摘要
研究目的
目前,同种器官移植已在临床广泛开展,对治疗器官衰竭、血液疾病以及恶性肿瘤发挥了不可替代的作用。然而发生于移植术后数周或数月的急性排斥反应(AR, Acute rejection)仍是导致治疗失败的主要因素。移植术后常规使用的免疫抑制性药物严重降低了患者机体的免疫功能,导致了感染、肝肾损伤,影响了移植受者的预后。为解决这一难题,研究者们利用多种细胞和生物因子及制剂,通过不同途径诱导机体产生特异性的免疫耐受以克服非特异性的免疫抑制剂的严重毒副作用。目前移植免疫耐受研究的热点之一是扩增调节性T细胞(Regulatory T cell, Treg)或增强其功能。既往研究表明,Treg细胞是参与维持外周耐受的重要细胞,应用Treg细胞诱导免疫耐受具有多种优势,因而有可能被发展为诱导移植免疫耐受和治疗器官移植排斥的重要工具细胞。
目前体外扩增的Treg进行细胞治疗方案的应用受到很多因素制约,而应用药物扩增受体机体内的Treg细胞比例或者增强Treg细胞抑制能力的方案具有更好的应用前景。人体内的Treg细胞主要存在于CD4+CD25hi(高表达)细胞亚群中,约占外周血CD4+T细胞的1%～2%。Treg细胞作为在体内外均具有调节免疫反应功能的重要细胞群,可根据其表达的细胞表面分子、产生的细胞因子及作用机制分为天然存在的CD4+CD25+Treg细胞(Naturally occurring CD4+CD25+Treg, nTreg)和诱导性CD4+CD25+Treg细胞(Induced CD4+CD25+Treg, iTreg)。大量动物移植模型的研究证实,两种Treg细胞对于维持移植物良好生存状态都具有重要作用,提高Treg细胞比例可以诱导免疫耐受、减轻排斥反应。在实体器官移植中,与等量nTreg相比,体内和体外同种异体抗原特异性诱导产生的iTreg能更好地延长移植物存活期及诱导免疫耐受。
Treg细胞的免疫调节机制主要包括以下几个方面：对抗原递呈细胞(Antigen presenting cell, APC)的抑制作用,介导靶细胞溶解、杀伤靶细胞,分泌TGF-β (Transformation growth factor-β,转化生长因子-p)和IL-10(Interleukin-10,白介素-10)发挥免疫抑制功能等。Treg细胞抑制CD4+效应T细胞的机制主要通过细胞间直接接触、分泌细胞因子、与APC结合后改变其代谢途径或分泌方式等途径。Treg细胞除高表达CD4和CD25之外,还表达一种特征性标志Foxp3(Forkhead transcription factors,叉头样转录因子3),其对Treg细胞的表型、发育和功能性维持具有重要作用。研究发现机体内Treg细胞比例和Foxp3的表达情况可以作为改善受体免疫状态及诱导耐受的重要指标。然而关于如何扩增Treg细胞,上调Foxp3表达以及增强Treg细胞抑制性效应的研究目前仍不够明确,因此仍需寻找能够有效扩增机体Treg细胞比例或增强其抑制能力的安全制剂。
研究发现,将人CD4+T细胞与基因重组鞭毛蛋白(rFliC)体外共培养后可显著增强CD4+CD25+Treg细胞的免疫抑制能力并提高Foxp3的表达。并且鞭毛蛋白可以保护机体免受化学、细菌、病毒以及放射性损伤。因此,鞭毛蛋白或可作为一种能够增强Treg细胞功能,诱导机体产生移植免疫耐受的制剂。鞭毛蛋白是细菌鞭毛的主要成分,是Toll样受体5(Toll-like receptor5, TLR5)的特异性配体。TLR5在人T细胞以及小鼠Treg细胞上均有表达。Toll样受体(TLR)家族是介导天然免疫的重要因素,可在病原体入侵机体的早期即启动天然免疫,继而激活获得性免疫应答,对免疫病理损伤有重要作用。鞭毛蛋白介导的免疫应答依赖于经典的TLR信号通路,可特异性激活TLR5, TLR5下游信号为MyD88依赖性并且与NF-κB或MAPK活化相关,MAPK通路受到多种因素的调控。
综上所述,本论文的科学假设是：rFliC可通过扩增同种移植受体CD4+CD25+Treg细胞比例,提高Foxp3表达,增强其增殖能力及抑制性效应,诱导同种移植免疫耐受。本研究试图阐明rFliC对同种移植物生存状态的影响,并且探讨rFliC此种作用的内在机制、信号途径及调控因素,从而为研究利用rFliC诱导移植免疫耐受提供理论和实验依据,为临床器官移植排斥的治疗提供新的制剂,因此具有重要的理论和实际意义。第一部分rFliC对小鼠同种皮肤移植物的影响及与TLR5表达的相关性研究方法1、提取并纯化基因重组鞭毛蛋白(rFliC)
利用携带GST-FliC-Burkholderia pseudomallei基因的pGEX4T-2质粒及BL21E.col感受态菌构建BL21E.coli-GST-FliC菌株；抗生素抗性筛选后,对目标菌株进行活化及诱导表达,然后利用Glutathione Sepharose4B凝胶提取基因重组鞭毛蛋白(rFliC),并使用ToxinEraser endotoxin removal resin去除细菌内毒素(Lipid polysacchride, LPS)污染。
2、构建小鼠同种皮肤移植模型
无菌条件下将制备的C57BL/6小鼠背部全厚度皮片移植至BALB/c小鼠背部并进行包扎,移植术后第7日拆包。在移植术进行第-1、3、5、7、9、11日向实验组小鼠i.p.注射rFliC (3mg/kg);同时设立等量PBS缓冲液注射组为对照组。
3、研究rFliC对同种皮肤移植物生存状态及生存期的影响
