis in DBA/1 mice was dependent on the production of P. gingivalis peptidylarginine deiminase (PPAD).
(A) Development of arthritis in DBA/1 mice (n = 7) inoculated with P. gingivalis wild-type W83 strain or a PPAD-complemented mutant (dPAD+). Mean severity of arthritis in mice followed for 5–6 weeks after immunization. B) Serum levels of IgG antibodies against CII were determined by ELISA on day 45 after immunization. Horizontal bar and error bars represent the mean and SEM, respectively. Statistical evaluation using two-way ANOVA with Bonferroni's post-test (* p<0.05, ** p<0.01).
Figure 5.
Inoculation with P. gingivalis strain W83 leads to citrullination in vivo and, in turn, production of antibody against citrullinated proteins (aCCP).
(A) Subcutaneously implanted titanium chambers were inoculated with: 1) 1×108 P. gingivalis, 2) PBS, 3) 5×107 and 4) 1×107 P. gingivalis. After 24 h, 20 µl of chamber fluid was collected and assayed for citrullinated proteins by immunoblotting with anti-modified citrulline antibody. B) Level of citrullinated proteins in chamber fluid 7 days post-inoculation. Pooled chamber fluid from 2 independent experiments was used. Horizontal bar and error bars represent the mean and SEM, respectively. C) Serum titers of aCCP IgG and D) IgG antibodies against CEP-1 were determined using an ELISA 45 days post-immunization with CII. Error bars represent the SEM. Statistical evaluation was carried out using one way ANOVA. * denotes p<0.05.