摘要
支气管哮喘(Asthma)是一种以肥大细胞、嗜酸性粒细胞和T淋巴细胞气道浸润,以黏液高分泌、气道高反应性、气道重构为特征的慢性变态反应性疾病。既往“卫生学说”很好地解释了近来哮喘的高发病率。减毒活菌卡介苗(Mycobacterum bovis bacillus calmette-guerin, BCG)是一种很强的Th1免疫刺激剂,动物和人体研究显示,BCG接种和／或感染可明显抑制哮喘的病理生理改变。我们前期研究发现,新生鼠BCG接种可以明显抑制成年后OVA致敏激发诱导的过敏性气道炎症和气道高反应性(airway hyperresponsiveness, AHR),这一结果令人鼓舞,为哮喘的预防提供了曙光。
一直来困扰的问题在于：BCG防治哮喘的具体机制是什么?过去的研究均以“卫生学说”来解释,认为BCG接种和／或感染刺激了机体Th1细胞因子的产生,降低了Th2细胞因子的释放,从而达到抑制哮喘的作用.随着新型Th细胞(Th17)以及调节性T细胞(Treg)的发现以及功能的不断研究,这些细胞是否会在BCG防治哮喘中起作用呢?IL-17能够促进Th1免疫,介导Th1细胞分泌IFN-γ,但在特定情形下也能抑制Th1免疫反应,考虑到Th17与Th1细胞之间的相互调节作用,Th17可能在BCG防治哮喘中起到一定的作用.虽然早期的研究认为微卡(Mycobacterium vaccae)能够通过诱导调节性T细胞抑制哮喘炎症,但是其调节性T细胞并不是目前比较公认的Foxp3+ T调节细胞；最近研究发现BCG以及延迟冷冻抽干BCG接种能够通过上调CD4+CD25+T细胞达到保护哮喘的作用,但是这些研究并没有通过流式检测CD4+CD25+Foxp3+T的百分比以及绝对数量,所以目前没有关于BCG通过Treg细胞达到抑制哮喘作用的可信服研究.基于此,Th17和Treg细胞是否参与BCG接种防治哮喘还有待于进一步证实.
关于BCG防治哮喘比较认可的观点仍然为：BCG促进了机体Th1免疫反应,从而达到了抑制哮喘的病理生理作用。既往的研究几乎局限于脾脏以及BALF的Th1免疫反应,近来有研究表明BCG或者BCG衍生物接种后,局部肺组织内增高的Th1细胞以及Th1免疫反应在哮喘的防治中起到重要的作用。BCG接种促进了机体肺组织内表达IFN-γ的CD4+和／或CD8+T细胞,这可能来自于CD4+和／或CD8+T细胞向肺组织的募集,或者肺内静息的记忆性CD4和／或CD8+T细胞在结核杆菌／OVA攻击后引起了二次免疫。目前尚没有“关于BCG接种后引起机体免疫系统(免疫功能)的变化以及这种变化在抑制哮喘的病理生理中的作用”的系统研究。一个非常重要但没有阐明的问题在于：BCG接种尾根部,其引流淋巴结为腹股沟淋巴结,而哮喘气道炎症发生的部位为肺脏,BCG接种诱发的Th1免疫应答如何在以肺脏Th2为主导的过敏性气道炎症中起到保护作用呢?也就是说BCG皮下接种后,OVA致敏攻击导致肺组织(或BALF)中增高的表达IFN-γ的CD4+T细胞的来源在哪里?BCG皮下接种后,活的细菌主要集中在引流淋巴结,少量迁移到脾脏,而肺组织基本检测不到BCG活菌的菌落数,由此肺组织内不可能产生效应性T细胞。而记忆性T细胞产生的一个必要条件是机体抗原的清除,而BCG接种后抗原在很长时间内不会清除,由此机体产生记忆性T细胞也是很困难的。由于效应性T细胞在机体静息状态下可以由淋巴组织向非淋巴组织迁移,一旦机体发生炎症反应,效应性细胞能够趋化募集到相应的炎症组织,发挥其生物学作用。为此,我们假设“BCG接种后,哮喘小鼠肺脏增高的Th1细胞和Th1免疫应答可能来自BCG接种部位的引流淋巴结”。
