肺炎嗜衣原体热休克蛋白经ERK、NF-κB信号通路调控HUVEC分泌炎症因子
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摘要
研究背景
肺炎嗜衣原体（Chlamydophila pneumoniae, Cpn）是一类重要的呼吸道病原体，造成全球约10%的社区获得性肺炎及5%支气管炎和窦炎病例的发生，还与冠心病、动脉粥样硬化、哮喘、反应性关节炎等多种慢性疾病相关。许多国家的人群Cpn感染率占全国人口的50%~70%，但人群感染该菌后多表现为持续的“隐蔽性”，患者病情易被忽视而导致Cpn感染反复迁延，造成机体不可逆的病理改变甚至严重并发症。研究Cpn的致病因素，阐明毒力相关蛋白的致病作用及机制对预防Cpn感染，控制其传播具有重要意义。
Cpn感染引起的炎症反应是多种疾病的发病基础。其中，Cpn热休克蛋白（Heat Shock Proteins, HSPs）在病原体介导的炎症反应中可能发挥重要作用。为研究CHSPs在Cpn诱导炎症反应中的功能及其相关受体及信号通路，进一步说明CHSPs在炎症/免疫病理损伤中的作用及其可能的致病机制，拟运用细胞微生物学分析Toll样受体（Toll-like receptor, TLR）2和TLR4、胞外信号调节激酶（Extracellular signal-regulated kinase, ERK）通路和核因子κB（Nuclear factor kB,NF-κB）信号通路在介导CHSPs诱生宿主细胞炎症反应中的作用。
研究目的
本研究试图探讨3种CHSPs在体外诱导人脐静脉内皮细胞（Human umbilicalvein endothelial cells, HUVEC）分泌前炎症细胞因子IL-6、IL-1β的功能，以及ERK信号通路与NF-κB信号通路在CHSPs诱生炎症因子过程中的作用，初步研究TLR2、TLR4与CHSPs诱生炎症因子及其炎症信号通路之间的关联，对进一步阐明Cpn的致病机制具有重要意义。
研究方法
从Genbank上获得目的蛋白的全基因序列，通过分子生物学技术，设计并合成特异性引物，从制备的Cpn基因组模板上PCR扩增获得目的基因全长，经双酶切后与pGEX-6p-2载体构建成重组质粒，经PCR、双酶切、测序鉴定后进一步转化E. coli XL1-Blue构建原核表达重组体。在IPTG诱导下表达目的蛋白，Western-blot鉴定蛋白，以GST·BindTMResin或Ni-NTA亲和层析纯化目的蛋白，超滤管浓缩纯化蛋白，并调整蛋白至1mg/mL的使用浓度。将该蛋白以polymyxinB agarose处理，或加入50μg/mL多粘菌素B于37℃孵育30min~1h去除内毒素。
培养HUVEC，将上述获得的无内毒素的纯化蛋白分别以0.5、1、5、10、15、20、30μg/mL的不同浓度刺激细胞，检测HUVEC分泌炎症因子IL-1β、IL-6的水平；并设2、6、12、24、36、48、60h不同的蛋白刺激时间，检测HUVEC分泌IL-1β、IL-6的水平；将纯化产物经热处理（56℃，20min或100℃，30min）或去蛋白处理后，再与细胞孵育以检测刺激物对细胞分泌炎症因子的影响。
将3种CHSPs按2、15、30、45、60min的不同时间作用宿主细胞，收集细胞裂解液，并以抗磷酸化ERK及抗总ERK单抗检测ERK通路的活化。设立LPS与Cpn阳性对照，观察比较CHSPs与阳性对照激活ERK的方式；观察比较3种CHSPs激活ERK的作用强弱；以ERK通路特异性抑制剂U0126预孵育细胞30min，CHSPs刺激后检测ERK通路的活化状态及炎症因子的水平。
将3种CHSPs按30min，60min，120min的作用时间孵育宿主细胞，收获细胞核蛋白提取物，与末端标记的核酸探针结合进行凝胶迁移滞后实验（Electrophoretic Mobility ShiftAssay, EMSA）检测，以PBS及GST蛋白为阴性对照，观察比较3种CHSPs与阴性对照的结果，鉴定CHSPs是否能激活HUVEC诱导NF-κB分子活化、活化分子的作用特点以及活化作用的强弱；进一步用特异性抗p65单抗进行supershift实验，验证上述EMSA实验核蛋白提取物中是否含有活化产物p65特异性抗原。以NF-κB通路特异性抑制剂PDTC预孵育细胞60min，CHSPs刺激后检测NF-κB通路的活化状态及炎症因子的水平。以报告基因pNF-κB-luc转染细胞，并设pRL-TK内参，进一步证实CHSPs在刺激HUVEC后能否活化NF-κB分子进入细胞核，并与靶基因上特定的κ位点结合以调节报告基因转录。
以TLR2、TLR4、CD14、MD2分子根据实验设计不转染或单独或共转染人胚肾细胞（Human embryonic kidney cell）HEK293，用20μg/mL的CHSPs分别刺激，Western-blot检测TLR2和/或TLR4在CHSPs激活ERK信号通路中的作用；同时，将这些分子按不同实验目的与pNF-κB-luc与pRL-TK质粒共转染HEK293细胞，荧光素酶报告基因检测TLR2和/或TLR4在CHSPs激活NF-κB转录活性中的作用。
研究结果
以Cpn标准株AR-39基因组为模板扩增得到Chsp60和Chsp70的基因全长，并构建了pGEX-6p-2的原核重组质粒，质粒经PCR、双酶切、测序鉴定为CHSP60及CHSP70蛋白的基因序列；重组质粒在E.coli XL1-blue中诱导表达出分子量约86kDa和98kDa的融合蛋白，且均以可溶性的方式存在，Western-blot检测为GST标记的目的蛋白。复苏CHSP10重组菌并诱导表达重组蛋白。分别用GST·BindTMResin及Ni-NTA亲和层析纯化获得高纯度的目的蛋白，经超滤装置可将目的蛋白浓缩至原浓度的几十倍，调整后使工作蛋白浓度为1mg/mL，经多粘菌素B处理可去除内毒素。
