【作者】 张一；

【导师】 黄波；

【作者基本信息】 华中科技大学 ， 生物化学与分子生物学， 2013， 博士

【摘要】 [目的]天然免疫和获得性免疫之间的相互作用在选择性清除病原体的过程中发挥着重要的作用。机体感染病原菌后天然免疫细胞首先发挥作用,当天然免疫不能够彻底清除病菌时,获得性免疫随之产生。巨噬细胞和树突状细胞这两种关键的免疫细胞,对于天然免疫向获得性免疫的转化至关重要,然而两者间的作用机制尚不清楚。以李斯特菌感染模型为例,机体产生抗李斯特菌的获得性免疫反应必须需要树突状细胞的参与,单独的巨噬细胞是不能够诱导出抗李斯特菌特异性免疫的。然而树突状细胞摄取李斯特菌的能力较巨噬细胞弱很多,这种对李斯特菌摄取能力的差异提示在细菌抗原加工、提呈并引起T细胞保护性免疫反应的过程中,巨噬细胞和树突状细胞可能承担着不同的职责和作用,了解两者之间的作用途径和机制尤为重要。近年来研究证明,微颗粒在细胞与细胞之间信息传递过程中发挥着重要作用,然而其在获得性免疫过程中的作用尚不清楚。本研究证明了树突状细胞介导的抗李斯特菌特异性免疫过程中巨噬细胞的重要性,进一步验证李斯特菌感染的巨噬细胞来源的微颗粒在树突状细胞介导的T细胞免疫反应中的作用,阐述巨噬细胞与树突状细胞间相互作用的新途径。[方法]1.检测巨噬细胞在诱导抗李斯特菌获得性免疫中的作用(1)清除体内巨噬细胞,检测过继T细胞增殖实验选择16g的雌性裸小鼠腹腔注射301μg抗F4/80的清除抗体,0.8mg Clodrolip,或PBS。每组6只,注射体积为200m,注射时间在Lm注射前48h和6h。将Lm感染过或未感染的BABL/c小鼠的脾脏T细胞磁珠分选出来,CFSE染色后通过尾静脉过继接种给处理过的裸小鼠,5×106个T细胞/只。6h后将1×103个活Lm经尾静脉注入裸小鼠体内,60h后取裸小鼠脾脏细胞流式检测过继T细胞的增殖情况。(2)体外实验检测巨噬细胞在李斯特菌刺激T细胞增殖过程中的作用将BABL/c小鼠骨髓来源的DCs与Lm共培养,加或不加腹腔巨噬细胞,培养时间分别为6h,12h,18h。再与Lm感染小鼠的脾脏T细胞按1：10共培养,72h后加入3H-胸腺嘧啶脱氧核苷,18h后收集细胞,液体闪烁仪计数。(3)体外实验检测李斯特菌感染的巨噬细胞上清对T细胞增殖的影响用Lm感染的巨噬细胞上清或Lm的培养上清与骨髓来源的DCs共培养,6h后再加入Lm感染小鼠的脾脏T细胞,72h后加入3H-胸腺嘧啶脱氧核苷,18h后收集细胞,液体闪烁仪计数。2.李斯特菌感染的巨噬细胞释放的微颗粒的检测(1)微颗粒的提取巨噬细胞与Lm共培养(补加100μg/ml的庆大霉素),培养12h后将上清液经过多步离心除去细胞、细胞碎片和大部分细菌,再用1.0μn的滤膜将上清液过滤完全除去Lm,最后用14000g离心60min,获得微颗粒沉淀。(2)微颗粒的标记用经过CFSE染色的Lm感染巨噬细胞,收集上清液中的微颗粒,流式检测。或者是将CFSE染色的Lm与PKH-26染色的巨噬细胞共培养,提取的微颗粒用双光子共聚焦显微镜观察。3.李斯特菌感染的巨噬细胞释放的微颗粒的功能检测(1)Lm感染的巨噬细胞来源的微颗粒刺激DCs活化的检测将DCs分别与Lm感染的巨噬细胞微颗粒,巨噬细胞微颗粒和PBS共培养,24h后分别用抗体CD80、CD86和MHCⅡ的流式抗体染色,流式检测表达情况。分时间段收取与微颗粒共培养的DCs蛋白样品,Western blot检测DCs活化信号ERK和Iκ.B的磷酸化水平。(2)Lm感染的巨噬细胞来源的微颗粒刺激T细胞增殖实验将不同数量级的Lm与巨噬细胞共培养,提取的微颗粒再与DCs共培养后加入Lm感染小鼠的脾脏T细胞,72h后加入3H-胸腺嘧啶脱氧核苷,18h后收集细胞,液体闪烁仪计数。4.动物实验选择6-8周龄的BABL/c小鼠,每组6只,用Lm感染的巨噬细胞微颗粒皮下免疫小鼠,对照为正常巨噬细胞微颗粒或Lm培养上清提取物免疫组。连续免疫7天后尾静脉注射1×105活的Lm,连续10天观察小鼠生存情况。[结果]1.体内实验证明巨噬细胞参与DCs诱导的抗Lm获得性免疫反应清除体内巨噬细胞的裸小鼠过继接种Lm感染小鼠的CFSE-T细胞,6h后尾静脉注射活的Lm,60h后检测发现清除了巨噬细胞组的小鼠脾脏中过继T细胞不发生增殖。将Lm感染的巨噬细胞注射到C57BL/6小鼠腹腔,7天后分离出脾脏细胞经过灭活的Lm再刺激,发现接种了感染的巨噬细胞组的脾脏细胞培养上清中IFN-γ和IL-22的表达均显著升高,同样流式检测CD11c+细胞IL-12的表达上调,CD4+细胞IFN-γ表达上调。这些结果说明诱导抗Lm T细胞免疫反应需要巨噬细胞的参与。2.体外实验证明Lm感染的巨噬细胞培养上清诱导DCs成熟并提呈抗原分别用Lm感染的巨噬细胞培养上清,正常巨噬细胞培养上清和Lm的培养上清来培养DCs,24h后检测DCs表面CD80、CD86和MHCⅡ的表达,发现Lm感染的巨噬细胞组DCs这三者表达均上调,表明DCs被激活。进一步通过T细胞增殖实验证明,Lm感染的巨噬细胞培养上清具有较强的刺激T细胞增殖的能力,提示Lm感染的巨噬细胞培养上清含有某种形式的Lm抗原,树突状细胞可以将其摄取并将抗原提呈给T细胞,诱导获得性免疫反应。3.Lm感染的巨噬细胞来源的微颗粒刺激DCs活化将Lm感染的巨噬细胞微颗粒提取出来,利用流式和双光子共聚焦显微镜检测,可以发现微颗粒中Lm成分的存在。通过流式检测DCs表面CD80、CD86和MHCⅡ的表达,Western Blot检测ERK和Iκd3的磷酸化水平发现含有Lm成分的微颗粒可以刺激DCs活化并成熟,说明微颗粒将其含有的Lm抗原传递给DCs。4.体外实验证明DCs摄取微颗粒并提呈Lm抗原的机制将Lm感染的巨噬细胞微颗粒与DCs共培养,检测DCs对微颗粒的摄取发现约有35%的DCs摄取了微颗粒。与正常情况下巨噬细胞释放的微颗粒相比,Lm感染的巨噬细胞释放的微颗粒表面Annexin-V表达更高,这可能是DCs更容易摄取含有Lm成分微颗粒的原因。为了了解DCs是通过何种抗原提呈途径提呈Lm抗原给T细胞的,我们用Lm感染MHC-Ⅰ-/-小鼠巨噬细胞,提取的微颗粒再与DCs和CD8+T细胞共培养,发现T细胞增殖能力减弱,提示DCs摄取微颗粒中的Lm抗原后可以直接将MHC-Ⅰ抗原肽复合物表达在细胞表面,提成给CD8+T细胞。而用MHC-Ⅱ-/-。小鼠的DCs与Lm感染的巨噬细胞微颗粒共培养,检测其对CD4+T细胞增殖的影响。我们发现T细胞增殖能力显著降低,这一结果说明DCs通过摄取微颗粒获得Lm抗原,加工后组合成MHC-Ⅱ-/-/-抗原肽复合物表达在细胞表面,再提成给CD4+T细胞。上述结果说明DCs可以通过多种方式摄取微颗粒,包括DCs与微颗粒间的细胞膜融合方式以及摄取后加工再提呈方式。而进一步利用MyD88-/-小鼠实验证明,DCs摄取Lm-MPs这一过程与TLR信号通路无关。