从拆包之日起,每日观察两组小鼠皮肤移植物的生长状态及排斥情况,记录移植皮片排斥百分比及生存期。在PBS对照组小鼠的移植皮片完全排斥当日,脱颈椎处死两组小鼠,完整取下供体皮片及受体移植床部位组织,进行甲醛固定并制备石蜡切片,应用HE染色观察移植物组织病理情况。
4、检测rFliC对同种皮肤移植物与移植受体TLR5表达的影响
在PBS对照组小鼠的移植皮片完全排斥当日,取受体移植床部位的移植皮片组织,进行甲醛固定并制备石蜡切片,应用抗-TLR5抗体进行免疫组化染色观察移植物TLR5表达情况。
研究结果
1、PBS对照组小鼠的皮肤移植物较早出现硬化、结痂、皱缩,拆包后至完全排斥进程较快。与对照组相比,rFliC处理组小鼠的皮肤移植物生长状况良好,排斥情况较轻,排斥进程较缓慢,皮肤移植物生存期明显长于对照组(p<0.05)。结果表示rFliC可以明显延长小鼠同种皮肤移植存活期,改善移植物生存状态。
2、移植物病理分析结果显示：对照组小鼠移植皮片皱缩、变薄,表皮及真皮明显坏死,移植物与受体组织间连接疏松,受体组织内存在大量以单个核细胞浸润为主的细胞浸润；而rFliC处理组小鼠的皮肤移植物平展、厚实,表皮及真皮未显示明显坏死,移植物与受体组织间连接紧密,受体组织内的炎性细胞浸润也较轻微。此结果进一步证实rFliC促进同种皮肤移植物生存。
3、移植物免疫组化染色结果显示：与对照组相比,rFliC处理组小鼠皮肤移植物的TLR5被激活,呈高表达状态,提示rFliC对同种皮肤移植物生存的影响可能与TLR5激活相关。
第二部分rFliC对小鼠同种移植受体Treg细胞的影响研究方法
1、构建小鼠同种皮肤移植模型
建立小鼠同种皮肤移植模型,随机分为rFliC注射组、对照组及TLR5阻断组。rFliC组：在移植术进行第-1、3、5、7、9、11日向小鼠i.p.注射rFliC (3mg/kg),对照组小鼠i.p.注射PBS缓冲液；TLR5阻断组：在移植术进行第-1日向小鼠注射rFliC前1h,向小鼠i.p.注射TLR5抗体(200μg/只),其它处理同rF1iC组。2、检测移植受体CD4+CD25+Foxp3+细胞亚群比例
PBS对照组小鼠移植皮片完全排斥当日,脱颈椎法处死三组小鼠,制备脾细胞悬液及腋窝淋巴结细胞悬液,然后应用CD4、CD25、Foxp3单克隆抗体进行荧光染色,使用流式细胞仪检测三组小鼠CD4+CD25+Foxp3+Treg细胞在CD4+细胞中的比例。
3、检测移植受体脾细胞Foxp3表达状况
在PBS对照组小鼠移植皮片完全排斥当日,脱颈椎处死三组小鼠,取出脾脏,进行甲醛固定并制备石蜡切片,应用抗-Foxp3抗体进行免疫组化染色检测受体小鼠脾Foxp3表达情况。4、检测移植受体Treg相关分子表达
PBS对照组小鼠移植皮片完全排斥当日,脱颈椎处死三组小鼠,取出脾脏,制备脾细胞悬液并应用免疫磁珠分离CD4+CD25+Treg细胞。应用实时定量RT-PCR技术检测TLR5、Foxp3. TGF-β1及IL-10表达水平。应用Western blot技术检测TLR5及Foxp3表达。
5、移植受体Treg细胞增殖实验
PBS对照组小鼠移植皮片完全排斥当日,脱颈椎处死三组小鼠,取出脾脏,制备脾细胞悬液,并进一步分离CD4+CD25+Treg细胞。将三组Treg细胞分别培养4d后,向培养基内掺入3H-TdR,继续孵育16h后,收集细胞,应用液体闪烁计数器检测各组细胞dpm (Decay per minute)计数。
6、移植受体Treg细胞抑制实验
PBS对照组小鼠移植皮片完全排斥当日,脱颈椎处死三组小鼠,取出脾脏,制备脾细胞悬液,并进一步分离CD4+CD25+Treg细胞及CD4+CD25" Teff细胞(Effector T cell,效应T细胞),同时使用野生型BALB/c小鼠制备Teff扭胞。分别将对照组、rFliC处理组及TLR5阻断组小鼠Treg细胞培养3d,然后将对照组、rFliC处理组及野生型小鼠Teff分别与三组Treg进行混合,继续孵育24h后向培养基内掺入3H-TdR,孵育16h后,收集细胞,应用液体闪烁计数器检测各组细胞dpm计数。
7、制备同种抗原
制备野生型B6小鼠脾细胞悬液后,加入40μg/ml丝裂霉素C避光孵育40min,PBS洗涤后,用培养液重悬,即为同种(异型)抗原递呈细胞(APC)
野生型B6小鼠的脾细胞进行超声破碎后离心收集上清,即为可溶性同种(异型)抗原。
8、检测体外同种抗原刺激下Treg细胞相关分子表达
制备野生型BALB/c小鼠CD4+CD25+Treg细胞,加入同种抗原刺激后收集细胞,应用半定量RT-PCR技术检测TLR5、Foxp3、TGF-β1及IL-10表达情况,应用Western blot技术检测TLR5及Foxp3表达情况。TLR5阻断组：将Treg细胞用TLR5阻断抗体进行预处理。
9、体外同种抗原刺激Treg细胞增殖实验
制备野生型BALB/c小鼠CD4+CD25+Treg细胞,分别加入APC细胞、APC+rFliC或rFliC进行刺激后,掺入3H-TdR检测细胞增殖水平。TLR5阻断组：将Treg细胞用抗TLR5阻断抗体进行预处理。
10、体外同种抗原刺激Treg田胞抑制功能实验
制备野生型BALB/c小鼠CD4+CD25+Treg细胞及CD4+CD25-Teff田胞,首先对Treg细胞进行APC或APC+rFliC刺激,然后洗涤,再将Treg与Teff共培养,最后检测共培养细胞增殖水平。TLR5阻断组：将Treg细胞应用阻断抗体进行预处理。
研究结果
