Induction of IL-10-producing CD4+ T-cells in chronic periodontitis

Abstract

Precise immunological aspects of inflamed gingival mucosa remain to be elucidated in the murine experimental periodontitis model; therefore, we have characterized the mucosal immune cells in the inflamed gingiva of mice with alveolar bone reduction. Mice were orally infected with Porphyromonas gingivalis 15 times over 2 weeks. Gingival mononuclear cells (GMCs) were isolated from P. gingivalis- and sham-infected mice 1, 7, 15, and 30 days after the last infection. Although the greatest degree of periodontitis was seen in P. gingivalis-infected mice at 30 days after infection, the highest levels of IL-6 and TNF-α production were noted in the GMCs isolated 7 days after infection. Further, the frequency of RANKL(+)CD4(+) T-cells in GMCs of inflamed gingiva peaked 15 days after infection. Importantly, the number of Foxp3(+)CD4(+) CD25(+) regulatory T (Treg)-cells was increased only in the experimental group 30 days after infection. Thus, intracellular cytokine analysis revealed an increased number of IL-10-producing CD4(+) T-cells in inflamed gingiva when compared with the control group. These results suggest that there are potential roles for Treg cells during the chronic stage of periodontitis in the regulation of gingival inflammation and alveolar bone loss.

Figure 1.

Induction of marginal gingival inflammation in mice infected with P. gingivalis. All mice were sacrificed by cervical dislocation after being anesthetized with ketamine and xylazine. (A) Histochemical analysis of gingival tissue and alveolar bone loss of mice. Thirty days after the last infection, lower jaws with gingival tissue from mice infected with or without P. gingivalis were stained with hematoxylin and eosin. (B) Detection of P. gingivalis-specific 16S rRNA. DNA was extracted from gingival tissues of P. gingivalis- and sham-infected mice 30 days after the last infection. DNA was amplified by real-time quantitative PCR with a pair of primers corresponding to P. gingivalis-specific 16S rRNA. Different numbers of DNA from P. gingivalis 33277 were used for generating a standard curve. All values are presented as the mean ± SEM of 25 mice in each group; *p < 0.05 when compared with sham-infected mice.

Figure 2.

The frequency of lymphocytes and bone loss in mice infected with P. gingivalis. All mice were sacrificed at each time-point as described in the legend to Fig. 1. (A) Time-points were assessed on days 1, 7, 15, and 30, after the last infection, and gingival mononuclear cells (GMCs) were stained for CD3, B220, CD11c, and CD11b expression. (B) Prolonged P. gingivalis infection induced alveolar bone loss. Thirty days after the last infection, horizontal bone loss around the maxillary molars was assessed by a morphometric method. Alveolar bone loss was measured at a total of 28 buccal sites per mouse (CEJ to ABC). Bone measurements were performed a total of 3 times by two evaluators using a random and blinded protocol. All values are presented as the mean ± SEM of 25 mice in each group at each time-point; *p < 0.05; **p < 0.01 when compared with sham-infected mice. ^†p < 0.05 when compared with mice before the infection.

Figure 3.

Cytokine profiles and the frequency of RANKL expression by CD4⁺ T-cells in mice orally infected with P. gingivalis. All mice were sacrificed at each time-point as described in the legend to Fig. 1. (A) GMCs (1 × 10⁶/mL) were isolated 1, 7, 15, and 30 days after the last infection and were then cultured for 3 days. The culture supernatants were collected and subjected to IL-6 and TNF-α specific ELISA. (B) One, 7, 15, and 30 days after the last infection with P. gingivalis, the frequency of RANKL⁺CD44⁺CD4⁺ T-cells was examined by FACSCalibur^TM. The values are presented as the mean ± SEM of 20 mice in each group at each time-point; *p < 0.05 when compared with sham-infected mice. ^†p < 0.05 when compared with mice one day after infection.

Figure 4.

Frequencies of interleukin-10-producing CD4⁺ T-cells and Foxp3-expressing CD25⁺CD4⁺ T-cells in inflamed gingiva. All mice were sacrificed 1, 7, 15, and 30 days after the last infection, as described in the legend to Fig. 1. (A) GMCs (1 × 10⁶/mL) were isolated and cultured for 3 days. The culture supernatants were collected and subjected to an IL-10-specific ELISA. (B) Thirty days after the last infection, GMCs were incubated with ionomycin (1 mg/mL) and phorbol 12-myristate 13-acetate (PMA, 25 ng/mL) for 3 hrs and then stained for the surface CD4 and intracellular IL-10 expression. (C) GMCs isolated from each time-point were stained with fluorescence-conjugated anti-CD4 and biotinylated anti-CD25 mAbs, followed by PerCP-Cy5.5-streptavidin. Samples were further stained intracellularly with PE-labeled anti-Foxp3 mAb. The values are presented as the mean ± SEM of 20 to 30 mice in each group at each time-point; *p < 0.05 when compared with sham-infected mice. ^†p < 0.05 when compared with mice one day after infection.