雷帕霉素与细胞自噬在哮喘气道炎症和嗜酸粒细胞分化中的作用研究
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摘要
支气管哮喘(简称哮喘)是一种以嗜酸性粒细胞(Eosinophils, Eos)、肥大细胞和T淋巴细胞浸润为主的气道慢性炎症性疾病,表现为气道高反应性和可逆性气流受限。其中嗜酸性粒细胞是哮喘过敏性气道炎症反应的主要效应细胞,Eos与哮喘发病之间存在直接因果关系。研究发现,嗜酸性粒细胞在哮喘发病过程中参与多种功能调控,包括抗原提呈,细胞因子、趋化因子、颗粒介质以及白三烯的释放等。嗜酸性粒细胞是由骨髓共同髓系祖细胞(CMP)经由粒-单核系祖细胞(GMP),由嗜酸性粒细胞祖细胞(EoP)定向分化成熟。嗜酸性粒细胞分化涉及多种转录因子(GATA-1等)和细胞因子,如白介素3(IL-3),IL-5,粒-单核集落刺激因子(GM-CSF),其中IL-5对Eos最终分化成熟起最主要的调节作用。然而,目前对于哮喘发病过程中Eos调控机制的研究并不多.
细胞自噬(autophagy)是机体一种重要的防御和保护机制。在某些特定的微环境条件(如饥饿,生长素缺乏)下,细胞内形成一种双层膜结构的囊泡,细胞自噬体(autophagosomes),并同时捕获细胞内的受损的细胞器(如线粒体,内质网)和变性的蛋白质等；然后该囊泡与溶酶体溶合,形成自噬溶酶体(autolysosomes)。在溶酶体水解酶等的作用下,自噬体所包含的各种细胞器和生物大分子得以消化和降解,从而为重新合成新的生物大分子提供原料和能量。因此从本义上讲,细胞自噬是机体保持内稳态(homeostasis)和适应微环境改变的一种重要的自我调节和保护机制。然而,在某些特定的条件下,细胞器和各种生物大分子的过量消耗最终会导致细胞的另一种程序性死亡,细胞自噬死亡(autophagic cell death)。由于细胞自噬能保护或者促进细胞死亡,因此在不同的疾病病变过程中,其功能截然不同。越来越多的研究表明,自噬在机体的免疫、感染、炎症、肿瘤、心血管病、神经退行性病的发病中具有十分重要的作用。然而,细胞自噬在呼吸系统的研究并不多。新近有研究开创性地阐明了细胞自噬在吸烟诱导的气道上皮细胞损伤以及慢性阻塞性肺疾病(COPD)形成过程中的重要调控作用。然而,细胞自噬在支气管哮喘分子发病机制中的作用鲜有研究报导。
雷帕霉素(rapamycin)是经典的细胞自噬诱导剂,是1975年从加拿大Easter岛上的吸水链霉菌中提取的一种大环内酯类抗生素。雷帕霉素的主要作用靶点,哺乳动物雷帕霉素靶蛋白(mammalian target of rapamycin, mTOR)能接受并整合生长因子、能量、氧气和氨基酸四大主要信号,参与调节能量代谢、蛋白质、脂质、细胞器的合成以及细胞自噬等生理过程,在细胞的生长、增殖、分化和凋亡中起着重要的调控作用,从而维持机体与细胞的稳态平衡。研究发现雷帕霉素在哮喘动物模型中的作用并不一致,有报道发现雷帕霉素能抑制过敏性气道炎症,气道高反应性,杯状细胞化生和IgE产生,但也有部分研究认为雷帕霉素对过敏性气道炎症和气道高反应性没有影响。然而,最关键的是这些研究均只简单利用雷帕霉素体内干预观察哮喘表型,并未阐明雷帕霉素及mTOR在哮喘发病机制中起到关键调控作用及其具体机制。
越来越多的研究发现细胞自噬和mTOR在造血干细胞分化和增殖中起到重要作用。自噬缺陷可抑制红系造血导致严重贫血、抑制T淋巴细胞、B淋巴细胞数目和功能,而mTOR通过调控T细胞分化、功能、代谢参与调控适应性免疫应答。由此,我们假设细胞自噬和mTOR也参与骨髓粒系祖细胞尤其是嗜酸性粒细胞分化的调控,并进一步探讨其在以嗜酸性粒细胞气道炎症为主要表现的整体哮喘模型中的可能作用与贡献,从而为哮喘分子发病机制的研究开拓新的视野,为哮喘临床的防治提供新的靶点。
本实验分两部分进行研究：(1)探求雷帕霉素及mTOR在哮喘气道炎症和骨髓嗜酸性粒细胞分化中的作用；(2)利用细胞自噬相关基因敲除小鼠Beclin1+/-和骨髓移植技术,分别阐明细胞自噬在气道上皮细胞损伤和骨髓嗜酸性粒细胞分化中的不同调控作用。
第一部分雷帕霉素在哮喘气道炎症和嗜酸粒细胞分化中的作用研究
目的：研究雷帕霉素干预对哮喘气道炎症的影响,并探讨雷帕霉素在OVA诱导肺组织T细胞免疫应答及骨髓嗜酸性粒细胞分化过程中的调控作用。
方法：健康雌性C57BL/6小鼠,随机分为四组：生理盐水对照组(SHAM组)、模型组(OVA组)、雷帕霉素对照组(SHAM+RAPA组)、雷帕霉素哮喘组(OVA+RAPA组)。以卵白蛋白(OVA)致敏和激发建立哮喘模型,生理盐水对照组以同等剂量的生理盐水代替OVA,雷帕霉素组在每次抗原激发前1小时进行腹腔注射(1mg/kg)。于最后一次抗原激发后24小时检测肺泡灌洗液(BALF)、外周血和骨髓中的嗜酸性粒细胞数；肺组织病理切片观察气道炎症细胞浸润情况,ELISA检测血清中IL-5,IL-13水平。流式细胞术检测和分析肺组织Th2, Th17, Treg各T细胞亚群的比例,同时检测骨髓中嗜酸性粒细胞祖细胞(EoP, Lin-Sca-l-CD34+IL-5Ra+c-Kitl0)的比例。Western blot检测骨髓嗜酸性粒细胞分化过程中mTOR水平变化,体外克隆形成试验及骨髓Eos诱导分化培养检测雷帕霉素干预对骨髓嗜酸性粒细胞分化及功能的影响。最后利用IL-5转基因小鼠NJ.1638,连续3天腹腔注射雷帕霉素,检测外周血和骨髓中嗜酸性粒细胞数,ELISA检测血清中IL-5水平,流式细胞术检测骨髓EoP的比例以及嗜酸性粒细胞凋亡情况。
结果：雷帕霉素干预后显著缓解OVA诱导的气道过敏性炎症,但是不影响肺组织中Th2细胞,Th17细胞和Treg水平。雷帕霉素抑制了气道局部以及外周血,骨髓嗜酸性粒细胞数目,但是并不改变血清IL-5水平。体外克隆形成试验和骨髓Eos诱导分化培养发现,雷帕霉素可以直接抑制IL-5介导的嗜酸性粒细胞分化及培养上清Eos分泌IL-6和IL-13的水平。离体骨髓克隆形成试验及骨髓流式细胞术检测结果提示,雷帕霉素对嗜酸性粒细胞分化的抑制最终导致骨髓嗜酸性粒细胞祖细胞的聚集。对IL-5转基因小鼠NJ.1638的研究同样证实了雷帕霉素对外周血和骨髓嗜酸性粒细胞数的抑制作用及骨髓EoP的聚集,并不依赖其血清高表达IL-5水平的改变,也不影响嗜酸性粒细胞的凋亡。
