Study Of The Role Of IL-17A On The Vasculopathy Pathogenesis In Patients With Systemic Scleroderma

Systemic sclerosis/scleroderma (SSc) is an autoimmune connective tissue disease with unknown etiology and complex pathogenesis. The main manifestations of the disease are cutaneous and internal organ fibrosis. The disease is associated with higher rates of disability and mortality, without satisfying treatment is available. Micro-angiopathy is the early clinical manifestation and along with process of the disease, plays important roles in pathogenesis, progression and prognosis. To date, the pathogenesis of micro-angiopathy remains unclear. So, the study of the mechanism of pathogenesis will facilitate early intervention and design the new anti-fibrosis therapeutic methods.In recent years, people have paid more attention of the role of Interlukin 17 secrating CD4+ T cells (Th17 cells) in the human disease. IL-17A is the main inflammatory factor secreted by Thl7 cells. Increasing data demonstrated that Th17 cells may involve in the pathogenesis of SSc. Some study suggested that the concentration of IL-17A in peripheral blood of SSc patients is associated with the disease activity. Our study observed that increasing number of IL-17+ cells infiltrate in the perivascular region of skin tissue in the early stage of SSc. We speculated that Th17 cells played a key role in the forming of micro-angiopathy. However, the research involved in the relationship between the Th17 cells and SSc was still elementary. Few study involved in the role Th17 cells in the pathogenesis of micro-angiopathy. Our study collected the blood specimens, skin tissue and culture cells of SSc patients and the healthy controls, used ELISA, RT-PCR, Western Blot, flow cytometry and immunohistochemistry technology to study the role and potential mechanism of Th17 cells involved in the injury and apoptosis of endothelial cells, and collagen overproduction.In this study, we showed that mRNA level of IL-17A was higher in PBMCs of SSc patients compared to the health controls (p<0.05). In addition, the concentration of IL-17A was increased in serum of SSc patients compared to the health controls (p<0.05). We observed that more IL-17+ lymphocytes infiltrated in the perivascular areas in the epidermal layer, dermal layer, and subcutaneous tissue of SSc patients. While, few Th17 cells was observed in every layers of skin of the health controls. IL-17A derived from SSc patient serum mediated endothelial cells inflammation by up-regulating chemokines and adhesion molecules and recruited more T cell-HUVEC adhesion. ERK cell signal pathway was also one of the cell signal pathways involved in vascular injury induced by IL-17A. The number of apoptosis endothelial cells and Th17 cells was increased in perivascular area of the skin tissue of SSc patients compared to health controls. We detected that IL-17A promotes apoptosis of endothelial cells, and can be neutralized by IL-17 antibody. IL-17A induce the human fibroblast cell proliferation and high expression of Collagen I and CollagenⅢ, in vitro. We observed that IL-17A promoted fibroblast change into myofibroblast expression of a-SMA. IL-17A activated the ERK cell signal pathway, and the expression ofa-SMA was blocked by ERK inhibitor. In conclusion, IL-17A mediated endothelial inflammation and apoptosis, promotes fibroblast change into myofibroblast and collagen overproduction in skin fibroblasts.Part Ⅰ The expression of IL-17A in peripheral blood and skin tissue of SSc patientsObjective:to explore the expression of IL-17A in peripheral blood and skin tissue of SSc patients and whether IL-17A involved in the pathogenesis of SSc.Methods:determination of the concentration of IL-17A in serum of 22 cases of SSc patients and 18 cases of health controls by ELISA; mRNA level of IL-17A in PBMCs of SSc patients and the healthy controls was measured by real-time PCR; Using the immunohistochemical staining technology to detect the expression of IL-17A and distribution of infiltrated IL-17+ cells in layers of skin tissue of SSc patients and the healthy controls.Results:the concentration of IL-17A (50.58±12.23 pg/ml) was increased in serum of SSc patients compared to the health controls (29.48±5.65pg/ml) (p<0.05). In addition, mRNA level of IL-17A was higher in PBMC of SSc patients compared to the health controls (p<0.05). Moreover, we observed