由此,本研究主要分两部分：(1)首先讨论在静息状态以及OVA攻击后,BCG接种对肺组织、脾脏以及引流淋巴结内辅助T细胞(Thl, Th17)以及Treg的改变,我们发现只有表达IFN-γ的CD4+T细胞在OVA攻击后向肺脏发生明显迁移,而且这种转移来自于BCG接种部位的引流淋巴结而不是脾脏；(2)接下来我们系统分析了引流淋巴结内CD4+T细胞的表型以及功能变化,通过过继试验和体内示踪证实引流淋巴结内的CD4+T细胞参与了肺组织的迁移以及抑制哮喘气道炎症的作用。本研究为BCG接种防治哮喘提供更有说服力的试验依据,同时为哮喘的免疫治疗提供了新的方向。
第一部分：BCG早期皮下接种抑制哮喘小鼠气道炎症以及气道高反应性,该作用与表达IFN-γ的CD4+T细胞向肺组织募集相关
目的：①探讨早期BCG接种对哮喘小鼠气道炎症和气道高反应性的保护作用；②探讨BCG接种后静息以及OVA攻击后机体的辅助T细胞以及Treg的变化。
方法：①新生鼠接种BCG,待到6-8W后,分为三组：正常对照组(saline/saline),哮喘组(saline/OVA)和BCG处理组(BCG/OVA).以OVA致敏和激发建立哮喘模型。最后一次抗原激发后24小时检测气道反应性,行支气管肺泡灌洗液(BALF)收集、肺组织病理切片观察炎症浸润以及黏液分泌,检测肺组织内IFN-γ、Foxp3以及IL-17A的mRNA表达,ELISA行BALF中IFN-γ测定；②为了系统观察BCG接种后静息以及OVA攻击后肺脏的辅助T细胞以及Treg变化,我们另行单独试验,小鼠分为四组：正常对照组(saline/saline), BCG接种静息状态组(BCG/saline),模型组(saline/OVA)以及BCG接种激发组(BCG/OVA)。于最后一次OVA激发48小时后,流式检测肺脏内表达IFN-γ以及IL-17的CD4+和CD8+T细胞,以及Foxp3+Treg细胞。同时流式检测肺门纵隔淋巴结(MLN),脾脏(spleen)以及腹股沟淋巴结(ILN)内表达IFN-γ的CD4+和CD8+T细胞。
结果：①saline/OVA组小鼠气道反应性,BALF中Eos总数,肺组织炎症浸润以及黏液分泌均较saline/saline明显增加(P<0.05).与saline/OVA组相比,早期接种BCG显著抑制了哮喘小鼠BALF中的Eos数,减轻了AHR,抑制了肺组织的气道炎症以及黏液分泌(P值均<0.05)。同时,BCG接种增加了肺组织中IFN-γ的mRNA表达,而不影响Foxp3和IL-17A的mRNA表达.②在静息状态下,BCG接种轻度增加了肺组织中表达IFN-γ的CD4+T细胞,而不改变表达IFN-γ的CD8+T细胞,表达IL-17的CD4+T细胞(Th17细胞)以及CD8+T细胞的比例,同样,BCG接种不增加肺组织内Foxp3+Treg细胞的比例；有趣而且令人惊奇的是,相对于静息状态而言,BCG接种小鼠在OVA致敏攻击后显著增加了肺组织内表达IFN-γ的CD4+T细胞(p<0.001).虽然也显著增加了表达IFN-γ的CD8+T细胞(p=0.026),但是,表达IFN-γ的CD8+T细胞仅仅为表达IFN-γ的CD4+T细胞的1／6。这提示OVA致敏攻击,导致大量表达IFN-γ的CD4+T细胞向BCG接种小鼠肺组织的募集。