Mir-10b调节K1f4介导的巨噬细胞极化并抑制T_H1T_H17细胞反应
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摘要
研究背景和目的
巨噬细胞在人类免疫系统中占有独特的位置,它是免疫系统第一个被确定的细胞成分,在机体正常生理过程和病理过程中发挥极其重要的作用。它在机体遭受损伤或者感染状态时,第一个到达病患处发挥重要的作用,是机体中重要的吞噬和抗原提呈细胞。临床研究及基础实验均已证实巨噬细胞在急性炎症(如感染,败血症等)和慢性炎症(胰岛素抵抗,动脉粥样硬化,慢性损伤,肿瘤的形成等)均起到关键性作用。全身各处的巨噬细胞以及树突状细胞,破骨细胞均来源于外周循环中的单核细胞。巨噬细胞是在细胞因子的刺激下由骨髓干细胞发育而来。在多集落刺激因子(multi-CSF,multi Colony Stimulating Factor)、粒细胞巨噬细胞集落刺激因子(GM-CSF,Granulocyte-Macrophage Colony Stimulating Factor)等刺激下,由骨髓干细胞发育成单核母细胞,后者进一步分化成为前单核细胞并进人外周血流,并在此处分化成为成熟的单核细胞。很多因素如前炎症状态,代谢,免疫都可以促使单核细胞向外周组织聚集,分化为巨噬细胞,实现宿主防御,机体自我修复与重塑等功能。
我们知道免疫效应细胞在免疫反应中并不是表型单一的活化细胞,在不同的微环境因素影响下,活化的细胞可以分化为不同表型的亚细胞型,这些亚型细胞不但适应局部微环境同时又是局部微环境不可缺少的组分。基于对Th细胞的极化的研究基础上,越来越多的研究开始关注巨噬细胞的极化现象。
目前研究发现,在不同的环境中,巨噬细胞可以分化为行使特定免疫功能的不同细胞亚群。巨噬细胞主要分为两种类型：由TH1型反应引起的IFN-γ依赖的经典途径激活的MI型巨噬细胞,即经典活化的巨噬细胞(classically activated macrophage),其可直接吞噬和杀伤病原微生物和肿瘤细胞,分泌促炎因子和趋化因子,并通过递呈抗原参与正向免疫应答,发挥免疫监视的功能和宿主防御的功能；由TH2型细胞因子、IL-4与IL-13引起的替代途径激活的M2型巨噬细胞,即替代性活化的巨噬细胞(alternatively activated macrophage)。其可在巨噬细胞集落刺激因子(M-CSF,Macrophage Colony-Stimulating Factor) IL-4、IL-13、IL-10、糖皮质激素、转化生长因子-β(TGF-β)、维生素D3和前列腺素E2(PGE2)等作用下诱导产生,其抗原递呈能力较低,并通过分泌IL-10、CCL17、CCL18、CCL22和TGF-β等抑制性细胞因子下调免疫应答。促进创伤修复和纤维变性。M2型巨噬细胞同肿瘤的发生发展有着密切的关系,肿瘤中的巨噬细胞主要是经微环境“教化”后的M2型巨噬细胞,具有促进肿瘤细胞侵袭转移的作用。同时大量研究证实M2型巨噬细胞可以减轻胰岛素抵抗,改善寄生虫感染,重塑脂肪组织,减轻体重。
MicroRNAs (miRNAs)是一类由内源基因编码的、长度约为18-25个核苷酸的、非编码单链小分子RNA,它们通过结合到靶基因的mRNA上,在转录后水平调控基因的表达,对增殖,分化,凋亡等基本细胞过程具有重要的调控作用。
目前越来越多的证据表明：microRNA(miRNA)参与了免疫调控,与适应性免疫系统的分化和功能维持关系密切,许多miRNAs在免疫反应中扮演着十分重要的角色。如研究证实TLR4配体脂多糖(Lipopolysaccharide,LPS)刺激后,可以通过调节肿瘤坏死因子受体相关蛋白6,白细胞介素1受体相关激酶1,来实现miR-146a在单核细胞中表达明显升高；在先天性免疫应答方面,miR-146a表达升高是内毒素耐受的重要表现之一。细菌病毒相关抗原可以激活TLR4,TLR2,TLR3或者TLR9等,进一步促进巨噬细胞表达miR-155。已经证实miR-155在免疫反应中既可以发挥促进免疫反应也可以表现出抑制免疫反应的作用。经LPS刺激后单核细胞可以表达miR-21,上调miR-21的表达可以抑制LPS所诱导的NF-κB调控的活化以及IL-6的表达,但可以提高IL-10的表达,同时miR-21可以通过TLR7和TLR8诱发免疫激活。
在各种miRNAs中,miR-10b引起了我们极大的兴趣。目前的研究主要致力于miR-10b同肿瘤之间的关系。miR-10b对肿瘤转移的作用是由Ma和同事等首次提出的。他们的研究发现miR-10b可以在高转移的乳腺癌细胞中表达,阻断miR-10b能够使MDA-MB-231细胞侵袭能力下降90%以上,而不具备转移能力的SUM149细胞过表达miR-10b后,在裸鼠体内发生远处转移。临床资料也表明,发生转移的乳腺癌患者,miR-10b表达高于非转移组。此外,Gabriely等的研究证明了在体外,抑制miR-10b的表达可以通过诱导细胞周期和凋亡,从而抑制恶性胶质瘤细胞的生长。并且与含miR-10家族水平低的胶质瘤患者相比,含miR-10家族水平高的患者其存活率明显降低。与注射安慰剂组相比,人胶质瘤小鼠模型在注射miR-10b抑制剂后,其肿瘤生长明显下降。这些均证明了miR-10b对肿瘤细胞生长的作用。
Kruppel样因子4(Klf4或称肠道富集的Kruppel样因子)是属于Kruppel蛋白家族的一种含有锌指结构的转录因子。Klf4具有多种生理功能,在肿瘤的发生发展中Klf4基因具有抑癌基因和癌基因的双重功能,可以影响细胞周期的调控、凋亡以及细胞分化。相当一部分研究发现在多种人类肿瘤,包括食管癌,胃癌,大肠癌和膀胱癌,Klf4的表达经常发生缺失,这提示其可能具有抑癌作用。据报道,Klf4可抑制多种癌症转移的发生,如大肠癌、食管癌、胰腺癌细胞等。并且在大肠癌中,Klf4可通过诱导细胞周期阻滞抑制细胞增殖；通过影响细胞增殖、凋亡和癌细胞的浸润,从而调节食管癌的发生。综合考虑上述结论,Klf4在癌细胞的转移和增殖过程中可能起抑癌基因的作用,这恰恰与miR-10b发挥的癌基因作用相反。目前已有研究探讨miR-10b通过调控Klf4,在大肠癌的进展过程中发挥促进增殖和转移的功能。本课题组在既往研究中构建了两个含有荧光素报告基因的质粒：Klf43'-UTR和Klf43'-UTR-mut。前者含有Klf4mRNA的全长3’UTR区域；后者3’非翻译区关键结合位点的第296-300位碱基被其互补碱基替代。在SW620细胞同时转染含有Klf43'-UTR或Klf43'-UTR-mut的荧光素酶报告质粒以及miR-lOb inhibitor或相应的对照。结果显示,下调miR-10b的表达去除了其对含Klf43'-UTR的荧光素酶活性的抑制作用,提示miR-10b可通过Klf43'-UTR来调节Klf4基因。而当Klf43’-UTR与miR-10b的结合位点发生突变,miR-10b表达抑制后对荧光素酶活性产生的作用将会受到影响。证实Klf43'UTR从295-301位核苷酸可以与miR-10b的5’端关键位点结合,并进一步证实Klf4是miR-10b的直接靶基因。
在巨噬细胞极化调节中,Klf4、Klf2、Klf1和Klf3被报道与单核巨噬细胞的分化或激活有关。其中研究得比较多的是Klf4。Klf4在单核细胞的谱系决定、分化以及激活过程中都是一个重要的调控因子。Klf4-/-嵌合体小鼠中,单核细胞的分化会受到严重影响,骨髓中的单核细胞、脾脏中的单核细出现大量减少,并且发现血液中循环的单核细胞的几近消失。Klf4不仅与巨噬细胞的分化有关,还与巨噬细胞的激活有关。最近的研究表明Klf4在M2型巨噬细胞的分化过程中具有重要作用。
随着1889年有学者提出“种子”与“土壤”假说,肿瘤微环境开始受到重视,炎症微环境已被列为肿瘤十大特征之一,肿瘤与炎症有着密不可分的关系,在肿瘤发展过程中,局部炎症环境促进肿瘤生长及基因突变。Klf4不仅参与炎症反应中在单核细胞的谱系决定、分化、激活过程,而且作为调节巨噬细胞极化中都是一个重要的调控因子。
综上所述,我们提出一个假说：miR10b是否能够通过调控KIf4而进一步调节巨噬细胞极化。为了验证这一假说,我们从如下几个方面入手：我们首先观察miR-10b分别在M1及M2型巨噬细胞的表达情况,分析巨噬细胞极性改变时miR-10b在巨噬细胞的表达是否有相应的变化；通过转染miR-10b inhibitor和miR-10b mimics,观察过表达miR-10b及抑制miR-10b是否能够调节巨噬细胞极化；巨噬细胞的除具有调节固有免疫之外,其中重要的作用是通过抗原提呈促进T辅助细胞极化调节继发性免疫,我们进一步观察抑制]miR-10b的表达能否减弱TH1及TH17细胞炎症反应；了解抑制miR-lOb是否降低小鼠对内毒素的敏感性及对细菌感染的清除能力；而后,采用CD4+CD45RhiT细胞诱导的肠炎模型模拟人类IBD炎症,观察抑制miR-10b能否减弱T细胞所介导的肠炎；最后通过荧光素酶报告基因证实小鼠中Klf4是miR-10b的靶基因,以探讨miR-10b—Klf4这一靶向调控关系对巨噬细胞极化的调节及其影响,对TH1及TH17细胞炎症反应的调控机制。
方法
为了明确miR-10b在M1型巨噬细胞极化过程中是否起到重要作用,我们采集健康志愿者外周静脉血,采用Ficoll密度梯度离心法,分离外周血单个核细胞,原代培养单个核细胞,采用GM-CSF(10ng/mL)诱导7天,然后采LPS(100ng/mL)和IFN-γ(20ng/mL)共刺激诱导巨噬细胞向M1型极化,分别取不同时间点0、12、24小时,采用实时荧光定量PCR检测M1型巨噬细胞中miR-10b的表达水平。我们也检测了在M2极化过程中miR-10b的表达水平,原代培养单个核细胞,采用IL-4(10ng/mL)和IL-13(10ng/mL)共刺激诱导M2型巨噬细胞,同样选择不同时间0、12、24小时,采用实时荧光定量PCR检测niR-10b的表达水平。我们检测是否在巨噬细胞发生极化改变时,：miR-10b的表达也随之发生变化。同样,我们分离了人外周血单个核细胞,采用GM-CSF(10ng/mL)诱导7天,然后采用LPS(100ng/mL)和IFN-γ(20ng/mL)共刺激诱导巨噬细胞向M1型极化24小时后,改用IL-4(10ng/mL)和IL-13(10ng/mL)共刺激24小时,同样,除去M2型极化因子后,再恢复M1型极化因子刺激24小时；然后采用实时荧光定量PCR检测miR-10b的表达。改变miR-10b的表达是否影响巨噬细胞的极化?我们外周血分离出单个核细胞,采用GM-CSF诱导7天,分别转染miR-10b inhibitor和miR-10b minic,48小时后,然后给予GM-CSF(20ng/mL)刺激4小时诱导M1型巨噬细胞,并检测M1型和M2型巨噬细胞特征性因子表达水平。iNOS是M1型巨噬细胞特征性因子,而Klf4和IL-4R是M2型特征性因子。采用荧光定量PCR检测以上因子mRNA水平。
为了说明抑制niR-10b表达能否减弱了TH1细胞的反应。从C57BL/6小鼠骨髓分离单个核细胞,采用GM-CSF(10ng/mL)诱导5天,转染miR-10b inhibitor,继续培养48小时,加入磁珠分选的小鼠CD4+T细胞混合培养,分别采用实时荧光定量PCR检测检测TH1转录因子T-bet mRNA表达,ELISA分析IFN-y表达,以及流式分析IFN-y表达。