1、rFliC处理组小鼠脾淋巴细胞及腋窝淋巴结细胞CD4+CD25+Foxp3+细胞亚群在CD4+T细胞中的比例均显著高于对照组。TLR5阻断组小鼠脾淋巴细胞及腋窝淋巴结细胞中三阳性Treg细胞比例均明显低于rFliC处理组。结果表明rFliC可以扩增同种移植受体Treg细胞比例,此作用与TLR5途径相关。
2、三组小鼠脾免疫组化染色结果显示：与PBS对照组相比,rFliC处理组小鼠Foxp3表达显著增加,而TLR5阻断组Foxp3;表达与rFliC处理组相比受到明显抑制。此结果表明,rFliC可通过TLR5相关性途径激活小鼠同种移植受体Treg细胞,上调Foxp3表达。
3、应用qRT-PCR技术检测移植受体Treg细胞TLR5、Foxp3、TGFβ1及IL-10的表达情况。结果显示：rFliC处理组四种分子的mRNA表达与PBS对照组相比均有显著提高；而应用TLR5阻断抗体之后,rFliC对四种分子表达的上调作用受到显著抑制。各组小鼠Treg细胞表达TLR5和Foxp3的情况通过Western blot技术进行了验证。以上结果表明rFliC对同种移植小鼠模型的刺激可增加Treg相关分子的表达,而此种作用与TLR5途径相关,结果进一步证实了rFliC对移植受体Treg细胞的激活作用。
4、移植受体Treg细胞增殖实验结果显示：rFliC处理组小鼠Treg细胞增殖水平与PBS对照组相比显著增高,而TLR5阻断组小鼠Treg细胞的增殖水平相比rFliC处理组受到明显抑制。此结果表明rFliC通过TLR5依赖性途径上调移植受体Treg细胞的增殖功能。
5、移植受体Treg细胞与Teff细胞共培养结果显示,三组Treg细胞中rFliC-Treg对Teff的抑制能力最强,而此作用在TLR5阻断后受到明显下调。以上结果表明rFliC对移植受体Treg细胞的功能起到激活上调作用,此作用与TLR5途径相关。
6、rFliC对体外同种抗原刺激的Treg细胞的分子表达及功能的影响与体内实验结果一致,从体外进一步证实了rFliC对同种抗原刺激的Treg细胞的作用。
第三部分rFliC对同种抗原激活的Treg细胞Foxp3表达影响的分子机理与调控
研究方法
1、检测rFliC对Treg细胞JNK、p38MAPK信号的活化情况
应用同种抗原刺激野生型BALB/c小鼠CD4+CD25+Treg细胞后,加入rFliC进行梯度时间刺激,用Western blot检测各时间点JNK及p38MAPK磷酸化情况。
2、检测rFliC活化的Treg细胞JNK、p38MAPK信号与Foxp3表达的相关性
应用同种抗原刺激Treg细胞,阻断JNK及p38MAPK信号,加入rFliC刺激后, Western blot检测Foxp3蛋白表达情况。3、检测rFliC对Treg细胞PI3K-Akt信号的活化情况
应用同种抗原刺激Treg细胞后,加入rFliC进行梯度时间刺激,用Western blot检测各时间点Akt磷酸化情况。
4、检测Treg细胞中rFIiC激活的PI3K信号对p38MAPK活化的影响
应用同种抗原刺激Treg细胞,阻断PI3K信号,加入rFliC刺激后,用Western blot检测不同时间点p38MAPK磷酸化情况。5、检测Treg细胞中rFIiC激活的PI3K信号与Foxp3表达的相关性
应用同种抗原刺激Treg细胞,阻断PI3K信号,加入rFliC刺激,应用Western blot及RT-PCR检测Foxp3表达情况；应用同种抗原刺激小鼠脾淋巴细胞,阻断PI3K信号,加入rFliC刺激,应用流式细胞术检测CD4+CD25+Foxp3+细胞在CD4+T细胞中的比例。
6、检测Treg细胞中rFliC激活的PI3K及p38MAPK信号与Foxp3表达的相关性
应用同种抗原刺激Treg细胞后,先阻断p38信号,再阻断PI3K信号,然后加入rFliC刺激,应用Vestern blot及RT-PCR检测Foxp3表达情况；应用同种抗原刺激小鼠脾淋巴细胞后,先阻断p38信号,再阻断PI3K信号,加入rFliC刺激后应用流式细胞术检测CD4+CD25+Foxp3+细胞在CD4+T细胞中的比例。
研究结果
1、rFliC作用于同种抗原激活的Treg细胞,可活化JNK及p38MAPK信号。而p38MAPK信号与rFliC引起的Foxp3表达上调相关,即rFliC对同种抗原激活Treg细胞Foxp3表达的作用通过rFliC-p38-Foxp3通路实现。
2. rFliC可以激活同种抗原刺激Treg细胞的PI3K-Akt信号。并且PI3K信号对p38MAPK活化具有负调节作用。
3、PI3K对rFliC-p38-Foxp3通路具有负调控作用,而调控方式通过抑制p38MAPK信号活化实现。全文结论1.rF1iC体内应用可以明显改善小鼠同种皮肤移植物生存状态,延长移植物生存期,诱导同种移植免疫耐受。并且rF1iC可激活移植物TLR5表达,提示
rF1iC诱导同种移植免疫耐受的作用与TLR5途径相关。2.rFliC可明显提高同种移植受体CD4+CD25+Foxp3+Treg细胞比例,上调Treg细胞相关分子Foxp3等表达水平,增强Treg细胞功能,并且这些作用都与TLR5途径相关。提示rFliC诱导同种移植免疫耐受的机制可能是通过TLR5相关途径上调Treg细胞功能。
3. rFliC刺激Treg细胞后对Foxp3表达的上调可通过rFliC-p38MAPK-Foxp3通路实现,而PI3K可通过抑制p38信号活化对此通路起负调控作用。
论文创新点
