IL-12~+重组卡介苗新生期接种干预实验性哮喘模型的作用及其机制研究
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摘要
背景与目的支气管哮喘是全球关注的公共卫生问题,其发病率在发达国家可达人群33%,且有持续上升的趋势。经典免疫学说认为哮喘的发病机制与辅助性T细胞(T helper cells,Th cells)亚群即Th1/Th2功能失衡密切相关,主要表现为Th2细胞功能亢进,分泌Th2细胞因子如IL-4、IL-5、IL-13等可以诱导IgE产生并刺激嗜酸性粒细胞(EOS)增殖、活化脱颗粒,进而分泌多种促炎介质引起气道慢性炎症。单纯Th1/Th2失衡不能解释所有哮喘的发病。
越来越多的研究表明哮喘具有异质性,至少分为嗜酸性粒细胞性哮喘和非嗜酸性粒细胞性哮喘,后者以中性粒细胞为主。新近发现的Th17是以表达IL-17为特征的CD4~+T细胞亚型,大量研究结果支持Th17/IL-17与哮喘气道慢性炎症有关:Th17通过分泌IL-17介导中性粒细胞性炎症,参与变应原暴露诱发的哮喘气道炎症。临床研究从分子和蛋白水平,在哮喘患者支气管黏膜、支气管肺泡灌洗液和诱导痰中都检测到IL-17表达增加,IL-17表达水平与气道中性粒细胞性炎症密切相关;动物实验进一步证实IL-17以及Th17细胞参与哮喘模型气道局部的炎症反应,肺局部过度表达IL-17可诱导哮喘表型,均提示IL-17在诱导哮喘气道炎症和气道高反应性中起到重要作用。然而国外相关研究发现通过不同途径单纯阻断哮喘模型IL-17作用,并不能完全改善哮喘模型气道炎症。在许多自身免疫性疾病和炎症疾病研究中,Th1和Th17细胞共同定位在炎症局部,并需要彼此才能动员和/或进入这一区域。在肺部,结核分枝杆菌疫苗免疫的小鼠:Th17细胞动员到肺部先于产IFN-γ的记忆细胞并且IL-17诱导的趋化因子表达对Th1效应因子快速聚集起重要作用。最新的研究发现同时表达IFN-γ和IL-17的Th17亚群,这类细胞同时具有T-be(tTh1转录因子)和ROR-γt(Th17转录因子),加入IL-12可以使Th17细胞转化为Th1细胞,提示Th1和Th17细胞共同分享一个通路。
我们前期研究表明儿童哮喘气道炎症细胞以中性粒细胞为主,新生期接种BCG能促进哮喘模型气道IFN-γ为代表的Th1反应,抑制气道IL-17表达显著改善气道炎症,提示新生期BCG接种抗实验性哮喘的机制可能与抑制IL-17作用同时加强IFN-γ作用,调节IFN-γ/IL-17平衡有关。
在前期研究基础上,我们拟通过观察卡介苗(BCG)/IL-12~+重组卡介苗(rBCG)新生期接种对小鼠肺脏和脾脏Th17/Th1平衡影响,探讨新生期BCG接种是否通过调节IL-17/IFN-γ平衡和Th17/Th1平衡发挥抗哮喘作用,采用新生期rBCG接种探讨IL-17/IFN-γ平衡和Th17/Th1平衡的影响因素,为进一步阐明哮喘的发病机制,从一级预防水平干预哮喘提供依据。
我们通过动物模型对以上假设分为三部分进行了初步探讨:
第一部分卡介苗/IL-12~+重组卡介苗新生期接种对小鼠肺脏和脾脏T细胞亚群发育和功能的影响
目的:观察BCG/rBCG新生期接种对小鼠肺部和脾脏T细胞亚群发育和Th17/Th1平衡的影响;通过体外分离培养小鼠肺组织单个核细胞和脾脏单个核细胞,使用儿童时期最常见的病毒病原RSV、细菌成分LPS和过敏原OVA刺激培养,探讨通过不同刺激对小鼠肺脏和脾脏免疫应答功能的影响。
方法:(1)新生清洁级BALB/c小鼠分为3组:对照组、BCG组和rBCG组,新生期接种4周后取肺和脾单个核细胞用流式细胞仪检测辅助性T细胞亚群Th1细胞、Th2细胞、Th17细胞、Treg细胞比例;(2)提取肺组织和脾组织总mRNA,逆转录为cDNA后使用real time PCR方法检测介导Th1细胞、Th2细胞、Th17细胞、Treg细胞分化的主要转录因子T-bet、GATA3、RORγt和Foxp3表达情况;(3)分离培养肺和脾单个核细胞,分别使用RSV、LPS、OVA刺激培养72小时后取上清,用ELISA方法检测培养上清中IL-17和IFN-γ水平。
结果:(1)对动物模型肺组织T细胞亚群的影响:rBCG组能显著增加转录因子RORγt和T-bet表达,CD3~+IL-17~+细胞和Th17细胞比例显著高于BCG组和对照组,BCG组仅T-bet水平显著高于对照组; BCG组和rBCG组肺单个核细胞刺激培养上清分泌IL-17、IFN-γ水平和IL-17/IFN-γ比值显著高于对照组。(2)对动物模型脾组织T细胞亚群及功能的影响:BCG组和rBCG组脾组织表达转录因子Foxp3和T-bet水平显著高于对照组,而表达转录因子RORγt水平却显著低于对照组;rBCG组脾单个核细胞中CD3~+IFN-γ~+细胞、Th1细胞以及CD4~+CD25~+Foxp3~+细胞百分率,Th1/Th17比值显著高于BCG组与对照组。
结论:(1)新生期rBCG接种在肺部诱导IL-17/Th17细胞优势发育,在肺部诱导抗感染免疫应答;(2)新生期rBCG接种在脾脏显著诱导产IFN-γ/Th1细胞和Treg细胞优势发育,加强脾脏作为中枢免疫器官的免疫调节能力;(3)新生期BCG和rBCG接种对实验动物T细胞亚群分化发育的影响具有组织特异性,提示IL-12在不同组织对Th1、Th17和Treg分化发育所起的作用不同。
第二部分卡介苗新生期接种通过调节IL-17/IFN-γ平衡干预实验性哮喘模型的作用及其机制研究
目的:建立小鼠OVA致敏模型、哮喘模型和BCG新生期接种的干预模型,探讨BCG在哮喘气道炎症不同阶段干预实验性哮喘模型是否是通过调节Th17/Th1平衡,并通过干预模型了解哮喘发生发展不同阶段的发病机制。
