Tregs调控疟疾保护性免疫应答和免疫病理效应机制的研究
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摘要
疟疾是疟原虫通过媒介昆虫蚊传播的人类最为严重的寄生原虫感染性疾病。《Nature》公布的统计数据显示,世界上100多个国家为疟疾流行区,约22亿人口受到疟疾的威胁,每年有300～500万疟疾临床病例,病死人数高达110～270万。为此,2003年WHO将疟疾作为优先重点防治的感染性疾病。在中国,尽管在控制疟疾流行、减少危害程度方面取得了显著成效,但由于耐药性原虫和耐杀虫剂性蚊的普遍出现,以及近年来流动人口剧增、气候变暖和生态环境变化等因素,疫情状况依然十分严峻。据最新统计显示,2000年以来我国疟疾发病人数呈现逐年上升趋势,现有21个省(直辖市、自治区)的907个县有疟疾病例报告,受疟疾威胁人口高达5.5亿。因此,有效的疟疾疫苗和抗疟新药的研制开发已经成为当今世界迫切需要解决的重点课题,而疟原虫感染宿主机体应答的免疫学、分子生物学等综合基础研究是其重要前提。
疟疾与其他大多数感染性疾病一样,需要通过免疫效应机制对其控制和消除。同时,由于过强的炎症反应引起脑疟等免疫病理损伤也是这一疾病的突出特征。关于红内期疟原虫感染的免疫机制,无论鼠疟模型和人体试验研究结果均已证实:①CD4~+T细胞在抵抗红内期疟原虫感染过程中发挥至关重要的作用。在感染早期,Th1型细胞经DC分泌的IL-12诱导活化,产生以IFN-γ为主的炎症性细胞因子,遏制疟原虫的爆发性增殖;随之,由Th2型细胞辅助B细胞产生特异性抗体,能够有效地清除疟原虫,防止复发和再燃。由此表明:抗疟保护性免疫有赖于Th1和Th2型免疫应答的有效建立和协调过渡。②Th1/Th2型免疫应答的发生时相和效应强度明显地影响着最终的感染结局。各种数据显示,致死型脑疟等严重并发症的出现与机体过强的Th1型炎症应答密切相关。由此提示:维持前炎症细胞因子和抗炎症细胞因子之间的动态平衡,控制炎症性细胞因子产生的时间和强度对疟疾感染甚为关键。打破免疫应答之间的平衡或扰乱免疫应答的时间进程,均可能导致慢性感染或者病情加重。换言之,在决定疟疾感染最终结局方面,调控疟疾感染的免疫应答与诱导有效的抗疟免疫同样重要。然而,目前关于保护性和病理性免疫应答动态平衡的调控机制尚未阐明。
CD4~+T细胞亚类CD4~+CD25~+调节性T细胞(Tregs)是一类具有独特免疫调节功能的细胞群,体内、外试验显示,Tregs具有广泛的免疫抑制性,通过表面膜分子与其它细胞直接接触、或分泌抑制性细胞因子IL-10和TGF-β两种方式,可抑制体内天然CD4~+T和CD8~+T细胞等多种细胞的活化和增殖。目前,对Tregs的研究主要集中于自身免疫性疾病、抗移植和抗肿瘤免疫等方面,而在疟疾等感染性疾病中作用的研究才刚刚起步,特别是关于Tregs对Th应答效应和Th1/Th2极化机制方面的研究尚未深入开展。有限的研究结果显示,在感染致死型伯氏疟原虫(Plasmodium.berghei NK65)或约氏疟原虫(P.yoelii17XL)之前,应用抗CD25单克隆抗体耗竭小鼠体内Tregs,可通过延缓疟原虫的增殖速率使其致死性感染得以控制。Tregs回输实验证实,P.yoelii 17XL感染能够特异性活化Tregs,介导疟原虫逃逸C57BL/6小鼠的T细胞应答。同样人体实验也表明恶性疟原虫(P.falciparum)感染诱导Tregs分化并伴有IL-10的升高。以上结果均提示,Tregs的活化是介导恶性疟原虫和致死型啮齿类疟原虫逃逸机体免疫应答的关键。
另外,人们研究发现在细胞亚群分化过程中,转录因子也具有重要的调节作用。T-bet和GATA3是两种细胞内的转录因子,分别特异性地表达于Th1和Th2细胞。T-bet正调控Th1细胞的发育,GATA3正调控Th2细胞的发育,二者最终决定Th0向Th1/Th2的转化。由此可以看出在疟疾感染过程中T-bet和GATA3两种转录因子也参与了Th1/Th2的极化过程。
为了解不同疟原虫感染过程中Th1/Th2应答的差异是否与Tregs的免疫调节作用具有一定的相关性,本研究选用致死型约氏疟原虫(P.yoelii 17XL,P.y17XL)、夏氏疟原虫(P.chabaudi AS,P.cAS)及其约氏+夏氏疟原虫(1:1)混合感染DBA/2和BALB/c小鼠,动态观察感染过程中Th1/Th2应答的差异、Tregs的数量变化和功能特点,并且在此基础上应用抗CD25单克隆抗体阻断技术,以期阐明Tregs在疟疾感染过程中的作用地位及其相关机制。
实验方法
1、实验动物及其模型构建
6-8周龄、雌性DBA/2和BALB/c小鼠,经腹腔分别感染1×10~6 P.y17XL、P.cAS和2×10~6 P.y17XL+P.cAS(1:1)寄生的红细胞(PRBC),构建不同的实验动物模型。
2、采用双抗体夹心ELISA法检测小鼠感染疟原虫后IFN-γ和IL-4的含量
无菌取出感染小鼠脾脏,制成细胞悬液并调整脾细胞终浓度为1×10~7/ml,24孔培养板上加入细胞悬液,500μl/孔,于37℃培养48h。350g室温离心10min,收集上清,-80℃保存,待IFN-γ和IL-4检测。
用双抗体夹心ELISA法对IFN-γ和IL-4的含量进行检测,酶标仪检测450nm处OD值,应用SoftMax Pro4.3.1 LS软件分析,绘制标准品标准曲线,计算细胞因子含量(pg/ml)。
3、采用RT-PCR方法检测转录因子T-bet和GATA3 mRNA的表达水平
(1)PCR引物利用引物设计软件Primer5设计T-bet、GATA3和β-actin特异性引物。
(2)实验方法采用TRIZOL一步法提取小鼠脾细胞总RNA,并经紫外分光光度仪检测纯度,RNAA_(260)/A_(280)的比值在1.8-2.0之间;PCR扩增产物用1.5%琼脂糖凝胶电泳检测,应用ID影像分析软件进行表达强度的分析。
4、采用流式细胞分析技术检测小鼠感染疟原虫后脾Tregs及CD4~+T细胞凋亡的百分比
(1)无菌取小鼠脾脏,制成1×10~7/ml脾细胞悬液。每份样品用抗CD4-FITC单抗和抗CD25-PE单抗进行双色分析,另设阴性对照管。流式细胞仪专用染色管预先加入抗FcγⅢ/Ⅱ受体抗体2μl,取新鲜制备的1×10~7/ml脾细胞悬液0.1ml,再加入抗CD4-FITC单抗和抗CD25-PE单抗各0.2μg。振荡器低速混匀,冰浴放置,避光反应30min。每管加入2ml 2%的FCS,水平转头离心机300g,离心5min,弃上清,洗涤两次,静置15 min上机。
(2)取binding buffer制备的1×10~6/ml脾细胞悬液0.1ml,加入流式细胞仪专用染色管中,每份样品设阴性对照管,另一管分别加入抗CD4-FITC单抗0.2μg,AnnexinV-PE和7AAD各5μl。25℃避光孵育15min,每管加binding buffer 400μl,上机。
(3)流式细胞仪激发波长为488nm,利用FACS CELLQUEST软件获取细胞,每个样品分析10,000个细胞,以二维点阵图显示,记录Tregs及CD4~+T细胞凋亡的百分比。
5、采用双抗体夹心ELISA法检测小鼠感染疟原虫后脾细胞培养上清抑制性细胞因子IL-10和TGF-β的含量
无菌取小鼠脾脏,制成1×10~7/ml脾细胞悬液。24孔培养板中加入细胞悬液,500μl/孔,于37℃培养48h。350g室温离心10min,收集上清。用双抗体夹心ELISA法对TGF-β和IL-10的含量进行检测,酶标仪检测450nm处OD值。应用SoftMax Pro4.3.1 LS软件分析,绘制标准品标准曲线,计算细胞因子含量(pg/ml)。
6、采用细胞内染色方法检测小鼠感染疟原虫后分泌IL-10的Tregs数量的百分比
无菌取小鼠脾脏,制成1×10~7/ml脾细胞悬液。于流式管中加入细胞悬液,500μl/管,37℃条件下PMA和伊屋诺霉素刺激2h后加入Golgi Stop共同培养4h,3%FCS洗涤后加入FITC-anti-CD4 and PE-anti-CD25荧光抗体,4℃孵育30min,3%FCS洗涤后加入固定透膜剂,4℃孵育20min,洗涤后加入APC-anti-IL-10(JES5-16E3)荧光抗体,4℃避光孵育30min,洗涤后上机。同时用FITC rat IgG2b作为同型对照。
7、应用抗CD25单克隆抗体阻断技术,研究Tregs在约氏疟原虫感染早期相关的作用机制
BALB/c小鼠分别在感染前1d和感染后1d腹腔注射1mg的抗CD25 mAb(7D4),腹腔注射PBS小鼠作为对照组。分别于感染0d、3d和5d常规制备脾细胞悬液,流式细胞仪检测Tregs体内消除后数量及其CD4~+T细胞凋亡数量变化;收集脾细胞培养上清待Tregs体内消除后IFN-γ和IL-10水平检测。
实验结果
1、不同疟原虫感染DBA/2和BALB/c小鼠脾细胞培养上清中IFN-γ和IL-4水平的比较
P.y17XL感染的BALB/c小鼠IFN-γ水平仅在感染后第3d出现一过性有意义的升高;而DBA/2小鼠IFN-γ水平在感染后第1-3d出现有意义的升高并达峰值,随后缓慢下降,且IFN-γ产生水平明显高于相应时间点的BALB/c小鼠。P.cAS感染的DBA/2小鼠IFN-γ水平仅在感染后第3d出现有意义的升高;而BALB/c小鼠IFN-γ水平在感染后第3d出现有意义的升高,第5d达峰值,随后下降至正常水平,且IFN-γ产生水平明显高于相应时间点的DBA/2小鼠。P.y17XL+P.cAS感染BALB/c和DBA/2小鼠IFN-γ水平均于感染后第3d达峰值,随后下降,且DBA/2小鼠IFN-γ产生水平明显高于相应时间点的BALB/c小鼠。
P.y17XL感染的BALB/c小鼠IL-4水平仅在感染后第3d出现有意义的升高;而DBA/2小鼠IL-4水平在感染后第5-8d升高并达峰值,此后缓慢下降,且IL-4产生水平明显高于相应时间点的BALB/c小鼠。P.cAS感染的DBA/2小鼠IL-4水平仅在感染后第5d出现一过性有意义的升高;而BALB/c小鼠IL-4水平在感染后第8-10d升高并达峰值,此后虽有回落但仍维持其高水平,且IL-4产生水平明显高于相应时间点的DBA/2小鼠。P.y17XL+P.cAS感染DBA/2和BALB/c小鼠IL-4水平于感染后第1-5d无明显变化,均于感染后第8d显著升高达峰值,并且DBA/2小鼠的IL-4水平明显高于相应时间点的BALB/c小鼠,随后DBA/2小鼠的IL-4水平缓慢下降,于感染后第15d下降至正常水平。
2、不同疟原虫感染DBA/2和BALB/c小鼠脾细胞中转录因子T-bet和GATA3的表达水平及其比值
.Py17XL感染的DBA/2和BALB/c小鼠于感染后第1dT-bet表达量均明显升高,GATA3表达量略有升高。DBA/2小鼠T-bet和GATA3的表达量均于感染后10d达峰值,而BALB/c小鼠于感染后3-5d无明显变化。DBA/2小鼠的T-bet/GATA3比值在感染后第3d达峰值,此后缓慢下降;而BALB/c小鼠的T-bet/GATA3比值在感染后第1d略有升高,随后迅速回落,小鼠死亡。
P.cAS感染的DBA/2和BALB/c小鼠于感染后第1dT-bet表达量均明显升高,GATA3表达量略有升高。DBA/2小鼠T-bet和GATA3的表达量均于感染后5d达峰值,随后迅速回落;而BALB/c小鼠T-bet和GATA3的表达量均于感染后3-5d无明显变化,此后缓慢回落。DBA/2小鼠T-bet/GATA3比值在感染后第1-8d没有明显下降趋势,感染后第10d出现陡然下降,小鼠死亡;而BALB/c小鼠T-bet/GATA3比值在感染后第3d达峰值,于感染后第8d迅速下降至最低点,随后于感染后第12d再次达峰值,感染后第15d再次回落至正常水平。
3、不同疟原虫感染DBA/2和BALB/c小鼠脾Tregs数量的比较
P.y17XL感染小鼠于感染后第1d Tregs均出现有意义的升高。随后DBA/2小鼠Tregs缓慢上升,第5d达峰值,然后缓慢下降;而BALB/c小鼠Tregs数量迅速上升,第5d达峰值,并且Tregs数量明显高于相应时间点的DBA/2小鼠。P.cAS感染的BALB/c小鼠于感染后第1d Tregs出现有意义的升高,第5d达峰值后缓慢下降;而DBA/2小鼠的Tregs于感染后迅速持续升高,第10d Tregs细胞数量达峰值,且Tregs数量明显高于相应时间点的BALB/c小鼠。P.y17XL+P.cAS感染的DBA/2和BALB/c小鼠于感染后第1d Tregs均出现有意义的升高。随后DBA/2小鼠Tregs缓慢上升,第5d达峰值后缓慢下降;BALB/c小鼠Tregs数量迅速上升,感染后第8d达峰值,而且Tregs数量明显高于相应时间点的DBA/2小鼠。
4、不同疟原虫感染DBA/2和BALB/c小鼠脾CD4~+T细胞凋亡百分比的比较
P.y17XL感染小鼠CD4~+T细胞凋亡数量均于感染后第3d出现升高。随后DBA/2小鼠CD4~+T细胞凋亡数量增加缓慢,而BALB/c小鼠则于感染后3d-5d迅速增加。P.cAS感染的BALB/c小鼠仅于感染后第8d CD4~+T细胞凋亡数量出现升高,而DBA/2小鼠CD4~+T细胞凋亡数量于感染后第5d出现升高,至感染后第8d达峰值,且CD4~+T细胞凋亡数量明显高于相应时间点的BALB/c小鼠。
5、不同疟原虫感染DBA/2和BALB/c小鼠脾细胞培养上清抑制性细胞因子IL-10和TGF-β含量的比较
P.y17XL感染的DBA/2小鼠于感染后第5d IL-10出现升高,随后缓慢下降;而BALB/c小鼠于感染后第1-5d IL-10明显升高后达峰值,且IL-10水平明显高于相应时间点的DBA/2小鼠。P.cAS感染的BALB/c小鼠于感染后第1d IL-10就出现升高,第8d达峰值,随后缓慢下降;DBA/2小鼠IL-10水平于感染后第3d出现升高,第10d达峰值,且IL-10水平明显高于相应时间点的BALB/c小鼠。
P.y17XL感染的DBA/2小鼠于感染后第10d和12d TGF-β出现升高,随后迅速下降至正常水平;而BALB/c小鼠仅于感染后第5d TGF-β出现升高。P.cAS感染的DBA/2小鼠TGF-β水平于感染5-8d升高后达峰值;而BALB/c小鼠于感染后第3d TGF-β出现一过性升高后于第8d下降至正常水平。
6、Anti-CD25mAb体内阻断后,P.y17XL感染早期BALB/c小鼠免疫应答模式的变化