结论：雷帕霉素可以缓解哮喘小鼠气道过敏性炎症,可能是通过抑制骨髓嗜酸性粒细胞分化及功能,而对Eos凋亡和肺组织T细胞免疫无明显影响。
第二部分细胞自噬在哮喘气道炎症和嗜酸粒细胞分化中的作用研究
目的：研究细胞自噬敲除对哮喘整体模型的影响,并探讨细胞自噬在OVA诱导的气道上皮细胞损伤和骨髓嗜酸性粒细胞分化过程中的不同调控作用。
方法：通过Western blot检测哮喘小鼠骨髓及体外嗜酸性粒细胞诱导分化过程中自噬水平变化,并利用细胞自噬相关基因缺陷小鼠Beclin1+/-,通过瑞士-吉姆萨染色分析其外周血和骨髓中嗜酸性粒细胞的水平,流式细胞术检测其骨髓嗜酸性粒细胞祖细胞水平,并通过甲基纤维素克隆形成试验检测其骨髓嗜酸性粒细胞分化能力。同时利用Beclin1+/-小鼠及其同窝生WT小鼠以OVA致敏和激发建立哮喘模型,随机分组如下：野生型生理盐水对照组(WT/NS),野生型哮喘模型组(WT/OVA),Beclin1+/-小鼠生理盐水对照组(BECN/NS)和Beclin1+/-小鼠哮喘模型组(BECN/OVA)。末次激发后24小时,采用有创方法测定小鼠气道反应性,并检测气道灌洗液中的白细胞总数和嗜酸性粒细胞数；肺组织病理切片观察气道炎症细胞浸润和粘液分泌情况,电镜观察哮喘小鼠气道上皮细胞自噬泡聚集情况。体外培养人正常肺上皮细胞(BEAS-2B),Western blot检测OVA体外干预过程中自噬水平变化,Q-PCR检测饥饿诱导自噬及IL-13体外干预对粘蛋白MUC5AC mRNA的表达情况。最后采用骨髓移植的方法,白硝胺+环磷酰胺联合化疗毁损野生型小鼠骨髓造血系统,然后将Beclin1+/-小鼠或同窝生野生型小鼠骨髓移植至野生型小鼠并重建其造血系统,OVA诱导建立哮喘模型,根据回输骨髓不同分两组：WT-WT-OVA组和BECN-WT-OVA组,观察骨髓局部自噬敲除对哮喘整体模型气道高反应和气道炎症的贡献。
结果：细胞自噬相关基因敲除小鼠Beclin1+/-,与同窝生野生型(WT)小鼠相比,外周血和骨髓中嗜酸性粒细胞数目显著增多,骨髓EoP水平虽有下降趋势,但无统计学意义。体外Eos诱导分化过程中LC3B蛋白水平的改变提示细胞自噬参与骨髓嗜酸性粒细胞分化。体外克隆形成试验直接证实,Beclin1+/-小鼠骨髓生成嗜酸性粒细胞克隆形成单位能力增加。但Beclin1+/-小鼠抗原激发后气道反应性、气道炎症、粘液高分泌,与野生型小鼠模型组相比并未恶化,反而出现明显减轻。同时发现,虽然哮喘小鼠骨髓中LC3B蛋白水平下降,但电镜可见哮喘气道上皮细胞中存在细胞自噬泡聚集。并且体外实验表明,OVA干预可上调LC3B蛋白水平,饥饿诱导自噬可进一步增强IL-13介导的BEAS-2B细胞MUC5AC mRNA的表达。在骨髓移植实验中,Beclin1+/"小鼠骨髓回输的哮喘小鼠(BECN-WT-OVA组),气道反应性和气道炎症都明显高于野生型小鼠骨髓回输的哮喘小鼠(WT-WT-OVA组)。
结论：细胞自噬敲除虽然在动物模型中整体表现为可以缓解哮嗤小鼠气道过敏性炎症,气道高反应性和粘液高分泌,但细胞自噬在气道局部损伤和骨髓Eos分化过程中可能存在两种完全相反的调控机制。自噬敲除抑制了OVA诱导的气道上皮细胞损伤从而缓解了哮喘表型,而在骨髓嗜酸性粒细胞分化过程中则促进了其分化能力从而恶化了哮喘气道炎症。我们的研究提示对细胞自噬和mTOR信号通路的研究有望为哮喘的防治提供新的靶点。
Asthma is a chronic airway inflammatory disease, which is characterized by reversible airway obstruction, bronchial hyperresponsiveness and airway inflammation. Among these, allergic airway inflammation in asthma is characterized by eosinophil infiltration. Studies have established that a causative relationship exists between eosinophils and the development of allergic asthma, and eosinophils participate in a variety of functions, including antigen presentation, cytokine production, chemokine production, secretion of granule mediators, and leukotriene secretion. Eosinophil differentiate from a common myeloid progenitor (CMP) in mice through an intermediate granulocyte/macrophage progenitor (GMP) and then via an eosinophil lineage-committed progenitor (EoP). The development of eosinophils is orchestrated by several transcription factors such as GATA-1, in the presence of certain cytokines, in particular Interleukin-3(IL-3), IL-5, and Granulocyte-macrophage colony stimulating factor (GM-CSF). Among these, IL-5seems to be more specific and efficient in eosinophil lineage development as it promotes the selective differentiation of eosinophils. However, little is known about the molecular mechanisms of eosinophil differentiation during asthma development.