方法:1)新生清洁级BALB/c小鼠分为3组:对照组、OVA组、BCG~+OVA组,新生期接种后分别在4周和6周除对照组外均进行OVA致敏,小鼠致敏期模型在进行激发前即7周进行相关检测;小鼠哮喘模型在第7周开始使用OVA雾化,连续雾化一周,最后一次雾化后24小时内进行相关检测;2)进行支气管肺泡灌洗,瑞氏染色观察BALF中白细胞数及分类,ELISA检测BALF中细胞因子含量和血清中OVA特异性IgE水平,real time PCR检测肺组织中四种辅助性T细胞转录因子表达;3)肺组织HE染色进行气道炎症病理分析,使用小鼠肺功能仪检测气道高反应性。
结果:(1)在致敏期:对照组、OVA组、BCG~+OVA组三组小鼠肺部均无明显炎症,肺泡间隔无增厚,血管周围和支气管细支气管周围无明显炎症细胞浸润;与对照组比较,各致敏组小鼠BALF中细胞总数、淋巴细胞、中性粒细胞均明显增加;和OVA组相比:新生期接种BCG气道高反性显著降低,BALF中细胞总数、淋巴细胞、中性粒细胞均明显降低;在转录因子水平RORγt和T-bet表达均上调;在细胞水平Th17/Th1比值较对照组上升;肺泡灌洗液中IL-17明显降低,IFN-γ升高,IL-17/IFN-γ比值显著降低,IL-10水平明显增高;(2)在激发期:各致敏组小鼠肺泡灌洗液中大量中性粒细胞和嗜酸性粒细胞浸润;新生期接种BCG干预后,和OVA组比较:肺泡灌洗液中炎症细胞总数、淋巴细胞、中性粒细胞和嗜酸性粒细胞比例,肺部炎症病理评分、气道高反应性均显著降低;Th1细胞比例显著升高,Tr细胞、Th17细胞、Th2细胞和OVA组无差异;肺泡灌洗液中,IL-17水平显著降低,IFN-γ水平显著增高,IL-17/IFN-γ比值显著降低;
结论:(1)在实验性哮喘模型的致敏期肺组织局部没有明显的炎症反应,但有炎症细胞动员增加和血清OVA特异性IgE水平显著增高,小鼠气道显示提高的气道高反应性;(2)在致敏期辅助性T细胞亚群中以Th2类细胞应答占优势,诱导血清OVA特异性IgE显著增加,肺泡灌洗液中IL-5和IL-10水平显著升高;(3)在致敏期主导炎症细胞动员和气道高反应性的是IL-17,Th2类反应起到的作用具有局限性;(4)新生期接种BCG能显著降低致敏期气道炎症细胞浸润和完全缓解气道高反应性,但对血清OVA特异性IgE水平无显著抑制作用,也并不能干预Th2类应答的优势,提示干预机制主要由IL-17/IFN-γ平衡介导;(5)在激发期,气道急性炎症和气道高反应性主要由IL-17介导;新生期接种BCG产生的干预作用由IL-17/IFN-γ平衡和调节性T细胞介导,可能Th17/Th1平衡只发挥次要作用。
第三部分IL-12~+重组BCG新生期接种干预实验性哮喘模型的作用及其机制研究
目的:建立rBCG新生期接种的小鼠致敏模型和哮喘模型,观察在生命早期进一步加强Th1类应答后机体在致敏和哮喘发作阶段气道炎症和气道高反应性受到的干预作用及其机制。
方法:(1)新生清洁级BALB/c小鼠分为3组:对照组、OVA组、rBCG~+OVA组,新生期接种后分别在4周和6周除对照组外均进行OVA致敏,小鼠致敏期模型在进行激发前即7周进行相关检测;小鼠哮喘模型在第7周开始使用OVA雾化,连续雾化一周,最后一次雾化后24小时内进行相关检测;(2)进行支气管肺泡灌洗,瑞氏染色观察BALF中白细胞数及分类,ELISA检测BALF中细胞因子含量和血清中OVA特异性IgE水平,real time PCR检测肺组织中四种辅助性T细胞转录因子表达;(3)肺组织HE染色进行气道炎症病理分析,使用小鼠肺功能仪检测气道高反应性。
结果:(1)在致敏期,与OVA组比较:新生期接种rBCG显著减少肺泡灌洗液中炎症细胞动员,完全缓解气道高反应性,降低OVA特异性IgE不显著;在转录因子水平上调T-bet表达,下调GATA3和Foxp3表达,但各辅助性T细胞亚群比例没有显著变化,Th17/Th1比值也没有变化;显著增加IFN-γ、IL-10、IL-12水平,下调IL-17/IFN-γ比值;(2)在激发期,与OVA组比较:新生期接种rBCG显著降低肺部炎症病理状况,减少中性粒细胞和嗜酸性粒细胞聚集,降低气道高反应性和血清OVA特异性IgE;在转录因子水平和细胞水平显著增加Th1,Tr亚群比例,显著降低Th17亚群比例,Th17/Th1比值降低;显著升高肺泡灌洗液中IFN-γ、IL-10、IL-12水平,降低IL-17/IFN-γ比值。
结论:(1)新生期接种rBCG进一步加强生命早期机体Th1类反应,能显著降低小鼠致敏模型和哮喘模型的气道炎症、气道高反应性和血清OVA特异性IgE水平;(2)rBCG发挥抗哮喘作用的机制和BCG有部分不同,是通过增加IFN-γ水平下调IL-17/IFN-γ平衡产生效应;(3)生命早期接触不同的微生物刺激影响哮喘的发生发展机制;(4)rBCG新生期接种在生命早期加强Th1类应答有利于在发生哮喘时诱导Tr1细胞发挥抗哮喘作用。
Background and Objective Bronchial asthma is the most common chronic airway inflammatory disease in childhood, which has become the public health problem in worldwide. Allergic asthma has been defined as a disease of immunodysregulation, where the pathology is a direct consequence of excessive T-helper type 2 immune responses (Th2) to allergens in the lungs. Disease is associated with increased secretion of type-2 cytokines such as interleukin (IL)-4, IL-5 and IL-13 which, among other things, promotes enhanced IgE levels, induces pulmonary eosinophilic inflammation and increased mucus production in the airways.
