【作者】 解奇；

【导师】 曲迅；

【作者基本信息】 山东大学 ， 肿瘤学， 2014， 博士

【摘要】 肿瘤(1tumor)是机体在各种致癌因素作用下,局部组织的某一个细胞在基因水平上失去对其生长的正常调控,导致其克隆性异常增生而形成的病变。目前的研究已经总结出癌症公认的六大显著特性：1.具有无限复制的潜能性；2.具有逃逸细胞程序性死亡的特性(programmed cell death, PCD);3.能够自身提供生长信号的特性；4.对生长抑制信号的不敏感性；5.具有持续促进血管生成的特性；6.具有组织侵袭和远处转移的特性。然而,越来越多的研究指出：炎症是癌症的第七个特性,被命名为癌症相关的炎症(Cancer-related inflammation, CRI)。炎症和癌症早已不是相互独立的两个事物,实际上早在19世纪,Rudolf Virchow研究发现：肿瘤内部存在大量的白细胞,因而作者提出假设：炎症和癌症之间可能存在关联,但是直到最近的二三十年,研究人员才获得确切的证据证实：炎症及炎性微环境在癌症的发生发展中发挥着重要的作用。有研究报道：种系突变诱发的癌症只占总癌症数的一小部分(约10%),与此对应的,体细胞突变和环境因素诱发的癌症占总癌症数的大部分(约90%),其中,慢性炎症参与形成诱发癌症的炎性微环境。Aggarwal等人在2009年统计研究指出：多达20%的癌症和慢性感染相关,诸如,目前公认的感染乙肝病毒(hepatitis B viruses, HBV)和丙肝病毒(hepatitis C viruses, HCV)增加肝癌发病风险；持续的幽门螺杆菌感染和胃癌发病密切相关；血吸虫或拟杆菌属种感染分别与膀胱癌和结肠癌密切相关。感染诱发的炎症反应属于正常的宿主免疫防御,目标旨在帮助机体清除病原体。然而,病原体破坏宿主免疫系统诱发持续感染,持续的慢性炎症促进肿瘤的发生发展。但值得注意的是,并非所有的慢性炎症都增加肿瘤发病风险,诸如,一项针对6238名50-75岁男性的统计研究发现：男性初期前列腺活检出现炎症的患者未来活检患有前列腺癌的风险降低；银屑病诱发的慢性炎症可能降低癌症发病风险。综上所述：炎症和肿瘤之间的复杂关系就像一把双刃剑,炎症既可以促进肿瘤的进展,又可以抑制肿瘤生长,然而目前决定两种差别过程的关键因素尚未完全阐明,其中涉及的机制还需要肿瘤免疫来解释。肿瘤微环境中,除了癌细胞和基质细胞,还包含参与固有性和适应性免疫应答的免疫细胞,诸如,单核细胞(monocyte)、巨噬细胞(macrophage)、中性粒细胞(neutrophi1)、骨髓来源的抑制性细胞(myeloid-derived suppressor cell, MDSC)、(?)(?)大细胞(mast cell)、自然杀伤细胞(natural killer cell, NK cell)、树突状细胞(dendritic cell, DC)、T淋巴细胞、B淋巴细胞等,这些细胞通过直接接触或者分泌细胞因子的方式相互沟通、相互影响。机体的固有性免疫应答和适应性免疫应答协同作用,在负责监视、清除突变肿瘤细胞的过程中发挥重要的作用。单核-巨噬细胞属于固有性免疫应答系统,肿瘤局部的巨噬细胞可以占到肿瘤比重的50%,提示巨噬细胞在肿瘤免疫中的重要作用。肿瘤相关的巨噬细胞(tumor associated macrophage, TAM)被认为是M2型巨噬细胞(替代性活化的巨噬细胞),能够促进肿瘤生长,血管生成,侵袭和转移。此外,TAM还参与抑制其他免疫细胞的抗肿瘤作用,肿瘤局部TAM的数量与肿瘤预后负相关。TAM高表达多种膜受体,如CD206、CD163、CXCR1、CXCR2、CCR2,并且分泌多种细胞因子,包括IL-10、IL-1受体拮抗剂(IL-1ra)、CCL17、CCL18和CCL2等。树突状细胞(DC)参与起始,维持和调节机体免疫应答,处于机体诱导免疫耐受或者免疫激活的中心环节,是目前已知的最强的,唯一能够活化初始T细胞(naive T cells)的抗原呈递细胞(antigen presenting cell, APC)。 DC能够诱导初始T细胞极化为两类功能不同的辅助性T细胞(T helper cell, Th cell),诸如,分泌IL-2、IL-12p70、IFN-γ等细胞因子的Th1细胞和分泌IL-4、IL-10、TGF-β等细胞因子的Th2细胞,两种细胞分别介导机体不同的炎症反应和细胞免疫；鉴于DC在抗肿瘤细胞免疫中的重要作用,目前以DC为基础的细胞免疫治疗(DC疫苗)逐渐成为肿瘤免疫治疗的热点。DC疫苗在体外实验中已经取得不错的抗肿瘤效果,然而DC疫苗在实际临床应用中却存在以下主要问题：诸如,DC疫苗诱发的机体抗肿瘤免疫应答较弱、不同个体间效果差异较大等。肿瘤局部DC极化成为耐受型DC,不能有效杀伤肿瘤细胞,伴随呈递肿瘤抗原能力降低,Th2型细胞因子分泌偏倚,DC凋亡增加等。DC疫苗在体外实验研究和临床治疗中显著性差异的抗肿瘤作用提示后来的研究者：机体内的肿瘤微环境参与诱导改变、甚至决定DC的性质和功能。pH值降低、营养缺乏和组织缺氧是肿瘤微环境的主要特征,然而,肿瘤微环境中的病原体在修饰和抑制DC功能中也发挥着不容忽视的重要作用。慢性感染过程中,阐明病原体及其特定的毒力分子对机体免疫系统调控结果及其机制,可以为阐明炎症的分子机制或者为肿瘤生物治疗提供借鉴意义。卡他莫拉菌(Moraxella catarrhalis, M. catarrhalis}最初被认为是呼吸道的正常菌群,但是后来的研究指出：卡他莫拉菌是一种致病细菌,能够诱导小儿中耳炎,在慢性阻塞性肺病(chronic obstructive pulmonary disease, COPD)患者病程加重过程中发挥重要作用,机制可能涉及卡他莫拉菌诱导肺泡上皮细胞凋亡。据研究统计：多达90%卡他莫拉菌能够产生β-内酰胺酶抵抗氨苄青霉素,因而研发针对卡他莫拉菌的疫苗能够解决日益严重的抗生素抵抗问题,但是目前针对卡他莫拉菌的疫苗研究进展缓慢,其中一个主要的因素在于缺乏用于研究的动物模型,小鼠肺清除模型是目前研究中广泛采用的、旨在评估肺部清除卡他莫拉菌的研究模型,但小鼠和人存在种系差别,小鼠在感染卡他莫拉菌的24小时内迅速清除细菌,不同于人呼吸道长期感染此菌的情况,因而该动物模型系统不能够模拟人类呼吸道细菌感染,鉴于此,寻求建立更好的模拟人呼吸道系统感染的动物模型以及进行卡他莫拉菌疫苗的体外实验研究显得尤为必要。实验证实：卡他莫拉菌感染后,大量单核细胞和树突状细胞被趋化因子募集至炎性位点。单核细胞以及DC都是高度异质性细胞群体,单核细胞可以被病原菌及其毒力分子诱导分化成为抗炎性的M1型巨噬细胞或者抑炎性的M2型巨噬细胞,而DC的成熟也受到微环境中病原菌及其他因素的影响,这些因素分别促进或者抑制DC的成熟。单核.巨噬细胞作为固有性免疫应答的一线哨兵细胞,而DC处于调控固有性和适应性免疫应答的核心环节,卡他莫拉菌对两群细胞的调节可能直接或间接的影响、甚至决定感染后肺部的免疫反应结果。但是,迄今为止,卡他莫拉菌对募集而来的单核细胞的分化和DC的成熟的影响尚未完全阐明,值得注意的是,虽然没有研究证实：卡他莫拉菌感染和肺癌之间存在阳性关联,但是针对肺癌患者病原菌筛查结果发现：约68%肺癌患者分离出革兰氏阴性菌,其中最主要的就是流感嗜血杆菌和卡他莫拉菌,这就提出一个新的问题：长期存在于肺部肿瘤患者体内的卡他莫拉菌是否参与调控患者的肿瘤免疫?单核细胞和DC可能遇到卡他莫拉菌表面的多种刺激物质,诸如外膜蛋白UspA1(ubiquitous surface proteins A1)和细菌细胞壁外膜的主要成分脂多糖(lipopolysaccharide, LPS)。LPS显著刺激单核细胞向M1型巨噬细胞分化,并且显著诱导DC的成熟,有文献报道：细菌毒力分子在诱导单核细胞向M2型巨噬细胞极化以及抑制DC成熟过程中发挥重要的作用。譬如：耶尔森菌(Yersinia sp.)表达毒力因子LcrV诱导单核细胞向M2型巨噬细胞分化；肠源性共生细菌产物通过刺激肝脏IL-6/STAT3通路活化抑制肝DC成熟。作为卡他莫拉菌毒力分子的外膜蛋白UspA1是否也诱导单核细胞的M2型极化并抑制DC的成熟?尤其值得注意的是,卡他莫拉菌外膜蛋白UspA1结合CEACAM1(Carcinoembryonic antigen-related cell adhesion molecule1)的N端结构域,CEACAM1通过免疫受体酪氨酸抑制基序活化的信号对T细胞活化发挥重要抑制作用。目前已经证实：UspA1交联表达CEACAM1的受体细胞,不仅调节上皮细胞的功能,还调节T细胞的功能。然而作为CEACAM1配体的卡他莫拉菌外膜蛋白UspA1(尤其是结合CEACAM1的UspA1来源的重组rD-7蛋白)是否也通过CEACAM1信号调控单核细胞分化及DC成熟?为了帮助揭示这一问题,我们从固有性免疫和适应性免疫的两个经典细胞群体(单核-巨噬细胞和DC)出发,采用来源于卡他莫拉菌外膜蛋白UspA1的重组蛋白rD-7体外刺激单核细胞和DC,研究该细菌的毒力分子对单核细胞分化及DC成熟的影响,从而揭示卡他莫拉菌UspA1分子对机体固有性和适应型免疫应答的调控,以期为肺癌患者合并卡他莫拉菌感染条件下的免疫治疗及卡他莫拉菌疫苗的研发提供借鉴意义。第一部分UspA1来源重组蛋白rD-7对人髓系单核细胞分化的影响及机制研究目的：研究卡他莫拉菌外膜蛋白UspA1来源重组蛋白rD-7对单核细胞分化的影响及其机制。方法：1.重组蛋白rD-7、rD-7/D和r6-8的制备与鉴定。通过pQE30表达系统制备重组蛋白rD-7及对照蛋白(rD-7/D和r6-8),Ni-NTA亲和层析法分离重组蛋白。采用抗His标签抗体和可溶性的CEACAM1-Fc通过Western blot实验验证rD-7的特异性及功能。多粘菌素B去除蛋白样品中的内毒素,0.2μm滤器过滤蛋白样本除菌,测定蛋白样品中内毒素含量低于1.0EU/μg。2.蛋白酶K消化处理重组蛋白rD-7和rD-7/D。采用终浓度为200μg/ml无菌蛋白酶K在37℃水浴条件下,消化重组蛋白rD-7和rD-7/D3小时。消化后的蛋白样本置于95℃水浴孵育1小时灭活蛋白酶K,SDS-PAGE检测rD-7和rD-7/D的消化结果。3.健康人外周血CD14+/CD15+细胞的分离和纯化。采集健康志愿者外周静脉血并收集于无菌采集袋中,密度梯度离心分离外周血单个核细胞(peripheral blood mononuclear cell, PBMC),采用免疫磁珠分选法阳性分选CD14+单核细胞和CD15+细胞。4.单核细胞的活化诱导。