1.首次利用小鼠同种皮肤移植模型,证实rFliC可明显延长小鼠同种皮肤移植物生存时间,诱导同种移植免疫耐受,从而为进一步研发诱导实体器官移植免疫耐受的新型制剂提供了实验依据。
2.首次证实rFliC可以提高小鼠同种移植受体CD4+CD25+Foxp3+Treg细胞比例及功能,并且作用机制与TLR5途径相关。为研究鞭毛蛋白诱导同种移植免疫耐受的细胞及分子机制提供了实验基础及理论依据。
3.首次证实rFliC上调Treg细胞Foxp3表达的作用与p38MAPK活化相关,即可通过rFliC-p38-Foxp3通路实现,证实了PI3K信号对此通路具有负调控作用。从而为研究鞭毛蛋白诱导同种移植免疫耐受的信号传导途径及调控靶点提供了新的思路。
Background and Objectives
Until now, allogeneic organ transplantation has been wildly used in treating organ failure, blood diseases or cancers. However, acute rejection, which occurs within the first few weeks or months, remains a major cause of treatment failure. The post-transplantant immunosuppressive drugs routinely used in clinic seriously affect patients' immunologic function along with side and toxic effects. To overcome this issue, great efforts have been paid to induce specific immune-tolerance in recipients and it is a hotspot to study how to induce immune-tolerance via augmenting frequency of regulatory T cells (Tregs) or enhance their functions. Treg is an important kind of cell subset for maintaining peripheral tolerance. It has many advantages of using Tregs for inducing immuno-tolerance; therefore Tregs maybe developed into a significant tool for inducing allotransplantation immune tolerance and transplantation rejection therapy.
At present, the cell therapy utilization of Tregs augmented in vitro is seriously limited by sterilization, cell amounts and so on, however, the method that using drugs to augment frequency of recipients' Tregs or enhance their suppressive functions showed a great implication prospects. Treg cells in human body mainly existed in CD4+CD25hi (highly-expressed) cell subset, which approximately occupied1%-2%of CD4+T cells in peripheral blood. As an important kind of cell subset which possessed function of negative-regulating immune response in vitro and in vivo, Treg could be classified into naturally occurring CD4+CD25+Treg (nTreg) and induced CD4+CD25+Treg (iTreg) according to their surface molecules, cytokine-produced and mechanism of action. It was confirmed in a large number of transplantation animal models that Tregs played a key role in keeping living condition of grafts; and augmented frequency of Tregs could induce immune tolerance and alleviate rejection reaction. In solid-organ transplantation, compared with equivalent nTregs, iTregs specifically induced by allogeneic antigens ex vivo and in vitro were more useful in prolonging allograft survival and inducing immune tolerance.
