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. 2016 Jan;42(1):120-6.
doi: 10.1016/j.joen.2015.09.022. Epub 2015 Nov 14.

Characterization of the Protective Role of Regulatory T Cells in Experimental Periapical Lesion Development and Their Chemoattraction Manipulation as a Therapeutic Tool

Carolina Favaro Francisconi  1 , Andreia Espindola Vieira  1 , Claudia Cristina Biguetti  1 , Andrew J Glowacki  2 , Ana Paula Favaro Trombone  3 , Ariadne Letra  4 , Renato Menezes Silva  4 , Charles S Sfeir  5 , Steven R Little  6 , Gustavo Pompermaier Garlet  7
Affiliations

Characterization of the Protective Role of Regulatory T Cells in Experimental Periapical Lesion Development and Their Chemoattraction Manipulation as a Therapeutic Tool

Carolina Favaro Francisconi et al. J Endod. 2016 Jan.

Abstract

Introduction: The pathogenesis of periapical lesions is determined by the balance between host proinflammatory immune response and counteracting anti-inflammatory and reparative responses, which include regulatory T cells (Tregs) as potential immunoregulatory agents. In this study, we investigated (in a cause-and-effect manner) the involvement of CCL22-CCR4 axis in Treg migration to the periapical area and the role of Tregs in the determination of outcomes in periapical lesions.

Methods: Periapical lesions were induced in C57Bl/6 (wild-type) and CCR4KO mice (pulp exposure and bacterial inoculation) and treated with anti-glucocorticoid-induced TNF receptor family regulated gene to inhibit Treg function or alternatively with CCL22-releasing, polylactic-glycolic acid particles to induce site-specific migration of Tregs. After treatment, lesions were analyzed for Treg influx and phenotype, overall periapical bone loss, and inflammatory/immunologic and wound healing marker expression (analyzed by real-time polymerase chain reaction array).

Results: Treg inhibition by anti-glucocorticoid-induced TNF receptor family regulated gene or CCR4 depletion results in a significant increase in periapical lesion severity, associated with upregulation of proinflammatory, T-helper 1, T-helper 17, and tissue destruction markers in parallel with decreased Treg and healing marker expression. The local release of CCL22 in the root canal system resulted in the promotion of Treg migration in a CCR4-dependent manner, leading to the arrest of periapical lesion progression, associated with downregulation of proinflammatory, T-helper 1, T-helper 17, and tissue destruction markers in parallel with increased Treg and healing marker expression.

Conclusions: Because the natural and CCL22-induced Treg migration switches active lesion into inactivity phenotype, Treg chemoattractant may be a promising strategy for the clinical management of periapical lesions.

Keywords: Apical lesions; T helper; cytokines; regulatory T cells; wound healing.

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Conflict of interest statement

The authors deny any conflicts of interest related to this study.

Figures

Figure 1
Figure 1. Tregs migration kinetics and its impact on the periapical bone loss and in the expression of inflammatory/immunological and healing markers in experimental periapical lesions in mice
C57Bl/6 (WT) mice were submitted to an experimental periapical lesion inducing protocol (pulp exposure and bacterial inoculation) and treated (or not) with anti-GITR to inhibit Tregs function. Samples from experimental and control groups were collected for histomorphometric and molecular analysis, and evaluated for: (A) Tregs (CD4+FOXp3+) cells count in periapical tissues analyzed by flow cytometry, at 0, 3, 7, 14 and 21 days post infection; depicted as the cell number ×104; (B) periapical lesion development, presented as periapical space area (mm2) increase after lesions induction, measured with ImageJ software in HE stained histological sections, or presented as (C) the lesion evolution index (fold increase in specific time intervals); the (D) correlation between the Tregs (CD4+FOXp3+) cells count and the lesion evolution index, performed with the data from WT group; (E) the expression of inflammatory/immunological and wound healing markers at 21d time point, measured quantitatively by RealTimePCRarray, presented as fold change relative to the control group after normalization by constitutive genes (GAPDH, ACTB, Hprt1) expression levels; and (F) cytokine levels in periapical lesions, measured by ELISA, presented as cytokine pg/mg of periapical tissue. In A panel, different letters represent statistically significant differences among the different time points within its respective groups (P<0.05; One-way ANOVA, Bonferroni post-test). In A, B, C, E and F panels, asterisks (*) represent statistically significant differences (P<0.05; One-way ANOVA, Bonferroni post-test) between the indicated group/time point vs control group, and the hashtag (#) represent statistically significant differences (P<0.05; One-way ANOVA, Bonferroni post-test) between the indicated group/time point vs WT group.
Figure 2
Figure 2. The role of CCR4 in Tregs migration kinetics and its impact on the periapical bone loss and in the expression of inflammatory/immunological and healing markers in experimental periapical lesions in mice
C57Bl/6 (WT) and CCR4KO mice were submitted to an experimental periapical lesion inducing protocol (pulp exposure and bacterial inoculation). Samples from WT and CCR4KO groups were collected for histomorphometric and molecular analysis, and evaluated for:(A) the phenotype of and Tregs (CD4+FOXp3+) from periapical lesions, evaluated by flow cytometry and depicted as the number of positive cells for each marker; (B) Tregs (CD4+FOXp3+) cells count in periapical tissues analyzed by flow cytometry, at 21 days post infection, depicted as the cell number ×104; (C) periapical lesion development, presented as periapical space area (mm2) increase after lesions induction, measured with ImageJ software in HE stained histological sections; (D) the expression of inflammatory/immunological and wound healing markers at 21d time point, measured quantitatively by RealTimePCRarray, presented as fold change relative to the control group after normalization by constitutive genes (GAPDH, ACTB, Hprt1) expression levels, and (E) cytokine levels in periapical lesions, measured by ELISA, presented as cytokine pg/mg of periapical tissue. Asterisks (*) represent statistically significant differences (P<0.05; One-way ANOVA, Bonferroni post-test) between the indicated group/time point vs control group, and the hashtag (#) represent statistically significant differences (P<0.05; One-way ANOVA, Bonferroni post-test) between the indicated group/time point vs WT group.
Figure 3
Figure 3. Involvement of CCL22 in Tregs migration kinetics and its impact on the periapical bone loss and in the expression of inflammatory/immunological and healing markers in experimental periapical lesions in mice
C57Bl/6 (WT) and CCR4KO mice were submitted to an experimental periapical lesion inducing protocol (pulp exposure and bacterial inoculation) and treated (or not) with CCL22 releasing PLGA particles to induce Tregs migration, by the direct injection of the particles at root canal system at 3d time point. Samples from WT and CCR4KO strains from control and experimental groups were collected for histomorphometric and molecular analysis, and evaluated for: (A) Tregs (CD4+FOXp3+) cells count in periapical tissues analyzed by flow cytometry at 0, 3, 7, 14 and 21 days post infection; depicted as the cell number ×104; (B) periapical lesion development, presented as periapical space area (mm2) increase after lesions induction, measured with ImageJ software in HE stained histological sections; (C) the expression of inflammatory/immunological and wound healing markers at 21d time point, measured quantitatively by RealTimePCRarray, presented as fold change relative to the control group after normalization by constitutive genes (GAPDH, ACTB, Hprt1) expression levels, and (D) cytokine levels in periapical lesions, measured by ELISA, presented as cytokine pg/mg of periapical tissue. Asterisks (*) represent statistically significant differences (P<0.05; One-way ANOVA, Bonferroni post-test) between the indicated group/time point vs control group, and the hashtag (#) represent statistically significant differences (P<0.05; One-way ANOVA, Bonferroni post-test) between the indicated group/time point vs WT group.
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