Antibody and T cell responses to Fusobacterium nucleatum and Treponema denticola in health and chronic periodontitis

Abstract

The characteristics of the T cell response to the members of oral flora are poorly understood. We characterized the antibody and T cell responses to FadA and Td92, adhesins from Fusobacterium nucleatum, an oral commensal, and Treponema denticola, a periodontal pathogen, respectively. Peripheral blood and saliva were obtained from healthy individuals and patients with untreated chronic periodontitis (CP, n = 11 paris) and after successful treatment of the disease (n = 9). The levels of antigen-specific antibody were measured by ELISA. In plasma, IgG1 was the most abundant isotype of Ab for both Ags, followed by IgA and then IgG4. The levels of FadA-specific salivary IgA (sIgA) were higher than Td92-specific sIgA and the FadA-specific IgA levels observed in plasma. However, the periodontal health status of the individuals did not affect the levels of FadA- or Td92-specific antibody. Even healthy individuals contained FadA- and Td92-specific CD4(+) T cells, as determined by the detection of intracytoplasmic CD154 after short-term in vitro stimulation of peripheral blood mononuclear cells (PBMCs) with the antigens. Patients with CP tended to possess increased numbers of FadA- and Td92-specific CD4(+) T cells but reduced numbers of Td92-specific Foxp3(+)CD4(+) Tregs than the healthy subjects. Both FadA and Td92 induced the production of IFNγ and IL-10 but inhibited the secretion of IL-4 by PBMCs. In conclusion, F. nucleatum induced Th3 (sIgA)- and Th1 (IFNγ and IgG1)-dominant immune responses, whereas T. denticola induced a Th1 (IFNγ and IgG1)-dominant response. This IFNγ-dominant cytokine response was impaired in CP patients, and the Td92-induced IFNγ levels were negatively associated with periodontal destruction in patients. These findings may provide new insights into the homeostatic interaction between the immune system and oral bacteria and the pathogenesis of periodontitis.

Figure 1. FadA-, Td92-, and TT-specific Abs in saliva and plasma.

The levels of FadA-, Td92-, and TT-specific-IgA in saliva (a) and the Ag-specific-IgA (b), -IgG1 (c), and -IgG4 (d) in plasma were determined by ELISA. Bars indicate median values. CP (B): patients with chronic periodontitis before treatment. CP (A): patients with chronic periodontitis after treatment.

Figure 2. Presence of FadA-, Td92-, and TT-specific CD4⁺ T cells and Ag-specific Foxp3⁺CD4⁺ Tregs in PBMCs PBMCs from healthy subjects (n = 7), patients with CP before (B) treatment (n = 8), and patients with CP after (A) treatment (n = 7) were stimulated with medium alone (No Ag), 30 µg/ml FadA, Td92, or TT for 16 hours in the presence of PE-conjugated anti-CD154 mAb, purified anti-CD28 mAb, and anti-CD49d mAb. Monensin was added during the last 4 h of culture.

The stimulated cells were stained with an APC-conjugated anti-CD4 mAb and a FITC-conjugated anti-Foxp3 mAb. (a) Lymphocytes gated based on FSC vs. SSC were plotted by CD4 vs. CD154 in the density plots (Top and the third rows). The percentage of CD154⁺ cells (R3) among the CD4⁺ T cells (R2) is presented. CD154⁺CD4⁺ T cells were gated and plotted by Foxp3 vs. CD4 (the second and the fourth rows). Numbers indicate the percentage of Foxp3⁺ cells among the gated CD154⁺CD4⁺ T cells. (b) The percentage of CD154⁺ cells out of the total CD4⁺ T cells was graphed (left panel). The percentage of Ag-specific cells out of the total CD4⁺ T cells was graphed after subtracting the number of background stained cells (right panel). (c) The percentage of Foxp3⁺CD4⁺ Tregs among the CD154⁺CD4⁺ T cells was graphed (left panel). The percentage of CD154⁺Foxp3⁺CD4⁺ T cells out of the total CD4⁺ T cells was graphed (right panel). Bars indicate median values. *, P<0.05 compared to No Ag.

Figure 3. Cytokine production by PBMCs in response to FadA, Td92, or TT PBMCs from healthy subjects (n = 9), patients with CP before (B) treatment (n = 9), and patients with CP after (A) treatment (n = 7) were stimulated with medium alone (No Ag), 30 µg/ml FadA, Td92, or TT for 48 hours in the presence of purified anti-CD28 mAb and anti-CD49d mAb.

(a–d) The amount of IFNγ, IL-4, IL-10, and IL-17 secreted into the supernatant was measured by ELISA. (e) The ratio of IFNγ/IL-4 was calculated and plotted. For the calculation of this ratio, half of the lowest detectable concentration (0.1 pg/ml) was arbitrarily assigned to IL-4 samples that had a zero value. Bars indicate median values. *, P<0.05; **, P<0.01 compared to No Ag.

Figure 4. Relative amounts of F. nucleatum and T. denticola in saliva.

The relative proportion of F. nucleatum or T. denticola among the total bacteria in saliva was determined by real-time PCR of 16S rRNA. Bars indicate median values.