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
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。
Asthma is a chronic inflammatory disorder of the airway which involves many cells. For a long time, the eosinophils (EOS) were regarded as the most important inflammatory effector cells which cause asthma. However, with on-going research into the causes for airway inflammations of asthma, two different types of asthma are identified, namely eosinophilic inflammation (EA) and non-eosinophilic inflammation (NEA), whereas for the NEA, more than half are characterized by neutrophilic asthma (NA). In our previous study, we had demonstrated delay of human neutrophil apoptosis is a potential mechanism contributing to airways neutrophilia in asthma, but the exact mechanism that underlie this phenomenon remains unknown.
Past animal experiments on asthma are completed on the traditional EA model, which failed to reflect the airway inflammations and hyperresponsiveness of NA. To avoid such shortcomings, in our study we established a NA model by sensitization through the airway. Briefly, mice were sensitized by airway delivery of100ug of the ovalbumin (OVA) solution complexed in O.lug lipopolysaccharide (LPS) on Days0,6and13. For challenges, mice were exposed to an aerosol of1%OVA in saline. It turned out that the mice showed symptoms and pathologic changes similar to asthma, neutrophilic airway inflammation, airway mucus hypersecretion which is similar to eosinophilic asthma model, and the more severe airway hyperresponsiveness (AHR). These demonstrated that we have successfully established the NA mice model and laid the foundation for further studies.
TH1/TH2paradigm is regarded as the most important immunological mechanism in asthma. T helper type2(TH2) cells play a key role in asthma pathogenesis by producing a variety of cytokines such as IL-4, IL-13, IL-5, which mediate airway eosinophilic inflammation and AHR. Recent studies showed that T helper type17(TH17) cells are a new subset of T helper cells which may be involved in the pathogenesis of asthma. TH17cells can secrete a variety of cytokines, the most important of which is the IL-17A (IL-17). IL-17send out signals to target cells, and induce target cells to produce a variety of cytokines such as IL-8, IL-6, granulocyte macrophage colony factor (GM-CSF), and CXC chemokine Ligand (CXCL), leading to chemotaxis and activation for neutrophils to infiltrate to inflammatory sites. Whether the two types of immunological mechanisms are involved respectively in EA and NA or both?
Our research showed that the mice experimented with the above-mentioned two asthmatic models have higher percentage of TH17and TH2cells in their spleens than mice in the control group. It suggested that the naive T cell differentiation towards TH17and TH2cells are intensified both in NA and EA mice, and that TH17and TH2cells are both involved in the mechanism of EA and NA. Based on further analysis on the proportion of TH2/TH17, we found that EA group was higher than the NA group, accompanied by significantly increased IL-17and slightly increased IL-5, suggesting that TH17cells play a more important role in the pathogenesis of NA mice. Differentiation of TH17cells advantage is achieved by increasing the positive regulation of the differentiation-specific transcription factor orphan nuclear receptor yt (RORyt) expression, elevated cytokines such as IL-6and transforming growth factor-β (TGF-β) formed in vivo microenvironment conducive to the differentiation of TH17cells. Moreover, the TH17cells differentiation advantages are relevant to down-regulated expression of suppressors of cytokine signaling3(SOCS3) in the NA mice, and the TH2cells differentiation advantages are relevant to up-regulated expression of SOCS3in the EA mice.
The modern immunology suggests that when activated T cells is again encountered with stimulus, they could cause activation induced cell deaths (AICD), so as to limit excessive immune response. Any exceptional condition in the AICD process may have resulted in the body abnormal performance and even disease. Therefore, the differentiation of TH17cells and the survival of the differentiated TH17cells is likely to affect the occurrence and development of airway neutrophilic inflammation. Our research found that in the spleens of NA and EA asthmatic mice, the Ki-67expression of the TH17cells was significantly higher than that in the control group, indicating that for asthmatic mice the TH17cells apoptosis is constrained, and that the deaths of TH17cells are delayed. Interleukin-7(IL-7) is a precious pleiotropic cytokine, whose main function is to promote the growth of B and T cells, T cell survival and proliferation. The question is, whether or not the TH17cell survival mechanism of asthmatic mice is dependent on the IL7-IL7receptor signaling pathways?