Autophagy is a regulated pathway for internal organelle or protein degradation. In this dynamic process, double-membraned autophagic vacuoles (AVs) or autophagosomes surround cytosolic organelles (e.g. endoplasmic reticulum, mitochondria) or protein, and subsequently fuse with lysosomes, where the engulfed components are degraded by lysosomal hydrolases. Autophagy provides essential functions in the maintenance of cellular homeostasis and adaptation to adverse environments. However, excessive autophagy may be associated with the activation of programmed cell death (i.e. apoptosis) through a cell-autodigestive process. The role of autophagy, whether protective or deleterious, in human diseases, or specifically in chronic lung disease remains obscure. It has been recently reported that autophagy plays an important role in smoking-induced epithelial cell injury and emphysema. However, the potential functions of autophagy in asthma, to our knowledge, have not been investigated so far.
Rapamycin is a macrolide product of Streptomyces hygroscopius that was initially in a soil sample from Easter Island (Rapa Nui) in the early1970s. Its major cellular target, mammalian target of rapamycin (mTOR), is a central regulator of cell growth/differentiation and survival in many cell types. Previous works using rapamycin in animal models of allergic asthma showed controversial results. In some studies rapamycin have been suggested to inhibit cardinal features of allergic asthma, including airway hyperresponsiveness (AHR), eosinophilic airway inflammation, goblet cell hyperplasia, and IgE production; however, there are other works have also shown that it has little effects on the allergic airway inflammation and AHR. Most importantly, the underlying mechanisms that mediated the effects of rapamycin in the process of allergic asthma remain largely unknown.
Recently, lots evidences showing that autophagy and mTOR play an important role in hematopoietic stem cell maintenance and differentiation. It has been shown that conditional deletion of ATG7or ATG5throughout the hematopoietic system in mice results in severe anemia and inhibition of lymphocyte survival and function. Also, mTOR provides a critical link between T cell differentiation, function and metabolism. We hypothesized that autophagy and mTOR would involve in the regulation of myeloid proliferation, especially in the eosinophil differentiation, and examined their contribution to its overall effect in allergic airway inflammation. Moreover, it will bring the new insight into the prevention of asthma and other allergic disorders for the public health.