同样为了探讨了抑制miR-10b表达能否减弱了TH17细胞的反应。同上述处理方法一致,分别采用实时荧光定量PCR检测检测TH17转录因子RORyt mRNA表达、ELISA分析IL-17表达,以及流式分析IL-17表达。
为了进一步探讨抑制miR-10b是否能明显抑制M1型极化,通过体外实验检测小鼠对内毒素的敏感性的变化。我们采用5%肉汤培养基冲洗小鼠腹腔,收集腹腔巨噬细胞,将收集的巨噬细胞种植于60mm培养皿,转染miR-10b inhibitor,按照2x106个注入小鼠腹腔,并按照1Omg/kg腹腔注射LPS,观察小鼠生存率；并且收集6血清。采用ELISA法检测血清中IL-6、TNF-a表达的变化。
抑制miR-10b能否减弱了对细菌感染清除能力?我们采用miR-10b antagomir尾静脉注入到小鼠体内,48小时后,通过尾静脉注入李斯特菌(2x104CFU/只),分别于24小时和48小时处死。每组小鼠取血,收集血清,采用ELISA检测IL-6、TNF-α水平；而且分别收集肝脏和脾脏,匀浆,涂抹在LB琼脂糖培养皿上,观察细菌的菌落形成。
最后我们检测抑制miR-10b能否减弱CD4+CD45RBhiT细胞诱导的肠炎。CD4+CD45RBhiT细胞诱导的肠炎类似于人类IBD炎症。我们采用miR-10bantagomir尾静脉注入到小鼠体内,每周两次,连续注射3周；采用流式细胞分选CD4+CD45RBhiT细胞,5x105细胞/只腹腔注入到Rag-/-小鼠体内,每周注射两次miR-10b antagomir。观察小鼠体重及活动。6周后处死小鼠,收取肠道组织制作石蜡病理切片,收取血液,离心收集血清,采用ELISA检测炎症因子。观察实验组和对照组小鼠体重变化情况,肠道炎症状况,免疫组化观察肠道炎性细胞侵入情况,流式细胞分析T辅助细胞TH1和TH17细胞分化。
我们采用生物信息学分析,通过构建Klf43'UTR包含预测结合位点的荧光素酶报告基因,验证鼠源miR-10b与Klf4是否有结合位点。
结果
巨噬细胞极化受局部微环境等多种因素的影响,本文意在探讨niR-10b对巨噬细胞M1/M2分型的影响及调节。在发生M1型巨噬细胞极化过程中,miR-10b表达明显升高,而在M2型极化过程,表达没有明显改变,这提示miR-10b在M1型巨噬细胞极化过程中可能起到重要作用。随着微环境或者外界刺激的改变,巨噬细胞的极性发生相应变化,与各亚型相关的标记分子也会发生表达改变,我们检测在巨噬细胞发生极化改变时,miR-10b的表达也随之发生变化,miR-10b在M1型极化时表达最高,在M2型极化因子刺激后表达明显下降,而重新采用M1型极化因子刺激后,miR-10b表达升高,这说明了巨噬细胞的极化可能影响了miR-10b的表达。反之miR-10b的表达也会影响巨噬细胞的极化,抑制miR-10b的表达能明显抑制M1型巨噬细胞特征性基因iNOS表达水平及M1巨噬细胞能产生致炎因子,如IL-6、TNF-α,M2型巨噬细胞特征性基因Klf4和IL-4R表达增加,增加miR-10b表达则提高了iNOS表达,Klf4和IL-4R表达下降,这说明了改变miR-10b的表达水平可以改变巨噬细胞极化状态,miR-10b促进M1型巨噬细胞极化,抑制miR-10b是的巨噬细胞M1型极化受阻。M1型巨噬细胞诱导TH1和TH17反应,M2型巨噬细胞诱导TH2反应。抑制miR-10b降低了T-bet mRNA,RORyt mRNA表达水平同时也降低了IFN-γ和IL-17表达水平,这说明了抑制miR-10b表达减弱了TH1和TH17细胞诱导的炎症反应。我们采用5%肉汤培养基冲洗小鼠腹腔,收集腹腔巨噬细胞,将收集的巨噬细胞转染miR-10b inhibitor,培养后注入小鼠腹腔,对比小鼠生存率：miR-10b inhibitor组生存率明显高于对照组,并且血清中IL-6、TNF-α表达也明显低于对照组。这说明了抑制miR-10b可以导致机体对内毒素敏感性下降,也说明了抑制了巨噬细胞向M1型极化,结合体外实验说明了抑制niR-10b能明显抑制M1型极化。巨噬细胞不同极化状态细菌入侵的反应不用,M1型巨噬细胞可以分泌iNOS等杀死细菌,而M2型巨噬细胞对细菌感染不敏感。我们采用miR-10b antagomir尾静脉注入到小鼠体内,再通过尾静脉注入李斯特菌(2x104CFU/只),收集血清、肝脏和脾脏,匀浆,涂抹在LB琼脂糖培养皿上,观察细菌的菌落形成。结果显示在miR-10b antagomir组肝脏和脾脏菌落比对照组明显增多,而且血清中IL-6和TNF-α表达也明显下降这说明了在体内抑制miR-10b的表达后,导致了M1向M2极化,致使对细菌的感染不敏感,对细菌的清楚能力下降。体外实验证明了抑制miR-10b能减弱TH1和TH17细胞的反应,TH1和TH17细胞被认为是人类IBD发病机制中介导炎症的重要细胞亚群,我们采用CD4+CD45RBhiT细胞诱导的肠炎模型模拟人类IBD炎症,观察抑制miR-10b可以减弱T细胞所介导的肠炎,这是由于M1型极化而导致TH1和TH17细胞反应减弱所致。通过小鼠骨髓来源单个核细胞,采用GM-CSF(10ng/mL)诱导5天,转染miR-10b mimic,更换为rIL-4/IL-13分别刺激4小时和24小时,结果表明在M2型巨噬细胞极化状态下,升高miR-10b能从转录和翻译水平抑制KLF4的表达。通过荧光素酶报告基因,结果证实了与人类miR-10b和Klf4结合相似,鼠miR-10b也能与KLF4特异性结合,。
结论
本研究第一次报道了miR-10b在M1型巨噬细胞的表达情况,揭示了miR-10b表达水平不仅可以随巨噬细胞极性改变而发生变化,反之通过改变miR-10b的表达可以调节巨噬细胞极化分型。抑制miR-10b明显抑制M1型极化,并减弱了TH1和TH17细胞的反应。同时抑制miR-10b可以导致机体对内毒素敏感性下降,对细菌清除能力下降,减弱T细胞所介导的肠炎。通过构建小鼠Klf43'UTR包含miR-10b结合位点的荧光素酶报告基因载体及相应的突变载体,小鼠niR-10b能与Klf4结合。
Background and Objection:
Macrophages have long been considered to be important immune effector cells.It plays an important role the in a broad spectrum of pathological and physiology procedure and respond to endogenous stimuli that are rapidly generated following injury or infection.which is first confirmed cell in the immune system. It is known that they are among the first cells to arrive at sites of wounding and/or infection and is characterize by a high capacity to present antigen,where they perform several functions. Clinical and experimental studies support an important role for the macrophage in a broad spectrum of acute (e.g., pathogen infection, sepsis) and chronic inflammatory conditions (e.g.insulin resistance, atherosclerosis, chronic wounds, and tumorigenesis).Circulating monocytes give rise to a variety of tissue resident macrophages throughout the body,as well as to specialized cells such as dendritic cells (DCs) and osteoclasts. With the stimulation of granulocyte-macrophage colony stimulating factor and multi-colony stimulating factor, bone marrow stem cell develops into Mononuclear cells.Monocytes are known to originate in the bone marrow from a common myeloid progenitor that is shared with neutrophils, and they are then released into the peripheral blood, where they circulate for several days before entering tissues and replenishing the tissue macrophage populations involved inflammation and host defense,,which are induced by pro-inflammatory and environmental signals.
Immunologic effector cell exhibit remarkable plasticity that allows them to develop into different type of cell and serve distinct functions in the regulation of the inflammatory response. As more and more studies about the molecular mechanisms that govern Th cell polarization, the regulation of macrophage polarization is paid huge attention to.
