调节性T细胞与Th17细胞在支气管哮喘中失衡表达的实验研究
摘要
研究背景
支气管哮喘(Asthma,Bronchial asthma,简称哮喘)是全球最常见的慢性疾病之一。据不完全统计,全世界目前大约有3亿哮喘患者,且有证据显示目前哮喘患病率以每年1%左右的速度递增,严重影响患者的日常生活,给患者家庭及社会带来了沉重的经济负担,因此对支气管哮喘进行深入的研究具有深远的社会意义。
支气管哮喘是由多种炎症细胞特别是肥大细胞、嗜酸性粒细胞和T淋巴细胞等共同参与的慢性气道炎症性疾病。炎症细胞在气道的局部聚集、炎症介质和细胞因子的释放等是形成和维持气道炎症并进而引起组织损伤和气道功能障碍的主要原因,其中T淋巴细胞在哮喘气道炎症中发挥着核心作用。根据T淋巴细胞在免疫应答中的功能不同,将T淋巴细胞分成辅助性T细胞(Th)、细胞毒性T细胞(Tc)和调节性T细胞(Treg),.其中Th细胞又分为Th1细胞和Th2细胞。
既往认为支气管哮喘是由过度激活的Th2细胞介导的气道慢性炎症性疾病,Th1反应被认为是过敏性疾病包括支气管哮喘的保护因素。针对过敏性疾病的免疫疗法可使Th2细胞因子表型转变为Th1细胞因子表型,同时临床症状得到改善。而且倾向产生Th1细胞因子的个体患过敏性疾病的可能性减少。因此哮喘中Th1/Th2失衡理论被广泛接受。
但近年来发现,哮喘的一些实验和临床现象并不能完全用Th1/Th2失衡理论来解释;并且有研究发现Th1细胞可以加重过敏症和哮喘,Th1细胞因子IFN-γ,与抗原诱导的气道高反应和嗜酸粒细胞浸润有关。而Treg既能抑制Th1细胞,也能抑制Th2细胞,用Treg细胞可更好的解释哮喘的发病机制,因此近年来成为研究的热点。Th17细胞是近来发现的不同于Th1和Th2的另一种CD4~+T细胞的新亚型,具有IL-23依赖性产生IL-17的特性,研究发现其与Treg之间存在复杂的相关关系,并且与支气管哮喘也有关。因此既然Th1/Th2细胞亚群比例和功能的失衡是支气管哮喘重要的免疫异常,是哮喘发病机制中的重要环节,那么是否在哮喘中同样也存在Treg/Th17亚群比例和功能的失衡,这个问题值得我们去深入研究。
目的
1、探讨调节性T细胞(Treg)与Th17细胞及相应转录因子Foxp3、RORγt在支气管哮喘中的失衡表达;2、探讨Treg与Th17细胞及转录因子Foxp3与RORγt表达量的比值与气道炎症的相关关系。
方法
1、小鼠哮喘模型的建立及评价
30只雌性SPF级Balb/c小鼠按随机数字表法分为正常对照组、哮喘组和地塞米松治疗组;哮喘组于第0d、7d、14d(腹腔+皮下)注射0.2ml致敏液(含20ug OVA,20mg AL(OH)_3),第21-27d雾化吸入5%OVA激发;地塞米松治疗组于21至27d雾化吸入5%OVA激发前30min腹腔注射地塞米松(1mg/Kg):正常对照组则用生理盐水替代OVA致敏和激发;利用无创小鼠体描仪通过全身性体积描记法观察各组小鼠气道反应性;通过肺组织石蜡切片苏木素-伊红(HE)染色观察各组气道病理改变;用酶联免疫吸附测定法检测小鼠支气管肺泡灌洗液(BALF)中IL-5、IFN-γ含量。
2、调节T细胞与Th17细胞在支气管哮喘小鼠中的失衡以及与气道炎症的相关性分析
用酶联免疫吸附测定法检测各组小鼠血清和支气管肺泡灌洗液中IL-10、IL-17含量;用逆转录-聚合酶链反应(RT-PCR)测定法检测肺组织IL-10mRNA、IL-17 mRNA的表达;通过各组小鼠脾脏制备脾细胞单细胞悬液,利用流式细胞检测技术检测各组CD4~+CD25~+Foxp3~+细胞和CD4~+IL-17~+细胞占CD4~+细胞的比例。
3、转录因子Foxp3/RORγt在支气管哮喘小鼠中失衡表达以及与气道炎症的相关性分析
用酶联免疫吸附测定法检测各组小鼠血清和支气管肺泡灌洗液中IL-10、IL-17含量;用逆转录.聚合酶链反应(RT-PCR)测定法检测肺组织IL-10mRNA、IL-17mRNA、Foxp3mRNA和RORytmRNA的表达;用免疫组化及免疫印迹(Western blot)法检测肺组织中Foxp3和RORγt蛋白的表达。
结果
1、成功建立小鼠哮喘模型。哮喘组所有小鼠在雾化过程中均出现不同程度的烦躁不安或安静少动、呼吸加快加深、点头呼吸、弓背直立、前肢缩抬,二便失禁等哮喘速发相反应表现。
同等剂量乙酰胆碱激发(3.125、6.25、12.5、25、50mg/m1)的气道反应性检测中,哮喘组小鼠%ΔPenh明显高于正常对照组。
哮喘组小鼠细支气管及伴行血管周围大量炎症细胞浸润,气道壁和肺组织中可见以嗜酸性粒细胞、淋巴细胞为主的大量炎症细胞浸润,支气管管壁增厚、管腔狭窄,支气管管腔内可见粘液栓,粘膜皱壁减少,杯状细胞增生,肺泡间隔不同程度增宽,部分断裂,形成肺气肿。
哮喘组小鼠肺泡灌洗液中细胞总数及嗜酸性粒细胞、中性粒细胞和淋巴细胞计数明显高于正常对照组(P＜0.01)。
哮喘组小鼠肺泡灌洗液中IL-5水平明显高于正常对照组(P＜0.01);与之相反,哮喘组小鼠肺泡灌洗液中IFN-γ,水平低于正常对照组(P＜0.01)。
2、同等剂量乙酰胆碱激发(3.125、6.25、12.5、25、50mg/ml)的气道反应性检测中,哮喘组小鼠%ΔPenh明显高于正常对照组小鼠(P＜0.01),地塞米松治疗组小鼠%ΔPenh较哮喘组明显降低(P＜0.01);在乙酰胆碱浓度为3.125、6.25、12.5mg/ml激发时,正常对照组和地塞米松治疗组小鼠%ΔPenh相似,差异无统计学意义(P＞0.05)。在乙酰胆碱浓度为25、50mg/ml激发时,地塞米松治疗组小鼠%ΔPenh高于正常对照组小鼠(P＜0.01)。
哮喘组肺泡灌洗液中细胞总数、中性粒细胞、嗜酸粒细胞和淋巴细胞计数均较正常对照组明显增多(P＜0.01);地塞米松治疗组与哮喘组相比中性粒细胞、嗜酸粒细胞和淋巴细胞计数明显减少(P＜0.01),与正常对照组比较仍较高(P＜0.01)。
哮喘组小鼠肺泡灌洗液、血清中IL-17水平较正常对照组明显升高(P＜0.01);地塞米松治疗组肺泡灌洗液、血清中IL-17水平较哮喘组有所下降,但与正常对照组比较仍较高(P＜0.01);与之相反,哮喘组小鼠肺泡灌洗液、血清中IL-10水平较正常对照组明显下降(P＜0.01);地塞米松治疗组肺泡灌洗液、血清中IL-10水平较哮喘组有所升高,但与正常对照组比较仍较低(P＜0.01)。
哮喘组小鼠肺组织中细胞因子IL-17mRNA的表达较正常对照组小鼠明显增强(P＜0.01);地塞米松治疗组小鼠肺组织IL-17mRNA的表达减弱,明显低于哮喘组小鼠(P＜0.01),但与正常对照组小鼠比较仍较高(P＜0.01)。哮喘组小鼠肺组织中细胞因子IL-10mRNA的表达较正常对照组小鼠明显减弱(P＜0.01);地塞米松治疗组小鼠肺组织IL-10mRNA的表达较哮喘组小鼠有所增强(P＜0.01),但与正常对照组小鼠比较仍较低(P＜0.01)。
流式细胞仪检测结果显示哮喘组CD4~+细胞中淋巴细胞亚群CD4~+CD25~+Foxp3~+细胞百分率显著低于正常对照组(P＜0.01);地塞米松治疗组CD4~+细胞中淋巴细胞亚群CD4~+CD25~+Foxp3~+细胞百分率较哮喘组小鼠有所增加(P＜0.01),但与正常对照组小鼠比较仍较低(P＜0.01)。哮喘组CD4~+细胞中淋巴细胞亚群CD4~+IL+17~+细胞百分率显著高于正常对照组(P＜0.01);地塞米松治疗组CD4~+细胞中淋巴细胞亚群CD4~+IL-17~+细胞百分率较哮喘组小鼠有所减少(P＜0.01),但与正常对照组小鼠比较仍较高(P＜0.01)。哮喘组CD4~+CD25~+Foxp3~+细胞/CD4~+IL+17~+细胞比例较正常对照组下降(P＜0.01);地塞米松治疗组CD4~+CD25~+Foxp3~+细胞/CD4~+IL-17~+细胞比例较哮喘组小鼠有所增加(P<0.01),但与正常对照组小鼠比较仍较低(P<0.01)。
CD4~+CD25~+Foxp3~+细胞/CD4~+IL-17~+细胞与气道炎性指标经相关性分析显示,CD4~+CD25~+Foxp3~+细胞/CD4~+IL-17~+细胞比值与气道反应性(%△Penh)呈负相关,用3.125、6.25、12.5、25、50mg/ml不同浓度乙酰胆碱激发时,相应的气道反应性(%△Penh)与CD4~+CD25~+Foxp3~+细胞/CD4~+IL-17~+细胞比值的相关系数分别是-0.810,-0.853,-0.796,-0.898,-0.838(P均<0.01);与嗜酸粒细胞、中性粒细胞、淋巴细胞数呈负相关(r=-0.873,-0.878,-0.855;P均<0.01);与血清、肺泡灌洗液中IL-10含量呈正相关(r=0.93,0.926;P均<0.01),而与血清、肺泡灌洗液中IL-17呈负相关(r=-0.878,0.828;P均<0.01)。