采用终浓度为2.5μg/ml重组蛋白rD-7、r6-8、rD-7/D或终浓度2μg/ml的LPS分别刺激新鲜分离的单核细胞24小时,流式细胞术检测单核细胞表型分化,收取上清-80℃冻存以备后续检测。在CEACAM1阻断实验中,采用终浓度为50p.g/ml抗CEACAM的多抗A0115封闭单核细胞1小时后,rD-7或对照蛋白rD-7/D刺激单核细胞23小时,收取处理后的单核细胞检测分化表型。5.流式细胞术检测表面分子表达。流式细胞术检测新鲜分离的单核细胞表面rD-7受体CEACAM1的表达；在单核活化实验中,流式细胞术检测重组蛋白rD-7、r6-8、rD-7/D或LPS刺激后CD14+细胞表面CD80、CD86、CD206的表达情况。6. rD-7在CD14+和CD15+细胞表面结合及A0115对结合作用的阻断效应检测。采用终浓度50μg/ml的抗CEACAM的阻断抗体A0115及IgG同型对照预先处理单核细胞30分钟,继而采用rD-7或rD-7/D刺激细胞1小时,流式细胞术定量分析rD-7和rD-7/D在CD14+或CD15+细胞表面的结合情况。7.液相芯片检测rD-7刺激单核细胞分泌IL-6、IL-10、IL-12p70和TNF-α水平。重组蛋白rD-7或rD-7/D刺激单核细胞24小时后,收取上清,采用MILLIPLEX (?)MAP Human Cytokine/Chemokine Magnetic Bead Panel Ⅰ试剂盒检测上清中IL-6、IL-10、IL-12p70和TNF-αα的水平并采用MAGPIX系统进行结果分析。8.蛋白芯片检测rD-7诱导单核细胞分泌的趋化因子/细胞因子谱。重组蛋白rD-7或rD-7/D刺激单核细胞24小时后,收取上清,采用Proteome Profiler human cytokine array Panel A试剂盒分析rD-7诱导单核细胞分泌的细胞因子和趋化因子谱。结果：1.卡他莫拉菌UspAl来源的重组蛋白rD-7修饰单核细胞的分化相比于未处理组,重组蛋白rD-7显著上调CD14+细胞CD206的表达(P<0.001),下调CD80表达(P0.05),不改变CD86表达；LPS显著上调CD14+细胞CD80(P<0.05)和CD206(P<0.001)的表达,显著下调CD86的表达(P<0.05)；r6-8不影响CD14+细胞CD206、CD80、CD86的表达。2.rD-7上调CD14+细胞CD206表达,诱导效应依赖完整蛋白而非内毒素相比未刺激组,rD-7显著上调CD14+细胞CD206的表达(P<0.001),然而相比于rD-7刺激组,蛋白酶K消化后的rD-7不能上调CD14+细胞CD206的表达(P<0.001)。3.CD14+单核细胞表达CEACAM1, rD-7的受体采用两种抗CEACAM1的抗体,一种是抗CEACAM1的多抗A0115,另一种是抗CEACAM1的单抗MAb2244,均检测到单核细胞表达CEACAM1,但是表达CEACAM1的细胞百分比较低。4.rD-7不依赖于绑定CEACAM1上调CD14+细胞CD206的表达采用rD-7和不结合CEACAM1的对照蛋白rD-7/D刺激单核细胞24小时,流式细胞术检测证实：相比于未刺激细胞,rD-7(P<0.001)和rD-7/D(P<0.001)均显著上调CD14+细胞CD206的表达,并且两者上调CD206表达水平相似。消化后的rD-7和rD-7/D均不能上调CD14+细胞CD206的表达；LPS显著上调CD14+细胞CD206的表达(P<0.001),而蛋白酶K消化后的LPS仍旧上调CD14+细胞CD206的表达(P<0.001)。流式细胞术检测证实：rD-7结合到CD15+细胞表面(P0.001),而rD-7/D不结合CD15+细胞。相比于IgG同型对照,抗CEACAM多抗A0115阻断CD15+细胞上CEACAM后,rD-7结合CD15+细胞能力显著降低(P<0.05)。5.rD-7结合单核细胞实验及CEACAMl阻断实验证实rD-7非CEACAM1依赖性上调CD206的表达。rD-7(P<0.05)与对照蛋白rD-7/D(P<0.05)均能够结合单核细胞,此外,相比同型对照IgG,阻断抗体A0115不能阻断rD-7结合单核细胞。抗体A0115阻断单核细胞CEACAM后检测rD-7对单核细胞分化的诱导作用,rD-7(P<0.01)和rD-7/D(P0.01)仍显著上调CD14+细胞CD206的表达。6.rD-7和rD-7/D诱导单核细胞IL-6、TNF-α、IL-10和IL-12p70的分泌情况终浓度2.5μg/ml rD-7或者rD-7/D刺激单核细胞24小时后,收取上清,液相悬浮芯片实验结果显示：相比于未刺激对照组,rD-7和rD-7/D不诱导单核细胞分泌IL-10、IL-12p70,也不显著诱导炎性因子IL-6和TNF-a的分泌。7.rD-7或rD-7/D修饰单核细胞的细胞因子/趋化因子表达谱rD-7(P<0.001)和rD-7/D(P<0.001)刺激单核细胞24小时后,IL-1ra的分泌水平显著升高,且IL-1ra的分泌水平类似于LPS刺激组。相比于未刺激对照组,rD-7(P<0.05)和rD-7/D(P<0.01)也显著增加单核细胞IL-8的分泌水平。在rD-7或rD-7/D组上清中,未检测到IL-2、IL-4、IL-5、IL-10、IL-12p70、IL-17、IL-32a、MCP-1、MIP-1β、CXCL10和CXCL12的表达。rD-7或rD-7/D比较未刺激组,C5a、CD40L、G-CSF、GM-CSF、CXCL1、CCL1、sICAM-1、 IFN-γ、IL-1α、IL-1β、IL-6、IL-13、IL-16、IL-17E、IL-23、IL-27、CXCL11、 MIF、CCL3、Serpin E1、CCL5、TNF-α、sTREM-1的分泌水平无显著差别。结论：卡他莫拉菌UspA1来源的重组蛋白rD-7修饰单核细胞的活化,调控单核细胞分化为CD14+CD206+表型的巨噬细胞,调控作用依赖完整蛋白而非内毒素；rD-7下调CD14+细胞CD80的表达；单核细胞表达rD-7受体CEACAM1,但rD-7通过非依赖CEACAMl途径结合单核细胞表面并调控单核细胞的分化；rD-7显著诱导单核细胞分泌抗炎性细胞因子IL-1ra。卡他莫拉菌UspA1来源重组蛋白rD-7显著修饰单核细胞分化,揭示了卡他莫拉菌毒力分子UspA1对机体固有性免疫应答的调控,为肺癌患者合并卡他莫拉菌感染条件下的免疫治疗及卡他莫拉菌疫苗的研发提供借鉴意义。第二部分UspAl来源重组蛋白rD-7对人髓系来源树突状细胞成熟的影响及机制研究目的：研究卡他莫拉菌外膜蛋白UspAl对未成熟DC的活化成熟及细胞因子分泌的影响及其机制。方法：1.重组蛋白rD-7和rD-7/D的制备。制备过程同第一部分,经验证重组蛋白rD-7和rD-7/D中内毒素的含量低于1.0EU/μg。2.健康人外周血CD14+单核细胞的分离和纯化。采集健康志愿者外周静脉血并收集于无菌采集袋中,密度梯度离心分离外周血单个核细胞,采用免疫磁珠分选法阳性分选CD14+单核细胞,流式细胞术检测CD14+细胞的纯度。3.DC的诱导与鉴定。配制含终浓度1000U/ml rh GM-CSF、500U/ml rh IL-4、10%人AB血清的DC培养基,DC培养基重悬新鲜分离的CD14+单核细胞,调整细胞浓度为5×105/ml。接种于二十四孔板,每孔2m1细胞悬液。将细胞置于37℃,饱和湿度,5%CO2的培养箱中,分别在第3/5天后换液200μl新鲜DC培养基。培养至第6天的未成熟DC,每孔加入终浓度2μg/mlLPS,孵箱中继续培养24小时诱导为成熟DC。4.DC活化实验。诱导新鲜分离的CD14+单核细胞6天为imDC,采用终浓度为2.5μg/ml rD-7或rD-7/D或者2μtg/ml LPS刺激imDC24小时,流式细胞术检测DC CD80、CD83、CD86、HLA-DR的表达。在阻断CEACAM实验中,加入终浓度为50μg/ml A0115或者IgG同型对照阻断imDC表面CEACAM1小时,继而采用终浓度为2.5μg/ml rD-7刺激DC23小时。5.酶联免疫吸附试验检测rD-7对DC分泌TNF-α、IL-10和IL-12p70的影响。采用终浓度为2.5μg/ml rD-7或rD-7/D或者2μg/ml LPS刺激imDC细胞24小时,收取上清,ELISA方法检测上清TNF-α、IL-10和IL-12p70的水平。在阻断CEACAM实验中,加入终浓度为50μg/ml A0115或者IgG同型对照阻断imDC表面CEACAM1小时,继而采用采用终浓度为2.5μg/ml rD-7刺激DC23小时后收取上清检测细胞因子分泌水平。结果：1.卡他莫拉菌UspAl来源的重组蛋白rD-7修饰DC活化成熟采用UspA1来源重组蛋白rD-7或LPS分别刺激未成熟DC24小时,流式细胞术检测证实：相比于未刺激组,rD-7显著上调DC CD86(P<0.001)和HLA-DR(P<0.01)的表达,rD-7虽然也上调DC CD83的表达(P<0.05),但是上调的程度远低于LPS组(P0.001)。虽然LPS显著增加DC CD80的表达(P<0.001),但是rD-7却显著下调DC CD80的表达(P<0.01)。2.DC表达rD-7的受体CEACAMlrh GM-CSF和rh IL-4诱导单核细胞6天获得未成熟DC,继而采用LPS刺激24小时获得成熟DC,未成熟DC和成熟DC表达CDllc比率均大于99%。采用抗CEACAM1的单抗MAb2244分别检测了imDC和mDC表面CEACAM1的表达,结果显示表达CEACAM1的imDC和mDC细胞百分比较低。3.rD-7不依赖于CEACAMl下调DC CD80的表达rD-7或rD-7/D分别刺激未成熟DC24小时后,rD-7(P<0.01)和rD-7/D(P<0.05)均显著下调DC CD80的表达,且两者对CD80表达下调作用无显著性差异,但是LPS显著上调DC CD80的表达(P<0.001)。此外,相比于IgG同型对照,A0115不能阻断rD-7下调DC CD80的表达。4.rD-7不依赖于CEACAMl上调DC CD83的表达rD-7和rD-7/D分别刺激未成熟DC24小时后,rD-7(P<0.05)和rD-7/D(P<0.01)均显著上调DC CD83的表达,且两者对CD83表达的上调作用无显著性差异,此外,阳性对照LPS显著上调CD83的表达(P<0.001),LPS对DC CD83的表达诱导能力显著高于rD-7或rD-7/D。相比于IgG同型对照,A0115也不能阻断rD-7上调DC CD83的表达。