5.体外证明含Lm成分微颗粒的产生受到细胞骨架变化的影响利用F-actin聚合抑制剂和肌球蛋白Ⅱ的ATP酶抑制剂作用Lm感染的巨噬细胞,发现含有Lm成分的微颗粒产量减少。这一结果提示肌球蛋白Ⅱ的触发和肌动蛋白丝产生的张力的变化都对巨噬细胞释放Lm微颗粒具有重要的作用。6.体内实验证明李斯特菌感染的巨噬细胞释放的微颗粒被DCs摄取将1×107个CFSE标记的Lm注射到小鼠腹腔,12h后冲洗腹腔收集微颗粒,大约有13%的微颗粒含有Lm成分,而这种Lm-MPs主要是由腹腔巨噬细胞释放的,因为清除了巨噬细胞后,含Lm成分的微颗粒比例下降到0.6%。将腹腔巨噬细胞释放的Lm-MPs再注射到新小鼠腹腔,6h后收集腹腔细胞染色CD11c,流式检测发现约有3%-6%的细胞CDllc+,而这些CDllc+细胞中有40%的细胞摄取了Lm-MPs。此结果说明体内感染Lm后巨噬细胞将其吞噬再以Lm-MPs的形式释放,DCs通过摄取Lm-MPs来获得Lm的抗原。7.Lm感染的巨噬细胞来源的微颗粒可以诱导抗Lm获得性免疫反应分别用巨噬细胞微颗粒和Lm感染的巨噬细胞微颗粒皮下免疫小鼠,Lm培养上清按提取微颗粒步骤处理后作为对照组,7天后尾静脉注射1×105个活Lm于小鼠体内。观察各组小鼠成活情况,发现Lm感染的巨噬细胞微颗粒免疫的小鼠生存率显著高于巨噬细胞微颗粒免疫小鼠和对照组小鼠。说明李斯特菌感染的巨噬细胞来源的微颗粒可以诱导抗李斯特菌保护性免疫反应。[结论]本研究证明诱导抗李斯特菌获得性免疫反应需要巨噬细胞的参与。巨噬细胞吞噬李斯特菌后释放出含有李斯特菌成分的微颗粒,树突状细胞主要是通过膜融合和内吞这两种方式摄取这些微颗粒,将抗原肽-MHC Ⅰ复合物直接提呈或加工抗原后再组装成抗原肽-MHC Ⅱ复合物提呈给T细胞使其活化增殖。以含有李斯特菌成分的巨噬细胞微颗粒作为疫苗免疫小鼠,可以使小鼠获得抗李斯特菌的能力。综上所述,李斯特菌感染后巨噬细胞以微颗粒形式将李斯特菌抗原传递给树突状细胞,进而提呈抗原激活T细胞,使机体获得抗李斯特菌特异性免疫。更多还原

【Abstract】 [Objective] The mutual interaction between innate and adaptive immune responses plays a crucial role in the optimal clearance of invading pathogens. Innate immune cells respond first to infection but are frequently insufficient to overcome the virulence mechanisms of pathogens; thus, the adaptive immune responses are activated. Macrophages and dendritic cells are two key innate immune cell types involved in phagocytosis and presentation of antigen respectively, upon bacterial infection. It is accepted that both macrophages and dendritic cells contribute to the activation of T cells; however, the interplay between these cells in the processing and presentation of bacterial antigens for the goal of activating T cells remains poorly understood. Using a Listeria monocytogenes (Lm) infection model, investigating a cross-talk between these two cell types. DCs play a critical role in priming adaptive immunity against Lm, whereas, macrophages fail to initiate anti-Listeria CTL responses in the absence of DC. However, DCs were not effective at directly capturing Lm, compared with macrophages. Such discrepancies suggest that a mutual interaction might exist between macrophages and dendritic cells in the processing, and presentation of antigens to T cells in the induction of protective immune response. Recent studies show that, microparticles play a important role in cell communications. It is not clear that the effection of microparticles in adaptive immune responses. In the present study, we show that both macrophages and DCs are essential for the induction of Lm-specific T cell responses but with different responsibilities. Macrophages phagocytose and release Lm antigens-containing MPs; which are subsequently captured by DCs leading to priming T cell responses.[Methods]1. To investigate the role of macrophages in eliciting the adaptive immunity against Lm infection.