3、哮喘组小鼠肺组织中转录因子RORγt mRNA的表达较正常对照组小鼠明显增强(P<0.01);地塞米松治疗组小鼠肺组织RORγt mRNA的表达减弱,明显低于哮喘组小鼠(P<0.01),但与正常对照组小鼠比较仍较高(P<0.01)。哮喘组小鼠肺组织中转录因子Foxp3 mRNA的表达较正常对照组小鼠明显减弱(P<0.01);地塞米松治疗组小鼠肺组织Foxp3 mRNA的表达较哮喘组小鼠有所增强(P<0.01),但与正常对照组小鼠比较仍较低(P<0.01);Foxp3/RORγt mRNA表达量比值哮喘组(0.18±0.05)较正常对照组(1.97±0.58)明显降低(P<0.01);地塞米松治疗组Foxp3/RORγt mRNA表达量比值为(0.59±0.14),与哮喘组Foxp3/RORγt mRNA表达量比值比较有所升高(P<0.01),但仍低于正常对照组(1.97±0.58)(P<0.01)。
免疫组化分析显示哮喘组小鼠转录因子Foxp3表达强度较正常对照组明显减弱,表达范围减少(P<0.01);转录因子Foxp3在地塞米松治疗组小鼠表达强度与哮喘组小鼠比较有所增高(P<0.01),与正常对照组比较强度和范围仍降低(P<0.01)。哮喘组小鼠转录因子RORγt表达强度较正常对照组小鼠异常增强,表达范围广泛(P<0.01);在地塞米松治疗组小鼠转录因子RORγt表达强度和表达范围高于正常对照组小鼠(P<0.01),与哮喘组小鼠相比,地塞米松治疗组小鼠RORγt表达强度和表达范围降低(P<0.01)。
Western blot结果分析显示转录因子Foxp3蛋白在正常对照组小鼠肺组织中有表达,而在哮喘组小鼠肺组织中低表达,两组比较差异有显著性(P＜0.01);予以地塞米松干预后,Foxp3蛋白在小鼠肺组织中表达增高,高于哮喘组小鼠,但低于正常对照组小鼠(P＜0.01)。转录因子RORγt蛋白在正常对照组小鼠肺组织中有低表达,而在哮喘组小鼠肺组织中异常高表达,两组比较差异有显著性(P＜0.01);在地塞米松干预治疗后,RORγt蛋白仍表达,低于哮喘组小鼠,但高于正常对照组小鼠(P＜0.01)。哮喘组Foxp3/RORγt蛋白表达量比值明显低于正常对照组(P＜0.01);地塞米松治疗组Foxp3/RORγt蛋白表达量比值较哮喘组有所升高,但仍低于正常对照组(P＜0.01)。
Foxp3/RORγt蛋白表达量的比值与气道炎症性指标作相关性分析,结果显示Foxp3/RORγt蛋白表达量的比值与气道反应性(%ΔPenh)呈负相关,用3.125、6.25、12.5、25、50mg/ml不同浓度乙酰胆碱激发时,相应的气道反应性(%△Penh)与Foxp3/RORγt蛋白表达量比值的相关系数分别是-0.708,-0.715,-0.702,-0.822,-0.846(P＜0.01);Foxp3/RORγt蛋白表达量比值与嗜酸性粒细胞数、淋巴细胞数和中性粒细胞数呈负相关(r＝-0.904,-0.881,-0.862,P＜0.01),与血清和肺泡灌洗液中IL-17含量呈负相关(r＝-0.880,-0.846,P＜0.01),而与血清和肺泡灌洗液中IL-10含量呈正相关(r＝0.718,0.816,P＜0.01)。
结论
1、通过对致敏剂及致敏方法的改进,使我们更容易成功建立小鼠哮喘动物模型,为随后进一步实验研究提供了一个很好的基础;利用无创小鼠体描仪通过全身性体积描记法可方便的观察实验小鼠气道反应性,为进一步研究支气管哮喘的病理生理学机制提供了很好的工具和手段。
2、在支气管哮喘中存在Treg与Th17的失衡,表现为CD4~+细胞中CD4~+CD25~+Foxp3~+细胞减少,CD4~+IL-17~+细胞增多,CD4~+CD25~+Foxp3~+细胞/CD4~+IL-17~+细胞比例低下,反映哮喘的免疫失衡;并且CD4~+CD25~+Foxp3~+细胞/CD4~+IL-17~+细胞比例与气道炎症各指标呈直线相关,Treg与Th17的失衡很可能是哮喘气道炎症形成的重要原因之一。
3、在支气管哮喘中,转录因子Foxp3/RORγt失衡表达,表现为转录因子Foxp3基因和蛋白的低表达,RORγt基因和蛋白的高表达,F0xp3/RORγt蛋白表达量比值与气道炎症各指标呈直线相关,Foxp3/RORγt的失衡表达很可能是哮喘中Treg与Th17失衡形成的主要机制。
4、地塞米松可能通过调节Treg与Th17的平衡来抑制气道炎症。
Background
Asthma is a very common disease in the world.It is now estimated that as many as 300 million people suffer from asthma and asthma attacks have increased by 1% per year.The burden of this disease to governments,health care systems,families,and patients is increasing worldwide,so there is a far-reaching society significance in the asthma research.
Asthna is a chronic inflammatory disease of the airways in which many inflammatory cells play a role,in particular,mast cells,eosinophils,T lymphocytes.The recruitment of inflammatory cells and the release of cytokines and mediators of inflammation are the major reason which cause and keep the airway inflamemation of asthma,and futher cause the tissue damage and airway disfunction.Among these inflammatory cells,T lymphocytes play a pivotal role in the pathogenesis of asthma.According to the function in immune response,we divided T lymphocytes into three subsets,help T cell(Th),cytotoxic T cell(Tc) and regulatory T cell(Treg).Th ceils divided into Th1 cell and Th2 cell.
Long time ago,asthma has believed as a chronic inflammatory disease of the airways orchestrated by activated Th2 cells.Th1 has believed as a protected factor of asthma.The immunotherapy aim to allergy could change the Th2 cell phenotype into Thl cell phenotype,and the clinical symptoms has improved.The individual which polarize to Th1 had a few possibility to suffer from asthma.So the theory of Th1/Th2 disequilibrium has got common received in asthma.
