胸腺基质淋巴细胞生成素在呼吸道合胞病毒感染诱发哮喘加重中的作用研究
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摘要
研究背景及意义:
支气管哮喘(简称哮喘)是一种严重危害人类健康的慢性呼吸道疾病,以气道慢性炎症、粘液分泌增加、气道反应性增高及气道重塑为主要特征。哮喘的发生与发展受多种因素的调控,从而决定了淋巴细胞分化,免疫反应类型,细胞因子合成及组织修复过程。尽管哮喘基础研究取得较大的进步,但目前临床上对于哮喘的急性加重以及重症哮喘的治疗仍然不是很满意。随着分子病毒学技术的发展和RT-PCR技术在临床研究中的应用,呼吸道病毒感染作为哮喘加重的最重要原因已被广泛认识。已经证实,病毒诱发的哮喘加重在学龄儿童中的比例约为80%-85%,而在成人中约为60%-75%。呼吸道合胞病毒(respiratorysyncytial virus,RSV)作为呼吸道单链RNA病毒的一种,是引起小儿呼吸道感染的最常见病原体,也是诱发婴幼儿哮喘的主要原因,在哮喘的发展和加重中被认为是一个重要的危险因子。而且,针对RSV的免疫是不完全的和暂时的,所以已经感染过RSV的成人可以再次被同株的RSV感染。
微生物感染对机体的破坏作用导致宿主防御机制的进化。在哺乳动物,有两套防御机制:固有免疫和继发性免疫。固有免疫识别是通过模式识别受体(pattern recognition receptors,PRRs)来完成的,每个受体都对微生物的保守和不变的特征识别具有广泛的特殊性。PRRs识别微生物是通过与病原体相关分子模式(pathogen-associated molecular patterns,PAMPs)的结合来完成的。在PRRs的病毒识别中,最具特征性的是Toll样受体(Toll like receptors,TLRs)。
呼吸道上皮细胞除了作为物理屏障,也能通过感受外界刺激因素启动固有免疫反应产生细胞因子,从而影响继发性免疫反应。在针对吸入性外来变应原和病原体的继发性免疫反应的起始中,局部浸润的树突状细胞(dendritic cells,DCs)起到关键性作用。气道上皮细胞影响DCs可以通过DCs激活因子胸腺基质淋巴细胞生成素(thymic stromal lymphopoietin,TSLP)的产生来调控Th细胞的局部分化。研究证实TSLP能诱导DCs调节初始CD4~+T淋巴细胞向Th2细胞分化,产生IL-4、IL-5、IL-13和TNF-α等细胞因子。在对气道上皮细胞生成TSL。P的刺激因子的研究中发现,TLR3的配体双链RNA(dsRNA)与TLR3受体结合产生大量的TSLP mRNA。这提示病毒在呼吸道上皮细胞内复制过程中形成的dsRNA中间体,可能通过TSLP的诱导,加重Th2炎症反应,从而进一步加重哮喘患者气道粘膜炎症。这个推论可以解释为什么呼吸道病毒感染加重变态反应性炎症,而从免疫学理论上讲,病毒诱导的Th1免疫反应能拮抗Th2调节的炎症反应。
本项研究主要通过研究RSV感染人支气管上皮细胞16HBE分析固有免疫反应受体TLR3的表达情况及对TSLP生成的影响,通过TLR3抗体阻断的方法研究是否可以引起TSLP的生成的改变,通过模拟气道局部Th1和Th2内环境加入IFN-γ和IL-4观察其对RSV感染诱发的TSLP生成影响,同时观察利巴韦林和地塞米松等药物干预对支气管上皮细胞生成TSLP的影响。为明确TSLP在哮喘动物体内的变化与哮喘的关系,通过RSV感染小鼠哮喘加重模型进一步分析在RSV感染情况下小鼠气道局部TSLP的表达与小鼠肺部炎症的相互关系,并观察不同药物治疗对RSV感染哮喘加重小鼠气道TSLP生成和肺部炎症的影响。
方法:
一、体外细胞学试验
1.不同浓度和不同时间人工合成dsRNA聚肌胞(PolyⅠ:C)对人支气管上皮细胞16HBE表达TSLP mRNA和TLR3 mRNA的影响
分别加入含0μg/ml、1μg/ml、5μg/ml、10μg/ml、25μg/ml、50μg/ml聚肌胞的10%DMEM培养基培养16HBE细胞,刺激3小时,实时荧光定量RT-PCR检测TSLP mRNA和TLR3 mRNA相对表达量。
10μg/ml聚肌胞加入10%DMEM培养基培养16HBE细胞,分别刺激1h、2h、3h、6h、12h、24h,实时荧光定量RT-PCR检测TSLP mRNA和TLR3 mRNA相对表达量。
2.RSV病毒感染对人支气管上皮细胞16HBE固有免疫受体TLR3表达的影响
RSV病毒感染Hep-2细胞进行病毒扩增;RSV在Hep-2细胞质内扩增的间接免疫荧光鉴定;Reed-Muench法测算RSV细胞毒力。RSV感染人支气管上皮细胞16HBE 24小时,间接免疫荧光检测细胞TLR3受体表达变化。
3.RSV病毒感染及干预因素对16HBE细胞TSLP mRNA和TLR 3mRNA表达水平和TSLP蛋白表达的影响
试验分6组:对照组,RSV组,RSV/Anti-TLR3组,RSV/IFN-γ组,RSV/IL-4组,RSV/地塞米松组;RSV感染6小时后实时荧光定量RT-PCR检测细胞TSLP mRNA和TLR3 mRNA相对表达量;RSV感染24小时后western blotting检测各组细胞TSLP蛋白表达水平。
4.利巴韦林抑制RSV病毒感染对16HBE细胞TSLP mRNA表达水平和TSLP蛋白表达的影响
试验分4组:对照组,RSV组,RSV/利巴韦林组,利巴韦林组;RSV感染6小时后实时荧光定量RT-PCR检测细胞TSLP mRNA相对表达量;RSV感染24小时后western blotting检测各组细胞TSLP蛋白表达水平。
二、动物实验
1.RSV病毒感染对哮喘加重小鼠气道TSLP分泌和肺部炎症的影响
32只BALB/c小鼠分为4组:A组(对照组),B组(OVA组),C组(RSV组),D组(OVA/RSV模型组)。
无创肺功能检测小鼠气道反应性:不同浓度乙酰甲胆碱激发,BUXCO无创肺功能检测仪检测Penh值。
小鼠血清ELISA法检测细胞因子:小鼠血清用于IL-4、IFN-γ、IL-5、IL-13细胞因子检测。
小鼠气管灌洗液细胞分类计数和ELISA检测TSLP:聚乙烯导管行小鼠气管插管,PBS灌洗,灌洗液上清ELISA法检测细胞因子TSLP;灌洗液离心沉淀细胞计数,瑞氏-吉姆萨染色细胞分类计数。
小鼠肺脏组织病理观察:苏木素—伊红(HE)染色观察小鼠肺脏病理变化;碘酸雪夫(PAS)染色观察气道上皮细胞粘液合成。
小鼠肺脏组织免疫组化观察气道上皮细胞TSLP表达,DAB显色,阳性显示棕黄色。
小鼠肺脏组织TSLP蛋白western blotting检测,Gel-Pro ANALYZER4.5软件分析TSLP的相对表达量。
2.不同药物治疗对RSV感染哮喘加重小鼠TSLP分泌和肺部炎症的作用
32只BALB/c小鼠分为4组:对照组(0VA/RSV组),聚肌胞组(OVA/RSV/PolyI:C组),利巴韦林组(OVA/RSV/Rib组),地塞米松组(OVA/RSV/Dex组)。
无创肺功能检测小鼠气道反应性;小鼠血清ELISA法检测细胞因子血清IL-4、IFN-γ、IL-5、IL-13浓度;小鼠气管灌洗液细胞分类计数和ELISA检测气管灌洗液TSLP浓度;小鼠肺脏组织病理观察;小鼠肺脏组织免疫组化观察TSLP表达;小鼠肺脏组织TSLP蛋白western blotting检测。(方法同上)。
三、统计学处理
采用SPSS13.0软件进行统计分析,计量资料以均数±标准差((?)±s)表示。各组间多重比较采用单向方差分析(one-way ANOVA),方差齐性时采用LSD法和SNK法检验,方差不齐时采用Dunnett's T3法检验。以P＜0.05为有统计学意义。
结果:
一、体外细胞学试验结果
1.随着聚肌胞浓度增加,16HBE细胞TSLP mRNA和TLR3 mRNA表达水平增高。当聚肌胞浓度为50μg/ml时,TSLP mRNA表达量最高,为对照组的29.84±5.32倍(P＜0.01);TLR3 mRNA表达量最高,为对照组的4.46±0.8倍(P=0.001)。
随着聚肌胞刺激时间延长,16HBE细胞TSLP mRNA和TLR3 mRNA表达水平先升高,后下降。当聚肌胞刺激3h,TSLP mRNA表达量达到最高(为聚肌胞刺激1h表达量4.48±0.35倍)(P＜0.001),随着时间延长TSLP mRNA表达下降;当聚肌胞刺激3h,TLR3 mRNA表达量达到最高(为聚肌胞刺激1h表达量1.66±0.12倍),随着时间延长TLR3 mRNA表达下降。
2.Hep-2细胞接种RSV后第5天细胞大片融合,并有细胞脱落;收集病毒,调整病毒毒力MOI为2;间接免疫荧光观察显示RSV在细胞内扩增;16HBE细胞接种RSV病毒24小时后间接免疫荧光观察细胞内TLR3受体表达情况显示:病毒感染细胞内TLR3受体表达明显增加,而阴性对照组细胞TLR3表达相对少。
3.RSV感染16HBE细胞可使细胞TSLP mRNA表达水平增高(为对照组表达量1.63±0.08倍)(P＜0.001),TLR3 mRNA表达水平增高(为对照组表达量1.54±0.34倍)(P=0.094)。加入重组人IFN-γ100μg/L,TSLP mRNA表达水平为对照组的0.83±0.18倍,比RSV组低(P＜0.001),TLR3 mRNA表达与RSV组比较无统计学意义(P=1.000);加入重组人IL-4 100μg/L,TSLP mRNA表达水平为对照组的2.61±0.45倍,比RSV组高(P=0.025),TLR3 mRNA表达与RSV组比较无统计学意义(P=1.000);加入Anti-TLR3阻断TLR3受体后细胞TSLP mRNA表达量(为对照组的1.23±0.2倍)相对于RSV感染组低(P=0.034),而TLR3 mRNA表达水平增高(为对照组细胞为2.06±0.5倍)。加入地塞米松,TSLP mRNA表达量比RSV组低(P＜0.001),而TLR3 mRNA表达水平相对于对照组仍高(P=0.042),与其它各组比较均无统计学意义(P＞0.05)。