应用anti-CD25mAb体内阻断后,BALB/c小鼠的原虫血症水平上升缓慢且峰值低,生存率明显延长,甚至部分小鼠存活;IFN-γ产生水平显著升高,IL-10产生水平明显减少,且CD~(4+)T细胞凋亡数量也明显下降。
结论
1、P.y17XL和P.cAS感染过程中,小鼠DBA/2和BALB/c免疫应答存在明显差异。
2、P.y17XL和P.cAS感染过程中,Th1和Th2型免疫应答的有效建立和协调过渡对抵抗疟疾感染至关重要,同时提示Th1和Th2型免疫应答的发生时相和效应强度可能最终影响着疟疾感染结局。
3、Tregs数量的过早升高与P.y17XL感染的BALB/c小鼠Th1型免疫应答未能有效建立密切相关;
4、Tregs数量的异常活化与P.cAS感染的DBA/2小鼠未能成功的从Th1向Th2型细胞免疫应答转化密切相关;
5、P.y17XL+P.cAS混合感染过程中,DBA/2和BALB/c小鼠免疫应答模式与P.y17XL单独感染DBA/2和BALB/c小鼠的应答模式相同;
6、P.y17XL+P.cAS混合感染过程中,Th1和Th2型免疫应答的有效建立和协调过渡对抵抗疟疾感染至关重要;
7、Tregs数量的持续升高与P.y17XL+P.cAS混合感染的BALB/c小鼠Th1型免疫应答未能有效建立及感染结局密切相关。
8、P.y17XL感染早期,Tregs是通过分泌抑制性细胞因子IL-10和诱导CD4~+T细胞的凋亡来介导免疫抑制作用,参与Th1型细胞免疫应答的调节。
Malaria infection is a major health problem to human beings even today.A recent survey has shown that,in 2002,roughly 2.2 billion people were at risk of P.falciparum infection,with a conservative estimate of 515 million individuals who had been infected.The prevention and therapy of such infectious diseases as Malaria,AIDS and tuberculosis have been taken as the focus of researches by WHO.However,Up till now, the mechanisms of immune response to malaria parasite infection are not fully understood yet.Thus,the rational development of effective anti-malarial vaccines and novel therapies to alleviate or prevent the symptoms of severe malaria infection requires a better understanding of the various mechanisms of immune responses to malaria parasite.
A series of studies have proved that immune effector mechanisms are required to eliminate malaria parasites,and Th1 immune effectors are crucial to control the early infection.Experiments in vivo and in vitro have demonstrated:(1) CD4~+ T cells are essential for the control of primary parasitemia in mouse models during the blood-stage of malaria infection.IFN-γplays an important role in the production and maintenance of Th1 in early response and control the outbreak proliferation of blood-stage malaria parasites.In addition,B cells secrete specific antibodies supporting by Th2 cells,which can effectively remove parasite,preventing the recidivation and recrudescence.Thus effective establishment of Th1 immune response and a successful switch to Th2 are crucial for educing protective immunity response in malaria.Balance and coordination of Th1/Th2 can not only control the crisis of the acute phase,but also help in the ultimate elimination of malaria parasite.(2) Phase and level of immune response might affect the final outcome of malaria infection.Related studies showed the serious complications of cerebral malaria were correlated with a strong Thl response.This indicated that it is critical for malaria infection to maintain the dynamic balance of inflammatory cytokines/anti-infiammatory cytokines and the control of inflammatory cytokine production time and intensity.Breaking or disturbing the balance and process of immune response,may be lead to chronic infection or the aggravation of pathogenetic condition.These observations also suggest the regulation of anti-malarial immune responses is as important as their induction in determining the final outcome of infection.However,the regulative mechanism on the dynamic balance of protective and pathologic immune response has not yet been clarified.
CD4~+CD25~+ regulatory T Cells(Tregs) are a T cell subgroup that has immunoregulatory effects different from Th1 and Th2.Tregs are widely accepted as potent suppressors to play important roles in immunotolerance,autoimmunity,and transplantation.Tregs could suppress proliferation of many types of immunocytes including CD4~+ T cells and CD8~+ T cells and inhibit secretion of cytokines.Some data demonstrated that the disorder of immune function in host correlated with the chronically tendency of diseases.Reduction or depletion of Tregs could enhance anti-infection immunity to a variety of pathogens such as bacteria,viruses,fungi and parasites.But the study on the role of Tregs in malaria and other infectious disease has just start.
In addition,people found that the transcription factor also has an important role during the cell subsets differentiation process.T-bet and GATA3 are two kinds of transcription factors within cells,specific expression on the surface of Th1/Th2 cells respectively.T-bet regulates the development of Th1 cells,GATA3 regulates the development of Th2 cells,and thus T-bet and GATA3 are the crucial factors that Th0 transform to Th1/Th2.
In present study we investigated the relation between the different of Th1/Th2 immune responses and effects of Tregs.Therefore,using rodent model of BALB/c and DBA/2 mice infected with P.y17XL,P.cAS and P.y17XL plus P.cAS,respectively.Then we determined the number of Tregs and level of cytokines as,IFN-γ,IL-4,IL-10 and TGF-βand percentage of apoptotic CD4~+ T cells,to investigate the role and the possible immunosuppressive mechanisms of Tregs.On the basis of it,in vivo CD25 depletion was performed to further determine the role and mechanisms of Tregs in early stage of malaria infection.
Methods
1.Experimental animal and models of construction
Female 6-8-weeks-old BALB/c and DBA/2 mice were infections with 1×10~6 Py17XL,P.cAS and 2×10~6 P.y17XL plus P.cAS(1:1) parasitized erythrocytes(PE) by intraperitoneal(i.p.),Constructed different experimental animal models.
2.Determination of contents of IFN-γand IL-4 in malaria parasite infected DBA/2 and BALB/c mice by double antibody sandwich ELISA
Spleen cells were harvested from mice and adjusted to a final concentration of 10~7cells/ml.Aliquots(500μl/well) of the cell suspension were incubated in 24-well plat-bottom tissue culture plates(FALCON) in triplicate for 48 hours at 37℃in a humidified 5%CO_2 incubator.Then the 24-well plates were centrifuged at 350g for 10 min at RT,supernatants were collected and stored at-80℃until the assayed for IFN-γand IL-4 levels.
3.Detection of transcription factors T-bet and GATA3 mRNA expression level by RT-PCR
(1) PCR primer
Using primer design software Primer5 design T-bet,GATA3 andβ-actin specific primers.
(2)RNA extraction
Using TRIZOL one-step method extract to total RNA in spleen cells of mice and the UV spectrophotometer measured purity,the ratio of RNAA_(260)/A_(280) is between 1.8 and 2.0.