that more IL-17+lymphocytes infiltrated in the perivascular areas in the epidermal layer, dermal layer, and subcutaneous tissue of SSc patients. While, few Th17 cells was observed in every layers of skin of the health controls.Conclusion:higher level of IL-17A in peripheral blood and skin tissue in SSc patients may involved in the pathogenesis of micro-angiopathy.Part Ⅱ. The relationship between endothelial cells injury and a high-expression of IL-17A in peripheral blood of SSc patientsObjective:to explore the mechanism of IL-17A induce the inflammation of vascular endothelial cellsMethods:cultured HUVECs and HMEC-1 cells were treated with serum of the SSc patients and IL-17A in vitro. Adhesion molecule and chemokine were measured by real-time PCR and Western Blot. Meanwhile, which signaling pathways were activated involve in IL-17A treated on HUVECs were explored by Western Blot; In addition, the relationship between signaling pathway and high expression of adhesion molecule and chemokine were determined. Moreover, whether IL-17A will induce the T cells adhere to HUVECs was study by T cells coculture with HUVECs.Results:IL-17A and serum of SSc patients stimulated the high expression of adhesion molecule VCAM-1、ICAM-1 and chemokine CCL-20 and CXCR-4 in endothelial cells HUVECs and HMEC-1.In addition, IL-17A and serum of SSc patients promoted the adherion of T cells to HUVECS. IL-17A activated p38 and ERK MAPK signaling pathway. ERK involved in the increased expression of adhesion molecule and chemokine as well as promotes T cells adhere to HUVECs.Conclusion:these data imply that ERK signal pathway might play a key role in the progression of endothelial injury induced by IL-17A in SSc.Part Ⅲ The relationship between the apoptosis of endothelial cells and the IL-17A and in SSc patientsObjective:to explore the role of IL-17A in the apoptosis of endothelial cells in SSc patients.Methods:determination the apoptosis of endothelial cells and IL-17+cells infiltration the perivascular area of of SSc patients and the health controls by TUNEL and immunohistochemistry. HUVECs were treated by IL-17A in vitro, the apoptosis HUVECs were analyzed by flow cytometry, and apoptosis genes expression of cascape-3, bcl-2 and bax were detected by Western blot.Results:the number of apoptosis endothelial cells and IL-17+ cells was increased in perivascular area of the skin tissue of SSc patients compared to the healthy controls. Flow cytometry technology detected that the IL-17A promotes apoptosis of endothelial cells. Western-blot results showed that expression of apoptosis gene caspase-3 and bax increased and anti-apoptosis gene bcl-2 decreased in endothelial cells treated by IL-17A. We detected that the function of IL-17A promotes apoptosis of endothelial cells can be blocked by IL-17 neutralizing antibody.Conclusion:IL-17A may induce apoptosis of endothelial cells in SSc patients.Part Ⅳ The role of IL-17A in the human fibroblast collagen overproduction in SSc patientsObjective:to investigate the role of IL-17A in collagen overproduction of human fibroblast cell and the fibroblast change into the myofibrolast.Methods:HUVECs were treated by the different concentrations of IL-17A for 1 day to 4 days, in vitro. The human fibroblast cell proliferation detected by CCK8 assay. The level of Collagen Ⅰ and Collagen Ⅲ were measured by WB and ELISA. Expression ofa-smooth muscle actin (a-SMA) was detected by immunocytofluorescent and WB. The human fibroblast cell signal pathways activated by IL-17A and the cell signal pathways inhibitor induced by the expression ofa-SMA detected by WB.Results:CCK8 analysed that the concentration of 5ng/ml,10Ong/ml and 20ng/ml of IL-17A treated on human fibroblast for 1 to 4 day, obviously induced the human fibroblast cell proliferation. And human fibroblast treated by the IL-17A(the concentration of 10ng/ml) for 24h in vitro, we observed that the increased expression of Collagen Ⅰ and CollagenⅢ detected by ELISA and Western Blot. The fibroblast treated by IL-17A (the concentration of lOng/ml) for 3 days could change into myofibroblast which specific expression of a-SMA. IL-17A treated on human fibroblast activated the ERK cell signal pathway, and the expression ofa-SMA was blocked by ERK inhibitor.Conclusion:IL-17A increased collagen synthesis of the human fibroblast cell and induced the fibroblast cell change into myofibroblast.