In this study, we have explored that:(1) Role of rapamycin in allergic airway inflammation and in eosinophil differentiation;(2) the different roles of autophagy in regulation of airway epithelial cell injury and eosinophil differentiation in bone marrow during asthma development.
Part I Mechanisms of rapamycin in airway inflammation and eosinophil differentiation in asthma
Objective:To investigate the effect of rapamycin in eosinophil differentiation and asthma airway inflammation, and to explore the underlying molecular mechanisms.
Methods:Female C57BL/6mice were randomly divided into four groups:SHAM group, OVA group, SHAM+RAPA group and OVA+RAPA group. In the OVA and OVA+RAPA group, mice sensitized and challenged with ovabumin (OVA).In SHAM and SHAM+RAPA group, mice were sensitized and challenged with normal saline by the same methods. In the RAPA and OVA+RAPA group, mice were treated with an intraperitoneal injection of rapamycin1hour before each aerosol OVA challenge.24hours after the final OVA challenge, mice were sacrificed and the number of eosinophils in bronchoalveolar fluid (BALF), blood and bone marrow were accessed. Lungs were processed for histologic staining (HE), and airway inflammation was analyzed. Levels of selected cytokines (IL-5, IL-13) in serum were assayed using ELISA kits. Lung lymphocytes were isolated and labeled with T cells surface Abs (CD4, CD8and CD25) and intracellular Abs (IL-4, IL-17A, Foxp3). Bone marrow Eosinophil progenitors (Eops) were determined as Lin-Sca-1-CD34+IL-5Ra+c-Kitlo cells. Data were collected and analyzed using a FACScan for seven-color flow cytometry. Western blot of phosphorylated S6(P-S6) ribosomal protein was analyzed to explore whether mTOR is involved in eosinophil differentiation. In vitro methylcellulose colony forming assays and liquid culture of mouse bone marrow-derived eosinophils (BM-Eos) were also performed to assess the ability of eosinophil differentiation and function. Finally, we used IL-5transgenic NJ.1638mice with treatment of rapamycin, to investigate the levels of eosinophils and apoptosis of Eos in blood and bone marrow, the levels of IL-5in serum, and Eops in BM were also analyzed.
Results:Rapamycin significantly attenuated OVA-induced allergic airway inflammation, without influence on the various subtypes of T helper cells. It markedly decreased the amount of eosinophils in local airways, peripheral blood, and bone marrow, independent on levels of IL-5. In vitro colony forming unit assay and liquid culture demonstrated that rapamycin directly inhibited IL-5-induced eosinophil differentiation. In addition, rapamycin reduced the production of IL-6and IL-13by eosinophils. Rapamycin was also capable of significantly reducing the eosinophil levels in IL-5transgenic NJ.1638mice, again regardless of the constitutive high levels of IL-5, and without influence on the eosinophil apoptosis. Interestingly, rapamycin inhibition of eosinophil differentiation in turn resulted in an accumulation of eosinophil progenitors in bone marrow.
Conclusion:Altogether these results clearly demonstrate a direct inhibitory role of rapamycin in eosinophil differentiation and function, and reemphasize the importance of rapamycin and possibly, mTOR in allergic airway disease.
Part II Mechanisms of autophagy in airway inflammation and eosinophil dfifferentiation in asthma
Objective:To investigate the effect of autophagy in eosinophil differentiation and asthma airway inflammation, and to explore the underlying molecular mechanisms.
Methods:Western blot of LC3B was analyzed to explore whether autophagy is involved in eosinophil differentiation. Eosibophil, Eosinophil progenitor level and the ability of eosinophil differentiation were assessed in Beclin-l+/-mice (autophagy related gene Beclin-1impaired). Beclin-1+/-and wild type (WT) mice were randomly divided into four group:WT/NS group, WT/OVA group, BECN/NS group and BECN/OVA group.24hours after the final OVA challenge airway hyperresponsiveness to inhaled methacholine, airway inflammation and airway mucus expression were measured. Lung tissue sections were also processed for transmission electron microscope (EM) and analyzed for formation of autophagic vacuoles (AVs). MUC5AC was induced by IL-13in BEAS-2B cells with or without starvation, and the mRNA expression was detected by Q-PCR. Finally, we transplanted the bone marrow of Beclin-1+/-mice to WT, and subjected these mice for the OVA asthma model to explore the contribution of local autophagy defect of bone marrow to its overall effect in asthma.
Results:The number of eosinophil in blood and bone marrow in vivo, and Eo-CFU in vitro colony forming assays were significantly increased in Beclin-1+/-mice. However, compared with WT/OVA group, BECN/OVA group showed significantly decreased AHR, airway inflammation and mucus overproduction. We found that OVA induced the accumulation of AVs in the airway epithelia cells as determined by EM. In vitro culture, starvation increased IL-13induced MUC5AC mRNA expression. In bone marrow transplantation test, AHR and airway inflammation were increased in BECN-WT-OVA group, compared with WT-WT-OVA group.