Mechanisms of Tregs'immunoregulation mainly include the following aspects: suppressing antigen presenting cells (APCs), mediating target cell dissolution and killing target cells, secreting TGF-β and IL-10for immuno-suppression, et al. The process of Tregs inhibiting CD4+effector T cells was by means of immediate contaction, secreting cytokine or combined to APCs and then changing their metabolic pathway or secrete way, et al. Treg cells highly expressed CD4and CD25and, in addition, a kind of characteristic marker-Foxp3, which was pivotal for phenotype, development and functional maintain of Tregs. It was reported that frequency of Tregs and expression level of Foxp3could act as an indicator for improving recipients' immunologic condition and inducing immune tolerance. However, research on how to augment frequency of Tregs, to improve expression of Foxp3or to enhance immunosuppressive function of Tregs was still inadequate. So, it is urgent to find a safe agent to augment frequency of Tregs in recipients or enhance their suppressive functions.
It was reported that co-culture of human CD4+T cells and recombinant flagellin (rFliC) in vitro significantly enhanced immunosuppressive ability of CD4+CD25+Treg cells and increased expression of Foxp3. Flagellin was also reported to be effective in protecting body from chemical, bacterial, viral and radioactive injury. Therefore, flagellin may act as a new agent to strengthen function of Tregs and thus to induce body's immune tolerance. Flagellin was a major ingredient of bacterial flagella, and the specific ligand of Toll-like receptor5(TLR5). It was found that TLR5was expressed both in human T cells and murine Treg cells. Toll-like receptor (TLR) family was a key factor in mediating innate immunity, and it activated innate immune response at the early time when pathogens invaded body; in addition, flagellin played an important role in pathogen-induced immuno-pathological injuries. Immune response mediated by flagellin was dependent on classic TLR signaling pathway. Flagellin could activate TLR5signaling specifically; the downstream signaling was MyD88-dependent and associated with activation of NF-kB or MAPK. The MAPK pathway was regulated by numerous factors.