RSV感染组细胞TSLP蛋白相对表达量(1.55±0.36)约为对照组的1.9倍(P＜0.001),加入IFN-γ可以降低RSV感染引起的TSLP表达升高(1.13±0.31)(与RSV组比较P=0.020),加入IL-4可以协同RSV感染引起TSLP表达升高(1.99±0.37),比RSV组表达量增加(P=0.014),加入TLR3抗体阻断TLR3受体可使TSLP表达降低(1.28±0.28),但与RSV感染组比较无统计学意义(P=0.114),加入地塞米松可以显著抑制RSV感染16HBE细胞TSLP表达(0.53±0.11)(与RSV组比较P＜0.001)。
4.RSV感染16HBE细胞可以使细胞TSLP mRNA表达水平增高(为对照组的1.99±0.25倍)(P=0.001);加入利巴韦林抑制RSV感染后TSLP mRNA表达水平下降,为对照组的1.41±0.18倍(P=0.012);利巴韦林治疗组细胞TSLP mRNA表达水平比RSV感染组低(P=0.006)。
利巴韦林可以显著抑制RSV感染引起的TSLP表达(1.16±0.17),与RSV组(1.97±0.19)比较有统计学意义(P＜0.001),但仍高于对照组(P=0.002),单独加入利巴韦林(0.99±0.15)与对照组细胞TSLP表达量比较无统计学意义(P=0.215)。
二、动物实验结果
1.随着乙酰甲胆碱浓度上升各组小鼠气道反应性检测指标Penh值均出现上升趋势,OVA/RSV组小鼠的气道反应性最高。乙酰甲胆碱激发浓度为3.125 mg/ml时,OVA/RSV组小鼠气道反应性Penh值(128.42±18.05)和OVA组(114.73±17.67)比对照组(19.48±4.44)高(P＜0.001);乙酰甲胆碱激发浓度为6.25mg/ml时,OVA/RSV组Penh值(318.66±50.87)大于OVA组(187.95±38.73)(P＜0.001),各药物治疗组小鼠气道反应性Penh值均低于OVA/RSV组(P＜0.01)。当乙酰甲胆碱激发浓度为50 mg/ml时,各治疗组小鼠气道反应性Penh值均低于OVA/RSV组(1335.46±196.61)(P＜0.05),地塞米松组(581.28±58.89)最低,与其它治疗组比较低(P＜0.001)。
2.OVA/RSV组小鼠血清IL-4浓度(274.24±103.67 pg/ml)比OVA组(94.18±26.4 pg/ml)高(P=0.001),聚肌胞组、利巴韦林组、地塞米松组均可降低血清IL-4水平(P＜0.05);地塞米松组(86.78±27.04 pg/ml)为治疗组内最低;OVA/RSV组小鼠血清IL-5(293.25±46.13 pg/ml)高于OVA组(239.57±33.84pg/ml)(P=0.003),各药物治疗组均比OVA/RSV组降低(P＜0.05),利巴韦林组IL-5水平(220.82±30.9 pg/ml)最低;OVA/RSV组小鼠血清IL-13(330±93.5pg/ml)比OVA组(187.04±62.66 pg/ml)高(P=0.02),聚肌胞组(219.91±60.88 pg/ml)、地塞米松组(194.65±73.27 pg/ml)均下降(P=0.008,P=0.001);OVA/RSV组小鼠血清IFN-γ(30.08±5.73 pg/ml)比OVA组(8.46±2.86 pg/ml)明显增高(P＜0.001),利巴韦林组(12.98±3.34 pg/ml)、地塞米松组(17.33±3.06 pg/ml)均有所下降(P＜0.01)。
3.小鼠支气管肺泡灌洗液(BALF)中:OVA/RSV组细胞总数(21.23±0.54)×10~6/ml最多(P＜0.001),各治疗组细胞总数均降低(P＜0.001);OVA/RSV组嗜酸性粒细胞数(4.87±0.37)×10~5/ml最多(P＜0.001),各治疗组均降低(P＜0.01);OVA/RSV组淋巴细胞数(5.45±0.16)×10~5/ml升高最为明显(P＜0.001),利巴韦林治疗组与之比较无统计学意义(P=0.051),余各治疗组下降;OVA/RSV组中性粒细胞(2.46±0.25)×10~5/ml升高最为明显,与OVA组比较有统计学意义(P＜0.01),聚肌胞组、地塞米松组有所下降(P=0.01,P=0.011),利巴韦林治疗与OVA/RSV组比较无统计学意义。
支气管肺泡灌洗液检测TSLP结果:OVA/RSV组小鼠支气管肺泡灌洗液TSLP(2.13±0.05 ng/ml)高于OVA组(1.99±0.05 ng/ml)(P=0.001),各药物治疗均使TSLP水平下降(P＜0.01)。
4.常规HE染色OVA/RSV组支气管上皮增生肥厚、粘液分泌增加、管腔明显狭窄,管壁及其周围大量淋巴细胞浸润,比OVA组加重,各治疗可使炎症减轻,地塞米松治疗组炎症反应最轻。PAS染色OVA/RSV组粘液分泌增加,杯状细胞增生最为明显,各治疗可一定程度减轻病变,地塞米松治疗组明显抑制上皮细胞粘液合成。免疫组化观察OVA/RSV组小鼠气道上皮细胞表达TSLP最为明显,各治疗组可减轻TSLP合成分泌。
5.Western blotting检测发现OVA/RSV组小鼠肺脏TSLP相对表达量(TSLP/GAPDH)(0.93±0.18)为最高,与对照组、OVA组、RSV组比较有统计学意义(P＜0.001,P=0.045,P=0.001);利巴韦林组小鼠肺脏TSLP表达量低于OVA/RSV组(P=0.031),地塞米松组为各治疗组中TSLP表达量最低,(P＜0.001)。
结论:
1.dsRNA可以激活人支气管上皮细胞16HBE固有免疫受体TLR3,促进TSLPmRNA表达量增加。
2.RSV感染后病毒复制促进固有免疫受体TLR3在人支气管上皮细胞16HBE内的表达。
3.RSV感染促进16HBE细胞表达TSLP;IFN-γ可起到抑制作用,IL-4则起到协同作用促进TSLP表达,表明不同Th1/Th2微环境对TSLP的表达有不同影响;TLR3受体阻断可以适度抑制TSLP表达,间接表明固有免疫受体TLR3在RSV感染的TSLP表达中起到一定作用;地塞米松可以显著降低RSV感染引起的TSLP表达,说明糖皮质激素在RSV感染引起的TSLP表达中起到抑制作用。
4.利巴韦林抗病毒治疗可以有效抑制16HBE细胞表达TSLP,说明抑制RSV在细胞内复制可以起到减少细胞表达TSLP的作用。
5.RSV感染可以促进OVA过敏性哮喘小鼠气道上皮细胞生成TSLP增加,并加重肺部炎症反应。
6.聚肌胞、利巴韦林和地塞米松药物治疗在一定程度上可以抑制RSV感染哮喘加重小鼠气道上皮细胞生成TSLP的增加,减轻小鼠肺部炎症反应,其中以地塞米松的相对作用最强。
BACKGROUND AND AIM:
Bronchial asthma(asthma) is a chronic respiratory disease characterized by chronic airway inflammation,mucus hypersecretion,airway hyperresponsiveness and airway remolding,which has a great harmful impact on health.The initiation and development of asthma are regulated by diverse factors,which determine the differentiation of lymphocytes,the type of immune responses,the synthesis of cytokines and the process of tissue's repair.In spite of the great progresses in fundamental researches of asthma,the current treatments to control the acute exacerbation of asthma and severe asthma are still unsatisfied.The development of molecular virology techniques,in particular of reverse transcription-polymerase chain reaction(RT-PCR) assays for the identification of respiratory viruses in biological samples,has provided a more sensitive tool to evaluate the role of respiratory viruses in the natural history of asthma.Clinical studies performed using RT-PCR suggest that the proportion of virus-induced asthma exacerbations is likely be around 80-85% in school-aged children and 60-75%in adults of the total number of exacerbations. respiratory syncytial virus(RSV) is a single strand negative-sense RNA virus,which is the most common pathogen causing the infection of children's airway.it's infection has been verified as a main cause of children's asthma,and was regarded as an important dangerous factor in development and exacerbation of asthma.But the human's immune response to RSV is incomplete and transient,the adults had been infected by RSV may be infected again by the same strain of RSV.