Conclusion:Our data suggested that autophagy mediated OVA-induced airway epithelial injury and Beclin-1defect protected the asthma features, including airway hyperresponsiveness, airway inflammation and airway mucus expression. On the other side, autophagy suppressed the eosinophil differentiation during asthma development. Targeting mTOR and autophagy may lead to effective therapeutics for asthma.
细胞自噬(autophagy)是机体一种重要的防御和保护机制。在某些特定的微环境条件(如饥饿,生长素缺乏)下,细胞内形成一种双层膜结构的囊泡,细胞自噬体(autophagosomes),并同时捕获细胞内的受损的细胞器(如线粒体,内质网)和变性的蛋白质等；然后该囊泡与溶酶体溶合,形成自噬溶酶体(autolysosomes)。在溶酶体水解酶等的作用下,自噬体所包含的各种细胞器和生物大分子得以消化和降解,从而为重新合成新的生物大分子提供原料和能量。因此从本义上讲,细胞自噬是机体保持内稳态(homeostasis)和适应微环境改变的一种重要的自我调节和保护机制。然而,在某些特定的条件下,细胞器和各种生物大分子的过量消耗最终会导致细胞的另一种程序性死亡,细胞自噬死亡(autophagic cell death)。由于细胞自噬能保护或者促进细胞死亡,因此在不同的疾病病变过程中,其功能截然不同。越来越多的研究表明,自噬在机体的免疫、感染、炎症、肿瘤、心血管病、神经退行性病的发病中具有十分重要的作用。然而,细胞自噬在呼吸系统的研究并不多。新近有研究开创性地阐明了细胞自噬在吸烟诱导的气道上皮细胞损伤以及慢性阻塞性肺疾病(COPD)形成过程中的重要调控作用。然而,细胞自噬在支气管哮喘分子发病机制中的作用鲜有研究报导。
雷帕霉素(rapamycin)是经典的细胞自噬诱导剂,是1975年从加拿大Easter岛上的吸水链霉菌中提取的一种大环内酯类抗生素。雷帕霉素的主要作用靶点,哺乳动物雷帕霉素靶蛋白(mammalian target of rapamycin, mTOR)能接受并整合生长因子、能量、氧气和氨基酸四大主要信号,参与调节能量代谢、蛋白质、脂质、细胞器的合成以及细胞自噬等生理过程,在细胞的生长、增殖、分化和凋亡中起着重要的调控作用,从而维持机体与细胞的稳态平衡。研究发现雷帕霉素在哮喘动物模型中的作用并不一致,有报道发现雷帕霉素能抑制过敏性气道炎症,气道高反应性,杯状细胞化生和IgE产生,但也有部分研究认为雷帕霉素对过敏性气道炎症和气道高反应性没有影响。然而,最关键的是这些研究均只简单利用雷帕霉素体内干预观察哮喘表型,并未阐明雷帕霉素及mTOR在哮喘发病机制中起到关键调控作用及其具体机制。
越来越多的研究发现细胞自噬和mTOR在造血干细胞分化和增殖中起到重要作用。自噬缺陷可抑制红系造血导致严重贫血、抑制T淋巴细胞、B淋巴细胞数目和功能,而mTOR通过调控T细胞分化、功能、代谢参与调控适应性免疫应答。由此,我们假设细胞自噬和mTOR也参与骨髓粒系祖细胞尤其是嗜酸性粒细胞分化的调控,并进一步探讨其在以嗜酸性粒细胞气道炎症为主要表现的整体哮喘模型中的可能作用与贡献,从而为哮喘分子发病机制的研究开拓新的视野,为哮喘临床的防治提供新的靶点。
本实验分两部分进行研究：(1)探求雷帕霉素及mTOR在哮喘气道炎症和骨髓嗜酸性粒细胞分化中的作用；(2)利用细胞自噬相关基因敲除小鼠Beclin1+/-和骨髓移植技术,分别阐明细胞自噬在气道上皮细胞损伤和骨髓嗜酸性粒细胞分化中的不同调控作用。
第一部分雷帕霉素在哮喘气道炎症和嗜酸粒细胞分化中的作用研究
目的：研究雷帕霉素干预对哮喘气道炎症的影响,并探讨雷帕霉素在OVA诱导肺组织T细胞免疫应答及骨髓嗜酸性粒细胞分化过程中的调控作用。
方法：健康雌性C57BL/6小鼠,随机分为四组：生理盐水对照组(SHAM组)、模型组(OVA组)、雷帕霉素对照组(SHAM+RAPA组)、雷帕霉素哮喘组(OVA+RAPA组)。以卵白蛋白(OVA)致敏和激发建立哮喘模型,生理盐水对照组以同等剂量的生理盐水代替OVA,雷帕霉素组在每次抗原激发前1小时进行腹腔注射(1mg/kg)。于最后一次抗原激发后24小时检测肺泡灌洗液(BALF)、外周血和骨髓中的嗜酸性粒细胞数；肺组织病理切片观察气道炎症细胞浸润情况,ELISA检测血清中IL-5,IL-13水平。流式细胞术检测和分析肺组织Th2, Th17, Treg各T细胞亚群的比例,同时检测骨髓中嗜酸性粒细胞祖细胞(EoP, Lin-Sca-l-CD34+IL-5Ra+c-Kitl0)的比例。Western blot检测骨髓嗜酸性粒细胞分化过程中mTOR水平变化,体外克隆形成试验及骨髓Eos诱导分化培养检测雷帕霉素干预对骨髓嗜酸性粒细胞分化及功能的影响。最后利用IL-5转基因小鼠NJ.1638,连续3天腹腔注射雷帕霉素,检测外周血和骨髓中嗜酸性粒细胞数,ELISA检测血清中IL-5水平,流式细胞术检测骨髓EoP的比例以及嗜酸性粒细胞凋亡情况。
结果：雷帕霉素干预后显著缓解OVA诱导的气道过敏性炎症,但是不影响肺组织中Th2细胞,Th17细胞和Treg水平。雷帕霉素抑制了气道局部以及外周血,骨髓嗜酸性粒细胞数目,但是并不改变血清IL-5水平。体外克隆形成试验和骨髓Eos诱导分化培养发现,雷帕霉素可以直接抑制IL-5介导的嗜酸性粒细胞分化及培养上清Eos分泌IL-6和IL-13的水平。离体骨髓克隆形成试验及骨髓流式细胞术检测结果提示,雷帕霉素对嗜酸性粒细胞分化的抑制最终导致骨髓嗜酸性粒细胞祖细胞的聚集。对IL-5转基因小鼠NJ.1638的研究同样证实了雷帕霉素对外周血和骨髓嗜酸性粒细胞数的抑制作用及骨髓EoP的聚集,并不依赖其血清高表达IL-5水平的改变,也不影响嗜酸性粒细胞的凋亡。
结论：雷帕霉素可以缓解哮喘小鼠气道过敏性炎症,可能是通过抑制骨髓嗜酸性粒细胞分化及功能,而对Eos凋亡和肺组织T细胞免疫无明显影响。
第二部分细胞自噬在哮喘气道炎症和嗜酸粒细胞分化中的作用研究
目的：研究细胞自噬敲除对哮喘整体模型的影响,并探讨细胞自噬在OVA诱导的气道上皮细胞损伤和骨髓嗜酸性粒细胞分化过程中的不同调控作用。