5.rD-7不依赖于CEACAMl上调DC CD86的表达rD-7和rD-7/D刺激imDC24小时后,rD-7(P<0.001)和rD-7/D(P0.001)均显著上调DC CD86的表达,且两者对CD86表达的上调作用无显著性差异,LPS也显著上调DC CD86的表达(P<0.001),但是,相比LPS,rD-7(P<0.05)和rD-7/D(P<0.01)对DC CD86的上调作用更加显著。此外,相比于IgG同型对照,A0115也不能阻断rD-7上调DC CD86的表达。6.rD-7不依赖于CEACAM1上调DC HLA-DR的表达rD-7和rD-7/D刺激imDC24小时后,rD-7(P<0.01)和rD-7/D(P<0.01)均显著上调DC HLA-DR的表达,且两者对DC HLA-DR表达的上调作用无显著性差异,LPS也显著上调DC HLA-DR的表达(P<0.01)。此外,相比于IgG同型对照,A0115不能阻断rD-7上调DC HLA-DR的表达。7.rD-7对DC分泌TNF-α.IL-10和IL-12p70的影响LPS显著诱导DC分泌TNF-α(P<0.05).IL-12p70和IL-10三种细胞因子。但是相比对照组,rD-7不诱导DC分泌IL-12p70和IL-10,虽然rD-7或者rD-7/D诱导DC分泌少量的TNF-a,但相比未刺激对照组无显著性差异。结论：卡他莫拉菌UspAl来源的重组蛋白rD-7显著诱导并修饰DC的活化成熟；rD-7诱导DC CD83.CD86和HLA-DR表达上调,不同于LPS活化的DC,rD-7活化的DC表现为CD80表达下调；DC低表达rD-7受体CEACAM1,但rD-7通过非依赖CEACAM1途径调控DC活化；不同于LPS,rD-7不显著诱导DC的分泌TNF-α、IL-10和IL-12p70。卡他莫拉菌UspAl来源重组蛋白rD-7显著修饰DC成熟,揭示了卡他莫拉菌毒力分子UspAl对机体适应性免疫应答的调控,为肺癌患者合并卡他莫拉菌感染条件下的免疫治疗及卡他莫拉菌疫苗的研发提供借鉴意义。更多还原

【Abstract】 The definition of tumor is that the abnormal growth of one cell in the tissue that lost its ability to regulate the cell growth on the gene level, resulting from uncontrolled, progressive multiplication of cells and serving no physiological function. Six hallmarks of cancer have been summarized through current research:1. the potential of unlimited growth;2. the characteristic to escape programmed cell death (PCD);3. the ability to provide growth signals for themselves;4. the insensitivity to growth-inhibitory signals;5. the properties to promote angiogenesis;6. with characteristics of tissue invasion and metastasis. However, much more researches recently indicate that inflammation is the seventh hallmark of cancer, named as cancer-related inflammation (CRI).Cancer is not independent of inflammation, in fact, as early as the19th century, Rudolf Virchow discovered the presence of a large number of leukocytes within the tumor, thus the author puts forward the hypothesis:the possible association exists between inflammation and cancer, but researchers did not obtain conclusive evidence confirming that inflammation and inflammatory microenvironment plays an important role in the development of cancer until recent two or three decades. It has been reported that cancers caused by germline mutations only account for a small fraction of all numbers (about10%), however, cancers caused by the somatic mutations and environmental factors account for the majority of total cancers (approximately90%), wherein chronic inflammation was involved in the formation of inflammatory microenvironment that results in the initiation and development of cancer. The statistical data from the research conducted by Aggarwal et al. in2009have shown that up to20%of cancers were related to chronic infection, for instance, it is the common view that hepatitis B viruses (HBV) and hepatitis C viruses (HCV) infection increases the risk of hepatocellular carcinoma (HCC); persistent H. pylori infection is closely related to gastric cancer; Schistosomiasis or Bacteroides infection is closely associated with bladder cancer or colon cancer, respectively. Infection-induced inflammation belongs to the normal host immune defense, aiming to eliminate pathogens. However, the functional destruction of host immune system caused by pathogens leads to persistent chronic inflammation that promotes occurrence and development of tumors, but it is noteworthy that not all of the chronic inflammation increased risk of cancer, for example, researchers have discovered that in the initial prostate biopsies patients who have signs of inflammation may have a reduced risk of being diagnosed with prostate cancer in prostate biopsies in future; psoriasis may reduce cancer risk. In summary, the complex relationship between inflammation and cancer is like a double-edged sword, as inflammation can promote tumor progression, however, it also inhibits the growth of tumor, so far, the key factors to determine the two different processes are not fully elucidated, and the mechanism still needs to be explained by tumor immunity.In tumor microenvironment, in addition to cancer cells and stromal cells, the immune cells are also important components which include monocytes, macrophages, neutrophils, myeloid-derived suppressor cells (MDSC), mast cells, natural killer cells (NK cells), dendritic cells (DC), T lymphocytes, B lymphocytes, which communicate with each other through cell-cell interaction or in a cytokine-regulation manner. Innate and adaptive immune responses both play an important role in