With a huge number of studies confirmed, it is well-accepted that macrophage is divided into M1macrophage (Classically activate) and M2macrophage (Alternatively activate). Macrophages stimulated with LPS or INF-γassume an M1pro-inflammatory phenotype characterized by a high expression level of iNOS, a high capacity to present antigen and production of pro-inflammatory cytokines such as TNF-a, IL-1β,monocyte chemoattractant protein-1(MCP-1). M1macrophages are critical effector cells that kill microorganisms and thus benefit the host. However, this pro-inflammatory activity must be carefully titrated, as sustained activation can predispose to chronic inflammatory states such as obesity and insulin resistance. M-CSF, IL-4, IL-13, IL-10, IL-4and TGF-βstimulation leads to M2macrophages.,which produce of anti-inflammatory cytokines such as IL-10、 CCL17,CCL18, CCL22和TGF-aand promote tissue repair and remodeling as well as tumor progression.Tumor-associated-macrophage is resembling M2macrophage,which is associated with cancer initiation and promotion. Accumulating evidence suggests that M2macrophages can protect against insulin resistance, eliminate parasites, and promote tissue remodeling and repair. Interestingly, it helps weight loss, known to improve insulin sensitivity and to induce remodeling of adipose tissue.
MicroRNAs (miRNAs), a newly discovered class of non-protein-coding small RNA, have been shown to play a vital role in processes such as development, cellular proliferation, and more recently apoptosis.It combine mRNA of target gene and supress gene expression post-transcriptionally.
Accumulating evidence suggests that miRNAs constitute an integrated part of the regulatory networks in innate immunity and adaptive. More than a dozen miRNA have been shown to play a critical role in regulating polarization of macrophage and Th-17cell. Studies found that miR-146a is rapidly upregulated in human monocytic cells stimulated with lipo-polysaccharide(LPS), a TLR4ligand, and acts as a negative feedback regulator of TLR signaling, presumably by targeting tumor necrosis factor receptor-associated factor6and interleukin(IL)-1receptor-associated kinase-1, miR-146a upregulation may be an essential component of endotoxin tolerance in innate immune response.miR-155is induced in macrophage in response to both bacterial and viral-derived antigens that activate TLR4,TLR2,TLR3or TLR9.It was estimated that miR-155plays both positive and negative regulator roles in immune responses. miR-21is induced in monocytic cells by LPS stimulation. Upregulated miR-21was found to dampen LPS-induced nuclear factor-κB activation and IL-6expression but enhance IL-10expression. miR-21can also trigger inflammatory responses by binding to TLR7and TLR8.
In all of miRNAs, miR-10b is of great interest to us. Because it not only initiates invasion and metastasis in cancers, but also controls cancer cell growthe by regulating cell-cycle and apoptosis.Ma first concerned that miR-10b promote tumor metastasis, and miR-10b has been expressed by sever breast cancer metastasis,.Inhibition of miR-10b can reduce MDA-MB-231cell invasion ability,and over-expression miR-10b of SUM149cell which is unable to metastasis can metasta distantly in nude mouse. Clinical studies also prove that expression of miR-10b of metastasis breast cancer is higher than no metastasis.Gabriely estimated that inhibition fo miR-10b can restrain the growth of malignant glioma cells by inducing cell apoptosis. The malignant glioma survival rate of more expression miR-10b is significant reduction. Compared with placebo group, tumor growth dropped significantly of glioma model in mice when injecting inhibition of miR-10b. All this studies concerns miR-10b promote tumor growth.
Kruppel-like factor4(Klf4; also called gut-enriched Kriippel-like factor) is a zinc finger-containing transcription factor, plays a role in cell cycle regulation, apoptosis, and differentiation. In accordance with these studies, Klf4expression is frequently lost in several human tumors, including esophageal, stomach, colorectal and bladder cancers, which indicates its tumor suppressor role. It has been reported to inhibit metastasis of several cancers, such as colorectal, esophageal, and pancreatic cancer cells. Moreover, Klf4inhibits cell proliferation by inducing cell cycle arrest in CRC; and regulates esophageal carcinogenesis by affecting proliferation, apoptosis, and cell invasion. Klf4is acting as cancer suppressor genes during tumor proliferation and metastasis,which is on the contrary of miR-10b. Recently studier estimated that miR-10b regulate colon cancer and promote tumor proliferation and metastasis by targeting Klf4.
Members of KLFs gene family have been shown to play important roles in a diverse array of cellular processes including hematopoiesis.It has been reported that Klf4,KLF2,KLF3is associated with macrophage macrophage polarization and activation.Huge studies have reported that Klf4is expressed in a stage-specific pattern during myelopoiesis and function to promote monocyte differentiation.In Klf4-/-mice,mononuclear cells differentiation is severely affected, the mononuclear cells from the bone marrow and spleen experience a substantial reduction and mononuclear cell of circulation almost disappear. It is identified that Klf4as an essential regulator of macrophage M1/M2polarization and attendant functions.
