日本血吸虫抗原诱导巨噬细胞极化的分子证据
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摘要
血吸虫病是一种严重危害人类健康的人兽共患寄生虫病，在世界范围内广泛分布，全球七十四个流行国家中至今仍有约七亿人口受到血吸虫病威胁，两亿人口受到血吸虫病感染，其中有两千万人有较重的临床症状并伴有不同程度的劳动力丧失，且每年至少还有两万人死于血吸虫病。在我国流行的是日本血吸虫病，虽然经过半个多世纪的努力，血吸虫病的流行程度已大大减低。但是，至2010年底，在全国七个流行省份的92个县中仍有三十余万的血吸虫病患者。因此，血吸虫病的防治和研究工作仍然面临着严峻的挑战。
作为一种危害严重的多细胞寄生蠕虫，日本血吸虫侵入宿主机体后可引发复杂的免疫应答。随着对日本血吸虫免疫学研究的不断深入，人类正在一层层揭开日本血吸虫与其适宜宿主人类之间关系的面纱。大量研究已归纳出日本血吸虫感染后宿主免疫应答的特征，即感染后最初的2-4周，宿主在移行中的童虫刺激下，呈现Th1型(IFN-g，TNF)优势应答；一旦成虫开始产卵，Th1型应答迅速下降，并持续呈现出Th2型（IL-4，IL-13，IL-10，IL-5）优势应答。可见虫卵抗原产生前后，宿主体内的细胞因子微环境也因此发生了变化。但到目前为止，对这一免疫现象背后更为深入的免疫机制还未被完全阐明。由此，继续探索这一免疫应答及其调节机制，从而通过人工调节，使免疫应答向着有利于人体的方向发展就显得极为重要。
巨噬细胞在人类免疫系统中占有独特的位置，其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。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角色的不同细胞亚群。在不同微环境中，巨噬细胞主要分为两种类型：M1型，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； M2型，即替代性活化的巨噬细胞(alternatively activatedmacrophage)。其可在M-CSF、 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。根据固有免疫参与并调控适应性免疫应答的启动，和影响特异性免疫应答的强度与类型，以及抗原刺激启动和调节适应性免疫应答的关键在于Mφ和DC等固有免疫细胞作用的认知，研究在血吸虫感染中固有免疫细胞与抗原相互作用可能发挥的免疫调节影响应是理所当然的关注重点。鉴于Mφ是血吸虫虫卵肉芽肿炎症中的主要细胞成分，而血吸虫感染全过程的宿主免疫应答经历了由I型（Th1）向II型（Th2）的漂移，漂移的结点正好是以成虫产卵为界，提示了Mφ在此过程中可能发挥重要的免疫调节功能并推论：通过成虫产卵前后抗原微环境的剧烈改变，从而诱导Mφ极化分型来指导和/或调节适应性免疫应答类型。本研究以这一设想作为我们关注的重点，通过体内体外实验证明血吸虫抗原刺激和巨噬细胞极化的相互关系，并探明Mφ极化分型的分子证据。
本研究实验设计的技术路线为：在体外使用经典的M1诱导剂IFN-γ/LPS和M2诱导剂IL-4诱导处于M0状态的小鼠巨噬细胞瘤系RAW264.7，建立以RAW264.7为试验用细胞的巨噬细胞极化属性识别系统，为了鉴定系统建立是否成功，分别对已报道的多个Mφ极化表型指标进行了检测；为了考察日本血吸虫生活史不同发育阶段来源抗原诱导下巨噬细胞的极化情况，在体外，使用不同的日本血吸虫抗原（正常尾蚴抗原NCA,辐照尾蚴抗原ACA,可溶性成虫抗原SWAP,可溶性虫卵抗原SEA）刺激RAW264.7，在体内，获得日本血吸虫自然感染不同阶段来源的腹腔巨噬细胞(感染前0w，感染后3w，6w，9w)，分别从精氨酸代谢途径，表面标志和细胞因子分泌三个方面对巨噬细胞极化类型进行了鉴定；为了证明不同极化状态下的巨噬细胞参与了血吸虫感染的免疫调节，运用ELISA方法分别从蛋白水平检测不同来源日本血吸虫抗原作用下巨噬细胞效应/抑制分子的表达；为了探明Mφ极化分型跟TLRs及相应通路的联系，采用抗体封闭实验考查TLR2和TLR4在巨噬细胞极化中的作用；此外，还运用流式细胞术检测M1/M2极化状态下，共刺激分子和共抑制分子的表达。主要研究结果如下：
1.在本实验室条件下，成功建立了有效的Mφ极化分型的识别系统。 M1诱导剂IFN-γ/LPS和M2诱导剂IL-4可诱导处于M0状态的小鼠巨噬细胞瘤RAW264.7，分别极化为M1和M2。
2.日本血吸虫不同发育阶段来源抗原可诱导Mφ向不同方向极化。日本血吸虫正常尾蚴抗原NCA和辐照尾蚴抗原ACA都可刺激RAW264.7细胞相对于未刺激组表达高水平的inos，CD16/32也呈现出高表达，IL-12产生显著增加(p<0.01)，但NCA和ACA间无差异；日本血吸虫虫卵抗原SEA刺激RAW264.7细胞相对于未刺激组,IL-10产生显著增加, CD206显示了高表达，表达高水平的arg1。结果表明NCA和ACA可诱导Mφ向M1极化，SEA可诱导Mφ向M2极化，SWAP中因为成分复杂（含有SEA）可同时诱导M1和M2增加。
3.日本血吸虫自然感染过程中存在Mφ极化漂移的现象。即随着感染发展的进程，M1细胞表面标志的CD16/32表达在3周达到最高，从6周开始逐渐降低；M2细胞的表面标志CD206的表达从3周开始逐渐升高，在第9周达到最高。但在3-9周，我们还观察到腹腔巨噬细胞根据F4/80荧光强度不同分为两群，因为我们未能找到合适的技术手段分离这两群巨噬细胞，并分别鉴定这两群细胞的极化属性，于是我们继续通过检测细胞因子IL-12、IL-10分泌和相关因子iNOS、Arg-1基因的表达来鉴定全部巨噬细胞的优势极化属性。结果表明，产卵前腹腔巨噬细胞IL-12的基础分泌量最高，之后表达逐渐减少，而IL-10的基础分泌量在6w后开始显著增加；inos的表达在3w达到最高，arg1在产卵后表达显著升高。证明了产卵前巨噬细胞以M1方向极化为主，产卵后趋于M2方向。
4.巨噬细胞极化后通过分泌细胞因子参与调节日本血吸虫感染过程中的免疫应答。巨噬细胞分泌的正向效应分子（TNF-α，IL-12）在产卵前占优势，而负向调节分子（IL-10，TGF-β）的分泌在产卵后占优势。
5. TLR4信号通路的存在会对巨噬细胞的极化产生影响。TLR2被拮抗后对CD16/32和CD206的表达，细胞因子IL-10和IL-12的分泌，iNOS和Arg-1的表达都没有显著性影响。而TLR4被拮抗后，NCA刺激Mφ后CD16/32和iNOS的表达与对照组相比显著降低（P<0.01），提示TLR4信号通路的存在对巨噬细胞向M1的极化有显著的影响。而SEA刺激的CD206，IL-10，Arg-1的表达与对照组相比表达变化没有显著性，原因待深入探索，可能与拮抗剂的剂量依赖性有关。
6.共刺激分子CD40，CD86和共抑制分子PD-L1在不同发育阶段来源的抗原刺激下表达均上调，故未发现正向和负向调节的共刺激分子可直接对应调节巨噬细胞极化属性的证据。但NCA刺激巨噬细胞表达的PD-L1显著高于SEA和SWA，NCA刺激巨噬细胞表达的CD86也显著高于SEA和SWAP，提示如果共刺激分子参与巨噬细胞的极化的话，并不单一地依赖正向或负向调节的共刺激分子的作用，而可能取决于我们尚未认识的正向或负向信号间微妙的平衡调节。关于共刺激信号参与巨噬细胞极化调节及调节机制，在本研究中还只是停留在对实验观察结果的直观描述和推论上，但却给我们留下了丰富的想像空间。
综上所述，本研究明确了抗原微环境的变化确实是巨噬细胞极化的关键。不同日本血吸虫抗原可以刺激巨噬细胞向不同类型极化，从而参与对日本血吸虫感染引起的机体免疫应答变化的调控，如对细胞免疫方面由Th1向Th2方向的极化有所贡献。这为未来从巨噬细胞极化属性调节入手，寻找可供发展新型免疫抑制剂的虫源性成分提供先导性实验论据。
Schistosomiasis is the2nd most common parasitic disease in the world, withalmost700million individuals at risk, about200million people infected and over halfof whom having various degrees of morbidity, which leads to a burden that might beas high as20million disability-adjusted life-years and results in more than20thousand deaths annually. In China, though great progress has been made incontrolling schistosomiasis japonica over the past50years, there still are more than300thousand cases of schistosomiasis reported in the seven endemic provinces by theend of2010. Therefore, prevention and control of schistosomiasis is still greatchallenges in China.