Th17 cells are a new subset of helper T cell characterized by the production of IL-17A, IL-17F, IL-6, IL-21 and IL-22. The proinflammatory cytokine IL-17 has previously been forwarded as a link between activated T-lymphocytes and the recruitment and activation of neutrophils in various types of airway inflammation. It has been shown that the concentration of IL-17 is increased in BALF, sputum, and blood from patients with asthma. Recently, it has been suggested that at least two inflammatory subtypes of asthma exist: the eosinophilic and the non-eosinophilic type. Neutrophils are specially prominent in acute, severe exacerbations of asthma and potentially contribute to airway gland hypersecretion, bronchial hyper-reactivity and to airway wall remodelling. Given that Th17 cells play a crucial role in the pathogenesis of neutrophilic inflammation, Th17 might be a predominant player in non-eosinophilic Asthma. However, the exact role of Th17 in asthma is not well known. In a series of mouse model of allergic airway inflammation, blockade of IL-17 exhibited different results in the airway. Both Th1 and Th17 cells co-localize within the region of inflammation and they may require each other for recruitment and/or entry to this region. In the lung, however, the reverse entry sequence has been reported in mice receiving a post-vaccination challenge of Mycobacteriumtuberculosis; Th17 cells were recruited to the lung prior to the appearance of IFN-γ-producing memory cells and IL-17-induced chemokine expression was essential for the rapid accumulation of these Th1 effectors.
In our preliminary data, RSV reversed the anti-asthma effect of neonatal BCG vaccination in BALB/c mice. IL-12~+ -recombined BCG inhibited the inflammatory response by decreasing IL-17 and regulating the imbalance between IL-17/IFN-γ.
The aim of this study is to investigate the effect of neonatal BCG/ IL-12~+ -recombined BCG vaccination on development of Th cells and Th17/Th1 balance, determine the effect and mechanism of neonatal BCG vaccination on murine asthma model through IL-17/IFN-γbalance and Th17/Th1 balance, elucidate the effect and mechanism of neonatal IL-12~+ -recombined BCG vaccination on murine asthma model through IL-17/IFN-γbalance and Th17/Th1 balance.
To test the hypothesis mentioned above, we conducted experiments in animal model in three parts.
Part one Effect of neonatal BCG and IL-12~+ -recombined BCG vaccination on development and function of Th cells
Objective: To explore the effects of neonatal BCG and IL-12~+ -recombined BCG vaccination on the development and function of lung and spleen Th cell subsets .
Methods: (1) Neonatal BALB/c mice were divided into 3 groups: control, BCG, and rBCG groups, which inoculated with BCG and rBCG subcutaneously within 2～3 days after birth. Four weeks later, lung cells and spleen cells of mice were isolated and the percentage of four groups of Th cells respectively were detected by flow cytometry at single cells level. (2) RNA was extracted from lung and spleen tissue, and then reverse transcribed into cDNA. Real time PCR were performed to identify the gene expression of transcription factors including RORγt, T-bet, GATA3 and Foxp3. (3) Single-cell suspensions of lung and spleen were prepared , plated in 12 well plates (2×106 cells/mL) in duplicate, and cultured with medium containing RSV/OVA/LPS at 37°C in 5% CO2 for 72 h. The supernatants were then collected and stored at -20°C until analysis. IFN-γand IL-17 were assayed by ELISA kits.
Results: (1) In comparison with mice in the BCG and control group , RORγt and T-bet mRNA and the percentages of CD3~+IL-17~+ cells and Th17 cells in the lung of mice in the rBCG group were increased. Compared with mice in the control group, T-bet mRNA in the lung of mice in the rBCG group was increased. The cultures of lung cells from mice in the BCG and rBCG group contained significantly higher levels of IL-17 , IFN-γand IL-17/IFN-γratio compared with the mice in the control group. (2) The mice in the BCG and rBCG group had a higher mRNA expression of T-bet, Foxp3 and lower mRNA expression of RORγt than those in the control group. In comparison with mice in the BCG and control group , the percentages of CD3~+IFN-γ~+ cells, Th1 cells, CD4~+CD25~+Foxp3~+ cells and ratio of Th1/Th17 in the lung of mice in the rBCG group were increased.