In recent years,the concept of a disturbed Th1/Th2 balance has failed to adequately explain many(pre)clinical observations.Some research found Th1 cells could aggratate allergy and asthma.Cytokine of Th1 cell INF-γis related with airway responsiveness and eosinophils.Treg cells not only repress the Th1 cells,but also repress the Th2 cells.So Treg cells can explain the pathogenesis better than the Th1/Th2,and become a focus in asthma reseach.Th17 cells have recently emerged as a new independent T cell subset that different from Th1,Th2 cells.There has a complicated relationship between Th17 and Treg cells.Th17 cells related with asthma.Since the disequilibrium of Th1/Th2 is an important immunologic abnormality in asthma,and also an important link in astham pathogenesis,so is there also exist a disequilibrium of Treg/Th17? This issue need us to have a further reseach.
Objective
To identify the unbalanced T cell-specific subpopulation Treg and Th17 in immune dysregulation of asthma,and the change of the gene and protein expression ratio of T cell-specific inflammation transcription factors Foxp3、RORγt in lung tissues,and to investigate the association between the unbalanced Treg/Th17 and the airway inflammation in asthmatic mice.
Methods
1.To establish and assess a modified mouse asthma model.
Thirty female BALB/c mice were randomly divided into a control group,an asthmatic group and a dexamethasone group.We immunized mice with OVA/alum (containing 20μg of OVA bound to 20mg of aluminum hydroxide),made up in 0.2ml sterile saline,injected intraperitoneally and subcutaneously on days 0,7 and 14. On days 21-27,mice were exposed to aerosolized OVA(5%) for 45 min.The control group were received an intraperitoneal and subcutaneous injection of 0.2 ml saline and inhaled saline.Airway responsiveness was measured by non-invasive whole-body plethysmography(WBP) method and the change of airway histology was observed by hematoxylin/eosin(HE) stain.Concentrations of IL-5 and IFN-γin bronchoalveolar lavage fluid(BALF) were measured by enzyme-linked immunosorbent assay(ELISA).
2.To identify the unbalanced T cell-specific subpopulation Treg and Th17 in immune dysregulation of asthma.
Concentrations of IL-10 and IL-17 in serum and in BALF were measured by ELISA.The mRNA expressions of IL-10 and IL-17 in the lungs were detected by RT-PCR.The frequencies of CD4~+CD25~+Foxp3~+ and CD4~+IL-17~+ cells were assessed by intracellular cytokine staining and flow cytometric analysis.
3.To identify the unbalanced T cell-specific transcription factors Foxp3,RORγt in immune dysregulation of asthma.
The mRNA expressions of IL-10,IL-17,Foxp3 and RORγt in the lungs were detected by RT-PCR.The expressions of Foxp3 and RORγt in tissues were determined immunohistochemical staining photographic analysis.The protein expressions of Foxp3 and RORγt in the lungs were detected by Western blot.
Results
1.The mice of asthmatic group demonstrated symptoms of acute asthma when exposed to aerosolized OVA,such as breathing deeply and fast,standing still,bowing the back,urinary and fecal incontinence,and so on;the mice of control group didn't show these symptoms of acute asthma.
Airway responsiveness was determined before and after aerosolizing of methacholine(3.125,6.25,12.5,25,50mg/ml),and airway responsiveness of the asthmatic group were significantly elevated compared with those of the control group(P＜0.01).
Numerous inflammatory cells,including eosinophils,lymphocyte and neutrophils et al,infiltrated around the bronchioles and blood vessels,the airway epithelium was thickened,and mucus had accumulated in the lumen of bronchioles,interalveolar septum collapse,construction disorder,alveolar space and interstitial congestion.
In bronchioalveolar Lavage Fluid,numbers of total cells,eosinophils,lymphocyte and neutrophils were were much higher in the asthma group than those in the control group(P＜0.01).
The levels of IL-5 in BALF were much higher in the asthma groups than those in the control group(P＜0.01);In contrast,the levels of IFN-γwas markedly lower in the asthma group as compared with the control group(P＜0.01).
2.Airway responsiveness was determined before and after aerosolizing of methacholine(3.125,6.25,12.5,25,50mg/ml),and airway responsiveness of the asthmatic group were significantly elevated compared with those of the control group and dexamethasone group(P＜0.01).Airway responsiveness of the dexamethasone group were significantly reduced compared with those of the asthmatic group(P＜0.01);there was no difference of airway responsiveness among the dexamethasone group and the control group before and after aerosolizing of methacholine(3.125,6.25,12.5mg/ml)(P＞0.05),but after aerosolizing of methacholine(25,50mg/ml),airway responsiveness of the dexamethasone group were significantly elevated compared with those of the control group(P＜0.01).
In bronchioalveolar Lavage Fluid,numbers of total cells,eosinophils,lymphocyte and neutrophils were were much higher in the asthma group than those in the Dexamethasone and control groups(P＜0.01),and there was also a significant difference between the Dexamethasone and control group(P＜0.01).
The levels of IL-17 in BALF and serum were much higher in the asthma group than those in the Dexamethasone and control groups(P＜0.01),and there was also a significant difference between the Dexamethasone and control group(P＜0.01).In contrast,the levels of IL-10 was markedly lower in the asthma group as compared with the Dexamethasone and control groups(P＜0.01),there was also a significant difference between the Dexamethasone and control group(P＜0.01).
The levels of IL-17 mRNA expression were much higher in the asthma group than those in the Dexamethasone and control groups(P＜0.01),and there was also a significant difference between the Dexamethasone and control group(P＜0.01).In contrast,the expression of IL-10 mRNA was markedly lower in the asthma group as compared with the Dexamethasone and control group(P＜0.01),there was also a significant difference between the Dexamethasone and control group(P＜0.01).
The frequencies of CD4~+CD25~+Foxp3~+ cells in asthmatic group were significantly lower than those of control group(P＜0.01),The frequencies of CD4~+CD25~+Foxp3~+ cells in Dexamethasone group were higher than those of asthma group(P＜0.01),but still lower than those of control group(P＜0.01);The frequencies of CD4~+IL-17~+ cells in asthmatic group were significantly higher than those of control group(P＜0.01),The frequencies of CD4~+IL-17~+ cells in Dexamethasone group were lower than those of asthma group(P＜0.01),but still higher than those of control group(P＜0.01).
The ratio of CD4~+CD25~+Foxp3~+ ceils/CD4~+IL-17~+ cells in asthmatic group were significantly lower than those of control group(P＜0.01),The ratio of CD4~+CD25~+Foxp3~+ cells/CD4~+IL-17~+ cells in Dexamethasone group were higher than those of asthma group(P＜0.01),but still lower than those of control group(P＜0.01);The ratio of CD4~+CD25~+Foxp3~+ cells/CD4~+IL-17~+ cells was negatively correlated with airway responsiveness,the number of eosinophils, lymphocytes and neutrophils,respectively.The ratio of CD4~+CD25~+Foxp3~+ cells/ CD4~+IL-17~+ cells was negatively correlated with the gene and protein expressions of IL-17 in lung tissues,but positively correlated with the gene and protein expressions of IL-10 in lung tissues,respectively.
3.The levels of RORγt mRNA expression were much higher in the asthma groups than those in the Dexamethasone and control groups(P＜0.01),and there was also a significant difference between the Dexamethasone and control group(P＜0.01).In contrast,the expression of Foxp3 mRNA was markedly lower in the asthma group as compared with the Dexamethasone and control group(P＜0.01),there was also a significant difference between the Dexamethasone and control group(P＜0.01).
The RORγt protein level were much higher in the asthma group than those in the Dexamethasone and control groups(P＜0.01),and there was also a significant difference between the Dexamethasone and control group(P＜0.01).In contrast,the Foxp3 protein level was markedly lower in the asthma group as compared with the Dexamethasone and control group(P＜0.01),there was also a significant difference between the Dexamethasone and control group(P＜0.01).In the control group,the ratios of gene and protein expressions of Foxp3 and RORγt were significantly increased compared with the asthmatic group(P＜0.01);In the dexamethasone group,the ratios of gene and protein expressions of Foxp3 and RORγt were significantly decreased compared with the control group(P＜0.01),and there was also a difference of the ratios of gene and protein expressions of Foxp3 and RORγt between the dexamethasone group and asthmatic group(P＜0.01).
The ratio of protein expression of Foxp3 and RORγt was negatively correlated with airway responsiveness,the numbers of eosinophils,lymphocytes and neutrophils in lung tissues,respectively.The ratio of protein expression of Foxp3 and RORγt was negatively correlated with the gene and protein expressions of IL-17 in lung tissues respectively,but positively correlated with the gene and protein expressions of IL-10 in lung tissues.