The destructive effects of microorganism invasion cause the evolution of host defense.In mammals,the immune system is composed of two branches,the innate immune system and the adaptive immune system,The innate immune response is the first line of host defense and is responsible for immediate recognition and control of microbial invasion.The innate immune response relies on evolutionarily ancient germline-encoded receptors,the pattern-recognition receptors(PRRs),which can recognize the conservative and invariant pathogen-associated molecular patterns (PAMPs) of microorganisms.Toll like receptors are the most distinctive receptors in the recognition of virus.
The airway epithelial cell was regarded as a line of physical barrier,which defense harmful inhalants and microorganisms.current researches have shown the airway epithelial cells play a key role in the initiation and acceleration of host airway defense.It can sense stimulation and initiate the innate immune response to induce cytokines production,which regulate the adaptive immune response by interacting with local dendritic cells(DCs).The airway epithelial cells can affect DCs through secreting thymic stromal lymphopoietin(TSLP),which can promote development of DCs to regulate naive CD4~+T cells' differentiation to Th2 type.The TSLP-DCs induced Th2 CD4~+T cells produce a very unique cytokine profile,including IL-4, IL-13,IL-5 and TNF-a.one study demonstrated that in all TLRs' ligands only TLR3 ligand(dsRNA) induced great TSLP mRNA expression in normal human bronchial epithelial cells.This suggest the formation of dsRNA during viral replication possibly induce TSLP expression to exacerbate the Th2 inflammation reaction,and aggravate the mucosal inflammation in airways of asthmatics.Although asthma is characterized by Th2-type inflammation,the normal CD4~+T cell response to viral infection is thought to be predominantly of the Th1 type.The abnormal reactive inflammation in respiratory viral infection may be skewed toward inappropriate and potentially harmful Th2 responses.
The purposes of this research are to investigate whether RSV has effects on the expression of innate immune response receptor TLR3 and Th2 cytokine TSLP during the virus' infection in human respiratory airway epithelial cells,to investigate whether anti-TLR3 antibody can inhibit the expression of TSLP in RSV-infected airway epithelial cells,to investigate whether local Th1/Th2 microenvironment has effects on the expression of TSLP by adding recombinant human IFN-γand recombinant human IL-4 into culture medium,and to investigate whether ribavirin and Dexamethasone have effects on the expression of TSLP in RSV-infected airway epithelial cells.subsequently to research whether the change of TSLP is concerned with asthma we investigate the expression of TSLP in RSV-induced asthma exacerbation and it's effects on airway inflammation in mice.finally we investigate different drugs' effects on expression of TSLP and airway inflammation in mice.
METHODS
In vitro tests
1.The comparative expression of TSLP mRNA and TLR3 mRNA in 16HBE after different concentration and time of PolyⅠ:C stimulation
Using 10%DMEM culture medium containing different concentration of PolyⅠ:C(0μg/ml,1μg/ml,5μg/ml,10μg/ml,25μg/ml,50μg/ml) to cultivate 16HBE for 3 hours,real-time RT-PCR was used to examine the comparative expression of TSLP mRNA and TLR3 mRNA.
Using 10%DMEM culture medium containing 10μg/ml PolyⅠ:C to cultivate 16HBE for different hours(1 h,2 h,3 h,6 h,12 h,24 h),real-time RT-PCR was used to examine the comparative expression of TSLP mRNA and TLR3 mRNA.
2.The expression of TLR3 in 16HBE after RSV infection
Hep-2 cells were inoculated to amplify RSV,indirect immunofluorescence assay was used to verify the amplification of RSV in 16HBE,Reed-Muench method was used to calculate RSV's virulence.indirect immunofluorescence assay was used to detect the expression of TLR3 in 16HBE after 24 hours of RSV infection.
3.The comparative expression of TSLP mRNA and TLR3 mRNA in 16HBE and production of TSLP protein after RSV infection and interventions
The experiment was designed to divide 6 groups:control group,RSV group, RSV/Anti-TLR3 group,RSV/IFN-γgroup,RSV/L-4 group,RSV/Dexamethasone group;real-time RT-PCR was used to examine the comparative expression of TSLP mRNA and TLR3 mRNA after 6 hours of RSV infection.western blotting was used to examine the production of TSLP protein after 24 hours of RSV infection.
4.The effect of ribavirin on the comparative expression of TSLP mRNA and production of TSLP protein in RSV-infected 16HBE
The experiment was designed to divide 4 groups:control group,RSV group; RSV/ribavirin group;ribavirin group;real-time RT-PCR was used to examine the comparative expression of TSLP mRNA and TLR3 mRNA after 6 hours of RSV infection.western blotting was used to examine the production of TSLP protein after 24 hours of RSV infection.
Animal experiments
1.The effects of RSV infection on production of TSLP and airway inflammation in asthmatic mice
32 BALB/c mice were divided into 4 groups:control group,OVA group,RSV group,OVA/RSV group.
Airway hyperresponsiveness to MeCh(Sigma;0,3.125,6.25,12.5,25 and 50 mg/ml in isotonic saline) was assessed by whole body plethysmography(Buxco), Penh value of different groups were recorded and compared.
Cytokines(IL-4、IFN-γ、IL-5、IL-13) in mice serum were examined by using ELISA assay.
To evaluate the pulmonary immune response,BAL fluid was isolated,total cells recovered were counted,and the cellular composition of BALF was determined using Giemsa's staining.TSLP in BALF was examined by using ELISA assay.
Pulmonary histopathology:On protocol day 26,mice lungs were excised and fixed.These tissues were then embedded in paraffin,cut sections and stained with hematoxylin and eosin(H&E);periodic acid-Schiff(PAS) to show mucus production in airway goblet cells.
TSLP Immunohistochemical staining of lung specimens was observed in different groups.
The production of TSLP was examined by using western blotting,Gel-Pro ANALYZER 4.5 software was used to analyze the comparative production of TSLP protein.
2.The effects of different drugs on secretion of TSLP and airway inflammation in RSV-induced exacerbation of asthma
32 BALB/c mice were divided into 4 groups:OVA/RSV group,PolyⅠ:C group, ribavirin group,Dexamethasone group.
Methods of examination in Airway hyperresponsiveness to MeCh,Cytokines (IL-4,IFN-γ,IL-5,IL-13,TSLP) assay,BALF's cell classification,pulmonary histopathology and TSLP's western blotting were described above.
Statistical analysis
Statistical analysis was performed using SPSS 13.0 software.All data were expressed as mean±SEM.One-way analysis of variance(one-way ANOVA) and LSD、SNK or Dunnett's T3 methods were used to determine differences between experimental groups according to test of homogeneity of variance.Significance was accepted when p＜0.05.
RESULTS:
Results of in vitro tests
1.The expressions of TSLP mRNA and TLR3 mRNA in 16HBE after different concentration of PolyⅠ:C stimulation were upregulated along with PolyⅠ:C concentration.Peak expression of TSLP mRNA was observed when the PolyⅠ:C concentration reach to 50μg/ml(29.84±5.32-fold compared to control,P＜0.01). Peak expression of TLR3 mRNA was observed when the PolyⅠ:C concentration reach to 50μg/ml(4.46±0.8-fold compared to control,P=0.001).