方法：通过Western blot检测哮喘小鼠骨髓及体外嗜酸性粒细胞诱导分化过程中自噬水平变化,并利用细胞自噬相关基因缺陷小鼠Beclin1+/-,通过瑞士-吉姆萨染色分析其外周血和骨髓中嗜酸性粒细胞的水平,流式细胞术检测其骨髓嗜酸性粒细胞祖细胞水平,并通过甲基纤维素克隆形成试验检测其骨髓嗜酸性粒细胞分化能力。同时利用Beclin1+/-小鼠及其同窝生WT小鼠以OVA致敏和激发建立哮喘模型,随机分组如下：野生型生理盐水对照组(WT/NS),野生型哮喘模型组(WT/OVA),Beclin1+/-小鼠生理盐水对照组(BECN/NS)和Beclin1+/-小鼠哮喘模型组(BECN/OVA)。末次激发后24小时,采用有创方法测定小鼠气道反应性,并检测气道灌洗液中的白细胞总数和嗜酸性粒细胞数；肺组织病理切片观察气道炎症细胞浸润和粘液分泌情况,电镜观察哮喘小鼠气道上皮细胞自噬泡聚集情况。体外培养人正常肺上皮细胞(BEAS-2B),Western blot检测OVA体外干预过程中自噬水平变化,Q-PCR检测饥饿诱导自噬及IL-13体外干预对粘蛋白MUC5AC mRNA的表达情况。最后采用骨髓移植的方法,白硝胺+环磷酰胺联合化疗毁损野生型小鼠骨髓造血系统,然后将Beclin1+/-小鼠或同窝生野生型小鼠骨髓移植至野生型小鼠并重建其造血系统,OVA诱导建立哮喘模型,根据回输骨髓不同分两组：WT-WT-OVA组和BECN-WT-OVA组,观察骨髓局部自噬敲除对哮喘整体模型气道高反应和气道炎症的贡献。
结果：细胞自噬相关基因敲除小鼠Beclin1+/-,与同窝生野生型(WT)小鼠相比,外周血和骨髓中嗜酸性粒细胞数目显著增多,骨髓EoP水平虽有下降趋势,但无统计学意义。体外Eos诱导分化过程中LC3B蛋白水平的改变提示细胞自噬参与骨髓嗜酸性粒细胞分化。体外克隆形成试验直接证实,Beclin1+/-小鼠骨髓生成嗜酸性粒细胞克隆形成单位能力增加。但Beclin1+/-小鼠抗原激发后气道反应性、气道炎症、粘液高分泌,与野生型小鼠模型组相比并未恶化,反而出现明显减轻。同时发现,虽然哮喘小鼠骨髓中LC3B蛋白水平下降,但电镜可见哮喘气道上皮细胞中存在细胞自噬泡聚集。并且体外实验表明,OVA干预可上调LC3B蛋白水平,饥饿诱导自噬可进一步增强IL-13介导的BEAS-2B细胞MUC5AC mRNA的表达。在骨髓移植实验中,Beclin1+/"小鼠骨髓回输的哮喘小鼠(BECN-WT-OVA组),气道反应性和气道炎症都明显高于野生型小鼠骨髓回输的哮喘小鼠(WT-WT-OVA组)。
结论：细胞自噬敲除虽然在动物模型中整体表现为可以缓解哮嗤小鼠气道过敏性炎症,气道高反应性和粘液高分泌,但细胞自噬在气道局部损伤和骨髓Eos分化过程中可能存在两种完全相反的调控机制。自噬敲除抑制了OVA诱导的气道上皮细胞损伤从而缓解了哮喘表型,而在骨髓嗜酸性粒细胞分化过程中则促进了其分化能力从而恶化了哮喘气道炎症。我们的研究提示对细胞自噬和mTOR信号通路的研究有望为哮喘的防治提供新的靶点。
Asthma is a chronic airway inflammatory disease, which is characterized by reversible airway obstruction, bronchial hyperresponsiveness and airway inflammation. Among these, allergic airway inflammation in asthma is characterized by eosinophil infiltration. Studies have established that a causative relationship exists between eosinophils and the development of allergic asthma, and eosinophils participate in a variety of functions, including antigen presentation, cytokine production, chemokine production, secretion of granule mediators, and leukotriene secretion. Eosinophil differentiate from a common myeloid progenitor (CMP) in mice through an intermediate granulocyte/macrophage progenitor (GMP) and then via an eosinophil lineage-committed progenitor (EoP). The development of eosinophils is orchestrated by several transcription factors such as GATA-1, in the presence of certain cytokines, in particular Interleukin-3(IL-3), IL-5, and Granulocyte-macrophage colony stimulating factor (GM-CSF). Among these, IL-5seems to be more specific and efficient in eosinophil lineage development as it promotes the selective differentiation of eosinophils. However, little is known about the molecular mechanisms of eosinophil differentiation during asthma development.
