Study On The Role And Mechanism Of Th17Cells And Related Cytokines In Graves’ Ophthalmopathy

BackgroundGraves’ ophthalmopathy (GO) is considered to be an organspecific autoimmune disease characterized by lymphocytes infiltrating orbital tissue. T cell and Macrophages product and release various cytokines to the tissue. The immune process in the orbit leads to proliferation of fibroblasts and stimulation of glycosaminoglycan (GAG) synthesis. Graves’ ophthalmopathy can leads to various clinical manifestations, such as ophthalmalgia, proptosis, periorbital edema, extraocular museles motor disorder, inflammation and vision damage. UP to now,20-25%patients with Graves’disease have got Graves’ ophthalmopathy in the world. The pathogenesis of GO is still unclear, and it also remains a therapeutic dilernilla.Current common view is that the pathology of GO is a kind of thymus derived cell mediated disease which is polygenic inheritance and caused by multi-factors. Its pathogenesis is complicated, the abnormity of CD4+T lymphocyte immunologic function is the most important. CD4+T helper(Th) cells play important roles in regulation of autoimmunity in vivo. Traditionally, CD4+Th cells had been divided into two types of subsets:type1and type2. Thl cytokines such as IFN-γ and IL-12influent the clean up of parasite and extracellular bacteria. IFN-γ and IL-12are symbolic cytokine of Thl and Th2cell subpopulation, respectively. Recently, the Thl/Th2dichotomy has been revisited and the third member of the CD4+Th cell falmily, now widely known as Th17cells, has substantially advanced our understanding of T cell-mediated immnunity. The development of Th17cells is regulated by IL-6and TGF-b and dependent on STAT3-dependent expression of the transcription factors ROR γ t and、ROR α. Th17cell responses have been demonstrated to be critical for inflammatory responses to tumors and contribute to the pathogenesis of many autoimmune diseases. And IL-23R is a key factor to Th17cell. IL-23is applied to Th17cell to maintain its expression. It is approved that IL-23stimulate the amplification and maintain of Th17cell. During differentiation of Th17celles, IL-23plays an important role in expansion and stabilization of them. IL-17A is the key cytokine of Thl7cells. So, some researchers draw a close attention to the role of the IL-23/IL-17axis in autoimmune diseases.In the early active phase of GO, lymphocytes and some other autoimmune cells infiltrate into the local site, accompanied by hydrophilia glycosaminoglycan cumulation in orbital connective tissue. While in the advanced stable phase, the fibrosis of retroorbital tissue is apparent. As special treatment for each GO patient in active or inactive phase, the outcomes may be different. Therefore evaluation of the condition of Patients correct and efficient is of great importance for the prognosis and treatment of this disease. Until now, the most common immunotherapy medicine of GO is glucocorticoid. Culminating evidences indieated that glucocorticoid treatment was effective for moderate-to-severe GO. In addition, the reported response rate to intravenous glucocorticoid pulse therapy in moderat-to-severe and active GO patients was about77%.This study intends to evaluate Graves’ophthalmopathy patient Thl7cells in the peripheral blood expression of PBMC and Graves’ophthalmopathy clinical relevance and significance, and watch the sugar cortical hormone (methylprednisolone) role in PBMC on\"eye disease Thl7cells and the related factors of influence, and further explore glucocorticoid treatment (methylprednisolone)\"eye new mechanism.Part1Peripheral change of Th17cells and the related cytokines and clinical correlations in Graves’ ophthalmopathy patients ObjectiveTo detect proportion of Thl7cells and the expression level of related cytokines IL-6, IL-17, IL-23in peripheral blood in patients with Graves’ophthalmopathy and then explore the differences in expression and clinical relevance of Thl7cells and related cytokines between Graves’ophthalmopathy patients and normal people and among patients in different period. We try to clear the mechanism and clinical significance of Thl7cells in the pathogenesis of Graves’ ophthalmopathy.Methods(1) We selected40untreated patients with Graves’ ophthalmopathy(GO group). The patients were divided into active GO group (CAS no less than3)and inactive GO group (CAS<3) according to the clinical activity score (CAS); Each group has20cases. At the same time wo choosed20untreated cases with Graves disease (GD) and20cases of normal control group,(2) The expression level of Th17cells in peripheral blood mononuclear cells (PBMC) was tested by the flow cytometry. The expression level of serum IL-6, IL17, IL-23were detected by ELISA. We analysed the relationship between IL-6, IL17, IL-23and indexes such as CAS, TSH, FT3, FT4, TRAb and exophthalmos respectively by clinical correlation analysis.