【作者】 李静；

【导师】 赵蔚明； 王红；

【作者基本信息】 山东大学 ， 病原生物学， 2017， 博士

【摘要】 第一部分鼠衣原体肺部感染中NK细胞对CD4+T细胞亚群的调节作用研究目的衣原体(Chlamydia)是一种严格细胞内寄生的原核细胞型微生物,可引起多种人类疾病。沙眼衣原体(Chlamydia trachomatis,C.trachomatis)是最常见的性传播性疾病的病原体,并可引起新生儿衣原体肺炎。肺炎衣原体(Chlamydia pneumoniae,C.pneuwoniae)是继肺炎链球尚、流感嗜血杆菌之后引起社区获得性肺炎的第三位主要病原体,在儿童、成人尤其是老人中引起急性非典型性肺炎。尽管多种广谱抗生素可用于治疗衣原体感染,但由于个体间不同的临床表现,药物耐药性等因素的存在影响了对衣原体病及时的诊断和有效治疗。目前尚无有效疫苗上市用于衣原体疾病的预防。因此,深入研究宿主抗衣原体感染中的免疫应答及免疫调控机制是探索新型防治策略的基石,对于进一步控制衣原体感染是必要的。衣原体感染后,宿主的固有免疫和适应性免疫共同参与抗感染免疫应答。衣原体感染可激活固有免疫细胞包括DC(树突状细胞,dendritic cell),巨噬细胞(macrophage,MΦ)和NK细胞(自然杀伤细胞,nature killer cell)等。这些细胞不仅能增强固有免疫应答也可以进一步影响适应性免疫应答。机体的抗衣原体适应性免疫以细胞免疫为主。CD4+T细胞在衣原体感染中具有重要作用,其中,以分泌IFN-γ为特征的CD4+T(Th1)细胞免疫应答在机体清除衣原体感染中发挥至关重要的保护性作用。Th17细胞通过产生IL-17和IL-22等细胞因子协同促进Th1细胞应答在机体清除衣原体感染中发挥重要作用。研究发现人和小鼠衣原体感染局部组织中Tregs应答增强且与组织病理损伤有关。NK细胞是固有免疫细胞的重要成员,在机体抵御病原体感染中起重要作用。除杀伤作用外,NK细胞的免疫调节作用近年来受到学者重视。越来越多的证据表明,在感染性疾病和炎症性疾病中NK细胞可调节T细胞应答。目前,尽管已有研究表明NK细胞在抗衣原体感染中起保护作用,但是有关NK细胞的免疫调节机制报道不多,尤其缺乏NK细胞对不同CD4+T细胞亚群免疫调控作用的研究。本研究采用BALB/c小鼠衣原体呼吸道感染模型,较系统的研究了 NK细胞对重要CD4+T细胞亚群的免疫调节作用。采用anti-asialo-GM1抗体特异性清除体内NK细胞。检测NK细胞去除对小鼠肺部衣原体清除能力、组织病理损伤的影响。利用ELISA、细胞内细胞因子染色以及荧光定量PCR等技术检测NK细胞对感染局部(肺组织)、免疫器官(脾脏和纵膈淋巴结)的不同CD4+T细胞亚群Th1、Th1 7以及Tregs的免疫调节作用,并进一步分析评估NK细胞对维持CD4+T细胞亚群免疫平衡的作用。研究方法1 NK细胞去除对小鼠衣原体肺部感染疾病的影响1.1鼠衣原体(C.muridaruw)的培养与纯化:培养Hep-2细胞,感染鼠衣原体,观察包涵体形成。玻璃珠方法收集感染细胞后,超声破碎释放衣原体,离心去除细胞碎片。超速离心纯化衣原体EB,-80℃保存1.2鼠肺部感染模型的建立:小鼠(6-8周龄,雌性BALB/c小鼠)麻醉后鼻吸入感染C.miridriarum(1 × 103IFU),建立C.muridarum肺部感染模型。1.3 NK细胞体内去除方法:小鼠于衣原体感染前一天,感染后一天尾静脉注射NK细胞封闭抗体(anti-asialo-GM1抗体),对照组注射同型对照抗体,之后间隔72h注射一次,直到实验结束。流式细胞术检测肺中NK细胞清除效率。正常对照组采用非感染的野生型小鼠。1.4小鼠疾病评估:小鼠经鼻吸入感染衣原体后,每隔24h记录体重;感染后不同时间(0、3、6、12和22天)取小鼠右肺中叶进行HE染色(hematoxylin-eosin staining),观察并评估肺组织病理损伤;取左肺制备肺匀浆,检测包涵体形成单位(IFU),观察肺中衣原体生长情况。2 NK细胞对T细胞亚群应答的调控作用2.1NK细胞对Th1、Th17和Tregs细胞分泌细胞因子的影响:小鼠于感染后6天和12天麻醉后处死,取肺(右肺上叶和下叶)、脾和纵膈淋巴结制备单个核细胞;心脏取血,获得血清。采用酶联免疫吸附实验(enzyme-linked immunosorbent assay,ELISA)检测小鼠肺、脾和纵膈淋巴结细胞培养上清(IFN-γ、IL-17A、IL-22和IL-10)和血清中(IFN-γ、IL-17A和IL-22)细胞因子分泌。2.2NK细胞对Th1、Th17和Tregs细胞群的影响:小鼠于感染后6天和12天麻醉后处死,制备小鼠肺、脾和纵膈淋巴结单个核细胞,细胞内因子染色,流式细胞术检测(CD3+CD4+IFN-γ+T)Th1、(CD3+CD4+IL-17+T)Th17 细胞和(CD4+CD25+Foxp3+T)Tregs 的比例和绝对数。3 NK细胞对Th1/Treg和Th17/Treg应答平衡的影响3.1 NK细胞对T细胞亚群转录因子表达的影响:取衣原体感染后6天和12天小鼠的脾和肺单个核细胞,提取总RNA,实时定量PCR检测Th1(T-bet)、Th2(GATA3)、Th17(RORγT)和Treg(Foxp3)细胞分化发育特异性转录因子的mRNA表达水平。3.2 NK细胞对感染局部T细胞分化相关细胞因子的影响:取衣原体感染后6天和12天小鼠的肺和纵膈淋巴结单个核细胞培养上清,采用ELISA方法检测IL-12、TGF-β和IL-6的表达变化。3.3 NK细胞对Th1/Treg和Th17/Treg比值的影响:根据以上流式数据和细胞计数结果,分析小鼠肺、脾和纵膈淋巴结中Th1、Th17和Tregs的绝对数,计算Th1/Treg 和 Th17/Treg 比值。研究结果1 NK细胞去除小鼠衣原体肺部感染加重1.1 Anti-asialo-GM1抗体可特异性去除NK细胞:于衣原体感染后第四天处死小鼠,检测小鼠肺中NK细胞比例。结果显示,注射anti-asialo-GM1抗体的小鼠肺中NK细胞数量和比例都显著降低,清除率为90%左右。1.2 NK细胞去除导致小鼠肺部感染加重:通过比较衣原体肺部感染后同型对照鼠和NK细胞去除鼠疾病变化发现,NK细胞去除鼠体重下降明显且恢复减慢(0-22天),肺部载菌量增加(感染后3、6、12和22天),HE染色结果显示,肺中支气管和血管周围有大量炎细胞浸润,组织病理损伤更重(感染后6天和12 天)。2衣原体肺部感染中,NK细胞增强Th1和Th17应答,抑制Tregs应答2.1 NK细胞促进IFN--γ、IL-17和IL-22表达:IFN-γ主要由Th1细胞分泌,而Th17细胞以产生IL-17和IL-22等细胞因子为特征。我们使用ELISA方法检测衣原体感染后6天和12天小鼠肺、脾和纵膈淋巴结细胞以及血清中IFN-γ、IL-17和IL-22的表达。结果显示,NK细胞去除后,以上器官中IFN-γ、IL-17和IL-22的表达水平均显著下降,血清中的浓度也明显降低。2.2 NK细胞去除导致Th1和Th17细胞的比例和绝对数降低:Th1细胞免疫应答在机体清除衣原体感染中发挥关键作用,Th17细胞协同促进Th1细胞应答。采用细胞内细胞因子染色,流式细胞术检测(CD3+CD4+IFN-γ+T)Th1和(CD3+CD4+IL-17+T)Th17细胞的比例和绝对数。分析NK细胞去除对Th1和Th17的影响。结果显示,与同型对照组相比,NK细胞去除组小鼠肺、脾和纵膈淋巴结中Th1、Th17细胞的比例和绝对数均明显降低,衣原体感染后6天和12天结果一致。说明NK细胞促进感染局部(肺)以及免疫器官(脾和纵膈淋巴结)屮Th1、Th17细胞的免疫应答。2.3 NK细胞去除导致Tregs的比例和绝对数明显升高:观察小鼠肺部感染过程中NK细胞去除对Tregs应答的影响。细胞内细胞因子染色,流式细胞术分析(CD4+CD25+Foxp3+T)Tregs的比例和绝对数。结果显示,与同型对照组相比,NK细胞去除组小鼠肺、脾和纵膈淋巴结中Tregs的比例和绝对数显著增加,尤其在衣原体感染早期(6天)肺中Tregs的升高比较显著。证明,在衣原体肺部感染中,NK细胞对Tregs应答具有抑制作用,尤其在感染早期(6天)较为明显。3衣原体肺部感染中,NK细胞可维持Th1/Treg和Th17/Treg应答平衡3.1NK细胞促进Th1和Th17细胞分化,抑制Tregs分化:实时定量PCR检测小鼠脾和肺细胞中T细胞亚群特异性转录因子的表达。