CHSPs刺激宿主细胞分泌前炎症因子的作用根据不同的蛋白剂量及不同刺激时间有所不同。CHSPs刺激HUVEC分泌IL-1β、IL-6的水平随蛋白浓度的升高而呈上升趋势，在浓度为0.5~30μg/mL的范围内，以30μg/mL的CHSPs诱生细胞分泌的炎症因子水平最高；在2~60h的作用时间内，随刺激时间的延长，诱生炎症因子的水平逐渐升高，CHSP10在刺激12h后诱生IL-1β、IL-6的量到达高峰，而CHSP60和CHSP70在刺激24h后诱生IL-1β、IL-6的量到达高峰，3种CHSPs相比，CHSP60诱生炎症因子的能力最强，CHSP70次之，CHSP10最弱。
3种CHSPs刺激HUVEC后，Western-blot能检测到明显增强的磷酸化ERK分子，CHSP10在刺激15min时就可出现明显的磷酸化蛋白条带，经bandscan扫描分析，其在30min磷酸化ERK达到峰值；CHSP60和CHSP70均在刺激30min时磷酸化ERK条带出现高峰值，随后逐渐降低；Cpn激活HUVEC的ERK通路也是在30min时达到峰值，其后下降。而LPS活化HUVEC的ERK通路则是在15min达到峰值。三种CHSPs相比，CHP60活化ERK通路的作用最强，20μg/mL浓度产生的效果仅比Cpn（MOI=0.5）的作用稍弱，CHSP10与CHSP70作用次之。用U0126预孵育后，Western-blot检测到每组磷酸化ERK的量显著降低，同时CHSPs刺激HUVEC分泌2种炎症因子的水平也明显减少。
3种CHSPs刺激HUVEC后，EMSA显示，实验组均出现蛋白与DNA结合的复合物迁移带，且在30min，60min，120min的不同时间点，以蛋白作用60min时浓度最深，与阴性对照组相比有明显区别；3组CHSPs相比，CHSP60的作用最强，20μg/mL浓度产生的效果与Cpn（MOI=0.5）的作用相当，CHSP70作用次之，CHSP10最弱。Supershift实验显示3组CHSPs中除了特异性蛋白/DNA复合物迁移带外，均出现一条特异性的抗原抗体复合物超迁移带。用PDTC预孵育细胞后，EMSA显示蛋白/DNA复合物迁移带明显减弱甚至消失，同时，CHSPs刺激HUVEC分泌的IL-1β、IL-6的水平也明显减少。荧光素酶报告基因检测显示，CHSPs可活化HUVEC的NF-κB分子。
以人TLR2、TLR4和CD14及MD2质粒分别转染HEK293细胞后，Western-blot显示转染TLR2和/或TLR4后的实验组细胞经CHSPs刺激后均可显著上调磷酸化ERK的表达；以共转染pNF-κB-luc、pRL-TK质粒的报告基因来检测TLR在介导NF-κB通路中的作用，结果显示，转染了TLR的实验组，经刺激后可显著激活NF-κB信号通路，其中，共转染2种受体的实验组通路活化的程度要高于单项转染组，在单项转染组中，以TLR4介导激活NF-κB活化的作用强于TLR2。
结论
1.成功构建了CHSP10、CHSP60、CHSP70的原核表达载体，经诱导表达及纯化后获得了1mg/mL高纯度的相对分子量分别为15kDa、86kDa、98kDa的可溶性纯化蛋白；
2. CHSPs能在体外刺激HUVEC分泌炎症因子IL-1β、IL-6，并呈现剂量依赖性与时间依赖性，说明CHSPs可能参与Cpn诱导的炎症反应；
3. CHSPs能通过活化HUVEC的ERK信号通路诱生炎症因子；
4. CHSPs能通过活化HUVEC的NF-κB信号通路诱生炎症因子；
5. TLR2与TLR4参与介导CHSPs激活ERK和NF-κB信号通路的过程。
Chlamydophila pneumoniae (C. pneumoniae) is an obligate intracellularpathogen which spread worldwidely. It is responsible for approximately10%of casesof community acquired pneumonia and5%of cases of bronchitis among adults. It cancause a pharyngitis, bronchitis, sinusitis and possibly atherosclerosis. The infection iscommon with200,000~300,000new cases reported annually, mostly in young adults.The seroprevalence in the adult population is about50~70%and nearly everybody hasbeen infected at least once during their lifetime. The asymptomatic infection whichoften be neglected and untreated usually causes chronic persistent infection andsevere complications. Therefore, research on the pathogenic factor and thenosogenesis has to be of great value in diagnosis and prevention/control theinfection.
The C. pneumoniae-related disease is characterized by the inflammations whichinduced with the bacteria, and the HSPs may play a key role in inducing it. In order toclarify the pathogenesis of the C. pneumoniae HSPs (CHSPs) in inflammatory/immunopathologic lesions, and identify the receptors as well as the signal pathwaysinvolved in it, we applied the celluar microbiology to analysing the interaction ofTLR2/4, ERK and NF-κB signal pathway with CHSPs inducing inflammatoryreactions.