S. japonicum causes serious damages of host organs, mainly by inducingimmune responses. Researchers are gradually uncovering the complex interplaybetween this helminth and its appropriate hosts including human. It has been reportedthat in the early stages of the infection, host immune responses are Th1-dominant andthen shifted to Th2-type associated with a series of immune suppressions after eggproduction by adult worms. But so far, the mechanism underlying this immunephenomenon has not been fully elucidated. In this respect, one of major concerns isthe regulatory role of macrophage plying in the immune responses induced by theinfection of the parasite, about which very little has so far been known.
It has been found that macrophage can differentiate into different subtypes in thedifferent micro-environments. There are two major subtypes of macrophage: M1andM2. M1is classically activated macrophages that can directly phagocytose anddestroy pathogens and malignant cells. It can also activate adaptive immuneresponses by secretion of proinflammatory cytokines and chemokines and acting asprofessional antigen-presenting cell. M2is alternatively activated macropahgesinduced by M-CSF, IL-4,-13,-10, glucocorticoid, TGF-beta, vitamin D3, PGE2, etc.M2has low antigen presentation capacity and can downregulate adapative immuneresponses by secretion of IL-10, CCL17, CCL18and TGF-beta. Obiviously, M1andM2can give rise to positive or negative regulatory signals respectively to the immuneresponses and it was important to determine whether the polarization of macrophagemay be linked to the change of antigen micro-environments before and after eggproduction during the infection of schistosome and then regulate the development ofadaptive immune responses, including Th polarization shift. In this study, we try tofind the molecular evidence of Mφ polarization associated with the change of antigenmicro-environments before and after egg production during the infection ofschistosome by use of experiments in vitro (macrophage cell line RAW264.7) and invivo (experimental infection of mice with S. japonicum).
The overall experimental design is：in vitro to stimulate RAW264.7(M0) withclassic M1inducer IFN-γ and M2inducer IL-4and then identify polarization propertyof RAW264.7with surface marks by FACS, cytokine secretion by ELISA and geneexpression by RT-PCR, to establish a Mφ polarization recognition system；Next, invitro to stimulate RAW264.7with S. japonicum antigens derived from differentdevelopmental stages，in vivo to obtain mouse peritoneal macrophages at differentstages of S. japonicum infection, then identify polarization property of these antigenstimulated macrophages and peritoneal macrophages through the arginine metabolicpathway, surface markers and cytokine secretion, to prove that antigens derived fromdifferent developmental stages of S. japonicum can induce macrophages to polarizeinto different subtypes; Then use ELISA to investigate the expression of effectory/inhibitory molecules of mouse peritoneal macrophages taken from different stages of S. japonicum infection; through TLRs blocking experiments, to explore therelationship of TLR4signal pathway and macrophage polarization; Finally, after S.japonicum antigens stimulation, the co-stimulatory molecules CD40, CD86andco-suppression molecular PD-L1expression of M1and M2will be detected.
The main results are as follows:
1. Under our laboratory conditions, we established a macrophage polarizationrecognition system successfully. M1inducer IFN-γ and M2inducer IL-4canstimulate RAW264.7to M1/M2polarization.
2. Antigens derived from different developmental stages of S. japonicuminduced Mφ polarization. CD16/32, IL-12and inos expression increasedsignificantly in RAW264.7following NCA and ACA stimulation compared withcontrol group(p<0.01), but there were no significant difference between NCA andACA stimulated groups. CD206, IL-10, arg1increased significantly (p<0.01) inRAW264.7following SEA stimulation compared with control group. These resultsshowed that ACA and NCA could induce M1polarization, SEA could induce M2polarization, SWAP could induce both M1and M2because of its complicatedingredients (contaminated with SEA).
3. Mφ polarization existed during the process of schistosome infection. Thehighest expression of M1surface marker CD16/32appeared at3weeks after infection,gradually reduced from6weeks after infection(p<0.01); M2surface marker CD206expression increased at3weeks after infection, reaching to the highest level at9weeks after infection. Interestingly, during3to9weeks after infection, we observedthat the peritoneal macrophage divided into two groups according to the F4/80fluorescent intensity. Because we could not find the right technology to separate thesetwo groups of macrophage, and identify the polarization properties of each group ofcells, we detected IL-12, IL-10secretion, inos, arg1mRNA expression to identifypolarization properties. The results showed that high level of IL-12was secretedbefore eggs laid, then gradually reduced after egg production, while IL-10secretion significantly increased at6weeks after infection. inos mRNA expression reached tothe highest level at3weeks after infection，arg1mRNA expression significantlyincreased after egg production. These results showed that M1polarization dominatedbefore egg production while M2dominated after egg production.
4. Macrophages polarization regulated immune responses through secretingcytokines after S. japonicum infection. Macrophages secreted cytokines of positiveeffect (TNF alpha, IL-12) in dominant before egg production, but molecules ofnegative effect (IL-10, TGF-beta) after egg production.
5. TLR4signal pathway may have effect on the macrophages polarization.When TLR2pathway was blocked, there were no significant influence in CD16/32，CD206expression, cytokines IL-10，IL-12secretion and inos，arg1expression. ButWhen TLR4pathway was blocked, NCA stimulated Mφ expressed lower level ofCD16/32and inos compared to the control group (P <0.01), prompting that TLR4signal pathway effect on the M1polarization. The expression of CD206, IL-10, arg1of macrophages after SEA stimulation have no significant changes compared to thecontrol group, the reason needs further exploration.
6. The modulation of balance between co-stimulatory molecule CD40, CD86and co-suppression molecule PD-L1might regulate macrophage polarization.Both INF-γ and IL-4could stimulate macrophages to express PD-L1, CD40andCD86; INF-γ stimulated macrophages to express significantly higher level of PD-L1,CD40and CD86than IL-4did(P <0.01); NCA stimulated macrophages expresssignificantly higher level of of PD-L1and CD86than SEA and SWAP stimulatedmacrophages; On the role of costimulatory signals involved in macrophagepolarization and regulation, in the present study, is only stay on the experimentalresults of intuitionistic description and inference and there are no direct evidence toprove the linkage of positive or negative co-stimulatory signals to the macrophagepolarization properties of regulation, which may take place at an unknown level involving the modulation of balance between co-stimulatory molecule andco-suppression molecule.
In summary, this study explored the relationship of S. japonicum antigens andformation of macrophage polarization and the impact of antigen environment changeduring schistosome infection on macrophage polarization modulation, for future frommacrophages polarization properties of regulation, looking for development of newimmunosuppressive agents based on the parasite components provide pilotexperimental arguments.