结果表明,NK细胞去除诱导脾和肺细胞中转录因子T-bet(Th1)和RORyT(Th17)表达降低,Foxp3(Tregs)和 GATA3(Th2)表达增加。3.2 NK细胞去除导致维持T细胞亚群分化的细胞因子环境改变:采用ELISA方法,检测感染局部肺和纵膈淋巴结细胞培养上清中维持T细胞亚群分化的细胞因子表达变化。结果显示,衣原体感染中,NK细胞去除鼠肺和淋巴结细胞中维持Th1和Th17细胞分化的IL-12p40和IL-6分泌降低,而诱导Tregs分化的TGF-β表达水平增加。说明,NK细胞去除导致细胞因子环境改变促进了 Tregs的分化,而不利于Th1和Th17细胞分化。3.3 NK细胞去除诱导Th1/Treg和Th17/Treg应答失衡:分析T细胞亚群的绝对数并计算Th1/Treg和Th17/Treg比值。结果发现,在衣原体肺部感染后第6天,NK细胞去除导致小鼠肺、脾和纵膈淋巴结中Th1/Treg和Th17/Treg比值均降低,机体免疫应答倾向于Treg方向。在感染后12天,尽管同型对照组Th1/Treg和Th17/Treg的比值很低,NK细胞去除组的比值仍然显著低于对照组。说明,衣原体肺部感染中,NK细胞通过调控Th1/Treg和Th17/Treg应答平衡,发挥其免疫保护性作用。研究结论与意义1.NK细胞在BALB/c小鼠抵御衣原体肺部感染中发挥重要保护作用,有助于清除衣原体感染,减轻肺组织病理损伤,加快疾病恢复。2.NK细胞可显著抑制衣原体感染局部(肺脏)和外周淋巴器官(脾和纵膈淋巴结)中的Tregs应答,有利于增强保护性免疫反应。3.NK细胞通过促进Th1和Th17应答,抑制Tregs应答,从而维持Th1/Treg和Th17/Treg免疫平衡,增强机体清除肺部衣原体感染的能力,减轻组织病理损伤。本研究系统的揭示了在衣原体肺部感染中NK细胞对重要CD4+T细胞亚群的免疫调节作用,尤其是首次发现NK细胞对Tregs应答具有明显抑制作用。NK细胞通过维持Th1/Treg和Th17/Treg免疫平衡(促进Th1和Th17应答,抑制Tregs应答),在机体抵御衣原体肺部感染中发挥重要的保护作用。本研究为NK细胞免疫调节机制的研究提供了新见解。第二部分鼠衣原体肺部感染中NK细胞对肺巨噬细胞极化的调节作用研究目的巨噬细胞是重要的固有免疫细胞,在维持免疫稳态和抵御病原体感染中起重要作用。巨噬细胞具有极强的可塑性,极化状态是其发挥不同功能的关键,巨噬细胞至少存在两种不同的极化状态,分为M1和M2型巨噬细胞。M1型巨噬细胞(又称为经典活化的巨噬细胞)可被IFN-γ、PAMPs、TNF-α等活化,产生前炎症细胞因子,高表达iNOS,可直接杀伤病原体。M2型巨噬细胞(又称为替代性活化巨噬细胞),由Th2型细胞因子例如IL-4、DAMPs和TGF-β等诱导激活,高表达精氨酸酶-1,YM-1,CD206,参与寄生虫的清除,免疫调节以及促进组织重建。与Th1/Th2极化相似,巨噬细胞M1与M2表型之间的转换同样由特异性转录因子控制。现已知,C/EBP-α、PU.1、NF-κB(P65/P50)、Stat1、IRF5和HIFα等分子参与调控M1型巨噬细胞激活;而Stat3、Stat6、IRF4和SOCS1等胞内信号分子则参与了对巨噬细胞M2型极化的调控。巨噬细胞的极化机制复杂,一直是最近研究的焦点。研究表明,巨噬细胞参与机体抵御与清除病原体感染,其不同极化状态在控制衣原体感染中的作用已有一定的了解:M1型巨噬细胞可限制衣原体在细胞内增殖,并通过分泌细胞因了增强固有免疫和适应性免疫应答,利于机体快速清除感染;衣原体可在M2型巨噬细胞中正常增殖,并转移感染机体其他器官。因此,诱导适度的M1型巨噬细胞应答有利于机体对衣原体感染的清除。NK细胞可根据细胞因子微环境以及与其他免疫细胞相互作用控制其对巨噬细胞的细胞毒性或正向免疫调节作用,在不同病原体感染中发挥不同的作用。在衣原体肺部感染中,NK细胞是否可调节巨噬细胞,尤其是肺局部巨噬细胞目前尚未见报道。MicroRNAs(miRNAs)是一类介导基因转录后沉默调控基因表达的内源性非编码RNA,已发现多种miRNAs可调控巨噬细胞极化中关键分子的转录,参与巨噬细胞极化的调控。在衣原体感染中,miRNAs是否参与NK细胞诱导的巨噬细胞极化过程尚不清楚。本研究拟探讨在衣原体肺部感染中NK细胞对肺巨噬细胞极化的调节作用及可能机制。采用BALB/c小鼠呼吸道感染模型,注射anti-asialo-GM1抗体特异性清除体内NK细胞。检测各组小鼠肺巨噬细胞极化状态,并进一步分析肺巨噬细胞中相关miRNAs及其靶分子的表达变化。研究方法1.巨噬细胞的纯化与流式分析1.1肺巨噬细胞纯化:BALB/c小鼠鼻吸入感染衣原体,实验分组同第一部分。于感染后第6天处死小鼠,取肺组织用胶原酶XI(10mg/ml)消化,percoll密度梯度离心,制备单个核细胞。采用anti-F4/80磁珠抗体,分离纯化肺巨噬细胞。1.2肺巨噬细胞纯度分析:取5×105个纯化的肺巨噬细胞,采用表面染色,流式细胞术检测分析F4/80+CD11 b+细胞的比例。2.NK细胞对巨噬细胞极化的影响2.1 NK细胞对肺巨噬细胞极化细胞因子表达的影响:磁珠分离纯化同型对照组和NK细胞去除组小鼠肺巨噬细胞(感染后第6天),提取总RNA,实时定量PCR检测TNF-α、IL-6(M1型细胞因子)和IL-10(M2型细胞因子)的mRNA表达。2.2 NK细胞对肺巨噬细胞极化标志分子的影响:取纯化的小鼠肺巨噬细胞,实时定量PCR检测M1型巨噬细胞标志物(iNOS、IRF5)和M2型巨噬细胞标志物(Arg-1、IRF4)mRNA表达;取小鼠左肺下叶,提取肺组织总蛋白,western blot方法检测iNOS、Arg-1、IRF5和IRF4蛋白表达;根据mRNA和蛋白相对表达水平计算iNOS/Arg-1和IRF5/IRF4比值。2.3 NK细胞对肺中M1和M2细胞比例的影响:取小鼠肺组织,制备单个核细胞,细胞内染色后,流式细胞术分析M1(CD45+F4/80+iNOS+)和M2(CD45+F4/80+CD206+)型巨噬细胞的比例。3.NK细胞调节肺巨噬细胞极化机制3.1 NK细胞影响肺巨噬细胞中miR-155和miR-223的表达:实验分组同上,于衣原体感染后第6天处死小鼠,制备肺单个核细胞,磁珠分离纯化肺巨噬细胞,提取小RNA,使用miR-155和miR-223特异性反转录引物将RNA反转录成cDNA,采用实时定量PCR方法检测miR-155和miR-223的表达。3.2 NK细胞对miR-155和miR-223相关靶分子表达的影响:提取肺巨噬细胞总RNA,实时定量PCR检测miR-155的靶分子SOCS1和miR-223的靶分子pknoxl的mRNA表达。研究结果1.衣原体肺部感染中,NK细胞促进肺局部M1,抑制M2型巨噬细胞极化1.1肺巨噬细胞纯度:采用anti-F4/80磁珠分离纯化肺巨噬细胞,流式细胞术分析巨噬细胞纯度。结果显示,磁珠分选后,F4/80+CD11b+巨噬细胞比例为96%以上。1.2衣原体肺部感染中,NK细胞促进肺巨噬细胞产生M1型极化细胞因子,抑制M2型细胞因子的产生:衣原体肺部感染后第六天,实时定量 PCR检测肺巨噬细胞中TNF-α、IL-6(M1型细胞因子)和IL-10(M2型细胞因子)mRNA表达。结果显示,NK细胞去除导致肺巨噬细胞中TNF-α,IL-6表达显著降低,而IL-1 0表达明显升高。1.3衣原体肺部感染中,NK细胞去除导致iNOS/Arg-1和IRF5/IRF4比值降低:采用实时定量PCR和western blot方法检测肺巨噬细胞或肺组织中iNOS、Arg-1、IRF5和IRF4mRNA、蛋白表达。结果显示,衣原体肺部感染中,NK细胞去除导致iNOS和IRF5表达略降低,而Arg-1和IRF4表达显著升高,mRNA、蛋白水平结果一致;分别使用mRNA相对表达和蛋白相对灰度值计算iNOS/Arg-1和IRF5/IRF4比值,结果显示,NK细胞去除导致iNOS/Arg-1和IRF5/IRF4比值显著降低,mRNA与蛋白水平结果一致。1.4衣原体肺部感染中,NK细胞缺失导致肺中M1型巨噬细胞减少,M2型巨噬细胞增加:采用流式细胞术,分析小鼠肺中M1和M2型巨噬细胞比例。结果显示,衣原体肺部感染后第6天,与同型对照组相比NK细胞去除组小鼠肺中M1型细胞(CD45+F4/80+iNOS+)的比例明显降低,而M2型巨噬细胞(CD45+F4/80+CD206+)比例显著增高。