Conclusions
1.We improved the sensitizing agent and the method of sensitization,and established a modified mouse asthma model successfully.Non-invasive whole-body plethysmography(WBP) method can detect airway responsiveness conveniently.This successful modified mouse asthma model provides useful tools in the subsequent research of asthma.
2.In asthmatic group,the unbalanced T cell-specific subpopulation Treg/Th17 contributes to both high frequencies of CD4~+IL-17~+ and low frequencies of CD4~+CD25~+Foxp3~+ cell,and the ratios of CD4~+CD25~+Foxp3~+ cell/CD4~+IL-17~+ cell were low,the ratios may evaluate the immune disbalance of asthma objectively.The ratio of CD4~+CD25~+Foxp3~+ cell/CD4~+IL-17~+ cell were negatively correlated with airway inflammation.Imbalance of Treg and Th17 may play a key role in the formation of airway inflammation of asthma.
3.In asthmatic group,the unbalanced T cell-specific transcription factors Foxp3 and RORγt contributes to both high expression of RORγt and low expression of Foxp3,and the ratios of gene and protein expressions of Foxp3/RORγt were low.The ratio of protein expressions of Foxp3/RORγt were negatively correlated with airway inflammation.Imbalance of transcription factors Foxp3 and RORγt may play a key role in the unbalance of Treg and Th17 in airway inflammation of asthma.
4.Dexamethasone treatment inhibits the airway inflammation of asthma by regulate the balance between Treg and Th17.
支气管哮喘(Asthma,Bronchial asthma,简称哮喘)是全球最常见的慢性疾病之一。据不完全统计,全世界目前大约有3亿哮喘患者,且有证据显示目前哮喘患病率以每年1%左右的速度递增,严重影响患者的日常生活,给患者家庭及社会带来了沉重的经济负担,因此对支气管哮喘进行深入的研究具有深远的社会意义。
支气管哮喘是由多种炎症细胞特别是肥大细胞、嗜酸性粒细胞和T淋巴细胞等共同参与的慢性气道炎症性疾病。炎症细胞在气道的局部聚集、炎症介质和细胞因子的释放等是形成和维持气道炎症并进而引起组织损伤和气道功能障碍的主要原因,其中T淋巴细胞在哮喘气道炎症中发挥着核心作用。根据T淋巴细胞在免疫应答中的功能不同,将T淋巴细胞分成辅助性T细胞(Th)、细胞毒性T细胞(Tc)和调节性T细胞(Treg),.其中Th细胞又分为Th1细胞和Th2细胞。
既往认为支气管哮喘是由过度激活的Th2细胞介导的气道慢性炎症性疾病,Th1反应被认为是过敏性疾病包括支气管哮喘的保护因素。针对过敏性疾病的免疫疗法可使Th2细胞因子表型转变为Th1细胞因子表型,同时临床症状得到改善。而且倾向产生Th1细胞因子的个体患过敏性疾病的可能性减少。因此哮喘中Th1/Th2失衡理论被广泛接受。
但近年来发现,哮喘的一些实验和临床现象并不能完全用Th1/Th2失衡理论来解释;并且有研究发现Th1细胞可以加重过敏症和哮喘,Th1细胞因子IFN-γ,与抗原诱导的气道高反应和嗜酸粒细胞浸润有关。而Treg既能抑制Th1细胞,也能抑制Th2细胞,用Treg细胞可更好的解释哮喘的发病机制,因此近年来成为研究的热点。Th17细胞是近来发现的不同于Th1和Th2的另一种CD4~+T细胞的新亚型,具有IL-23依赖性产生IL-17的特性,研究发现其与Treg之间存在复杂的相关关系,并且与支气管哮喘也有关。因此既然Th1/Th2细胞亚群比例和功能的失衡是支气管哮喘重要的免疫异常,是哮喘发病机制中的重要环节,那么是否在哮喘中同样也存在Treg/Th17亚群比例和功能的失衡,这个问题值得我们去深入研究。
目的
1、探讨调节性T细胞(Treg)与Th17细胞及相应转录因子Foxp3、RORγt在支气管哮喘中的失衡表达;2、探讨Treg与Th17细胞及转录因子Foxp3与RORγt表达量的比值与气道炎症的相关关系。
方法
1、小鼠哮喘模型的建立及评价
30只雌性SPF级Balb/c小鼠按随机数字表法分为正常对照组、哮喘组和地塞米松治疗组;哮喘组于第0d、7d、14d(腹腔+皮下)注射0.2ml致敏液(含20ug OVA,20mg AL(OH)_3),第21-27d雾化吸入5%OVA激发;地塞米松治疗组于21至27d雾化吸入5%OVA激发前30min腹腔注射地塞米松(1mg/Kg):正常对照组则用生理盐水替代OVA致敏和激发;利用无创小鼠体描仪通过全身性体积描记法观察各组小鼠气道反应性;通过肺组织石蜡切片苏木素-伊红(HE)染色观察各组气道病理改变;用酶联免疫吸附测定法检测小鼠支气管肺泡灌洗液(BALF)中IL-5、IFN-γ含量。
2、调节T细胞与Th17细胞在支气管哮喘小鼠中的失衡以及与气道炎症的相关性分析
用酶联免疫吸附测定法检测各组小鼠血清和支气管肺泡灌洗液中IL-10、IL-17含量;用逆转录-聚合酶链反应(RT-PCR)测定法检测肺组织IL-10mRNA、IL-17 mRNA的表达;通过各组小鼠脾脏制备脾细胞单细胞悬液,利用流式细胞检测技术检测各组CD4~+CD25~+Foxp3~+细胞和CD4~+IL-17~+细胞占CD4~+细胞的比例。
3、转录因子Foxp3/RORγt在支气管哮喘小鼠中失衡表达以及与气道炎症的相关性分析
用酶联免疫吸附测定法检测各组小鼠血清和支气管肺泡灌洗液中IL-10、IL-17含量;用逆转录.聚合酶链反应(RT-PCR)测定法检测肺组织IL-10mRNA、IL-17mRNA、Foxp3mRNA和RORytmRNA的表达;用免疫组化及免疫印迹(Western blot)法检测肺组织中Foxp3和RORγt蛋白的表达。
结果
1、成功建立小鼠哮喘模型。哮喘组所有小鼠在雾化过程中均出现不同程度的烦躁不安或安静少动、呼吸加快加深、点头呼吸、弓背直立、前肢缩抬,二便失禁等哮喘速发相反应表现。
同等剂量乙酰胆碱激发(3.125、6.25、12.5、25、50mg/m1)的气道反应性检测中,哮喘组小鼠%ΔPenh明显高于正常对照组。
哮喘组小鼠细支气管及伴行血管周围大量炎症细胞浸润,气道壁和肺组织中可见以嗜酸性粒细胞、淋巴细胞为主的大量炎症细胞浸润,支气管管壁增厚、管腔狭窄,支气管管腔内可见粘液栓,粘膜皱壁减少,杯状细胞增生,肺泡间隔不同程度增宽,部分断裂,形成肺气肿。
哮喘组小鼠肺泡灌洗液中细胞总数及嗜酸性粒细胞、中性粒细胞和淋巴细胞计数明显高于正常对照组(P＜0.01)。
哮喘组小鼠肺泡灌洗液中IL-5水平明显高于正常对照组(P＜0.01);与之相反,哮喘组小鼠肺泡灌洗液中IFN-γ,水平低于正常对照组(P＜0.01)。
2、同等剂量乙酰胆碱激发(3.125、6.25、12.5、25、50mg/ml)的气道反应性检测中,哮喘组小鼠%ΔPenh明显高于正常对照组小鼠(P＜0.01),地塞米松治疗组小鼠%ΔPenh较哮喘组明显降低(P＜0.01);在乙酰胆碱浓度为3.125、6.25、12.5mg/ml激发时,正常对照组和地塞米松治疗组小鼠%ΔPenh相似,差异无统计学意义(P＞0.05)。在乙酰胆碱浓度为25、50mg/ml激发时,地塞米松治疗组小鼠%ΔPenh高于正常对照组小鼠(P＜0.01)。