(1) To detect the the proliferation of adoptively transferred T cells in depleting macrophages mice in vivo.BALB/c nude mice (n=6) were i.p. injected with clodrolip or anti-F4/80depleting mAb for macrophage depletion. Then mice were adoptively transferred with CFSE-labeled T cells isolated from the spleens of Lm-infected mice or naive mice (control), and1.0×103viable Lm were injected into these mice after6h.60h later, the proliferation of adoptively transferred T cells in the spleen was determined by flow cytometry.(2) To investigate the role of macrophages in the proliferation of anti-Lm T cells in vitro.Bone marrow-derived DCs (BALB/c background) were incubated with viable Lm (100μg/ml gentamycin added) in the presence or absence of peritoneal macrophages for various time intervals (6,12, and18h).1×105splenic T cells, isolated from the spleens of Lm-infected BALB/c mice, were co-cultured with those DCs (1×104) or Lm-infected macrophages.[3H] thymidine was added after cuturing72h, then T cell proliferation was measured by liquid scintillation analyser18h later.(3) Supernatants from Lm-infected macrophage cell cultures stimulated the proliferation of T cells.T cells, isolated from the spleens of Lm-infected BALB/c mice, were incubated with DCs that had been treated with the supernatants of Lm-infected macrophages, Lm-infected DCs, or untreated DCs.[3H] thymidine was added after cuturing72h, then T cell proliferation was measured by liquid scintillation analyser18h later.2. To detect MPs from Lm-infected macrophage cell(1) Isolation of microparticlesSupernatants of cultured macrophages were used to isolate MPs as described before.16Briefly, supernatants were centrifuged at300gx5min,500gx5min,1500gx5min and5000gx5min to remove of cells and debris. The supernatant was passed through a1.2μm filter in order to remove bacteria, and then further centrifuged for60min at14000g to pellet MPs.(2) Labelling of microparticlesBacteria were stained with CFSE and used to infect macrophages. MPs isolated from macrophages were labeled with a red-fluorescent cell linker (PKH26, Sigma) according to the manufacturer’s protocol. Such fluorescent MPs were observed under two-photon fluorescent microscopy or analyzed by flow cytometr.3. Functions of microparticles(1) MPs drived from Lm-infected macrophage stimulated DCs activation.DCs were incubated with PBS, MPs from Lm infected or control macrophages for24h and were stained with CD80, CD86or MHC class Ⅱ mAb. and analyzed by flow cytometry. Bone marrow-derived DCs were stimulated with MPs from Lm-infected or control macrophages for various time intervals (0-60min). Western blot was performed for analysis of MAPK ERK and IkB phosphorylation.(2) MPs drived from Lm-infected macrophage stimulated Tcell proliferation.Macrophages were treated with different MOIs, and the isolated MPs were used to treat DCs for T-cell proliferation. 