相反,Th17,表达IL-17A的CD8+T细胞以及Foxp3+ Treg细胞没有出现与Thl细胞相似的变化。③静息状态下,BCG接种没有改变脾脏、纵隔淋巴结内Thl细胞和表达IFN-γ的CD8+T细胞。出乎意料的是,OVA致敏攻击后,BCG接种显著增加了脾脏内表Th1细胞(p=0.001)),但不改变表达IFN-γ的CD8+T细胞；与之对应的是,BCG接种后静息状态下ILN内增高的Thl细胞在OVA致敏攻击后明显减少(p=0.035),而表达IFN-γ的CD8+T细胞没有观察到相似的变化。而在纵隔淋巴结内,单独OVA致敏激发导致Thl细胞和表达IFN-γ的CD8+T细胞显著性降低；与BCG/saline相比,Thl细胞和表达IFN-γ的CD8+T在BCG/OVA组并不增加,反而这些细胞出现降低(虽然统计学上没有显著性的差异)。
结论：①BCG皮下尾根部接种能够抑制哮喘小鼠的气道炎症以及气道高反应性,这种抑制作用与迅速导致Th1细胞向炎症肺组织的募集相关,而与Th17,表达IL-17A的CD8+T细胞以及Foxp3+ Treg无关；③OVA致敏攻击后,BCG接种小鼠肺内增高的Th1细胞来自于ILN,通过脾脏向肺内迁移,从而达到抑制哮喘气道炎症的作用。
第二部分：BCG接种部位的局部引流淋巴结内CD4+T细胞的表型及体外功能研究
目的：进一步探讨BCG接种后局部引流淋巴结(ILN)内CD4+T细胞的表型以及功能。
方法：BCG接种方法同前。小鼠分为两组：正常对照组(saline)和BCG接种组(BCG)。动态观察BCG接种后ILN和脾脏的细胞数改变；由于第一部分我们已经发现BCG接种12W后ILN内Th1细胞增高,为了进一步证实这一现象,继续观察BCG接种18W以及30W后ILN和脾脏中表达IFN-γ的CD4+T和CD8+T细胞的变化；以12W为时间点,流式检测ILN和脾脏内表达IL-17A的CD4+T和CD8+T细胞以及Foxp3+Treg细胞比例。以12W为时间点,定量PCR测定ILN内IFN-γ、IL-17A以及Foxp3的mRNA水平,同时分析ILN内淋巴细胞的细胞周期、CD4+T细胞的活化程度以及表型；以MTT法以及CFSE稀释法检测ILN内淋巴细胞和CD4+T细胞的体外增殖能力,同时ELISA检测培养上清内IFN-γ的水平。
结果：①BCG接种促进了ILN细胞数呈指数级增长,但不改变脾脏的细胞数。②无论18W还是30W, BCG接种均促进了ILN内表达IFN-γ的CD4+T细胞比例的增加(p=0.027, p=0.04, respectively).而对于脾脏细胞,BCG接种均不改变表达IFN-γ的CD4+T细胞和CD8+T细胞的百分比。同样,BCG接种不改变ILN和脾脏内Th17细胞和表达IL-17的CD8+T细胞,以及Foxp3+CD4+T细胞。与之相一致的是：BCG接种促进了ILN内IFN-γ和转录因子T-bet的mRNA表达,而不改变IL-17A和Foxp3转录因子的nRNA水平；③BCG接种轻度增加了ILN内效应和／或效应记忆CD4+T细胞(Tef/em) (p=0.042),而对于处女型T细胞(Tnai)和中央记忆型T细胞CD4+T细胞(Tcm)没有明显改变；BCG接种不改变ILN细胞的增殖指数,不改变ILN内CD4+T细胞的活化程度。但是体外刺激后,BCG接种后的ILN细胞表现为活化程度显的著增高,体外增殖能力的增强以及IFN-γ分泌能力的上升.
