TH17细胞及其功能状态在中性粒细胞哮喘发病中的作用及相关分子机制

发布时间：2018-03-12 09:19

  本文选题：哮喘　切入点：中性粒细胞　出处：《广西医科大学》2013年博士论文　论文类型：学位论文

【摘要】：支气管哮喘(哮喘)是多种细胞参与的慢性气道炎症,长期以来嗜酸性粒细胞被认为是哮喘最重要的炎症效应细胞,但随着哮喘气道炎症的深入研究,发现存在嗜酸细胞性哮喘(EA)及非嗜酸细胞性哮喘两种不同的亚型,非嗜酸细胞性哮喘中半数以上为中性粒细胞性哮喘(NA),我们前期对哮喘患者的研究发现哮喘气道中性粒细胞炎症与气道中性粒细胞凋亡障碍有关,其发生机制仍在探讨中。 哮喘发病机制的研究很大程度上需借助动物模型来进行,但既往哮喘的动物研究多采用传统的嗜酸细胞性哮喘动物模型,并不能真正反映中性粒细胞性哮喘的气道炎症状态及气道高反应的特征。因此,本研究采用脂多糖联合卵清蛋白气道滴入致敏,卵清蛋白雾化激发建立中性粒细胞性哮喘小鼠模型。结果显示小鼠出现了类似哮喘发作的症状及病理改变,以中性粒细胞为主的气道炎症,与嗜酸细胞性哮喘模型相似的气道粘液高分泌及更严重的气道高反应(AHR)；提示该方法成功地建立了中性粒细胞性哮喘小鼠模型,为后续研究奠定了基础。 目前认为TH1/TH2细胞失衡所致TH2细胞优势是介导嗜酸细胞性哮喘发病最重要的免疫学机制,但最近的研究表明TH17细胞作为一类新发现的辅助性T细胞也参与哮喘发病。TH17细胞能够分泌多种细胞因子,其中最重要的是IL-17A(亦称IL-17), IL-17通过受体介导的信号途径可诱导多种细胞产生IL-8、IL-6、GM-CSF、CXCL等细胞因子,趋化并激活中性粒细胞在局部浸润。基于TH17细胞及IL-17受体信号途径所产生的细胞因子对中性粒细胞的生物学功能,我们推测TH17细胞参与了中性粒细胞性哮喘发病,然而也有研究显示TH17细胞参与了嗜酸细胞性哮喘发病。这两种免疫学机制是分别或共同参与中性粒细胞性哮喘、嗜酸细胞性哮喘发病? 本研究结果显示两种哮喘小鼠脾脏TH17细胞比例、TH2细胞比例均高于对照组,提示TH17、TH2细胞均参与NA、EA小鼠发病；进一步分析TH2/TH17比例,我们发现NA组低于EA组,且NA组支气管肺泡灌洗液(BALF)中IL-17水平高于EA组,而IL-5水平明显低于EA组,提示NA组小鼠产生IL-17的功能亢进,存在强烈的TH17细胞免疫反应、中度TH2细胞免疫反应,TH17细胞在中性粒细胞性哮喘小鼠发病中起着更重要的作用。同时研究结果还表明增高的IL-6、TGF-β形成了有利于中性粒细胞性哮喘小鼠TH17细胞优势的体内微环境,正调控TH17细胞分化的特异性转录因子RORyt的表达增强、负调控TH17细胞分化的SOCS3表达下调与中性粒细胞性哮喘小鼠体内初始T细胞向TH17方向分化增强有关；而SOCS3表达增强是形成嗜酸细胞性哮喘小鼠TH2细胞优势的机制之一。 现有的免疫学理论认为,激活的T细胞再次遇到刺激原时可诱导其进入凋亡途径,即激活诱导细胞死亡(activation-induced cell death, AICD),藉此限制免疫应答的过度发展,AICD过程中任何异常都有可能造成机体的不正常表现甚至疾病。因此,已分化TH17细胞的凋亡与存活状态很可能会影响哮喘气道中性粒细胞炎症的发生、发展。我们的研究发现NA组和EA组小鼠脾脏TH17细胞Ki-67的表达均明显高于对照组,说明哮喘小鼠体内已分化的TH17细胞的凋亡受到抑制,处于持续存活状态,因此,哮喘小鼠体内除初始T细胞向TH17方向分化增强外,TH17细胞持续存活是导致其TH17细胞优势的另一重要机制。 白介素-7(IL-7)主要的功能是促进B和T细胞生长、T细胞存活及增殖。哮喘小鼠TH17细胞持续存活的机制是否依赖于IL7-IL7受体信号途径?本研究检测了脾脏及BALF中IL-7的表达,发现两种哮喘小鼠模型IL-7表达均增高,高表达的IL-7提供了延长TH17细胞存活的微环境。与对照组相比,哮喘小鼠脾脏TH17细胞上STAT5的表达均明显增高,且NA组高于EA组,提示哮喘小鼠TH17细胞存活依赖于JAK/STAT5信号途径激活,且中性粒细胞性哮喘小鼠TH17细胞STAT5信号途径激活程度更高；NA组TH17细胞上BCL-2的表达增高,提示中性粒细胞性哮喘小鼠TH17细胞通过上调抗凋亡蛋白BCL-2,抑制TH17细胞凋亡；两组哮喘小鼠脾脏TH17细胞上凋亡终末剪切酶Caspase-3的表达均明显增高,说明TH17的细胞凋亡是依赖Caspase途径的细胞凋亡,在启动抗凋亡机制的同时也激活了凋亡途径；但Caspase-3上升的幅度明显低于BCL-2,表明在促凋亡与抗凋亡的相互作用下抗凋亡的作用更强以致TH17细胞持续存活。IL-7信号通路可以被一些负调控机制所干扰,如SOCS-1作为细胞因子信号抑制剂可抑制STAT5信号转导而维持适度的炎性反应,本研究发现两种哮喘小鼠SOCS1表达均下调,可能使机体出现IL-7信号的高反应性,导致TH17细胞长期存活。 这些功能增强的TH17细胞如何参与哮喘气道中性粒细胞炎症的形成?