The expressions of TSLP mRNA and TLR3 mRNA in 16HBE after different time of PolyⅠ:C stimulation were induced to peak levels at 3h,corresponding copies of TSLP mRNA was increased to 4.48±0.35-fold compared to 1h stimulation(P＜0.001),TLR3 mRNA was increased to 1.66±0.12-fold compared to 1h stimulation(P＜0.001).
2.Hep-2 cells inoculated by RSV show great typical cytopathic effect,the supernatant of culture medium was collected to get viruses,the virulence of virus was adjusted to MOI=2.indirect immunofluorescence assay show RSV amplication in Hep-2 cells.
16HBE cells inoculated by RSV were cultured subsequently for 24 hours, indirect immunofluorescence assay show higher production of TLR3 when compared to control.
3.The expression of TSLP mRNA and TLR3 mRNA in 16HBE after 6 hours of RSV infection were increased(1.63±0.08-fold and 1.54±0.34-fold compared to control respectively).The expression of TSLP mRNA(0.83±0.18-fold compared to control) was decreased when recombinant human IFN-γwas added,lower than RSV group(P＜0.001).The expression of TSLP mRNA(2.61±0.45-fold compared to control) was enhanced synergistically when recombinant human IL-4 was added, higher than RSV group(P=0.025).The expression of TSLP mRNA(1.23±0.2-fold compared to control) was decreased when anti-TLR3 antibody was added,lower than RSV group(P=0.034).Dexamethasone inhibited the increased level of TSLP mRNA expression in RSV-infected cells(P＜0.001).different interventions have no evident effects in expression of TLR3 mRNA except for Dexamethasone(inhibited the increased level of TLR3 mRNA expression in RSV-infected cells).
The comparative production of TSLP protein in 16HBE after 24 hours of RSV infection were increased(1.55±0.36)(1.9-fold compared to control,P＜0.001).The comparative production of TSLP protein(1.13±0.31) was decreased when recombinant human IFN-γwas added,lower than RSV group(P=0.020).The comparative production of TSLP protein(1.99±0.37) was enhanced synergistically when recombinant human IL-4 was added,higher than RSV group(P=0.014).The comparative production of TSLP protein(1.28±0.28) was decreased when anti-TLR3 antibody was added,but no significance was found when compared to RSV group(P=0.114).Dexamethasone significantly inhibited the increased production of TSLP protein in RSV-infected cells(0.53±0.11)(P＜0.001).
4.The expression of TSLP mRNA in 16HBE after 6 hours of RSV infection was increased.Ribavirin inhibited the increased level of expression.TSLP mRNA expression in ribavirin group(1.41±0.18-fold compared to control) was lower than RSV group(1.99±0.25-fold compared to control)(P=0.006).
The comparative production of TSLP protein in 16HBE after 24 hours of RSV infection was increased.Ribavirin significantly inhibited the increased level of TSLP protein.The comparative production of TSLP protein in ribavirin group(1.16±0.17) was lower than RSV group(1.97±0.19)(P＜0.001).
Results of animal experiments
1.Airway responsiveness to MeCh increased along with its concentration.Mice in the OVA/RSV group showed reduced airway function with the most high Penh compared to mice in other groups.a significantly increased Penh compared to OVA mice from 6.25 mg/ml MeCh(318.66±50.87 vs 187.95±38.73,P＜0.001),different drug treatments somewhat inhibited the enhancement of Penh(P＜0.01).a significantly decreased Penh in mice of different treatment groups compared to OVA/RSV mice at 50 mg/ml MeCh(P＜0.05),Dexamethasone is the most effective drug in decreasing Penh compared to other treatment groups(P＜0.001).
2.Cytokine levels in mice serum were examined to study the Th1/Th2 polarization in different groups.levels of Th1/Th2 cytokine(IL-4,IL-5,IL-13,IFN-γ, TSLP) were elevated in OVA/RSV mice,higher than OVA mice(P=0.001, P=0.003,P=0.02,P＜0.001 respectively).All treatments inhibited the production of IL-4 and IL-5(P＜0.05),PolyⅠ:C and dexamethasone inhibited the production of IL-13(P=0.008,P=0.001 respectively),ribavirin and dexamethasone inhibited the production of IFN-γ(P＜0.01).
3.numbers of total cells,eosinophils,neutrophils and lymphocytes in OVA/RSV group were the highest compared to all other groups(P＜0.01).Treatment groups all inhibited the elevation of total cells and eosinophils(P＜0.01).PolyⅠ:C and dexamethasone decreased elevation of lymphocytes,but ribavirin group showed no significant difference with OVA/RSV group(P=0.051).PolyⅠ:C and dexamethasone inhibited elevation of neutrophils when compared to OVA/RSV group(P=0.01,P=0.011 respectively).
4.H&E staining and PAS staining showed the most severe Airway inflammation in mice lung of OVA/RSV group,epithelial cells hypertrophy /hyperplasia,mucus hypersecretion,tremendous lymphocytes infiltration were observed.All treatment groups inhibited the airway inflammation to some degree, dexamethasone is the most effective drug in inhibiting inflammation. Immunohistochemical staining showed the most significant production of TSLP in OVA/RSV mice,treatment groups showed decreased production of TSLP.
5.TSLP protein in mice lung was examined,the comparative production of TSLP protein in OVA/RSV group(0.93±0.18) was the highest compared to other groups.Ribavirin inhibited production of TSLP protein(P=0.031),dexamethosone was the most effective drug in inhibiting production of TSLP protein(P＜0.001).
CONCLUSIONS:
1.dsRNA promotes expression of TSLP mRNA by stimulating innate immune receptor TLR3 in human bronchial epithelial cells 16HBE..
2.RSV replication after infection promotes the production of TLR3 in 16HBE.
3.RSV infection stimulates 16HBE to product TSLP;IFN-γinhibit production of TSLP induced by RSV infection;but IL-4 synergistically enhance its production, these results demonstrate different Th1/Th2 microenvironments have different effects on RSV-induced TSLP production;TLR3 blockade can inhibit RSV-induced TSLP production moderately,this result demonstrates indirectly TLR3 is an important factor in RSV-induced TSLP production;dexamethosone significantly inhibit RSV-induced TSLP production,this result supports the useful effect of corticosteroids on treating RSV-induced TSLP production.
4.Anti-virus drug ribavirin can inhibit RSV-induced TSLP production in 16HBE,this result indicates ribavirin may has therapeutical effect on treating RSV-induced asthma exacerbation.
5.RSV infection promotes the production of TSLP in OVA sensitized asthmatic mice and aggravates inflammation in mice lung.
6.PolyⅠ:C,ribavirin and dexamethasone can inhibit the production of TSLP to some degree in OVA sensitized asthmatic mice,and alleviate inflammation in mice lung.
