Effect of Fusobacterium nucleatum on the T and B cell responses to Porphyromonas gingivalis in a mouse model

Abstract

T cell cytokine profiles and specific serum antibody levels in five groups of BALB/c mice immunized with saline alone, viable Fusobacterium nucleatum ATCC 25586, viable Porphyromonas gingivalis ATCC 33277, F. nucleatum followed by P. gingivalis and P. gingivalis followed by F. nucleatum were determined. Splenic CD4 and CD8 cells were examined for intracytoplasmic interleukin (IL)-4, interferon (IFN)-gamma and IL-10 by dual colour flow cytometry and the levels of serum anti-F. nucleatum and anti-P. gingivalis antibodies determined by an ELISA. Both Th1 and Th2 responses were demonstrated by all groups, and while there were slightly lower percentages of cytokine positive T cells in mice injected with F. nucleatum alone compared with the other groups immunized with bacteria, F. nucleatum had no effect on the T cell production of cytokines induced by P. gingivalis in the two groups immunized with both organisms. However, the percentages of cytokine positive CD8 cells were generally significantly higher than those of the CD4 cells. Mice immunized with F. nucleatum alone had high levels of serum anti-F. nucleatum antibodies with very low levels of P. gingivalis antibodies, whereas mice injected with P. gingivalis alone produced anti-P. gingivalis antibodies predominantly. Although the levels of anti-F. nucleatum antibodies in mice injected with F. nucleatum followed by P. gingivalis were the same as in mice immunized with F. nucleatum alone, antibody levels to P. gingivalis were very low. In contrast, mice injected with P. gingivalis followed by F. nucleatum produced equal levels of both anti-P. gingivalis and anti-F. nucleatum antibodies, although at lower levels than the other three groups immunized with bacteria, respectively. Anti-Actinobacillus actinomycetemcomitans, Bacteroides forsythus and Prevotella intermedia serum antibody levels were also determined and found to be negligible. In conclusion, F. nucleatum immunization does not affect the splenic T cell cytokine response to P. gingivalis. However, F. nucleatum immunization prior to that of P. gingivalis almost completely inhibited the production of anti-P. gingivalis antibodies while P. gingivalis injection before F. nucleatum demonstrated a partial inhibitory effect by P. gingivalis on antibody production to F. nucleatum. The significance of these results with respect to human periodontal disease is difficult to determine. However, they may explain in part differing responses to P. gingivalis in different individuals who may or may not have had prior exposure to F. nucleatum. Finally, the results suggested that P. gingivalis and F. nucleatum do not induce the production of cross-reactive antibodies to other oral microorganisms.

Fig. 1

Five groups of BALB/c mice were immunized with saline (Group 1), F. nucleatum alone (Group 2), P. gingivalis alone (Group 3), F. nucleatum followed by P. gingivalis (Group 4) and P. gingivalis followed by F. nucleatum (Group 5) and the mean percentage (± standard error of the mean) IL-4⁺ CD4 and CD8 cells determined by dual colour flow cytometry. 1*, 2*, 3* 4*: the percentage of IL-4⁺ CD8 cells was increased in Groups 2, 3 and 4 in comparison with the percentage of IL-4⁺ CD4 cells (P=0·004, 0·000 and 0·02, respectively). The percentage of IL-4⁺ CD8⁺ cells in Groups 2, 3, 4 and 5 were also higher than those in the control Group 1 (P=0·005, 0·000, 0·000 and 0·004, respectively). 5*, 6*: the percentage of IL-4⁺ CD4 cells was increased in Groups 4 and 5 compared with control Group 1 (P=0·027 and 0·008, respectively). ▪, CD4; □, CD8.

Fig. 2

Five groups of BALB/c mice were immunized with saline (Group 1), F. nucleatum alone (Group 2), P. gingivalis alone (Group 3), F. nucleatum followed by P. gingivalis (Group 4) and P. gingivalis followed by F. nucleatum (Group 5) and the mean percentage (±standard error of the mean) IFN-gamma⁺ CD4 and CD8 cells determined by dual colour flow cytometry. 1*, 2*, 3*: the percentage of IFN-gamma⁺ CD8 cells was increased in Groups 3, 4 and 5 in comparison with the percentage of IFN-gamma⁺ CD4 cells (P=0·000, 0·006 and 0·004, respectively). 4*, 5*, 6*: the percentage of IFN-gamma⁺ CD8 cells was increased in Groups 3, 4 and 5 in comparison with control Group 1 (P=0·000, 0·000 and 0·01, respectively). The percentage of IFN-gamma⁺ CD8 cells was also increased in Group 5 in comparison with Group 2 (P=0·027). ▪, CD4; □, CD8.

Fig. 3

Five groups of BALB/c mice were immunized with saline (Group 1), F. nucleatum alone (Group 2), P. gingivalis alone (Group 3), F. nucleatum followed by P. gingivalis (Group 4) and P. gingivalis followed by F. nucleatum (Group 5) and the mean percentage (± standard error of the mean) of IL-10⁺ CD4 and CD8 cells determined by dual colour flow cytometry. 1*, 2*: the percentage of IL-10⁺ CD8 cells was increased in Groups 3 and 4 in comparison with the percentage of IL-10⁺ CD4 cells (P=0·002). 3*, 4*, 5*: the percentage of IL-10⁺ CD4 cells in Group 5 and the percentage of IL-10⁺ CD8 cells in Groups 3 and 4 were increased in comparison with control Group 1 (P=0·029, 0·007 and 0·004, respectively). ▪, CD4; □, CD8.

Fig. 4

Mean (± standard error of the mean) anti-F. nucleatum (Fn), P gingivalis (Pg), A. actinomycetemcomitans (Aa), B. forsythus (Bf) and P. intermedia (Pi) antibodies equivalent to μg/ml IgG in the serum samples of five groups of BALB/c mice immunized with saline (Group 1), F. nucleatum alone (Group 2), P. gingivalis alone (Group 3), F. nucleatum followed by P. gingivalis (Group 4) and P. gingivalis followed by F. nucleatum (Group 5) were determined by ELISA. 1*, 2*, 3*: anti-F. nucleatum antibodies were increased in Groups 2, 4 and 5 compared with Group 1 (P=0·000) and the levels in Groups 2 and 4 were higher than in Group 5 (P=0·000). Levels in Groups 2 and 4 were also higher than all other specific antibodies and in Group 5 higher than all except P. gingivalis (P=0·000). 4*, 5*, 6*: anti-P. gingivalis antibodies were increased in Groups 3, 4 and 5 compared with Groups 1 and 2 (P=0·000). Group 3 levels were increased compared with Groups 4 and 5 (P=0·000) and Group 5 levels were higher than those in Group 4 (P=0·000). Group 3 levels were higher than all other specific antibodies and in Group 5, higher than all except anti-F. nucleatum antibodies (P=0·000). ▪, pg; □, Fn; , Aa; , Bf; , Pi.