4.Determination by flow cytometry of the proportion of Tregs and apoptotic CD4~+ T cells in malaria parasite infected DBA/2 and BALB/c mice
Spleen cells were aliquoted into staining tubes at approximately 1×10~6 cells per tube and incubated with Mouse-block to block non-specific binding of fluorochrome-labeled antibodies.Two-color staining was then performed using FITC-labeled anti-mouse CD4 mAb and PE-labeled anti-mouse CD25 mAb simultaneously;The antibodies were diluted in FASC buffer and incubated with the cells for 30 min on ice without light.FACS analyses were performed on a FACSCalibur instrument run with the CELLQuest programme.
Spleen cells diluted with binding buffer were aliquoted into staining tubes at approximately 1×10~5 cells per tube.Three-color staining was then performed using FITC-labeled anti-mouse CD4 mAb,PE-labeled anti-mouse Annexin V and 7AAD simultaneously,and incubated with the cells for 15 min at 25℃without light.FACS analyses were performed on a FACSCalibur instrument run with the CELLQuest programme
Flow cytometric analysis utilized linear forward light scatter(FS),linear side light scatter(SS) and log fluorescence parameters.
5.Determination of the contents of IL-10 and TGF-βin malaria parasite infected DBA/2 and BALB/c mice by double antibody sandwich ELISA
All cells used were spleen cells harvested from mice and concentration of cells were adjusted to 1×10~7/ml and 500μl cells per well were plated on the plastic 24-well plates with RPMI 1640 medium supplemented with 10%heat-inactivated FCS in 95% air-5%CO_2 at 37℃for 48 hours.Supernatants were collected after centrifugation at room temperature,350g.Supernatants above were tested in duplicate using ELISA kits for IL-10 and TGF-β.
6.Intracytoplasmic staining
Spleen cells from BALB/c and DBA/2 mice were collected at different time point after infection.After stimulated for 2 hours with PMA and ionomycin at 37℃,Golgi Stop was added to each reaction(1:500,vol/vol).After continued co-culture for 4 hours at 37℃,cells were washed with 3%FCS and then resuspended in 100μl of 3%FCS. FITC-anti-CD4 and PE-anti-CD25 were added for surface staining.Then cells were fixed and permeabilized,and intracytoplasmic staining was performed using allophycocyanin(APC)-anti-IL-10.FITC rat IgG2b was used as the isotype control.
7.CD25 depletion in vivo
Depletion of Tregs in BALB/c mice was carried out respectively by i.p. administration of 1mg anti-mouse CD25 mAb(7D4) 1 day before parasite challenge and on day 1 pi.Control group was carried out by i.p.administration of PBS.Tregs depletion was confirmed by flow cytometry.Spleen cell cultures harvested from Py17XL-infected BALB/c mice on days 0,3 and 5 pi was used to analyze level of cytokines IFN-γand IL-10 and FCSA were performed to determine the change number of Tregs and apoptosic CD4~+ T cells after in vivo depletion of Tregs.
Results
1.Comparison of the concentration of IFN-γand IL-4 from spleen supernatants in malaria parasite infected DBA/2 and BALB/c mice
The level of IFN-γincreased rapidly and reached the peak on day 3 pi and then decreased slowly in P.y17XL-infected DBA/2 mice,while in BALB/c mice,the level of IFN-γonly increased significantly on day 3 pi,which was followed by a sharp reduction.In contrast,the level of IFN-γonly transient increased significantly on day 3 pi in P.cAS-infected DBA/2 mice,while in BALB/c mice,the level of IFN-γincreased significantly on day 3 pi,and peaked on day 5 pi;The level of IFN-γreached peak on day 3 pi in mixed-species infected DBA/2 and BALB/c mice.
The level of IL-4 only increased significantly on day 3 pi in P.y17XL-infected BALB/c mice,while in DBA/2 mice,the level of IL-4 increased and reached the peak on day 5-8 pi,then reduction slowly.In contrast,the level of IL-4 only transient increased significantly on day 5 pi in P.cAS-infected DBA/2 mice,while in BALB/c mice,the level of IL-4 increased and peaked on day 8-10 pi,followed decreased,but still higher than normal.There was no notable change of IL-4 on day 1-5 pi in mixed-species infected DBA/2 and BALB/c mice,and peaked on day 8 pi,then decreased down to normal.
2.Comparison of the expression and the ratio of T-bet and GATA3 from spleen cells in malaria parasite infected DBA/2 and BALB/c mice
The expression of T-bet mRNA in both P.y17XL and P.cAS infected DBA/2 mice increased and was higher than that of GATA3,the ratio of T-bet/GATA3 increased significantly in early phase of infection.In contrast,the expression of T-bet and GATA3 mRNA in both P.y17XL and P.cAS infected BALB/c mice have no obviously change on day 3-5 pi,the ratio of T-bet/GATA3 increased significantly on day 1 and 3 pi respectively.
3.Comparison of differences in proportion of Tregs between malaria parasite infected DBA/2 and BALB/c mice
The percentage of Tregs raised significantly on day 1 pi in P.y17XL-infected mice, and the percentage of Tregs increased slowly,peaked on day 5 pi and then decreased; while the percentage of Tregs increased rapidly and reached the peak on day 5 pi.In contrast,the percentage of Tregs reached the peak on day 5 pi and 10 pi in P.cAS-infected DBA/2 and BALB/c mice respectively.The percentage of Tregs increased slowly and peaked on day 5 pi,followed decreased in mixed-infected DBA/2 mice,while the percentage of Tregs rapidly raise and reached the peak on day 8 pi.
4.Comparison of differences in proportion of CD4~+ T cells between malaria parasite infected DBA/2 and BALB/c mice
The percentage of apoptotic CD4~+ T cells increased slowly in P.y17XL-infected DBA/2 mice,while the percentage of apoptotic CD4~+ T cells increased rapidly on day 3-5 pi in BALB/c mice;In contrast,the percentage of apoptotic CD4~+T cells raised significantly only on day 8 pi in P.cAS-infected BALB/c mice,while in DBA/2 mice, the percentage of apoptotic CD4~+T cells reached the peak on day 8 pi.
5.Comparison of the contents of IL-10 and TGF-βin malaria parasite infected DBA/2 and BALB/c mice
The concentration of IL-10 increased significantly and kept an elevated level from day 1 to day 5 in P.y17XL-infected DBA/2 and BALB/c mice.In contrast,the level of IL-10 reached the peak on day 8 pi and 10 pi in P.cAS-infected BALB/c and DBA/2 mice.
The level of TGF-βraised significantly on day 10 pi and 12 pi in P.y17XL -infected DBA/2 mice and then decreased down to normal;while in BALB/c mice,the level of TGF-βincreased significantly only on day 5 pi.In contrast,the level of TGF-βraised and peaked on day 8 pi in P.cAS-infected DBA/2 mice,while in BALB/c mice, the level of TGF-βtransient increased significantly on day 3 pi and then decreased down to normal on day 8 pi.
6.The change of immune response mode in BALB/c mice during the early stage of P.y17XL infection after CD25 depletion in vivo
In CD25-depleted BALB/c mice,parasitemia increased slowly and reached the low-level peak,survival rate was obviously prolonged,and even part of CD25-depleted mice survival;The level of IFN-γmarkedly elevated,while the level of IL-10 markedly decreased.
Conclusions
1.Immune response mode is different in DBA/2 and BALB/c mice during P.y17XL and P.cAS infection respectively.
2.It is essential for the establishment of effective Th1 and Th2 responses and a successful switch of immune response from Th1 to Th2 in DBA/2 and BALB/c mice during P.y17XL and P.cAS infection respectively.And phase and level of immune response might affect the final outcome of malaria infection.
3.The increase of Tregs amount in P.y17XL-infected BALB/c mice is closely correlated with failure to establishment and maintenance of Th1 response in the early stage of infection.
4.The abnormal activation of Tregs is related with failure to switch of immune response from Th1 to Th2 in P.cAS-infected DBA/2 mice.
5.The immune response mode in mixed-species infected DBA/2 and BALB/c mice is the same as that of P.y17XL-infected mice.
6.The protective immunity against mixed-species malaria infections depends on the establishment of effective Th1 and Th2 responses and a successful switch of immune response from Th1 to Th2.
7.The persistent increase of Tregs amount is correlated with failure to establishment and maintenance of Th1 response in mixed-species infected BALB/c mice,and seemed to significantly affect the final outcome of malaria infection through its regulation of the Th1 and Th2 response.
8.Tregs have a crucial role to regulate Th1 responses,potential regulatory mechanisms include IL-10-dependent manner and induce CD4~+T cells apoptosis during the early stage of P.y17XL infection.