支气管哮喘(简称哮喘)是一种严重危害人类健康的慢性呼吸道疾病,以气道慢性炎症、粘液分泌增加、气道反应性增高及气道重塑为主要特征。哮喘的发生与发展受多种因素的调控,从而决定了淋巴细胞分化,免疫反应类型,细胞因子合成及组织修复过程。尽管哮喘基础研究取得较大的进步,但目前临床上对于哮喘的急性加重以及重症哮喘的治疗仍然不是很满意。随着分子病毒学技术的发展和RT-PCR技术在临床研究中的应用,呼吸道病毒感染作为哮喘加重的最重要原因已被广泛认识。已经证实,病毒诱发的哮喘加重在学龄儿童中的比例约为80%-85%,而在成人中约为60%-75%。呼吸道合胞病毒(respiratorysyncytial virus,RSV)作为呼吸道单链RNA病毒的一种,是引起小儿呼吸道感染的最常见病原体,也是诱发婴幼儿哮喘的主要原因,在哮喘的发展和加重中被认为是一个重要的危险因子。而且,针对RSV的免疫是不完全的和暂时的,所以已经感染过RSV的成人可以再次被同株的RSV感染。
微生物感染对机体的破坏作用导致宿主防御机制的进化。在哺乳动物,有两套防御机制:固有免疫和继发性免疫。固有免疫识别是通过模式识别受体(pattern recognition receptors,PRRs)来完成的,每个受体都对微生物的保守和不变的特征识别具有广泛的特殊性。PRRs识别微生物是通过与病原体相关分子模式(pathogen-associated molecular patterns,PAMPs)的结合来完成的。在PRRs的病毒识别中,最具特征性的是Toll样受体(Toll like receptors,TLRs)。
呼吸道上皮细胞除了作为物理屏障,也能通过感受外界刺激因素启动固有免疫反应产生细胞因子,从而影响继发性免疫反应。在针对吸入性外来变应原和病原体的继发性免疫反应的起始中,局部浸润的树突状细胞(dendritic cells,DCs)起到关键性作用。气道上皮细胞影响DCs可以通过DCs激活因子胸腺基质淋巴细胞生成素(thymic stromal lymphopoietin,TSLP)的产生来调控Th细胞的局部分化。研究证实TSLP能诱导DCs调节初始CD4~+T淋巴细胞向Th2细胞分化,产生IL-4、IL-5、IL-13和TNF-α等细胞因子。在对气道上皮细胞生成TSL。P的刺激因子的研究中发现,TLR3的配体双链RNA(dsRNA)与TLR3受体结合产生大量的TSLP mRNA。这提示病毒在呼吸道上皮细胞内复制过程中形成的dsRNA中间体,可能通过TSLP的诱导,加重Th2炎症反应,从而进一步加重哮喘患者气道粘膜炎症。这个推论可以解释为什么呼吸道病毒感染加重变态反应性炎症,而从免疫学理论上讲,病毒诱导的Th1免疫反应能拮抗Th2调节的炎症反应。
本项研究主要通过研究RSV感染人支气管上皮细胞16HBE分析固有免疫反应受体TLR3的表达情况及对TSLP生成的影响,通过TLR3抗体阻断的方法研究是否可以引起TSLP的生成的改变,通过模拟气道局部Th1和Th2内环境加入IFN-γ和IL-4观察其对RSV感染诱发的TSLP生成影响,同时观察利巴韦林和地塞米松等药物干预对支气管上皮细胞生成TSLP的影响。为明确TSLP在哮喘动物体内的变化与哮喘的关系,通过RSV感染小鼠哮喘加重模型进一步分析在RSV感染情况下小鼠气道局部TSLP的表达与小鼠肺部炎症的相互关系,并观察不同药物治疗对RSV感染哮喘加重小鼠气道TSLP生成和肺部炎症的影响。
方法:
一、体外细胞学试验
1.不同浓度和不同时间人工合成dsRNA聚肌胞(PolyⅠ:C)对人支气管上皮细胞16HBE表达TSLP mRNA和TLR3 mRNA的影响
分别加入含0μg/ml、1μg/ml、5μg/ml、10μg/ml、25μg/ml、50μg/ml聚肌胞的10%DMEM培养基培养16HBE细胞,刺激3小时,实时荧光定量RT-PCR检测TSLP mRNA和TLR3 mRNA相对表达量。
10μg/ml聚肌胞加入10%DMEM培养基培养16HBE细胞,分别刺激1h、2h、3h、6h、12h、24h,实时荧光定量RT-PCR检测TSLP mRNA和TLR3 mRNA相对表达量。
2.RSV病毒感染对人支气管上皮细胞16HBE固有免疫受体TLR3表达的影响
RSV病毒感染Hep-2细胞进行病毒扩增;RSV在Hep-2细胞质内扩增的间接免疫荧光鉴定;Reed-Muench法测算RSV细胞毒力。RSV感染人支气管上皮细胞16HBE 24小时,间接免疫荧光检测细胞TLR3受体表达变化。
3.RSV病毒感染及干预因素对16HBE细胞TSLP mRNA和TLR 3mRNA表达水平和TSLP蛋白表达的影响
试验分6组:对照组,RSV组,RSV/Anti-TLR3组,RSV/IFN-γ组,RSV/IL-4组,RSV/地塞米松组;RSV感染6小时后实时荧光定量RT-PCR检测细胞TSLP mRNA和TLR3 mRNA相对表达量;RSV感染24小时后western blotting检测各组细胞TSLP蛋白表达水平。
4.利巴韦林抑制RSV病毒感染对16HBE细胞TSLP mRNA表达水平和TSLP蛋白表达的影响
试验分4组:对照组,RSV组,RSV/利巴韦林组,利巴韦林组;RSV感染6小时后实时荧光定量RT-PCR检测细胞TSLP mRNA相对表达量;RSV感染24小时后western blotting检测各组细胞TSLP蛋白表达水平。
二、动物实验
1.RSV病毒感染对哮喘加重小鼠气道TSLP分泌和肺部炎症的影响
32只BALB/c小鼠分为4组:A组(对照组),B组(OVA组),C组(RSV组),D组(OVA/RSV模型组)。
无创肺功能检测小鼠气道反应性:不同浓度乙酰甲胆碱激发,BUXCO无创肺功能检测仪检测Penh值。
小鼠血清ELISA法检测细胞因子:小鼠血清用于IL-4、IFN-γ、IL-5、IL-13细胞因子检测。
小鼠气管灌洗液细胞分类计数和ELISA检测TSLP:聚乙烯导管行小鼠气管插管,PBS灌洗,灌洗液上清ELISA法检测细胞因子TSLP;灌洗液离心沉淀细胞计数,瑞氏-吉姆萨染色细胞分类计数。
小鼠肺脏组织病理观察:苏木素—伊红(HE)染色观察小鼠肺脏病理变化;碘酸雪夫(PAS)染色观察气道上皮细胞粘液合成。
小鼠肺脏组织免疫组化观察气道上皮细胞TSLP表达,DAB显色,阳性显示棕黄色。
小鼠肺脏组织TSLP蛋白western blotting检测,Gel-Pro ANALYZER4.5软件分析TSLP的相对表达量。
2.不同药物治疗对RSV感染哮喘加重小鼠TSLP分泌和肺部炎症的作用
32只BALB/c小鼠分为4组:对照组(0VA/RSV组),聚肌胞组(OVA/RSV/PolyI:C组),利巴韦林组(OVA/RSV/Rib组),地塞米松组(OVA/RSV/Dex组)。
无创肺功能检测小鼠气道反应性;小鼠血清ELISA法检测细胞因子血清IL-4、IFN-γ、IL-5、IL-13浓度;小鼠气管灌洗液细胞分类计数和ELISA检测气管灌洗液TSLP浓度;小鼠肺脏组织病理观察;小鼠肺脏组织免疫组化观察TSLP表达;小鼠肺脏组织TSLP蛋白western blotting检测。(方法同上)。
三、统计学处理
采用SPSS13.0软件进行统计分析,计量资料以均数±标准差((?)±s)表示。各组间多重比较采用单向方差分析(one-way ANOVA),方差齐性时采用LSD法和SNK法检验,方差不齐时采用Dunnett's T3法检验。以P＜0.05为有统计学意义。
结果:
一、体外细胞学试验结果
1.随着聚肌胞浓度增加,16HBE细胞TSLP mRNA和TLR3 mRNA表达水平增高。当聚肌胞浓度为50μg/ml时,TSLP mRNA表达量最高,为对照组的29.84±5.32倍(P＜0.01);TLR3 mRNA表达量最高,为对照组的4.46±0.8倍(P=0.001)。
随着聚肌胞刺激时间延长,16HBE细胞TSLP mRNA和TLR3 mRNA表达水平先升高,后下降。当聚肌胞刺激3h,TSLP mRNA表达量达到最高(为聚肌胞刺激1h表达量4.48±0.35倍)(P＜0.001),随着时间延长TSLP mRNA表达下降;当聚肌胞刺激3h,TLR3 mRNA表达量达到最高(为聚肌胞刺激1h表达量1.66±0.12倍),随着时间延长TLR3 mRNA表达下降。
2.Hep-2细胞接种RSV后第5天细胞大片融合,并有细胞脱落;收集病毒,调整病毒毒力MOI为2;间接免疫荧光观察显示RSV在细胞内扩增;16HBE细胞接种RSV病毒24小时后间接免疫荧光观察细胞内TLR3受体表达情况显示:病毒感染细胞内TLR3受体表达明显增加,而阴性对照组细胞TLR3表达相对少。
3.RSV感染16HBE细胞可使细胞TSLP mRNA表达水平增高(为对照组表达量1.63±0.08倍)(P＜0.001),TLR3 mRNA表达水平增高(为对照组表达量1.54±0.34倍)(P=0.094)。加入重组人IFN-γ100μg/L,TSLP mRNA表达水平为对照组的0.83±0.18倍,比RSV组低(P＜0.001),TLR3 mRNA表达与RSV组比较无统计学意义(P=1.000);加入重组人IL-4 100μg/L,TSLP mRNA表达水平为对照组的2.61±0.45倍,比RSV组高(P=0.025),TLR3 mRNA表达与RSV组比较无统计学意义(P=1.000);加入Anti-TLR3阻断TLR3受体后细胞TSLP mRNA表达量(为对照组的1.23±0.2倍)相对于RSV感染组低(P=0.034),而TLR3 mRNA表达水平增高(为对照组细胞为2.06±0.5倍)。加入地塞米松,TSLP mRNA表达量比RSV组低(P＜0.001),而TLR3 mRNA表达水平相对于对照组仍高(P=0.042),与其它各组比较均无统计学意义(P＞0.05)。
RSV感染组细胞TSLP蛋白相对表达量(1.55±0.36)约为对照组的1.9倍(P＜0.001),加入IFN-γ可以降低RSV感染引起的TSLP表达升高(1.13±0.31)(与RSV组比较P=0.020),加入IL-4可以协同RSV感染引起TSLP表达升高(1.99±0.37),比RSV组表达量增加(P=0.014),加入TLR3抗体阻断TLR3受体可使TSLP表达降低(1.28±0.28),但与RSV感染组比较无统计学意义(P=0.114),加入地塞米松可以显著抑制RSV感染16HBE细胞TSLP表达(0.53±0.11)(与RSV组比较P＜0.001)。