哮喘组肺泡灌洗液中细胞总数、中性粒细胞、嗜酸粒细胞和淋巴细胞计数均较正常对照组明显增多(P＜0.01);地塞米松治疗组与哮喘组相比中性粒细胞、嗜酸粒细胞和淋巴细胞计数明显减少(P＜0.01),与正常对照组比较仍较高(P＜0.01)。
哮喘组小鼠肺泡灌洗液、血清中IL-17水平较正常对照组明显升高(P＜0.01);地塞米松治疗组肺泡灌洗液、血清中IL-17水平较哮喘组有所下降,但与正常对照组比较仍较高(P＜0.01);与之相反,哮喘组小鼠肺泡灌洗液、血清中IL-10水平较正常对照组明显下降(P＜0.01);地塞米松治疗组肺泡灌洗液、血清中IL-10水平较哮喘组有所升高,但与正常对照组比较仍较低(P＜0.01)。
哮喘组小鼠肺组织中细胞因子IL-17mRNA的表达较正常对照组小鼠明显增强(P＜0.01);地塞米松治疗组小鼠肺组织IL-17mRNA的表达减弱,明显低于哮喘组小鼠(P＜0.01),但与正常对照组小鼠比较仍较高(P＜0.01)。哮喘组小鼠肺组织中细胞因子IL-10mRNA的表达较正常对照组小鼠明显减弱(P＜0.01);地塞米松治疗组小鼠肺组织IL-10mRNA的表达较哮喘组小鼠有所增强(P＜0.01),但与正常对照组小鼠比较仍较低(P＜0.01)。
流式细胞仪检测结果显示哮喘组CD4~+细胞中淋巴细胞亚群CD4~+CD25~+Foxp3~+细胞百分率显著低于正常对照组(P＜0.01);地塞米松治疗组CD4~+细胞中淋巴细胞亚群CD4~+CD25~+Foxp3~+细胞百分率较哮喘组小鼠有所增加(P＜0.01),但与正常对照组小鼠比较仍较低(P＜0.01)。哮喘组CD4~+细胞中淋巴细胞亚群CD4~+IL+17~+细胞百分率显著高于正常对照组(P＜0.01);地塞米松治疗组CD4~+细胞中淋巴细胞亚群CD4~+IL-17~+细胞百分率较哮喘组小鼠有所减少(P＜0.01),但与正常对照组小鼠比较仍较高(P＜0.01)。哮喘组CD4~+CD25~+Foxp3~+细胞/CD4~+IL+17~+细胞比例较正常对照组下降(P＜0.01);地塞米松治疗组CD4~+CD25~+Foxp3~+细胞/CD4~+IL-17~+细胞比例较哮喘组小鼠有所增加(P<0.01),但与正常对照组小鼠比较仍较低(P<0.01)。
CD4~+CD25~+Foxp3~+细胞/CD4~+IL-17~+细胞与气道炎性指标经相关性分析显示,CD4~+CD25~+Foxp3~+细胞/CD4~+IL-17~+细胞比值与气道反应性(%△Penh)呈负相关,用3.125、6.25、12.5、25、50mg/ml不同浓度乙酰胆碱激发时,相应的气道反应性(%△Penh)与CD4~+CD25~+Foxp3~+细胞/CD4~+IL-17~+细胞比值的相关系数分别是-0.810,-0.853,-0.796,-0.898,-0.838(P均<0.01);与嗜酸粒细胞、中性粒细胞、淋巴细胞数呈负相关(r=-0.873,-0.878,-0.855;P均<0.01);与血清、肺泡灌洗液中IL-10含量呈正相关(r=0.93,0.926;P均<0.01),而与血清、肺泡灌洗液中IL-17呈负相关(r=-0.878,0.828;P均<0.01)。
3、哮喘组小鼠肺组织中转录因子RORγt mRNA的表达较正常对照组小鼠明显增强(P<0.01);地塞米松治疗组小鼠肺组织RORγt mRNA的表达减弱,明显低于哮喘组小鼠(P<0.01),但与正常对照组小鼠比较仍较高(P<0.01)。哮喘组小鼠肺组织中转录因子Foxp3 mRNA的表达较正常对照组小鼠明显减弱(P<0.01);地塞米松治疗组小鼠肺组织Foxp3 mRNA的表达较哮喘组小鼠有所增强(P<0.01),但与正常对照组小鼠比较仍较低(P<0.01);Foxp3/RORγt mRNA表达量比值哮喘组(0.18±0.05)较正常对照组(1.97±0.58)明显降低(P<0.01);地塞米松治疗组Foxp3/RORγt mRNA表达量比值为(0.59±0.14),与哮喘组Foxp3/RORγt mRNA表达量比值比较有所升高(P<0.01),但仍低于正常对照组(1.97±0.58)(P<0.01)。
免疫组化分析显示哮喘组小鼠转录因子Foxp3表达强度较正常对照组明显减弱,表达范围减少(P<0.01);转录因子Foxp3在地塞米松治疗组小鼠表达强度与哮喘组小鼠比较有所增高(P<0.01),与正常对照组比较强度和范围仍降低(P<0.01)。哮喘组小鼠转录因子RORγt表达强度较正常对照组小鼠异常增强,表达范围广泛(P<0.01);在地塞米松治疗组小鼠转录因子RORγt表达强度和表达范围高于正常对照组小鼠(P<0.01),与哮喘组小鼠相比,地塞米松治疗组小鼠RORγt表达强度和表达范围降低(P<0.01)。
Western blot结果分析显示转录因子Foxp3蛋白在正常对照组小鼠肺组织中有表达,而在哮喘组小鼠肺组织中低表达,两组比较差异有显著性(P＜0.01);予以地塞米松干预后,Foxp3蛋白在小鼠肺组织中表达增高,高于哮喘组小鼠,但低于正常对照组小鼠(P＜0.01)。转录因子RORγt蛋白在正常对照组小鼠肺组织中有低表达,而在哮喘组小鼠肺组织中异常高表达,两组比较差异有显著性(P＜0.01);在地塞米松干预治疗后,RORγt蛋白仍表达,低于哮喘组小鼠,但高于正常对照组小鼠(P＜0.01)。哮喘组Foxp3/RORγt蛋白表达量比值明显低于正常对照组(P＜0.01);地塞米松治疗组Foxp3/RORγt蛋白表达量比值较哮喘组有所升高,但仍低于正常对照组(P＜0.01)。
Foxp3/RORγt蛋白表达量的比值与气道炎症性指标作相关性分析,结果显示Foxp3/RORγt蛋白表达量的比值与气道反应性(%ΔPenh)呈负相关,用3.125、6.25、12.5、25、50mg/ml不同浓度乙酰胆碱激发时,相应的气道反应性(%△Penh)与Foxp3/RORγt蛋白表达量比值的相关系数分别是-0.708,-0.715,-0.702,-0.822,-0.846(P＜0.01);Foxp3/RORγt蛋白表达量比值与嗜酸性粒细胞数、淋巴细胞数和中性粒细胞数呈负相关(r＝-0.904,-0.881,-0.862,P＜0.01),与血清和肺泡灌洗液中IL-17含量呈负相关(r＝-0.880,-0.846,P＜0.01),而与血清和肺泡灌洗液中IL-10含量呈正相关(r＝0.718,0.816,P＜0.01)。
结论
1、通过对致敏剂及致敏方法的改进,使我们更容易成功建立小鼠哮喘动物模型,为随后进一步实验研究提供了一个很好的基础;利用无创小鼠体描仪通过全身性体积描记法可方便的观察实验小鼠气道反应性,为进一步研究支气管哮喘的病理生理学机制提供了很好的工具和手段。
2、在支气管哮喘中存在Treg与Th17的失衡,表现为CD4~+细胞中CD4~+CD25~+Foxp3~+细胞减少,CD4~+IL-17~+细胞增多,CD4~+CD25~+Foxp3~+细胞/CD4~+IL-17~+细胞比例低下,反映哮喘的免疫失衡;并且CD4~+CD25~+Foxp3~+细胞/CD4~+IL-17~+细胞比例与气道炎症各指标呈直线相关,Treg与Th17的失衡很可能是哮喘气道炎症形成的重要原因之一。
3、在支气管哮喘中,转录因子Foxp3/RORγt失衡表达,表现为转录因子Foxp3基因和蛋白的低表达,RORγt基因和蛋白的高表达,F0xp3/RORγt蛋白表达量比值与气道炎症各指标呈直线相关,Foxp3/RORγt的失衡表达很可能是哮喘中Treg与Th17失衡形成的主要机制。
4、地塞米松可能通过调节Treg与Th17的平衡来抑制气道炎症。
Background
Asthma is a very common disease in the world.It is now estimated that as many as 300 million people suffer from asthma and asthma attacks have increased by 1% per year.The burden of this disease to governments,health care systems,families,and patients is increasing worldwide,so there is a far-reaching society significance in the asthma research.