疟疾与其他大多数感染性疾病一样,需要通过免疫效应机制对其控制和消除。同时,由于过强的炎症反应引起脑疟等免疫病理损伤也是这一疾病的突出特征。关于红内期疟原虫感染的免疫机制,无论鼠疟模型和人体试验研究结果均已证实:①CD4~+T细胞在抵抗红内期疟原虫感染过程中发挥至关重要的作用。在感染早期,Th1型细胞经DC分泌的IL-12诱导活化,产生以IFN-γ为主的炎症性细胞因子,遏制疟原虫的爆发性增殖;随之,由Th2型细胞辅助B细胞产生特异性抗体,能够有效地清除疟原虫,防止复发和再燃。由此表明:抗疟保护性免疫有赖于Th1和Th2型免疫应答的有效建立和协调过渡。②Th1/Th2型免疫应答的发生时相和效应强度明显地影响着最终的感染结局。各种数据显示,致死型脑疟等严重并发症的出现与机体过强的Th1型炎症应答密切相关。由此提示:维持前炎症细胞因子和抗炎症细胞因子之间的动态平衡,控制炎症性细胞因子产生的时间和强度对疟疾感染甚为关键。打破免疫应答之间的平衡或扰乱免疫应答的时间进程,均可能导致慢性感染或者病情加重。换言之,在决定疟疾感染最终结局方面,调控疟疾感染的免疫应答与诱导有效的抗疟免疫同样重要。然而,目前关于保护性和病理性免疫应答动态平衡的调控机制尚未阐明。
CD4~+T细胞亚类CD4~+CD25~+调节性T细胞(Tregs)是一类具有独特免疫调节功能的细胞群,体内、外试验显示,Tregs具有广泛的免疫抑制性,通过表面膜分子与其它细胞直接接触、或分泌抑制性细胞因子IL-10和TGF-β两种方式,可抑制体内天然CD4~+T和CD8~+T细胞等多种细胞的活化和增殖。目前,对Tregs的研究主要集中于自身免疫性疾病、抗移植和抗肿瘤免疫等方面,而在疟疾等感染性疾病中作用的研究才刚刚起步,特别是关于Tregs对Th应答效应和Th1/Th2极化机制方面的研究尚未深入开展。有限的研究结果显示,在感染致死型伯氏疟原虫(Plasmodium.berghei NK65)或约氏疟原虫(P.yoelii17XL)之前,应用抗CD25单克隆抗体耗竭小鼠体内Tregs,可通过延缓疟原虫的增殖速率使其致死性感染得以控制。Tregs回输实验证实,P.yoelii 17XL感染能够特异性活化Tregs,介导疟原虫逃逸C57BL/6小鼠的T细胞应答。同样人体实验也表明恶性疟原虫(P.falciparum)感染诱导Tregs分化并伴有IL-10的升高。以上结果均提示,Tregs的活化是介导恶性疟原虫和致死型啮齿类疟原虫逃逸机体免疫应答的关键。
另外,人们研究发现在细胞亚群分化过程中,转录因子也具有重要的调节作用。T-bet和GATA3是两种细胞内的转录因子,分别特异性地表达于Th1和Th2细胞。T-bet正调控Th1细胞的发育,GATA3正调控Th2细胞的发育,二者最终决定Th0向Th1/Th2的转化。由此可以看出在疟疾感染过程中T-bet和GATA3两种转录因子也参与了Th1/Th2的极化过程。
为了解不同疟原虫感染过程中Th1/Th2应答的差异是否与Tregs的免疫调节作用具有一定的相关性,本研究选用致死型约氏疟原虫(P.yoelii 17XL,P.y17XL)、夏氏疟原虫(P.chabaudi AS,P.cAS)及其约氏+夏氏疟原虫(1:1)混合感染DBA/2和BALB/c小鼠,动态观察感染过程中Th1/Th2应答的差异、Tregs的数量变化和功能特点,并且在此基础上应用抗CD25单克隆抗体阻断技术,以期阐明Tregs在疟疾感染过程中的作用地位及其相关机制。
实验方法
1、实验动物及其模型构建
6-8周龄、雌性DBA/2和BALB/c小鼠,经腹腔分别感染1×10~6 P.y17XL、P.cAS和2×10~6 P.y17XL+P.cAS(1:1)寄生的红细胞(PRBC),构建不同的实验动物模型。
2、采用双抗体夹心ELISA法检测小鼠感染疟原虫后IFN-γ和IL-4的含量
无菌取出感染小鼠脾脏,制成细胞悬液并调整脾细胞终浓度为1×10~7/ml,24孔培养板上加入细胞悬液,500μl/孔,于37℃培养48h。350g室温离心10min,收集上清,-80℃保存,待IFN-γ和IL-4检测。
用双抗体夹心ELISA法对IFN-γ和IL-4的含量进行检测,酶标仪检测450nm处OD值,应用SoftMax Pro4.3.1 LS软件分析,绘制标准品标准曲线,计算细胞因子含量(pg/ml)。
3、采用RT-PCR方法检测转录因子T-bet和GATA3 mRNA的表达水平
(1)PCR引物利用引物设计软件Primer5设计T-bet、GATA3和β-actin特异性引物。
(2)实验方法采用TRIZOL一步法提取小鼠脾细胞总RNA,并经紫外分光光度仪检测纯度,RNAA_(260)/A_(280)的比值在1.8-2.0之间;PCR扩增产物用1.5%琼脂糖凝胶电泳检测,应用ID影像分析软件进行表达强度的分析。
4、采用流式细胞分析技术检测小鼠感染疟原虫后脾Tregs及CD4~+T细胞凋亡的百分比
(1)无菌取小鼠脾脏,制成1×10~7/ml脾细胞悬液。每份样品用抗CD4-FITC单抗和抗CD25-PE单抗进行双色分析,另设阴性对照管。流式细胞仪专用染色管预先加入抗FcγⅢ/Ⅱ受体抗体2μl,取新鲜制备的1×10~7/ml脾细胞悬液0.1ml,再加入抗CD4-FITC单抗和抗CD25-PE单抗各0.2μg。振荡器低速混匀,冰浴放置,避光反应30min。每管加入2ml 2%的FCS,水平转头离心机300g,离心5min,弃上清,洗涤两次,静置15 min上机。
(2)取binding buffer制备的1×10~6/ml脾细胞悬液0.1ml,加入流式细胞仪专用染色管中,每份样品设阴性对照管,另一管分别加入抗CD4-FITC单抗0.2μg,AnnexinV-PE和7AAD各5μl。25℃避光孵育15min,每管加binding buffer 400μl,上机。
(3)流式细胞仪激发波长为488nm,利用FACS CELLQUEST软件获取细胞,每个样品分析10,000个细胞,以二维点阵图显示,记录Tregs及CD4~+T细胞凋亡的百分比。
5、采用双抗体夹心ELISA法检测小鼠感染疟原虫后脾细胞培养上清抑制性细胞因子IL-10和TGF-β的含量
无菌取小鼠脾脏,制成1×10~7/ml脾细胞悬液。24孔培养板中加入细胞悬液,500μl/孔,于37℃培养48h。350g室温离心10min,收集上清。用双抗体夹心ELISA法对TGF-β和IL-10的含量进行检测,酶标仪检测450nm处OD值。应用SoftMax Pro4.3.1 LS软件分析,绘制标准品标准曲线,计算细胞因子含量(pg/ml)。
6、采用细胞内染色方法检测小鼠感染疟原虫后分泌IL-10的Tregs数量的百分比
无菌取小鼠脾脏,制成1×10~7/ml脾细胞悬液。于流式管中加入细胞悬液,500μl/管,37℃条件下PMA和伊屋诺霉素刺激2h后加入Golgi Stop共同培养4h,3%FCS洗涤后加入FITC-anti-CD4 and PE-anti-CD25荧光抗体,4℃孵育30min,3%FCS洗涤后加入固定透膜剂,4℃孵育20min,洗涤后加入APC-anti-IL-10(JES5-16E3)荧光抗体,4℃避光孵育30min,洗涤后上机。同时用FITC rat IgG2b作为同型对照。
7、应用抗CD25单克隆抗体阻断技术,研究Tregs在约氏疟原虫感染早期相关的作用机制
BALB/c小鼠分别在感染前1d和感染后1d腹腔注射1mg的抗CD25 mAb(7D4),腹腔注射PBS小鼠作为对照组。分别于感染0d、3d和5d常规制备脾细胞悬液,流式细胞仪检测Tregs体内消除后数量及其CD4~+T细胞凋亡数量变化;收集脾细胞培养上清待Tregs体内消除后IFN-γ和IL-10水平检测。
实验结果
1、不同疟原虫感染DBA/2和BALB/c小鼠脾细胞培养上清中IFN-γ和IL-4水平的比较
P.y17XL感染的BALB/c小鼠IFN-γ水平仅在感染后第3d出现一过性有意义的升高;而DBA/2小鼠IFN-γ水平在感染后第1-3d出现有意义的升高并达峰值,随后缓慢下降,且IFN-γ产生水平明显高于相应时间点的BALB/c小鼠。P.cAS感染的DBA/2小鼠IFN-γ水平仅在感染后第3d出现有意义的升高;而BALB/c小鼠IFN-γ水平在感染后第3d出现有意义的升高,第5d达峰值,随后下降至正常水平,且IFN-γ产生水平明显高于相应时间点的DBA/2小鼠。P.y17XL+P.cAS感染BALB/c和DBA/2小鼠IFN-γ水平均于感染后第3d达峰值,随后下降,且DBA/2小鼠IFN-γ产生水平明显高于相应时间点的BALB/c小鼠。
P.y17XL感染的BALB/c小鼠IL-4水平仅在感染后第3d出现有意义的升高;而DBA/2小鼠IL-4水平在感染后第5-8d升高并达峰值,此后缓慢下降,且IL-4产生水平明显高于相应时间点的BALB/c小鼠。P.cAS感染的DBA/2小鼠IL-4水平仅在感染后第5d出现一过性有意义的升高;而BALB/c小鼠IL-4水平在感染后第8-10d升高并达峰值,此后虽有回落但仍维持其高水平,且IL-4产生水平明显高于相应时间点的DBA/2小鼠。P.y17XL+P.cAS感染DBA/2和BALB/c小鼠IL-4水平于感染后第1-5d无明显变化,均于感染后第8d显著升高达峰值,并且DBA/2小鼠的IL-4水平明显高于相应时间点的BALB/c小鼠,随后DBA/2小鼠的IL-4水平缓慢下降,于感染后第15d下降至正常水平。
2、不同疟原虫感染DBA/2和BALB/c小鼠脾细胞中转录因子T-bet和GATA3的表达水平及其比值
.Py17XL感染的DBA/2和BALB/c小鼠于感染后第1dT-bet表达量均明显升高,GATA3表达量略有升高。DBA/2小鼠T-bet和GATA3的表达量均于感染后10d达峰值,而BALB/c小鼠于感染后3-5d无明显变化。DBA/2小鼠的T-bet/GATA3比值在感染后第3d达峰值,此后缓慢下降;而BALB/c小鼠的T-bet/GATA3比值在感染后第1d略有升高,随后迅速回落,小鼠死亡。
P.cAS感染的DBA/2和BALB/c小鼠于感染后第1dT-bet表达量均明显升高,GATA3表达量略有升高。DBA/2小鼠T-bet和GATA3的表达量均于感染后5d达峰值,随后迅速回落;而BALB/c小鼠T-bet和GATA3的表达量均于感染后3-5d无明显变化,此后缓慢回落。DBA/2小鼠T-bet/GATA3比值在感染后第1-8d没有明显下降趋势,感染后第10d出现陡然下降,小鼠死亡;而BALB/c小鼠T-bet/GATA3比值在感染后第3d达峰值,于感染后第8d迅速下降至最低点,随后于感染后第12d再次达峰值,感染后第15d再次回落至正常水平。
3、不同疟原虫感染DBA/2和BALB/c小鼠脾Tregs数量的比较
P.y17XL感染小鼠于感染后第1d Tregs均出现有意义的升高。随后DBA/2小鼠Tregs缓慢上升,第5d达峰值,然后缓慢下降;而BALB/c小鼠Tregs数量迅速上升,第5d达峰值,并且Tregs数量明显高于相应时间点的DBA/2小鼠。P.cAS感染的BALB/c小鼠于感染后第1d Tregs出现有意义的升高,第5d达峰值后缓慢下降;而DBA/2小鼠的Tregs于感染后迅速持续升高,第10d Tregs细胞数量达峰值,且Tregs数量明显高于相应时间点的BALB/c小鼠。P.y17XL+P.cAS感染的DBA/2和BALB/c小鼠于感染后第1d Tregs均出现有意义的升高。随后DBA/2小鼠Tregs缓慢上升,第5d达峰值后缓慢下降;BALB/c小鼠Tregs数量迅速上升,感染后第8d达峰值,而且Tregs数量明显高于相应时间点的DBA/2小鼠。
4、不同疟原虫感染DBA/2和BALB/c小鼠脾CD4~+T细胞凋亡百分比的比较
P.y17XL感染小鼠CD4~+T细胞凋亡数量均于感染后第3d出现升高。随后DBA/2小鼠CD4~+T细胞凋亡数量增加缓慢,而BALB/c小鼠则于感染后3d-5d迅速增加。P.cAS感染的BALB/c小鼠仅于感染后第8d CD4~+T细胞凋亡数量出现升高,而DBA/2小鼠CD4~+T细胞凋亡数量于感染后第5d出现升高,至感染后第8d达峰值,且CD4~+T细胞凋亡数量明显高于相应时间点的BALB/c小鼠。
5、不同疟原虫感染DBA/2和BALB/c小鼠脾细胞培养上清抑制性细胞因子IL-10和TGF-β含量的比较
P.y17XL感染的DBA/2小鼠于感染后第5d IL-10出现升高,随后缓慢下降;而BALB/c小鼠于感染后第1-5d IL-10明显升高后达峰值,且IL-10水平明显高于相应时间点的DBA/2小鼠。P.cAS感染的BALB/c小鼠于感染后第1d IL-10就出现升高,第8d达峰值,随后缓慢下降;DBA/2小鼠IL-10水平于感染后第3d出现升高,第10d达峰值,且IL-10水平明显高于相应时间点的BALB/c小鼠。
P.y17XL感染的DBA/2小鼠于感染后第10d和12d TGF-β出现升高,随后迅速下降至正常水平;而BALB/c小鼠仅于感染后第5d TGF-β出现升高。P.cAS感染的DBA/2小鼠TGF-β水平于感染5-8d升高后达峰值;而BALB/c小鼠于感染后第3d TGF-β出现一过性升高后于第8d下降至正常水平。
6、Anti-CD25mAb体内阻断后,P.y17XL感染早期BALB/c小鼠免疫应答模式的变化
应用anti-CD25mAb体内阻断后,BALB/c小鼠的原虫血症水平上升缓慢且峰值低,生存率明显延长,甚至部分小鼠存活;IFN-γ产生水平显著升高,IL-10产生水平明显减少,且CD~(4+)T细胞凋亡数量也明显下降。
结论
1、P.y17XL和P.cAS感染过程中,小鼠DBA/2和BALB/c免疫应答存在明显差异。
2、P.y17XL和P.cAS感染过程中,Th1和Th2型免疫应答的有效建立和协调过渡对抵抗疟疾感染至关重要,同时提示Th1和Th2型免疫应答的发生时相和效应强度可能最终影响着疟疾感染结局。
3、Tregs数量的过早升高与P.y17XL感染的BALB/c小鼠Th1型免疫应答未能有效建立密切相关;
4、Tregs数量的异常活化与P.cAS感染的DBA/2小鼠未能成功的从Th1向Th2型细胞免疫应答转化密切相关;
5、P.y17XL+P.cAS混合感染过程中,DBA/2和BALB/c小鼠免疫应答模式与P.y17XL单独感染DBA/2和BALB/c小鼠的应答模式相同;
6、P.y17XL+P.cAS混合感染过程中,Th1和Th2型免疫应答的有效建立和协调过渡对抵抗疟疾感染至关重要;
7、Tregs数量的持续升高与P.y17XL+P.cAS混合感染的BALB/c小鼠Th1型免疫应答未能有效建立及感染结局密切相关。
8、P.y17XL感染早期,Tregs是通过分泌抑制性细胞因子IL-10和诱导CD4~+T细胞的凋亡来介导免疫抑制作用,参与Th1型细胞免疫应答的调节。
Malaria infection is a major health problem to human beings even today.A recent survey has shown that,in 2002,roughly 2.2 billion people were at risk of P.falciparum infection,with a conservative estimate of 515 million individuals who had been infected.The prevention and therapy of such infectious diseases as Malaria,AIDS and tuberculosis have been taken as the focus of researches by WHO.However,Up till now, the mechanisms of immune response to malaria parasite infection are not fully understood yet.Thus,the rational development of effective anti-malarial vaccines and novel therapies to alleviate or prevent the symptoms of severe malaria infection requires a better understanding of the various mechanisms of immune responses to malaria parasite.
