T cell-derived leptin contributes to increased frequency of T helper type 17 cells in female patients with Hashimoto's thyroiditis

Abstract

Leptin modulates T cell function and plays an important role in autoimmune diseases. Our study aimed to explore the role of leptin and T helper type 17 (Th17) cells in Hashimoto's thyroiditis patients. Twenty-seven patients with Hashimoto's thyroiditis (HT) and 20 healthy controls were enrolled into the current study. A modest increase of plasma leptin in HT patients and the CD4(+) T cell-derived leptin from HT patients was stronger than that from healthy controls. In HT patients, there are no statistically significant correlations between plasma leptin concentrations and the percentage of Th17 cells or the level of retinoic acid-related orphan receptor γt (RORγt), but strong positive correlations were observed between CD4(+) T cell-derived leptin and the percentage of Th17 cells or the level of RORγt mRNA, and additionally significantly up-regulated leptin, interleukin (IL)17 and RORγt mRNA levels in the thyroid tissue. Furthermore, neutralization of leptin decreases the frequency of Th17 cells in vitro. Current study has revealed an increased leptin involvment in Hashimoto's thyroiditis associated with an increased number of Th17 cells.

Fig. 1

Increased serum and CD4⁺ T cell-derived leptin in Hashimoto's thyroiditis (HT) patients. Peripheral blood was obtained from 27 HT patients and 20 healthy controls. (a) Plasma leptin levels were determined by enzyme-linked immunosorbent assay (ELISA) from HT patients and healthy controls. Correlation between the level of plasma leptin and body mass index (BMI) in healthy controls (b) and HT patients (c), respectively. (d) The level of leptin in culture of CD4⁺ T cells from HT patients was determined by ELISA from HT patients and healthy controls. Each data point represents an individual subject; results are expressed as mean ± standard deviation, Student's unpaired t-test.

Fig. 2

The correlation between leptin and T helper type 17 (Th17) cells in Hashimoto's thyroiditis (HT) patients. (a) The percentages of CD3⁺CD8⁻ Th17 cells were analysed by flow cytometry, representative flow cytometry plots of Th17 cells. (b) Collective analysis of results from HT patients and control groups. Results are expressed as mean ± standard deviation, n = 12, Student's unpaired t-test. (c,d) Correlation between plasma leptin concentrations and the percentage of Th17 cells or the level of retinoic acid-related orphan receptor γt (RORγt) in HT patients. (e,f) Correlation between CD4⁺ T cell-derived leptin concentrations and the percentage of Th17 cells or the level of RORγt in HT patients.

Fig. 3

Expression of leptin, interleukin (IL)-17 and retinoic acid-related orphan receptor γt (RORγt) mRNA in thyroid tissue. Expression of leptin, IL-17 and RORγt mRNA in thyroid mononuclear cells (TMCs) from six Hashimoto's thyroiditis (HT) patients compared with three patients with simple goitre (control). The levels of β-actin, leptin, IL-17 and RORγt mRNA were determined by polymerase chain reaction (PCR). Levels of greyscale were then tested and corresponding values are shown.

Fig. 4

Neutralization of leptin decreases the percentage of T helper type 17 (Th17) cells in vitro. Human CD4⁺ T cells were purified from peripheral blood mononuclear cells (PBMCs) by magnetic beads and cultured with RPMI-1640 medium supplemented with 10% fetal bovine serum; 10 μg/ml human leptin-neutralizing monoclonal antibody (mAb) was administered in CD4⁺ T cell culture in the presence of soluble anti-human CD3 mAb and anti-human CD28 mAb; the irrelevant isotype-matched antibody was used as control. The percentages of CD4⁺ Th17 cells were analysed by flow cytometry (a); retinoic acid-related orphan receptor γt (RORγt) mRNA were determined by quantitative PCR (b).