Relationship Between The Differentiation Features Of Th17 Cells And Pulmonary Vascular Reconstruction Under Hypobaric Hypoxia

Background:Hypoxia Pulmonary Hypertension is a chronic progressive disease which caused by hypoxia, characterized by elevated pulmonary artery pressure and associated with right ventricular hypertrophy and pulmonary vascular remodeling. High altitude heart disease is a common disease on the plateau area, and is a leading cause of death in patients with chronic altitude sickness. Therefore, the study of the pathogenesis of HPH is always the key point and hot spots of the high altitude medical research. Because the pathogenesis of HPH is not entirely clear, there are still no effective prevention and control measures. Previous studies have shown that inflammatory immune responses played an important role in HPH, but the detail mechanism is still not very clear. Th17 cells play a strong role in proinf lammatory responses. Th17 cell is different from Th1 and Th2 cell, is a new subunit belonging to the CD4+ T helper cell. Hypoxia inducible factor(HIF-1) is the key elements in the oxygen sensing signal pathway that mediated a lot of hypoxic responses including hypoxic pulmonary vascular remodeling. The upregulation of HIF-1 can promote the naive T cells differentiation to the Th17 cell by the means of direct activation of Th17 cell specific transcription factor retinoid related orphan receptor(RORγt). Therefore, hypoxia promotes the differentiation of naive T cells differentiation to Th17 by upregulation of HIF-1. Interleukin-17 is the main effect factor of Th17 cell. Th17 cells promote the inflammatory reaction by producing IL-17, which can promote T cell activation, recruitment and activation of neutrophils, stimulating the fibrocyte and fibroblast proliferation and collagen deposition. Several studies show that, Th17 cells play an important role in chronic obstructive pulmonary disease(COPD), asthma and other chronic inflammatory diseases and autoimmune diseases. We put mice in hypobaric hypoxic chamber to set the HPH model to study the changes of Th17 cells after hypobaric hypoxia exposure and its relationship with pulmonary vascular remodeling. The study plans to provide clues and evidences for revealing the pathogenesis of HPH and seeking for novel targets of effective prevention and therapy of HPH.Method:1. Animals and hypoxiaMale balb/c mice(n=50) were randomly divided into 0(control group), 3, 7, 14, 28 d hypobaric hypoxia groups. The mice in hypobaric hypoxia group were continuously housed in a hypobaric hypoxia chamber(simulated altitude of 6000 m) for 0, 3, 7, 14 or 28 d.2. MethodsThe hemodynamic data such as RVSP pulmonary artery pressure were gathered by cardiac catheterization. The RVHI was evaluated by the ratio of weight of the RV over the weight of the LV plus VS and RV weight over body weight. Collage deposit in pulmonary of was observed by masson staining on the lung tissue paraffin section. The lung and spleen was collected and the proportions of the Th17(CD4+IL-17+RORγt+) cells were detected by flow cytometry; The expression of IL-4, IL-6 and IL-17 in serum and the change of IL-17 in lung tissue were assayed by ELISA. The mRNA level of RORγt in the spleen and lung tissues was measured by RT-qPCR.Results:1. Hemodynamic changesRVSP and RVHI were significantly increased in hypoxic group(hypoxia 7, 14, and 28 d) than the control group and the difference was statistically significant(P<0.05 or P<0.01).2. Changes of serum cytokines in miceELISA testing results showed that the levels of IL-4、IL-6 and IL-17 in serum of mice in hypobaric hypoxia groups(hypoxia 7, 14, 28 d) were significantly increased than thecontrol group, and the difference was statistically significant(P<0.05).3. Changes of Th17 cells proportion in spleen tissueFlow cytometry results show that the percentage of IL-17+ RORγt+ CD4+T cell in spleen tissue was significantly increased than the control group, and increases over time, group 14 d, 28 d compare with control group had significant difference, and the difference was statistically significant(P<0.01).4. RORγt mRNA level in lung tissueThe RT-PCR results showed that the RORγt mRNA level in the lung tissue was significantly increased in 7, 14 and 28 d hypoxia groups, and the difference was statistical significance(P<0.05 or P<0.01).5. The level of IL-17 in lung tissueThe level of IL-17 in lung tissue in hypobaric hypoxia groups was significantly increased than the control group, and the difference was statistically significant(P<0.01).6. The level of RORγt protein expression in lung tissueWestern blot showed that the level of RORγt protein expression in lung tissue in hypobaric hypoxia groups was significantly increased than the control group, and the difference was statistically significant(P<0.05 or P<0.01).7. Changes of Th17 cell proportion in lung tissueThe percentage of IL-17+ RORγt+ CD4+T cell in lung tissue was significantly increased on an time-dependent way. than the control group, and increases over time, group 7, 14, 28 d compare with control group had significant difference, and the difference was statistically significant(P<0.05 or P<0.01).8. Correlation analysisThe RORγt m RNA and the IL-17 protein expression levels in the lung tissue were significantly positively related with RVSP and RVHIConclusion:1. The RVSP and RVHI were increased obviously, and the collagen deposition around pulmonary vascular surrounding was increased in mice after simulated high altitude(6000 m) for 7 d.2. Hypoxia promotes differentiation of Th0 cells to Th17 cells in mice.3. Hypobaric hypoxia promotes the infiltration of Th17 cells to lung tissue, which was involved in pulmonary vascular reconstruction.