作为一种危害严重的多细胞寄生蠕虫，日本血吸虫侵入宿主机体后可引发复杂的免疫应答。随着对日本血吸虫免疫学研究的不断深入，人类正在一层层揭开日本血吸虫与其适宜宿主人类之间关系的面纱。大量研究已归纳出日本血吸虫感染后宿主免疫应答的特征，即感染后最初的2-4周，宿主在移行中的童虫刺激下，呈现Th1型(IFN-g，TNF)优势应答；一旦成虫开始产卵，Th1型应答迅速下降，并持续呈现出Th2型（IL-4，IL-13，IL-10，IL-5）优势应答。可见虫卵抗原产生前后，宿主体内的细胞因子微环境也因此发生了变化。但到目前为止，对这一免疫现象背后更为深入的免疫机制还未被完全阐明。由此，继续探索这一免疫应答及其调节机制，从而通过人工调节，使免疫应答向着有利于人体的方向发展就显得极为重要。
巨噬细胞在人类免疫系统中占有独特的位置，其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。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角色的不同细胞亚群。在不同微环境中，巨噬细胞主要分为两种类型：M1型，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； M2型，即替代性活化的巨噬细胞(alternatively activatedmacrophage)。其可在M-CSF、 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。根据固有免疫参与并调控适应性免疫应答的启动，和影响特异性免疫应答的强度与类型，以及抗原刺激启动和调节适应性免疫应答的关键在于Mφ和DC等固有免疫细胞作用的认知，研究在血吸虫感染中固有免疫细胞与抗原相互作用可能发挥的免疫调节影响应是理所当然的关注重点。鉴于Mφ是血吸虫虫卵肉芽肿炎症中的主要细胞成分，而血吸虫感染全过程的宿主免疫应答经历了由I型（Th1）向II型（Th2）的漂移，漂移的结点正好是以成虫产卵为界，提示了Mφ在此过程中可能发挥重要的免疫调节功能并推论：通过成虫产卵前后抗原微环境的剧烈改变，从而诱导Mφ极化分型来指导和/或调节适应性免疫应答类型。本研究以这一设想作为我们关注的重点，通过体内体外实验证明血吸虫抗原刺激和巨噬细胞极化的相互关系，并探明Mφ极化分型的分子证据。
本研究实验设计的技术路线为：在体外使用经典的M1诱导剂IFN-γ/LPS和M2诱导剂IL-4诱导处于M0状态的小鼠巨噬细胞瘤系RAW264.7，建立以RAW264.7为试验用细胞的巨噬细胞极化属性识别系统，为了鉴定系统建立是否成功，分别对已报道的多个Mφ极化表型指标进行了检测；为了考察日本血吸虫生活史不同发育阶段来源抗原诱导下巨噬细胞的极化情况，在体外，使用不同的日本血吸虫抗原（正常尾蚴抗原NCA,辐照尾蚴抗原ACA,可溶性成虫抗原SWAP,可溶性虫卵抗原SEA）刺激RAW264.7，在体内，获得日本血吸虫自然感染不同阶段来源的腹腔巨噬细胞(感染前0w，感染后3w，6w，9w)，分别从精氨酸代谢途径，表面标志和细胞因子分泌三个方面对巨噬细胞极化类型进行了鉴定；为了证明不同极化状态下的巨噬细胞参与了血吸虫感染的免疫调节，运用ELISA方法分别从蛋白水平检测不同来源日本血吸虫抗原作用下巨噬细胞效应/抑制分子的表达；为了探明Mφ极化分型跟TLRs及相应通路的联系，采用抗体封闭实验考查TLR2和TLR4在巨噬细胞极化中的作用；此外，还运用流式细胞术检测M1/M2极化状态下，共刺激分子和共抑制分子的表达。主要研究结果如下：
1.在本实验室条件下，成功建立了有效的Mφ极化分型的识别系统。 M1诱导剂IFN-γ/LPS和M2诱导剂IL-4可诱导处于M0状态的小鼠巨噬细胞瘤RAW264.7，分别极化为M1和M2。
2.日本血吸虫不同发育阶段来源抗原可诱导Mφ向不同方向极化。日本血吸虫正常尾蚴抗原NCA和辐照尾蚴抗原ACA都可刺激RAW264.7细胞相对于未刺激组表达高水平的inos，CD16/32也呈现出高表达，IL-12产生显著增加(p<0.01)，但NCA和ACA间无差异；日本血吸虫虫卵抗原SEA刺激RAW264.7细胞相对于未刺激组,IL-10产生显著增加, CD206显示了高表达，表达高水平的arg1。结果表明NCA和ACA可诱导Mφ向M1极化，SEA可诱导Mφ向M2极化，SWAP中因为成分复杂（含有SEA）可同时诱导M1和M2增加。
3.日本血吸虫自然感染过程中存在Mφ极化漂移的现象。即随着感染发展的进程，M1细胞表面标志的CD16/32表达在3周达到最高，从6周开始逐渐降低；M2细胞的表面标志CD206的表达从3周开始逐渐升高，在第9周达到最高。但在3-9周，我们还观察到腹腔巨噬细胞根据F4/80荧光强度不同分为两群，因为我们未能找到合适的技术手段分离这两群巨噬细胞，并分别鉴定这两群细胞的极化属性，于是我们继续通过检测细胞因子IL-12、IL-10分泌和相关因子iNOS、Arg-1基因的表达来鉴定全部巨噬细胞的优势极化属性。结果表明，产卵前腹腔巨噬细胞IL-12的基础分泌量最高，之后表达逐渐减少，而IL-10的基础分泌量在6w后开始显著增加；inos的表达在3w达到最高，arg1在产卵后表达显著升高。证明了产卵前巨噬细胞以M1方向极化为主，产卵后趋于M2方向。
4.巨噬细胞极化后通过分泌细胞因子参与调节日本血吸虫感染过程中的免疫应答。巨噬细胞分泌的正向效应分子（TNF-α，IL-12）在产卵前占优势，而负向调节分子（IL-10，TGF-β）的分泌在产卵后占优势。
5. TLR4信号通路的存在会对巨噬细胞的极化产生影响。TLR2被拮抗后对CD16/32和CD206的表达，细胞因子IL-10和IL-12的分泌，iNOS和Arg-1的表达都没有显著性影响。而TLR4被拮抗后，NCA刺激Mφ后CD16/32和iNOS的表达与对照组相比显著降低（P<0.01），提示TLR4信号通路的存在对巨噬细胞向M1的极化有显著的影响。而SEA刺激的CD206，IL-10，Arg-1的表达与对照组相比表达变化没有显著性，原因待深入探索，可能与拮抗剂的剂量依赖性有关。
6.共刺激分子CD40，CD86和共抑制分子PD-L1在不同发育阶段来源的抗原刺激下表达均上调，故未发现正向和负向调节的共刺激分子可直接对应调节巨噬细胞极化属性的证据。但NCA刺激巨噬细胞表达的PD-L1显著高于SEA和SWA，NCA刺激巨噬细胞表达的CD86也显著高于SEA和SWAP，提示如果共刺激分子参与巨噬细胞的极化的话，并不单一地依赖正向或负向调节的共刺激分子的作用，而可能取决于我们尚未认识的正向或负向信号间微妙的平衡调节。关于共刺激信号参与巨噬细胞极化调节及调节机制，在本研究中还只是停留在对实验观察结果的直观描述和推论上，但却给我们留下了丰富的想像空间。
综上所述，本研究明确了抗原微环境的变化确实是巨噬细胞极化的关键。不同日本血吸虫抗原可以刺激巨噬细胞向不同类型极化，从而参与对日本血吸虫感染引起的机体免疫应答变化的调控，如对细胞免疫方面由Th1向Th2方向的极化有所贡献。这为未来从巨噬细胞极化属性调节入手，寻找可供发展新型免疫抑制剂的虫源性成分提供先导性实验论据。
Schistosomiasis is the2nd most common parasitic disease in the world, withalmost700million individuals at risk, about200million people infected and over halfof whom having various degrees of morbidity, which leads to a burden that might beas high as20million disability-adjusted life-years and results in more than20thousand deaths annually. In China, though great progress has been made incontrolling schistosomiasis japonica over the past50years, there still are more than300thousand cases of schistosomiasis reported in the seven endemic provinces by theend of2010. Therefore, prevention and control of schistosomiasis is still greatchallenges in China.
S. japonicum causes serious damages of host organs, mainly by inducingimmune responses. Researchers are gradually uncovering the complex interplaybetween this helminth and its appropriate hosts including human. It has been reportedthat in the early stages of the infection, host immune responses are Th1-dominant andthen shifted to Th2-type associated with a series of immune suppressions after eggproduction by adult worms. But so far, the mechanism underlying this immunephenomenon has not been fully elucidated. In this respect, one of major concerns isthe regulatory role of macrophage plying in the immune responses induced by theinfection of the parasite, about which very little has so far been known.
It has been found that macrophage can differentiate into different subtypes in thedifferent micro-environments. There are two major subtypes of macrophage: M1andM2. M1is classically activated macrophages that can directly phagocytose anddestroy pathogens and malignant cells. It can also activate adaptive immuneresponses by secretion of proinflammatory cytokines and chemokines and acting asprofessional antigen-presenting cell. M2is alternatively activated macropahgesinduced by M-CSF, IL-4,-13,-10, glucocorticoid, TGF-beta, vitamin D3, PGE2, etc.M2has low antigen presentation capacity and can downregulate adapative immuneresponses by secretion of IL-10, CCL17, CCL18and TGF-beta. Obiviously, M1andM2can give rise to positive or negative regulatory signals respectively to the immuneresponses and it was important to determine whether the polarization of macrophagemay be linked to the change of antigen micro-environments before and after eggproduction during the infection of schistosome and then regulate the development ofadaptive immune responses, including Th polarization shift. In this study, we try tofind the molecular evidence of Mφ polarization associated with the change of antigenmicro-environments before and after egg production during the infection ofschistosome by use of experiments in vitro (macrophage cell line RAW264.7) and invivo (experimental infection of mice with S. japonicum).
The overall experimental design is：in vitro to stimulate RAW264.7(M0) withclassic M1inducer IFN-γ and M2inducer IL-4and then identify polarization propertyof RAW264.7with surface marks by FACS, cytokine secretion by ELISA and geneexpression by RT-PCR, to establish a Mφ polarization recognition system；Next, invitro to stimulate RAW264.7with S. japonicum antigens derived from differentdevelopmental stages，in vivo to obtain mouse peritoneal macrophages at differentstages of S. japonicum infection, then identify polarization property of these antigenstimulated macrophages and peritoneal macrophages through the arginine metabolicpathway, surface markers and cytokine secretion, to prove that antigens derived fromdifferent developmental stages of S. japonicum can induce macrophages to polarizeinto different subtypes; Then use ELISA to investigate the expression of effectory/inhibitory molecules of mouse peritoneal macrophages taken from different stages of S. japonicum infection; through TLRs blocking experiments, to explore therelationship of TLR4signal pathway and macrophage polarization; Finally, after S.japonicum antigens stimulation, the co-stimulatory molecules CD40, CD86andco-suppression molecular PD-L1expression of M1and M2will be detected.