Since1889, some scholars put forward the "seeds" and "soil" hypothesis, tumor microenvironment began to be taken seriously, inflammatory microenvironment has been listed as one of the top ten characteristics of tumor. Tumor has a closely relation with inflammation. During tumor initiation, they create an inflammatory environment that is mutagenic and promotes growth.It is identified that Klf4as an essential regulator of macrophage M1/M2polarization and attendant functions. Meanwhile Klf4is target gene of miR-10b,which regulate tumor progression and metastases.Base on that,we put a forward the hypothesis that wether miR-10b could regulate macrophage polarization by modulating Klf4. To improve the hypothesis, wo do reacher as follows:to determine wether different macrophage polarization could change the expression of miR-10b in macrophage, we evaluated the expression of miR-10Ob in M1and M2macrophage;then we transfect miR-10b inhibitor and miR-10b mimics to answer wether over-expression and inhibition of miR-10b could regulate macrophage polarization. One of remarkable ability of macrophage is presenting antigen to accelerate T helper cell polarization.We next analyze wether inhibition miR-10b could weak Thl cell and Th17cell polarization response and reduce the sensitive of endotoxin in mice and the ability of lear the bacterial infection in mice. At last,we produce the CD4+CD45RBhiT cells colitis mice model,which is resembling human inflammatory brown disease, to explore inhibition miR-10b could release the T cell induced colitis.
Methods and materials
To explore wether miR-10b modulate M1macrophage polarization and macrophage polarization is consistent with miR-10b expression.Human monocytes were obtained from the periphery blood5-10ml from healthy donors by density gradient centrifugation(Ficoll centrifugation). The monocytes was cultured7days with stimulation of GM-CSF(10ng/mL), then co-stimulated by LPS (100ng/mL)and IFN-γ(20ng/mL) to polarize M1macrophage.RT-PCR analyze of miR-10b mRNA in M1macrophage at0h,12h,24h.We also evaluated expression of miR-10b in miR-10b.The monocytes from healthy donors was co-stimulated by IL-4(10ng/mL)and L13(10ng/mL),RT-PCR analyze of miR-10b mRNA in Ml macrophage at0h,12h,24h. Next we speculated that wether varying expression of miR-10b has influence on macrophage polarization.The monocytes was cultured7days with stimulation of GM-CSF(10ng/mL), then co-stimulated by LPS (100ng/mL)and IFN-γ(20ng/mL) to polarize M1macrophage for24h to form M1macrophage, then was removed the culture medium,and was washed by PBS for3time, which eliminated the influence of M1polarization factor. The cell was co-stimulated by IL-4(10ng/mL)and L13(10ng/mL)for24h to polarize M2macrophage.Then they were removed the influence of M2polarization factor as the same way and simulated into M1macrophage. RT-PCR analyze of miR-10b mRNA in M1,M2.The monocytes was stimulated into Ml macrophage as mentioned above, than transfected miR-10b inhibitor and miR-10b mimic for48h,then was simulated with GM-CSF(20ng/mL)for4h to polarize M1macrophage.RT-PCR analyze of highly specific macrophage marker expression of M1,M2,which iNOS is marker of M1macrophage.and Arg-1,Klf4and IL-4R are marker of M2macrophage.
To explore wether inhibition expression of miRlOb in M1macrophage could reduce TH1cell response. The monocytes was isolated from C57BL/6mice and was cultured with simulation GM-CSF(10ng/mL)for5days,then was transfected miR-10b inhibitor for48h.Then the cells were co-cultured with CD4+T cell which was screening by magnetic cell sorting;magnetic activated cell sorting. RT-PCR analyzed expression of T-bet mRNA; Elisa and Flow Injection Analysis analyzed IFN-y level.
To explore wether inhibition expression of miR10b in M1macrophage could reduce TJ17cell response. The monocytes was isolated from C57BL/6mice and was cultured with simulation GM-CSF(10ng/mL)for5days,then was transfected miR-10b inhibitor for48h.Then the cells were co-cultured with CD4+T cell which was screening by magnetic cell sorting; magnetic activated cell sorting. RT-PCR analyzed expression of RORyt mRNA;Elisa and Flow Injection Analysis analyzed IL-17level.
We monitored the sensitive of endotoxin in mice to discuss wether inhibition miR-10b could restrain M1macrophage polarization in vitro experiment. Rinsed the mice abdominal with5%broth medium, peritoneal macrophage were collected and were planted on the60mm culture dish, after2h were removed suspension cell. The adherent cells were planted into6holes culture plate,2×106per hole and were transfected with miR-10b inhibitor.10mouse were divided into2group.The cells were injected into mice enterocoelia for48h.The experimental group was injected LPS via enterocoelia. Two groups of Survival rate was observed,blood serum was collected after6h for Elisa analyzing IL-6, TNF-a level.
To explain inhibition of miR-10b could reduce the ability of lear the bacterial infection in mice, we performed injection miR-10b antagomir via tail vein in20mouse(2x104CFU per one),after48h, injection Listeria bacteria via tail vein of10mouse in experimental group.At24h,48h,every5mouse was put to execution respectively and collected blood serum for Elisa of IL-6, TNF-alevel.Liver and spleen were collected to homogenate, daub on the LB agarose petri dishes, observe the bacteria colony formation.
At last, we verify whether inhibition of miR-10b could reduce CD4+CD45RBhiT cell induced colitis,which is resembling human inflammatory brown disease. miR-10b antagomir is injected into mice via tail vein,twice a week for3weeks. Screening CD4+CD45RBhiT cells by flow cytom and MACS,5x105cells each mice were injected via enterocoelia in Rag-/-mice,miR-10b antagomir were injected twice a week,for6weeks.Observe two groups of mice's body weight,color, motion and change in stool, colonic inflammation histological pathology.
To explore wether miR-10b could regulate Klf4to modulate M1/M2polarization, we isolate monocyte from mice bone marrow, Stimulate with GM-CSF(10ng/mL) for5day,then transfect miR-10b mimic for48h,then Stimulate with rIL-4/IL-13for4h and24respectively. Detection protein and mRNA of Klf4. The cells were transiently cotransfected in96-well plates with luciferase reporter vector containing3'-UTR variants and either control or miR-10b inhibitor. After48h, luciferase activity was measured with Dual-Glo Luciferase Assay System (Promega, WI), and Renilla luciferase activity was normalized to Firefly luciferase activity.
Result
Macrophage polarization is affected by many factors such as microenvironment. This article is discussed that miR-10b can regulate M1/M2polarization by targeting Klf4for first time. Expression of miR-lOb is elevated in M1macrophage and that is not found in M2macrophage. We also found that expression of miR-10b varied with macrophage polarization. The highest expression of miR-10b in M1macrophage,which was dramatic declined in M2macrophage,but it recovered high expression again after stimulation of influence factor of M1macrophage. The result concerns that macrophage polarization can affect expression of miR-10b and on the contrary the expression of miR-10b can also influence the polarization of macrophage. The remarkable factor of Ml macrophage such as iNOS is reduce as inhibition of miR-10b but that of M2macrophage is elevated. It means that changing expression of miR-10b can affect polarization of macrophage. M1macrophage induced TH1and TH17cell response and M2macrophage induce TH2cell response. Inhibition of expression of miR-10b can reduce level of T-bet mRNA, ROR t mRNA expression and IFN-γ,IL-17level.In vitro experiment, survival rate experimental group is significantly higher than control group,and IL-6,TNF-aof blood serum is lower than cntrol group. We also find experimental group has more the bacteria colony formation than control group. The result emphasize on the inhibition of miR-10b can restrain M1macrophage polarization and reduce THland TH17cell response by vitro experiment. TH1and TH17cell play a crucial role on human inflammatory brown disease, we find inhibition of miR-10b can release T cell induced colitis which is resembling IBD. It estimate that inhibition of miR-10b can restrain M1macrophage polarization and reduce TH1and TH17cell response. We also found elevated miR-10b can reduce mRNA and protein level of Klf4in M2macrophage. Klf4is the direct target of miR-10b, and the expression of Klf4is regulated by miR-10b.
Conclusion
This article first report expression of miR-10b in M1and m2macrophage,which indicate that expression of miR-10b not only take an adoptive change with macrophage polarization,but also it could modulate polarization of macrophage.Inhibition miR-10b can reduce Thl and Th2cell response.In vitrvo experiment, it estimate that inhibition miR-10b can restrain M1polarization. In vivo experimentation, inhibition miR-10b can reduce the sensitive of endotoxin in mice and the ability of lear the bacterial infection in mice. It also release T cell induce colitis. All the data estimate the hypothesis that miR-10b could target Klf4to regulate M1/M2polarization.