monitoring and clearing mutant tumor cells. Monocytes and macrophages are considered as important parts of innate immune system. It has been reported that macrophages in local tumor can account for50%of the proportion of tumors, suggesting the highly important role of macrophages in tumor immunity. Tumor associated macrophages (TAMs) have been propagated as M2type macrophage (also named as alternatively activated macrophages), promoting tumor growth, angiogenesis, invasion and metastasis. TAMs were also involved in the inhibition of anti tumor effect of other immune cells, and significant negative correlation exists between high numbers of TAM in local tumor and prognosis. M2macrophages were characterized with special membrane receptors and chemokine receptors, such as mannose receptor (CD206), CD163, CXCR1, CXCR2, CCR2and also with secretion of several cytokines and chemokines, including IL-10, IL-1receptor antagonist (IL-lra), CCL17, CCL18, and CCL22.Dendritic cells (DCs) participate in the initiation, maintenance and regulation of the immune response, and play the key role in immune tolerance or immune activation. DCs are considered as only antigen-presenting cells (APC) those could activate naive T cells. DC could induce naive T cells into T helper cells1(Thl cells) or Th2cells, such as Thl cells with secretion of IL-2, IL-12p70, IFN-y, and Th2cells with the secretion of IL-4, IL-10, TGF-P, which are involved in inflammatory response and cell-mediated immune response. Given the important role of DC in anti-tumor immunity, DC-based immunotherapy (also known as DC vaccine) has become the hot topic in tumor immunotherapy and researchers has obtained good results from experiments in vitro, but there exists many problems in clinical applications, including weak anti-tumor immune response, great difference among individuals and so on. In fact, DCs in local tumor are tailored into tolerogenic DC with reduced antigen-presenting ability and secretion of Th2-type cytokines which can not effectively kill tumor cells. Significant differences on anti-tumor therapeutic effect of DC vaccines in vitro and in vivo remind the researchers that tumor microenvironment in vivo may modulate and even dominate the nature and function of DC. The main characteristics of the tumor microenvironment contains low pH values, hypoxia, and nutrient deficiencies, in addition, modification on DC function by pathogens in the tumor microenvironment also play a crucial and unignored role in inhibiting DC function.During the chronic infection process, to clarify how did pathogens and their specific virulence factors regulate the immune system and their mechanisms will provide direct insight into molecular mechanisms of inflammation or cancer biotherapy. Moraxella catarrhalis was originally considered as normal flora of the respiratory tract, but a growing number of studies indicate that Moraxella catarrhalis is one kind of pathogenic bacteria that can induce otitis media in children, and it is also responsible for the exacerbation with chronic obstructive pulmonary disease (COPD) through inducing apoptosis of alveolar epithelial cells. More than90%of M. catarrhalis produce β-lactamase that is resistant to ampicillin, so the research and development of M. catarrhali vaccine can solve the problem that is about increasingly serious antibiotic resistance, but the progress of M. catarrhalis vaccine research is slow, and one of the main factors is the lack of animal models for research. Mouse pulmonary clearance model is the widely used model, which is designed to measure clearance of bacteria from the lungs following bacterial challenge directly into the airways, but there is difference between mouse and human, as M. catarrhalis was cleared in mouse within24hours, but with the long-term existence in human respiratory tract after infection, so this model can not simulate the human respiratory tract infection. In view of this, it is necessary to seek better animal model to simulate human respiratory system infection and to conduct the research on M. catarrhalis vaccine in vitro is particularly necessary.Researchers have discovered that a lot of monocytes and dendritic cells were recruited to inflammation sites by chemokines after M. catarrhalis infection. Monocytes and DCs are highly heterogeneous populations, and monocytes can be polarized to into pro-inflammatory M1macrophages or anti-inflammatory M2macrophages by pathogen and its virulence factor, also, DC maturation was