A series of studies have proved that immune effector mechanisms are required to eliminate malaria parasites,and Th1 immune effectors are crucial to control the early infection.Experiments in vivo and in vitro have demonstrated:(1) CD4~+ T cells are essential for the control of primary parasitemia in mouse models during the blood-stage of malaria infection.IFN-γplays an important role in the production and maintenance of Th1 in early response and control the outbreak proliferation of blood-stage malaria parasites.In addition,B cells secrete specific antibodies supporting by Th2 cells,which can effectively remove parasite,preventing the recidivation and recrudescence.Thus effective establishment of Th1 immune response and a successful switch to Th2 are crucial for educing protective immunity response in malaria.Balance and coordination of Th1/Th2 can not only control the crisis of the acute phase,but also help in the ultimate elimination of malaria parasite.(2) Phase and level of immune response might affect the final outcome of malaria infection.Related studies showed the serious complications of cerebral malaria were correlated with a strong Thl response.This indicated that it is critical for malaria infection to maintain the dynamic balance of inflammatory cytokines/anti-infiammatory cytokines and the control of inflammatory cytokine production time and intensity.Breaking or disturbing the balance and process of immune response,may be lead to chronic infection or the aggravation of pathogenetic condition.These observations also suggest the regulation of anti-malarial immune responses is as important as their induction in determining the final outcome of infection.However,the regulative mechanism on the dynamic balance of protective and pathologic immune response has not yet been clarified.
CD4~+CD25~+ regulatory T Cells(Tregs) are a T cell subgroup that has immunoregulatory effects different from Th1 and Th2.Tregs are widely accepted as potent suppressors to play important roles in immunotolerance,autoimmunity,and transplantation.Tregs could suppress proliferation of many types of immunocytes including CD4~+ T cells and CD8~+ T cells and inhibit secretion of cytokines.Some data demonstrated that the disorder of immune function in host correlated with the chronically tendency of diseases.Reduction or depletion of Tregs could enhance anti-infection immunity to a variety of pathogens such as bacteria,viruses,fungi and parasites.But the study on the role of Tregs in malaria and other infectious disease has just start.
In addition,people found that the transcription factor also has an important role during the cell subsets differentiation process.T-bet and GATA3 are two kinds of transcription factors within cells,specific expression on the surface of Th1/Th2 cells respectively.T-bet regulates the development of Th1 cells,GATA3 regulates the development of Th2 cells,and thus T-bet and GATA3 are the crucial factors that Th0 transform to Th1/Th2.
In present study we investigated the relation between the different of Th1/Th2 immune responses and effects of Tregs.Therefore,using rodent model of BALB/c and DBA/2 mice infected with P.y17XL,P.cAS and P.y17XL plus P.cAS,respectively.Then we determined the number of Tregs and level of cytokines as,IFN-γ,IL-4,IL-10 and TGF-βand percentage of apoptotic CD4~+ T cells,to investigate the role and the possible immunosuppressive mechanisms of Tregs.On the basis of it,in vivo CD25 depletion was performed to further determine the role and mechanisms of Tregs in early stage of malaria infection.
Methods
1.Experimental animal and models of construction
Female 6-8-weeks-old BALB/c and DBA/2 mice were infections with 1×10~6 Py17XL,P.cAS and 2×10~6 P.y17XL plus P.cAS(1:1) parasitized erythrocytes(PE) by intraperitoneal(i.p.),Constructed different experimental animal models.
2.Determination of contents of IFN-γand IL-4 in malaria parasite infected DBA/2 and BALB/c mice by double antibody sandwich ELISA
Spleen cells were harvested from mice and adjusted to a final concentration of 10~7cells/ml.Aliquots(500μl/well) of the cell suspension were incubated in 24-well plat-bottom tissue culture plates(FALCON) in triplicate for 48 hours at 37℃in a humidified 5%CO_2 incubator.Then the 24-well plates were centrifuged at 350g for 10 min at RT,supernatants were collected and stored at-80℃until the assayed for IFN-γand IL-4 levels.
3.Detection of transcription factors T-bet and GATA3 mRNA expression level by RT-PCR
(1) PCR primer
Using primer design software Primer5 design T-bet,GATA3 andβ-actin specific primers.
(2)RNA extraction
Using TRIZOL one-step method extract to total RNA in spleen cells of mice and the UV spectrophotometer measured purity,the ratio of RNAA_(260)/A_(280) is between 1.8 and 2.0.
4.Determination by flow cytometry of the proportion of Tregs and apoptotic CD4~+ T cells in malaria parasite infected DBA/2 and BALB/c mice
Spleen cells were aliquoted into staining tubes at approximately 1×10~6 cells per tube and incubated with Mouse-block to block non-specific binding of fluorochrome-labeled antibodies.Two-color staining was then performed using FITC-labeled anti-mouse CD4 mAb and PE-labeled anti-mouse CD25 mAb simultaneously;The antibodies were diluted in FASC buffer and incubated with the cells for 30 min on ice without light.FACS analyses were performed on a FACSCalibur instrument run with the CELLQuest programme.
Spleen cells diluted with binding buffer were aliquoted into staining tubes at approximately 1×10~5 cells per tube.Three-color staining was then performed using FITC-labeled anti-mouse CD4 mAb,PE-labeled anti-mouse Annexin V and 7AAD simultaneously,and incubated with the cells for 15 min at 25℃without light.FACS analyses were performed on a FACSCalibur instrument run with the CELLQuest programme
Flow cytometric analysis utilized linear forward light scatter(FS),linear side light scatter(SS) and log fluorescence parameters.
5.Determination of the contents of IL-10 and TGF-βin malaria parasite infected DBA/2 and BALB/c mice by double antibody sandwich ELISA
All cells used were spleen cells harvested from mice and concentration of cells were adjusted to 1×10~7/ml and 500μl cells per well were plated on the plastic 24-well plates with RPMI 1640 medium supplemented with 10%heat-inactivated FCS in 95% air-5%CO_2 at 37℃for 48 hours.Supernatants were collected after centrifugation at room temperature,350g.Supernatants above were tested in duplicate using ELISA kits for IL-10 and TGF-β.
6.Intracytoplasmic staining
Spleen cells from BALB/c and DBA/2 mice were collected at different time point after infection.After stimulated for 2 hours with PMA and ionomycin at 37℃,Golgi Stop was added to each reaction(1:500,vol/vol).After continued co-culture for 4 hours at 37℃,cells were washed with 3%FCS and then resuspended in 100μl of 3%FCS. FITC-anti-CD4 and PE-anti-CD25 were added for surface staining.Then cells were fixed and permeabilized,and intracytoplasmic staining was performed using allophycocyanin(APC)-anti-IL-10.FITC rat IgG2b was used as the isotype control.
7.CD25 depletion in vivo
Depletion of Tregs in BALB/c mice was carried out respectively by i.p. administration of 1mg anti-mouse CD25 mAb(7D4) 1 day before parasite challenge and on day 1 pi.Control group was carried out by i.p.administration of PBS.Tregs depletion was confirmed by flow cytometry.Spleen cell cultures harvested from Py17XL-infected BALB/c mice on days 0,3 and 5 pi was used to analyze level of cytokines IFN-γand IL-10 and FCSA were performed to determine the change number of Tregs and apoptosic CD4~+ T cells after in vivo depletion of Tregs.
Results
1.Comparison of the concentration of IFN-γand IL-4 from spleen supernatants in malaria parasite infected DBA/2 and BALB/c mice
The level of IFN-γincreased rapidly and reached the peak on day 3 pi and then decreased slowly in P.y17XL-infected DBA/2 mice,while in BALB/c mice,the level of IFN-γonly increased significantly on day 3 pi,which was followed by a sharp reduction.In contrast,the level of IFN-γonly transient increased significantly on day 3 pi in P.cAS-infected DBA/2 mice,while in BALB/c mice,the level of IFN-γincreased significantly on day 3 pi,and peaked on day 5 pi;The level of IFN-γreached peak on day 3 pi in mixed-species infected DBA/2 and BALB/c mice.
The level of IL-4 only increased significantly on day 3 pi in P.y17XL-infected BALB/c mice,while in DBA/2 mice,the level of IL-4 increased and reached the peak on day 5-8 pi,then reduction slowly.In contrast,the level of IL-4 only transient increased significantly on day 5 pi in P.cAS-infected DBA/2 mice,while in BALB/c mice,the level of IL-4 increased and peaked on day 8-10 pi,followed decreased,but still higher than normal.There was no notable change of IL-4 on day 1-5 pi in mixed-species infected DBA/2 and BALB/c mice,and peaked on day 8 pi,then decreased down to normal.
2.Comparison of the expression and the ratio of T-bet and GATA3 from spleen cells in malaria parasite infected DBA/2 and BALB/c mice
The expression of T-bet mRNA in both P.y17XL and P.cAS infected DBA/2 mice increased and was higher than that of GATA3,the ratio of T-bet/GATA3 increased significantly in early phase of infection.In contrast,the expression of T-bet and GATA3 mRNA in both P.y17XL and P.cAS infected BALB/c mice have no obviously change on day 3-5 pi,the ratio of T-bet/GATA3 increased significantly on day 1 and 3 pi respectively.