Conclusion: (1) Neonatal BCG and rBCG vaccination have impact on the development of Th cells, but the target tissue itself participates actively in creating the site-specific milieu and contribute to the development of Th cells. (2) Neonatal BCG rBCG vaccination promotes enhanced IL-17/Th17 development in lung and induced Th1 cell, Tr in spleen. These data suggest IL-12 play an important role in the development of Th1, Th17 and Tr.
Part two Effect and mechanism of neonatal BCG vaccination on murine asthma model through IL-17/IFN-γbalance
Objective: To determine the effect and mechanism of neonatal BCG vaccination on OVA sensitized mice and OVA sensitized and challenged mice, and investigate the role of Th17/Th1 balance in the pathogenesis of asthma.
Methods: (1) Neonatal BALB/c mices were divided into 3 groups: control, OVA, BCG~+OVA groups. Mice in BCG~+OVA group inoculated with BCG subcutaneously within 2～3 days after birth. Then Using the intraperitoneal OVA with alum sensitization protocol at 4-week and 6-week as mice in OVA group. Mice which were used to perform asthma model were challenged by an aerosol of OVA at 7- week for one week. (2) Bronchoalveolar lavage was performed before challenge. Cells in BALF were counted. Cytokines in BALF and serum OVA-specific IgE were detected by ELISA and Real time PCR were performed to identify the gene expression of transcription factors including RORγt, T-bet, GATA3 and Foxp3. (3) Inflammatory characteristics of lungs was scored by staining with hematoxylin and eosin, and airway hyperresponsiveness to methacholine in conscious, spontaneously breathing animals was measured by enhanced pause.
Results: (1) After sensitization, none of the mice in the three groups showed obvious asthmatic manifestation. Difference was not found in peribronchiolitis, perivasculitis and alveolitis among the mice in the three groups. The numbers of total white cells,lymphocytes, neutrophils in the BALF of the mice from all OVA-sensitized groups (OVA, BCG~+OVA) were significantly greater than those in the control group. In comparison with OVA group, the numbers of total white cells, lymphocytes, neutrophils in the BALF were decreased in the BCG~+OVA group. Mice in the BCG~+OVA group had lower IL-17 concentration and higher IL-10, IL-12, IFN-γconcentration in BALF than mice in the OVA group. Ratio of IL-17/IFN-γwas reduced in mice of the BCG~+OVA group. (2) OVA-sensitizated/challenged mice showed obvious asthmatic manifestation. The numbers of total white cells and percentages of lymphocytes, neutrophils and eosinophils in the BALF of the mice from all OVA-sensitized/challenged groups (OVA, BCG~+OVA) were significantly greater than those in the control group. Mice in BCG~+OVA group had significantly lower total white cells and percentages of lymphocytes, neutrophils and eosinophils in BALF and airway hyperresponsiveness than those in the OVA group. Mice in the BCG~+OVA group had lower concentration of IL-17 and higher concentration of IFN-γin BALF than those in the OVA group. Ratio of IL-17/IFN-γwas downregulated in mice of BCG~+OVA group.
Conclusion: (1) After OVA sensitization airway inflammation was not found in murine model, but inflammatory cells were recruited, increased OVA-specific IgE level and airway hyperreactivity were exist. (2) Enhanced Th2 response was showed after OVA sensitization, which induced increased OVA-specific IgE level and IL-5, IL-10 level in BALF. (3) After OVA sensitization inflammatory cells recruitment and airway hyperreactivity were mediated by IL-17. (4) Neonatal BCG vaccination reduce airway inflammatory cells recruitment and airway hyperreactivity through the regulation of the balance of IL-17/IFN-γ, but have not impact on enhanced Th2 response and increased OVA-specific IgE level. (5) After challenge, airway inflammatory cells recruitment and airway hyperreactivity were reduced in the neonatal BCG vaccinated mice by .the regulation of the balance of IL-17/IFN-γ.
Part three Effect and mechanism of neonatal IL-12~+ -recombined BCG vaccination on murine asthma model
Objective: To investigate Effect and mechanism of neonatal IL-12~+ -recombined BCG vaccination on murine asthma model.
Methods: (1) Neonatal BALB/c mice were divided three groups: control, OVA and rBCG~+OVA groups, which inoculated with rBCG subcutaneously within 2～3 days after birth. Then Using the intraperitoneal OVA with alum sensitization protocol at 4-week and 6-week as mice in OVA group. Mice which were used to perform asthma model were challenged by an aerosol of OVA at 7- week for one week. (2) Bronchoalveolar lavage was performed before challenge. Cells in BALF were counted. Cytokines in BALF and serum OVA-specific IgE were detected by ELISA and Real time PCR were performed to identify the gene expression of transcription factors including RORγt, T-bet, GATA3 and Foxp3. (3) Inflammatory characteristics of lungs was scored by staining with hematoxylin and eosin, and airway hyperresponsiveness to methacholine in conscious, spontaneously breathing animals was measured by enhanced pause.
Results: (1) After sensitization, in comparison with OVA group inflammatory cell recruitment and airway hyperresponsiveness were decreased in the rBCG~+OVA group. T-bet mRNA was increased and GATA3, Foxp3 mRNA were decreased in the lung of mice in the rBCG group compared with mice in the OVA group. (2) Mice in the BCG~+OVA group had significantly lower total white cells and percentages of lymphocytes, neutrophils and eosinophils in BALF and airway hyperresponsiveness, OVA specific IgE than those in the OVA group. Mice in the BCG~+OVA group had higher percentages of Th1 cell and Tr cell than those in the OVA group. Ratio of IL-17/IFN-γwas downregulated in mice of BCG~+OVA group by increased level of IFN-γin BALF.