In conclusion, we proposed a scientific hypothesis in this paper that rFliC was effective in inducing allotransplantation immune tolerance by means of augmenting frequency of recipient CD4+CD25+Tregs, increasing expression of Foxp3and enhancing functions and suppressive ability of Treg cells, et al. The objectives of this research were to clarify the impact of rFliC on allografts' living condition and survival time, to explore the undergoing mechanism and molecular regulation of rFliC's effects, and thus, to provide theoretical and experimental basis for studying flagellin-induced-transplantation tolerance and offer a new candidate agent for clinical transplantation rejection therapy. So this research was of theoretical and practical significance.
Part One Impact of rFliC on murine skin allograft and relationship with TLR5expression
Methods
1. Preparation of purified recombinant flagellin (rFliC) Firstly, BL21E.coli-GST-FliC bacterial strain was established by transferring GST-FliC-Burkholderia pseudomallei-pGEX4T-2plasmid into competent BL21E.coli. Established bacterial strain was screened according to antibiotics resistance characteristic and then activated and induced to express recombinant flagellin (rFliC). Flagellin was purified by Glutathione Sepharose4B gelatum and the LPS contaminant was cleared away by ToxinEraser endotoxin removal resin.
2. Establishment of murine skin allograft models The dorsal skin of C57BL/6mice was transplanted onto the BALB/c mice under sterile conditions. The bandage was removed on day7post-transplantation. rFliC (3mg/kg) was i.p. injected into the models on day-1.3.5.7.9and11of the operation, PBS was used as the control.
3. Examining the impact of rFliC on living condition and survival time of allografts
From day7post-transplantation, survival condition of transplantation models in two groups was observed; rejection percentage and survival time was recorded. On the day that grafts of control group were totally rejected, two groups of model mice were euthanatized and the grafts were prepared to be paraffin sections for HE staining.
4. Examing impact of rFliC on TLR5expression in allografts and recipients
On the day that grafts of control group totally rejected, two groups of model mice were euthanatized and the grafts were prepared to be paraffin sections for immunohistochemistry (IHC) staining using anti-TLR5antibody.
Results
1. Allografts of control mice were early rejected characterized by massive epidermal damage, scab formation and a rapid loss of graft volume. Compared with control, living condition of grafts in rFliC-treated group was much better and the survival time was longer. The result indicated that rFliC was effective in prolonging allograft survival and improving living condition.
2. HE staining of allografts showed that the skin graft sections from the control mice exhibited a clear border between thehost and grafted skin, epidermal and dermal necrosis and extensive inflammatory cell infiltration. By contrast, the grafts from the rFliC-treated hosts showed healthy growth, which was manifested as an imprecise boundary between the host and grafted skin and less inflammation and skin appendage degeneration.This result proved the effect of rFliC on allograft survival.
3. IHC staining of allografts showed that high TLR5expression was induced by rFliC treatment compared with PBS control. These data suggested that rFliC treatment in vivo improved allograft survival and this effect was correlated with TLR5activation.
Part Two Effect of rFliC on Treg cells of allotransplanted recipients Methods
1. Establishment of murine skin allograft models
The dorsal skin of C57BL/6mice was transplanted onto the BALB/c mice under sterile conditions. rFliC (3mg/kg) was i.p. injected into the models on day-1.3.5.7.9and11of the operation, PBS was used for control. One hour before rFliC administered, anti-TLR5antibody was injected into model mice to set up as TLR5-blocking group.
2. Frequency of recipient CD4+CD25+Foxp3+Tregs
On the day that grafts of control group were totally rejected, three groups of model mice were euthanatized to prepare splenocytes and axillary lymph node cells. Cells were stained by CD4, CD25and Foxp3antibodies, and then, frequency of CD4+CD25+Foxp3+Tregs in CD4+T cells was tested.
3. Examining Foxp3expression in recipient spleen section
On the day that grafts of control group were totally rejected, three groups of model mice were euthanatized and the spleen was prepared to be paraffin sections for IHC staining using anti-Foxp3antibody.