3.Comparison of differences in proportion of Tregs between malaria parasite infected DBA/2 and BALB/c mice
The percentage of Tregs raised significantly on day 1 pi in P.y17XL-infected mice, and the percentage of Tregs increased slowly,peaked on day 5 pi and then decreased; while the percentage of Tregs increased rapidly and reached the peak on day 5 pi.In contrast,the percentage of Tregs reached the peak on day 5 pi and 10 pi in P.cAS-infected DBA/2 and BALB/c mice respectively.The percentage of Tregs increased slowly and peaked on day 5 pi,followed decreased in mixed-infected DBA/2 mice,while the percentage of Tregs rapidly raise and reached the peak on day 8 pi.
4.Comparison of differences in proportion of CD4~+ T cells between malaria parasite infected DBA/2 and BALB/c mice
The percentage of apoptotic CD4~+ T cells increased slowly in P.y17XL-infected DBA/2 mice,while the percentage of apoptotic CD4~+ T cells increased rapidly on day 3-5 pi in BALB/c mice;In contrast,the percentage of apoptotic CD4~+T cells raised significantly only on day 8 pi in P.cAS-infected BALB/c mice,while in DBA/2 mice, the percentage of apoptotic CD4~+T cells reached the peak on day 8 pi.
5.Comparison of the contents of IL-10 and TGF-βin malaria parasite infected DBA/2 and BALB/c mice
The concentration of IL-10 increased significantly and kept an elevated level from day 1 to day 5 in P.y17XL-infected DBA/2 and BALB/c mice.In contrast,the level of IL-10 reached the peak on day 8 pi and 10 pi in P.cAS-infected BALB/c and DBA/2 mice.
The level of TGF-βraised significantly on day 10 pi and 12 pi in P.y17XL -infected DBA/2 mice and then decreased down to normal;while in BALB/c mice,the level of TGF-βincreased significantly only on day 5 pi.In contrast,the level of TGF-βraised and peaked on day 8 pi in P.cAS-infected DBA/2 mice,while in BALB/c mice, the level of TGF-βtransient increased significantly on day 3 pi and then decreased down to normal on day 8 pi.
6.The change of immune response mode in BALB/c mice during the early stage of P.y17XL infection after CD25 depletion in vivo
In CD25-depleted BALB/c mice,parasitemia increased slowly and reached the low-level peak,survival rate was obviously prolonged,and even part of CD25-depleted mice survival;The level of IFN-γmarkedly elevated,while the level of IL-10 markedly decreased.
Conclusions
1.Immune response mode is different in DBA/2 and BALB/c mice during P.y17XL and P.cAS infection respectively.
2.It is essential for the establishment of effective Th1 and Th2 responses and a successful switch of immune response from Th1 to Th2 in DBA/2 and BALB/c mice during P.y17XL and P.cAS infection respectively.And phase and level of immune response might affect the final outcome of malaria infection.
3.The increase of Tregs amount in P.y17XL-infected BALB/c mice is closely correlated with failure to establishment and maintenance of Th1 response in the early stage of infection.
4.The abnormal activation of Tregs is related with failure to switch of immune response from Th1 to Th2 in P.cAS-infected DBA/2 mice.
5.The immune response mode in mixed-species infected DBA/2 and BALB/c mice is the same as that of P.y17XL-infected mice.
6.The protective immunity against mixed-species malaria infections depends on the establishment of effective Th1 and Th2 responses and a successful switch of immune response from Th1 to Th2.
7.The persistent increase of Tregs amount is correlated with failure to establishment and maintenance of Th1 response in mixed-species infected BALB/c mice,and seemed to significantly affect the final outcome of malaria infection through its regulation of the Th1 and Th2 response.
8.Tregs have a crucial role to regulate Th1 responses,potential regulatory mechanisms include IL-10-dependent manner and induce CD4~+T cells apoptosis during the early stage of P.y17XL infection.
引文
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22 Yazdani SS,Mukherjee P,Chauhan VS,et al.Immune responses to asexual blood-stages of malaria parasites.Curr Mol Med,2006; 6:187-203.
23 Taylor-Robinson AW,Phillips RS.Infective dose modulates the balance between Thl-and Th2-regulated immune responses during blood-stage malaria infection.Scand J Immunol.1998;48:527-534.
24 Sakaguchi,S.N.,M.Itoh et al.Immunologic self-tolerance maintained by activated T cells expressing IL-2 receptor a-chains(CD25).Breakdown of a single mechanism of self-tolerance causes various autoimmune diseases.J.Immunol.1995; 160:1151-1164.
25 Robertson SJ,Messer RJ,Carmody AB,et al.In Vitro Suppression of CD8+ T Cell Function by Friend Virus-Induced Regulatory T Cells.J Immunol.2006; 176:3342-3349.
26 Martin B,Banz A,Bienvenu B,et al.Suppression of CD4+T lymphocyte effector functions by CD4+CD25+ cells in vivo.J Immunol.2004; 172:3391-3398.
27 Mendez S,Reckling SK,Piccirillo CA,et al.Role for CD4(+) CD25(+) regulatory T cells in reactivation of persistent leishmaniasis and control of concomitant immunity J Exp Med.2004;200:201-210.
28 Long,T.T.,Nakazawa,S.,Onizuka,S.,et al.Influence of CD4+CD25+T cells on Plasmodium berghei NK65 infection in BALB/c mice.International journal for parasitology.2003;33:175-183.
29 Nie,C.Q.,Bernard,N.J.,Schofield,L.,et al.CD4+CD25+ regulatory T cells suppress CD4+T-cell function and inhibit the development of Plasmodium berghei-specific TH1 responses involved in cerebral malaria pathogenesis.Infection and Immunity.2007;75:2275-2282.
30 Hisaeda H,Maekawa Y,Iwakawa D,et al.Escape of malaria parasites from host immunity requires CD4~+ CD25~+regulatory T cells.Nat Med.2004; 10:29-30.
31 Gondek DC,Lu LF,Quezada SA,et al.Cutting edge:contact-mediated suppression by CD4~+CD25~+ regulatory cells involves a granzyme B-dependent,perforin- independent mechanism.J Immunol.2005; 174:1783-1786.
32 Zhao DM,Thornton AM,Dipaolo RJ,et al.Activated CD4~+CD25~+T cells selectively kill B lymphocytes.Blood.2006; 107:3925-3932.
33 Sakaguchi,S.,N.Sakaguchi,M.Itoh,et al.Immunologic self-tolerance maintained by activated T cells expressing IL-2 receptor a-chains(CD25).Breakdown of a single mechanism of self-tolerance causes various autoimmune diseases.J.Immunol.1995; 160:1151-1164.
34 Garra AO,Bieia P.Regulatory T cells and mechanisms of immune system control.Nat Med,2004; 10:801-805.
35 Couper KN,Blount DG,de Souza JB,et al.Incomplete depletion and rapid regeneration of Foxp3+ regulatory T cells following anti-CD25 treatment in malaria-infected mice.J Immunol.2007; 178:4136-46.
36 Fontenot JD,Gavin MA,Runensky AY,et al.Foxp3 programs the development and function of CD4+CD25+ regulatory T cell.Nat Immunol.2003; 4:330-336.
37 Zhang SG,Wang Jh,Stowh W,et al.TGF-beta Requires CTLA-4 Early after T Cell Activation to Induce FoxP3 and Generate Adaptive CD4+CD25+ Regulatory Cells.J Immunol.2006; 176:3321-3329.
38 Hisaeda H,Hamano S,Mitoma-Obata C,et al.Resistance of regulatory T cells to glucocorticoid-viduced TNFR family-related protein(GITR) during Plasmodium yoelii infection.Eur.J.Immunol.2005; 35:3516-3524.
39 Fontenot,J.D.,M.A.Gavin,and A.Y.Rudensky.Foxp3 programs the development and function of CD4+CD25+regulatory T cells.Nat.Immunol.2003; 4:330-336.
40 Fontenot,J.D.,J.P.Rasmussen,L.M.Williams,et al.Regulatory T cell lineage specification by the forkhead transcription factor Foxp3.Immunity.2005; 22:329-341.
41 Fontenot,J.D.,and A.Y.Rudensky.A well adapted regulatory contrivance:regulatory T cell development and the forkhead family transcription factor Foxp3.Nat.Immunol.2005; 6:331-337.
42 Hori,S.,T.Nomura,and S.Sakaguchi.Control of regulatory T cell development by the transcription factor Foxp3.Science.2003; 299:1057-1061.
43 Khattri,R.,T.Cox,S.A.Yasayko,et al.An essential role for Scurfin in CD4~+CD25~+T regulatory cells.A/at.Immunol.2003; 4:337-342.
44 Cambos,M.,B(?)langer,B.,Jacques,A.,et al.Natural regulatory(CD4~+CD25~+FOXP~+) T cells control the production of pro-inflammatory cytokines during Plasmodium chabaudi adami infection and do not contribute to immune evasion.International journal for parasitology.2007;38:229-238.
45 Couper KN,Blount DG,de Souza JB,et al.Incomplete depletion and rapid regeneration of Foxp3+ regulatory T cells following anti-CD25 treatment in malaria-infected mice.J Immunol.2007; 178:4136-4146.
46 Weidanz WP,Batchelder JM,Flaherty P,et al.Plasmodium chabaudi adami:use of the B-cell-deficient mouse to define possible mechanisms modulating parasitemia of chronic malaria.Exp Parasitol.2005; 111:97-104.
47 Li C,Sanni LA,Omer F,et al.Pathology of Plasmodium chabaudi chabaudi infection and mortality in interleukin-10-deficient mice are ameliorated by anti-tumor necrosis factor alpha and exacerbated by anti-transforming growth factor beta antibodies.Infect Immun.2003;71:4850-4856.
48 Belkaid,Y.,Rouse,B.T.Natural regulatory T cells in infectious diseases.Nature Immunology.2005; 6:353-360.
49 Xingmin Zhang,Djordje N.Koldzic,Leonid lzikson,et al.IL-10 is involved in the suppression of experimental autoimmune encephalomyelitis by CD25+CD4+ regulatory T cells.Int.Immunol.2004; 16:249-256.
50 Subash Babu,V.Kumaraswami,and Thomas B.Nutmanl Transcriptional Control of Impaired Thl Responses in PatentLymphatic Filariasis by T-Box Expressedin T Cells and Suppressor of Cytokine Signaling Genes.Infect Immun.2005; 73:3394-3401.
51 Yasmine Belkaid,Ciriaco A.Piccirillo,Susana Mendez,et al.CD4~+CD25~+regulatory T cells control Leishmania major persistence and immunity.Nature.2002; 420:502-507.
52 Brustoski,K.,U.Moller,M.Kramer,et al.Reduced cord blood immune effector-cell responsiveness mediated by CD4+ cells induced in utero as a consequence of placental Plasmodium falciparum infection.The Journal of Infectious Diseases.2006; 193:146-154.
1 Snow RW,Guerra CA,Noor AM,et al.The global distribution of clinical episodes of Plasmodium falciparum malaria.Nature,2005,434:214-217.