结论：①BCG接种促进了ILN而不是脾脏的增生并且导致ILN内储存大量表达IFN-γ的CD4+T细胞,而不改变表达IFN-γ的CD8+T细胞,Th17细胞,表达IL-17的CD8+T细胞,以及Foxp3+CD4+T细胞比例.②BCG接种导致的ILN内的CD4+T细胞处在一种相对静息的状态,但是一旦给予刺激,表现出增强的活化增殖以及分泌效应因子能力。
第三部分：BCG接种诱导的局部引流淋巴结内淋巴细胞参与OVA激发后向肺组织的募集以及发挥对哮喘小鼠气道炎症的保护作用
目的：进一步探讨BCG接种以后富集表达IFN-γ的CD4+T细胞的ILN细胞是否参与了OVA致敏激发后向肺脏组织的募集以及这些淋巴细胞是否参与了抑制哮喘气道炎症的作用。
方法：①本部分试验分两步走,早期接种BCG,这些小鼠待作过继免疫时的“供者小鼠”(donor mice)。同时另选一批小鼠用作哮喘模型,作为细胞过继的“受者小鼠”(recipient mice)。小鼠8W开始建立哮喘模型。“受者小鼠”在OVA激发前一天,接受来自于“供者小鼠”的ILN单个核细胞悬液,后诱发哮喘模型。小鼠分为3组：BCG接种ILN细胞过继OVA攻击组(BCG-ILN/OVA),生理盐水对照ILN细胞过继OVA攻击组(saline-ILN/OVA)和过继PBS对照OVA攻击组(PBS/OVA)。于最后一次抗原激发后24小时检测气道反应性,48小时行支气管肺泡灌洗、肺组织病理切片观察炎症浸润以及黏液分泌情况以及检测Thl细胞因子的表达。在另一单独试验中,过继脾脏细胞,分组基本同ILN细胞过继,分为：BCG-spleen/OVA, saline-spleen/OVA以及PBS/OVA,后检测气道反应性、支气管肺泡灌洗液、肺组织病理切片观察炎症浸润以及黏液分泌情况。②另外,为直接证实BCG接种后的ILN内的淋巴细胞以及CD4+T细胞是否具有向炎症肺组织迁移的优先能力,我们设立三组：CFSE标记的BCG接种ILN细胞过继OVA攻击组(BCG-ILN/OVA), CFSE标记的saline对照ILN细胞过继OVA攻击组(saline-ILN/OVA)以及CFSE标记的BCG接种ILN细胞过继saline攻击对照组(BCG-ILN/OVA).细胞体外CFSE标记,行尾静脉过继,诱发哮喘模型。流式检测CFSE阳性的淋巴细胞以及CD4+T细胞数。③分别取8W时BCG接种以及正常对照组的ILN组织,定量PCR检测趋化因子受体CXCR3,CCR5,CXCR6, CCR6,CCR4和CCR8的mRNA水平；另外,分别检测OVA致敏攻击组和生理盐水对照组的肺组织,定量PCR检测CXCR3相匹配的趋化因子CXCL9,CXCL10和CXCL11的mRNA水平。
结果：①过继BCG接种后的ILN细胞,表现为气道反应性的降低,BALF中Eos数的下调,以及肺组织炎症和黏液分泌的减轻,值得注意的是,这些小鼠同时伴随着肺组织增高的IFN-γmRNA水平以及BALF中IFN-γ的表达的增加；②无论过继BCG接种或者对照组的脾脏细胞悬液,均不能降低哮喘小鼠的病理生理改变；③与saline-ILN/OVA相比,CFSE阳性的淋巴细胞(p=0.022)以及CD4+T细胞(p=0.045)在BCG-ILN/OVA组肺组织中显著增高。④BCG接种后ILN组织选择性的高表达Thl细胞表面趋化因子受体CXCR3,而不增高其他T细胞相关的趋化因子受体。与之相对应的是,OVA致敏攻击后的肺组织高表达与CXCR3匹配的趋化因子CXCL9、CXCL10和CXCL11。
结论：BCG接种引起的增生引流淋巴结(ILN)具有直接保护哮喘气道炎症和气道高反应性的作用,而脾脏细胞不具有这样的保护作用,这种保护作用与ILNCD4+T细胞优先向肺部炎症组织迁移相关。
Allergic asthma has been defined as a disease of immunodysregulation, which associated with pulmonary eosinophilic inflammation and increased mucus production in the airways. An attractive explanation is offered by the "hygiene hypothesis", which suggests a decrease of or an altered exposure to microbes in the environment results in alteration of immune-regulation. BCG is considered as a strong inducer of T-helper type 1 immune response and modulates the development of asthma both in animal models and human beings. Importantly, our previous study has shown that neonatal BCG vaccination elicited long-term protection on allergic airway inflammation in young and aged mice, which provides a new insight for the prevention of asthma.