Autophagy is a regulated pathway for internal organelle or protein degradation. In this dynamic process, double-membraned autophagic vacuoles (AVs) or autophagosomes surround cytosolic organelles (e.g. endoplasmic reticulum, mitochondria) or protein, and subsequently fuse with lysosomes, where the engulfed components are degraded by lysosomal hydrolases. Autophagy provides essential functions in the maintenance of cellular homeostasis and adaptation to adverse environments. However, excessive autophagy may be associated with the activation of programmed cell death (i.e. apoptosis) through a cell-autodigestive process. The role of autophagy, whether protective or deleterious, in human diseases, or specifically in chronic lung disease remains obscure. It has been recently reported that autophagy plays an important role in smoking-induced epithelial cell injury and emphysema. However, the potential functions of autophagy in asthma, to our knowledge, have not been investigated so far.
Rapamycin is a macrolide product of Streptomyces hygroscopius that was initially in a soil sample from Easter Island (Rapa Nui) in the early1970s. Its major cellular target, mammalian target of rapamycin (mTOR), is a central regulator of cell growth/differentiation and survival in many cell types. Previous works using rapamycin in animal models of allergic asthma showed controversial results. In some studies rapamycin have been suggested to inhibit cardinal features of allergic asthma, including airway hyperresponsiveness (AHR), eosinophilic airway inflammation, goblet cell hyperplasia, and IgE production; however, there are other works have also shown that it has little effects on the allergic airway inflammation and AHR. Most importantly, the underlying mechanisms that mediated the effects of rapamycin in the process of allergic asthma remain largely unknown.
Recently, lots evidences showing that autophagy and mTOR play an important role in hematopoietic stem cell maintenance and differentiation. It has been shown that conditional deletion of ATG7or ATG5throughout the hematopoietic system in mice results in severe anemia and inhibition of lymphocyte survival and function. Also, mTOR provides a critical link between T cell differentiation, function and metabolism. We hypothesized that autophagy and mTOR would involve in the regulation of myeloid proliferation, especially in the eosinophil differentiation, and examined their contribution to its overall effect in allergic airway inflammation. Moreover, it will bring the new insight into the prevention of asthma and other allergic disorders for the public health.
In this study, we have explored that:(1) Role of rapamycin in allergic airway inflammation and in eosinophil differentiation;(2) the different roles of autophagy in regulation of airway epithelial cell injury and eosinophil differentiation in bone marrow during asthma development.
Part I Mechanisms of rapamycin in airway inflammation and eosinophil differentiation in asthma
Objective:To investigate the effect of rapamycin in eosinophil differentiation and asthma airway inflammation, and to explore the underlying molecular mechanisms.