The main results are as follows:
1. Under our laboratory conditions, we established a macrophage polarizationrecognition system successfully. M1inducer IFN-γ and M2inducer IL-4canstimulate RAW264.7to M1/M2polarization.
2. Antigens derived from different developmental stages of S. japonicuminduced Mφ polarization. CD16/32, IL-12and inos expression increasedsignificantly in RAW264.7following NCA and ACA stimulation compared withcontrol group(p<0.01), but there were no significant difference between NCA andACA stimulated groups. CD206, IL-10, arg1increased significantly (p<0.01) inRAW264.7following SEA stimulation compared with control group. These resultsshowed that ACA and NCA could induce M1polarization, SEA could induce M2polarization, SWAP could induce both M1and M2because of its complicatedingredients (contaminated with SEA).
3. Mφ polarization existed during the process of schistosome infection. Thehighest expression of M1surface marker CD16/32appeared at3weeks after infection,gradually reduced from6weeks after infection(p<0.01); M2surface marker CD206expression increased at3weeks after infection, reaching to the highest level at9weeks after infection. Interestingly, during3to9weeks after infection, we observedthat the peritoneal macrophage divided into two groups according to the F4/80fluorescent intensity. Because we could not find the right technology to separate thesetwo groups of macrophage, and identify the polarization properties of each group ofcells, we detected IL-12, IL-10secretion, inos, arg1mRNA expression to identifypolarization properties. The results showed that high level of IL-12was secretedbefore eggs laid, then gradually reduced after egg production, while IL-10secretion significantly increased at6weeks after infection. inos mRNA expression reached tothe highest level at3weeks after infection，arg1mRNA expression significantlyincreased after egg production. These results showed that M1polarization dominatedbefore egg production while M2dominated after egg production.
4. Macrophages polarization regulated immune responses through secretingcytokines after S. japonicum infection. Macrophages secreted cytokines of positiveeffect (TNF alpha, IL-12) in dominant before egg production, but molecules ofnegative effect (IL-10, TGF-beta) after egg production.
5. TLR4signal pathway may have effect on the macrophages polarization.When TLR2pathway was blocked, there were no significant influence in CD16/32，CD206expression, cytokines IL-10，IL-12secretion and inos，arg1expression. ButWhen TLR4pathway was blocked, NCA stimulated Mφ expressed lower level ofCD16/32and inos compared to the control group (P <0.01), prompting that TLR4signal pathway effect on the M1polarization. The expression of CD206, IL-10, arg1of macrophages after SEA stimulation have no significant changes compared to thecontrol group, the reason needs further exploration.
6. The modulation of balance between co-stimulatory molecule CD40, CD86and co-suppression molecule PD-L1might regulate macrophage polarization.Both INF-γ and IL-4could stimulate macrophages to express PD-L1, CD40andCD86; INF-γ stimulated macrophages to express significantly higher level of PD-L1,CD40and CD86than IL-4did(P <0.01); NCA stimulated macrophages expresssignificantly higher level of of PD-L1and CD86than SEA and SWAP stimulatedmacrophages; On the role of costimulatory signals involved in macrophagepolarization and regulation, in the present study, is only stay on the experimentalresults of intuitionistic description and inference and there are no direct evidence toprove the linkage of positive or negative co-stimulatory signals to the macrophagepolarization properties of regulation, which may take place at an unknown level involving the modulation of balance between co-stimulatory molecule andco-suppression molecule.
In summary, this study explored the relationship of S. japonicum antigens andformation of macrophage polarization and the impact of antigen environment changeduring schistosome infection on macrophage polarization modulation, for future frommacrophages polarization properties of regulation, looking for development of newimmunosuppressive agents based on the parasite components provide pilotexperimental arguments.
引文
1. Steinmann, P., et al., Schistosomiasis and water resources development:systematic review, meta-analysis, and estimates of people at risk. LancetInfect Dis,2006.6(7): p.411-25.
2. Colley, D.G. and W.E. Secor, A schistosomiasis research agenda. PLoS NeglTrop Dis,2007.1(3): p. e32.
3. Fenwick, A., Waterborne infectious diseases--could they be consigned tohistory? Science,2006.313(5790): p.1077-81.
4.赵慰先，高淑芬主编，王锐等编写,实用血吸虫病学.1996: p.247.
5.周晓农主编,全球血吸虫防治研究进展与展望.世界卫生组织血吸虫病科学工作组报告,2008: p.1-43,76-99.
6. Jia, T.W., et al., Assessment of the age-specific disability weight of chronicschistosomiasis japonica. Bull World Health Organ,2007.85(6): p.458-65.
7. King, C.H., K. Dickman, and D.J. Tisch, Reassessment of the cost of chronichelmintic infection: a meta-analysis of disability-related outcomes in endemicschistosomiasis. Lancet,2005.365(9470): p.1561-9.
8. Bethony, J.M. and A. Loukas, The schistosomiasis research agenda--whatnow? PLoS Negl Trop Dis,2008.2(2): p. e207.
9.汪天平，操治国，林丹丹，周晓农,“十二五”期间我国血吸虫病科学研究重点和方向.中国血吸虫病防治杂志,2011.23(2): p.111-113.
10. Wiest, P.M., et al., Impact of annual screening and chemotherapy withpraziquantel on schistosomiasis japonica on Jishan Island, People's Republicof China. Am J Trop Med Hyg,1994.51(2): p.162-9.
11. Butterworth, A.E., Vaccines against schistosomiasis: where do we stand?Trans R Soc Trop Med Hyg,1992.86(1): p.1-2.
12. Ross, A.G., et al., Schistosomiasis. N Engl J Med,2002.346(16): p.1212-20.
13. Gryseels, B., et al., Human schistosomiasis. Lancet,2006.368(9541): p.1106-18.
14. Doherty, J.F., A.H. Moody, and S.G. Wright, Katayama fever: an acutemanifestation of schistosomiasis. Bmj,1996.313(7064): p.1071-2.
15. Ross, A.G., et al., Katayama syndrome. Lancet Infect Dis,2007.7(3): p.218-24.
16. Pearce, E.J. and A.S. MacDonald, The immunobiology of schistosomiasis. NatRev Immunol,2002.2(7): p.499-511.
17. Ji, M.J., et al., Gene expression profile of CD4+T cells reveals an interferonsignaling suppression associated with progression of experimentalSchistosoma japonicum infection. Cell Immunol,2003.224(1): p.55-62.
18. Ji, M.J., et al., Characterization of CD4+T cell responses in mice infectedwith Schistosoma japonicum. Acta Biochim Biophys Sin (Shanghai),2006.38(5): p.327-34.
19. Domingo, E.O. and K.S. Warren, Endogenous desensitization: changing hostgranulomatou response to schistosome eggs at different stages of infectionwith schistosoma mansoni. Am J Pathol,1968.52(2): p.369-79.
20. Warren, K.S., D.I. Grove, and R.P. Pelley, The Schistosoma japonicum egggranuloma. II. Cellular composition, granuloma size, and immunologicconcomitants. Am J Trop Med Hyg,1978.27(2Pt1): p.271-5.
21. Boros, D.L., R.P. Pelley, and K.S. Warren, Spontaneous modulation ofgranulomatous hypersensitivity in schistosomiasis mansoni. J Immunol,1975.114(5): p.1437-41.
22. Warren, K.S., Modulation of immunopathology and disease in schistosomiasis.Am J Trop Med Hyg,1977.26(6Pt2): p.113-9.
23. Fallon, P.G. and D.W. Dunne, Tolerization of mice to Schistosoma mansoniegg antigens causes elevated type1and diminished type2cytokine responsesand increased mortality in acute infection. J Immunol,1999.162(7): p.4122-32.
24. Zheng, X., et al., Soluble egg antigen from Schistosoma japonicum modulatesthe progression of chronic progressive experimental autoimmuneencephalomyelitis via Th2-shift response. J Neuroimmunol,2008.194(1-2): p.107-14.
25. Wang, X., et al., CD4+CD25+Treg induction by an HSP60-derived peptideSJMHE1from Schistosoma japonicum is TLR2dependent. Eur J Immunol,2009.39(11): p.3052-65.
26. Stavitsky, A.B., Regulation of granulomatous inflammation in experimentalmodels of schistosomiasis. Infect Immun,2004.72(1): p.1-12.
27. Mosser, D.M., The many faces of macrophage activation. J Leukoc Biol,2003.73(2): p.209-12.
28. Gordon, S., Alternative activation of macrophages. Nat Rev Immunol,2003.3(1): p.23-35.
29. Smith, P., et al., Schistosoma mansoni worms induce anergy of T cells viaselective up-regulation of programmed death ligand1on macrophages. JImmunol,2004.173(2): p.1240-8.
