Study Of Th17Cells In Patients With Graves Disease

Objective: Graves disease (GD) is one of the organ specificautoimmune thyroid disease (AITD) caused by of autoimmunity. The mainclinical manifestations of GD are various degrees of diffuse goiter, thyrotoxia,ophthalmopathy, and less commonly pretibial myxedema. Production ofthyroid autoantibodies and lymphocytic infiltrate in the thyroid are theimportangt characteristics in GD, but the mechanisms of autoimmuneresponses in the pathogenesis of GD are largely unknown.T helper17(Th17) cell is a new subset of CD4~+T cells which secretinginterleukin-17(IL-17). The predominant function of IL-17is thought to be asa proinflammatory mediator. IL-17stimulates production of chemokines.Granulocyte-macrophage colony stimulating factor (GM-CSF), tumournecrosis factor (TNF)-α, IL-6and mediates chemotaxis of neutrophils andmonocytes to sites of inflammationin, leads to progression and amplificationof local inflammation. Several studies have suggested that the Th17population is important in mediating autoimmune diseases such as rheumatoidarthritis, multiple sclerosis, and systemic lupus erythematosus in human andanimals. The pathogenesis of GD is not completely elucidated. In this study,the proportion of CD4~+IL-17~+T (Th17) cells in peripheral blood mononuclearcells (PBMC) was assessed by flow cytometry. The levels of IL-17in PBMCcultivated supernatants were determined by ELISA kit. The expression levelsof ROR-γt mRNA, IFN-γ mRNA and IL-4mRNA in PBMC were detected byqRT-PCR. The IL-17~+cells in thyroid gland were observed byimmunohistochemistry. The aim of this study is to explore the role of Th17cells in the pathogenesis of GD.Methods:1Objects of study30new-onset and untreated GD patients were enrolled in this study. Diagnostic criteria refer to〈Guide line for the diagnosis and treatment ofthyroid disease in China〉(Chinese Society of Endocrinology,2008),30age-and sex-matched healthy subjects were as controls. Thyroid glands wereobtained from6patients with GD who had received surgery, norml thyroidtissue was obtained from3healthy controls dead from trauma.2Methods:2.1The serum concentrations of TSH, FT3, FT4The serum levels of thyroid stimulating hormone (TSH), free thyroxine(FT4), free triiodothyronine (FT3) were measured with a commercialelectrochemiluminescence (ECL) Kit.2.2The number of Th17cells in PBMCPBMC were isolated from fresh blood by density gradient centrifugationnew, after stimulating by anti-CD3mAb and anti-CD28mAb for24h. Theproportion of circulating CD4~+IL-17~+T (Th17) cells from on-set GD patientsand healthy control subjects was assessed by flow cytometry.2.3The levels of IL-17in PBMC cultivated supernatantPBMC were isolated from fresh blood by density gradient centrifugation,after stimulating by anti-CD3mAb and anti-CD28mAb for24h, the culturesupernatant were celected. The levels of IL-17in PBMC culture supernatantswere determined by enzyme-linked immunosorbent assay (ELISA) kit.2.4The levels of ROR-γt, IFN-γ and IL-4mRNA in PBMCTotal RNA were extracted from2.2mentioned remanent PBMC, afterreverse transcription, the expression levels of ROR-γt, IFN-γ and IL-4mRNAwere detected by qRT-PCR. Human GAPDH gene was as internal control.2.5Distribution and number of IL-17~+cells in thyroid glandIL-17~+cells in thyroid gland were observed by immunohistochemistry.2.6Statistical methodsStatistical analysis was performed using SPSS13.0. All data waspresented as mean±SD. The differences between two groups were comparedby the Student's t-test, and double groups were proceeded correlation test bybivariate correlation. p-value less than0.05is considered statistically significant.Result:1The serum concentrations of TSH, FT3, FT4In the30on-set GD cases, the serum concentrations of TSH were lower,while the levels of thyroid hormone FT3and FT4were higher than healthcontrols.2The proportion of Th17cells in PBMCAfter stimulating by anti-CD3mAb and anti-CD28mAb for24h, theproportion of the CD4~+IL-17~+T (Th17) cells in PBMC were significantlyincreased in GD patients (21.95±3.62%) than healthy control subjects(9.26±5.85%).(P<0.01)3The PBMC cultivated supernatant levels of IL-17After stimulating by anti-CD3mAb and anti-CD28mAb for24h, thelevels of IL-17in PBMC culture supernatant from on-set GD patients(80.56±40.59pg/ml) were significantly higher than healthy control subjects(15.05±7.82pg/ml).(P<0.01)4The levels of ROR-γt, IFN-γ and IL-4mRNA in PBMCAfter stimulating by anti-CD3mAb and anti-CD28mAb for24h, higherexpression levels of ROR-γt mRNA (1.67±0.93) was observed in PBMC fromGD patients compared to healthy control subjects (0.93±0.17)(P<0.05). Incontrast, the levels of IFN-γ mRNA (0.31±0.08vs0.69±0.20, P<0.01), andIL-4mRNA (2.53±0.70vs7.29±0.83, P<0.01) were lower in PBMC from GDthan health controls.5Distribution of IL-17~+cells in thyroid glandImmunohistochemical analysis of thyroid sections revealed that IL-17~+cells appeared in thyroid gland from GD patients but not in healthy controlsubjects.6Correlation analysisThe proportion of CD4~+IL-17~+T (Th17) cells correlated negatively withthe serum concentrations of TSH (r=-0.4205, P＜0.05), but correlatedpositively with the the serum concentrations of FT3(r=0.5930, P＜0.01) and FT4(r=0.5580, P＜0.01). The proportion of Th17cells correlated positivelywith the expression levels of ROR-γt mRNA (r=0.5047, P＜0.05), butcorrelated negatively with the the expression levels of IFN-γ mRNA(r=-0.5085, P＜0.01) and IL-4mRNA (r=-0.5372, P＜0.01).Conclusions:1In GD patients, the proportion of Th17cells in PBMC and the levels ofIL-17in PBMC culture supernatant were significantly higher.The proportion of Th17cells correlated negatively with the serumconcentrations of TSH, but correlated positively with the serumconcentrations of FT3and FT4. There were IL-17~+cells appeared inthyroid gland from GD patients. These results indicate that Th17cells mayparticipate in the pathogenesis of GD.2In GD patients, the expression level of ROR-γt mRNA was significanthigher, while the IFN-γ mRNA and IL-4mRNA expression were lower inPBMC stimulated by anti-CD3and anti-CD8antibodys. It can be proposedthat CD4~+T cells tent to develop into Th17cells after being activatd in theGD patients.