Th17/Treg cells imbalance and GITRL profile in patients with Hashimoto's thyroiditis

Abstract

Hashimoto's thyroiditis (HT) is an organ-specific immune disease characterized by the presence of lymphocytic infiltration and serum autoantibodies. Previous studies have confirmed the critical role of Th17 cells in the pathopoiesis of HT patients. Additionally, regulatory T cells (Treg) display a dysregulatory function in autoimmune disease. The purpose of this study is to investigate the alteration of Th17 and Treg cells in HT patients and explore contributing factors. We found there was an increased ratio of Th17/Treg in HT patients and a positive correlation with autoantibodies (anti-TgAb). In addition, there was an increased level of GITRL, which has been demonstrated to be correlated with the increassement of Th17 cells in the serum and thyroid glands of HT patients; the upregulated serum level of GITRL has a positive correlation with the percentage of Th17 cells in HT patients. In summary, an increase in GITRL may impair the balance of Th17/Treg, and contribute to the pathopoiesis of Hashimoto's thyroiditis.

Figure 1

Enhancement of Th17 cells in peripheral blood from HT patients. PBMCs from HT patients and healthy controls were incubated with PMA/ionomycin, stained for cell surface ijms CD3 and CD8 as well as intracellular IL-17 and analyzed by flow cytometry. (a) Representative dot plots from HT patient and healthy control are shown. Values correspond to the percentage of Th17 cells. We used isotype controls to determine the positive cells, and all the values are gated on the CD3⁺CD8⁻ cells; (b) Percentages of Th17 cells were compared between HT patients and healthy controls; (c) Levels of ROR-γt mRNA in PBMCs from HT patients and healthy controls; (d) Serum levels of IL-6 were determined by ELISA in serum samples from HT patients and healthy controls and (e) Serum levels of IL-23 were determined by ELISA in serum samples from HT patients and healthy controls. ** p < 0.01; *** p < 0.001.

Figure 1

Enhancement of Th17 cells in peripheral blood from HT patients. PBMCs from HT patients and healthy controls were incubated with PMA/ionomycin, stained for cell surface ijms CD3 and CD8 as well as intracellular IL-17 and analyzed by flow cytometry. (a) Representative dot plots from HT patient and healthy control are shown. Values correspond to the percentage of Th17 cells. We used isotype controls to determine the positive cells, and all the values are gated on the CD3⁺CD8⁻ cells; (b) Percentages of Th17 cells were compared between HT patients and healthy controls; (c) Levels of ROR-γt mRNA in PBMCs from HT patients and healthy controls; (d) Serum levels of IL-6 were determined by ELISA in serum samples from HT patients and healthy controls and (e) Serum levels of IL-23 were determined by ELISA in serum samples from HT patients and healthy controls. ** p < 0.01; *** p < 0.001.

Figure 2

Reduction of regulatory T cells in HT patients. PBMCs from HT patients and healthy controls were stained for cell surface molecule CD4, CD25, CD127 and analyzed by flow cytometry. (a) Representative dot plots from HT patients and healthy controls are shown. Values correspond to the percentage of Treg cells. We used isotype controls to determine the positive cells, and all the values are gated on the CD4⁺ cells; (b) Percentages of Treg cells were compared between HT patients and healthy controls and (c) Levels of Foxp3 mRNA in PBMCs from HT patients and healthy controls. * p < 0.05; ** p < 0.01.

Figure 2

Reduction of regulatory T cells in HT patients. PBMCs from HT patients and healthy controls were stained for cell surface molecule CD4, CD25, CD127 and analyzed by flow cytometry. (a) Representative dot plots from HT patients and healthy controls are shown. Values correspond to the percentage of Treg cells. We used isotype controls to determine the positive cells, and all the values are gated on the CD4⁺ cells; (b) Percentages of Treg cells were compared between HT patients and healthy controls and (c) Levels of Foxp3 mRNA in PBMCs from HT patients and healthy controls. * p < 0.05; ** p < 0.01.

Figure 3

Correlation between Th17/Treg balance and TgAb levels in HT patients. (a) The ratio of Th17/Treg in patients with HT compared with healthy controls and (b) The correlation between the ratio of Th17/Treg and TgAb levels in HT patients. ** p < 0.01.

Figure 4

High levels of GITRL in patients with HT. (a) Serum levels of GITRL were determined by ELISA in serum samples from HT patients and controls; (b) The correlation between the percentage of Th17 cellsand serum levels of GITRL in HT patients and (c) The correlation between the percentage of Treg cells and serum levels of GITRL in HT patients. * p < 0.05.

Figure 5

Higher expression of IL-17, ROR-γt and GITRL mRNA in thyroid tissue from HT patients. (a) The levels of IL-17 mRNA in thyroid glands were detected by real-time PCR from HT patients, and patients with simple goiter; (b) The levels of ROR-γt mRNA in thyroid glands were detected by real-time PCR from HT patients, and patients with simple goiter and (c) The levels of GITRL mRNA in thyroid glands were detected by real-time PCR from HT patients, and patients with simple goiter. * p < 0.05.