30. Keir, M.E., et al., PD-1and its ligands in tolerance and immunity. Annu RevImmunol,2008.26: p.677-704.
31. Sharpe, A.H., et al., The function of programmed cell death1and its ligandsin regulating autoimmunity and infection. Nat Immunol,2007.8(3): p.239-45.
32. Herbert, D.R., et al., Alternative macrophage activation is essential forsurvival during schistosomiasis and downmodulates T helper1responses andimmunopathology. Immunity,2004.20(5): p.623-35.
33. Mantovani, A., A. Sica, and M. Locati, Macrophage polarization comes of age.Immunity,2005.23(4): p.344-6.
34. Rauh, M.J., et al., SHIP represses the generation of alternatively activatedmacrophages. Immunity,2005.23(4): p.361-74.
35. Arnold, L., et al., Inflammatory monocytes recruited after skeletal muscleinjury switch into antiinflammatory macrophages to support myogenesis. JExp Med,2007.204(5): p.1057-69.
36. Lumeng, C.N., J.L. Bodzin, and A.R. Saltiel, Obesity induces a phenotypicswitch in adipose tissue macrophage polarization. J Clin Invest,2007.117(1):p.175-84.
37. Biswas, S.K., et al., A distinct and unique transcriptional program expressedby tumor-associated macrophages (defective NF-kappaB and enhancedIRF-3/STAT1activation). Blood,2006.107(5): p.2112-22.
38. Mantovani, A., A. Sica, and M. Locati, New vistas on macrophagedifferentiation and activation. Eur J Immunol,2007.37(1): p.14-6.
39. Namangala, B., et al., Trypanosoma brucei brucei infection impairs MHCclass II antigen presentation capacity of macrophages. Parasite Immunol,2000.22(7): p.361-70.
40. Vats, D., et al., Oxidative metabolism and PGC-1beta attenuatemacrophage-mediated inflammation. Cell Metab,2006.4(1): p.13-24.
41. Mantovani, A., et al., The chemokine system in diverse forms of macrophageactivation and polarization. Trends Immunol,2004.25(12): p.677-86.
42. Gordon, S., Macrophage heterogeneity and tissue lipids. J Clin Invest,2007.117(1): p.89-93.
43. Viana, I.R., et al., Interferon-gamma production by peripheral bloodmononuclear cells from residents of an area endemic for Schistosomamansoni. Trans R Soc Trop Med Hyg,1994.88(4): p.466-70.
44. Odegaard, J.I., et al., Macrophage-specific PPARgamma controls alternativeactivation and improves insulin resistance. Nature,2007.447(7148): p.1116-20.
45. Janeway, C.A., Jr., Approaching the asymptote? Evolution and revolution inimmunology. Cold Spring Harb Symp Quant Biol,1989.54Pt1: p.1-13.
46. Mountford, A.P., S. Anderson, and R.A. Wilson, Induction of Th1cell-mediated protective immunity to Schistosoma mansoni byco-administration of larval antigens and IL-12as an adjuvant. J Immunol,1996.156(12): p.4739-45.
47.李康，郭强，王翠妮，陈敏，徐薇，熊思东, M1和M2型巨噬细胞表型的比较分析.现代免疫学,2008.28(3): p.177-183.
48. Bonecchi, R., et al., Divergent effects of interleukin-4and interferon-gammaon macrophage-derived chemokine production: an amplification circuit ofpolarized T helper2responses. Blood,1998.92(8): p.2668-71.
49. Martinon, F. and J. Tschopp, NLRs join TLRs as innate sensors of pathogens.Trends Immunol,2005.26(8): p.447-54.
50. Todd, C.W., R.W. Goodgame, and D.G. Colley, Immune responses duringhuman schistosomiasis mansoni. V. Suppression of schistosomeantigen-specific lymphocyte blastogenesis by adherent/phagocytic cells. JImmunol,1979.122(4): p.1440-6.
51. Atochina, O., et al., A schistosome-expressed immunomodulatoryglycoconjugate expands peritoneal Gr1(+) macrophages that suppress naiveCD4(+) T cell proliferation via an IFN-gamma and nitric oxide-dependentmechanism. J Immunol,2001.167(8): p.4293-302.
52. Welch, J.S., et al., TH2cytokines and allergic challenge induce Ym1expression in macrophages by a STAT6-dependent mechanism. J Biol Chem,2002.277(45): p.42821-9.
53. Loke, P., et al., IL-4dependent alternatively-activated macrophages have adistinctive in vivo gene expression phenotype. BMC Immunol,2002.3: p.7.
54. Hesse, M., et al., Differential regulation of nitric oxide synthase-2andarginase-1by type1/type2cytokines in vivo: granulomatous pathology isshaped by the pattern of L-arginine metabolism. J Immunol,2001.167(11): p.6533-44.
55. Iwasaki, A. and R. Medzhitov, Toll-like receptor control of the adaptiveimmune responses. Nat Immunol,2004.5(10): p.987-95.
56. Trinchieri, G. and A. Sher, Cooperation of Toll-like receptor signals in innateimmune defence. Nat Rev Immunol,2007.7(3): p.179-90.
57. Murawski, M.R., et al., Respiratory syncytial virus activates innate immunitythrough Toll-like receptor2. J Virol,2009.83(3): p.1492-500.
58. Feng, C.G., et al., Mice lacking myeloid differentiation factor88displayprofound defects in host resistance and immune responses to Mycobacteriumavium infection not exhibited by Toll-like receptor2(TLR2)-andTLR4-deficient animals. J Immunol,2003.171(9): p.4758-64.
59. Pearl, J.I., et al., Role of the Toll-like receptor pathway in the recognition oforthopedic implant wear-debris particles. Biomaterials.32(24): p.5535-42.
60. Gelani, V., et al., The role of toll-like receptor2in the recognition ofAggregatibacter actinomycetemcomitans. J Periodontol,2009.80(12): p.2010-9.
61. Fuse, E.T., et al., Role of Toll-like receptor2in recognition of Legionellapneumophila in a murine pneumonia model. J Med Microbiol,2007.56(Pt3):p.305-12.
62. Mun, H.S., et al., TLR2as an essential molecule for protective immunityagainst Toxoplasma gondii infection. Int Immunol,2003.15(9): p.1081-7.
63. Kropf, P., et al., Toll-like receptor4contributes to efficient control of infectionwith the protozoan parasite Leishmania major. Infect Immun,2004.72(4): p.1920-8.
64. Kropf, P., et al., Infection of C57BL/10ScCr and C57BL/10ScNCr mice withLeishmania major reveals a role for Toll-like receptor4in the control ofparasite replication. J Leukoc Biol,2004.76(1): p.48-57.
65. Campos, M.A., et al., Activation of Toll-like receptor-2byglycosylphosphatidylinositol anchors from a protozoan parasite. J Immunol,2001.167(1): p.416-23.
66. Oliveira, A.C., et al., Expression of functional TLR4confers proinflammatoryresponsiveness to Trypanosoma cruzi glycoinositolphospholipids and higherresistance to infection with T. cruzi. J Immunol,2004.173(9): p.5688-96.
67. Netea, M.G., et al., Toll-like receptor2suppresses immunity against Candidaalbicans through induction of IL-10and regulatory T cells. J Immunol,2004.172(6): p.3712-8.
68. Kurt-Jones, E.A., et al., Herpes simplex virus1interaction with Toll-likereceptor2contributes to lethal encephalitis. Proc Natl Acad Sci U S A,2004.101(5): p.1315-20.
69. Zhang, M., et al., Toll-like receptor (TLR)2and TLR4deficiencies exertdifferential in vivo effects against Schistosoma japonicum. Parasite Immunol.33(4): p.199-209.
70. Porta, C., et al., Tolerance and M2(alternative) macrophage polarization arerelated processes orchestrated by p50nuclear factor kappaB. Proc Natl AcadSci U S A,2009.106(35): p.14978-83.
71. Subudhi, S.K., et al., Local expression of B7-H1promotes organ-specificautoimmunity and transplant rejection. J Clin Invest,2004.113(5): p.694-700.
72. Shin, T., et al., In vivo costimulatory role of B7-DC in tuning T helper cell1and cytotoxic T lymphocyte responses. J Exp Med,2005.201(10): p.1531-41.
73. Barber, D.L., et al., Restoring function in exhausted CD8T cells duringchronic viral infection. Nature,2006.439(7077): p.682-7.
74. Wabnitz, G.H., et al., Phosphatidylinositol3-kinase functions as a Ras effectorin the signaling cascade that regulates dephosphorylation of theactin-remodeling protein cofilin after costimulation of untransformed humanT lymphocytes. J Immunol,2006.176(3): p.1668-74.
