Regulatory T Cell Phenotype and Function in Periodontal Inflammation

Objectives: The immune homeostasis associated with periodontal health requires a regulated immuno-inflammatory response. The presence of regulatory T cells (Tregs) is critical to minimize collateral tissue damage. Tregs are phenotypically unstable under sustained inflammation; they may lose their suppressive functions and become pro-inflammatory and osteoclastogenic. This work aimed to determine the impact of periodontal inflammation on the phenotypic characteristics and anti-osteoclastogenic functions of regulatory T cells in vivo.
Methods: Experimental periodontitis was induced with ligatures on WT and Foxp3^eGFP mice for 10 days. Animals without ligatures were used as controls. The gingival mucosa was processed to analyze mRNA levels of Treg/Th17-associated cytokines, transcription factors, and receptors by q-PCR. The frequency and the total number of IL-17-producing Tregs in the gingiva, cervical lymph nodes, and spleen were analyzed by flow cytometry. Tregs from cervical lymph nodes were sorted, and mRNA was purified for RNA sequencing. Tregs (5x10⁴) were co-cultured with CD11b⁺ bone-marrow-derived pre-osteoclasts (2.5x10⁵) in the presence of M-CSF and RANKL for 4 days; then stained for TRAP to evaluate their osteoclastic differentiation.
Results: Ligature-induced periodontitis was associated with the upregulation of Th17-related mediators and the infiltration of both Treg and Th17 cells in the gingiva. Tregs from cervical lymph nodes had reduced expression of Foxp3 (>20% MFI loss) and increased expression of IL-17 (>15%), compared with Tregs from spleen and healthy controls. Tregs transcriptome analysis showed a differential signature between health and disease, with increased expression of Th17-associated factors in periodontitis-derived Tregs. The suppression capacity of Tregs on osteoclastic differentiation was significantly lower in Tregs obtained from periodontally diseased animals compared to the controls (P<0.05) as identified by the increased number of TRAP⁺ osteoclasts (P<0.01) in the Tregs/Pre-osteoclast co-cultures.
Conclusions: Tregs lose their suppressive capacity and acquire Th17-like functions during periodontitis, which could explain the mechanism of increased bone loss.