4. In vivo Listeria monocytogenes protection assay5×106macrophages in2ml culture media were treated with PBS or5x107viable Lm (100μg/ml gentamycin added30min later) for48h. Additionally,5×107viable Lm were incubated in2ml culture media alone with gentamycin. Each2ml supernatants were used for MP isolation and the quantity was used for one mouse injection. Mice were immunized subcutaneously with MPs mixed with rehydragel adjuvants for7days and challenged by i.v. injection of1×105viable Lm. Survival was monitored for10days. Six mice were used per group.[Results]1. Macrophages are required for DC-elicited anti-Lm T cell response in vivoSplenic macrophages, but not DCs, were depleted in mice by i.p. injecting liposomal clodronate or anti-F4/80antibody. Under such condition, BALB/c nude mice were adoptively transferred with T cells isolated from the spleens of Lm-infected mice. We found that the depletion of macrophages abrogated the in vivo proliferative response of adoptively transferred T cells60h after the i.v. injection of1.0×103viable Lm. To confirm these results, we transferred the Lm-infected macrophages into naive C57BL/6mice for7days and the spleen cells were cultured with killed bacteria for measurement of cytokine production by ELISA assay. As expected, the inoculation of Lm-infected macrophages into C57BL/6mice strongly induced the production of IFN-y and IL-22, two potent mediators of cellular inflammatory responses against bacterial pathogens. In addition, IFN-y-producing CD4+T cells were analyzed by FACS. These findings suggested that the initial infection of macrophages by Lm is required for the generation of protective immune responses. Thus, although macrophages do not directly present Lm peptides, they seem to participate in the induction of Lm specific T cell responses.2. Supernatants from Lm-infected macrophage cell cultures confer DC maturation and presentation of Lm antigensMacrophages were infected with viable Lm (gentamycin was added30min later) for24h and supernatants were harvested by centrifugation and filtration. And then the supernatants were incubated with DCs for24h. The expression of CD80, CD86, and MHC class II on DCs was upregulated by the supernatants from Lm-infected macrophages compared to cells incubated with supernatants from uninfected macrophages. Such DC activation was not ascribed to the bacterial contamination, since the supernatant from the above Lm alone did not affect DC maturation. Moreover, we found that DCs treated with supernatants of Lm-infected macrophages effectively induced T cell proliferation. These findings suggested that factors released from Lm-infected macrophages are capable of eliciting the maturation and conferring the immunogenicity of DCs against Lm infection.3. Microparticles shed by Lm-infected macrophages are the source for DC immunogenicityMacrophages were infected with CFSE-labeled Lm and the released MPs were isolated from the supernatants. The fluorescence was observed in MPs via both flow cytometry (27.4%CFSE positive MPs) and