affected by pathogen and other factors in the micro-environment, which promote or inhibit DC maturation. Monocytes/macrophages are regarded as the sentinel cells of innate immune response, while DCs play the key role in regulating innate and adaptive immune response, thus regulation of M. catarrhalis on two cell populations may be directly or indirectly affects, even determines results of the immune response in lung after infection. However, the regulation on monocyte differentiation and DC maturation induced by M. catarrhalis has not been fully elucidated. Although there is no proof to support the existence of the positive association between M. catarrhalis and lung cancer, yet it is worth noting that the Gram-negative bacteria were isolated in68%patients who suffer from lung cancer, of which the most important are Haemophilus influenzae and M. catarrhalis. This fact raises a new question:whether M. catarrhalis that exist in the lung of patients who also suffer from lung cancer regulate or modulate tumor immunity?Monocytes and DCs may encounter multiple stimulants presented on the bacterial surface, such as outer membrane protein UspAl (ubiquitous surface protein A1) of M. catarrhalis and lipopolysaccharide (LPS), the major component of the outer membrane of Gram-negative bacteria including M. catarrhali. Monocytes can be induced to M1macrophage with LPS stimulation and DC maturation are also enhanced by LPS. It has been reported that pathogen and its virulence factor play an important role in M2polarization and the inhibition of DC maturation. For example, monocytes were polarized to M2macrophages by virulence factor LcrV of Yersinia sp.; hepatic DC maturation was inhibited by intestinal commensal bacteria products through the activation of IL-6/STAT3pathway, thus the question is that whether M. catarrhalis UspAl also induce M2polarization and the inhibition of DC maturation as other virulence factors as mentioned above? M. catarrhalis UspAl binds with N terminal domain of carcinoembryonic antigen-related cell adhesion molecule1(CEACAM1) and it has been proved that T cell activation and function were inhibited by CEACAM1signal through ITIM activation. Engagement of UspAl with CEACAM1has been reported to be involved in the regulation not only of epithelial function but also of T cell function upon CEACAM1cross-linking. However, it is not clear that whether M. catarrhalis UspAl (and in particular the recombinant UspAl fragment rD-7that binds to CEACAM1) affects monocyte differentiation and DC maturation through UspAl-CEACAM1signal. To reveal this question, the two classical cell populations of innate and adaptive immune system (monocyte/macrophages and dendritic cells) were stimulated with recombinant protein rD-7based on outer membrane protein UspAl of Moraxella catarrhalis and the effects on monocyte differentiation and DC maturation were determined respectively in vitro. Our study may provide the insight for the lung cancer patients with M. catarrhalis infections who were also treated with DC vaccine and the development of M. catarrhali vaccine.Part One The study on monocytes differentiation modulated by Moraxella catarrhalis adhesin UspAl-derived recombinant fragment rD-7and mechanism of rD-7-driven differentiationObjective:To investigate the effect of Moraxella catarrhalis adhesin UspAl-derived recombinant fragment rD-7on human monocyte differentiation and the mechanism of rD-7-driven differentiation.Methods:1. Production and identification of recombinant rD-7, rD-7/D and r6-8proteins. Recombinant protein rD-7and control protein (rD-7/D and r6-8) were produced using the pQE30expression system. Recombinant proteins carrying a His tag were purified by affinity chromatography using Ni-NTA agarose. Purified proteins were detected using an anti-His-tag antibody and in the case of rD-7, also a soluble CEACAMl-Fc construct by western blotting. The samples were treated with Polymyxin B to remove endotoxin and then sterilized with0.2μm filter. The endotoxin levels in all purified proteins were less than1.0EU/μg as determined by Limulus Amoebocyte Lysate method.2. Digestions of rD-7and rD-7/D with Proteinase K. Samples of the purified recombinant molecules were digested using200μg/ml of Proteinase K (PK) at37℃for3h and the digested samples were heated at95℃for1h to inactive the enzyme. The absence of intact proteins in the relevant samples was confirmed using SDS-PAGE.3. Isolation of CD14+/CD15+cells from healthy human peripheral blood. Peripheral bloods from healthy volunteers under inform consent was collected in the sterile collection bag, and PBMCs were prepared with density gradient centrifugation using Histopaque-1077. CD14+monocytes and CD15+cells were positively selected from PBMCs by using CD14and CD15immunomagnetic beads respectively. 