巨噬细胞在人类免疫系统中占有独特的位置,它是免疫系统第一个被确定的细胞成分,在机体正常生理过程和病理过程中发挥极其重要的作用。它在机体遭受损伤或者感染状态时,第一个到达病患处发挥重要的作用,是机体中重要的吞噬和抗原提呈细胞。临床研究及基础实验均已证实巨噬细胞在急性炎症(如感染,败血症等)和慢性炎症(胰岛素抵抗,动脉粥样硬化,慢性损伤,肿瘤的形成等)均起到关键性作用。全身各处的巨噬细胞以及树突状细胞,破骨细胞均来源于外周循环中的单核细胞。巨噬细胞是在细胞因子的刺激下由骨髓干细胞发育而来。在多集落刺激因子(multi-CSF,multi Colony Stimulating Factor)、粒细胞巨噬细胞集落刺激因子(GM-CSF,Granulocyte-Macrophage Colony Stimulating Factor)等刺激下,由骨髓干细胞发育成单核母细胞,后者进一步分化成为前单核细胞并进人外周血流,并在此处分化成为成熟的单核细胞。很多因素如前炎症状态,代谢,免疫都可以促使单核细胞向外周组织聚集,分化为巨噬细胞,实现宿主防御,机体自我修复与重塑等功能。
我们知道免疫效应细胞在免疫反应中并不是表型单一的活化细胞,在不同的微环境因素影响下,活化的细胞可以分化为不同表型的亚细胞型,这些亚型细胞不但适应局部微环境同时又是局部微环境不可缺少的组分。基于对Th细胞的极化的研究基础上,越来越多的研究开始关注巨噬细胞的极化现象。
目前研究发现,在不同的环境中,巨噬细胞可以分化为行使特定免疫功能的不同细胞亚群。巨噬细胞主要分为两种类型：由TH1型反应引起的IFN-γ依赖的经典途径激活的MI型巨噬细胞,即经典活化的巨噬细胞(classically activated macrophage),其可直接吞噬和杀伤病原微生物和肿瘤细胞,分泌促炎因子和趋化因子,并通过递呈抗原参与正向免疫应答,发挥免疫监视的功能和宿主防御的功能；由TH2型细胞因子、IL-4与IL-13引起的替代途径激活的M2型巨噬细胞,即替代性活化的巨噬细胞(alternatively activated macrophage)。其可在巨噬细胞集落刺激因子(M-CSF,Macrophage Colony-Stimulating Factor) IL-4、IL-13、IL-10、糖皮质激素、转化生长因子-β(TGF-β)、维生素D3和前列腺素E2(PGE2)等作用下诱导产生,其抗原递呈能力较低,并通过分泌IL-10、CCL17、CCL18、CCL22和TGF-β等抑制性细胞因子下调免疫应答。促进创伤修复和纤维变性。M2型巨噬细胞同肿瘤的发生发展有着密切的关系,肿瘤中的巨噬细胞主要是经微环境“教化”后的M2型巨噬细胞,具有促进肿瘤细胞侵袭转移的作用。同时大量研究证实M2型巨噬细胞可以减轻胰岛素抵抗,改善寄生虫感染,重塑脂肪组织,减轻体重。
MicroRNAs (miRNAs)是一类由内源基因编码的、长度约为18-25个核苷酸的、非编码单链小分子RNA,它们通过结合到靶基因的mRNA上,在转录后水平调控基因的表达,对增殖,分化,凋亡等基本细胞过程具有重要的调控作用。
目前越来越多的证据表明：microRNA(miRNA)参与了免疫调控,与适应性免疫系统的分化和功能维持关系密切,许多miRNAs在免疫反应中扮演着十分重要的角色。如研究证实TLR4配体脂多糖(Lipopolysaccharide,LPS)刺激后,可以通过调节肿瘤坏死因子受体相关蛋白6,白细胞介素1受体相关激酶1,来实现miR-146a在单核细胞中表达明显升高；在先天性免疫应答方面,miR-146a表达升高是内毒素耐受的重要表现之一。细菌病毒相关抗原可以激活TLR4,TLR2,TLR3或者TLR9等,进一步促进巨噬细胞表达miR-155。已经证实miR-155在免疫反应中既可以发挥促进免疫反应也可以表现出抑制免疫反应的作用。经LPS刺激后单核细胞可以表达miR-21,上调miR-21的表达可以抑制LPS所诱导的NF-κB调控的活化以及IL-6的表达,但可以提高IL-10的表达,同时miR-21可以通过TLR7和TLR8诱发免疫激活。
在各种miRNAs中,miR-10b引起了我们极大的兴趣。目前的研究主要致力于miR-10b同肿瘤之间的关系。miR-10b对肿瘤转移的作用是由Ma和同事等首次提出的。他们的研究发现miR-10b可以在高转移的乳腺癌细胞中表达,阻断miR-10b能够使MDA-MB-231细胞侵袭能力下降90%以上,而不具备转移能力的SUM149细胞过表达miR-10b后,在裸鼠体内发生远处转移。临床资料也表明,发生转移的乳腺癌患者,miR-10b表达高于非转移组。此外,Gabriely等的研究证明了在体外,抑制miR-10b的表达可以通过诱导细胞周期和凋亡,从而抑制恶性胶质瘤细胞的生长。并且与含miR-10家族水平低的胶质瘤患者相比,含miR-10家族水平高的患者其存活率明显降低。与注射安慰剂组相比,人胶质瘤小鼠模型在注射miR-10b抑制剂后,其肿瘤生长明显下降。这些均证明了miR-10b对肿瘤细胞生长的作用。
Kruppel样因子4(Klf4或称肠道富集的Kruppel样因子)是属于Kruppel蛋白家族的一种含有锌指结构的转录因子。Klf4具有多种生理功能,在肿瘤的发生发展中Klf4基因具有抑癌基因和癌基因的双重功能,可以影响细胞周期的调控、凋亡以及细胞分化。相当一部分研究发现在多种人类肿瘤,包括食管癌,胃癌,大肠癌和膀胱癌,Klf4的表达经常发生缺失,这提示其可能具有抑癌作用。据报道,Klf4可抑制多种癌症转移的发生,如大肠癌、食管癌、胰腺癌细胞等。并且在大肠癌中,Klf4可通过诱导细胞周期阻滞抑制细胞增殖；通过影响细胞增殖、凋亡和癌细胞的浸润,从而调节食管癌的发生。综合考虑上述结论,Klf4在癌细胞的转移和增殖过程中可能起抑癌基因的作用,这恰恰与miR-10b发挥的癌基因作用相反。目前已有研究探讨miR-10b通过调控Klf4,在大肠癌的进展过程中发挥促进增殖和转移的功能。本课题组在既往研究中构建了两个含有荧光素报告基因的质粒：Klf43'-UTR和Klf43'-UTR-mut。前者含有Klf4mRNA的全长3’UTR区域；后者3’非翻译区关键结合位点的第296-300位碱基被其互补碱基替代。在SW620细胞同时转染含有Klf43'-UTR或Klf43'-UTR-mut的荧光素酶报告质粒以及miR-lOb inhibitor或相应的对照。结果显示,下调miR-10b的表达去除了其对含Klf43'-UTR的荧光素酶活性的抑制作用,提示miR-10b可通过Klf43'-UTR来调节Klf4基因。而当Klf43’-UTR与miR-10b的结合位点发生突变,miR-10b表达抑制后对荧光素酶活性产生的作用将会受到影响。证实Klf43'UTR从295-301位核苷酸可以与miR-10b的5’端关键位点结合,并进一步证实Klf4是miR-10b的直接靶基因。
在巨噬细胞极化调节中,Klf4、Klf2、Klf1和Klf3被报道与单核巨噬细胞的分化或激活有关。其中研究得比较多的是Klf4。Klf4在单核细胞的谱系决定、分化以及激活过程中都是一个重要的调控因子。Klf4-/-嵌合体小鼠中,单核细胞的分化会受到严重影响,骨髓中的单核细胞、脾脏中的单核细出现大量减少,并且发现血液中循环的单核细胞的几近消失。Klf4不仅与巨噬细胞的分化有关,还与巨噬细胞的激活有关。最近的研究表明Klf4在M2型巨噬细胞的分化过程中具有重要作用。
随着1889年有学者提出“种子”与“土壤”假说,肿瘤微环境开始受到重视,炎症微环境已被列为肿瘤十大特征之一,肿瘤与炎症有着密不可分的关系,在肿瘤发展过程中,局部炎症环境促进肿瘤生长及基因突变。Klf4不仅参与炎症反应中在单核细胞的谱系决定、分化、激活过程,而且作为调节巨噬细胞极化中都是一个重要的调控因子。
综上所述,我们提出一个假说：miR10b是否能够通过调控KIf4而进一步调节巨噬细胞极化。为了验证这一假说,我们从如下几个方面入手：我们首先观察miR-10b分别在M1及M2型巨噬细胞的表达情况,分析巨噬细胞极性改变时miR-10b在巨噬细胞的表达是否有相应的变化；通过转染miR-10b inhibitor和miR-10b mimics,观察过表达miR-10b及抑制miR-10b是否能够调节巨噬细胞极化；巨噬细胞的除具有调节固有免疫之外,其中重要的作用是通过抗原提呈促进T辅助细胞极化调节继发性免疫,我们进一步观察抑制]miR-10b的表达能否减弱TH1及TH17细胞炎症反应；了解抑制miR-lOb是否降低小鼠对内毒素的敏感性及对细菌感染的清除能力；而后,采用CD4+CD45RhiT细胞诱导的肠炎模型模拟人类IBD炎症,观察抑制miR-10b能否减弱T细胞所介导的肠炎；最后通过荧光素酶报告基因证实小鼠中Klf4是miR-10b的靶基因,以探讨miR-10b—Klf4这一靶向调控关系对巨噬细胞极化的调节及其影响,对TH1及TH17细胞炎症反应的调控机制。
方法
为了明确miR-10b在M1型巨噬细胞极化过程中是否起到重要作用,我们采集健康志愿者外周静脉血,采用Ficoll密度梯度离心法,分离外周血单个核细胞,原代培养单个核细胞,采用GM-CSF(10ng/mL)诱导7天,然后采LPS(100ng/mL)和IFN-γ(20ng/mL)共刺激诱导巨噬细胞向M1型极化,分别取不同时间点0、12、24小时,采用实时荧光定量PCR检测M1型巨噬细胞中miR-10b的表达水平。我们也检测了在M2极化过程中miR-10b的表达水平,原代培养单个核细胞,采用IL-4(10ng/mL)和IL-13(10ng/mL)共刺激诱导M2型巨噬细胞,同样选择不同时间0、12、24小时,采用实时荧光定量PCR检测niR-10b的表达水平。我们检测是否在巨噬细胞发生极化改变时,：miR-10b的表达也随之发生变化。同样,我们分离了人外周血单个核细胞,采用GM-CSF(10ng/mL)诱导7天,然后采用LPS(100ng/mL)和IFN-γ(20ng/mL)共刺激诱导巨噬细胞向M1型极化24小时后,改用IL-4(10ng/mL)和IL-13(10ng/mL)共刺激24小时,同样,除去M2型极化因子后,再恢复M1型极化因子刺激24小时；然后采用实时荧光定量PCR检测miR-10b的表达。改变miR-10b的表达是否影响巨噬细胞的极化?我们外周血分离出单个核细胞,采用GM-CSF诱导7天,分别转染miR-10b inhibitor和miR-10b minic,48小时后,然后给予GM-CSF(20ng/mL)刺激4小时诱导M1型巨噬细胞,并检测M1型和M2型巨噬细胞特征性因子表达水平。iNOS是M1型巨噬细胞特征性因子,而Klf4和IL-4R是M2型特征性因子。采用荧光定量PCR检测以上因子mRNA水平。
为了说明抑制niR-10b表达能否减弱了TH1细胞的反应。从C57BL/6小鼠骨髓分离单个核细胞,采用GM-CSF(10ng/mL)诱导5天,转染miR-10b inhibitor,继续培养48小时,加入磁珠分选的小鼠CD4+T细胞混合培养,分别采用实时荧光定量PCR检测检测TH1转录因子T-bet mRNA表达,ELISA分析IFN-y表达,以及流式分析IFN-y表达。
同样为了探讨了抑制miR-10b表达能否减弱了TH17细胞的反应。同上述处理方法一致,分别采用实时荧光定量PCR检测检测TH17转录因子RORyt mRNA表达、ELISA分析IL-17表达,以及流式分析IL-17表达。
