Regulatory T cells in mouse periapical lesions

Abstract

Introduction: T-regulatory (Treg, CD4+ FOXP3+) cells constitute a unique subpopulation of CD4+ T cells that inhibit T-cell responses and prevent disease development/exacerbation in models of autoimmunity. In the present study, we tested the hypothesis that Treg cells are induced in periapical lesions by dental pulp infection.

Methods: In situ hybridization (ISH) was used to localize FOXP3+ cells on day 21 after pulp exposure of the first molar teeth and infection with bacteria from the oral environment. FOXP3/GFP knock-in transgenic mice were used to quantify FOXP3+ Treg cells that infiltrate into periapical lesions by flow cytometry on days 7, 14, and 21 after infection. Periodontal ligament from uninfected teeth served as a negative control.

Results: ISH showed strong signals that showed the presence of FOXP3+ cells mainly at the periphery of periapical lesions. In contrast, no positive cells were present in the periodontal ligament of uninfected controls. Flow cytometry showed an increase in the number of FOXP3+ Treg beginning between day 7 and day 14 (0.69% of the infiltrate) after infection and increased to day 21 (0.94%) (p < 0.05 and p < 0.001, respectively, vs uninfected controls). Treg were also increased in number in draining cervical lymph nodes after pulpal infection.

Conclusions: These results show that Treg cells are induced to infiltrate into periapical lesions by pulpal infection and suggest that they increase in a time-dependent manner.

Figure 1. In situ hybridization of F0XP3+ cells in periapical lesions

Representative sections of (A): F0XP3 anti-sense probe, control unexposed pulp, first mandibular molar (×40); (B): higher magnification (×l00 of box in A); (C): FOXP3 anti-sense probe, exposed pulp (×40); (D): higher magnification (×l00) from C; (E): higher magnification (×400) from D; (F): FOXP3 sense probe, control unexposed pulp; (G): higher magnification (×400) from F; (H): FOXP3 sense probe, exposed pulp; (I): higher magnification (×l00) from H; R: root; PA: periapical lesion; B: bone. Eosin counterstain.

Figure 2. Quantitation of Treg in periapical lesions after pulp infection

Cells were extracted from lesions at the indicated times, and CD4+/FOXP3/GFP+ cells were quantified by flow cytometry. Control: unexposed, uninfected pulp; other groups are exposed and infected. Bars represent mean ± SEM.* p < 0.05; ** p < 0.001 by one way-ANOVA and Tukey’s multiple comparison test.

Figure 3. Treg numbers in cervical lymph nodes (CLN)

CD4+FOXP3/GFP+ cells were quantified by flow cytometry in CLN 14 and 21 days after pulp exposure and compared to unexposed pulps. Bars represent mean ± SEM. *p < 0.05 by t test.