We detected the IL-7expression in the spleen and bronchoalveolar lavage fluid (BALF), and found that the IL-7expression was up-regulated in both EA and NA asthma mice which in turn provides a favorable microenvironment to extend the survival of TH17cells. Compared with the control group, NA mice showed increased expression of transducer and activator of transcription-5(STAT5), B-cell lymphoma-2(BCL-2) and cysteine-containing aspartate-specific proteases3(Caspase-3) expression in TH17cells of the spleen, suggesting that prolonged survival of TH17cells in NA mice by the way of IL7-IL7receptors depends on Janus kinase-signal transducer and activator of transcription-5(JAK-STAT5) signal pathway activation, and by up-regulating anti-apoptotic protein BCL-2, inhibition of apoptosis of TH17cells; at the same time activate apoptosis terminal shear enzyme Caspase-3, suggesting that Th17cells apoptosis depends on caspase pathway. But the increasing range of BCL-2was greater than that of Caspase-3, suggesting that the anti-apoptotic action of BCL-2predominated in the interaction of anti-apoptotic effect and pro-apoptotic effect. Moreover, IL-7signaling pathway can be interrupted by some negative regulation mechanisms. For example, suppressors of cytokine signaling1(SOCS1) as inhibitors of cytokine signaling, could suppress STAT5signal transmission while maintaining a moderate inflammatory reaction. In our study we found that reduced SOCS1expression in NA and EA asthmatic mice may cause IL-7signal hypersensitivity reaction in the body, resulting in the long-term survival of TH17cells.
How are the TH17cells with enhanced expression engaged in the formation of the airway neutrophilic inflammation? Similar to previous studies, we found higher average IL-17levels in BALF of EA and NA asthmatic mice. We also found that the level of chemokines CXCL1, CXCL5, CXCL8/IL-8was also significantly increased, and was positively correlated with the IL-17level, suggesting that the TH17cells, at least partially by the way of inducing these chemokine expression, cause the gathering of neutrophils where the airway inflammation occurs. Consistent with our previous study on asthma patients, through NA asthmatic mice study we also found a positive correlation between the enhanced airway neutrophils expression and delayed airway neutrophil apoptosis. Moreover for asthmatic mice with nebulization for14days, we also found positive correlation between IL-8levels in BALF and AHR-suggesting a possibility that the TH17cells, by stimulating target cells to secrete a variety of cytokines through IL-17, is involved in airway neutrophils inflammation and AHR formation.
Glucocorticoid is currently the most effective first-line drugs to control airway inflammations in asthma. Systemic dexamethasone application on asthmetic mice mitigates pathological changes of lung tissue, significantly reduces the number of BALF cells and neutrophils, but the level of such cells is still higher than that of the control group. TH17cell numbers and related cytokines IL-17levels are decreased, demonstrating that dexamethasone may have alleviated the airway neutrophilic inflammation symptom by reducing the TH17cell numbers and cytokine levels. However, dexamethasone does not affect the airway neutrophil apoptosis rate of NA asthmatic mice, nor could it inhibit the TH17cell survival in both EA and NA asthmatic mice.
In summary, our research successfully established a neutrophilic asthma mice model, confirmed on the basis of this model that we elucidated that the NA mice were primed only modest TH2responses, but strong Th17responses. The TH17cell differentiation advantages is regulated by positive adjustment (RORyt) and a negative adjustment (SOCS3), and the elevated IL-6and TGF-β formed in vivo microenvironment conducive to the differentiation of TH17cells. Differentiated TH17cells depend on the IL7-IL7receptor signaling pathway to maintain their survival status. Increased IL-17participates in airway neutrophilic inflammation and AHR formation through a variety of ways. Dexamethasone can reduce airway neutrophilic inflammation by down-regulating the number of TH17cells and cytokine levels; but has no significant effect on airway neutrophil apoptosis rate and prolonged survival status of TH17cells; it may be one of the molecular mechanisms of steroid-resistance (SR) of asthma. This study elucidates the potential contribution of TH17cell survival to neutrophil airway inflammation in neutrophilic asthma and the related molecular mechanisms, it will provide a novel scientific basis for the asthma therapy.