与以往研究一致,本研究中两种哮喘小鼠BALF中IL-17水平均增高；此外趋化因子CXCL1、CXCL5、CXCL8/IL-8也明显增高且与IL-17水平呈正相关,提示TH17细胞可能通过IL-17诱导趋化因子的表达,募集中性粒细胞至气道炎症局部聚集。与我们在哮喘患者的研究一致,中性粒细胞性哮喘小鼠气道中性粒细胞增高与气道中性粒细胞凋亡受到抑制有关；此外我们还证实哮喘小鼠BALF中IL-8水平与AHR呈正相关。因此,TH17细胞可能通过IL-17促进多种细胞因子分泌参与哮喘气道中性粒细胞炎症及AHR形成。 糖皮质激素是目前控制哮喘气道炎症最有效的一线药物,通过全身应用地塞米松干预后肺组织病理改变减轻,哮喘小鼠BALF细胞总数、中性粒细胞均明显下降,但仍然高于对照组；TH17细胞数量及相关细胞因子IL-17水平降低,说明地塞米松通过下调TH17细胞数量及相关细胞因子水平以减轻哮喘气道中性粒细胞炎症。但地塞米松不影响NA小鼠气道中性粒细胞凋亡率,也未能影响两种哮喘小鼠TH17细胞存活延长的状态。 综上所述,本研究成功建立了中性粒细胞性哮喘小鼠模型,在此基础上证实中性粒细胞性哮喘小鼠存在强烈的TH17细胞免疫、中度TH2细胞免疫反应,增高的IL-6、TGF-β形成了有利于中性粒细胞性哮喘小鼠TH17细胞优势的体内微环境,并通过RORγt和SOCS3两种途径共同调控TH17细胞的优势分化。除初始T细胞向TH17方向分化增强外,中性粒细胞性哮喘小鼠体内已分化的TH17细胞在机体高IL-7微环境下,依赖JAK/STAT5信号途径激活维持其存活状态,高表达的IL-17通过促进多种细胞因子分泌参与哮喘气道中性粒细胞炎症及AHR形成。地塞米松下调TH17细胞数量及相关细胞因子水平是减轻哮喘气道中性粒细胞炎症的机制之一；但地塞米松对气道中性粒细胞凋亡率及TH17细胞存活延长的状态无明显影响,可能是哮喘激素抵抗的免疫学机制之一。本研究初步阐明TH17细胞及其功能状态在哮喘发病中的作用及相关分子机制,为寻找哮喘治疗靶点提供了新线索和理论依据。
[Abstract]:Bronchial asthma (asthma) is a chronic airway inflammation in various cells, long-term since eosinophils are thought to be the most important effector cells in asthma, but with the in-depth study of airway inflammation in asthma, found eosinophilic asthma (EA) and non eosinophilic asthma in two different subtypes and non eosinophilic asthma in more than half of neutrophilic asthma (NA), we study on the early asthmatic patients found that airway neutrophilic inflammation is associated with airway neutrophil apoptosis, its mechanism is still under discussion.
Study on the pathogenesis of asthma is largely to use animal models, but animal studies asthma use more eosinophilic asthma animal model of tradition, and can not truly reflect the neutrophilic asthma airway inflammation and airway hyperresponsiveness characteristics. Therefore, this study uses lipopolysaccharide and airway instillation of ovalbumin sensitized ovalbumin inhalation to establish a mouse model of neutrophilic asthma. Results showed that the mice developed asthma like symptoms and pathological changes of airway inflammation in neutrophils, similar to eosinophilic asthma airway mucus hypersecretion and more severe airway hyperresponsiveness (AHR) showed the method; successfully established a mouse model of neutrophilic asthma, laid the foundation for further study.