4.RSV感染16HBE细胞可以使细胞TSLP mRNA表达水平增高(为对照组的1.99±0.25倍)(P=0.001);加入利巴韦林抑制RSV感染后TSLP mRNA表达水平下降,为对照组的1.41±0.18倍(P=0.012);利巴韦林治疗组细胞TSLP mRNA表达水平比RSV感染组低(P=0.006)。
利巴韦林可以显著抑制RSV感染引起的TSLP表达(1.16±0.17),与RSV组(1.97±0.19)比较有统计学意义(P＜0.001),但仍高于对照组(P=0.002),单独加入利巴韦林(0.99±0.15)与对照组细胞TSLP表达量比较无统计学意义(P=0.215)。
二、动物实验结果
1.随着乙酰甲胆碱浓度上升各组小鼠气道反应性检测指标Penh值均出现上升趋势,OVA/RSV组小鼠的气道反应性最高。乙酰甲胆碱激发浓度为3.125 mg/ml时,OVA/RSV组小鼠气道反应性Penh值(128.42±18.05)和OVA组(114.73±17.67)比对照组(19.48±4.44)高(P＜0.001);乙酰甲胆碱激发浓度为6.25mg/ml时,OVA/RSV组Penh值(318.66±50.87)大于OVA组(187.95±38.73)(P＜0.001),各药物治疗组小鼠气道反应性Penh值均低于OVA/RSV组(P＜0.01)。当乙酰甲胆碱激发浓度为50 mg/ml时,各治疗组小鼠气道反应性Penh值均低于OVA/RSV组(1335.46±196.61)(P＜0.05),地塞米松组(581.28±58.89)最低,与其它治疗组比较低(P＜0.001)。
2.OVA/RSV组小鼠血清IL-4浓度(274.24±103.67 pg/ml)比OVA组(94.18±26.4 pg/ml)高(P=0.001),聚肌胞组、利巴韦林组、地塞米松组均可降低血清IL-4水平(P＜0.05);地塞米松组(86.78±27.04 pg/ml)为治疗组内最低;OVA/RSV组小鼠血清IL-5(293.25±46.13 pg/ml)高于OVA组(239.57±33.84pg/ml)(P=0.003),各药物治疗组均比OVA/RSV组降低(P＜0.05),利巴韦林组IL-5水平(220.82±30.9 pg/ml)最低;OVA/RSV组小鼠血清IL-13(330±93.5pg/ml)比OVA组(187.04±62.66 pg/ml)高(P=0.02),聚肌胞组(219.91±60.88 pg/ml)、地塞米松组(194.65±73.27 pg/ml)均下降(P=0.008,P=0.001);OVA/RSV组小鼠血清IFN-γ(30.08±5.73 pg/ml)比OVA组(8.46±2.86 pg/ml)明显增高(P＜0.001),利巴韦林组(12.98±3.34 pg/ml)、地塞米松组(17.33±3.06 pg/ml)均有所下降(P＜0.01)。
3.小鼠支气管肺泡灌洗液(BALF)中:OVA/RSV组细胞总数(21.23±0.54)×10~6/ml最多(P＜0.001),各治疗组细胞总数均降低(P＜0.001);OVA/RSV组嗜酸性粒细胞数(4.87±0.37)×10~5/ml最多(P＜0.001),各治疗组均降低(P＜0.01);OVA/RSV组淋巴细胞数(5.45±0.16)×10~5/ml升高最为明显(P＜0.001),利巴韦林治疗组与之比较无统计学意义(P=0.051),余各治疗组下降;OVA/RSV组中性粒细胞(2.46±0.25)×10~5/ml升高最为明显,与OVA组比较有统计学意义(P＜0.01),聚肌胞组、地塞米松组有所下降(P=0.01,P=0.011),利巴韦林治疗与OVA/RSV组比较无统计学意义。
支气管肺泡灌洗液检测TSLP结果:OVA/RSV组小鼠支气管肺泡灌洗液TSLP(2.13±0.05 ng/ml)高于OVA组(1.99±0.05 ng/ml)(P=0.001),各药物治疗均使TSLP水平下降(P＜0.01)。
4.常规HE染色OVA/RSV组支气管上皮增生肥厚、粘液分泌增加、管腔明显狭窄,管壁及其周围大量淋巴细胞浸润,比OVA组加重,各治疗可使炎症减轻,地塞米松治疗组炎症反应最轻。PAS染色OVA/RSV组粘液分泌增加,杯状细胞增生最为明显,各治疗可一定程度减轻病变,地塞米松治疗组明显抑制上皮细胞粘液合成。免疫组化观察OVA/RSV组小鼠气道上皮细胞表达TSLP最为明显,各治疗组可减轻TSLP合成分泌。
5.Western blotting检测发现OVA/RSV组小鼠肺脏TSLP相对表达量(TSLP/GAPDH)(0.93±0.18)为最高,与对照组、OVA组、RSV组比较有统计学意义(P＜0.001,P=0.045,P=0.001);利巴韦林组小鼠肺脏TSLP表达量低于OVA/RSV组(P=0.031),地塞米松组为各治疗组中TSLP表达量最低,(P＜0.001)。
结论:
1.dsRNA可以激活人支气管上皮细胞16HBE固有免疫受体TLR3,促进TSLPmRNA表达量增加。
2.RSV感染后病毒复制促进固有免疫受体TLR3在人支气管上皮细胞16HBE内的表达。
3.RSV感染促进16HBE细胞表达TSLP;IFN-γ可起到抑制作用,IL-4则起到协同作用促进TSLP表达,表明不同Th1/Th2微环境对TSLP的表达有不同影响;TLR3受体阻断可以适度抑制TSLP表达,间接表明固有免疫受体TLR3在RSV感染的TSLP表达中起到一定作用;地塞米松可以显著降低RSV感染引起的TSLP表达,说明糖皮质激素在RSV感染引起的TSLP表达中起到抑制作用。
4.利巴韦林抗病毒治疗可以有效抑制16HBE细胞表达TSLP,说明抑制RSV在细胞内复制可以起到减少细胞表达TSLP的作用。
5.RSV感染可以促进OVA过敏性哮喘小鼠气道上皮细胞生成TSLP增加,并加重肺部炎症反应。
6.聚肌胞、利巴韦林和地塞米松药物治疗在一定程度上可以抑制RSV感染哮喘加重小鼠气道上皮细胞生成TSLP的增加,减轻小鼠肺部炎症反应,其中以地塞米松的相对作用最强。
BACKGROUND AND AIM:
Bronchial asthma(asthma) is a chronic respiratory disease characterized by chronic airway inflammation,mucus hypersecretion,airway hyperresponsiveness and airway remolding,which has a great harmful impact on health.The initiation and development of asthma are regulated by diverse factors,which determine the differentiation of lymphocytes,the type of immune responses,the synthesis of cytokines and the process of tissue's repair.In spite of the great progresses in fundamental researches of asthma,the current treatments to control the acute exacerbation of asthma and severe asthma are still unsatisfied.The development of molecular virology techniques,in particular of reverse transcription-polymerase chain reaction(RT-PCR) assays for the identification of respiratory viruses in biological samples,has provided a more sensitive tool to evaluate the role of respiratory viruses in the natural history of asthma.Clinical studies performed using RT-PCR suggest that the proportion of virus-induced asthma exacerbations is likely be around 80-85% in school-aged children and 60-75%in adults of the total number of exacerbations. respiratory syncytial virus(RSV) is a single strand negative-sense RNA virus,which is the most common pathogen causing the infection of children's airway.it's infection has been verified as a main cause of children's asthma,and was regarded as an important dangerous factor in development and exacerbation of asthma.But the human's immune response to RSV is incomplete and transient,the adults had been infected by RSV may be infected again by the same strain of RSV.