75. Isogawa, M., Y. Furuichi, and F.V. Chisari, Oscillating CD8(+) T cell effectorfunctions after antigen recognition in the liver. Immunity,2005.23(1): p.53-63.
76. Lukens, J.R., et al., Blockade of PD-1/B7-H1interaction restores effectorCD8+T cell responses in a hepatitis C virus core murine model. J Immunol,2008.180(7): p.4875-84.
77. Wiesemann, E., et al., Effects of interferon-beta on co-signaling molecules:upregulation of CD40, CD86and PD-L2on monocytes in relation to clinicalresponse to interferon-beta treatment in patients with multiple sclerosis. MultScler,2008.14(2): p.166-76.
[1] Blood monocytes consist of two principal subsets with distinct migratoryproperties. Geissmann F et al. Immunity,2003,19:71
[2] Human IL-23-producing type1macrophages promote but IL-10-producingtype2macrophages subvert immunity to (myco)bacteria.Verreck FA et al. ProcNatl Acad Sci USA,2004,101:4560
[3] Alternative activation of macrophages. Gordon S et al. Nat Rev Immunol,2003,3:23
[4] Other functions, other genes: alternative activation of antigen-presenting cells.Goerdt S et al. Immunity,1999,10:137
[5] The many faces of macrophage activation. Mosser DM et al. J Leukoc Biol,2003,73:209
[6] The chemokine system in diverse forms of macrophage activation andpolarization. Mantovani A et al. Trends Immunol,2004,25:677
[7] SHIP represses the generation of alternatively activated macrophages. RauhMJ et al. Immunity,2005,23:361
[8] Macrophages from IL-12p40-deficient mice have a bias toward the M2activation profile. Bastos KR et al. J Leukoc Biol,2002,71:271
[9] Notch-1up-regulation and signaling following macrophage activationmodulates gene expression patterns known to affect antigen-presenting capacityand cytotoxic activity. Monsalve E et al. J Immunol,2006,176:5362
[10] B7-H4expression identifies a novel suppressive macrophage population inhuman ovarian carcinoma. Kryczek I et al. J Exp Med,2006,203:871
[11] Tumour-derived microvesicles carry several surface determinants and mRNAof tumour cells and transfer some of these determinants to monocytes.Baj-Krzyworzeka M et al. Cancer Immunol Immunother,2006,55:808
[12] Macrophage polarization: tumor-associated macrophages as a paradigm forpolarized M2mononuclear phagocytes. Mantovani A et al. Trends Immunol,2002,23:549
[13] Interleukin-1and interleukin-1antagonism. Dinarello CA et al. Blood,1991,77:1627
[14] Shaping gene expression in activated and resting primary macrophages byIL-10. Lang R et al. J Immunol,2002,169:2253
[15] Expression profiling of IL-10-regulated genes in human monocytes andperipheral blood mononuclear cells from psoriatic patients during IL-10therapy. Jung M et al. Eur J Immunol,2004,34:481
[16] Acidic mammalian chitinase in asthmatic Th2inflammation and IL-13pathway activation. Zhu Z et al. Science,2004,304:1678
[17] Receptor mediated subversion of macrophage cytokine production byintracellular pathogens. Mosser DM et al. Curr Opin Immunol,1999,11:406
[18] Selective depletion of macrophages reveals distinct, opposing roles duringliver injury and repair. Duffield JS et al. J Clin Invest,2005,115:56
[19] Alternative macrophage activation is essential for survival duringschistosomiasis and downmodulates T helper1responses andimmunopathology. Herbert DR et al. Immunity,2004,20:623
[20] The origin and function of tumor-associated macrophages. Mantovani A et al.Immunol Today,1992,13:265
[21] Inflammation and cancer: back to Virchow? Balkwill F et al. Lancet2001,357:539
[22] Association of macrophage infiltration with angiogenesis and prognosis ininvasive breast carcinoma. Leek RD et al. Cancer Res,1996,56:4625
[23] Necrosis correlates with high vascular density and focal macrophageinfiltration in invasive carcinoma of the breast. Leek RD et al. Br J Cancer,1999,79:991
[24] NF-kappa B, chemokine gene transcription and tumour growth. Richmond A.Nat Rev Immunol,2002,2:664
[25] Requirement for STAT1in LPS-induced gene expression in macrophages.Ohmori Y et al. J Leukoc Biol,2001,69:598
[26] Toll-like receptor signaling. Akira S. J Biol Chem,2003,278:38105
[27] Interleukin-10inhibits expression of both interferon alpha-and interferongamma-induced genes by suppressing tyrosine phosphorylation of STAT1.Ito S et al. Blood,1999,93:1456
[28] Eotaxin-2generation is differentially regulated by lipopolysaccharide andIL-4in monocytes and macrophages. Watanabe K et al.J Immunol,2002,168:1911
[29] Divergent effects of interleukin-4and interferon-gamma onmacrophage-derived chemokine production: an amplification circuit ofpolarized T helper2responses. Bonecchi R et al. Blood,1998,92:2668
[30] Control of TH2polarization by the chemokine monocyte chemoattractantprotein-1. Gu L et al. Nature,2000,404:407
[31] CCL17and IL-10as effectors that enable alternatively activated macrophagesto inhibit the generation of classically activated macrophages. Katakura T et al.J Immunol,2004,172:1407
[32] Increased responsiveness of murine eosinophils to MIP-1beta (CCL4) andTCA-3(CCL1) is mediated by their specific receptors, CCR5and CCR8Oliveira. SH et al. J Leukoc Biol,2002,71:1019
[33] Phenotype, localization, and mechanism of suppression of CD4(+)CD25(+)human thymocytes. Annunziato F et al. J Exp Med,2002,196:379
[34] Selective up-regulation of chemokine receptors CCR4and CCR8uponactivation of polarized human type2Th cells. D et al. J Immunol,
[35] Unique chemotactic response profile and specific expression of chemokinereceptors CCR4and CCR8by CD4(+)CD25(+) regulatory T cells. Iellem A etal. J Exp Med,2001,194:847
[36] A skin-selective homing mechanism for human immune surveillance T cells.Schaerli P et al. J Exp Med,2004,99:1265
[37] Trypanosoma brucei brucei infection impairs MHC class II antigenpresentation capacity of macrophages. Namangala B et al. ParasiteImmunol.2000,22:361
[38] Th2cytokines and allergic challenge induce Ym1expression in macrophagesby a STAT6dependent mechanism.Welch J S et al. J Biol Chem2002,277:42821
[39] The chemokine system in diverse forms of macrophage activation andpolarization. Mantovani A et al. Trends Immunol.2004,25:677
2. Colley, D.G. and W.E. Secor, A schistosomiasis research agenda. PLoS NeglTrop Dis,2007.1(3): p. e32.
3. Fenwick, A., Waterborne infectious diseases--could they be consigned tohistory? Science,2006.313(5790): p.1077-81.
4.赵慰先，高淑芬主编，王锐等编写,实用血吸虫病学.1996: p.247.
5.周晓农主编,全球血吸虫防治研究进展与展望.世界卫生组织血吸虫病科学工作组报告,2008: p.1-43,76-99.
6. Jia, T.W., et al., Assessment of the age-specific disability weight of chronicschistosomiasis japonica. Bull World Health Organ,2007.85(6): p.458-65.
7. King, C.H., K. Dickman, and D.J. Tisch, Reassessment of the cost of chronichelmintic infection: a meta-analysis of disability-related outcomes in endemicschistosomiasis. Lancet,2005.365(9470): p.1561-9.
8. Bethony, J.M. and A. Loukas, The schistosomiasis research agenda--whatnow? PLoS Negl Trop Dis,2008.2(2): p. e207.
9.汪天平，操治国，林丹丹，周晓农,“十二五”期间我国血吸虫病科学研究重点和方向.中国血吸虫病防治杂志,2011.23(2): p.111-113.
10. Wiest, P.M., et al., Impact of annual screening and chemotherapy withpraziquantel on schistosomiasis japonica on Jishan Island, People's Republicof China. Am J Trop Med Hyg,1994.51(2): p.162-9.
11. Butterworth, A.E., Vaccines against schistosomiasis: where do we stand?Trans R Soc Trop Med Hyg,1992.86(1): p.1-2.
12. Ross, A.G., et al., Schistosomiasis. N Engl J Med,2002.346(16): p.1212-20.
13. Gryseels, B., et al., Human schistosomiasis. Lancet,2006.368(9541): p.1106-18.
14. Doherty, J.F., A.H. Moody, and S.G. Wright, Katayama fever: an acutemanifestation of schistosomiasis. Bmj,1996.313(7064): p.1071-2.
15. Ross, A.G., et al., Katayama syndrome. Lancet Infect Dis,2007.7(3): p.218-24.
16. Pearce, E.J. and A.S. MacDonald, The immunobiology of schistosomiasis. NatRev Immunol,2002.2(7): p.499-511.
17. Ji, M.J., et al., Gene expression profile of CD4+T cells reveals an interferonsignaling suppression associated with progression of experimentalSchistosoma japonicum infection. Cell Immunol,2003.224(1): p.55-62.