Asthna is a chronic inflammatory disease of the airways in which many inflammatory cells play a role,in particular,mast cells,eosinophils,T lymphocytes.The recruitment of inflammatory cells and the release of cytokines and mediators of inflammation are the major reason which cause and keep the airway inflamemation of asthma,and futher cause the tissue damage and airway disfunction.Among these inflammatory cells,T lymphocytes play a pivotal role in the pathogenesis of asthma.According to the function in immune response,we divided T lymphocytes into three subsets,help T cell(Th),cytotoxic T cell(Tc) and regulatory T cell(Treg).Th ceils divided into Th1 cell and Th2 cell.
Long time ago,asthma has believed as a chronic inflammatory disease of the airways orchestrated by activated Th2 cells.Th1 has believed as a protected factor of asthma.The immunotherapy aim to allergy could change the Th2 cell phenotype into Thl cell phenotype,and the clinical symptoms has improved.The individual which polarize to Th1 had a few possibility to suffer from asthma.So the theory of Th1/Th2 disequilibrium has got common received in asthma.
In recent years,the concept of a disturbed Th1/Th2 balance has failed to adequately explain many(pre)clinical observations.Some research found Th1 cells could aggratate allergy and asthma.Cytokine of Th1 cell INF-γis related with airway responsiveness and eosinophils.Treg cells not only repress the Th1 cells,but also repress the Th2 cells.So Treg cells can explain the pathogenesis better than the Th1/Th2,and become a focus in asthma reseach.Th17 cells have recently emerged as a new independent T cell subset that different from Th1,Th2 cells.There has a complicated relationship between Th17 and Treg cells.Th17 cells related with asthma.Since the disequilibrium of Th1/Th2 is an important immunologic abnormality in asthma,and also an important link in astham pathogenesis,so is there also exist a disequilibrium of Treg/Th17? This issue need us to have a further reseach.
Objective
To identify the unbalanced T cell-specific subpopulation Treg and Th17 in immune dysregulation of asthma,and the change of the gene and protein expression ratio of T cell-specific inflammation transcription factors Foxp3、RORγt in lung tissues,and to investigate the association between the unbalanced Treg/Th17 and the airway inflammation in asthmatic mice.
Methods
1.To establish and assess a modified mouse asthma model.
Thirty female BALB/c mice were randomly divided into a control group,an asthmatic group and a dexamethasone group.We immunized mice with OVA/alum (containing 20μg of OVA bound to 20mg of aluminum hydroxide),made up in 0.2ml sterile saline,injected intraperitoneally and subcutaneously on days 0,7 and 14. On days 21-27,mice were exposed to aerosolized OVA(5%) for 45 min.The control group were received an intraperitoneal and subcutaneous injection of 0.2 ml saline and inhaled saline.Airway responsiveness was measured by non-invasive whole-body plethysmography(WBP) method and the change of airway histology was observed by hematoxylin/eosin(HE) stain.Concentrations of IL-5 and IFN-γin bronchoalveolar lavage fluid(BALF) were measured by enzyme-linked immunosorbent assay(ELISA).
2.To identify the unbalanced T cell-specific subpopulation Treg and Th17 in immune dysregulation of asthma.
Concentrations of IL-10 and IL-17 in serum and in BALF were measured by ELISA.The mRNA expressions of IL-10 and IL-17 in the lungs were detected by RT-PCR.The frequencies of CD4~+CD25~+Foxp3~+ and CD4~+IL-17~+ cells were assessed by intracellular cytokine staining and flow cytometric analysis.
3.To identify the unbalanced T cell-specific transcription factors Foxp3,RORγt in immune dysregulation of asthma.
The mRNA expressions of IL-10,IL-17,Foxp3 and RORγt in the lungs were detected by RT-PCR.The expressions of Foxp3 and RORγt in tissues were determined immunohistochemical staining photographic analysis.The protein expressions of Foxp3 and RORγt in the lungs were detected by Western blot.
Results
1.The mice of asthmatic group demonstrated symptoms of acute asthma when exposed to aerosolized OVA,such as breathing deeply and fast,standing still,bowing the back,urinary and fecal incontinence,and so on;the mice of control group didn't show these symptoms of acute asthma.
Airway responsiveness was determined before and after aerosolizing of methacholine(3.125,6.25,12.5,25,50mg/ml),and airway responsiveness of the asthmatic group were significantly elevated compared with those of the control group(P＜0.01).
Numerous inflammatory cells,including eosinophils,lymphocyte and neutrophils et al,infiltrated around the bronchioles and blood vessels,the airway epithelium was thickened,and mucus had accumulated in the lumen of bronchioles,interalveolar septum collapse,construction disorder,alveolar space and interstitial congestion.
In bronchioalveolar Lavage Fluid,numbers of total cells,eosinophils,lymphocyte and neutrophils were were much higher in the asthma group than those in the control group(P＜0.01).
The levels of IL-5 in BALF were much higher in the asthma groups than those in the control group(P＜0.01);In contrast,the levels of IFN-γwas markedly lower in the asthma group as compared with the control group(P＜0.01).
2.Airway responsiveness was determined before and after aerosolizing of methacholine(3.125,6.25,12.5,25,50mg/ml),and airway responsiveness of the asthmatic group were significantly elevated compared with those of the control group and dexamethasone group(P＜0.01).Airway responsiveness of the dexamethasone group were significantly reduced compared with those of the asthmatic group(P＜0.01);there was no difference of airway responsiveness among the dexamethasone group and the control group before and after aerosolizing of methacholine(3.125,6.25,12.5mg/ml)(P＞0.05),but after aerosolizing of methacholine(25,50mg/ml),airway responsiveness of the dexamethasone group were significantly elevated compared with those of the control group(P＜0.01).
In bronchioalveolar Lavage Fluid,numbers of total cells,eosinophils,lymphocyte and neutrophils were were much higher in the asthma group than those in the Dexamethasone and control groups(P＜0.01),and there was also a significant difference between the Dexamethasone and control group(P＜0.01).
The levels of IL-17 in BALF and serum were much higher in the asthma group than those in the Dexamethasone and control groups(P＜0.01),and there was also a significant difference between the Dexamethasone and control group(P＜0.01).In contrast,the levels of IL-10 was markedly lower in the asthma group as compared with the Dexamethasone and control groups(P＜0.01),there was also a significant difference between the Dexamethasone and control group(P＜0.01).
The levels of IL-17 mRNA expression were much higher in the asthma group than those in the Dexamethasone and control groups(P＜0.01),and there was also a significant difference between the Dexamethasone and control group(P＜0.01).In contrast,the expression of IL-10 mRNA was markedly lower in the asthma group as compared with the Dexamethasone and control group(P＜0.01),there was also a significant difference between the Dexamethasone and control group(P＜0.01).
The frequencies of CD4~+CD25~+Foxp3~+ cells in asthmatic group were significantly lower than those of control group(P＜0.01),The frequencies of CD4~+CD25~+Foxp3~+ cells in Dexamethasone group were higher than those of asthma group(P＜0.01),but still lower than those of control group(P＜0.01);The frequencies of CD4~+IL-17~+ cells in asthmatic group were significantly higher than those of control group(P＜0.01),The frequencies of CD4~+IL-17~+ cells in Dexamethasone group were lower than those of asthma group(P＜0.01),but still higher than those of control group(P＜0.01).
The ratio of CD4~+CD25~+Foxp3~+ ceils/CD4~+IL-17~+ cells in asthmatic group were significantly lower than those of control group(P＜0.01),The ratio of CD4~+CD25~+Foxp3~+ cells/CD4~+IL-17~+ cells in Dexamethasone group were higher than those of asthma group(P＜0.01),but still lower than those of control group(P＜0.01);The ratio of CD4~+CD25~+Foxp3~+ cells/CD4~+IL-17~+ cells was negatively correlated with airway responsiveness,the number of eosinophils, lymphocytes and neutrophils,respectively.The ratio of CD4~+CD25~+Foxp3~+ cells/ CD4~+IL-17~+ cells was negatively correlated with the gene and protein expressions of IL-17 in lung tissues,but positively correlated with the gene and protein expressions of IL-10 in lung tissues,respectively.