The destructive effects of microorganism invasion cause the evolution of host defense.In mammals,the immune system is composed of two branches,the innate immune system and the adaptive immune system,The innate immune response is the first line of host defense and is responsible for immediate recognition and control of microbial invasion.The innate immune response relies on evolutionarily ancient germline-encoded receptors,the pattern-recognition receptors(PRRs),which can recognize the conservative and invariant pathogen-associated molecular patterns (PAMPs) of microorganisms.Toll like receptors are the most distinctive receptors in the recognition of virus.
The airway epithelial cell was regarded as a line of physical barrier,which defense harmful inhalants and microorganisms.current researches have shown the airway epithelial cells play a key role in the initiation and acceleration of host airway defense.It can sense stimulation and initiate the innate immune response to induce cytokines production,which regulate the adaptive immune response by interacting with local dendritic cells(DCs).The airway epithelial cells can affect DCs through secreting thymic stromal lymphopoietin(TSLP),which can promote development of DCs to regulate naive CD4~+T cells' differentiation to Th2 type.The TSLP-DCs induced Th2 CD4~+T cells produce a very unique cytokine profile,including IL-4, IL-13,IL-5 and TNF-a.one study demonstrated that in all TLRs' ligands only TLR3 ligand(dsRNA) induced great TSLP mRNA expression in normal human bronchial epithelial cells.This suggest the formation of dsRNA during viral replication possibly induce TSLP expression to exacerbate the Th2 inflammation reaction,and aggravate the mucosal inflammation in airways of asthmatics.Although asthma is characterized by Th2-type inflammation,the normal CD4~+T cell response to viral infection is thought to be predominantly of the Th1 type.The abnormal reactive inflammation in respiratory viral infection may be skewed toward inappropriate and potentially harmful Th2 responses.
The purposes of this research are to investigate whether RSV has effects on the expression of innate immune response receptor TLR3 and Th2 cytokine TSLP during the virus' infection in human respiratory airway epithelial cells,to investigate whether anti-TLR3 antibody can inhibit the expression of TSLP in RSV-infected airway epithelial cells,to investigate whether local Th1/Th2 microenvironment has effects on the expression of TSLP by adding recombinant human IFN-γand recombinant human IL-4 into culture medium,and to investigate whether ribavirin and Dexamethasone have effects on the expression of TSLP in RSV-infected airway epithelial cells.subsequently to research whether the change of TSLP is concerned with asthma we investigate the expression of TSLP in RSV-induced asthma exacerbation and it's effects on airway inflammation in mice.finally we investigate different drugs' effects on expression of TSLP and airway inflammation in mice.
METHODS
In vitro tests
1.The comparative expression of TSLP mRNA and TLR3 mRNA in 16HBE after different concentration and time of PolyⅠ:C stimulation
Using 10%DMEM culture medium containing different concentration of PolyⅠ:C(0μg/ml,1μg/ml,5μg/ml,10μg/ml,25μg/ml,50μg/ml) to cultivate 16HBE for 3 hours,real-time RT-PCR was used to examine the comparative expression of TSLP mRNA and TLR3 mRNA.
Using 10%DMEM culture medium containing 10μg/ml PolyⅠ:C to cultivate 16HBE for different hours(1 h,2 h,3 h,6 h,12 h,24 h),real-time RT-PCR was used to examine the comparative expression of TSLP mRNA and TLR3 mRNA.
2.The expression of TLR3 in 16HBE after RSV infection
Hep-2 cells were inoculated to amplify RSV,indirect immunofluorescence assay was used to verify the amplification of RSV in 16HBE,Reed-Muench method was used to calculate RSV's virulence.indirect immunofluorescence assay was used to detect the expression of TLR3 in 16HBE after 24 hours of RSV infection.
3.The comparative expression of TSLP mRNA and TLR3 mRNA in 16HBE and production of TSLP protein after RSV infection and interventions
The experiment was designed to divide 6 groups:control group,RSV group, RSV/Anti-TLR3 group,RSV/IFN-γgroup,RSV/L-4 group,RSV/Dexamethasone group;real-time RT-PCR was used to examine the comparative expression of TSLP mRNA and TLR3 mRNA after 6 hours of RSV infection.western blotting was used to examine the production of TSLP protein after 24 hours of RSV infection.
4.The effect of ribavirin on the comparative expression of TSLP mRNA and production of TSLP protein in RSV-infected 16HBE
The experiment was designed to divide 4 groups:control group,RSV group; RSV/ribavirin group;ribavirin group;real-time RT-PCR was used to examine the comparative expression of TSLP mRNA and TLR3 mRNA after 6 hours of RSV infection.western blotting was used to examine the production of TSLP protein after 24 hours of RSV infection.
Animal experiments
1.The effects of RSV infection on production of TSLP and airway inflammation in asthmatic mice
32 BALB/c mice were divided into 4 groups:control group,OVA group,RSV group,OVA/RSV group.
Airway hyperresponsiveness to MeCh(Sigma;0,3.125,6.25,12.5,25 and 50 mg/ml in isotonic saline) was assessed by whole body plethysmography(Buxco), Penh value of different groups were recorded and compared.
Cytokines(IL-4、IFN-γ、IL-5、IL-13) in mice serum were examined by using ELISA assay.
To evaluate the pulmonary immune response,BAL fluid was isolated,total cells recovered were counted,and the cellular composition of BALF was determined using Giemsa's staining.TSLP in BALF was examined by using ELISA assay.
Pulmonary histopathology:On protocol day 26,mice lungs were excised and fixed.These tissues were then embedded in paraffin,cut sections and stained with hematoxylin and eosin(H&E);periodic acid-Schiff(PAS) to show mucus production in airway goblet cells.
TSLP Immunohistochemical staining of lung specimens was observed in different groups.
The production of TSLP was examined by using western blotting,Gel-Pro ANALYZER 4.5 software was used to analyze the comparative production of TSLP protein.
2.The effects of different drugs on secretion of TSLP and airway inflammation in RSV-induced exacerbation of asthma
32 BALB/c mice were divided into 4 groups:OVA/RSV group,PolyⅠ:C group, ribavirin group,Dexamethasone group.
Methods of examination in Airway hyperresponsiveness to MeCh,Cytokines (IL-4,IFN-γ,IL-5,IL-13,TSLP) assay,BALF's cell classification,pulmonary histopathology and TSLP's western blotting were described above.
Statistical analysis
Statistical analysis was performed using SPSS 13.0 software.All data were expressed as mean±SEM.One-way analysis of variance(one-way ANOVA) and LSD、SNK or Dunnett's T3 methods were used to determine differences between experimental groups according to test of homogeneity of variance.Significance was accepted when p＜0.05.
RESULTS:
Results of in vitro tests
1.The expressions of TSLP mRNA and TLR3 mRNA in 16HBE after different concentration of PolyⅠ:C stimulation were upregulated along with PolyⅠ:C concentration.Peak expression of TSLP mRNA was observed when the PolyⅠ:C concentration reach to 50μg/ml(29.84±5.32-fold compared to control,P＜0.01). Peak expression of TLR3 mRNA was observed when the PolyⅠ:C concentration reach to 50μg/ml(4.46±0.8-fold compared to control,P=0.001).
The expressions of TSLP mRNA and TLR3 mRNA in 16HBE after different time of PolyⅠ:C stimulation were induced to peak levels at 3h,corresponding copies of TSLP mRNA was increased to 4.48±0.35-fold compared to 1h stimulation(P＜0.001),TLR3 mRNA was increased to 1.66±0.12-fold compared to 1h stimulation(P＜0.001).
2.Hep-2 cells inoculated by RSV show great typical cytopathic effect,the supernatant of culture medium was collected to get viruses,the virulence of virus was adjusted to MOI=2.indirect immunofluorescence assay show RSV amplication in Hep-2 cells.
16HBE cells inoculated by RSV were cultured subsequently for 24 hours, indirect immunofluorescence assay show higher production of TLR3 when compared to control.
3.The expression of TSLP mRNA and TLR3 mRNA in 16HBE after 6 hours of RSV infection were increased(1.63±0.08-fold and 1.54±0.34-fold compared to control respectively).The expression of TSLP mRNA(0.83±0.18-fold compared to control) was decreased when recombinant human IFN-γwas added,lower than RSV group(P＜0.001).The expression of TSLP mRNA(2.61±0.45-fold compared to control) was enhanced synergistically when recombinant human IL-4 was added, higher than RSV group(P=0.025).The expression of TSLP mRNA(1.23±0.2-fold compared to control) was decreased when anti-TLR3 antibody was added,lower than RSV group(P=0.034).Dexamethasone inhibited the increased level of TSLP mRNA expression in RSV-infected cells(P＜0.001).different interventions have no evident effects in expression of TLR3 mRNA except for Dexamethasone(inhibited the increased level of TLR3 mRNA expression in RSV-infected cells).