under the microscope, indicating the presence of Lm-derived bacterial components in MPs. Consistently, Lm component-containing MPs stimulated DC maturation and resulted in T cell proliferation. Here, we also used Lm with different multiplicity of infection (MOI) to treat macrophages and assayed the effect of isolated MPs on T cell proliferation. We found that even low numbers of Lm could result in T cell proliferationvia DC antigen presentation and that increased numbers of Lm further promoted T cell proliferation. To further analyze the effect of MPs on DCs, we examined the activation of MAP kinases and NF-κB, two critical signaling pathways involved in DC activation. DCs were stimulated with MPs from Lm-infected or control macrophages for various time intervals (10,30,60min). The activation of MAP kinase and NF-κB by MPs from Lm-infected macrophages was confirmed by the induction of ERK and IκB phosphorylation.4. Macrophages/microparticles/DCs form an axis to transfer Lm antigenicityNext, we wondered how MPs by Lm-infected macrophages transferred Lm antigenicity to DCs. By using PKH26membrane dye to stain MPs, we found that35%DCs presented red fluorescence. DCs are known to have the capacity to take up apoptotic cells. Here, we also found that phosphatidylserine was translocated to the outer layer of the membrane of MPs, the marker of apoptosis. In addition, we wanted to clarify DCs acquiring antigens pathway. MHC class I-deficient macrophages were infected with Lm to generate MHC class I-deficient MPs. These MPs significantly reduced the presentation of Lm antigen by DCs to CD8+T cells. We also found that MyD88deficiency in macrophages did not affect the activation of T cells, but MHC class II deficiency in DCs significantly reduced the presentation of MP-derived Lm antigen by DCs to CD4+T cells. These data also suggested that DCs may capture MPs through different pathways, including the membrane fusion between DCs and MPs and DC uptake and processing.5. Actin filament is required for the generation of microparticles containing Lm components by macrophagesIn this regard, we wondered whether cytoskeleton was required for the production of Lm-induced MPs by macrophages. Thus, we treated macrophages with cytochalasin D, an inhibitor of F-actin polymerization, and found that the impairment of actin filament formation resulted in the decreased of MP release by Lm-treated macrophages. We then further treated macrophages with blebbistatin, an inhibitor of myosin II ATPase activity. Similarly, the inhibition of actin filament motility also led to the decreased MP release. Consistently, it was found that after the cytochalasin D or blebbistatin treatment MPs attenuated the effect of Lm components on DCs eliciting T cell proliferation. Therefore, these findings suggest that myosin Ⅱ-triggered, actin filament-generated tension might mediate the production of Lm