Conclusions: (1) After sensitization or challenge, airway inflammatory cells recruitment and airway hyperreactivity were reduced in the neonatal BCG vaccinated mice. (2) Neonatal rBCG vaccination elicited protection in murine asthma model by downregulation of IL-17/IFN-γratio, which is different from BCG. (3) Neonatal rBCG vaccination induced Tr recruitment to mediate protection in murine asthma model.(4) Diversity neonatal inmmune stimulation had different impact on the prognosis of asthma.
越来越多的研究表明哮喘具有异质性,至少分为嗜酸性粒细胞性哮喘和非嗜酸性粒细胞性哮喘,后者以中性粒细胞为主。新近发现的Th17是以表达IL-17为特征的CD4~+T细胞亚型,大量研究结果支持Th17/IL-17与哮喘气道慢性炎症有关:Th17通过分泌IL-17介导中性粒细胞性炎症,参与变应原暴露诱发的哮喘气道炎症。临床研究从分子和蛋白水平,在哮喘患者支气管黏膜、支气管肺泡灌洗液和诱导痰中都检测到IL-17表达增加,IL-17表达水平与气道中性粒细胞性炎症密切相关;动物实验进一步证实IL-17以及Th17细胞参与哮喘模型气道局部的炎症反应,肺局部过度表达IL-17可诱导哮喘表型,均提示IL-17在诱导哮喘气道炎症和气道高反应性中起到重要作用。然而国外相关研究发现通过不同途径单纯阻断哮喘模型IL-17作用,并不能完全改善哮喘模型气道炎症。在许多自身免疫性疾病和炎症疾病研究中,Th1和Th17细胞共同定位在炎症局部,并需要彼此才能动员和/或进入这一区域。在肺部,结核分枝杆菌疫苗免疫的小鼠:Th17细胞动员到肺部先于产IFN-γ的记忆细胞并且IL-17诱导的趋化因子表达对Th1效应因子快速聚集起重要作用。最新的研究发现同时表达IFN-γ和IL-17的Th17亚群,这类细胞同时具有T-be(tTh1转录因子)和ROR-γt(Th17转录因子),加入IL-12可以使Th17细胞转化为Th1细胞,提示Th1和Th17细胞共同分享一个通路。
我们前期研究表明儿童哮喘气道炎症细胞以中性粒细胞为主,新生期接种BCG能促进哮喘模型气道IFN-γ为代表的Th1反应,抑制气道IL-17表达显著改善气道炎症,提示新生期BCG接种抗实验性哮喘的机制可能与抑制IL-17作用同时加强IFN-γ作用,调节IFN-γ/IL-17平衡有关。
在前期研究基础上,我们拟通过观察卡介苗(BCG)/IL-12~+重组卡介苗(rBCG)新生期接种对小鼠肺脏和脾脏Th17/Th1平衡影响,探讨新生期BCG接种是否通过调节IL-17/IFN-γ平衡和Th17/Th1平衡发挥抗哮喘作用,采用新生期rBCG接种探讨IL-17/IFN-γ平衡和Th17/Th1平衡的影响因素,为进一步阐明哮喘的发病机制,从一级预防水平干预哮喘提供依据。
我们通过动物模型对以上假设分为三部分进行了初步探讨:
第一部分卡介苗/IL-12~+重组卡介苗新生期接种对小鼠肺脏和脾脏T细胞亚群发育和功能的影响
目的:观察BCG/rBCG新生期接种对小鼠肺部和脾脏T细胞亚群发育和Th17/Th1平衡的影响;通过体外分离培养小鼠肺组织单个核细胞和脾脏单个核细胞,使用儿童时期最常见的病毒病原RSV、细菌成分LPS和过敏原OVA刺激培养,探讨通过不同刺激对小鼠肺脏和脾脏免疫应答功能的影响。
方法:(1)新生清洁级BALB/c小鼠分为3组:对照组、BCG组和rBCG组,新生期接种4周后取肺和脾单个核细胞用流式细胞仪检测辅助性T细胞亚群Th1细胞、Th2细胞、Th17细胞、Treg细胞比例;(2)提取肺组织和脾组织总mRNA,逆转录为cDNA后使用real time PCR方法检测介导Th1细胞、Th2细胞、Th17细胞、Treg细胞分化的主要转录因子T-bet、GATA3、RORγt和Foxp3表达情况;(3)分离培养肺和脾单个核细胞,分别使用RSV、LPS、OVA刺激培养72小时后取上清,用ELISA方法检测培养上清中IL-17和IFN-γ水平。
结果:(1)对动物模型肺组织T细胞亚群的影响:rBCG组能显著增加转录因子RORγt和T-bet表达,CD3~+IL-17~+细胞和Th17细胞比例显著高于BCG组和对照组,BCG组仅T-bet水平显著高于对照组; BCG组和rBCG组肺单个核细胞刺激培养上清分泌IL-17、IFN-γ水平和IL-17/IFN-γ比值显著高于对照组。(2)对动物模型脾组织T细胞亚群及功能的影响:BCG组和rBCG组脾组织表达转录因子Foxp3和T-bet水平显著高于对照组,而表达转录因子RORγt水平却显著低于对照组;rBCG组脾单个核细胞中CD3~+IFN-γ~+细胞、Th1细胞以及CD4~+CD25~+Foxp3~+细胞百分率,Th1/Th17比值显著高于BCG组与对照组。
结论:(1)新生期rBCG接种在肺部诱导IL-17/Th17细胞优势发育,在肺部诱导抗感染免疫应答;(2)新生期rBCG接种在脾脏显著诱导产IFN-γ/Th1细胞和Treg细胞优势发育,加强脾脏作为中枢免疫器官的免疫调节能力;(3)新生期BCG和rBCG接种对实验动物T细胞亚群分化发育的影响具有组织特异性,提示IL-12在不同组织对Th1、Th17和Treg分化发育所起的作用不同。
第二部分卡介苗新生期接种通过调节IL-17/IFN-γ平衡干预实验性哮喘模型的作用及其机制研究
目的:建立小鼠OVA致敏模型、哮喘模型和BCG新生期接种的干预模型,探讨BCG在哮喘气道炎症不同阶段干预实验性哮喘模型是否是通过调节Th17/Th1平衡,并通过干预模型了解哮喘发生发展不同阶段的发病机制。