Objectives
The research is carried out to investigate①the functions of CHSPs in inducing thepro-inflammtory cytokines including interleukin-lβ (IL-1β) and IL-6in HUVEC;②the correlation between activation of ERK and NF-κB signal pathways withproduction of cytokines;③the role of TLR2and TLR4involving in activation ofERK and NF-κB signal pathways with CHSPs stimulation. These laid the foundationfor the pathogenesis research of C. pneumoniae.
Methods
The genetic sequence of CHSP60and CHSP70obtained from Genbank, and thespecific primers designed were used to amplifie the targeted genes from C.pneumoniae complete genome by polymerase chain reactions (PCR). The PCRproducts were ligased into the prokaryotic expression vector pGEX-6p-2to constructthe recombinant plasmid, and then were transformed into E. coli XL1-Blue. Afterinduced with IPTG, the three recombinant proteins were expressed and identified withwestern-blot, and purified using affinity chromatography. The purified proteins wereconcentrated into1mg/mL, and were excluded the possible contaminated endotoxinby using polymyxin B agarose or50μg/mL polymyxin B, then the samples weredetermined the endotoxin activity with Limulus amoebocyte lysate (LAL) test kit.
HUVEC were cultivated in24well plates and stimulated with purified samplesobtained above, the stimulus concentrations of CHSPs varied from0.5to30μg/mL(0.5、1、5、10、15、20、30μg/mL) and the secretion of IL-1β and IL-6aredose-dependent, the stimulus time of CHSPs varied from2to60h (2、6、12、24、36、48、60h) and the secretion of IL-1β and IL-6are time-dependent. To furtherconfirmed the ability of CHSPs inducing cytokines, the purified sample wereperformed by heating at56℃for30min or100℃for20min, or deproteinizingtreatments, respectively, and then were used to stimulate the cells for detection of thelevels of cytokines.
HUVEC were incubated with CHSPs in different time (2、15、30、45、60min).The cells lysate were collected to react with anti-phospho-ERK and anti-ERKantiboties in order to determine the activation of ERK signal pathway by western-blot.LPS and C. pneumoniae stimulation were used as positive controls to compare themanner in activating ERK pathway, and PBS and GST were used as negative controls.The ability in activating ERK pathway between CHSPs were compared. ERKinhibitor U0126were used to preincubated with cells, the level of IL-1β, IL-6and theactivation of ERK pathway were determined after CHSPs stimulation.