2 卫生部《2006-2015年全国疟疾防治规划》
3 Xu H,Hodder AN,Yan H,et al.CD4~+T cell sacting independently of antibody contribute to protective immunity to Plasmodium chabaudi infection after apical membrane antigen 1immunization.J Immunol,2000,165(1):389-396.
4 Burns JM,Flaherty PR,Nanavati P,et al.Protection against Plasmodium chabaudi malaria induced by immunization with apical membrane antigen 1 and merozoite surface protein 1 in the absence of gamma interferon or interleukin-4.Infect Immun,2004,72(10):5605-5612.
5 Wipasa J,Xu H,Makobongo M,et al.Nature and specificity of the required protective immune response that develops post challenge in mice vaccinated with the 19 kilodalton fragment of Plasmodium yoelii merozoite surface proteinl.Infect Immun,2002,70(11):6013-6020.
6 Langhorne J,Albano FR,Hensmann M,et al.Dendritic cells,proinflammatory responses,and antigen presentation in a rodent malaria infection.Immunol Rev,2004,201:35-47.
7 Urban BC,Ferguson DJ,Pain A,et al.Plasmodium falciparum infecte derythrocytes modulate the maturation of dendritic cells.Nature,1999,400(6739):73-77.
8 Urban BC,M wangi A,Ross S,et al.Peripheral blood dendritic cells in children with acute Plasmodium falciparum malaria.Blood,2001,98:2859-2861.
9 James A.Perry,Adam Rush,Randy J.Wilson,Dendritic Cells from Malaria Infected Mice Are Fully Functional APC.The Journal of Immunology,2004,172:475 -482.
10 Roberto Maldonado-L(?)pez and Muriel Moser,Dendritic cell subsets and the regulation of Th1/Th2 responses.Immunology,2001,13:275-282.
11 McCall MB,Netea MG,Hermsen CC,et al.Plasmodium falciparum Infection Causes Proinflammatory Priming of Human TLR Responses.J Immunol,2007,179(1):162-171.
12 James A.Perry,Christine S.Olver,Robert C.Burnett,et al.Cutting Edge:The Acquisition of TLR Tolerance during Malaria Infection Impacts T Cell Activationl.The Journal of Immunology,2005,174:5921-5925.
13 Pichyangkul S,Kosol Yongvanitchit,Utaiwan Kum-arb,et al.Malaria Blood Stage Parasite Activate Human Plasmacytoid Dendritic Cells and Murine Dendritic Cells through a Toll Like Receptor 9-Dependent Pathway.The Journal of Immunology,2004,172:4926-4933.
14 Mohan K,Stevenson MM.Interleukin-12 corrects severe anemia during blood stage Plasmodium chabaudiAS in susceptible A/J mice.Exp Hematol,1998,26(1):45-52
15 Luty AJ,Perkins DJ,Lell B,et al.Low interleukin-12 acibity in severe Plasmodium falciparum malaria.lnfectlmmun,2000,68:3909-3915.
16 Anggard E.Nitric oxide:mediator,murderer and medicine.Lancet,1994,343(8907):1199-1206.
17 Darrah PA,Hondalus MK,Chen Q,et al.Cooperation between reactive oxygen and nitrogen intermediates in killing of Rhodococcusequi by activated macrophages.Infect Immun,2000,68(6):3587-3593.
18 Ritter U,Moll H.Monocyte chemotactic protein-1 stimulates the killing of leishmania major by human monocytes,acts synergistically with IFN-gamma and is antagonized by IL-4.Eur J Immunol,2000,30(11):3111-3120.
19 Oosterwegel MA,Mandelbrot DA,Boyd SD,et al.The role of CTLA-4 in regulating Th2 differentiation.J Immunol,1999,163:26-34
20 Zhang SG,Wang Jh,Stowh W,et al.TGF-beta Requires CTLA-4 Early after T Cell Activation to Induce FoxP3 and Generate Adaptive CD4+CD25+Regulatory Cells.J Immunol,2006,176(6):3321-3329.
21 Hisaeda H,Hamano S,Mitoma-Obata C,et al.Resistance of regulatory T cells to glucocorticoid-viduced TNFa family-related protein(GITR) during Plasmodium yoelii infection.Eur J Immunol,2005,35:3516-3524.
22 Fontenot JD,Gavin MA,Runensky AY,et al.Foxp3 programs the development and function of CD4~+CD25~+ regulatory T cell.Nat Immunol,2003,4:330-336.
23 Mendez S,Reckling SK,Piccirillo CA,et al.Role for CD4(+) CD25(+) regulatory T cells in reactivation of persistent leishmaniasis and control of concomitant immunity.J Exp Med,2004,200(2):201-210.
24 Hisaeda H,Maekawa Y,Iwakawa D,et al.Escape of malaria parasites from host immunity requires CD4+CD25+regulatory T cells.Nat Med,2004,10(1):29-30.
25 Walther M,Tongren JE,Andrews L,et al.Upregulation of TGF-beta,FOXP3,and CD4~+CD25~+ regulatory T cells correlates with more rapid parasite growth in human malaria infection.Immunity,2005,23(3):287-296.
26 Nie CQ,Bernard NJ,Schofield L,et al.CD4~+CD25~+Regulatory T Cells Suppress CD4~+T Cell Function and Inhibit the Development of Plasmodium berghei Specific TH1 Responses Involved in Cerebral Malaria Pathogenesis.Infect Immun,2007,75(5):2275-2282.
27 Szabo SJ,Kim ST,Costa GL,et al.A novel transcription factor,T-bet,directs Thl lineage co mmitment.Cell,2000,100:655-669.
28 Afkarian M,Sedy JR,Yang J,et al.T-bet is a STAT-1 induced regulator of IL-12R expression in naive CD4+T cells.Nat Immunol,2002,3:549-557.
29 Dong C,Flavell RA.Thl and Th2 cells.Curr Opin Hematol,2001,8(1):47-51.
30 Glimcher LH,Murphy KM.Lineage commitment in the immune system:the T helper lymphocyte grows up.Gene Dev,2000,14:1693-1711.
31 Usui T,Nishikomori R,Kitani A,et al.GATA-3 suppresses Thl development by down regulation of Stat4 and not through effects on IL-12R beta-chain or T-bet.Immunity,2003,18:415-428.
32 Farrar JD,Asnagli H,Murphy KM.T helper subset development:roles of instruction,selection,and transcriptionJ Clin Invest,2002,109:431-435.
2 Taylor-Robinson AW,Phillips RS.B cells are required for the switch from Thl to Th2 regulated immune responses to Plasmodium chabaudi infection Infect Immun.1994; 62:490-498.
3 Yazdani SS,Mukherjee P,Chauhan VS,et al.Immune responses to asexual blood-stages of malaria parasites[J].Curr Mol Med.2006; 6:187-203.
4 Seixas E,Ostler D.Plasmodium chabaudi chabaudi(AS):differential cellular responses to infection in resistant and susceptible mice.Exp Parasitol.2005; 110:394-405.
5 Couper KN,Phillips RS,Brombacher F,et al.Parasite-specific IgM plays a significant role in the protective immune response to asexual erythrocytic stage Plasmodium chabaudi AS infection[J].Parasite Immunol.2005; 27:171-180.
6 Duque S,Montenegro-James S,Arevalo-Herrera M,et al.Expression of cytokine genes in Aotus monkeys immunized with synthetic and recombinant Plasmodium vivax and Plasmodium falciparum antigens.Ann Trop Med Parasitol.1998; 92:553-559.
7 Souza De,Williamson K.E,Otani T,et al.Early gamma interferon responses in lethal and nonlethal murine blood-stage malaria.Infect Immun.1997; 65:1593-1598.
8 Torre D,Speranza F,Giola M,et al.Role of Thl and Th2 cytokines in immune response to uncomplicated Plasmodium falciparum malaria.Clin Diagn Lab Immunol.2002; 9:348-351.
9 Malaguarnera L,Imbesi RM,Dignatelli S,et al.Increased levels of nterleukin-12 in Plasmodium falciparum malaria.Parasite Immunol.2002; 24:387-389.
10 Dodoo D,Omer FM,Todd J,et al.Absolute levels and ratios of proinflammatory and anti-inflammatory cytokine procuction in vitro predict clinical immunity to Plasmodium falciparum malaria.J Infect Dis.2002; 185:971-979.
11 Richardo Rosa,Henrique silveira,Elsa Seixas,et al.The effect of chloroquine on the production of interferon-y,interleukin(IL)-4,IL-6,and IL-10 in Plasmodium chabaudi chabaudi in infected C57BL6 mice.J.Parasitol.1999; 85:956-960.
12 Taylor-Robinson A W,Phillips RS,Severn A,et al.The role of Thl and Th2 cells in a rodent malaria infection[J].Science.1993; 260:1931-1934.
13 Malhotra I,Mungai P,Muchiri E,et al.Distinct Thl- and Th2-Type prenatal cytokine responses to Plasmodium falciparum erythrocyte invasion ligands.Infect Immun.2005;73:3462-3470.
14 Cheesman S,Raza A,Carter R.Mixed strain infections and strain-specific protective immunity in the rodent malaria parasite Plasmodium chabaudi chabaudi in mice.Infection and Immunity.2006; 74:2996-3001.
15 Snounou,G.,T.Bourne,W.Jarra,et al.Assessment of parasite population dynamics in mixed infections ofrodent plasmodia.Parasitology.1992; 105:363-374.
16 Tatiana Voza,Ana M.Viga'rio,Elodie Belnoue,et al.Species-Specific Inhibition of Cerebral Malaria in Mice Coinfected with Plasmodium spp.Infection and Immunity.2005;73:4777-4786.
17 Glimcher LH,Murphy KM.Lineage commitment in the immune system:the T helper lymphocyte grows up.Gene Dev,2000; 14:1693-1711.
18 Chakir H,Wang H,Lefebvre DE,et al.T-bet/GATA-3 ratio as a measure of the Thl/Th2 cytokine profile in mixed cell populations:predominant role of GATA-3.J Immunol Methods.2003;278:157-169.
19 Perkins D,Weinberg J & Kremsner P.Reduced interleukin-12 and transforming growth factor-pi in severe childhood malaria:relationship of cytokine balance with disease severity[J].J Infect Dis.2000; 182:988-992.
20 Dodoo D,Omer FM,Todd J,et al.Absolute levels and ratios of pro-inflammatory and anti-inflammatory cytokine production in vitro predict clinical immunity to P.falciparum malaria[J].J Infect Dis.2002; 185:971-979.
21 Martins GA,Hutchins AS,Reiner SL.Transcriptional Activators of Helper T Cell Fate Are Required for Establishment but Not Maintenance of Signature Cytokine Expressionl[J].The Journal of Immunology.2005; 175:5981-5985.
22 Yazdani SS,Mukherjee P,Chauhan VS,et al.Immune responses to asexual blood-stages of malaria parasites.Curr Mol Med,2006; 6:187-203.
23 Taylor-Robinson AW,Phillips RS.Infective dose modulates the balance between Thl-and Th2-regulated immune responses during blood-stage malaria infection.Scand J Immunol.1998;48:527-534.
24 Sakaguchi,S.N.,M.Itoh et al.Immunologic self-tolerance maintained by activated T cells expressing IL-2 receptor a-chains(CD25).Breakdown of a single mechanism of self-tolerance causes various autoimmune diseases.J.Immunol.1995; 160:1151-1164.
25 Robertson SJ,Messer RJ,Carmody AB,et al.In Vitro Suppression of CD8+ T Cell Function by Friend Virus-Induced Regulatory T Cells.J Immunol.2006; 176:3342-3349.