方法:1)新生清洁级BALB/c小鼠分为3组:对照组、OVA组、BCG~+OVA组,新生期接种后分别在4周和6周除对照组外均进行OVA致敏,小鼠致敏期模型在进行激发前即7周进行相关检测;小鼠哮喘模型在第7周开始使用OVA雾化,连续雾化一周,最后一次雾化后24小时内进行相关检测;2)进行支气管肺泡灌洗,瑞氏染色观察BALF中白细胞数及分类,ELISA检测BALF中细胞因子含量和血清中OVA特异性IgE水平,real time PCR检测肺组织中四种辅助性T细胞转录因子表达;3)肺组织HE染色进行气道炎症病理分析,使用小鼠肺功能仪检测气道高反应性。
结果:(1)在致敏期:对照组、OVA组、BCG~+OVA组三组小鼠肺部均无明显炎症,肺泡间隔无增厚,血管周围和支气管细支气管周围无明显炎症细胞浸润;与对照组比较,各致敏组小鼠BALF中细胞总数、淋巴细胞、中性粒细胞均明显增加;和OVA组相比:新生期接种BCG气道高反性显著降低,BALF中细胞总数、淋巴细胞、中性粒细胞均明显降低;在转录因子水平RORγt和T-bet表达均上调;在细胞水平Th17/Th1比值较对照组上升;肺泡灌洗液中IL-17明显降低,IFN-γ升高,IL-17/IFN-γ比值显著降低,IL-10水平明显增高;(2)在激发期:各致敏组小鼠肺泡灌洗液中大量中性粒细胞和嗜酸性粒细胞浸润;新生期接种BCG干预后,和OVA组比较:肺泡灌洗液中炎症细胞总数、淋巴细胞、中性粒细胞和嗜酸性粒细胞比例,肺部炎症病理评分、气道高反应性均显著降低;Th1细胞比例显著升高,Tr细胞、Th17细胞、Th2细胞和OVA组无差异;肺泡灌洗液中,IL-17水平显著降低,IFN-γ水平显著增高,IL-17/IFN-γ比值显著降低;
结论:(1)在实验性哮喘模型的致敏期肺组织局部没有明显的炎症反应,但有炎症细胞动员增加和血清OVA特异性IgE水平显著增高,小鼠气道显示提高的气道高反应性;(2)在致敏期辅助性T细胞亚群中以Th2类细胞应答占优势,诱导血清OVA特异性IgE显著增加,肺泡灌洗液中IL-5和IL-10水平显著升高;(3)在致敏期主导炎症细胞动员和气道高反应性的是IL-17,Th2类反应起到的作用具有局限性;(4)新生期接种BCG能显著降低致敏期气道炎症细胞浸润和完全缓解气道高反应性,但对血清OVA特异性IgE水平无显著抑制作用,也并不能干预Th2类应答的优势,提示干预机制主要由IL-17/IFN-γ平衡介导;(5)在激发期,气道急性炎症和气道高反应性主要由IL-17介导;新生期接种BCG产生的干预作用由IL-17/IFN-γ平衡和调节性T细胞介导,可能Th17/Th1平衡只发挥次要作用。
第三部分IL-12~+重组BCG新生期接种干预实验性哮喘模型的作用及其机制研究
目的:建立rBCG新生期接种的小鼠致敏模型和哮喘模型,观察在生命早期进一步加强Th1类应答后机体在致敏和哮喘发作阶段气道炎症和气道高反应性受到的干预作用及其机制。
方法:(1)新生清洁级BALB/c小鼠分为3组:对照组、OVA组、rBCG~+OVA组,新生期接种后分别在4周和6周除对照组外均进行OVA致敏,小鼠致敏期模型在进行激发前即7周进行相关检测;小鼠哮喘模型在第7周开始使用OVA雾化,连续雾化一周,最后一次雾化后24小时内进行相关检测;(2)进行支气管肺泡灌洗,瑞氏染色观察BALF中白细胞数及分类,ELISA检测BALF中细胞因子含量和血清中OVA特异性IgE水平,real time PCR检测肺组织中四种辅助性T细胞转录因子表达;(3)肺组织HE染色进行气道炎症病理分析,使用小鼠肺功能仪检测气道高反应性。
结果:(1)在致敏期,与OVA组比较:新生期接种rBCG显著减少肺泡灌洗液中炎症细胞动员,完全缓解气道高反应性,降低OVA特异性IgE不显著;在转录因子水平上调T-bet表达,下调GATA3和Foxp3表达,但各辅助性T细胞亚群比例没有显著变化,Th17/Th1比值也没有变化;显著增加IFN-γ、IL-10、IL-12水平,下调IL-17/IFN-γ比值;(2)在激发期,与OVA组比较:新生期接种rBCG显著降低肺部炎症病理状况,减少中性粒细胞和嗜酸性粒细胞聚集,降低气道高反应性和血清OVA特异性IgE;在转录因子水平和细胞水平显著增加Th1,Tr亚群比例,显著降低Th17亚群比例,Th17/Th1比值降低;显著升高肺泡灌洗液中IFN-γ、IL-10、IL-12水平,降低IL-17/IFN-γ比值。
结论:(1)新生期接种rBCG进一步加强生命早期机体Th1类反应,能显著降低小鼠致敏模型和哮喘模型的气道炎症、气道高反应性和血清OVA特异性IgE水平;(2)rBCG发挥抗哮喘作用的机制和BCG有部分不同,是通过增加IFN-γ水平下调IL-17/IFN-γ平衡产生效应;(3)生命早期接触不同的微生物刺激影响哮喘的发生发展机制;(4)rBCG新生期接种在生命早期加强Th1类应答有利于在发生哮喘时诱导Tr1细胞发挥抗哮喘作用。
Background and Objective Bronchial asthma is the most common chronic airway inflammatory disease in childhood, which has become the public health problem in worldwide. Allergic asthma has been defined as a disease of immunodysregulation, where the pathology is a direct consequence of excessive T-helper type 2 immune responses (Th2) to allergens in the lungs. Disease is associated with increased secretion of type-2 cytokines such as interleukin (IL)-4, IL-5 and IL-13 which, among other things, promotes enhanced IgE levels, induces pulmonary eosinophilic inflammation and increased mucus production in the airways.