4. Monocytes activation. Freshly isolated monocytes were stimulated with2.5μg/ml of the recombinant proteins rD-7, r6-8or rD-7/D, or with LPS (2μg/ml) respectively. After24h stimulation, cells were harvested and the surface molecules expression was analyzed by flow cytometry and culture supernatants were collected and stored at-80℃for cytokine detection. To block the binding of rD-7to CEACAM1in some experiments, monocytes were treated with polyclonal anti CEACAM antibody A0115or IgG control (50μg/ml) for1h prior to the addition of the recombinant molecules as above. After additional23h, cells were collected and the surface molecules expressions were detected by using flow cytometry.5. The detection of molecular expression on monocytes by using flow cytometry. The CEACAM1expression on freshly isolated monocytes was determined using flow cytometry. In monocyte activation experiment, monocytes stimulated with rD-7, r6-8, rD-7/D or LPS were collected and CD80, CD86and CD206expressions on cells were detected by using flow cytometry.6. The study of the binding of rD-7on CD14+or CD15+cells and the blocking effect of A0115on rD-7binding on cells. For detection of surface-bound rD-7or rD-7/D and their inhibition by anti-CEACAM antibody, freshly isolated monocytes or CD15+cells were first incubated in the presence of A0115(50μg/ml) or its IgG control (50μg/ml) for30min on ice and then with the recombinant bacterial molecules (2.5μg/ml) for1h on ice. After treatment, the surface binding of rD-7on CD14+or CD15+cells were analyzed by using flow cytometry.7. Detection of the levels of IL-6, IL-10, IL-12p70and TNF-a from monocytes stimulated by rD-7with Luminex assay. Freshly isolated monocytes were stimulated by rD-7or rD-7/D for24h, and then the supernatants were collected. The levels of IL-6, IL-10, IL-12p70and TNF-a were detected using MILLIPLEX (?) MAP Human Cytokine/Chemokine Magnetic Bead Panel I and results were analyzed using MAGPIX system.8. Analysis of chemokines/cytokine profiles from monocytes stimulated by rD-7or rD-7/D with proteome profiler antibody arrays. Freshly isolated monocytes were stimulated by rD-7or rD-7/D for24h, the supernatants were collected and the chemokines/cytokine profiles were detected using Proteome Profiler human cytokine array Panel A kit.Results:1. Modulation of monocyte differentiation by M. catarrhalis UspAl-derived recombinant fragment rD-7. Compared to the unstimulated group, CD206expression on CD14+cells was significantly increased by the recombinant protein rD-7(P<0.001), however, CD80expression was significantly decreased (P<0.05) and CD86expression was not significantly changed; CD80(P<0.05) and CD206(P<0.001) expression on CD14+cells were significantly enhanced by LPS but CD86expression was reduced (P<0.05); the expressions of CD80, CD206, CD86on CD14+cells were not significantly affected by r6-8.2. The increased MFI of CD14+cells expressing CD206modulated by rD-7was dependent on the intact protein. CD206expression on CD14+cells was significantly increased by rD-7(P<0.001) compared with unstimulated cells, whilst digested rD-7lost the ability to increase CD206expression on monocytes (P<0.001) compared with monocytes stimulated with rD-7, suggesting the increased MFI of CD14+cells expressing CD206modulated by rD-7was dependent on the intact protein instead of endotoxin.3. CD14+monocytes express CEACAM1, the receptor of rD-7. The expression of CEACAM1on CD14+monocytes was detected by flow cytometry using the polyclonal antibody A0115as well as an anti-CEACAM1mAb (R and D Systems MAb2244). Low numbers of monocytes expressing CEACAM1were detected by both antibodies.4. rD-7-induced CD206expression on monocytes is independent of the ability to bind CEACAM1. Compared to the unstimulated control, CD206expression on CD14+monocytes was significantly increased by rD-7(P<0.001) or rD-7/D (P< 0.001) at a similar level. Digested rD-7and digested rD-7/D lost the ability to increase CD206expression on CD14+cells. CD206expression on CD14+cells was significantly increased by LPS (P<0.001); however, CD206expression was still enhanced by LPS treated with Proteinase K (P<0.001). Flow cytometry analysis