4. Checking expression of Treg-related molecules in recipients
On the day that skin grafts of control group were totally rejected, splenocytes were freshly prepared and CD4+CD25+Tregs were sorted. Then, expression of TLR5, Foxp3, TGF-β1and IL-10was tested by qRT-PCR. Expression of TLR5and Foxp3was additionally examined by Western blot.
5. Proliferation of recipient Treg cells
When skin grafts of control group were totally rejected, CD4+CD25+Tregs were sorted using freshly prepared splenocytes. Three groups of Tregs were cultured for4d, then3H-TdR was incorporated and dpm was tested after16h.
6. Suppression of recipient Treg cells
When skin grafts of control group were totally rejected, CD4+CD25+Tregs and CD4+CD25" effector T cells (Teffs) were sorted using freshly prepared splenocytes. Teffs from naive BALB/c mice were also prepared. Firstly, CD4+CD25+Tregs from rFliC-treated, PBS-control and TLR5-blocking groups were cultured for3d. Then, CD4+CD25" Teffs from rFliC-treated, PBS-control and naive BALB/c groups were added into Treg cultures for another24h. At last,3H-TdR was incorporated and dpm was tested after16h.
7. Preparation of antigen presenting cells (APCs) and allogeneic antigen
APCs were prepared by treating splenocytes from naive B6mice with40μg/ml Mitomycin C for40min at37℃in5%CO2.
Naive B6mice splenocytes were sonic disrupted and centrifuged to get supernatants as allogeneic antigen.
8. Examining molecule expression of alloantigen-stimulated Tregs in vitro
CD4+CD25+Tregs were prepared from naive BALB/c mice and stimulated by alloantigen and rFliC. Expression of TLR5, Foxp3, TGF-β1and IL-10was examined by semi-quantitative RT-PCR. Expression of TLR5and Foxp3was additionally tested by Western blot.
9. Proliferation of alloantigen-stimulated Tregs in vitro
CD4+CD25+Tregs were prepared from naive BALB/c mice firstly. Then, APC, APC+rFliC or rFliC was added into the culture to stimulate Tregs. At last,3H-TdR was incorporated and dpm was tested. TLR5-blocking group was set up by pre-treating Tregs with anti-TLR5antibody.
10. Suppression of alloantigen-stimulated Tregs in vitro
CD4+CD25+Tregs and CD4+CD25" Teffs were prepared from naive BALB/c mice. Firstly, Tregs were stimulated by APC or APC+rFliC. Then, Teffs were added for co-culture. At last,3H-TdR was incorporated and dpm was tested. TLR5-blocking group was set up by pre-treating Tregs with anti-TLR5antibody.
Results
1. For both of splenocytes and axillary lymph node cells in rFliC-treated mice, frequencyof CD4+CD25+Foxp3+cells in CD4+T cells was significantly high than control. However, frequency of triple positive cells in TLR5-blocked mice was obviously lower than that in rFliC-treated mice. This result showed that rFliC administration could augment frequency of recipient Treg cells and this effect was associated with TLR5signaling pathway.
2. IHC staining of spleen section showed that expression of Foxp3in rFliC-treated mice was significantly higher than that in PBS-control mice; Foxp3expression in TLR5-blocked mice was much lower than that in rFliC-treated group. The result indicated that rFliC might activate allotransplantation recipient Treg cells and enhance Foxp3expression via a TLR5-dependent manner.
3. The mRNA expression of TLR5, Foxp3, TGF-βl and IL-10in rFliC-treated recipient Tregs was significantly higher than that in control. In TLR5-blocked mice, expression of these four molecules was attenuated, compared with rFliC-treated mice. The expression condition of TLR5and Foxp3was confirmed by Western blot. The data suggested that treatment of rFliC increased expression of Treg-related molecules in a TLR5-correlated way.
4. Proliferation of rFliC-treated recipient Tregs was obviously stronger than that of control mice. Proliferation of TLR5-blocked recipient Tregs was inhibited compared with rFliC-treated group. This result indicated that rFliC up-regulated proliferation of recipient Tregs in a TLR5-dependent manner.
5. Treg cells from rFliC-treated hosts possessed the strongest suppressive ability on Teffs, and the ability was inhibited when TLR5pathway was blocked. This data revealed that rFliC enhanced suppressive function of recipient Tregs and this effect was related with TLR5activation.
6. Molecules expression and functions of alloantigen stimulated Tregs in vitro was also up-regulated by rFliC stimulation, which was consistent with results in vivo.