BCG vaccination/infection stimulates the production of Th1 cytokines and decreases Th2 cytokine secretion in both BALF and the splenetic supernatants. However, the exact mechanism underlying the BCG vaccination preventing the development of asthma remains obscure. With the discovery and functional analysis of Th17 and Treg, we want to know whether and how these new subsets of T cells involve in the process of protective effects of BCG on asthma. It has been reported that IL-17 is required to induce optimum Th1 in some condition, while inhibits the Th1 immune response at other condition. Given the mutual effects of Th17 and Thl, we proposed that Th17 may be play a role in the prevention of BCG on asthma.BCG has been reported to induce immune-regulatory responses. However, the role of Treg in the effects of BCG on allergic inflammation has been little studied. M. vaccae regulates the allergic host immune responses through the induction of Treg (IL-10 and TGF-β), rather than affecting the Th1/Th2 balance. Indeed, BCG vaccinations ameliorate de novo local eosinophilic inflammation induced by allergen and increase the numbers of CD4+CD25+ Treg cells and Foxp3 expression. Collectively, these data suggest Treg may involve in the protection against allergic asthma. Based on the aforementioned studies, whether Th17 and Treg involve in the protective effect of BCG vaccination on asthma needs to further confirm.
The most acceptable view is the enhanced Th1 immune response by BCG vaccination play a central role in the inhibition of asthma. Previous studies focused on the increased IFN-γlevel of BALF and/or cultured supernatant of spleen, yet the local enhanced IFN-γ+ CD4 T has been considered to play a critical role against allergic airway inflammation. The migration of FN-y-positive CD4+T and CD8+T cells from circulation and/or the rapid expansion of the resting memory CD4+ T and CD8+ T cells in the local lung contribute to the accumulation. It is more acceptable for the former, as effector T cell can migrate into non-lymph tissue in a low-frequency pattern in the stable condition, while they can rapidly influx into inflamed tissues. In fact, it is hard to establish a real memory immune in BCG vaccination or M.tb choronical infection as the substantial antigen. However, an unresolved issue in these studies is where the IFN-y-positive Thl cells induced by BCG vaccination stem and how these cells recruit into the flamed site of pulmonary after OVA challenge. Thus, we put forward to the hypothesis that "the increased Thl in the draining lymph node for the site of BCG vaccination contribute to enhanced Thl cells in lung after OVA challenge and yield to a protective effect on asthma".
To confirm this hypothesis, we first detect the changes of Thl, Th17 and Treg in the lung after BCG vaccination both in stable-state and in OVA-challenged condition, we found BCG vaccination significantly increased Thl cells after OVA challenge, while other T subset have no differences. Furthermore, we found the increased Thl in the lung resulted from the ILN (not from spleen), and the ILN serves as a pool of Thl cells and migrated into the lung directly to alleviate the OVA-induced airway inflammation and AHR. These data support further that neonatal BCG vaccination elicits a protection of asthma, and provide a new insight of immune vaccination.
Part 1:Neonatal BCG vaccination elicits a protection of airway inflammation and airway hyperresponsiveness via the recruitment of IFN-γ-expression CD4+T cells in the lung.
Objective:To investigate whether neonatal BCG vaccination at the base of tail elicits the protective effects from the development of asthma and to examine the changes of Thl, Th17 and Treg in the lung in both resting and OVA challenge condition.