The comparative production of TSLP protein in 16HBE after 24 hours of RSV infection were increased(1.55±0.36)(1.9-fold compared to control,P＜0.001).The comparative production of TSLP protein(1.13±0.31) was decreased when recombinant human IFN-γwas added,lower than RSV group(P=0.020).The comparative production of TSLP protein(1.99±0.37) was enhanced synergistically when recombinant human IL-4 was added,higher than RSV group(P=0.014).The comparative production of TSLP protein(1.28±0.28) was decreased when anti-TLR3 antibody was added,but no significance was found when compared to RSV group(P=0.114).Dexamethasone significantly inhibited the increased production of TSLP protein in RSV-infected cells(0.53±0.11)(P＜0.001).
4.The expression of TSLP mRNA in 16HBE after 6 hours of RSV infection was increased.Ribavirin inhibited the increased level of expression.TSLP mRNA expression in ribavirin group(1.41±0.18-fold compared to control) was lower than RSV group(1.99±0.25-fold compared to control)(P=0.006).
The comparative production of TSLP protein in 16HBE after 24 hours of RSV infection was increased.Ribavirin significantly inhibited the increased level of TSLP protein.The comparative production of TSLP protein in ribavirin group(1.16±0.17) was lower than RSV group(1.97±0.19)(P＜0.001).
Results of animal experiments
1.Airway responsiveness to MeCh increased along with its concentration.Mice in the OVA/RSV group showed reduced airway function with the most high Penh compared to mice in other groups.a significantly increased Penh compared to OVA mice from 6.25 mg/ml MeCh(318.66±50.87 vs 187.95±38.73,P＜0.001),different drug treatments somewhat inhibited the enhancement of Penh(P＜0.01).a significantly decreased Penh in mice of different treatment groups compared to OVA/RSV mice at 50 mg/ml MeCh(P＜0.05),Dexamethasone is the most effective drug in decreasing Penh compared to other treatment groups(P＜0.001).
2.Cytokine levels in mice serum were examined to study the Th1/Th2 polarization in different groups.levels of Th1/Th2 cytokine(IL-4,IL-5,IL-13,IFN-γ, TSLP) were elevated in OVA/RSV mice,higher than OVA mice(P=0.001, P=0.003,P=0.02,P＜0.001 respectively).All treatments inhibited the production of IL-4 and IL-5(P＜0.05),PolyⅠ:C and dexamethasone inhibited the production of IL-13(P=0.008,P=0.001 respectively),ribavirin and dexamethasone inhibited the production of IFN-γ(P＜0.01).
3.numbers of total cells,eosinophils,neutrophils and lymphocytes in OVA/RSV group were the highest compared to all other groups(P＜0.01).Treatment groups all inhibited the elevation of total cells and eosinophils(P＜0.01).PolyⅠ:C and dexamethasone decreased elevation of lymphocytes,but ribavirin group showed no significant difference with OVA/RSV group(P=0.051).PolyⅠ:C and dexamethasone inhibited elevation of neutrophils when compared to OVA/RSV group(P=0.01,P=0.011 respectively).
4.H&E staining and PAS staining showed the most severe Airway inflammation in mice lung of OVA/RSV group,epithelial cells hypertrophy /hyperplasia,mucus hypersecretion,tremendous lymphocytes infiltration were observed.All treatment groups inhibited the airway inflammation to some degree, dexamethasone is the most effective drug in inhibiting inflammation. Immunohistochemical staining showed the most significant production of TSLP in OVA/RSV mice,treatment groups showed decreased production of TSLP.
5.TSLP protein in mice lung was examined,the comparative production of TSLP protein in OVA/RSV group(0.93±0.18) was the highest compared to other groups.Ribavirin inhibited production of TSLP protein(P=0.031),dexamethosone was the most effective drug in inhibiting production of TSLP protein(P＜0.001).
CONCLUSIONS:
1.dsRNA promotes expression of TSLP mRNA by stimulating innate immune receptor TLR3 in human bronchial epithelial cells 16HBE..
2.RSV replication after infection promotes the production of TLR3 in 16HBE.
3.RSV infection stimulates 16HBE to product TSLP;IFN-γinhibit production of TSLP induced by RSV infection;but IL-4 synergistically enhance its production, these results demonstrate different Th1/Th2 microenvironments have different effects on RSV-induced TSLP production;TLR3 blockade can inhibit RSV-induced TSLP production moderately,this result demonstrates indirectly TLR3 is an important factor in RSV-induced TSLP production;dexamethosone significantly inhibit RSV-induced TSLP production,this result supports the useful effect of corticosteroids on treating RSV-induced TSLP production.
4.Anti-virus drug ribavirin can inhibit RSV-induced TSLP production in 16HBE,this result indicates ribavirin may has therapeutical effect on treating RSV-induced asthma exacerbation.
5.RSV infection promotes the production of TSLP in OVA sensitized asthmatic mice and aggravates inflammation in mice lung.
6.PolyⅠ:C,ribavirin and dexamethasone can inhibit the production of TSLP to some degree in OVA sensitized asthmatic mice,and alleviate inflammation in mice lung.
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[20]Ying S,O'Connor B,Ratoff J,et al.Thymic stromal lymphopoietin expression is increased in asthmatic airways and correlates with expression of Th2-attracting chemokines and disease severity[J].J Immunol,2005,174(12):8183-8190.
[21]Al-Shami A,Spolski R,Kelly J,et al.A role for TSLP in the development of inflammation in an asthma model[J].J Exp Med,2005;202(6):829 -839.
[22]Zhou B,Comeau MR,De Smedt T,et al.Thymic stromal lymphopoietin as a key initiator of allergic airway inflammation in mice[J].Nat Immunol,2005,6(10):1047-1053.
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[1]Johnston SL,Pattemore PK,Sanderson G,et al.Community study of role of viral infections in exacerbations of asthma in 9-11 year old children[J].BMJ,1995,310(6989):1225-29.
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[15]Liu YJ.Thymic stromal lymphopoietin:master switch for allergic inflammation [J].J Exp Med,2006,203(2):269-273.
[16]Liu YJ,Soumelis V,Watanabe N,et al.TSLP:an epithelial cell cytokine that regulates T cell differentiation by conditioning dendritic cell maturation[J].Annu Rev Immunol,2007,25:193-219.
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[1]Johnston SL,Pattemore PK,Sanderson G,et al.Community study of role of viral infections in exacerbations of asthma in 9-11 year old children[J].BMJ,1995,310(6989):1225-29.
[2]Wark PA,Johnston SL,Moric I,et al.Neutrophil degranulation and cell lysis is associated with clinical severity in vires-induced asthma[J].Eur Respir J,2002,19(1):68-75.
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[5]Ying S,O'Connor B,Ratoff J,et al.Thymic stromal lymphopoietin expression is increased in asthmatic airways and correlates with expression of Th2-attracting chemokines and disease severity[J].J Immunol,2005,174(12):8183-90.
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[8]Reche PA,Soumelis V,Gorman DM,et al.Human thymic stromal lymphopoietin preferentially stimulates myeloid cells[J].J Immunol,2001,167(1):336-43.
[9]Pandey A,Ozaki K,Baumann H,et al.Cloning of a receptor subunit required for signaling by thymic stromal lymphopoietin[J].Nat Immunol,2000,1(1):59 -64.
[10]Fujio K,Nosaka T,Kojima T,et al.Molecular cloning of a novel type 1 cytokine receptor similar to the common gamma chain[J].Blood,2000,95(7):2204-10.
[11]Watanabe N,Wang YH,Lee HK,et al.Hassall's corpuscles instruct dendritic cells to induce CD4+ CD25 + regulatory T cells in human thymus[J].Nature,2005,436(7054):1181 - 85.
[12]Soumelis V,Reche PA,Kanzler H,et al.Human epithelial cells trigger dendritic cell mediated allergic inflammation by producing TSLP[J].Nat Immunol,2002,3(7):673 - 80.
[13]Zhou B,Comeau MR,De Smedt T,et al.Thymic stromal lymphopoietin as a key initiator of allergic airway inflammation in mice[J].Nat Immunol,2005,6(10):1047-53.
[14]Isaksen DE,Baumann H,Zhou B,et al.Uncoupling of proliferation and Stat5activation in thymic stromal lymphopoietin mediated signal transduction[J]J Immunol,2002,168(7):3288-94.
[15]Ito T,Wang YH,Duramad O,et al.TSLP2activated dendritic cells induce an inflammatory T helper type 2 cell response through OX40 ligand[J].J Exp Med,2005,202(9):1213-23.
[16]Omori M,Ziegler S.Induction of IL-4 expression in CD4+T cells by thymic stromal lymphopoietin[J].J Immunol,2007,178(3):1396-404.
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