证明,在衣原体肺部感染中,NK细胞可通过调节肺巨噬细胞极化(促进M1型极化,抑制M2型极化),发挥免疫保护作用。2.衣原体肺部感染中,miR-155和miR-223参与NK细胞诱导的肺巨噬细胞极化2.1 NK细胞去除鼠肺巨噬细胞中miR-155表达降低,miR-223表达升高:实时定量PCR检测衣原体感染后同型对照组和NK细胞去除组小鼠肺巨噬细胞中miRNAs表达。结果显示,衣原体肺部感染后,NK细胞去除导致小鼠肺巨噬细胞中miR-155表达显著降低,而miR-223表达显著升高,miR-155的表达变化与肺中M1型巨噬细胞比例变化一致,而miR-223的表达变化与M2型巨噬细胞一致。证明,在衣原体肺部感染模型中miR-155和miR-223参与了了肺巨噬细胞极化过程。2.2NK细胞去除鼠肺巨噬细胞中SOCS1表达升高,pknox1表达降低:实时定量PCR检测以上两组小鼠肺巨噬细胞中,miR-155的靶分了 SOCS1和miR-223靶分子pknox1的表达。结果显示,衣原体肺部感染中,NK细胞去除导致SOCS1表达升高,而pknox1表达降低。证明,在衣原体肺部感染模型中,miR-155和miR-223可分别抑制其相应靶分子SOCS1和pknox1的转录,调节肺巨噬细胞极化。以上研究为阐明衣原体感染中NK细胞调控肺巨噬细胞极化的作用及其分子机制提供了实验依据。研究结论与意义1.在衣原体肺部感染中,NK细胞对肺巨噬细胞的极化具有调控作用,促进肺M1型巨噬细胞极化,抑制M2型巨噬细胞极化,发挥其免疫保护作用。2.NK细胞去除导致衣原体感染鼠的肺巨噬细胞中miR-155表达降低(其靶分子SOSC1升高)而miR-223表达升高(其靶分子pknox1降低)。说明,miR-155、miR-223及其靶分子参与了 NK细胞诱导的肺巨噬细胞极化过程。本研究首次揭示了衣原体肺部感染中NK细胞对肺巨噬细胞极化的正向免疫调节作用及可能机制,并发现miR-155、miR-223及其靶分子SOCS1和pknox1参与了肺巨噬细胞极化的调控。为阐明NK细胞和肺巨噬细胞相互间的免疫调节作用提供了新的实验依据。更多还原

【Abstract】 Part one:Natural killer cells regulate CD4+T cell subsets in chlamydial lung infectionObjectiveChlamydica is a strict intracellular parasitic prokaryotic cell microbes that can cause a variety of human diseases.Chlamydia trachomatis(C.truchomatis)is the most common pathogen of sexually transmitted diseases and can cause neonatal chlamydial pneumonia.Chloamydia pneumoniae(C.pneumoniae)is the third major pathogen of community-acquired pneumonia after Streptococcus pneumoniae and Haemophilus influenzae.which causes acute atypical pneumonia in children,adults.especially the elderly.Although a variety of broad-spectrum antibiotics can be used to treat chlamydial infection,but due to the different clinical manifestations of individuals.drug resistance and other factors affect the timely diagnosis and effective treatment of’chlamydial disease.Currently,there is no effective vaccine for the prevention of chlamydial disease.Therefore,it is necessary to study the immune response and immune regulation mechanism in order to further control chlamydial infection.Numerous studies have shown that innate and adaptive immune response play an important role in host defense chlamydial infection.Chlamydial infection can activate innate immune cells including DC(dendritic cells),macrophages(macrophages,MΦ),NK cells(nature killer cells)and so on.These cells not only induce an innate immune response but also affect the adaptive immune response.Cellular-mediated immune response is the main adaptive immunity in host defense chlamydial infection.CD4+T cell immune response(IFN-γ;Th1)for the host to clear the chlamydial infection plays a primary protective role.Th17 cells play an important role in the clearance of chlamydial infection by producing cytokines such as IL-17 and IL-22.Previous studies have found that human and mouse chlamydial infection induced Tregs response increased in local tissue and associated with histopathological damage.NK cells are an important member of innate lymphocytes and play an important role in the body’s first line of defense against pathogen invasion.NK cells without sensitization can directly identify and kill infected cells,in addition to cytotoxicity,more attention has been paid to NK cell immune regulation role.More and more studies have shown that NK cells can regulate T cell responses in infectious and inflammatory diseases.At present,studies have shown that NK cells play a protective role in host defense against chlamydial infection,the reported studies on the immunoregulation role of NK cell in modulating CD4+T cell subset are limited.BALB/c mice chlamydial respiratory tract infection model has been widely used to