HUVEC were collected after30min,60min and120min incubation of CHSPs.Nuclear protein extracts were mixed with the labeling DNA probe and the activatedtranscription factors that bind DNA will migrate differently than free DNA. In electrophoretic mobility shift assay (EMSA), PBS and GST were used as negativecontrols to evaluate the ability of CHSPs in activating NF-κB by observingDNA-NF-κB interactions. Supershift assay were performed to further analyze thespecific p65antigen in extracts. NF-κB inhibitor PDTC were used to preincubatedwith cells, the level of IL-1β, IL-6and the activation of NF-κB pathway weredetermined after CHSPs stimulation. Dual-Luciferase reporter system was set up bytransfection with pNF-κB-luc and pRL-TK, the expression of luciferase in HUVECwere measured quantitatively to confirm the transcriptional activation induced withNF-κB.
HEK293cells were cotransiently transfected with or without different amountsof plasmids including human TLR2, TLR4as well as CD14and MD2according toresearch design, together with pNF-kB-luc and pRL-TK for normalization. Aferstimulation with CHSPs, the expression of luciferase in HUVEC were measuredquantitatively to confirm the TLR2/4involvment in activating signal pathway andinducing cytokines.
Results
The recombinant plasmids of targeted gene with pGEX-6p-2vector wereconstructed successfully and expressed in E. coli which induced with IPTG.Restriction enzyme digestion analysis and sequencing suggested the insertedfragments were targeted gene, which show100%similarity with sequence reported inGenbank. The results obtained by western blot analysis showed a band at the expectedmolecular weight, confirming correct insertion of the encoding gene. Purified proteinswere obtained with affinity chromatography and then concentrated into1mg/mLsamples. After removing the possible effects of LPS with polymyxin B agarose, theendotoxin activity of purified samples was determined <0.04EU/μg by using the LALassay kit.
Dose-dependent curve was observed in presence of various concentrations ofCHSPs (0.5,1,5,10,15,20,30μg/mL) in production of inflammatory cytokines IL-1β and IL-6in HUVEC. Low concentrations of prepared protein induced lowlevels of cytokine secretion and high concentrations induced high levels of cytokineproduction. Of increasing concentrations tested,20or30μg/mL CHSPs was thestronger inducers of cytokine production in HUVEC compared with0.5,1,5,10,15μg/mL protein. The pro-inflammatory activity was also time-dependent. From2to60h, the amounts of IL-6and IL-1β peaked at12h after stimulation with CHSP10, whilepeaked at24h after stimulation with CHSP60or CHSP70. Among CHSPs, the abilityof CHSP60inducing cytokines is strongest, CHSP10triggered low synthesis ofinflammatory cytokines comparable with CHSP60and CHSP70.
After stimulation with CHSPs, the phosphorylated ERK were tested time-dependently and the peak occurred at30min, which is consistent with thecircumstance of C. pneumoniae. The phosphorylated ERK peaked after15minstimulation with LPS and then decreased. Compared with other two CHSPs, theactiviation of CHSP60to ERK pathway is the strongest, while CHSP10and CHSP70is lower than it. U0126can not only inhibit the phosphorylation of ERK but alsodecreased the secretion of IL-1β and IL-6.
In EMSA assay, after incubated with CHSPs, the cell lysates can react with DNAprobes and show DNA-protein complex which migrate differently than free DNA.The activated NF-κB is the strongest which induced with CHSP60and peaked at60min with all the CHSPs. In addition to specific migrant band of DNA-proteincomplex, supershift test showed another supershift band of p65antigen-antibodycomplex. PDTC can not only inhibit the activation of NF-κB but also decreased thesecretion of IL-1β and IL-6. The reportor gene also comfirmed the activation ofNF-κB which induced with CHSPs in HUVEC.
After transfection with different amounts of indicated plasmids including TLR2or TLR4, CD14and MD2, the phosphorylated ERK increased in TLR2and/or TLR4transfected cells with CHSPs incubation. Dual luciferase reporter assay system thattransfected HEK293cells together with pNF-κB-luc and pRL-TK was to analyze therole of TLRs in activation of NF-κB pathway. It showed the effect of TLR4whichinvolved in activating NF-κB in HEK293cells was better than TLR2, and the effect of two TLRs was more effective than transfection of one TLR.
Conclusion
1. Prokaryotic expression vector of CHSPs were successfully constructed, and thesoluble purified proteins with molecular weight about15kDa,86kDa,98kDawere obtained.
2. CHSPs can induce secretion of proinflammation cytokines IL-1β and IL-6inHUVEC dose-dependently and time dependently, and may be implicated in thepathogenesis of inflammation which induced with C. pneumoniae.
