The Role Of Th17 Cells In Intractable Graves Disease

Graves disease(GD) is the most commont organ-specific autoimmune disease characterized by reactivity to self-thyroid antigens and lymphocytic infiltrate within the thyroid.CD4⁺T helper lymphocytes play a key rote in the pathogenesis of GD.Th17 a new CD4⁺helper T cell subset which produce IL-17.IL-17 a proinflammatory cytokine signals via IL-17R that induces inflammatory response.IL-17 plays a major role in numerous autoimmune disease.The proportions of Th17 cells were higher in patients with intractable GD than those with GD in remission. none of IL-17(-/-) NOD-H2(h4) mice became hyperthyroid.It is show that the role of Th17 cells for the pathogenesis of GD.Peroxisome proliferator-actived receptory-gamma(PPARγ) agonist pioglitazone has anti-inflammatory effects and selectively inhibits Th17 differentiation.SO we investigated the role of Th17 cells in GD especially in intractable GD and the effect of PPARγagonist on IL-17 inducible inflammatory cytokines secretion in primary human thyroid cultures.Hope that Th17/IL-17 could become a promising target in the treatment of GD.The status of Th17,IL-17 and IL-17R in patients with GDObjective:To explore the serum levels of IL-17 in patients with GD and the status of Th17,IL-17and IL-17R in the immunopathogenesis of GD.Method:1.ELISA were used to measure the serum leves of IL-17 of GD.The studied subjects inclue 38 case of untreated GD patients,36 case of intractable GD,34 case of GD patients in remission and 37 case of healthy donors as normal controls.2.Immunohistochemictry were used to access in vitro synthesis of IL-17 and IL-17R in thyroid glands of 13 case of intractable GD and 11 case of normal controls.3.Real-time PCR were used to detect the IL-17mRNA of intrathyroid lymphocytes and IL-17RmRNA of thyrocytes.4.Flow cytometry and immunofluorescence were used to evaluated the expression of IL-17R protein in human thyrocytes.Results:1.As compared with the healthy controls,the leves of IL-17 in untreaed GD and intractable GD significantly increased ,and the leves of IL-17 in intractable GD was higher than that in untread GD.2.The levels of IL-17mRNA in intrathyroid lymphocytes of intractable GD was higher than that in the healthy controls.3.The expression of IL-17 was detected in intrathyroid lymphocytes of intractable GD and IL-17R protein was also detected in thyrocytes of intractable GD.The effects of IL-17 on cells viability and the functions of human thyrocytes ,as well as the expression of inflammatory cytokines,and the activation of signaling pathways in IL-17-induced.Objective:To explore the effects of IL-17 on cells viability and the functions of thyrocytes of intractable GD and healthy controls,as well as the expression of IL-6,CXCL10 and ICAM-1,and the activation of signaling pathways in IL-17-induced.Method:1.MTT were used to access the cells viability.2.Chemiluminescence were used to measure T3,T4 secretions by thyrocytes.3.Real-time PCR investigated the expression of IL-6,CXCL10,ICAM-1mRNA with stimulus on cells.4.ELISA were used to measure the leves of IL-6 and CXCL10 secretions .5.Flow cytometry were used to evaluated the expression of IL-17R and ICAM-1 protein on human thyrocytes.6.Westernblot were used to evaluate the signaling molecules of P38MAPK,JNK,ERK1/2 and NF-κB.7.Immunofluorescence were used to evaluated the nuclear transloeation of NF-κBp65.Results:1.There were no effects of IL-17 on cells viability and the secretions of T₃,T₄ in thyrocytes of intractable GD and healthy controls.2.the expressions of IL-6,CXCL10 and ICAM-1 in intractable GD thyrocytes were much higher than that of normal thyrocytes.3.IL-17 up-regulated the expressions of IL-6,CXCL10 and ICAM-1 at a time-and dose-dependent manners in thyrocytes of intractable GD.4.IL-17 combin with TSAb the leves of IL-6,CXCL10 and ICAM-1 was remarkedly increased not only in thyrocytes of intractable GD,but also in healthy controls.4.IL-17 has no effects on the expressions of IL-17R in two type thyrocytes.TSAb up-regulated the expressions of IL-17R in thyrocytes of healthy controls. 5.The expressions of inflammatory mediators is through IL-17R,P38MAPK and NF-κB ,but no JNK and ERK1/2.The effects of IL-17 in thyrocytes of intractable GD are modulated by pioglitazoneObjective:To explore whether the expressions of IL-6,CXCL10,ICAM-1 and MAPK,NF-κB activation induced by IL-17 are modulated by pioglitazone Method:1.MTT were used to access the cell cells viability.2.ELISA were used to measure the leves of IL-6 and CXCL10 secretions .3.Flow cytometry were used to evaluated the expression of ICAM-1 protein in human thyrocytes. 4.Real-time PCR investigated the expression of IL-6,CXCL10,ICAM-1mRNA with stimulus on cells.5.Westernblot were used to evaluate the signaling molecules of P38MAPK,JNK,ERK1/2 and NF-κB modulated by pioglitazone.Results:1.There were no effects of IL-17(0-50μM) on cells viability ,but IL-17(100μM) inhibited the thyrocytes growth.2. Pioglitazone inhibited the gene and protein levels of IL-6 CXCL10 and ICAM-1 induced by IL-17 in thyrocytes of intractable GD.3. It is the activations of P38MAPK and NF-κB ,but no JNK ERK1/2 induced by IL-17 were inhibited by pioglitazone .Conclusion1. The leves of IL-17 in untreaed GD and intractable GD significantly increased .Not only Th17 cells could be detected in the thyroid glands of intractable GD,but also IL-17R were positive expression in the thyrocytes of intractable GD.It suggested that Th17 was involved in pathological process of intractable GD.2. IL-17 can up-regulated the expressions of IL-6,CXCL10 and ICAM-1 via p38MAPK and NF-κB.The effects of IL-17 on thyrocytes of intractable GD can be enhanced by TSAb.3. pioglitazone inhibits IL-17-stimulated secretions of inflammatory cytokines by blocking P38MAPK and NF-κB signaling pathways.In summary Th17 may play an important role in the pathophysiology of intractable GD .Pioglitazone inhibits IL-17-stimulated secretions of inflammatory cytokines in thyrocytes of intractable GD.It suggested that PPARγagonists could be a potential choice of cure to intractable GD.