study the immunoregulatory effect of NK cells on the important CD4+T cell subsets.In current study.we used a NK cell-specific antibody(anti-asialo GM1)to specifically eliminate NK cells to investigate the eff-ect of NK cell on the clearing ability of infected chlamydial and histopathological damage.The immunoregulation effect of NK cells on different CD4+T cell subsets(Th1.Th17 and Tregs)in infected local(lung tissue)and immune organs(spleen and mediastinal lymph nodes)were detected by ELISA,intracellular cytokine staining and real-time quantitative PCR.Furthermore,we analyzed the effect of NK cells on the maintenance of CD4+T cell subsets immune balance.Methods1.The effect of NK cells depletion on chlamydial diseases1.1 Chlamydia:Hep-2 cells were cultured and infected with C.muridarum.Glass bead method to collect infected cells,then ultrasonically broken to release chlamydia,centrifugal removal of cell debris.After ultra-centrifugation purification EBs were stored at-80 ℃.1.2 Establishment of mice lung infection model:For mouse infection,1×103 inclusion-forming units(IFUs)of live C.muridarum organisms in 40 μl SPG buffer were used to inoculate mice(6-8 weeks,female)intranasally.1.3 NK cell depletion in vivo:Mice received an intravenous tail-vein injection of 20μl anti-asialo GM1 or control normal rabbit IgG antibody(isotype)in 50 μl PBS 1 day before and 1 day after C..muridarum infection,then every 3 or 5 day injected 10μl anti-asialo GM1 or isotype in 50 μl PBS until the end of the test.Flow cytometry was used to detect NK cell clearance efficiency in lung.1.4 Assessment of mice disease:Body weights of mice were monitored daily.Lung injury was assessed by HE staining(hematoxylin-eosin staining).The lung C.muridarum burden was assessed by immunostaining of chlamydial inclusions(IFUs).2.NK cells regulate CD4+T cell subsets immune response2.1 Effects of NK cells on cytokines secreted by Th1,Th17 and Tregs cells:At predetermined days after inoculation(day 6 and 12).the mice were euthanized under light anesthesia with isoflurane.The supernatant of spleen.lung and mediastinal lymph nodes cells was harvested after 72h culture,and the production of cytokines(IFN-γ,IL-17A,IL-22,IL-10)was measured by ELISA(enzyme-linked immunosorbent assay).Cytokines(IFN-γ.IL-17 and IL-22)levels in serum were assayed by ELISA.2.2 Effects of NK cells on Th1,Th17 and Tregs cell populations:Splenocytes,lung mononuclear cells and mediastinal lymph node cells were incubated and intracellular cytokine stained.The percentage and number of CD3+CD4+IFN-γ+T(Th1).CD3+CD4+IL-17A+T(Th17)and CD4+CD25+Foxp3+T(Treg)cells in the spleen,lung and mediastinal lymph nodes were detected by flow cytometry.3.NK cells regulate Th1/Treg and Th17/Treg balance3.1 Effects of NK cells on the expression of transcription factor in T cell subsets:RNA was extracted from mouse splenocytes and lung mononuclear cells.The expression of Th1(T-bet),Th2(GATA3),Th17(RORγT)and Treg(Foxp3)transcription factor mRN A expression was detected by real-time quantitative PCR.3.2 Effects of NK cells on T cell differentiation related cytokines:At day 6 and 12 after infection,the supernatant of lung and mediastinal lymph nodes cells was harvested after 72h culture.and the production of cytokines(IL-6,TGF-β and IL-12p40)were assayed by ELISA.3.3 Effects of NK cells on Th1/Treg and Th17/Treg ratio:Th1/Treg and Th17/Treg ratio in splenocytes,lung mononuclear cells and mediastinal lymph node cells were calculated by the numbers of Th1 or Th17 cells by the number of Treg cells,respectively.Results1.NK cell-depleted mice show