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。
Asthma is a chronic inflammatory disorder of the airway which involves many cells. For a long time, the eosinophils (EOS) were regarded as the most important inflammatory effector cells which cause asthma. However, with on-going research into the causes for airway inflammations of asthma, two different types of asthma are identified, namely eosinophilic inflammation (EA) and non-eosinophilic inflammation (NEA), whereas for the NEA, more than half are characterized by neutrophilic asthma (NA). In our previous study, we had demonstrated delay of human neutrophil apoptosis is a potential mechanism contributing to airways neutrophilia in asthma, but the exact mechanism that underlie this phenomenon remains unknown.
Past animal experiments on asthma are completed on the traditional EA model, which failed to reflect the airway inflammations and hyperresponsiveness of NA. To avoid such shortcomings, in our study we established a NA model by sensitization through the airway. Briefly, mice were sensitized by airway delivery of100ug of the ovalbumin (OVA) solution complexed in O.lug lipopolysaccharide (LPS) on Days0,6and13. For challenges, mice were exposed to an aerosol of1%OVA in saline. It turned out that the mice showed symptoms and pathologic changes similar to asthma, neutrophilic airway inflammation, airway mucus hypersecretion which is similar to eosinophilic asthma model, and the more severe airway hyperresponsiveness (AHR). These demonstrated that we have successfully established the NA mice model and laid the foundation for further studies.
TH1/TH2paradigm is regarded as the most important immunological mechanism in asthma. T helper type2(TH2) cells play a key role in asthma pathogenesis by producing a variety of cytokines such as IL-4, IL-13, IL-5, which mediate airway eosinophilic inflammation and AHR. Recent studies showed that T helper type17(TH17) cells are a new subset of T helper cells which may be involved in the pathogenesis of asthma. TH17cells can secrete a variety of cytokines, the most important of which is the IL-17A (IL-17). IL-17send out signals to target cells, and induce target cells to produce a variety of cytokines such as IL-8, IL-6, granulocyte macrophage colony factor (GM-CSF), and CXC chemokine Ligand (CXCL), leading to chemotaxis and activation for neutrophils to infiltrate to inflammatory sites. Whether the two types of immunological mechanisms are involved respectively in EA and NA or both?
Our research showed that the mice experimented with the above-mentioned two asthmatic models have higher percentage of TH17and TH2cells in their spleens than mice in the control group. It suggested that the naive T cell differentiation towards TH17and TH2cells are intensified both in NA and EA mice, and that TH17and TH2cells are both involved in the mechanism of EA and NA. Based on further analysis on the proportion of TH2/TH17, we found that EA group was higher than the NA group, accompanied by significantly increased IL-17and slightly increased IL-5, suggesting that TH17cells play a more important role in the pathogenesis of NA mice. Differentiation of TH17cells advantage is achieved by increasing the positive regulation of the differentiation-specific transcription factor orphan nuclear receptor yt (RORyt) expression, elevated cytokines such as IL-6and transforming growth factor-β (TGF-β) formed in vivo microenvironment conducive to the differentiation of TH17cells. Moreover, the TH17cells differentiation advantages are relevant to down-regulated expression of suppressors of cytokine signaling3(SOCS3) in the NA mice, and the TH2cells differentiation advantages are relevant to up-regulated expression of SOCS3in the EA mice.
The modern immunology suggests that when activated T cells is again encountered with stimulus, they could cause activation induced cell deaths (AICD), so as to limit excessive immune response. Any exceptional condition in the AICD process may have resulted in the body abnormal performance and even disease. Therefore, the differentiation of TH17cells and the survival of the differentiated TH17cells is likely to affect the occurrence and development of airway neutrophilic inflammation. Our research found that in the spleens of NA and EA asthmatic mice, the Ki-67expression of the TH17cells was significantly higher than that in the control group, indicating that for asthmatic mice the TH17cells apoptosis is constrained, and that the deaths of TH17cells are delayed. Interleukin-7(IL-7) is a precious pleiotropic cytokine, whose main function is to promote the growth of B and T cells, T cell survival and proliferation. The question is, whether or not the TH17cell survival mechanism of asthmatic mice is dependent on the IL7-IL7receptor signaling pathways?