Methods:Female C57BL/6mice were randomly divided into four groups:SHAM group, OVA group, SHAM+RAPA group and OVA+RAPA group. In the OVA and OVA+RAPA group, mice sensitized and challenged with ovabumin (OVA).In SHAM and SHAM+RAPA group, mice were sensitized and challenged with normal saline by the same methods. In the RAPA and OVA+RAPA group, mice were treated with an intraperitoneal injection of rapamycin1hour before each aerosol OVA challenge.24hours after the final OVA challenge, mice were sacrificed and the number of eosinophils in bronchoalveolar fluid (BALF), blood and bone marrow were accessed. Lungs were processed for histologic staining (HE), and airway inflammation was analyzed. Levels of selected cytokines (IL-5, IL-13) in serum were assayed using ELISA kits. Lung lymphocytes were isolated and labeled with T cells surface Abs (CD4, CD8and CD25) and intracellular Abs (IL-4, IL-17A, Foxp3). Bone marrow Eosinophil progenitors (Eops) were determined as Lin-Sca-1-CD34+IL-5Ra+c-Kitlo cells. Data were collected and analyzed using a FACScan for seven-color flow cytometry. Western blot of phosphorylated S6(P-S6) ribosomal protein was analyzed to explore whether mTOR is involved in eosinophil differentiation. In vitro methylcellulose colony forming assays and liquid culture of mouse bone marrow-derived eosinophils (BM-Eos) were also performed to assess the ability of eosinophil differentiation and function. Finally, we used IL-5transgenic NJ.1638mice with treatment of rapamycin, to investigate the levels of eosinophils and apoptosis of Eos in blood and bone marrow, the levels of IL-5in serum, and Eops in BM were also analyzed.
Results:Rapamycin significantly attenuated OVA-induced allergic airway inflammation, without influence on the various subtypes of T helper cells. It markedly decreased the amount of eosinophils in local airways, peripheral blood, and bone marrow, independent on levels of IL-5. In vitro colony forming unit assay and liquid culture demonstrated that rapamycin directly inhibited IL-5-induced eosinophil differentiation. In addition, rapamycin reduced the production of IL-6and IL-13by eosinophils. Rapamycin was also capable of significantly reducing the eosinophil levels in IL-5transgenic NJ.1638mice, again regardless of the constitutive high levels of IL-5, and without influence on the eosinophil apoptosis. Interestingly, rapamycin inhibition of eosinophil differentiation in turn resulted in an accumulation of eosinophil progenitors in bone marrow.
Conclusion:Altogether these results clearly demonstrate a direct inhibitory role of rapamycin in eosinophil differentiation and function, and reemphasize the importance of rapamycin and possibly, mTOR in allergic airway disease.
Part II Mechanisms of autophagy in airway inflammation and eosinophil dfifferentiation in asthma
Objective:To investigate the effect of autophagy in eosinophil differentiation and asthma airway inflammation, and to explore the underlying molecular mechanisms.
Methods:Western blot of LC3B was analyzed to explore whether autophagy is involved in eosinophil differentiation. Eosibophil, Eosinophil progenitor level and the ability of eosinophil differentiation were assessed in Beclin-l+/-mice (autophagy related gene Beclin-1impaired). Beclin-1+/-and wild type (WT) mice were randomly divided into four group:WT/NS group, WT/OVA group, BECN/NS group and BECN/OVA group.24hours after the final OVA challenge airway hyperresponsiveness to inhaled methacholine, airway inflammation and airway mucus expression were measured. Lung tissue sections were also processed for transmission electron microscope (EM) and analyzed for formation of autophagic vacuoles (AVs). MUC5AC was induced by IL-13in BEAS-2B cells with or without starvation, and the mRNA expression was detected by Q-PCR. Finally, we transplanted the bone marrow of Beclin-1+/-mice to WT, and subjected these mice for the OVA asthma model to explore the contribution of local autophagy defect of bone marrow to its overall effect in asthma.
Results:The number of eosinophil in blood and bone marrow in vivo, and Eo-CFU in vitro colony forming assays were significantly increased in Beclin-1+/-mice. However, compared with WT/OVA group, BECN/OVA group showed significantly decreased AHR, airway inflammation and mucus overproduction. We found that OVA induced the accumulation of AVs in the airway epithelia cells as determined by EM. In vitro culture, starvation increased IL-13induced MUC5AC mRNA expression. In bone marrow transplantation test, AHR and airway inflammation were increased in BECN-WT-OVA group, compared with WT-WT-OVA group.
Conclusion:Our data suggested that autophagy mediated OVA-induced airway epithelial injury and Beclin-1defect protected the asthma features, including airway hyperresponsiveness, airway inflammation and airway mucus expression. On the other side, autophagy suppressed the eosinophil differentiation during asthma development. Targeting mTOR and autophagy may lead to effective therapeutics for asthma.
引文
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7. Beth Levine, Guido Kroemer. Autophagy in the Pathogenesis of Disease. Cell 2008;132:27-42.
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