revealed the rD-7binding on CD15+cells (P<0.001) instead of rD-7/D. Compared with IgG control, A0115blocked the rD-7binding on CD15+cells (P<0.05).5. Confirmation of CEACAM-independent binding and stimulation of CD206expression on CD14+cells by rD-7and rD-7/D by antibody inhibition assays. Both rD-7(P<0.05) and rD-7/D (P<0.05) bound to the surface of CD14+monocytes, and A0115did not block the binding as observed compared with IgG control. Freshly isolated monocytes were blocked with A0115before stimulation with rD-7or rD-7/D for24h. Both rD-7(P<0.01) and rD-7/D (P<0.01) still significantly enhanced CD206expression on CD14+cells and this remained unaffected in the presence of A0115.6. IL-6, TNF-a, IL-10and IL-12p70secretion from monocytes stimulated by rD-7and rD-7/D. IL-10, IL-12p70, IL-6and TNF-a in supernatants from monocytes stimulated with2.5μg/ml rD-7or rD-7/D were quantified by Luminex assays, confirming that rD-7and rD-7/D did not increase the secretion of IL-10or IL-12p70, whilst neither rD-7nor rD-7/D induced proinflammatory cytokines IL-6and TNF-a secretion compared with the unstimulated group from the four donors.7. Cytokines and chemokine expression profiles from monocytes modulated by rD-7or rD-7/D. A significant increase in IL-1ra secretion produced by monocytes stimulated with rD-7(P<0.001) or rD-7/D (P<0.001) for24h was observed, and the quantity of IL-lra released following treatment with rD-7or rD-7/D was similar to that from the LPS-stimulated group. In addition, IL-8secretion was also increased by either rD-7(P<0.05) or rD-7/D (P<0.01) when compared to the unstimulated group. IL-2, IL-4, IL-5, IL-10, IL-12p70, IL-17, IL-32a, MCP-1, MIP-1β, CXCL10and CXCL12were undetectable in rD-7-or rD-7/D-stimulated groups. There was no significant change for C5a, CD40L, G-CSF, GM-CSF, CXCL1, CCL1, sICAM-1, IFN-γ, IL-1α, IL-1β, IL-6, IL-13, IL-16, IL-17E, IL-23, IL-27, CXCL11, MIF, CCL3, Serpin E1, CCL5, TNF-a, sTREM-1secretion between the rD-7or rD-7/D group and the unstimulated group.Conclusions:Moraxella catarrhalis adhesin UspAl-derived recombinant fragment rD-7modulated monocyte differentiation to CD14+CD206+phenotype; the modulation was dependent on intact rD-7protein instead of endotoxin; CD80expression on CD14+cells were decreased by rD-7; CEACAM1, the receptor of rD-7was expressed on monocyte, however, monocytes differentiation modulated by rD-7was independent of CE AC AMI; the secretion of the anti-inflammatory cytokine IL-1ra was significantly induced by rD-7. Monocyte differentiation was significantly modified by Moraxella catarrhalis UspAl-derived recombinant fragment rD-7, revealing the regulation of Moraxella catarrhalis virulence factor UspAl on innate immune response, thus our study will provide the insight for the lung cancer patients with M. catarrhalis infections who were also treated with DC vaccine and the development of M. catarrhali vaccine. Part Two The study on dendritic cells maturation modulated by Moraxella catarrhalis adhesin UspAl-derived recombinant fragment rD-7and mechanism of rD-7-driven DC maturationObjective:To investigate the effect of Moraxella catarrhalis adhesin UspAl-derived recombinant fragment rD-7on human DC maturation and the mechanism of rD-7-driven maturation. Methods:1. Production and identification of recombinant rD-7and rD-7/D proteins. The procedure of production of recombinant proteins has been described in part one. The endotoxin levels in all purified proteins were less than1.0EU/μg as determined by Limulus Amoebocyte Lysate method.2. Isolation of CD14+monocytes from healthy human peripheral blood. Peripheral bloods from healthy volunteers under inform consent was collected in the sterile collection bag, and PBMCs were prepared with density gradient centrifugation using Histopaque-1077. CD14+monocytes were positively selected from PBMCs by using CD14immunomagnetic beads.3. Induction and identification of DC. DC medium containing1000U/ml rh GM-CSF,500U/ml rh IL-4,10%human AB serum was prepared, and freshly isolated CD14+monocytes were resuspended by DC medium to adjust the cell concentration of5x105/ml.2ml cell suspension were grown in24-well plate and plated in the humidified incubator at37℃with5%CO2.200μl fresh DC medium were changed on day3and day5. Immature DCs were yielded on day6, and imDCs were induced to mDCs by LPS (2μg/ml) for additional24h.4. DC activation. Freshly isolated CD14+monocytes was induced to imDCs on Day6with rh GM-CSF and rh IL-4, and imDCs were