为了进一步探讨抑制miR-10b是否能明显抑制M1型极化,通过体外实验检测小鼠对内毒素的敏感性的变化。我们采用5%肉汤培养基冲洗小鼠腹腔,收集腹腔巨噬细胞,将收集的巨噬细胞种植于60mm培养皿,转染miR-10b inhibitor,按照2x106个注入小鼠腹腔,并按照1Omg/kg腹腔注射LPS,观察小鼠生存率；并且收集6血清。采用ELISA法检测血清中IL-6、TNF-a表达的变化。
抑制miR-10b能否减弱了对细菌感染清除能力?我们采用miR-10b antagomir尾静脉注入到小鼠体内,48小时后,通过尾静脉注入李斯特菌(2x104CFU/只),分别于24小时和48小时处死。每组小鼠取血,收集血清,采用ELISA检测IL-6、TNF-α水平；而且分别收集肝脏和脾脏,匀浆,涂抹在LB琼脂糖培养皿上,观察细菌的菌落形成。
最后我们检测抑制miR-10b能否减弱CD4+CD45RBhiT细胞诱导的肠炎。CD4+CD45RBhiT细胞诱导的肠炎类似于人类IBD炎症。我们采用miR-10bantagomir尾静脉注入到小鼠体内,每周两次,连续注射3周；采用流式细胞分选CD4+CD45RBhiT细胞,5x105细胞/只腹腔注入到Rag-/-小鼠体内,每周注射两次miR-10b antagomir。观察小鼠体重及活动。6周后处死小鼠,收取肠道组织制作石蜡病理切片,收取血液,离心收集血清,采用ELISA检测炎症因子。观察实验组和对照组小鼠体重变化情况,肠道炎症状况,免疫组化观察肠道炎性细胞侵入情况,流式细胞分析T辅助细胞TH1和TH17细胞分化。
我们采用生物信息学分析,通过构建Klf43'UTR包含预测结合位点的荧光素酶报告基因,验证鼠源miR-10b与Klf4是否有结合位点。
结果
巨噬细胞极化受局部微环境等多种因素的影响,本文意在探讨niR-10b对巨噬细胞M1/M2分型的影响及调节。在发生M1型巨噬细胞极化过程中,miR-10b表达明显升高,而在M2型极化过程,表达没有明显改变,这提示miR-10b在M1型巨噬细胞极化过程中可能起到重要作用。随着微环境或者外界刺激的改变,巨噬细胞的极性发生相应变化,与各亚型相关的标记分子也会发生表达改变,我们检测在巨噬细胞发生极化改变时,miR-10b的表达也随之发生变化,miR-10b在M1型极化时表达最高,在M2型极化因子刺激后表达明显下降,而重新采用M1型极化因子刺激后,miR-10b表达升高,这说明了巨噬细胞的极化可能影响了miR-10b的表达。反之miR-10b的表达也会影响巨噬细胞的极化,抑制miR-10b的表达能明显抑制M1型巨噬细胞特征性基因iNOS表达水平及M1巨噬细胞能产生致炎因子,如IL-6、TNF-α,M2型巨噬细胞特征性基因Klf4和IL-4R表达增加,增加miR-10b表达则提高了iNOS表达,Klf4和IL-4R表达下降,这说明了改变miR-10b的表达水平可以改变巨噬细胞极化状态,miR-10b促进M1型巨噬细胞极化,抑制miR-10b是的巨噬细胞M1型极化受阻。M1型巨噬细胞诱导TH1和TH17反应,M2型巨噬细胞诱导TH2反应。抑制miR-10b降低了T-bet mRNA,RORyt mRNA表达水平同时也降低了IFN-γ和IL-17表达水平,这说明了抑制miR-10b表达减弱了TH1和TH17细胞诱导的炎症反应。我们采用5%肉汤培养基冲洗小鼠腹腔,收集腹腔巨噬细胞,将收集的巨噬细胞转染miR-10b inhibitor,培养后注入小鼠腹腔,对比小鼠生存率：miR-10b inhibitor组生存率明显高于对照组,并且血清中IL-6、TNF-α表达也明显低于对照组。这说明了抑制miR-10b可以导致机体对内毒素敏感性下降,也说明了抑制了巨噬细胞向M1型极化,结合体外实验说明了抑制niR-10b能明显抑制M1型极化。巨噬细胞不同极化状态细菌入侵的反应不用,M1型巨噬细胞可以分泌iNOS等杀死细菌,而M2型巨噬细胞对细菌感染不敏感。我们采用miR-10b antagomir尾静脉注入到小鼠体内,再通过尾静脉注入李斯特菌(2x104CFU/只),收集血清、肝脏和脾脏,匀浆,涂抹在LB琼脂糖培养皿上,观察细菌的菌落形成。结果显示在miR-10b antagomir组肝脏和脾脏菌落比对照组明显增多,而且血清中IL-6和TNF-α表达也明显下降这说明了在体内抑制miR-10b的表达后,导致了M1向M2极化,致使对细菌的感染不敏感,对细菌的清楚能力下降。体外实验证明了抑制miR-10b能减弱TH1和TH17细胞的反应,TH1和TH17细胞被认为是人类IBD发病机制中介导炎症的重要细胞亚群,我们采用CD4+CD45RBhiT细胞诱导的肠炎模型模拟人类IBD炎症,观察抑制miR-10b可以减弱T细胞所介导的肠炎,这是由于M1型极化而导致TH1和TH17细胞反应减弱所致。通过小鼠骨髓来源单个核细胞,采用GM-CSF(10ng/mL)诱导5天,转染miR-10b mimic,更换为rIL-4/IL-13分别刺激4小时和24小时,结果表明在M2型巨噬细胞极化状态下,升高miR-10b能从转录和翻译水平抑制KLF4的表达。通过荧光素酶报告基因,结果证实了与人类miR-10b和Klf4结合相似,鼠miR-10b也能与KLF4特异性结合,。
结论
本研究第一次报道了miR-10b在M1型巨噬细胞的表达情况,揭示了miR-10b表达水平不仅可以随巨噬细胞极性改变而发生变化,反之通过改变miR-10b的表达可以调节巨噬细胞极化分型。抑制miR-10b明显抑制M1型极化,并减弱了TH1和TH17细胞的反应。同时抑制miR-10b可以导致机体对内毒素敏感性下降,对细菌清除能力下降,减弱T细胞所介导的肠炎。通过构建小鼠Klf43'UTR包含miR-10b结合位点的荧光素酶报告基因载体及相应的突变载体,小鼠niR-10b能与Klf4结合。
Background and Objection:
Macrophages have long been considered to be important immune effector cells.It plays an important role the in a broad spectrum of pathological and physiology procedure and respond to endogenous stimuli that are rapidly generated following injury or infection.which is first confirmed cell in the immune system. It is known that they are among the first cells to arrive at sites of wounding and/or infection and is characterize by a high capacity to present antigen,where they perform several functions. Clinical and experimental studies support an important role for the macrophage in a broad spectrum of acute (e.g., pathogen infection, sepsis) and chronic inflammatory conditions (e.g.insulin resistance, atherosclerosis, chronic wounds, and tumorigenesis).Circulating monocytes give rise to a variety of tissue resident macrophages throughout the body,as well as to specialized cells such as dendritic cells (DCs) and osteoclasts. With the stimulation of granulocyte-macrophage colony stimulating factor and multi-colony stimulating factor, bone marrow stem cell develops into Mononuclear cells.Monocytes are known to originate in the bone marrow from a common myeloid progenitor that is shared with neutrophils, and they are then released into the peripheral blood, where they circulate for several days before entering tissues and replenishing the tissue macrophage populations involved inflammation and host defense,,which are induced by pro-inflammatory and environmental signals.
Immunologic effector cell exhibit remarkable plasticity that allows them to develop into different type of cell and serve distinct functions in the regulation of the inflammatory response. As more and more studies about the molecular mechanisms that govern Th cell polarization, the regulation of macrophage polarization is paid huge attention to.