component-containing MPs by macrophages.6. Generation of microparticles containing Lm components by macrophages and uptake by DCs in vivoNext, we validated the in vivo generation of MPs by macrophages under the condition of Lm infection. Using a peritoneal infection model, we i.p. injected1x107CFU CFSE-labeled Lm to mice.12h later, the peritoneal lavage was applied to isolate the MPs. As expected, we found that13%MPs contained Lm components by flow cytometry. However, if we previously depleted peritoneal macrophages we found that Lm infection only resulted in0.6%CFSE positive MPs, suggesting that MPs containing Lm components are mainly generated by macrophages after peritoneal Lm infection. To clarify DCs taking up these MPs, MPs were isolated from peritoneal lavage12h after CFSE-labeled Lm peritoneal infection, and then i.p. injected into naive mice.6hours later, We harvested peritoneal cells and found that3～6%of peritoneal cells were CD11c+DCs and～40%of these cells were CFSE positive. Consistently, the result of confocal microscopy also showed that DCs took up Lm components. These findings suggested that during Lm infection in vivo, macrophages phagocytose Lm and release Lm component-packaging MPs, leading to the subsequent uptake of the released MPs by DCs.7. Microparticles from Lm-infected macrophages elicit protective immunityFinally, we wondered whether the Lm antigenicity of MPs is able to elicit protective immune response in vivo. To verify this, Mice were immunized subcutaneously with MPs from Lm-infected macrophages mixed with rehydragel adjuvant, for7days followed by challenge by i.v. injection of1×105CFU viable Lm. The results showed that most mice immunized with MPs from Lm-infected macrophages survived, as opposed to the mice immunized with control MPs. However, such protective immunity could not be ascribed to the contamination of Lm in MPs, since after the filtration of the supernatants of single Lm incubation, the centrifugated pellets had no protective effect against Lm challenge. Furthermore, in these in vivo experiments, we also used gentamicin to treat mice concomitant with MP injection. Together, these data suggested that MPs containing Lm components elicit protective immune responses.[Conclusions] In the present study, we demonstrated that DCs require the participation of macrophages to generate protective immune responses against Lm infection. MPs were released from Lm-infected macrophages, might contain Lm components. DCs may capture Lm-MPs through different pathways, including the membrane fusion between DCs and MPs and DC uptake and processing. DCs directly present MHC class Ⅰ-peptide complexes derived from Lm-MPs or process Lm antigens and assemble MHC class Ⅱ-peptidecomplexes to T cells.In conclusion, Microparticles released by Listeria Monocytogenes infected macrophages are required for dendritic cell-elicited protective immunity.更多还原