Methods:①C57BL/6 neonates were vaccinated with BCG At age of 6-8 weeks, these mice were invided into three groups:negative control mice (saline/saline), asthma model mice (saline/OVA) and BCG-treated mice (BCG/OVA). The protocol for asthma model described as previous. After the last OVA challenge, AHR, total cell counts and cell differentiation were detected and the airway inflammation and mucus secretion were examined. Then mRNA level of IFN-γ, Foxp3 and IL-17A in the lung and IFN-γconcentration in BALF were determined by real-time PCR and ELISA respectively.②In a reparatory experiment, mice were divided into 4 groups:saline/saline for negative control, saline/OVA for asthma model control, BCG/saline and BCG/OVA. Within 24 h after the last OVA challenge, cell suspensions of lung were obtained and decteced the frequency of Th1, Th17 and Treg via FCM. Finally, cell suspensions were isolated from MLN, spleen and ILN and decteced the percentage of IFN-γ-expession CD4+ T and CD8+T via FCM.
Results:①Neonatal BCG vaccination at the base of tail decreased AHR, inhibited eosinophils infiltration and mucus overproduction (P< 0.05).②In resting state, the IFN-y-positive CD4+T cells were significantly increased in the lung compared with that in saline/saline mice, while no differences were observed about IFN-γ-positive CD8+T, Th17, IL-17-positive CD8+T and Treg in the lung. Interestingly, the frequency of Thl cells was significantly increased in BCG/OVA mice in compared to that in BCG/saline(p< 0.001). Although IFN-y-positive CD8+T was also significantly enhanced in the lung of BCG/OVA mice (p=0.026), the major of IFN-γ-positive T cells was CD4+T cells. However, these changes were not observed for Th17, IL-17-expression CD8+T and Treg.③In resting condition, BCG vaccination have no effects on the changes of IFN-y-expression CD4+T and CD8+T in the MLN and spleen. In contrast, the frequency of Thl was significantly increased in ILN compared to that in saline/saline mice (p=0.002). To our surprise, OVA challenge enhanced significantly the frequency of Th1 in the spleen from BCG/OVA mice compared to BCG/saline mice, which was accompanied with the reduced frequency of IFN-γ-expression CD4+T in the ILN in BCG/OVA mice compared to BCG/saline mice (p=0.035). Although the differences were not significant, the frequency of IFN-γ-expression CD4+T and CD8+T in MLN were indeed reduced in BCG/OVA mice compared with those in BCG/saline mice.
Conclusion:①Neonatal BCG vaccination elicits the protective effects on the airway inflammation and AHR in an experimental asthma model, which were corrected with the recruitment of large IFN-γ-expression CD4+T cells but not the Th17 and Treg cells into the lung.②The enhanced Thl cells in the lung after OVA challenge in mice vaccinated with BCG were result from ILN via spleen pathway.
Part 2:phenotype and functional analysis of CD4+T cells in ILN
Objective:To investigate whether BCG vaccination influences the phenotype and function of CD4+T cells in ILN for the site of BCG vaccination.
Methods:C57BL/6 neonates were vaccinated with BCG as described in Part 1. We dynastically observed the cell counts of spleen and ILN at the age of 4W,12W and 30W. To confirm further IFN-γ-expression CD4+ T was indeed increased in ILN of mice vaccinated with BCG, we also dectected the frequency of IFN-γ-expression CD4+ T and CD8+T cells in ILN at the age of 18W and 30W. At the age of 12W, cell counts of MLN, MLN and thymus were also counted, and mRNA level of IFN-γ, IL-17A and Foxp3 in ILN were evaluated by real-time PCR. In addition, the frequencies of Th17, IL-17A-expression CD8+ T and Treg were dectected via FCM in spleen and ILN. Subsequently, cell cycle of ILN, activation and cell phenotype of CD4+T cells in ILN were also illusiated by FCM. The prolification capacity of lymphocytes and CD4+T cells were determined by MTT and CFSE dilution analysis. Finally, the secretary capacity of cytokine was examined by ELISA.