stimulated with rD-7(2.5μg/ml), rD-7/D (2.5μg/ml) or LPS (2μg/ml) for24h, and the expression of CD80, CD83, CD86, HLA-DR on DCs were detected by flow cytometry. In blocking CEACAM experiment, imDCs were blocked with A0115or IgG isotype control (50μg/ml) for1h, and cells were stimulated with rD-7or rD-7/D for additional23h.5. Detection the levels of TNF-a, IL-10and IL-12p70. imDCs were stimulated with rD-7(2.5μg/ml), rD-7/D (2.5μg/ml) or LPS (2μg/ml) for24h, and then supernatants were collected and the levels of TNF-a, IL-10and IL-12p70were determined by ELISA. In blocking CEACAM experiment, imDCs were blocked with A0115or IgG isotype control (50μg/ml) for1h, and cells were stimulated with rD-7or rD-7/D for additional23h.Results:1. Modulation of DC maturation by M. catarrhalis UspAl-derived recombinant fragment rD-7. imDCs on day6were incubated with the CEACAM-binding recombinant molecule rD-7or LPS. The mean fluorescence intensities (MFIs) of cells expressing CD86(P<0.001) and HLA-DR (P<0.01) were significantly increased by rD-7compared with untreated imDCs. rD-7also increased MFI of cells expressing CD83(P<0.05) but to a lesser extent than LPS (P<0.001). Additionally, while LPS increased CD80expression (P<0.001), rD-7decreased its expression on DCs (P<0.01).2. CEACAM1, the receptor of rD-7, was expressed on human DCs. Myeloid-derived monocytes were induced by rh GM-CSF and rh IL-4for6days to yield immature DC or mature DC with additional24h LPS stimulation. The percentages of cells expressing CD11c were almost100%. CEACAM1was detected on imDCs and mDCs with monoclonal anti CEACAM1antibody MAb2244whilst the percentage of CEACAM1+DCs was low by using flow cytometry.3. Decreased CD80expression on DC modulated by rD-7was independent of CEACAM1signal. After imDCs were stimulated with either rD-7or rD-7/D for24h, CD80expression on DCs was significantly decreased to similar levels by rD-7(P<0.01) or rD-7/D (P<0.05), but positive control stimulation with LPS alone significantly increased CD80expression as observed (P<0.001). However, the decrease of CD80expression induced by rD-7was not blocked by using anti CEACAM antibody A0115.4. Increased CD83expression on DC modulated by rD-7was independent of CEACAM1signal. CD83expression on DCs was increased by rD-7/D (P<0.01) to similar levels in each case with using rD-7(P<0.05). Furthermore, control LPS also significantly increased CD83expression (P<0.001). Also, the increase of CD83expression induced by rD-7was not blocked by A0115 compared with IgG control.5. Increased CD86expression on DC modulated by rD-7was independent of CEACAM1signal. CD86expression on DCs was also increased by rD-7/D (P<0.001) to similar levels in each case with using rD-7(P<0.001). Furthermore, control LPS also significantly increased CD86expression (P<0.001). Compared with LPS, the increased CD86expression induced by rD-7(P<0.05) and rD-7/D (P<0.01) were more significant. Also, the increase of CD86expression induced by rD-7was not blocked by A0115compared with IgG control.6. Increased HLA-DR expression on DC modulated by rD-7was independent of CEACAM1signal. HLA-DR expression on DCs was all increased by rD-7(P<0.01), rD-7/D (P<0.01), LPS (P<0.01) to similar levels in each case and A0115could not block the increase of HLA-DR expression on DCs induced by rD-7compared with IgG control.7. TNF-a, IL-10and IL-12p70secretions from DCs stimulated by rD-7. TNF-a secretions was significantly increased by positive control LPS (P<0.05). TNF-a produced by DCs was slightly increased by rD-7and rD-7D to a similar level. Furthermore, IL-10and IL-12p70were not detectable using ELISA after rD-7or rD-7/D stimulation.Conclusions:Moraxella catarrhalis adhesin UspAl-derived recombinant fragment rD-7modulated DC activation and maturation. CD83, CD86and HLA-DR expressions were increased by rD-7. Unlike LPS, CD80expression on DC was decreased by rD-7. CEACAM1, the receptor of rD-7was expressed on DC; however, DC maturation modulated by rD-7was independent of CEACAM1. Unlike LPS, TNF-a, IL-10and IL-12p70secretion was not significantly affected by rD-7. DC maturation was significantly modified by Moraxella catarrhalis UspAl-derived recombinant fragment rD-7, revealing the regulation of Moraxella catarrhalis virulence factor UspAl on adaptive immune response, thus our study will provide the insight for the lung cancer patients with M. catarrhalis infections who were also treated with DC vaccine and the development of M. catarrhali vaccine.更多还原