more severe disease to chlamydial lung infection 1.1 Anti-asialo-GM1 antibody deplete NK cells specifically:The percentage of NK cells in the lungs was measured at day 4 after chlamydial infection.The results showed that the number and proportion of NK cells in the lungs of mice injected with anti-asialo-GM1 antibody were significantly decreased,and the clearance rate was about 90%.1.2 NK cell-depleted mice show more severe disease:Following chlamydial lung infection,as compared to isotype control mice.NK cell-depleted mice showed greater body weight loss and slower recovery,higher chlamydial loads(IFUs)in the lung and more intense pathologic changes.2.NK cells enhance Th1 and Th17 responses but inhibit Tregs response2.1 NK cell promote IFN-γ,IL-17 and IL-22 secretion with chlamydial lung infection:IFN-γ is mainly secreted by Thl cells,whereas Th17 cells are characterized by cytokines such as IL-17 and IL-22.Use the ELISA method to detect the expression of IFN-γ,IL-17 and IL-22 in spleen,lung,mediastinal lymph nodes and serum at day 6 and 12.The results showed that the levels of IFN-γ,IL-17 and IL-22 in the above organs were significantly decreased after NK cells depleted,and the serum concentration was also decreased.2.2 NK cell-depletion lead to decreased Th1 and Th17 cells populations:Th1 immune response plays a key role in host defense against chlamydial infection,Th17 cells synergistically promote Thl cell response.Flow cytometry was used to analyze the effect of NK cell-depletion on the ratio and absolute number of CD3+CD4+IFN-γ+T(Th1)and CD3+CD4+IL-17A+T(Th17)cells.The results showed that the proportion and absolute number of Th1 and Th17 cells in the spleen,lung and mediastinal lymph nodes of NK cells depleted group were significantly lower than those of the control group at day6 and day 12.Indicating that NK cells promote the immune response of Th1 and Th17 cells in local(lung)and immune organs(spleen and mediastinal lymph nodes).2.3 NK cell-depletion lead to significant increase proportion and absolute number of Tregs:To observe the effect of NK cell removal on Tregs response in mouse lung infection,intracellular cytokine staining and low cytometry analysis the proportion and absolute number of(CD4+CD25+Foxp3+T)Tregs.The results showed that the proportion and absolute number of Tregs in the lung,spleen and mediastinal lymph nodes of NK cells depleted mice were significantly higher than control mice,especially in the early stage of chlamydial infection(6 days).It was proved that NK cells had inhibitory effect on Tregs response in Chlamydial infection.especially in the early stages of infection(6 days).3.Effect of NK cell on Th1/Treg,Th17/Treg balance:3.1 NK cells promote Thl and Th17 cell differentiation but inhibit Tregs differentiation:Real-time quantitative PCR detect the expression of T cell subsets specific transcription factors in mouse spleen and lung cells.The results showed that the expression of T-bet(Th1)and RORγT(Th17)was decreased and the expression of Foxp3(Tregs)and GATA3(Th2)was increased in NK cells depleted mice.3.2 NK cell depleted mice shown lower IL-12p40,IL-6 and higher TGF-βexpression:The expression of T cell subsets differentiation related cytokines was observed in the supernatant of local lung and mediastinal lymph nodes cells by ELISA.The results showed that the secretion of IL-12p40(Th1)and IL-6(Th17)was decreased and the expression of TGF-β(Tregs)was increased in the lung and lymph node cells of NK cells depleted mice.3.3 NK cell depletion leads to imbalanced Th1/Treg and Th17/Treg responses:The absolute