3. CHSPs can activate ERK signal pathway to produce inflammatory cytokines inHUVEC.
4. CHSPs can activate NF-κB signal pathway to produce inflammatory cytokines inHUVEC.
5. TLR2and TLR4may be involved in activation of ERK and NF-κB signalpathways induced with CHSPs.
肺炎嗜衣原体（Chlamydophila pneumoniae, Cpn）是一类重要的呼吸道病原体，造成全球约10%的社区获得性肺炎及5%支气管炎和窦炎病例的发生，还与冠心病、动脉粥样硬化、哮喘、反应性关节炎等多种慢性疾病相关。许多国家的人群Cpn感染率占全国人口的50%~70%，但人群感染该菌后多表现为持续的“隐蔽性”，患者病情易被忽视而导致Cpn感染反复迁延，造成机体不可逆的病理改变甚至严重并发症。研究Cpn的致病因素，阐明毒力相关蛋白的致病作用及机制对预防Cpn感染，控制其传播具有重要意义。
Cpn感染引起的炎症反应是多种疾病的发病基础。其中，Cpn热休克蛋白（Heat Shock Proteins, HSPs）在病原体介导的炎症反应中可能发挥重要作用。为研究CHSPs在Cpn诱导炎症反应中的功能及其相关受体及信号通路，进一步说明CHSPs在炎症/免疫病理损伤中的作用及其可能的致病机制，拟运用细胞微生物学分析Toll样受体（Toll-like receptor, TLR）2和TLR4、胞外信号调节激酶（Extracellular signal-regulated kinase, ERK）通路和核因子κB（Nuclear factor kB,NF-κB）信号通路在介导CHSPs诱生宿主细胞炎症反应中的作用。
研究目的
本研究试图探讨3种CHSPs在体外诱导人脐静脉内皮细胞（Human umbilicalvein endothelial cells, HUVEC）分泌前炎症细胞因子IL-6、IL-1β的功能，以及ERK信号通路与NF-κB信号通路在CHSPs诱生炎症因子过程中的作用，初步研究TLR2、TLR4与CHSPs诱生炎症因子及其炎症信号通路之间的关联，对进一步阐明Cpn的致病机制具有重要意义。
研究方法
从Genbank上获得目的蛋白的全基因序列，通过分子生物学技术，设计并合成特异性引物，从制备的Cpn基因组模板上PCR扩增获得目的基因全长，经双酶切后与pGEX-6p-2载体构建成重组质粒，经PCR、双酶切、测序鉴定后进一步转化E. coli XL1-Blue构建原核表达重组体。在IPTG诱导下表达目的蛋白，Western-blot鉴定蛋白，以GST·BindTMResin或Ni-NTA亲和层析纯化目的蛋白，超滤管浓缩纯化蛋白，并调整蛋白至1mg/mL的使用浓度。将该蛋白以polymyxinB agarose处理，或加入50μg/mL多粘菌素B于37℃孵育30min~1h去除内毒素。
培养HUVEC，将上述获得的无内毒素的纯化蛋白分别以0.5、1、5、10、15、20、30μg/mL的不同浓度刺激细胞，检测HUVEC分泌炎症因子IL-1β、IL-6的水平；并设2、6、12、24、36、48、60h不同的蛋白刺激时间，检测HUVEC分泌IL-1β、IL-6的水平；将纯化产物经热处理（56℃，20min或100℃，30min）或去蛋白处理后，再与细胞孵育以检测刺激物对细胞分泌炎症因子的影响。
将3种CHSPs按2、15、30、45、60min的不同时间作用宿主细胞，收集细胞裂解液，并以抗磷酸化ERK及抗总ERK单抗检测ERK通路的活化。设立LPS与Cpn阳性对照，观察比较CHSPs与阳性对照激活ERK的方式；观察比较3种CHSPs激活ERK的作用强弱；以ERK通路特异性抑制剂U0126预孵育细胞30min，CHSPs刺激后检测ERK通路的活化状态及炎症因子的水平。
将3种CHSPs按30min，60min，120min的作用时间孵育宿主细胞，收获细胞核蛋白提取物，与末端标记的核酸探针结合进行凝胶迁移滞后实验（Electrophoretic Mobility ShiftAssay, EMSA）检测，以PBS及GST蛋白为阴性对照，观察比较3种CHSPs与阴性对照的结果，鉴定CHSPs是否能激活HUVEC诱导NF-κB分子活化、活化分子的作用特点以及活化作用的强弱；进一步用特异性抗p65单抗进行supershift实验，验证上述EMSA实验核蛋白提取物中是否含有活化产物p65特异性抗原。以NF-κB通路特异性抑制剂PDTC预孵育细胞60min，CHSPs刺激后检测NF-κB通路的活化状态及炎症因子的水平。以报告基因pNF-κB-luc转染细胞，并设pRL-TK内参，进一步证实CHSPs在刺激HUVEC后能否活化NF-κB分子进入细胞核，并与靶基因上特定的κ位点结合以调节报告基因转录。
以TLR2、TLR4、CD14、MD2分子根据实验设计不转染或单独或共转染人胚肾细胞（Human embryonic kidney cell）HEK293，用20μg/mL的CHSPs分别刺激，Western-blot检测TLR2和/或TLR4在CHSPs激活ERK信号通路中的作用；同时，将这些分子按不同实验目的与pNF-κB-luc与pRL-TK质粒共转染HEK293细胞，荧光素酶报告基因检测TLR2和/或TLR4在CHSPs激活NF-κB转录活性中的作用。
研究结果
以Cpn标准株AR-39基因组为模板扩增得到Chsp60和Chsp70的基因全长，并构建了pGEX-6p-2的原核重组质粒，质粒经PCR、双酶切、测序鉴定为CHSP60及CHSP70蛋白的基因序列；重组质粒在E.coli XL1-blue中诱导表达出分子量约86kDa和98kDa的融合蛋白，且均以可溶性的方式存在，Western-blot检测为GST标记的目的蛋白。复苏CHSP10重组菌并诱导表达重组蛋白。分别用GST·BindTMResin及Ni-NTA亲和层析纯化获得高纯度的目的蛋白，经超滤装置可将目的蛋白浓缩至原浓度的几十倍，调整后使工作蛋白浓度为1mg/mL，经多粘菌素B处理可去除内毒素。
