Fas信号激活树突状细胞炎性复合体形成的生物学意义与分子机制研究

发布时间：2018-05-18 20:42

本文选题：树突状细胞 + Fas　；参考：《第二军医大学》2010年博士论文

【摘要】： Fas(CD95/Apo-1)属于肿瘤坏死因子(TNF)受体超家族,一旦和其配体FasL或者活化性抗体相互作用后便能够触发细胞的凋亡。Fas介导的细胞凋亡在维持脑、眼睛和睾丸器官的免疫自稳和免疫豁免中起着至关重要的作用。Fas介导的细胞凋亡既往被认为是一个非炎症性的过程,能够引起免疫反应和炎症反应的消退。然而,越来越多的实验表明Fas也能够启动触发增殖性的和活化性的信号来促进炎症反应。已有报告显示,Fas信号能够引起一些趋化性细胞因子和炎性细胞因子的分泌。例如,Fas信号能够引起树突状细胞(dendritic cells, DCs)、早期人胎星形胶质细胞(early passage fetal human astrocytes)和人血管平滑肌细胞分泌趋化性细胞因子。而且,Fas信号也能促进巨噬细胞、纤维母细胞、上皮细胞和滑膜细胞分泌炎性细胞因子如肿瘤坏死因子α(tumor necrosis factorα, TNF-α)、IL-6和IL-8。另外,Fas信号也能引起关键性炎性细胞因子IL-1β的分泌,但其机制尚不清楚。与其他炎症性细胞因子不同的是,IL-1p是以前体的形式存在于细胞浆中的,其被Caspase-1加工、剪接后方能成为有活性的成熟形式。IL-18和IL-33也需要经过Caspase-1的加工、剪接才能成为有活性的成熟形式。最近几年的大量研究表明Caspase-1的活化需要一个称为炎性复合体(inflammasome)的分子复合物的参与。炎性复合体能识别特定的配体包括MDP (muramyl dipeptide)、ATP和尿酸,最终导致分子复合物构象改变和Caspase-1的激活。危险信号分子ATP受到关注较多,它能够结合P2X7R (purinergic P2X7 receptor)而激活NALP3炎性复合体。NLR(NOD-like receptor)家族成员包括NALP3、NALP1或IPAF参与识别危险信号以及PAMP (pathogen-associated molecular pattern)。 DCs是体内已知的效能最强的抗原提呈细胞,在启动免疫应答的过程中起着至关重要的作用。多个研究表明,DCs细胞表面表达Fas但抵抗Fas介导的凋亡,而且不依赖于它们的成熟状态。这可能和DCs高表达凋亡抵抗蛋白FLIP(FLICE-inhibitory protein)相关。我们实验室以往的研究也证明Fas信号作用于DCs后诱导了DCs的成熟和存活。因此,Fas信号在DCs中触发了不同于经典凋亡途径的信号转导,其不诱导DCs凋亡可能对于DCs在体内免疫应答启动的关键步骤即抗原提呈中发挥重要作用是有益的,但是,对于其详细的作用机制仍不甚了解。我们实验室以往的研究显示Fas信号能够诱导DCs分泌IL-1β,这提示我们Fas信号可能和炎性复合体存在关联。在本研究中,我们发现Fas的活化性抗体Jo-2作用于DCs后,引起内源性的ATP释放。释放的ATP以自分泌的方式作用于DCs细胞表面的P2X7R,触发胞内信号转导引起NALP3炎性复合体的激活,最终导致 Caspase-1的活化、IL-1β的加工剪接与分泌,导致DCs的成熟与活化。 1.Fas信号通过激活NALP3炎性复合体而诱导DCs分泌IL-1β 为详细全面地研究Fas信号对DCs功能的影响,我们首先检测了Fas信号是否诱导DCs的凋亡。在我们的试验体系中,我们使用Fas的活化性抗体Jo-2作用于经CD11c磁珠阳性分选过的DCs。与以前的报道结果一样,我们发现imDCs(immatureDCs)和mDCs(mature DCs)均对Fas介导的细胞凋亡不敏感,高浓度的Jo-2作用24h也不能诱导DCs的凋亡。而作为阳性对照的胸腺细胞对Jo-2诱导的凋亡很敏感,并且Fas缺陷小鼠来源的胸腺细胞对Jo-2诱导的细胞凋亡表现为抵抗。这说明与胸腺细胞不同,小鼠来源的DCs无论成熟状态与否均对Fas介导的细胞凋亡不敏感。接着我们观察了Jo-2对DCs分泌细胞因子的影响。结果发现Jo-2不能诱导炎性细胞因子TNF-α和IL-6的分泌,但能够特异性地诱导IL-1β的分泌,对照抗体对细胞因子分泌没有任何影响,FasL诱导细胞因子分泌的效应和Jo-2相似。Jo-2诱导的IL-1β分泌可以被Caspase-1特异性的抑制剂YVAD所抑制。我们利用Westernblot的方法证实了Jo-2作用于DCs后可以引起Caspase-1的活化,由于Caspase-1的活化是炎性复合体激活的标志,这提示Fas信号和炎性复合体相关联。