3.The levels of RORγt mRNA expression were much higher in the asthma groups than those in the Dexamethasone and control groups(P＜0.01),and there was also a significant difference between the Dexamethasone and control group(P＜0.01).In contrast,the expression of Foxp3 mRNA was markedly lower in the asthma group as compared with the Dexamethasone and control group(P＜0.01),there was also a significant difference between the Dexamethasone and control group(P＜0.01).
The RORγt protein level were much higher in the asthma group than those in the Dexamethasone and control groups(P＜0.01),and there was also a significant difference between the Dexamethasone and control group(P＜0.01).In contrast,the Foxp3 protein level was markedly lower in the asthma group as compared with the Dexamethasone and control group(P＜0.01),there was also a significant difference between the Dexamethasone and control group(P＜0.01).In the control group,the ratios of gene and protein expressions of Foxp3 and RORγt were significantly increased compared with the asthmatic group(P＜0.01);In the dexamethasone group,the ratios of gene and protein expressions of Foxp3 and RORγt were significantly decreased compared with the control group(P＜0.01),and there was also a difference of the ratios of gene and protein expressions of Foxp3 and RORγt between the dexamethasone group and asthmatic group(P＜0.01).
The ratio of protein expression of Foxp3 and RORγt was negatively correlated with airway responsiveness,the numbers of eosinophils,lymphocytes and neutrophils in lung tissues,respectively.The ratio of protein expression of Foxp3 and RORγt was negatively correlated with the gene and protein expressions of IL-17 in lung tissues respectively,but positively correlated with the gene and protein expressions of IL-10 in lung tissues.
Conclusions
1.We improved the sensitizing agent and the method of sensitization,and established a modified mouse asthma model successfully.Non-invasive whole-body plethysmography(WBP) method can detect airway responsiveness conveniently.This successful modified mouse asthma model provides useful tools in the subsequent research of asthma.
2.In asthmatic group,the unbalanced T cell-specific subpopulation Treg/Th17 contributes to both high frequencies of CD4~+IL-17~+ and low frequencies of CD4~+CD25~+Foxp3~+ cell,and the ratios of CD4~+CD25~+Foxp3~+ cell/CD4~+IL-17~+ cell were low,the ratios may evaluate the immune disbalance of asthma objectively.The ratio of CD4~+CD25~+Foxp3~+ cell/CD4~+IL-17~+ cell were negatively correlated with airway inflammation.Imbalance of Treg and Th17 may play a key role in the formation of airway inflammation of asthma.
3.In asthmatic group,the unbalanced T cell-specific transcription factors Foxp3 and RORγt contributes to both high expression of RORγt and low expression of Foxp3,and the ratios of gene and protein expressions of Foxp3/RORγt were low.The ratio of protein expressions of Foxp3/RORγt were negatively correlated with airway inflammation.Imbalance of transcription factors Foxp3 and RORγt may play a key role in the unbalance of Treg and Th17 in airway inflammation of asthma.
4.Dexamethasone treatment inhibits the airway inflammation of asthma by regulate the balance between Treg and Th17.
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37.Wong CK,Ho CY,Ko FW,et al.Proinflammatory cytokines(IL-17,IL-6,IL-18 and IL-12) and Th cytokines(IFN-γ,IL-4,IL-10 and IL-13) in patients with allergic asthma.[TJ.Clin Exp Immunol,2001,125(2):177-183.
38.Laan M,Palmberg L,Larsson K,et al.Free,soluble interleukin-17 protein during severe inflammation in human airways[J].Eur Respir J,2002,19(3):534-537.
39.Barczyk A,Pierzchala W,Sozanska E.Interleukin-17 in sputum correlates with airway hyperresponsiveness to methacholine[J].Respir Med,2003,97(6):726-733.
40.Sun YC,Zhou QT,Yao WZ.Sputum interleukin-17 is increased and associated with airway neutrophilia in patients with severe asthma[J].Chin Med J,2005,118(11):953-956.
41.Bullens DM,Truyen E,Coteur L,et al.IL-17 mRNA in sputum of asthmatic patients:linking T cell driven inflammation and granulocytic influx?[J].Respir Res,2006,7:135.
42.Oboki K,Ohno T,Saito H,et al.Th17 and allergy.[J].Allergol Int,2008;57(2):121-134
43.Nakae S,Komiyama Y,Nambu A,et al.Antigen-specific T cell sensitization is impaired in IL-17-deficient mice,causing suppression of allergic cellular and humoral responses[J].Immunity,2002,17(3):375-387.
44.Molet S,Hamid Q,Davoine F,et al.IL-17 is increased in asthmatic airways and induces human bronchial fibroblasts to produce cytokines[J].J Allergy Clin Immunol,2001,108(3):430-438.
45.Aggarwal S,Gurney AL.IL-17:prototype member of an emerging cytokine family[J].J Leukoc Biol,2002,71(1):1-8.
46.Bettelli E,Carder Y,Gao W,et al.Reciprocal developmental pathways for the generation of pathogenic effector Th17 and regulatory T cells[J].Nature,2006,441(7090):235-238.
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48.Chen X,Murakami T,Oppenheim JJ,et al.Differential response of murine CD4~+CD25~+ and CD4~+CD25~- T cells to dexamethasone-induced cell death[J].Eur J Immunol,2004,34(3):859-869
49.Dao Nguyen X,Robinson DS.Fluticasone propionate increases CD4~+CD25~+ T regulatory cell suppression of allergen-stimulated CD4~+CD25~+T cells by an IL- 10-dependent mechanism[J].J Allergy Clin Immunol,2004,114(2):296-301.
1.Li MO,Wan YY,Flavell RA.T cell-produced transforming growth factor betal controls T cell tolerance and regulates Th1 and Th17-cell differentiation[J].Immunity,2007,26(5):579-591.
2.Harrington LE,Mangan PR,Weaver CT.Expanding the effector CD4 T-cell repertoire:the Thl 7 lineage[J].Curr Opin Immunol,2006,18(3):349-356
3.Weaver CT,Harrington LE,Mangan PR,et al.Thl7:an effector CD4 T cell lineage with regulatory T cell ties[J].Immtmity,2006,24(6):677-688
4.Murphy KM,Reiner SL.The lineage decisions of helper T cells[J].Nat Rev Immunol,2002,2(12):933-944.
5.Farrar JD,Asnagli H,Murphy KM.T helper subset development:roels of instruction,selection,and transcription[J].J Clin Invest,2002,109(4):431-435.
6.Ho IC,Glimcher LH.Transcription:tantalizing times for T cells[J].Cell,2002,109(Suppl):S 109-S 120.
7.Brunkow ME,Jeffery EW,Hjerrild KA,et al.Disruption of a new forkhead/winged-helix protein,scurfm,results in the fatal lymphoproliferative disorder of the scurfy mouse[J].Nat Genet,2001,27(1):68-73.
8.Walker MR,Kasprowicz D J,Gersuk VH,et al.Induction of FoxP3 and acquisition of T regulatory activity by stimulated human CD4~+CD25~- T cells[J].J Clin Invest,2003,112(9):1437-1443.
9.Hori S,Nomura T,Sakaguchi S.Control of regulatory T cell development by the transcription factor FOXP3[J].Science,2003,299(5609):1057-1061.
10.Fontenot JD,Gavin MA,Rudensky AY.Foxp3 programs the development and function of CD4~+CD25~+ regulatory T cells[J].Nat Immunol,2003,4(4):330-336.
11.Khattri R,Cox T,Yasayko SA,et al.An essential role for Scurfin in CD4~+CD25~+T regulatory cells[J].Nat Immunol,2003,4(4):337-342.
12.Sakaguchi S.Naturally arising Foxp3-expressing CD25~+CD4~+ regulatory T cells in immunological tolerance to self and non-self[J].Nat Immunol,2005;6(4):345 -352.
13.Fontenot JD,Rudensky AY.A well adapted regulatory contrivance:regulatory T cell development and the forkhead family transcription factor Foxp3[J].Nat Immunol,2005,6(4):331-337.
14.Jetten AM.Recent advances in the mechanisms of action and physiological functions of the retinoid-related orphan receptors(RORs)[J].Curr Drug Targets Inflamm Allergy,2004,3(4):395-412.
15.Ivanov Ⅱ,McKenzie BS,Zhou Let al.The orphan nuclear receptor RORgammat directs the differentiation program of proinflammatory IL-17~+ T helper cells[J].Cell,2006,126(6):1121-1133.