Part Three Mechanism of rFliC regulating Foxp3expression in alloantigen stimulated Tregs and the molecular modulation
Methods
1. Examining activation of JNK and p38MAPK in Tregs under rFliC stimulation
CD4+CD25+Tregs from naive BALB/c mice were stimulated with alloantigen. Then, rFliC stimulation was added for gradient times and phosphorylation of JNK and p38MAPK was tested by Western blot.
2. Checking the relevance between activated JNK, p38MAPK and Foxp3expression
Alloantigen stimulated Tregs were pre-treated to block JNK and p38MAPK signaling. Then, rFliC was added for stimulation, and expression of Foxp3was tested by Western blot assay.
3. Examining activation of PI3K-Akt signaling in Tregs under rFliC stimulation
CD4+CD25+Tregs were stimulated by alloantigen. Then, rFliC was added and phosphorylation of Akt was tested by Western blot.
4. Checking the impact of activated PI3K-Akt on p38MAPK signaling
Alloantigen stimulated Tregs were prepared and PI3K signaling was blocked. Then, rFliC was added for gradient times and phosphorylation of p38MAPK was tested by Western blot assay.
5. Testing the relevance between activated PI3K signaling and Foxp3expression
Alloantigen stimulated Tregs were prepared and PI3K signaling was blocked, then, rFliC was added for stimulation. Expression of Foxp3was examined by Western blot and semi-quantitative RT-PCR assay. Alloantigen stimulated splenocytes were prepared and PI3K signaling was blocked, then, rFliC was added for stimulation. Frequency of CD4+CD25+Foxp3+cells in CD4+T cells was tested by flow cytometry (FCM).
6. Examining the relationship between activated PI3K and p38MAPK signaling and Foxp3expression
Prepared alloantigen stimulated Tregs was pre-treated to block p38signaling firstly. After that, PI3K signaling was blocked too. Then, rFliC was added for stimulation. Expression of Foxp3was examined by Western blot and RT-PCR. Prepared alloantigen stimulated Tregs was pre-treated to block p38and PI3K signaling and then stimulated by rFliC. Frequency of CD4+CD25+Foxp3+cells in CD4+T cells was tested by FCM.
Results
1. rFliC could activate JNK and p38MAPK signaling in alloantigen stimulated Tregs. In addition, p38signaling was associated with rFliC-induced up-regulation of Foxp3expression. In other words, effect of rFliC on Foxp3expression in alloantigen stimulated Tregs was achieved via an rFliC-p38-Foxp3pathway in alloantigen stimulated Tregs, and PI3K signaling negatively regulated p38activation.
2. PI3K signaling negatively regulated rFliC-p38-Foxp3pathway via inhibiting p38activation.
Conclusions
1. rFliC administration in vivo significantly prolonged survival time and improved living condition of murine skin allograft, and furthermore, induced allotransplantation immune tolerance. In addition, rFliC activated TLR5expression in allografts, indicating that effect of rFliC in inducing allograft tolerance was associated with TLR5pathway.
2. rFliC improved frequency of recipient CD4+CD25+Foxp3+Tregs, enhanced expression of Treg-related molecules and strengthened function of Tregs, in a TLR5dependent manner. It was indicated that rFliC-induced allotransplantation tolerance may be achieved by rFliC up-regulating Tregs in a TLR5-dependent manner.
3. Improvement of Foxp3expression in Tregs stimulated by rFliC was mediated by an rFliC-p38MAPK-Foxp3passageway; PI3K signaling negatively regulated this pathway by inhibiting p38MAPK activation.
Points of Innovation
1. It is the first time to prove the effect of rFliC in inducing allotransplantation immune tolerance in a murine skin allograft model. These results provided an experimental basis for investigating new candidate agent for inducing immune tolerance in solid-organ transplantation.
2. It is the first time to verify that rFliC could improve frequency and function of CD4+CD25+Foxp3+Treg cells in allografted recipients. Furthermore, this kind of effect was in a TLR5-dependent manner.
3. It is the first time to prove that effect of rFliC in up-regulating expression of Foxp3could be mediated by activating p38MAPK signaling, which means that Foxp3expression in Tregs was improved by rFliC in a rFliC-p38MAPK-Foxp3 passageway. In addition, the negatively regulation of PI3K signaling on this pathway was demonstrated. These results provided a new idea and regulating target for studying signaling pathway in rFliC inducing allotransplantation immune tolerance.
引文
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