(3) Statistical analysis:Measurement datas are measured by mean±standard deviation. Statistical analysis were performed using the SPSS16.0software Package. Statistical significance of differences among groups was evaluated by one-way ANOVA. Multiple comparisons were carried out using the Least-significant Difference (LSD) method. Welch method was used when equal variances not assumed. Multiple comparisons was analyzed by Dunnett’s T3method. The normal distribution data was analyzed using Pearson correlation analysis and the non-normal distribution data was analyzed using Spearman rank correlation analysis. P<0.05is considered statistically significant.Results(1) The flow cytometry assay showed that Thl7cells ratio was higher in the patients with GO group than that of normal control, The difference was statistically significant (1.24±0.18%vs0.55±0.09%P<0.05;0.99±0.13%vs0.55±0.09%P<0.05). Th17cells ratio was higher in patients with active GO group than that of inactive GO group (1.24±0.18%vs0.99±0.13%; P<0.05) and GD group (1.24±0.18%vs0.97±0.16%; P<0.05); There was no significant statistically difference between patients with inactive GO and patients with GD (P>0.05). Correlation test analysis showed that the expression level of Th17cells in GO group was positively correlated with clinical activity score (CAS), P<0.05, and has no significant correlation with indexes such as TSH, FT3, FT4, TRAb and exophthalmos (P>0.05).(2) ELISA test result showed that the expression level of serum IL-6, IL-17and IL-23in active GO group was higher than that of control (P<0.05). There was no significant statistically difference in the expression level of IL-17and IL-23of between inactive GO group and GD group. There was no significant statistically difference in the expression level of IL-6among GO group,GD group and control(P>0.05). Correlation analysis showed that the expression level of IL-17and IL-23of GO group was positively correlated with clinical activity score (CAS)(P<0.05), while there was no significant correlation between IL-17、IL-23and indexes such as TSH, FT3, FT4, TRAb and exophthalmos (P>0.05).ConclusionThe expression level of Th17cells and related cytokines IL-6, IL-17, IL-23in patients with GO was higher than that of normal control groupand active patients was higher than the inactive patients. It was positively correlated with clinical activity score (CAS), The results showe that Th17cells and related cytokines may play an important role in development of GO. It can be considered as an new index to evaluat GO activety. Part2Peripheral expression of IL-17、the ROR-γt mRNA related to Th17cells and clinical correlations in patients with Graves’ophthalmopathy ObjectiveTo research expression of IL-17, the ROR-γt mRNA related to Th17cells in peripheral blood of patient with Graves’ ophthalmopathy and differences among different periods of disease and analysis clinical correlation. To further explore the role of Th17cells in the pathogenesis of GO.Methods(1) We selected40untreated patients with Graves’ ophthalmopathy(GO group). The patients were divided into active GO group (CAS no less than3)and inactive GO group (CAS<3) according to the clinical activity score (CAS); Each group has20cases. At the same time wo choosed20untreated cases with Graves disease (GD) and20cases of normal control group,(2) The expression level of IL-17、ROR γt mRNA in peripheral blood mononuclear cells (PBMC) was detected using real-time fluorescent PCR. Then We analysed the relationship between IL-17、ROR γ t mRNA and indexes such as CAS, TSH, FT3, FT4, TRAb and exophthalmos respectively by clinical correlation analysis.