16.He YW,Deftos ML,Ojala EW,et al.RORγt,a novel isoform of an orphan receptor,negatively regulates Fas ligand expression and IL-2 production in T cells[J].Immunity,1998,9(6):797-806.
17.Eberl G,Littman DR.The role of the nuclear hormone receptor RORgammat in the development of lymph nodes and Peyer's patches[J].Immunol Rev,2003,195(1):81-90.
18.Yang XO,Panopoulos AD,Nurieva R,et al.STAT3 regulates cytokine-mediated generation of inflammatory helper T cells[J].J Biol Chem,2007,282(13):9358-9363.
19.Jetten AM,Kurebayashi S,Ueda E.The ROR nuclear orphan receptor subfamily: critical regulators of multiple biological processes[J].Prog Nucleic Acid Res Mol Biol,2001,69:205-247.
20.Manga PR,Harrington LE,O'Quinn DB,et al.Transforming growth factor-β induces development of the Th17 lineage[J].Nature,2006,441(7090):231-234.
21.Kolls JK,Linden A.Interleukin-17 family members and inflammation[J].Immunity,2004,21(4):467-476.
22.Ichiyama K,Yoshida H,Wakabayashi Y,et al.Foxp3 inhibits RORgammat-mediated IL-17A mRNA transcription through direct interaction with RORgammat[J].J Biol Chem,2008,283(25):17003-17008.
23.Bettelli E,Carrier Y,Gao W,et al.Reciprocal developmental pathways for the generation of pathogenic effector Th17 and regulatory T cells[J].Nature,2006,441(7090):235-238.
24.Zhou L,Lopes JE,Chong MM,et al.TGF-P-induced Foxp3 inhibits Th17 cell differentiation by antagonizing RORγt function[J].Nature,2008,453(7192):236-240.
25.Gronemeyer H.Control of transcription activation by steroid hormone receptors[J].FASEBJ,1992,6(8):2524-2529.
26.Wilson SJ,Wallin A,Della-Cioppa G,et al.Effects of budesonide and formoterol on NF-kappa B,adhesion molecules,and cytokines in asthma[J].Am J Respir Crit Care Med,2001,164(6):1047-1052.
27.Vacca A,Felli MP,Farina AR,et al.Glucocorticoid receptor-mediated suppression of the interleukin 2 gene expression through impairment of the cooperativity between nuclear factor of activated T cells and AP-1 enhancer elements[J].J Exp Med,1992,175(3):637-646.
28.Paliogianni F,Raptis A,Ahuja SS,et al.Negative transcriptional regulation of interleukin 2(IL-2) gene by glucocorticoids through interference with nuclear transcriptional factors AP-1 and NF-AT[J].J Clin Invest,1993,91(4):1481-1489.
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47.杨世杰.药理学[M].北京:人民卫生出版社,2001.422-423
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1.Li MO,Wan YY,Flavell RA.T cell-produced transforming growth factor betal controls T cell tolerance and regulates Th1 and Th17-cell differentiation[J].Immunity,2007,26(5):579-591.
2.Harrington LE,Mangan PR,Weaver CT.Expanding the effector CD4 T-cell repertoire:the Thl 7 lineage[J].Curr Opin Immunol,2006,18(3):349-356
3.Weaver CT,Harrington LE,Mangan PR,et al.Thl7:an effector CD4 T cell lineage with regulatory T cell ties[J].Immtmity,2006,24(6):677-688
4.Murphy KM,Reiner SL.The lineage decisions of helper T cells[J].Nat Rev Immunol,2002,2(12):933-944.
5.Farrar JD,Asnagli H,Murphy KM.T helper subset development:roels of instruction,selection,and transcription[J].J Clin Invest,2002,109(4):431-435.
6.Ho IC,Glimcher LH.Transcription:tantalizing times for T cells[J].Cell,2002,109(Suppl):S 109-S 120.
7.Brunkow ME,Jeffery EW,Hjerrild KA,et al.Disruption of a new forkhead/winged-helix protein,scurfm,results in the fatal lymphoproliferative disorder of the scurfy mouse[J].Nat Genet,2001,27(1):68-73.
8.Walker MR,Kasprowicz D J,Gersuk VH,et al.Induction of FoxP3 and acquisition of T regulatory activity by stimulated human CD4~+CD25~- T cells[J].J Clin Invest,2003,112(9):1437-1443.
9.Hori S,Nomura T,Sakaguchi S.Control of regulatory T cell development by the transcription factor FOXP3[J].Science,2003,299(5609):1057-1061.
10.Fontenot JD,Gavin MA,Rudensky AY.Foxp3 programs the development and function of CD4~+CD25~+ regulatory T cells[J].Nat Immunol,2003,4(4):330-336.
11.Khattri R,Cox T,Yasayko SA,et al.An essential role for Scurfin in CD4~+CD25~+T regulatory cells[J].Nat Immunol,2003,4(4):337-342.
12.Sakaguchi S.Naturally arising Foxp3-expressing CD25~+CD4~+ regulatory T cells in immunological tolerance to self and non-self[J].Nat Immunol,2005;6(4):345 -352.
13.Fontenot JD,Rudensky AY.A well adapted regulatory contrivance:regulatory T cell development and the forkhead family transcription factor Foxp3[J].Nat Immunol,2005,6(4):331-337.
14.Jetten AM.Recent advances in the mechanisms of action and physiological functions of the retinoid-related orphan receptors(RORs)[J].Curr Drug Targets Inflamm Allergy,2004,3(4):395-412.
15.Ivanov Ⅱ,McKenzie BS,Zhou Let al.The orphan nuclear receptor RORgammat directs the differentiation program of proinflammatory IL-17~+ T helper cells[J].Cell,2006,126(6):1121-1133.
16.He YW,Deftos ML,Ojala EW,et al.RORγt,a novel isoform of an orphan receptor,negatively regulates Fas ligand expression and IL-2 production in T cells[J].Immunity,1998,9(6):797-806.
17.Eberl G,Littman DR.The role of the nuclear hormone receptor RORgammat in the development of lymph nodes and Peyer's patches[J].Immunol Rev,2003,195(1):81-90.
18.Yang XO,Panopoulos AD,Nurieva R,et al.STAT3 regulates cytokine-mediated generation of inflammatory helper T cells[J].J Biol Chem,2007,282(13):9358-9363.
19.Jetten AM,Kurebayashi S,Ueda E.The ROR nuclear orphan receptor subfamily: critical regulators of multiple biological processes[J].Prog Nucleic Acid Res Mol Biol,2001,69:205-247.
20.Manga PR,Harrington LE,O'Quinn DB,et al.Transforming growth factor-β induces development of the Th17 lineage[J].Nature,2006,441(7090):231-234.
21.Kolls JK,Linden A.Interleukin-17 family members and inflammation[J].Immunity,2004,21(4):467-476.
22.Ichiyama K,Yoshida H,Wakabayashi Y,et al.Foxp3 inhibits RORgammat-mediated IL-17A mRNA transcription through direct interaction with RORgammat[J].J Biol Chem,2008,283(25):17003-17008.
23.Bettelli E,Carrier Y,Gao W,et al.Reciprocal developmental pathways for the generation of pathogenic effector Th17 and regulatory T cells[J].Nature,2006,441(7090):235-238.
24.Zhou L,Lopes JE,Chong MM,et al.TGF-P-induced Foxp3 inhibits Th17 cell differentiation by antagonizing RORγt function[J].Nature,2008,453(7192):236-240.
25.Gronemeyer H.Control of transcription activation by steroid hormone receptors[J].FASEBJ,1992,6(8):2524-2529.
26.Wilson SJ,Wallin A,Della-Cioppa G,et al.Effects of budesonide and formoterol on NF-kappa B,adhesion molecules,and cytokines in asthma[J].Am J Respir Crit Care Med,2001,164(6):1047-1052.
27.Vacca A,Felli MP,Farina AR,et al.Glucocorticoid receptor-mediated suppression of the interleukin 2 gene expression through impairment of the cooperativity between nuclear factor of activated T cells and AP-1 enhancer elements[J].J Exp Med,1992,175(3):637-646.
28.Paliogianni F,Raptis A,Ahuja SS,et al.Negative transcriptional regulation of interleukin 2(IL-2) gene by glucocorticoids through interference with nuclear transcriptional factors AP-1 and NF-AT[J].J Clin Invest,1993,91(4):1481-1489.