number of T cell subsets was analyzed and the Th1/Treg and Th17/Treg ratios were calculated.The results showed that the removal of NK cells resulted in decreased Th1/Treg and Th17/Treg ratios in the lung,spleen and mediastinal lymph nodes at day6 after chlamydial infection.At day 12,the ratios of Th1/Treg and Th17/Treg was low in the isotype control group,and the ratio of NK cell depleted group was still significantly lower than that of the control group.Conclusions and Significance1.NK cells play an important protective role in BALB/c mice against chlamydial pulmonary infection.NK Cells contribute to BALB/c mice to clear the chlamydial in the lungs,reduce lung tissue pathology and accelerate disease recovery.2.We demonstrated that NK cells can inhibit Tregs responses and help to enhance the protective immune response in chlamydial infection.The results are consistent in the the local infection site of(lung)and lymphoid organs(spleen and lymph nodes).3.NK cells can inhibit the Tregs response,promoting Thl and Th17 responses,and maintain the immune balance of Th1/Treg and Th17/Treg,which is beneficial to the rapid and effective chlamydial infection and reduce the pathological damage.The above study systematically studied the immunoregulatory effect of NK cells on CD4+T cell subsets in chlamydial pulmonary infection,especially revealed the significant inhibitory effect of NK cells on Tregs response for the first time.We found NK cells play an important protective role in the host defense against chlamydial pulmonary infection by maintaining Th1/Treg and Th17/Treg balance.This study provides a new insight into the mechanism of NK cell immune regulation.Part two:Natural killer cells regulate pulmonary macrophage polarization in chlamydial lung infectionObjectiveMacrophages are important innate immune cells,which play an important role in maintaining immune homeostasis and resisting pathogen infection.Macrophages have a strong plasticity.and polarization state is the key to play a different function,Macrophages have at least two different polarization state,classically activated Ml macrophages and alternatively activated M2 macrophages.M1 macrophages can be activated by IFN-γ.PAMPs.TNFα and so on.M1 produce proinflammatory cytokines and overexpress iNOS.thus contributing to clearing pathogens.On the other hand,M2 was activated by Th2 type cytokines such as IL-4,DAMPs and TGF-β,characterized by the relatively high expression of arginase-1,YM-1 and CD206 which are involved in promoting phagocytosis and clearance of parasites,participate in immunomodulation and promote tissue remodeling and matrix precipitation the repair or remodeling of tissues.Similar to Th1/Th2 polarization,the conversion between macrophages M1 and M2 phenotype is also controlled by specific transcription factors.It is known that molecules such as C/EBP-α,PU.1,NF-κB(P65/P50),Statl,IRF5 and HIFa are involved in the regulation of Ml macrophage activation;Stat3,Stat6,IRF4 and SOCS1 Signaling molecules are involved in the regulation of macrophage M2 type polarization.The polarization mechanism of macrophages is complex and has been the focus of recent research.Some research works showed that macrophages involved in host defense and clear pathogen infection,and its role in the control of chlamydial infection has a certain understanding.M1 macrophages can limit the proliferation of chlamydial in the cell,and enhance the innate immune and adaptive immune response through cytokines secretion,which will help the body to quickly remove the infection;Chlamydial can grow normally in the M2 