We detected the IL-7expression in the spleen and bronchoalveolar lavage fluid (BALF), and found that the IL-7expression was up-regulated in both EA and NA asthma mice which in turn provides a favorable microenvironment to extend the survival of TH17cells. Compared with the control group, NA mice showed increased expression of transducer and activator of transcription-5(STAT5), B-cell lymphoma-2(BCL-2) and cysteine-containing aspartate-specific proteases3(Caspase-3) expression in TH17cells of the spleen, suggesting that prolonged survival of TH17cells in NA mice by the way of IL7-IL7receptors depends on Janus kinase-signal transducer and activator of transcription-5(JAK-STAT5) signal pathway activation, and by up-regulating anti-apoptotic protein BCL-2, inhibition of apoptosis of TH17cells; at the same time activate apoptosis terminal shear enzyme Caspase-3, suggesting that Th17cells apoptosis depends on caspase pathway. But the increasing range of BCL-2was greater than that of Caspase-3, suggesting that the anti-apoptotic action of BCL-2predominated in the interaction of anti-apoptotic effect and pro-apoptotic effect. Moreover, IL-7signaling pathway can be interrupted by some negative regulation mechanisms. For example, suppressors of cytokine signaling1(SOCS1) as inhibitors of cytokine signaling, could suppress STAT5signal transmission while maintaining a moderate inflammatory reaction. In our study we found that reduced SOCS1expression in NA and EA asthmatic mice may cause IL-7signal hypersensitivity reaction in the body, resulting in the long-term survival of TH17cells.
How are the TH17cells with enhanced expression engaged in the formation of the airway neutrophilic inflammation? Similar to previous studies, we found higher average IL-17levels in BALF of EA and NA asthmatic mice. We also found that the level of chemokines CXCL1, CXCL5, CXCL8/IL-8was also significantly increased, and was positively correlated with the IL-17level, suggesting that the TH17cells, at least partially by the way of inducing these chemokine expression, cause the gathering of neutrophils where the airway inflammation occurs. Consistent with our previous study on asthma patients, through NA asthmatic mice study we also found a positive correlation between the enhanced airway neutrophils expression and delayed airway neutrophil apoptosis. Moreover for asthmatic mice with nebulization for14days, we also found positive correlation between IL-8levels in BALF and AHR-suggesting a possibility that the TH17cells, by stimulating target cells to secrete a variety of cytokines through IL-17, is involved in airway neutrophils inflammation and AHR formation.
Glucocorticoid is currently the most effective first-line drugs to control airway inflammations in asthma. Systemic dexamethasone application on asthmetic mice mitigates pathological changes of lung tissue, significantly reduces the number of BALF cells and neutrophils, but the level of such cells is still higher than that of the control group. TH17cell numbers and related cytokines IL-17levels are decreased, demonstrating that dexamethasone may have alleviated the airway neutrophilic inflammation symptom by reducing the TH17cell numbers and cytokine levels. However, dexamethasone does not affect the airway neutrophil apoptosis rate of NA asthmatic mice, nor could it inhibit the TH17cell survival in both EA and NA asthmatic mice.
In summary, our research successfully established a neutrophilic asthma mice model, confirmed on the basis of this model that we elucidated that the NA mice were primed only modest TH2responses, but strong Th17responses. The TH17cell differentiation advantages is regulated by positive adjustment (RORyt) and a negative adjustment (SOCS3), and the elevated IL-6and TGF-β formed in vivo microenvironment conducive to the differentiation of TH17cells. Differentiated TH17cells depend on the IL7-IL7receptor signaling pathway to maintain their survival status. Increased IL-17participates in airway neutrophilic inflammation and AHR formation through a variety of ways. Dexamethasone can reduce airway neutrophilic inflammation by down-regulating the number of TH17cells and cytokine levels; but has no significant effect on airway neutrophil apoptosis rate and prolonged survival status of TH17cells; it may be one of the molecular mechanisms of steroid-resistance (SR) of asthma. This study elucidates the potential contribution of TH17cell survival to neutrophil airway inflammation in neutrophilic asthma and the related molecular mechanisms, it will provide a novel scientific basis for the asthma therapy.
引文
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