Leptin receptor antagonist attenuates experimental autoimmune thyroiditis in mice by regulating Treg/Th17 cell differentiation

Abstract

Leptin has been found to be involved in the development and progression of many autoimmune diseases. As an organ-specific autoimmune disease, the pathogenesis of Hashimoto's thyroiditis has not been fully elucidated. It has been reported that serum leptin level is increased in Hashimoto's thyroiditis, but other studies have not shown any difference. We replicated a mouse model of experimental autoimmune thyroiditis (EAT) with a high-iodine diet and found that injection of the leptin receptor antagonist Allo-aca reduced thyroid follicle destruction and inflammatory cell infiltration in EAT mice, and thyroxine and thyroid autoimmune antibody levels. Further investigation revealed that Allo-aca promotes the differentiation of Treg cells and inhibits the differentiation of Th17 cells. We believe that Allo-aca can alter the differentiation of Treg/Th17 cells by inhibiting the leptin signaling pathway, thereby alleviating thyroid injury in EAT mice. Interfering with the leptin signaling pathway may be a novel new approach to treat treating and ameliorating Hashimoto's thyroiditis.

Keywords: JAK2/STAT3 pathway; NOD/ShiLtJ mouse; experimental autoimmune thyroiditis (EAT); leptin; leptin receptor antagonist.

Figure 1

Experimental Procedure. NOD mice were divided into the control group, the experimental autoimmune thyroiditis (EAT) group and the Allo-aca treatment group. The EAT model was replicated by 0.05% iodine water intaking for 8 weeks. The Allo-aca treatment group received a subcutaneous injection of Allo-aca daily. The peripheral blood, thyroid and spleen of the experimental animals were used in the subsequent experiments.

Figure 2

Experimental autoimmune thyroiditis (EAT) in mice. (A) The EAT mouse model was replicated by ingestion of 0.05% sodium iodine water. The thyroid follicular epithelial cells were necrotic, and a large number of lymphocytes infiltrated the thyroid gland of EAT group. (B) Compared with the NC group, the serum level of thyroid autoantibody TgAb was significantly increased in the EAT group. The above features indicate that the EAT model was successfully replicated. ***P < 0.001.

Figure 3

Allo-aca alleviates autoimmune thyroiditis caused by high iodine intake. (A) In normal mouse thyroid, the follicles were distributed evenly and there were no infiltrated inflammatory cells. High iodine intake caused autoimmune thyroiditis (EAT) in NOD mice, which was characterized by structural damage to thyroid follicles. Whereas treatment with Allo-aca attenuated the destruction of thyroid follicular cells. (B) Allo-aca significantly reduced the number of lymphocytes in the thyroid gland according to the lymphocyte infiltration score. (C) The body weight of mice was continuously monitored during the experimental period, and there was no statistical difference in body weight among the three groups. n = 4, data are expressed as mean ± standard deviation, **P < 0.01, ***P < 0.001.

Figure 4

Serum leptin, thyroid hormone, and thyroid autoimmune antibody levels in experimental mice. Serum leptin, T3, T4, TSH, TPO-Ab, and TgAb expressions were all up-regulated in high iodine-induced EAT mice. Allo-aca reduced serum T3, T4, TPO-Ab, and TgAb levels in EAT mice. n=10, data are expressed as mean ± standard deviation, ns: not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 5

Allo-aca altered the expression of FOXP3 and IL -17A in the thyroid gland of EAT mice. (A) Allo-aca increased the level of FOXP3 in the thyroid gland of EAT mice. (B) The level of IL -17A was increased in the thyroid gland of EAT mice and was significantly attenuated by Allo-aca. FOX3P was labeled in green, IL -17A was labeled in pink, and nuclei were stained with DAPI. Scale bar: 20µm. n=3, data are expressed as mean ± standard deviation, *P < 0.05, **P < 0.01.

Figure 6

Effects of leptin receptor antagonists on inflammatory factors associated with hyper iodine-induced autoimmune thyroiditis in mice. (A-C). The levels of STAT3, RORγt and IL-17 in thyroid of EAT mice increased and Allo-aca decreased their levels. (D). Allo-aca increased the level of FOXP3 in thyroid of EAT mice. (E-F). Allo-aca mildly increased the levels of TGF-β and IL-10 in thyroid of EAT mice, but the change was not statistically significant. n=6. Data is expressed as mean ± SEM. ns: not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 7

The levels of IL-17 and IL-10 in EAT mouse serum and the effect of Allo-aca on them. (A) Serum IL-17 levels in EAT mice were higher than in control mice, but Allo-aca had no significant effect on it. (B) Allo-aca significantly up-regulated serum IL-10 levels in EAT mice. n=10 in NC and EAT group, n=8 in Allo-aca group. Data is expressed as mean ± SEM. ns: not significant, *P < 0.05, ***P < 0.001.

Figure 8

Mechanism of leptin receptor antagonists attenuating experimental autoimmune thyroiditis. Leptin binds to the leptin receptor (ObR) on the surface of T CD4+ lymphocytes, transmits signals to the nucleus through the JAK2/STAT3 pathway (47), promotes the transcription of RORγt, and then promotes the differentiation of T CD4+ lymphocytes into Th17 cells. Th17 cells mediate downstream immune responses through the production of IL-17, thereby leading to apoptosis/necrosis of thyroid cells. Treg can alleviate inflammatory response by inhibiting Th1, Th2 and Th17 cells. Treg and Th17 cells have antagonistic developmental programs and the differentiation of Treg inhibits Th17 differentiation (43, 44). On the other hand, Allo-aca can also bind to ObR of Treg cells to maintain Treg proliferation (46). Therefore, leptin receptor antagonists alleviate thyroid cell apoptosis/necrosis in experimental autoimmune thyroiditis by inhibiting Th17 cell differentiation and promoting Treg differentiation and proliferation.