With a huge number of studies confirmed, it is well-accepted that macrophage is divided into M1macrophage (Classically activate) and M2macrophage (Alternatively activate). Macrophages stimulated with LPS or INF-γassume an M1pro-inflammatory phenotype characterized by a high expression level of iNOS, a high capacity to present antigen and production of pro-inflammatory cytokines such as TNF-a, IL-1β,monocyte chemoattractant protein-1(MCP-1). M1macrophages are critical effector cells that kill microorganisms and thus benefit the host. However, this pro-inflammatory activity must be carefully titrated, as sustained activation can predispose to chronic inflammatory states such as obesity and insulin resistance. M-CSF, IL-4, IL-13, IL-10, IL-4and TGF-βstimulation leads to M2macrophages.,which produce of anti-inflammatory cytokines such as IL-10、 CCL17,CCL18, CCL22和TGF-aand promote tissue repair and remodeling as well as tumor progression.Tumor-associated-macrophage is resembling M2macrophage,which is associated with cancer initiation and promotion. Accumulating evidence suggests that M2macrophages can protect against insulin resistance, eliminate parasites, and promote tissue remodeling and repair. Interestingly, it helps weight loss, known to improve insulin sensitivity and to induce remodeling of adipose tissue.
MicroRNAs (miRNAs), a newly discovered class of non-protein-coding small RNA, have been shown to play a vital role in processes such as development, cellular proliferation, and more recently apoptosis.It combine mRNA of target gene and supress gene expression post-transcriptionally.
Accumulating evidence suggests that miRNAs constitute an integrated part of the regulatory networks in innate immunity and adaptive. More than a dozen miRNA have been shown to play a critical role in regulating polarization of macrophage and Th-17cell. Studies found that miR-146a is rapidly upregulated in human monocytic cells stimulated with lipo-polysaccharide(LPS), a TLR4ligand, and acts as a negative feedback regulator of TLR signaling, presumably by targeting tumor necrosis factor receptor-associated factor6and interleukin(IL)-1receptor-associated kinase-1, miR-146a upregulation may be an essential component of endotoxin tolerance in innate immune response.miR-155is induced in macrophage in response to both bacterial and viral-derived antigens that activate TLR4,TLR2,TLR3or TLR9.It was estimated that miR-155plays both positive and negative regulator roles in immune responses. miR-21is induced in monocytic cells by LPS stimulation. Upregulated miR-21was found to dampen LPS-induced nuclear factor-κB activation and IL-6expression but enhance IL-10expression. miR-21can also trigger inflammatory responses by binding to TLR7and TLR8.
In all of miRNAs, miR-10b is of great interest to us. Because it not only initiates invasion and metastasis in cancers, but also controls cancer cell growthe by regulating cell-cycle and apoptosis.Ma first concerned that miR-10b promote tumor metastasis, and miR-10b has been expressed by sever breast cancer metastasis,.Inhibition of miR-10b can reduce MDA-MB-231cell invasion ability,and over-expression miR-10b of SUM149cell which is unable to metastasis can metasta distantly in nude mouse. Clinical studies also prove that expression of miR-10b of metastasis breast cancer is higher than no metastasis.Gabriely estimated that inhibition fo miR-10b can restrain the growth of malignant glioma cells by inducing cell apoptosis. The malignant glioma survival rate of more expression miR-10b is significant reduction. Compared with placebo group, tumor growth dropped significantly of glioma model in mice when injecting inhibition of miR-10b. All this studies concerns miR-10b promote tumor growth.
Kruppel-like factor4(Klf4; also called gut-enriched Kriippel-like factor) is a zinc finger-containing transcription factor, plays a role in cell cycle regulation, apoptosis, and differentiation. In accordance with these studies, Klf4expression is frequently lost in several human tumors, including esophageal, stomach, colorectal and bladder cancers, which indicates its tumor suppressor role. It has been reported to inhibit metastasis of several cancers, such as colorectal, esophageal, and pancreatic cancer cells. Moreover, Klf4inhibits cell proliferation by inducing cell cycle arrest in CRC; and regulates esophageal carcinogenesis by affecting proliferation, apoptosis, and cell invasion. Klf4is acting as cancer suppressor genes during tumor proliferation and metastasis,which is on the contrary of miR-10b. Recently studier estimated that miR-10b regulate colon cancer and promote tumor proliferation and metastasis by targeting Klf4.
Members of KLFs gene family have been shown to play important roles in a diverse array of cellular processes including hematopoiesis.It has been reported that Klf4,KLF2,KLF3is associated with macrophage macrophage polarization and activation.Huge studies have reported that Klf4is expressed in a stage-specific pattern during myelopoiesis and function to promote monocyte differentiation.In Klf4-/-mice,mononuclear cells differentiation is severely affected, the mononuclear cells from the bone marrow and spleen experience a substantial reduction and mononuclear cell of circulation almost disappear. It is identified that Klf4as an essential regulator of macrophage M1/M2polarization and attendant functions.