26 Martin B,Banz A,Bienvenu B,et al.Suppression of CD4+T lymphocyte effector functions by CD4+CD25+ cells in vivo.J Immunol.2004; 172:3391-3398.
27 Mendez S,Reckling SK,Piccirillo CA,et al.Role for CD4(+) CD25(+) regulatory T cells in reactivation of persistent leishmaniasis and control of concomitant immunity J Exp Med.2004;200:201-210.
28 Long,T.T.,Nakazawa,S.,Onizuka,S.,et al.Influence of CD4+CD25+T cells on Plasmodium berghei NK65 infection in BALB/c mice.International journal for parasitology.2003;33:175-183.
29 Nie,C.Q.,Bernard,N.J.,Schofield,L.,et al.CD4+CD25+ regulatory T cells suppress CD4+T-cell function and inhibit the development of Plasmodium berghei-specific TH1 responses involved in cerebral malaria pathogenesis.Infection and Immunity.2007;75:2275-2282.
30 Hisaeda H,Maekawa Y,Iwakawa D,et al.Escape of malaria parasites from host immunity requires CD4~+ CD25~+regulatory T cells.Nat Med.2004; 10:29-30.
31 Gondek DC,Lu LF,Quezada SA,et al.Cutting edge:contact-mediated suppression by CD4~+CD25~+ regulatory cells involves a granzyme B-dependent,perforin- independent mechanism.J Immunol.2005; 174:1783-1786.
32 Zhao DM,Thornton AM,Dipaolo RJ,et al.Activated CD4~+CD25~+T cells selectively kill B lymphocytes.Blood.2006; 107:3925-3932.
33 Sakaguchi,S.,N.Sakaguchi,M.Itoh,et al.Immunologic self-tolerance maintained by activated T cells expressing IL-2 receptor a-chains(CD25).Breakdown of a single mechanism of self-tolerance causes various autoimmune diseases.J.Immunol.1995; 160:1151-1164.
34 Garra AO,Bieia P.Regulatory T cells and mechanisms of immune system control.Nat Med,2004; 10:801-805.
35 Couper KN,Blount DG,de Souza JB,et al.Incomplete depletion and rapid regeneration of Foxp3+ regulatory T cells following anti-CD25 treatment in malaria-infected mice.J Immunol.2007; 178:4136-46.
36 Fontenot JD,Gavin MA,Runensky AY,et al.Foxp3 programs the development and function of CD4+CD25+ regulatory T cell.Nat Immunol.2003; 4:330-336.
37 Zhang SG,Wang Jh,Stowh W,et al.TGF-beta Requires CTLA-4 Early after T Cell Activation to Induce FoxP3 and Generate Adaptive CD4+CD25+ Regulatory Cells.J Immunol.2006; 176:3321-3329.
38 Hisaeda H,Hamano S,Mitoma-Obata C,et al.Resistance of regulatory T cells to glucocorticoid-viduced TNFR family-related protein(GITR) during Plasmodium yoelii infection.Eur.J.Immunol.2005; 35:3516-3524.
39 Fontenot,J.D.,M.A.Gavin,and A.Y.Rudensky.Foxp3 programs the development and function of CD4+CD25+regulatory T cells.Nat.Immunol.2003; 4:330-336.
40 Fontenot,J.D.,J.P.Rasmussen,L.M.Williams,et al.Regulatory T cell lineage specification by the forkhead transcription factor Foxp3.Immunity.2005; 22:329-341.
41 Fontenot,J.D.,and A.Y.Rudensky.A well adapted regulatory contrivance:regulatory T cell development and the forkhead family transcription factor Foxp3.Nat.Immunol.2005; 6:331-337.
42 Hori,S.,T.Nomura,and S.Sakaguchi.Control of regulatory T cell development by the transcription factor Foxp3.Science.2003; 299:1057-1061.
43 Khattri,R.,T.Cox,S.A.Yasayko,et al.An essential role for Scurfin in CD4~+CD25~+T regulatory cells.A/at.Immunol.2003; 4:337-342.
44 Cambos,M.,B(?)langer,B.,Jacques,A.,et al.Natural regulatory(CD4~+CD25~+FOXP~+) T cells control the production of pro-inflammatory cytokines during Plasmodium chabaudi adami infection and do not contribute to immune evasion.International journal for parasitology.2007;38:229-238.
45 Couper KN,Blount DG,de Souza JB,et al.Incomplete depletion and rapid regeneration of Foxp3+ regulatory T cells following anti-CD25 treatment in malaria-infected mice.J Immunol.2007; 178:4136-4146.
46 Weidanz WP,Batchelder JM,Flaherty P,et al.Plasmodium chabaudi adami:use of the B-cell-deficient mouse to define possible mechanisms modulating parasitemia of chronic malaria.Exp Parasitol.2005; 111:97-104.
47 Li C,Sanni LA,Omer F,et al.Pathology of Plasmodium chabaudi chabaudi infection and mortality in interleukin-10-deficient mice are ameliorated by anti-tumor necrosis factor alpha and exacerbated by anti-transforming growth factor beta antibodies.Infect Immun.2003;71:4850-4856.
48 Belkaid,Y.,Rouse,B.T.Natural regulatory T cells in infectious diseases.Nature Immunology.2005; 6:353-360.
49 Xingmin Zhang,Djordje N.Koldzic,Leonid lzikson,et al.IL-10 is involved in the suppression of experimental autoimmune encephalomyelitis by CD25+CD4+ regulatory T cells.Int.Immunol.2004; 16:249-256.
50 Subash Babu,V.Kumaraswami,and Thomas B.Nutmanl Transcriptional Control of Impaired Thl Responses in PatentLymphatic Filariasis by T-Box Expressedin T Cells and Suppressor of Cytokine Signaling Genes.Infect Immun.2005; 73:3394-3401.
51 Yasmine Belkaid,Ciriaco A.Piccirillo,Susana Mendez,et al.CD4~+CD25~+regulatory T cells control Leishmania major persistence and immunity.Nature.2002; 420:502-507.
52 Brustoski,K.,U.Moller,M.Kramer,et al.Reduced cord blood immune effector-cell responsiveness mediated by CD4+ cells induced in utero as a consequence of placental Plasmodium falciparum infection.The Journal of Infectious Diseases.2006; 193:146-154.
1 Snow RW,Guerra CA,Noor AM,et al.The global distribution of clinical episodes of Plasmodium falciparum malaria.Nature,2005,434:214-217.
2 卫生部《2006-2015年全国疟疾防治规划》
3 Xu H,Hodder AN,Yan H,et al.CD4~+T cell sacting independently of antibody contribute to protective immunity to Plasmodium chabaudi infection after apical membrane antigen 1immunization.J Immunol,2000,165(1):389-396.
4 Burns JM,Flaherty PR,Nanavati P,et al.Protection against Plasmodium chabaudi malaria induced by immunization with apical membrane antigen 1 and merozoite surface protein 1 in the absence of gamma interferon or interleukin-4.Infect Immun,2004,72(10):5605-5612.
5 Wipasa J,Xu H,Makobongo M,et al.Nature and specificity of the required protective immune response that develops post challenge in mice vaccinated with the 19 kilodalton fragment of Plasmodium yoelii merozoite surface proteinl.Infect Immun,2002,70(11):6013-6020.
6 Langhorne J,Albano FR,Hensmann M,et al.Dendritic cells,proinflammatory responses,and antigen presentation in a rodent malaria infection.Immunol Rev,2004,201:35-47.
7 Urban BC,Ferguson DJ,Pain A,et al.Plasmodium falciparum infecte derythrocytes modulate the maturation of dendritic cells.Nature,1999,400(6739):73-77.
8 Urban BC,M wangi A,Ross S,et al.Peripheral blood dendritic cells in children with acute Plasmodium falciparum malaria.Blood,2001,98:2859-2861.
9 James A.Perry,Adam Rush,Randy J.Wilson,Dendritic Cells from Malaria Infected Mice Are Fully Functional APC.The Journal of Immunology,2004,172:475 -482.
10 Roberto Maldonado-L(?)pez and Muriel Moser,Dendritic cell subsets and the regulation of Th1/Th2 responses.Immunology,2001,13:275-282.
11 McCall MB,Netea MG,Hermsen CC,et al.Plasmodium falciparum Infection Causes Proinflammatory Priming of Human TLR Responses.J Immunol,2007,179(1):162-171.
12 James A.Perry,Christine S.Olver,Robert C.Burnett,et al.Cutting Edge:The Acquisition of TLR Tolerance during Malaria Infection Impacts T Cell Activationl.The Journal of Immunology,2005,174:5921-5925.
13 Pichyangkul S,Kosol Yongvanitchit,Utaiwan Kum-arb,et al.Malaria Blood Stage Parasite Activate Human Plasmacytoid Dendritic Cells and Murine Dendritic Cells through a Toll Like Receptor 9-Dependent Pathway.The Journal of Immunology,2004,172:4926-4933.
14 Mohan K,Stevenson MM.Interleukin-12 corrects severe anemia during blood stage Plasmodium chabaudiAS in susceptible A/J mice.Exp Hematol,1998,26(1):45-52
15 Luty AJ,Perkins DJ,Lell B,et al.Low interleukin-12 acibity in severe Plasmodium falciparum malaria.lnfectlmmun,2000,68:3909-3915.
16 Anggard E.Nitric oxide:mediator,murderer and medicine.Lancet,1994,343(8907):1199-1206.
17 Darrah PA,Hondalus MK,Chen Q,et al.Cooperation between reactive oxygen and nitrogen intermediates in killing of Rhodococcusequi by activated macrophages.Infect Immun,2000,68(6):3587-3593.
18 Ritter U,Moll H.Monocyte chemotactic protein-1 stimulates the killing of leishmania major by human monocytes,acts synergistically with IFN-gamma and is antagonized by IL-4.Eur J Immunol,2000,30(11):3111-3120.
19 Oosterwegel MA,Mandelbrot DA,Boyd SD,et al.The role of CTLA-4 in regulating Th2 differentiation.J Immunol,1999,163:26-34
20 Zhang SG,Wang Jh,Stowh W,et al.TGF-beta Requires CTLA-4 Early after T Cell Activation to Induce FoxP3 and Generate Adaptive CD4+CD25+Regulatory Cells.J Immunol,2006,176(6):3321-3329.
21 Hisaeda H,Hamano S,Mitoma-Obata C,et al.Resistance of regulatory T cells to glucocorticoid-viduced TNFa family-related protein(GITR) during Plasmodium yoelii infection.Eur J Immunol,2005,35:3516-3524.
22 Fontenot JD,Gavin MA,Runensky AY,et al.Foxp3 programs the development and function of CD4~+CD25~+ regulatory T cell.Nat Immunol,2003,4:330-336.
23 Mendez S,Reckling SK,Piccirillo CA,et al.Role for CD4(+) CD25(+) regulatory T cells in reactivation of persistent leishmaniasis and control of concomitant immunity.J Exp Med,2004,200(2):201-210.
24 Hisaeda H,Maekawa Y,Iwakawa D,et al.Escape of malaria parasites from host immunity requires CD4+CD25+regulatory T cells.Nat Med,2004,10(1):29-30.
25 Walther M,Tongren JE,Andrews L,et al.Upregulation of TGF-beta,FOXP3,and CD4~+CD25~+ regulatory T cells correlates with more rapid parasite growth in human malaria infection.Immunity,2005,23(3):287-296.
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