18. Ji, M.J., et al., Characterization of CD4+T cell responses in mice infectedwith Schistosoma japonicum. Acta Biochim Biophys Sin (Shanghai),2006.38(5): p.327-34.
19. Domingo, E.O. and K.S. Warren, Endogenous desensitization: changing hostgranulomatou response to schistosome eggs at different stages of infectionwith schistosoma mansoni. Am J Pathol,1968.52(2): p.369-79.
20. Warren, K.S., D.I. Grove, and R.P. Pelley, The Schistosoma japonicum egggranuloma. II. Cellular composition, granuloma size, and immunologicconcomitants. Am J Trop Med Hyg,1978.27(2Pt1): p.271-5.
21. Boros, D.L., R.P. Pelley, and K.S. Warren, Spontaneous modulation ofgranulomatous hypersensitivity in schistosomiasis mansoni. J Immunol,1975.114(5): p.1437-41.
22. Warren, K.S., Modulation of immunopathology and disease in schistosomiasis.Am J Trop Med Hyg,1977.26(6Pt2): p.113-9.
23. Fallon, P.G. and D.W. Dunne, Tolerization of mice to Schistosoma mansoniegg antigens causes elevated type1and diminished type2cytokine responsesand increased mortality in acute infection. J Immunol,1999.162(7): p.4122-32.
24. Zheng, X., et al., Soluble egg antigen from Schistosoma japonicum modulatesthe progression of chronic progressive experimental autoimmuneencephalomyelitis via Th2-shift response. J Neuroimmunol,2008.194(1-2): p.107-14.
25. Wang, X., et al., CD4+CD25+Treg induction by an HSP60-derived peptideSJMHE1from Schistosoma japonicum is TLR2dependent. Eur J Immunol,2009.39(11): p.3052-65.
26. Stavitsky, A.B., Regulation of granulomatous inflammation in experimentalmodels of schistosomiasis. Infect Immun,2004.72(1): p.1-12.
27. Mosser, D.M., The many faces of macrophage activation. J Leukoc Biol,2003.73(2): p.209-12.
28. Gordon, S., Alternative activation of macrophages. Nat Rev Immunol,2003.3(1): p.23-35.
29. Smith, P., et al., Schistosoma mansoni worms induce anergy of T cells viaselective up-regulation of programmed death ligand1on macrophages. JImmunol,2004.173(2): p.1240-8.
30. Keir, M.E., et al., PD-1and its ligands in tolerance and immunity. Annu RevImmunol,2008.26: p.677-704.
31. Sharpe, A.H., et al., The function of programmed cell death1and its ligandsin regulating autoimmunity and infection. Nat Immunol,2007.8(3): p.239-45.
32. Herbert, D.R., et al., Alternative macrophage activation is essential forsurvival during schistosomiasis and downmodulates T helper1responses andimmunopathology. Immunity,2004.20(5): p.623-35.
33. Mantovani, A., A. Sica, and M. Locati, Macrophage polarization comes of age.Immunity,2005.23(4): p.344-6.
34. Rauh, M.J., et al., SHIP represses the generation of alternatively activatedmacrophages. Immunity,2005.23(4): p.361-74.
35. Arnold, L., et al., Inflammatory monocytes recruited after skeletal muscleinjury switch into antiinflammatory macrophages to support myogenesis. JExp Med,2007.204(5): p.1057-69.
36. Lumeng, C.N., J.L. Bodzin, and A.R. Saltiel, Obesity induces a phenotypicswitch in adipose tissue macrophage polarization. J Clin Invest,2007.117(1):p.175-84.
37. Biswas, S.K., et al., A distinct and unique transcriptional program expressedby tumor-associated macrophages (defective NF-kappaB and enhancedIRF-3/STAT1activation). Blood,2006.107(5): p.2112-22.
38. Mantovani, A., A. Sica, and M. Locati, New vistas on macrophagedifferentiation and activation. Eur J Immunol,2007.37(1): p.14-6.
39. Namangala, B., et al., Trypanosoma brucei brucei infection impairs MHCclass II antigen presentation capacity of macrophages. Parasite Immunol,2000.22(7): p.361-70.
40. Vats, D., et al., Oxidative metabolism and PGC-1beta attenuatemacrophage-mediated inflammation. Cell Metab,2006.4(1): p.13-24.
41. Mantovani, A., et al., The chemokine system in diverse forms of macrophageactivation and polarization. Trends Immunol,2004.25(12): p.677-86.
42. Gordon, S., Macrophage heterogeneity and tissue lipids. J Clin Invest,2007.117(1): p.89-93.
43. Viana, I.R., et al., Interferon-gamma production by peripheral bloodmononuclear cells from residents of an area endemic for Schistosomamansoni. Trans R Soc Trop Med Hyg,1994.88(4): p.466-70.
44. Odegaard, J.I., et al., Macrophage-specific PPARgamma controls alternativeactivation and improves insulin resistance. Nature,2007.447(7148): p.1116-20.
45. Janeway, C.A., Jr., Approaching the asymptote? Evolution and revolution inimmunology. Cold Spring Harb Symp Quant Biol,1989.54Pt1: p.1-13.
46. Mountford, A.P., S. Anderson, and R.A. Wilson, Induction of Th1cell-mediated protective immunity to Schistosoma mansoni byco-administration of larval antigens and IL-12as an adjuvant. J Immunol,1996.156(12): p.4739-45.
47.李康，郭强，王翠妮，陈敏，徐薇，熊思东, M1和M2型巨噬细胞表型的比较分析.现代免疫学,2008.28(3): p.177-183.
48. Bonecchi, R., et al., Divergent effects of interleukin-4and interferon-gammaon macrophage-derived chemokine production: an amplification circuit ofpolarized T helper2responses. Blood,1998.92(8): p.2668-71.
49. Martinon, F. and J. Tschopp, NLRs join TLRs as innate sensors of pathogens.Trends Immunol,2005.26(8): p.447-54.
50. Todd, C.W., R.W. Goodgame, and D.G. Colley, Immune responses duringhuman schistosomiasis mansoni. V. Suppression of schistosomeantigen-specific lymphocyte blastogenesis by adherent/phagocytic cells. JImmunol,1979.122(4): p.1440-6.
51. Atochina, O., et al., A schistosome-expressed immunomodulatoryglycoconjugate expands peritoneal Gr1(+) macrophages that suppress naiveCD4(+) T cell proliferation via an IFN-gamma and nitric oxide-dependentmechanism. J Immunol,2001.167(8): p.4293-302.
52. Welch, J.S., et al., TH2cytokines and allergic challenge induce Ym1expression in macrophages by a STAT6-dependent mechanism. J Biol Chem,2002.277(45): p.42821-9.
53. Loke, P., et al., IL-4dependent alternatively-activated macrophages have adistinctive in vivo gene expression phenotype. BMC Immunol,2002.3: p.7.
54. Hesse, M., et al., Differential regulation of nitric oxide synthase-2andarginase-1by type1/type2cytokines in vivo: granulomatous pathology isshaped by the pattern of L-arginine metabolism. J Immunol,2001.167(11): p.6533-44.
55. Iwasaki, A. and R. Medzhitov, Toll-like receptor control of the adaptiveimmune responses. Nat Immunol,2004.5(10): p.987-95.
56. Trinchieri, G. and A. Sher, Cooperation of Toll-like receptor signals in innateimmune defence. Nat Rev Immunol,2007.7(3): p.179-90.
57. Murawski, M.R., et al., Respiratory syncytial virus activates innate immunitythrough Toll-like receptor2. J Virol,2009.83(3): p.1492-500.
58. Feng, C.G., et al., Mice lacking myeloid differentiation factor88displayprofound defects in host resistance and immune responses to Mycobacteriumavium infection not exhibited by Toll-like receptor2(TLR2)-andTLR4-deficient animals. J Immunol,2003.171(9): p.4758-64.
59. Pearl, J.I., et al., Role of the Toll-like receptor pathway in the recognition oforthopedic implant wear-debris particles. Biomaterials.32(24): p.5535-42.
60. Gelani, V., et al., The role of toll-like receptor2in the recognition ofAggregatibacter actinomycetemcomitans. J Periodontol,2009.80(12): p.2010-9.
61. Fuse, E.T., et al., Role of Toll-like receptor2in recognition of Legionellapneumophila in a murine pneumonia model. J Med Microbiol,2007.56(Pt3):p.305-12.
62. Mun, H.S., et al., TLR2as an essential molecule for protective immunityagainst Toxoplasma gondii infection. Int Immunol,2003.15(9): p.1081-7.
63. Kropf, P., et al., Toll-like receptor4contributes to efficient control of infectionwith the protozoan parasite Leishmania major. Infect Immun,2004.72(4): p.1920-8.