Th17 cells are a new subset of helper T cell characterized by the production of IL-17A, IL-17F, IL-6, IL-21 and IL-22. The proinflammatory cytokine IL-17 has previously been forwarded as a link between activated T-lymphocytes and the recruitment and activation of neutrophils in various types of airway inflammation. It has been shown that the concentration of IL-17 is increased in BALF, sputum, and blood from patients with asthma. Recently, it has been suggested that at least two inflammatory subtypes of asthma exist: the eosinophilic and the non-eosinophilic type. Neutrophils are specially prominent in acute, severe exacerbations of asthma and potentially contribute to airway gland hypersecretion, bronchial hyper-reactivity and to airway wall remodelling. Given that Th17 cells play a crucial role in the pathogenesis of neutrophilic inflammation, Th17 might be a predominant player in non-eosinophilic Asthma. However, the exact role of Th17 in asthma is not well known. In a series of mouse model of allergic airway inflammation, blockade of IL-17 exhibited different results in the airway. Both Th1 and Th17 cells co-localize within the region of inflammation and they may require each other for recruitment and/or entry to this region. In the lung, however, the reverse entry sequence has been reported in mice receiving a post-vaccination challenge of Mycobacteriumtuberculosis; Th17 cells were recruited to the lung prior to the appearance of IFN-γ-producing memory cells and IL-17-induced chemokine expression was essential for the rapid accumulation of these Th1 effectors.
In our preliminary data, RSV reversed the anti-asthma effect of neonatal BCG vaccination in BALB/c mice. IL-12~+ -recombined BCG inhibited the inflammatory response by decreasing IL-17 and regulating the imbalance between IL-17/IFN-γ.
The aim of this study is to investigate the effect of neonatal BCG/ IL-12~+ -recombined BCG vaccination on development of Th cells and Th17/Th1 balance, determine the effect and mechanism of neonatal BCG vaccination on murine asthma model through IL-17/IFN-γbalance and Th17/Th1 balance, elucidate the effect and mechanism of neonatal IL-12~+ -recombined BCG vaccination on murine asthma model through IL-17/IFN-γbalance and Th17/Th1 balance.
To test the hypothesis mentioned above, we conducted experiments in animal model in three parts.
Part one Effect of neonatal BCG and IL-12~+ -recombined BCG vaccination on development and function of Th cells
Objective: To explore the effects of neonatal BCG and IL-12~+ -recombined BCG vaccination on the development and function of lung and spleen Th cell subsets .
Methods: (1) Neonatal BALB/c mice were divided into 3 groups: control, BCG, and rBCG groups, which inoculated with BCG and rBCG subcutaneously within 2～3 days after birth. Four weeks later, lung cells and spleen cells of mice were isolated and the percentage of four groups of Th cells respectively were detected by flow cytometry at single cells level. (2) RNA was extracted from lung and spleen tissue, and then reverse transcribed into cDNA. Real time PCR were performed to identify the gene expression of transcription factors including RORγt, T-bet, GATA3 and Foxp3. (3) Single-cell suspensions of lung and spleen were prepared , plated in 12 well plates (2×106 cells/mL) in duplicate, and cultured with medium containing RSV/OVA/LPS at 37°C in 5% CO2 for 72 h. The supernatants were then collected and stored at -20°C until analysis. IFN-γand IL-17 were assayed by ELISA kits.
Results: (1) In comparison with mice in the BCG and control group , RORγt and T-bet mRNA and the percentages of CD3~+IL-17~+ cells and Th17 cells in the lung of mice in the rBCG group were increased. Compared with mice in the control group, T-bet mRNA in the lung of mice in the rBCG group was increased. The cultures of lung cells from mice in the BCG and rBCG group contained significantly higher levels of IL-17 , IFN-γand IL-17/IFN-γratio compared with the mice in the control group. (2) The mice in the BCG and rBCG group had a higher mRNA expression of T-bet, Foxp3 and lower mRNA expression of RORγt than those in the control group. In comparison with mice in the BCG and control group , the percentages of CD3~+IFN-γ~+ cells, Th1 cells, CD4~+CD25~+Foxp3~+ cells and ratio of Th1/Th17 in the lung of mice in the rBCG group were increased.
Conclusion: (1) Neonatal BCG and rBCG vaccination have impact on the development of Th cells, but the target tissue itself participates actively in creating the site-specific milieu and contribute to the development of Th cells. (2) Neonatal BCG rBCG vaccination promotes enhanced IL-17/Th17 development in lung and induced Th1 cell, Tr in spleen. These data suggest IL-12 play an important role in the development of Th1, Th17 and Tr.
