Down-regulated Treg cells in exacerbated periodontal disease during pregnancy

Abstract

Pregnancy is a special period marked with complicated changes in various immune responses. Although pregnant women are prone to developing gingival inflammation, its immunological mechanism remains to be clarified. In a modified ligature-induced periodontal disease murine model, pregnant mice developed more severe alveolar bone loss. Using this model, we investigated the Treg responses during exacerbated periodontal disease in pregnant mice. We tested Treg-associated molecules in gingival tissues by quantitative real-time PCR and found decreased gingival expression of Foxp3, TGFβ, CTLA-4, and CD28 in pregnant mice after periodontal disease induction. We further confirmed that lower number of Treg cells were present in the cervical lymph nodes of pregnant periodontitis mice. Treg cells from the cervical lymph nodes of ligated pregnant mice and non-pregnant mice were tested for their suppressive function in vitro. We manifested that Treg suppressive function was also down-regulated in the pregnant mice. Additionally, we demonstrated that more inflammatory Th17 cells were present in the cervical lymph nodes of ligated pregnant mice. Therefore, impaired Treg development and function, together with upregulated Th17 response, may contribute to the exacerbated periodontal disease during pregnancy.

Keywords: Alveolar bone loss; Periodontal disease; Porphyromonas gingivalis; Pregnancy; Th17; Treg.

Fig. 1

Periodontal bone loss in ligated pregnant and non-pregnant mice. (A) Representative images from the maxillae of ligated (lower panels) or non-ligated (upper panels) non-pregnant (Non-preg; left panels) and pregnant (Preg; right panels) mice. (B) The mm distance from the cementoenamel junction (CEJ) to the alveolar bone crest (ABC) was measured at 6 most affected maxillary palatal sites and the readings were totaled for each mouse. The CEJ-ABC reading of each mouse was represented by each dot. The data from CEJ-ABC readings were transformed to indicate bone loss, as outlined in Materials and Methods. Asterisks indicate statistically significant (***, p < 0.001) differences between different groups.

Fig. 2

Relative expression of Treg-related cytokines and molecules in the gingivae of ligated pregnant and non-pregnant mice. Quantitative real-time PCR (qPCR) was used to determine gingival mRNA expression levels for the indicated molecules (normalized against GAPDH mRNA levels). The gingivae used were excised from pregnant (Preg) and non-pregnant (Non-Preg) C57/BL6 mice. Results are shown as fold change relative to non-pregnant sham-ligated mice. Each group represents the mean ± SD of at least 5 separate expression values, corresponding to qPCR analysis of individual mice. Asterisks indicate statistically significant (*, p < 0.05; **, p < 0.01) differences between different groups.

Fig. 3

Treg cells in the cervical lymph nodes of ligated pregnant and non-pregnant mice. (A) Representative flow cytometry images of the gated CD3+CD4+ cells from non-ligated (upper panels) or ligated (lower panels), non-pregnant (left panels) and pregnant (right panels) mice; (B) the frequency of Treg cells were shown as percentage of CD3+CD4+Foxp3+ cells in CD3+CD4+ cells. Each mouse was represented by each dot. Asterisks indicate statistically significant (*, p < 0.05;) differences between different groups.

Fig. 4

Treg cells from ligated pregnant mice exhibited less potent regulatory function in vitro than those from ligated non-pregnant mice. After periodontal disease induction in pregnant mice and non-pregnant mice, GFP⁺ cells (Treg cells) were sorted from cervical lymphocytes by fluorescence-activated cell sorter and tested for regulatory function in vitro. For this assay, 25,000 freshly harvested CD4⁺ CD25⁻ cells (effector cells) were purified and cocultured at varying (Treg/effector cells) ratios with GFP⁺ cells from pregnant mice (Preg) or non-pregnant mice (Non-preg) in the presence of irradiated spleen cells (APC) and anti-CD3 for 3 days. Asterisks indicate statistically significant (*, p < 0.05;) differences between different groups. #, indicate statistically significant difference from the group without Treg (group 0).

Fig. 5

Th17 cells in the cervical lymph nodes of ligated pregnant and non-pregnant mice. (A) Representative flow cytometry images of the gated CD3+CD4+ cells from non-ligated (upper panels) or ligated (lower panels), non-pregnant (left panels) and pregnant (right panels) mice; (B) the frequency of Th17 cells were shown as percentage of CD3+CD4+RORγt+ cells in CD3+CD4+ cells. Each mouse was represented by each dot. Asterisks indicate statistically significant (*, p < 0.05; **, p < 0.01) differences between different groups.