CHSPs刺激宿主细胞分泌前炎症因子的作用根据不同的蛋白剂量及不同刺激时间有所不同。CHSPs刺激HUVEC分泌IL-1β、IL-6的水平随蛋白浓度的升高而呈上升趋势，在浓度为0.5~30μg/mL的范围内，以30μg/mL的CHSPs诱生细胞分泌的炎症因子水平最高；在2~60h的作用时间内，随刺激时间的延长，诱生炎症因子的水平逐渐升高，CHSP10在刺激12h后诱生IL-1β、IL-6的量到达高峰，而CHSP60和CHSP70在刺激24h后诱生IL-1β、IL-6的量到达高峰，3种CHSPs相比，CHSP60诱生炎症因子的能力最强，CHSP70次之，CHSP10最弱。
3种CHSPs刺激HUVEC后，Western-blot能检测到明显增强的磷酸化ERK分子，CHSP10在刺激15min时就可出现明显的磷酸化蛋白条带，经bandscan扫描分析，其在30min磷酸化ERK达到峰值；CHSP60和CHSP70均在刺激30min时磷酸化ERK条带出现高峰值，随后逐渐降低；Cpn激活HUVEC的ERK通路也是在30min时达到峰值，其后下降。而LPS活化HUVEC的ERK通路则是在15min达到峰值。三种CHSPs相比，CHP60活化ERK通路的作用最强，20μg/mL浓度产生的效果仅比Cpn（MOI=0.5）的作用稍弱，CHSP10与CHSP70作用次之。用U0126预孵育后，Western-blot检测到每组磷酸化ERK的量显著降低，同时CHSPs刺激HUVEC分泌2种炎症因子的水平也明显减少。
3种CHSPs刺激HUVEC后，EMSA显示，实验组均出现蛋白与DNA结合的复合物迁移带，且在30min，60min，120min的不同时间点，以蛋白作用60min时浓度最深，与阴性对照组相比有明显区别；3组CHSPs相比，CHSP60的作用最强，20μg/mL浓度产生的效果与Cpn（MOI=0.5）的作用相当，CHSP70作用次之，CHSP10最弱。Supershift实验显示3组CHSPs中除了特异性蛋白/DNA复合物迁移带外，均出现一条特异性的抗原抗体复合物超迁移带。用PDTC预孵育细胞后，EMSA显示蛋白/DNA复合物迁移带明显减弱甚至消失，同时，CHSPs刺激HUVEC分泌的IL-1β、IL-6的水平也明显减少。荧光素酶报告基因检测显示，CHSPs可活化HUVEC的NF-κB分子。
以人TLR2、TLR4和CD14及MD2质粒分别转染HEK293细胞后，Western-blot显示转染TLR2和/或TLR4后的实验组细胞经CHSPs刺激后均可显著上调磷酸化ERK的表达；以共转染pNF-κB-luc、pRL-TK质粒的报告基因来检测TLR在介导NF-κB通路中的作用，结果显示，转染了TLR的实验组，经刺激后可显著激活NF-κB信号通路，其中，共转染2种受体的实验组通路活化的程度要高于单项转染组，在单项转染组中，以TLR4介导激活NF-κB活化的作用强于TLR2。
结论
1.成功构建了CHSP10、CHSP60、CHSP70的原核表达载体，经诱导表达及纯化后获得了1mg/mL高纯度的相对分子量分别为15kDa、86kDa、98kDa的可溶性纯化蛋白；
2. CHSPs能在体外刺激HUVEC分泌炎症因子IL-1β、IL-6，并呈现剂量依赖性与时间依赖性，说明CHSPs可能参与Cpn诱导的炎症反应；
3. CHSPs能通过活化HUVEC的ERK信号通路诱生炎症因子；
4. CHSPs能通过活化HUVEC的NF-κB信号通路诱生炎症因子；
5. TLR2与TLR4参与介导CHSPs激活ERK和NF-κB信号通路的过程。
Chlamydophila pneumoniae (C. pneumoniae) is an obligate intracellularpathogen which spread worldwidely. It is responsible for approximately10%of casesof community acquired pneumonia and5%of cases of bronchitis among adults. It cancause a pharyngitis, bronchitis, sinusitis and possibly atherosclerosis. The infection iscommon with200,000~300,000new cases reported annually, mostly in young adults.The seroprevalence in the adult population is about50~70%and nearly everybody hasbeen infected at least once during their lifetime. The asymptomatic infection whichoften be neglected and untreated usually causes chronic persistent infection andsevere complications. Therefore, research on the pathogenic factor and thenosogenesis has to be of great value in diagnosis and prevention/control theinfection.
The C. pneumoniae-related disease is characterized by the inflammations whichinduced with the bacteria, and the HSPs may play a key role in inducing it. In order toclarify the pathogenesis of the C. pneumoniae HSPs (CHSPs) in inflammatory/immunopathologic lesions, and identify the receptors as well as the signal pathwaysinvolved in it, we applied the celluar microbiology to analysing the interaction ofTLR2/4, ERK and NF-κB signal pathway with CHSPs inducing inflammatoryreactions.