Results:①Cell counts of ILN from BCG mice increased early at 4 W, and peaked at 12 W, then formed a plat until 30 W. In contrast, cell counts of spleen, thymus and non-draining Lymph node (LN) such as MLN and MLN were no difference between in BCG and saline mice.②Besides at the age of 12 W(as described in part 1), the enhanced Th1 cells were also observed in ILN in BCG mice compared to that in saline mice whether at age of 18W or at 30W(p=0.027 at age of 18W; p=0.04 at age of 30 W, respectively).However, the similar changes were not obseaved for IFN-γ-expression CD8+T cells.In contrast, the frequency of Thl and IFN-γ-expression CD8+T cells in spleen were similar whether BCG vaccination or not. The percentage of Th17, IL-17A-expression CD8+T and Foxp3+CD4+T in spleen and ILN were not changed between in BCG and saline mice. The mRNA level of IFN-γand the transcriptor T-bet were significanted increased in lung of BCG mice compared with that in saline mice, while the mRNA level of IL-17A and the transcriptor Foxp3 were similar regardless of BCG vaccination.③Cell prolification index of ILN, the CD69 and CD25 molecular expression on CD4+T cells in ILN in BCG mice were similar to that in saline mice in steady-state condition. In contrast, CD25 expression on CD4+T cells in ILN of BCG mice was significantly increased in the stimulator compared to that in saline mice. The percentage of Tef/em in ILN in BCG mice was significantly but slighter than that in saline mice. The proliferation of CD4+T cells in ILN of BCG mice measured by MTT and CFSE-dilution was enhanced significantly in contrast to that in saline mice, which was accompanied with the increased IFN-y concentration in cultured supenarant of lymphocyte from ILN of BCG mice.
Conclusion:①BCG vaccination promotes the prolification of ILN but not spleen, and much Thl but IFN-γ-expression CD8+T, Th17, IL-17-expression CD8+T and Treg was enriched in ILN of mice vaccinated with BCG.②CD4+ T cells in ILN of mice vaccinated with BCG was in a resting state in steady-state condition, which have a potential capacity of activation, prolification and secretion.
Part 3:Lymphocyte isolated from ILN of BCG mice elicits directly protective effects on airway inflammation and AHR via a protential of migration into lung after OVA challeng.
Objiective:To investigate directly the lymphocyte enriched with Thl cells in ILN of BCG mice elicits protective effects on airway inflammation and AHR in a mouse model of asthma.
Methods:For adoptive transfer test of ILN cell suspensions, mice were divided into three groups:BCG-ILN/OVA, saline-ILN/OVA and PBS/OVA. Single-cell suspensions were obtained from ILN, and transferred into recipient mice. After the consequent 3 days challenge, AHR were assayed and samples were collected for inflammation measurement.In addition, the IFN-γmRNA expression in the lung and IFN-γconcentration in BAL fluid were analyzed by real-time PCR and ELISA respectively. Together, we also performed the adoptive transfer test of spleen cell suspensions. In other experiment for directly detect the migration capacity of CD4+T cells of ILN, single-cell suspensions of ILN were stained with CFSE in vitro, and injected into recipient mice. The percentage of CFSE-positive cells and CD4 T cells were assayed using FACS analysis. Furthermore, we detected the mRNA expression of associated chemokine receptors of T cells in ILN and matched chemokine in Lung.
Results:①Adoptive transfer of ILN cells from BCG mice inhibited AHR, attenuated total numbers of eosinophils in BALF, and decreased pulmonary inflammation and mucus secretion. The IFN-γmRNA expression in the lung was enhanced from BCG-ILN/OVA mice compared with that from saline-ILN/OVA mice, which accompanied with the enhanced IFN-γselection in BALF. However, adoptive transferring of spleen cells from BCG mice has no benefit.In support, CFSE-positive CD4+T cells in BCG-ILN/OVA were enhanced in lung compared to those in saline-ILN/OVA (p=0.045). As expected, the mRNA level of CXCR3 in ILN of BCG mice was higher significantly than that in saline mice. Intriguingly, the matched chemokine CXCL9,10 and 11 for CXCR3 in Lung challenged with OVA were also significantly increased compared to saline mice.
Conclusion:Lymphocyte enriched with Thl cells in ILN of BCG mice elicits protective effects on airway inflammation and AHR in a mouse model of asthma. Moreover, the protective effect was associated with the potential migration of CD4+T into inflamed lung. Moreover, the high expression of CXCR3 in ILN contributes to the potential migration of CD4+T cells.
引文
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