macrophages and further infect other organs.Therefore,induction of modest M1 macrophage response is conducive to clear chlamydial infection.NK cells can act on cytokine microenvironment and interact with other immune cells to control their cytotoxicity or positive immune regulation of macrophages and play a different role in different pathogen infections.In chlamydial lung infection,wheher NK cells can regulate macrophages,especially lung local macrophages are not clear.MicroRNAs(miRNAs)are endogenous non-coding RNAs that involved in the regulation of gene expression at the post-translational level.miRNAs can regulate the transcription of key molecules in macrophage polarization and influence macrophage polarization.Whether or not miRNAs are involved in NK cell-induced macrophage polarization in chlamydial infection is still unclear.This study aimed to investigate the effect of NK cells on pulmonary macrophage polarization and its possible mechanism in chlarmydial lung infection.BALB/c mice were treated with anti-asialo GM1 antibody to specifically eliminate NK cells in vivo,The expression of miRNAs and their target molecules in lung macrophages were detected.The macrophage polarization status and the expression of miRNAs and their target molecules in lung macrophages were analyzed.Methods1.Purification and flow cytometry analysis of macrophages1.1 Lung macrophage purification:BALB/c mice inhale chlamydial intranasally,experimental group as same as the first part.Mice were sacrificed on day 6 after infection.Lung tissue was digested with collagenase Ⅺ(10 mg/ml)and centrifuged at percoll density gradient to prepare mononuclear cells.Anti-F4/80 magnetic beads antibody was used to isolate and purify lung macrophages.1.2 lung macrophage purity analysis:5 × 105 purified lung macrophages were used for analysis the percentage of F4/80+CD11 b+cells by flow cytometry.2.Effects of NK cells on macrophage polarization2.1 Effects of NK cells on the expression of polarized cytokines in lung macrophages:The total RNA was extracted from lung macrophage of isotype control and NK cell depleted mice.The mRNA expression of TNF-α,IL-6(M1-type cytokines)and IL-10(M2-type cytokine)were detected by real-time quantitative PCR.2.2 Effects of NK cells on the polarization of M1/M2 macrophages:The mRNA and protein level of M1(iNOS.IRF5)and M2 macrophage markers(Arg-1,IRF4)were detected by real-time quantitative PCR and western blot.The ratio of iNOS/Arg-1 and IRF5/IRF4 were calculated according to the relative expression level of mRNA and protein.2.3 Effects of NK cells on the percentage of M1 and M2 cells in lungs:The percentages of M1(CD45+F4/80+iNOS+)and M2(CD45+F4/80+CD206+)were detected in lung mononuclear cells by flow cytometry.3.The mechanism of NK cells regulate macrophage polarization3.1 NK cells affect the expression of miR-155 and miR-223:The lung macrophages were isolated and purified by magnetic beads at day 6 after chlamydial infection from isotype control and NK cells depleted mice.Total miRNA was extracted and reverse transcribed into cDNA using miR-155 and miR-223 specific reverse transcription primers.The expression of miR-155 and miR-223 was detected by real-time quantitative PCR.3.2 Effects of NK cells on miRNA target expression:Total RNA was extracted from lung macrophages,and real-time quantitative PCR was used to detect the expression of SOCS1(target of miR-155)and pknoxl(target of miR-223).Results1.NK cells promote M1 but inhibit M2 macrophage polarization in chlamydial infection1.1 Lung macrophage purity:We isolated purified lung macrophages by