Objectives
The research is carried out to investigate①the functions of CHSPs in inducing thepro-inflammtory cytokines including interleukin-lβ (IL-1β) and IL-6in HUVEC;②the correlation between activation of ERK and NF-κB signal pathways withproduction of cytokines;③the role of TLR2and TLR4involving in activation ofERK and NF-κB signal pathways with CHSPs stimulation. These laid the foundationfor the pathogenesis research of C. pneumoniae.
Methods
The genetic sequence of CHSP60and CHSP70obtained from Genbank, and thespecific primers designed were used to amplifie the targeted genes from C.pneumoniae complete genome by polymerase chain reactions (PCR). The PCRproducts were ligased into the prokaryotic expression vector pGEX-6p-2to constructthe recombinant plasmid, and then were transformed into E. coli XL1-Blue. Afterinduced with IPTG, the three recombinant proteins were expressed and identified withwestern-blot, and purified using affinity chromatography. The purified proteins wereconcentrated into1mg/mL, and were excluded the possible contaminated endotoxinby using polymyxin B agarose or50μg/mL polymyxin B, then the samples weredetermined the endotoxin activity with Limulus amoebocyte lysate (LAL) test kit.
HUVEC were cultivated in24well plates and stimulated with purified samplesobtained above, the stimulus concentrations of CHSPs varied from0.5to30μg/mL(0.5、1、5、10、15、20、30μg/mL) and the secretion of IL-1β and IL-6aredose-dependent, the stimulus time of CHSPs varied from2to60h (2、6、12、24、36、48、60h) and the secretion of IL-1β and IL-6are time-dependent. To furtherconfirmed the ability of CHSPs inducing cytokines, the purified sample wereperformed by heating at56℃for30min or100℃for20min, or deproteinizingtreatments, respectively, and then were used to stimulate the cells for detection of thelevels of cytokines.
HUVEC were incubated with CHSPs in different time (2、15、30、45、60min).The cells lysate were collected to react with anti-phospho-ERK and anti-ERKantiboties in order to determine the activation of ERK signal pathway by western-blot.LPS and C. pneumoniae stimulation were used as positive controls to compare themanner in activating ERK pathway, and PBS and GST were used as negative controls.The ability in activating ERK pathway between CHSPs were compared. ERKinhibitor U0126were used to preincubated with cells, the level of IL-1β, IL-6and theactivation of ERK pathway were determined after CHSPs stimulation.