64. Kropf, P., et al., Infection of C57BL/10ScCr and C57BL/10ScNCr mice withLeishmania major reveals a role for Toll-like receptor4in the control ofparasite replication. J Leukoc Biol,2004.76(1): p.48-57.
65. Campos, M.A., et al., Activation of Toll-like receptor-2byglycosylphosphatidylinositol anchors from a protozoan parasite. J Immunol,2001.167(1): p.416-23.
66. Oliveira, A.C., et al., Expression of functional TLR4confers proinflammatoryresponsiveness to Trypanosoma cruzi glycoinositolphospholipids and higherresistance to infection with T. cruzi. J Immunol,2004.173(9): p.5688-96.
67. Netea, M.G., et al., Toll-like receptor2suppresses immunity against Candidaalbicans through induction of IL-10and regulatory T cells. J Immunol,2004.172(6): p.3712-8.
68. Kurt-Jones, E.A., et al., Herpes simplex virus1interaction with Toll-likereceptor2contributes to lethal encephalitis. Proc Natl Acad Sci U S A,2004.101(5): p.1315-20.
69. Zhang, M., et al., Toll-like receptor (TLR)2and TLR4deficiencies exertdifferential in vivo effects against Schistosoma japonicum. Parasite Immunol.33(4): p.199-209.
70. Porta, C., et al., Tolerance and M2(alternative) macrophage polarization arerelated processes orchestrated by p50nuclear factor kappaB. Proc Natl AcadSci U S A,2009.106(35): p.14978-83.
71. Subudhi, S.K., et al., Local expression of B7-H1promotes organ-specificautoimmunity and transplant rejection. J Clin Invest,2004.113(5): p.694-700.
72. Shin, T., et al., In vivo costimulatory role of B7-DC in tuning T helper cell1and cytotoxic T lymphocyte responses. J Exp Med,2005.201(10): p.1531-41.
73. Barber, D.L., et al., Restoring function in exhausted CD8T cells duringchronic viral infection. Nature,2006.439(7077): p.682-7.
74. Wabnitz, G.H., et al., Phosphatidylinositol3-kinase functions as a Ras effectorin the signaling cascade that regulates dephosphorylation of theactin-remodeling protein cofilin after costimulation of untransformed humanT lymphocytes. J Immunol,2006.176(3): p.1668-74.
75. Isogawa, M., Y. Furuichi, and F.V. Chisari, Oscillating CD8(+) T cell effectorfunctions after antigen recognition in the liver. Immunity,2005.23(1): p.53-63.
76. Lukens, J.R., et al., Blockade of PD-1/B7-H1interaction restores effectorCD8+T cell responses in a hepatitis C virus core murine model. J Immunol,2008.180(7): p.4875-84.
77. Wiesemann, E., et al., Effects of interferon-beta on co-signaling molecules:upregulation of CD40, CD86and PD-L2on monocytes in relation to clinicalresponse to interferon-beta treatment in patients with multiple sclerosis. MultScler,2008.14(2): p.166-76.
[1] Blood monocytes consist of two principal subsets with distinct migratoryproperties. Geissmann F et al. Immunity,2003,19:71
[2] Human IL-23-producing type1macrophages promote but IL-10-producingtype2macrophages subvert immunity to (myco)bacteria.Verreck FA et al. ProcNatl Acad Sci USA,2004,101:4560
[3] Alternative activation of macrophages. Gordon S et al. Nat Rev Immunol,2003,3:23
[4] Other functions, other genes: alternative activation of antigen-presenting cells.Goerdt S et al. Immunity,1999,10:137
[5] The many faces of macrophage activation. Mosser DM et al. J Leukoc Biol,2003,73:209
[6] The chemokine system in diverse forms of macrophage activation andpolarization. Mantovani A et al. Trends Immunol,2004,25:677
[7] SHIP represses the generation of alternatively activated macrophages. RauhMJ et al. Immunity,2005,23:361
[8] Macrophages from IL-12p40-deficient mice have a bias toward the M2activation profile. Bastos KR et al. J Leukoc Biol,2002,71:271
[9] Notch-1up-regulation and signaling following macrophage activationmodulates gene expression patterns known to affect antigen-presenting capacityand cytotoxic activity. Monsalve E et al. J Immunol,2006,176:5362
[10] B7-H4expression identifies a novel suppressive macrophage population inhuman ovarian carcinoma. Kryczek I et al. J Exp Med,2006,203:871
[11] Tumour-derived microvesicles carry several surface determinants and mRNAof tumour cells and transfer some of these determinants to monocytes.Baj-Krzyworzeka M et al. Cancer Immunol Immunother,2006,55:808
[12] Macrophage polarization: tumor-associated macrophages as a paradigm forpolarized M2mononuclear phagocytes. Mantovani A et al. Trends Immunol,2002,23:549
[13] Interleukin-1and interleukin-1antagonism. Dinarello CA et al. Blood,1991,77:1627
[14] Shaping gene expression in activated and resting primary macrophages byIL-10. Lang R et al. J Immunol,2002,169:2253
[15] Expression profiling of IL-10-regulated genes in human monocytes andperipheral blood mononuclear cells from psoriatic patients during IL-10therapy. Jung M et al. Eur J Immunol,2004,34:481
[16] Acidic mammalian chitinase in asthmatic Th2inflammation and IL-13pathway activation. Zhu Z et al. Science,2004,304:1678
[17] Receptor mediated subversion of macrophage cytokine production byintracellular pathogens. Mosser DM et al. Curr Opin Immunol,1999,11:406
[18] Selective depletion of macrophages reveals distinct, opposing roles duringliver injury and repair. Duffield JS et al. J Clin Invest,2005,115:56
[19] Alternative macrophage activation is essential for survival duringschistosomiasis and downmodulates T helper1responses andimmunopathology. Herbert DR et al. Immunity,2004,20:623
[20] The origin and function of tumor-associated macrophages. Mantovani A et al.Immunol Today,1992,13:265
[21] Inflammation and cancer: back to Virchow? Balkwill F et al. Lancet2001,357:539
[22] Association of macrophage infiltration with angiogenesis and prognosis ininvasive breast carcinoma. Leek RD et al. Cancer Res,1996,56:4625
[23] Necrosis correlates with high vascular density and focal macrophageinfiltration in invasive carcinoma of the breast. Leek RD et al. Br J Cancer,1999,79:991
[24] NF-kappa B, chemokine gene transcription and tumour growth. Richmond A.Nat Rev Immunol,2002,2:664
[25] Requirement for STAT1in LPS-induced gene expression in macrophages.Ohmori Y et al. J Leukoc Biol,2001,69:598
[26] Toll-like receptor signaling. Akira S. J Biol Chem,2003,278:38105
[27] Interleukin-10inhibits expression of both interferon alpha-and interferongamma-induced genes by suppressing tyrosine phosphorylation of STAT1.Ito S et al. Blood,1999,93:1456
[28] Eotaxin-2generation is differentially regulated by lipopolysaccharide andIL-4in monocytes and macrophages. Watanabe K et al.J Immunol,2002,168:1911
[29] Divergent effects of interleukin-4and interferon-gamma onmacrophage-derived chemokine production: an amplification circuit ofpolarized T helper2responses. Bonecchi R et al. Blood,1998,92:2668
[30] Control of TH2polarization by the chemokine monocyte chemoattractantprotein-1. Gu L et al. Nature,2000,404:407
[31] CCL17and IL-10as effectors that enable alternatively activated macrophagesto inhibit the generation of classically activated macrophages. Katakura T et al.J Immunol,2004,172:1407
[32] Increased responsiveness of murine eosinophils to MIP-1beta (CCL4) andTCA-3(CCL1) is mediated by their specific receptors, CCR5and CCR8Oliveira. SH et al. J Leukoc Biol,2002,71:1019
[33] Phenotype, localization, and mechanism of suppression of CD4(+)CD25(+)human thymocytes. Annunziato F et al. J Exp Med,2002,196:379
[34] Selective up-regulation of chemokine receptors CCR4and CCR8uponactivation of polarized human type2Th cells. D et al. J Immunol,
[35] Unique chemotactic response profile and specific expression of chemokinereceptors CCR4and CCR8by CD4(+)CD25(+) regulatory T cells. Iellem A etal. J Exp Med,2001,194:847
[36] A skin-selective homing mechanism for human immune surveillance T cells.Schaerli P et al. J Exp Med,2004,99:1265
[37] Trypanosoma brucei brucei infection impairs MHC class II antigenpresentation capacity of macrophages. Namangala B et al. ParasiteImmunol.2000,22:361
[38] Th2cytokines and allergic challenge induce Ym1expression in macrophagesby a STAT6dependent mechanism.Welch J S et al. J Biol Chem2002,277:42821
[39] The chemokine system in diverse forms of macrophage activation andpolarization. Mantovani A et al. Trends Immunol.2004,25:677