using anti-F4/80 magnetic beads.Flow cytometry analysis results showed that the percentage of F4/80+CD11b+ macrophages was more than 96%after magnetic bead sorting.1.2 NK cells promote M1 cytokines but inhibit M2 cytokines in lung macrophage:The expression of TNF-α,IL-6(M1-type cytokine)and IL-10(M2-type cytokine)mRNA in lung macrophages on the sixth day after Chlamydial infection were detected by real-time quantitative PCR.The results showed that the expression of TNF-α and IL-6 in lung macrophages was significantly decreased and the expression of IL-10 was significantly increased in NK cell depleted mice.1.3 NK cell depletion leads to decrease iNOS/Arg-1 and IRF5/IRF4 ratios in chlamydial lung infection:Real-time quantitative PCR and western blot were used to detect the expression of iNOS,Arg-1,IRF5 and IRF4 in lung macrophages or lung tissues.The results showed that the expression of iNOS and IRF5 decreased slightly and the expression of Arg-1 and IRF4 was significantly increased in NK cell depleted group in both mRNA and protein levels.Furthermore,NK cell depletion leaded to the significantly decreased ratios of iNOS/Arg-1 and IRF5/IRF4 both in nRNA and protein level.1.4 NK cells depletion induced increased M2 but decreased M2 macrophages in chlamydial lung infection:was used to the percentage of M1 and M2 macrophages in the lungs of mice were analyze by flow cytometry.The results showed that the proportion of M1 cells(CD45+F4/80+iNOS+)was significantly lower(P<0.05).and the proportion of M2 macrophages(CD45+F4/80+CD206+)significantly increased in the lungs of NK cells depleted mice.2.miR-155 and miR-223 involved in NK cell-induced pulmonary macrophage polarization during chlamydial lung infection2.1 NK cells depletion induced increased miR-223 but decreased miR-155 expression in lung macrophages during chlamydial infection:Real-time quantitative PCR was used to detect the expression of miRNAs in lung macrophages in isotype control mice and NK cells depleted mice after chlamydial infection.The results showed that the expression of lung macrophages miR-155 was significantly decreased and miR-223 was significantly higher in NK cell-depleted mice than those in control group.2.2 NK cells depletion induced increased SOCS1 but decreased pknoxl in lung macrophages during chlamydial infection:The expression of SOCS1(target of miR-155)and pknox1(target of miR-223)was detected by Real-time quantitative PCR.The results showed that NK cells depletion induced increased SOCS1 but decreased pknox1 expression in lung macrophages during chlamydial infection.Conclusions and Significance1.In chlamydial lung infection,NK cells have a regulation effect on the polarization of lung macrophages.NK cells can induce M1 macrophage polarization but inhibit M2 macrophage polarization.2.NK cell depletion induced decreased mir-155 and increased expression of its target SOSC1.but increased expression of miR-223 and decreased expression of its target pknox1 in pulmonary macrophages of C.muridarum infected mice.These results suggested that miR-155.miR-223 and their target molecules are involved in NK cell-induced pulmonary macrophage polarization process.The findings of current study provide the first report,to our knowledge,on the immunoregulatory effect and possible mechanism of NK cells in macrophages polarization in chlamydial lung infection.We found that miR-155 and miR-223 are involved in the regulation of pulmonary macrophage polarization.Our findings provide a new experimental basis for elucidating the immune regulation between NK cells and lung macrophages.更多还原