HUVEC were collected after30min,60min and120min incubation of CHSPs.Nuclear protein extracts were mixed with the labeling DNA probe and the activatedtranscription factors that bind DNA will migrate differently than free DNA. In electrophoretic mobility shift assay (EMSA), PBS and GST were used as negativecontrols to evaluate the ability of CHSPs in activating NF-κB by observingDNA-NF-κB interactions. Supershift assay were performed to further analyze thespecific p65antigen in extracts. NF-κB inhibitor PDTC were used to preincubatedwith cells, the level of IL-1β, IL-6and the activation of NF-κB pathway weredetermined after CHSPs stimulation. Dual-Luciferase reporter system was set up bytransfection with pNF-κB-luc and pRL-TK, the expression of luciferase in HUVECwere measured quantitatively to confirm the transcriptional activation induced withNF-κB.
HEK293cells were cotransiently transfected with or without different amountsof plasmids including human TLR2, TLR4as well as CD14and MD2according toresearch design, together with pNF-kB-luc and pRL-TK for normalization. Aferstimulation with CHSPs, the expression of luciferase in HUVEC were measuredquantitatively to confirm the TLR2/4involvment in activating signal pathway andinducing cytokines.
Results
The recombinant plasmids of targeted gene with pGEX-6p-2vector wereconstructed successfully and expressed in E. coli which induced with IPTG.Restriction enzyme digestion analysis and sequencing suggested the insertedfragments were targeted gene, which show100%similarity with sequence reported inGenbank. The results obtained by western blot analysis showed a band at the expectedmolecular weight, confirming correct insertion of the encoding gene. Purified proteinswere obtained with affinity chromatography and then concentrated into1mg/mLsamples. After removing the possible effects of LPS with polymyxin B agarose, theendotoxin activity of purified samples was determined <0.04EU/μg by using the LALassay kit.
Dose-dependent curve was observed in presence of various concentrations ofCHSPs (0.5,1,5,10,15,20,30μg/mL) in production of inflammatory cytokines IL-1β and IL-6in HUVEC. Low concentrations of prepared protein induced lowlevels of cytokine secretion and high concentrations induced high levels of cytokineproduction. Of increasing concentrations tested,20or30μg/mL CHSPs was thestronger inducers of cytokine production in HUVEC compared with0.5,1,5,10,15μg/mL protein. The pro-inflammatory activity was also time-dependent. From2to60h, the amounts of IL-6and IL-1β peaked at12h after stimulation with CHSP10, whilepeaked at24h after stimulation with CHSP60or CHSP70. Among CHSPs, the abilityof CHSP60inducing cytokines is strongest, CHSP10triggered low synthesis ofinflammatory cytokines comparable with CHSP60and CHSP70.
After stimulation with CHSPs, the phosphorylated ERK were tested time-dependently and the peak occurred at30min, which is consistent with thecircumstance of C. pneumoniae. The phosphorylated ERK peaked after15minstimulation with LPS and then decreased. Compared with other two CHSPs, theactiviation of CHSP60to ERK pathway is the strongest, while CHSP10and CHSP70is lower than it. U0126can not only inhibit the phosphorylation of ERK but alsodecreased the secretion of IL-1β and IL-6.
In EMSA assay, after incubated with CHSPs, the cell lysates can react with DNAprobes and show DNA-protein complex which migrate differently than free DNA.The activated NF-κB is the strongest which induced with CHSP60and peaked at60min with all the CHSPs. In addition to specific migrant band of DNA-proteincomplex, supershift test showed another supershift band of p65antigen-antibodycomplex. PDTC can not only inhibit the activation of NF-κB but also decreased thesecretion of IL-1β and IL-6. The reportor gene also comfirmed the activation ofNF-κB which induced with CHSPs in HUVEC.
After transfection with different amounts of indicated plasmids including TLR2or TLR4, CD14and MD2, the phosphorylated ERK increased in TLR2and/or TLR4transfected cells with CHSPs incubation. Dual luciferase reporter assay system thattransfected HEK293cells together with pNF-κB-luc and pRL-TK was to analyze therole of TLRs in activation of NF-κB pathway. It showed the effect of TLR4whichinvolved in activating NF-κB in HEK293cells was better than TLR2, and the effect of two TLRs was more effective than transfection of one TLR.
Conclusion
1. Prokaryotic expression vector of CHSPs were successfully constructed, and thesoluble purified proteins with molecular weight about15kDa,86kDa,98kDawere obtained.
2. CHSPs can induce secretion of proinflammation cytokines IL-1β and IL-6inHUVEC dose-dependently and time dependently, and may be implicated in thepathogenesis of inflammation which induced with C. pneumoniae.
3. CHSPs can activate ERK signal pathway to produce inflammatory cytokines inHUVEC.
4. CHSPs can activate NF-κB signal pathway to produce inflammatory cytokines inHUVEC.
5. TLR2and TLR4may be involved in activation of ERK and NF-κB signalpathways induced with CHSPs.
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