Part two Effect and mechanism of neonatal BCG vaccination on murine asthma model through IL-17/IFN-γbalance
Objective: To determine the effect and mechanism of neonatal BCG vaccination on OVA sensitized mice and OVA sensitized and challenged mice, and investigate the role of Th17/Th1 balance in the pathogenesis of asthma.
Methods: (1) Neonatal BALB/c mices were divided into 3 groups: control, OVA, BCG~+OVA groups. Mice in BCG~+OVA group inoculated with BCG subcutaneously within 2～3 days after birth. Then Using the intraperitoneal OVA with alum sensitization protocol at 4-week and 6-week as mice in OVA group. Mice which were used to perform asthma model were challenged by an aerosol of OVA at 7- week for one week. (2) Bronchoalveolar lavage was performed before challenge. Cells in BALF were counted. Cytokines in BALF and serum OVA-specific IgE were detected by ELISA and Real time PCR were performed to identify the gene expression of transcription factors including RORγt, T-bet, GATA3 and Foxp3. (3) Inflammatory characteristics of lungs was scored by staining with hematoxylin and eosin, and airway hyperresponsiveness to methacholine in conscious, spontaneously breathing animals was measured by enhanced pause.
Results: (1) After sensitization, none of the mice in the three groups showed obvious asthmatic manifestation. Difference was not found in peribronchiolitis, perivasculitis and alveolitis among the mice in the three groups. The numbers of total white cells,lymphocytes, neutrophils in the BALF of the mice from all OVA-sensitized groups (OVA, BCG~+OVA) were significantly greater than those in the control group. In comparison with OVA group, the numbers of total white cells, lymphocytes, neutrophils in the BALF were decreased in the BCG~+OVA group. Mice in the BCG~+OVA group had lower IL-17 concentration and higher IL-10, IL-12, IFN-γconcentration in BALF than mice in the OVA group. Ratio of IL-17/IFN-γwas reduced in mice of the BCG~+OVA group. (2) OVA-sensitizated/challenged mice showed obvious asthmatic manifestation. The numbers of total white cells and percentages of lymphocytes, neutrophils and eosinophils in the BALF of the mice from all OVA-sensitized/challenged groups (OVA, BCG~+OVA) were significantly greater than those in the control group. Mice in BCG~+OVA group had significantly lower total white cells and percentages of lymphocytes, neutrophils and eosinophils in BALF and airway hyperresponsiveness than those in the OVA group. Mice in the BCG~+OVA group had lower concentration of IL-17 and higher concentration of IFN-γin BALF than those in the OVA group. Ratio of IL-17/IFN-γwas downregulated in mice of BCG~+OVA group.
Conclusion: (1) After OVA sensitization airway inflammation was not found in murine model, but inflammatory cells were recruited, increased OVA-specific IgE level and airway hyperreactivity were exist. (2) Enhanced Th2 response was showed after OVA sensitization, which induced increased OVA-specific IgE level and IL-5, IL-10 level in BALF. (3) After OVA sensitization inflammatory cells recruitment and airway hyperreactivity were mediated by IL-17. (4) Neonatal BCG vaccination reduce airway inflammatory cells recruitment and airway hyperreactivity through the regulation of the balance of IL-17/IFN-γ, but have not impact on enhanced Th2 response and increased OVA-specific IgE level. (5) After challenge, airway inflammatory cells recruitment and airway hyperreactivity were reduced in the neonatal BCG vaccinated mice by .the regulation of the balance of IL-17/IFN-γ.
Part three Effect and mechanism of neonatal IL-12~+ -recombined BCG vaccination on murine asthma model
Objective: To investigate Effect and mechanism of neonatal IL-12~+ -recombined BCG vaccination on murine asthma model.
Methods: (1) Neonatal BALB/c mice were divided three groups: control, OVA and rBCG~+OVA groups, which inoculated with rBCG subcutaneously within 2～3 days after birth. Then Using the intraperitoneal OVA with alum sensitization protocol at 4-week and 6-week as mice in OVA group. Mice which were used to perform asthma model were challenged by an aerosol of OVA at 7- week for one week. (2) Bronchoalveolar lavage was performed before challenge. Cells in BALF were counted. Cytokines in BALF and serum OVA-specific IgE were detected by ELISA and Real time PCR were performed to identify the gene expression of transcription factors including RORγt, T-bet, GATA3 and Foxp3. (3) Inflammatory characteristics of lungs was scored by staining with hematoxylin and eosin, and airway hyperresponsiveness to methacholine in conscious, spontaneously breathing animals was measured by enhanced pause.
Results: (1) After sensitization, in comparison with OVA group inflammatory cell recruitment and airway hyperresponsiveness were decreased in the rBCG~+OVA group. T-bet mRNA was increased and GATA3, Foxp3 mRNA were decreased in the lung of mice in the rBCG group compared with mice in the OVA group. (2) Mice in the BCG~+OVA group had significantly lower total white cells and percentages of lymphocytes, neutrophils and eosinophils in BALF and airway hyperresponsiveness, OVA specific IgE than those in the OVA group. Mice in the BCG~+OVA group had higher percentages of Th1 cell and Tr cell than those in the OVA group. Ratio of IL-17/IFN-γwas downregulated in mice of BCG~+OVA group by increased level of IFN-γin BALF.
Conclusions: (1) After sensitization or challenge, airway inflammatory cells recruitment and airway hyperreactivity were reduced in the neonatal BCG vaccinated mice. (2) Neonatal rBCG vaccination elicited protection in murine asthma model by downregulation of IL-17/IFN-γratio, which is different from BCG. (3) Neonatal rBCG vaccination induced Tr recruitment to mediate protection in murine asthma model.(4) Diversity neonatal inmmune stimulation had different impact on the prognosis of asthma.
引文
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