(3) Statistical analysis:Measurement datas are measured by mean±standard deviation. Statistical analysis were performed using the SPSS16.0software Package. Statistical significance of differences among groups was evaluated by one-way ANOVA. Multiple comparisons were carried out using the Least-significant Difference (LSD) method. Welch method was used when equal variances not assumed. Multiple comparisons was analyzed by Dunnett’s T3method. The normal distribution data was analyzed using Pearson correlation analysis and the non-normal distribution data was analyzed using Spearman rank correlation analysis. P<0.05is considered statistically significant.ResultsThe expression levels of IL-17, the ROR-γ t mRNA was higher in the patients with GO group and GD group than that of control, The difference was statistically significant. It was higher in patients with active GO group than that in inactive GO group. There was no significant statistically difference between patients with inactive GO and patients with GD (P>0.05). Correlation t analysis showed that expression level of ROR γ t mRNA was significantly positive correlated to IL-17mRNA. The expression level of IL-17、ROR γ t mRNA was positively correlated with clinical activity score (CAS), P<0.05, and it had no significant correlation with laboratory indexes (TSH, FT3, FT4, TRAb) and exophthalmos (P>0.05).ConclusionThe expression levels of IL-17, the ROR-γ t mRNA was higher in the patients with GO group than that of control. It was higher in patients with active GO group than that in inactive GO group and was positively correlated with clinical activity score (CAS). The results shows that the development of the GO is associated with increases of IL-17, ROR-γ tmRNA. Part3The effect of methylprednisolone on Th17cells and its related cytokines in peripheral blood of active Graves’ophthalmopathyObjectiveThe purpose of this study is to observe the effect of different doses methylprednisolone in vitro on Th17cells and the related cytokines of patients with active Graves’ophthalmopathy. It can provide us an new theoretical basis to treat with active Graves’ophthalmopathy using methylprednisolone for clinic treatment.Methods(1) We elected5cases of untreated patients with active Graves’ophthalmopathy and5cases of normal healthy controls.Then we separated PBMC in vitro and the PBMC was stimulated for12h with different concentrations of methylprednisolone (0,1,5,25,125ug/ml). We collected the culture supernatant after12h.(2) We detected the proportion of Th17cells of mononuclear cells (PBMC) stimulated by methylprednisolone using flow cytometry. The expression level of IL17of culture supernatant was detected using ELISA.(3) Statistical analysis:Measurement datas are measured by mean±standard deviation. Statistical analysis were performed using the SPSS16.0software Package. Repeated data was measured by repeated measures analysis of variance. P<0.05is considered statistically significant.Results(1) By repeated measures analysis of variance, the results showed that the difference was statistically significant between the active GO group and the normal control group (F=402.728, P<0.001). The difference was statistically significant between different concentrations of group (F=50.870, P<0.001). We can observed interaction between the different groups and the different concentration (F=4.208, P <0.001). It suggested that different group with the increase of the concentration of methylprednisolone, the proportion of Thl7cells trend. Further separate effects analysis showed that the differences were statistically significant (P<0.05), the two groups in different concentrations of methylprednisolone after two groups within different concentrations difference was statistically significant (P<0.05).(2) The results showed that the difference was statistically significant between the active GO group and the normal control group by repeated measures analysis of variance (F=392.181, P<0.001). The difference was statistically significant between different concentrations of group (F=28.691, P<0.001). We can observed interaction between the different groups and the different concentration (F=6.705, P<0.001). It suggested that different group with the increase of the concentration of methylprednisolone, the proportion of IL-17trend. Further separate effects analysis showed that the differences were statistically significant (P<0.05), the two groups in different concentrations of methylprednisolone after two groups within different concentrations difference was statistically significant (P<0.05).ConclusionMethylprednisolone can obviously inhibit the expression level of Thl7cells and IL-17of active Graves’ophthalmopathy and it provided a theoretical basis and laboratory evidence for the study of the clinical application of glucocorticoid methylprednisolone.