Now that the TH1/TH2 cells caused by the imbalance of TH2 cells is mediated by the advantage of eosinophilic asthma most important immunological mechanisms, but recent studies suggest that TH17 cells as a kind of new T helper cells are found in.TH17 cells of asthma can secrete a variety of cytokines, the most important of which is IL-17A (also called IL-17). IL-17 signaling pathway mediated by receptors can produce IL-8, many cells induced IL-6, GM-CSF, CXCL and other cytokines, chemotaxis and activation of neutrophil infiltration in the local. The biological function of TH17 cells and IL-17 receptor signaling pathway based on cytokines produced by the neutrophil, we speculate that TH17 cells involved in the pathogenesis of neutrophilic asthma, however, studies have shown that TH17 cells are involved in the pathogenesis of eosinophilic asthma. Addicted to these two kinds of immunological mechanisms are respectively or jointly participate in neutrophilic asthma Asthma, eosinophilic asthma?
The results of this study showed that two kinds of asthma mice spleen TH17 cell ratio, TH2 cell ratio was higher than the control group, suggesting that TH17 cells are involved in NA, TH2, EA of mouse disease; further analysis of the proportion of TH2/TH17, we found that NA group than in EA group, NA group and bronchoalveolar lavage fluid (BALF) IL-17 levels higher than the EA group. While the IL-5 level was significantly lower than that of group EA, suggesting that NA mice hyperfunction of IL-17, TH17 cells in presence of a strong immune response, immune TH2 cells TH17 cells in response to moderate, neutrophils in the pathogenesis of asthma mice plays a more important role. The results also showed that the increased IL-6, TGF- beta form in favor of neutrophilic asthma mice TH17 cells in vivo microenvironment, enhance the positive regulation of RORyt expression of specific transcription factor of TH17 cell differentiation, negative regulation of TH17 cell differentiation by down-regulation of SOCS3 expression and neutrophil In asthmatic mice, the initial T cells were enhanced to differentiate into TH17, and SOCS3 expression was one of the mechanisms of TH2 cell dominance in eosinophilic asthmatic mice.
Think that the existing theory of immunology, the activation of T cells again encountered stimuli can induce the apoptosis in ways that activation induced cell death (activation-induced cell, death, AICD), so as to limit the excessive development of the immune response, AICD in the process of any anomalies may be caused by the body is not normal and disease. Therefore, has the differentiation of TH17 cell apoptosis and survival status may affect airway neutrophil inflammation. We found that the expression of NA group and EA group of mice spleen TH17 cells of Ki-67 were significantly higher than the control group, indicating apoptosis in asthmatic mice differentiated TH17 cells was inhibited, in a sustained survival state. Therefore, in addition to the initial T cells in asthmatic mice to TH17 differentiation increased, TH17 cell survival is another important mechanism leading to the TH17 cells.
Interleukin -7 (IL-7) the main function is to promote the growth of B and T cells, T cell survival and proliferation. The mechanism of TH17 cells in asthmatic mice continue to survive depends on IL7-IL7 receptor signal pathway? This study examined the expression of IL-7 in spleen and BALF, found that two kinds of mouse model of asthma IL-7 expression increased, high expression the IL-7 provides extended microenvironment for TH17 cell survival. Compared with the control group, TH17 cells of asthmatic mice spleen STAT5 expression were significantly increased, and the NA group than in EA group, suggesting that TH17 cells of asthmatic mice survival depends on JAK/STAT5 signaling pathway activation, and neutrophilic asthma mice TH17 cell activation of STAT5 signaling pathway a higher degree; the expression of BCL-2 NA in TH17 cells, suggesting that neutrophils of asthmatic mice TH17 cells by up regulation of anti apoptotic protein BCL-2, inhibiting the apoptosis of TH17 cells; two groups of spleen TH17 in asthmatic mice The expression of apoptosis cells end shear enzyme Caspase-3 were significantly increased, indicating TH17 apoptosis is Caspase dependent apoptosis pathway, at the start of the anti apoptosis mechanism also activate apoptosis pathway; but the Caspase-3 increase was significantly lower than that of BCL-2, indicated that in the pro apoptotic and anti apoptotic interactions of anti apoptosis a stronger effect that TH17 cells survive.IL-7 signaling pathway may be some interference of negative regulatory mechanism, such as SOCS-1 cytokine signaling inhibitors can inhibit STAT5 signaling and maintain moderate inflammatory reaction, the study found that two of asthmatic mice SOCS1 expression were down regulated, high reactivity may make the body appear IL-7 signals, resulting in long-term the survival of TH17 cells.
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