Since1889, some scholars put forward the "seeds" and "soil" hypothesis, tumor microenvironment began to be taken seriously, inflammatory microenvironment has been listed as one of the top ten characteristics of tumor. Tumor has a closely relation with inflammation. During tumor initiation, they create an inflammatory environment that is mutagenic and promotes growth.It is identified that Klf4as an essential regulator of macrophage M1/M2polarization and attendant functions. Meanwhile Klf4is target gene of miR-10b,which regulate tumor progression and metastases.Base on that,we put a forward the hypothesis that wether miR-10b could regulate macrophage polarization by modulating Klf4. To improve the hypothesis, wo do reacher as follows:to determine wether different macrophage polarization could change the expression of miR-10b in macrophage, we evaluated the expression of miR-10Ob in M1and M2macrophage;then we transfect miR-10b inhibitor and miR-10b mimics to answer wether over-expression and inhibition of miR-10b could regulate macrophage polarization. One of remarkable ability of macrophage is presenting antigen to accelerate T helper cell polarization.We next analyze wether inhibition miR-10b could weak Thl cell and Th17cell polarization response and reduce the sensitive of endotoxin in mice and the ability of lear the bacterial infection in mice. At last,we produce the CD4+CD45RBhiT cells colitis mice model,which is resembling human inflammatory brown disease, to explore inhibition miR-10b could release the T cell induced colitis.
Methods and materials
To explore wether miR-10b modulate M1macrophage polarization and macrophage polarization is consistent with miR-10b expression.Human monocytes were obtained from the periphery blood5-10ml from healthy donors by density gradient centrifugation(Ficoll centrifugation). The monocytes was cultured7days with stimulation of GM-CSF(10ng/mL), then co-stimulated by LPS (100ng/mL)and IFN-γ(20ng/mL) to polarize M1macrophage.RT-PCR analyze of miR-10b mRNA in M1macrophage at0h,12h,24h.We also evaluated expression of miR-10b in miR-10b.The monocytes from healthy donors was co-stimulated by IL-4(10ng/mL)and L13(10ng/mL),RT-PCR analyze of miR-10b mRNA in Ml macrophage at0h,12h,24h. Next we speculated that wether varying expression of miR-10b has influence on macrophage polarization.The monocytes was cultured7days with stimulation of GM-CSF(10ng/mL), then co-stimulated by LPS (100ng/mL)and IFN-γ(20ng/mL) to polarize M1macrophage for24h to form M1macrophage, then was removed the culture medium,and was washed by PBS for3time, which eliminated the influence of M1polarization factor. The cell was co-stimulated by IL-4(10ng/mL)and L13(10ng/mL)for24h to polarize M2macrophage.Then they were removed the influence of M2polarization factor as the same way and simulated into M1macrophage. RT-PCR analyze of miR-10b mRNA in M1,M2.The monocytes was stimulated into Ml macrophage as mentioned above, than transfected miR-10b inhibitor and miR-10b mimic for48h,then was simulated with GM-CSF(20ng/mL)for4h to polarize M1macrophage.RT-PCR analyze of highly specific macrophage marker expression of M1,M2,which iNOS is marker of M1macrophage.and Arg-1,Klf4and IL-4R are marker of M2macrophage.
To explore wether inhibition expression of miRlOb in M1macrophage could reduce TH1cell response. The monocytes was isolated from C57BL/6mice and was cultured with simulation GM-CSF(10ng/mL)for5days,then was transfected miR-10b inhibitor for48h.Then the cells were co-cultured with CD4+T cell which was screening by magnetic cell sorting;magnetic activated cell sorting. RT-PCR analyzed expression of T-bet mRNA; Elisa and Flow Injection Analysis analyzed IFN-y level.
To explore wether inhibition expression of miR10b in M1macrophage could reduce TJ17cell response. The monocytes was isolated from C57BL/6mice and was cultured with simulation GM-CSF(10ng/mL)for5days,then was transfected miR-10b inhibitor for48h.Then the cells were co-cultured with CD4+T cell which was screening by magnetic cell sorting; magnetic activated cell sorting. RT-PCR analyzed expression of RORyt mRNA;Elisa and Flow Injection Analysis analyzed IL-17level.
We monitored the sensitive of endotoxin in mice to discuss wether inhibition miR-10b could restrain M1macrophage polarization in vitro experiment. Rinsed the mice abdominal with5%broth medium, peritoneal macrophage were collected and were planted on the60mm culture dish, after2h were removed suspension cell. The adherent cells were planted into6holes culture plate,2×106per hole and were transfected with miR-10b inhibitor.10mouse were divided into2group.The cells were injected into mice enterocoelia for48h.The experimental group was injected LPS via enterocoelia. Two groups of Survival rate was observed,blood serum was collected after6h for Elisa analyzing IL-6, TNF-a level.
To explain inhibition of miR-10b could reduce the ability of lear the bacterial infection in mice, we performed injection miR-10b antagomir via tail vein in20mouse(2x104CFU per one),after48h, injection Listeria bacteria via tail vein of10mouse in experimental group.At24h,48h,every5mouse was put to execution respectively and collected blood serum for Elisa of IL-6, TNF-alevel.Liver and spleen were collected to homogenate, daub on the LB agarose petri dishes, observe the bacteria colony formation.
At last, we verify whether inhibition of miR-10b could reduce CD4+CD45RBhiT cell induced colitis,which is resembling human inflammatory brown disease. miR-10b antagomir is injected into mice via tail vein,twice a week for3weeks. Screening CD4+CD45RBhiT cells by flow cytom and MACS,5x105cells each mice were injected via enterocoelia in Rag-/-mice,miR-10b antagomir were injected twice a week,for6weeks.Observe two groups of mice's body weight,color, motion and change in stool, colonic inflammation histological pathology.
To explore wether miR-10b could regulate Klf4to modulate M1/M2polarization, we isolate monocyte from mice bone marrow, Stimulate with GM-CSF(10ng/mL) for5day,then transfect miR-10b mimic for48h,then Stimulate with rIL-4/IL-13for4h and24respectively. Detection protein and mRNA of Klf4. The cells were transiently cotransfected in96-well plates with luciferase reporter vector containing3'-UTR variants and either control or miR-10b inhibitor. After48h, luciferase activity was measured with Dual-Glo Luciferase Assay System (Promega, WI), and Renilla luciferase activity was normalized to Firefly luciferase activity.
Result
Macrophage polarization is affected by many factors such as microenvironment. This article is discussed that miR-10b can regulate M1/M2polarization by targeting Klf4for first time. Expression of miR-lOb is elevated in M1macrophage and that is not found in M2macrophage. We also found that expression of miR-10b varied with macrophage polarization. The highest expression of miR-10b in M1macrophage,which was dramatic declined in M2macrophage,but it recovered high expression again after stimulation of influence factor of M1macrophage. The result concerns that macrophage polarization can affect expression of miR-10b and on the contrary the expression of miR-10b can also influence the polarization of macrophage. The remarkable factor of Ml macrophage such as iNOS is reduce as inhibition of miR-10b but that of M2macrophage is elevated. It means that changing expression of miR-10b can affect polarization of macrophage. M1macrophage induced TH1and TH17cell response and M2macrophage induce TH2cell response. Inhibition of expression of miR-10b can reduce level of T-bet mRNA, ROR t mRNA expression and IFN-γ,IL-17level.In vitro experiment, survival rate experimental group is significantly higher than control group,and IL-6,TNF-aof blood serum is lower than cntrol group. We also find experimental group has more the bacteria colony formation than control group. The result emphasize on the inhibition of miR-10b can restrain M1macrophage polarization and reduce THland TH17cell response by vitro experiment. TH1and TH17cell play a crucial role on human inflammatory brown disease, we find inhibition of miR-10b can release T cell induced colitis which is resembling IBD. It estimate that inhibition of miR-10b can restrain M1macrophage polarization and reduce TH1and TH17cell response. We also found elevated miR-10b can reduce mRNA and protein level of Klf4in M2macrophage. Klf4is the direct target of miR-10b, and the expression of Klf4is regulated by miR-10b.
Conclusion
This article first report expression of miR-10b in M1and m2macrophage,which indicate that expression of miR-10b not only take an adoptive change with macrophage polarization,but also it could modulate polarization of macrophage.Inhibition miR-10b can reduce Thl and Th2cell response.In vitrvo experiment, it estimate that inhibition miR-10b can restrain M1polarization. In vivo experimentation, inhibition miR-10b can reduce the sensitive of endotoxin in mice and the ability of lear the bacterial infection in mice. It also release T cell induce colitis. All the data estimate the hypothesis that miR-10b could target Klf4to regulate M1/M2polarization.
引文
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