The Association Between Type 2 Innate Lymphoid Cells And The Pathogenesis Of Graves Disease

Objective:Graves Disease is a common organ specific autoimmune disease,it’s characterized by the presence of thyroid autoantibody,the infiltration of the thyroid gland by autoactivated lymphocytes and,occasionally with the infiltration of orbital and retrobulbar tissues.Most patients of Graves disease have some hypermetabolism symptoms,thyroid enlargement,decreased levels of TSH and elevated levels of thyroxine（T4）and triiodothyronine（T3）.The pathogenesis of Graves Disease remains obscure,but a large number of studies suggested that Graves Disease was an autoimmune response mediated by humoral immunity.Meanwhile,type 2 innate lymphoid cell（ILC2）is a new type of lymphocyte that is developed by common lymphoid progenitor cells and it is a non-B and non-T cell,which is capable of producing large amounts of IL-5,IL-4,IL-9,IL-13 and amphiregulin when stimulated by IL-25 and IL-33.ILC2 mainly participates in regulation of the Th2 immune response,and mediates multiple signaling pathways by producing various cytokines,it plays an important role in many immune diseases such as asthma,atopic dermatitis,eosinophilic gastroenteritis.Some researches suggested that Graves Disease was an autoimmunity disease mediated by Th2 immunity response and it was found that the infiltrating lymphocyte in thyroid of Graves Disease patients can secrete Th2 cytokines such as IL-4,IL-5,IL-10,IL-13.So,whethe ILC2 involve in the development of Graves disease by regulating Th2 immune response or not.This study aims to explore the association between ILC2 and the pathogenesis of Graves Disease.Methods:1.Collecting the peripheral blood of patients of Graves Disease and Graves ophthalmopathy and healthy volunteers and their information of age,gender,blood routine examination,thyroid function tests.Besides,isolating of individual mononuclear cell and collecting the peripheral blood mononuclear cell（PBMC）and the upper plasma.2.Cells culture and experimental groups:Human normal thyroid follicular epithelium cell were cultured in vitro,stimulating normal thyroid cell for 24 hour with 50ng/ml rhIL-4 or 25ng/ml rhIL-13respectively.Collecting the supernatant and extracting the total RNA from the cell after stimulation.3.Detections:（1）Observe the frequency of ILC2 in PBMC by flow cytometry in each group;（2）Observe the level of the IL-4,IL-5,IL-13 in serum:ELISA detected the level of IL-4,IL-5,IL-13 in plasma.（3）Observe the level of the thyroid hormones in supernatant:ELISA detected the level of thyroxine and triiodothyronine in the supernatant after respectively stimulation of 50ng/ml rhIL-4 and25ng/ml rhIL-13.（4）Observe the relative expression of PKA、CREB、STAT6、BCl-2 in thyroid cell:qRT-PCR detected the relative expression of PKA、CREB、STAT6、BCl-2 in thyroid cell under stimulation of50ng/ml rhIL-4 and 25ng/ml rhIL-13.4.Statistical Analysis:Test the normality of the data,the measured data of normal distribution are indicated as mean±standard deviation（SD）.The measured data of abnormal distribution are indicated as the median and the upper and lower quartile M(P₂₅,P₇₅).All data are analyzed by SPSS 20.0 statistical software.The data is tested for homogeneity of variance,two groups measurement data of homogeneity of variance are analyzed using independent-samples T test,Multi groups measurement data are analyzed using one-way analysis of variance（ANOVA）,followed by the LSD post hoc test for multiple comparisons.Multi groups measurement data of heterogeneity of variance are analyzed using non-parametric test,followed by the Kruskal-Wallis post hoc test for multiple comparisons.Binary variable are analyzed using chi-square test.The single-factor correlation analysis used Pearson correlation coefficient to represent the linear relationship between variables and independent variables.P<0.05was considered significant.Results:（1）Compared with normal control group,the frequency of ILC2 in patients of GD and GO group was enhanced obviously（P<0.05）,and the frequency of ILC2 in patients of GO group was a bit higher than GD group,but there was no significantly difference（P>0.05）.（2）Compared with normal control group,the level of IL-4,IL-5,IL-13 in plasma in patients of GD and GO group was enhanced obviously（P<0.05）,and the level of IL-4,IL-5,IL-13 in plasmain patients of GO group was a bit higher than GD group,but there wasnosignificantlydifference（P>0.05）.（3）Therewas no correlation between the frequency of ILC2 and white blood cells,neutrophils,lymphocytes.There was a correlation between ILC2and TSH,FT4,FT3,aTG,aTPO,but it’s low correlation.There was a positive correlation between ILC2 and the level of IL-4,IL-5,IL-13 in plasma.（4）Compared with normal control group,the level of thyroxine and triiodothyronine in the supernatant under stimulation of 50ng/ml rhIL-4 and stimulation of 25ng/ml rhIL-13 were enhanced obviously（P<0.05）.（5）Compared with normal control group,the relative expression of PKA、CREB、STAT6、BCl-2 mRNA of cell under stimulation of 50ng/ml rhIL-4 and stimulation of 25ng/ml rhIL-13 was enhanced obviously（P<0.05）.Conclusion:（1）The elevated frequency of ILC2 in PBMC and the elevated Th2 cytokines in plasma may activate Th2 immunity response in patients with Graves disease.（2）Th2 cytokines secreted from ILC2 may directly stimulate thyroid hormone secretion of thyroid cells,and inhibit thyroid cells apoptosis,finally lead to occurrence and development of Graves Disease.