有趣的是,我们发现DCs用LPS处理16h后再用Jo-2刺激6h可以更加显著地增强IL-1β的分泌,并且Jo-2的此种增强效应可以被Caspase-1特异性的抑制剂YVAD或者ATP通道拮抗剂oATP所抑制。众所周知,LPS可以显著促进NALP3炎性复合体前体成分的合成,Jo-2的这种显著增强LPS处理过DCs分泌IL-1β的效应显然和炎性复合体的活化有关并且和ATP密切关联。为了进一步明确Fas信号和炎性复合体的关系,我们利用实时定量PCR的方法检测了Jo-2对炎性复合体成分表达的影响。结果发现,Jo-2可以明显促进NALP3炎性复合体中NALP3和IL-1β的表达,而对ASC和Caspase-1的表达没有影响。而且Jo-2诱导的NALP3和IL-1βmRNA的表达可以被NF-κB特异性抑制剂PDTC所抑制,这提示Jo-2通过活化NF-κB信号通路来促进炎性复合体相关成分的表达。我们利用Western blot的方法证实了Jo-2作用于DCs后可以引起NF-κB上游抑制蛋白IκB的磷酸化以及降解,这就导致NF-κB与IκB的解离,并使得NF-κB进入细胞核中与相应的靶序列结合促进基因的表达。为了进一步明确Fas信号与NALP3炎性复合体的关系,我们利用基因沉默的方法干扰NALP3炎性复合体相关成分的表达,然后观察Jo-2诱导的IL-1β分泌是否依赖于NALP3炎性复合体。结果发现,用siRNA干扰NALP3炎性复合体的表达后,LPS和Jo-2诱导的IL-1β分泌受到明显的抑制。表明Fas信号诱导的IL-1β分泌是通过NALP3炎性复合体来实现的。 2.Fas信号通过促进内源性ATP的释放而激活NALP3炎性复合体由于我们的试验结果提示Jo-2诱导的IL-1β分泌和ATP相关,因此我们进一步·用报告基因的方法检测了Fas信号对ATP分泌的影响。结果发现LPS能够引起ATP的释放,而FasL或Jo-2刺激能够引起大量的ATP释放,pan Caspase抑制剂zVAD对Jo-2引起的ATP分泌没有影响,但ATP酶的抑制剂ARL能够增强Jo-2引起的ATP分泌一倍左右。这提示Jo-2能够引起内源性的ATP释放,并和细胞死亡没有关联。相应的,我们也发现LPS和Jo-2能够IL-1p的分泌,并且被pan Caspase抑制剂zVAD抑制,这可能和Caspase-1活化受抑制有关。而ARL相应的增强IL-1β的产生。LDH释放结果显示LPS和Jo-2不能引起LDH的释放,以上结果提示Jo-2在不影响细胞存活的情况下能够特异性诱导细胞内源性的ATP释放。胞外的ATP作用于细胞表面的P2X7R后能够触发细胞内的信号转导通路,最终引起NALP3炎性复合体的活化。我们接下来研究了P2X7R在Fas信号诱导IL-1β分泌中的作用。我们发现预处理P2X7R抑制剂后,Jo-2诱导的IL-1p分泌受到明显的抑制,同时伴随着DCs的成熟(lab,CD86)也受到抑制。我们通过试验证实了IL-1β在Jo-2诱导的DCs成熟中起着至关重要的作用,而P2X7R抑制剂即是通过抑制IL-1β分泌来影响Jo-2诱导的DCs成熟。接下来我们研究了ATP作用于DCs后引起的细胞信号通路变化。我们通过Western blot的方法证实了Jo-2作用于DCs后可以引起ERK信号通路显著活化。而给予P2X7R抑制剂后能够剂量依赖性的降低Jo-2引起的ERK信号通路的活化以及Caspase-1的活化,这提示ATP信号作用于ERK和Caspase-1的上游。另外,给予ERK信号通路抑制剂PD98059能剂量依赖性的抑制ERK以及Caspase-1的活化,可见ERK信号作用于Caspase-1的上游。相应的PD98059和Caspase-1特异性的抑制剂YVAD能够特异性的抑制IL-1β的分泌和DCs的成熟。综上,ATP-P2X7R-ERK-Caspase-1通路在Fas信号诱导DCs分泌IL-1β以及由其引起的DCs成熟中发挥着至关重要的作用。 3.Fas信号通过诱导内源性ATP释放和IL-1β产生而参与DC激活T细胞的作用体内ATP的来源一直不是很清楚,因为ATP一旦分泌到细胞外便被胞外的ATP酶迅速降解,其浓度受到严格的控制。由于我们发现Fas信号可以促进大量ATP的释放,而DC-T相互作用时也有FasL-Fas之间的相互作用,因此我们推测在DC-T相互作用时可能会引起大量ATP的释放并参与DC-T相互作用。我们在抗原肽特异性T细胞增殖体系中研究了DC-T相互作用时ATP可能起的作用。我们在DC-T共培养体系中加入了P2X7R抑制剂KN-62以及ATP酶apyrase,培养48h后检测上清中IL-1β的水平。结果发现KN-62和apyrase能够明显抑制IL-1β的分泌,提示在DC-T相互作用时ATP参与IL-1β的产生。另外,KN-62和apyrase也明显抑制了IL-2和IFN-7的分泌,而且KN-62的抑制效应可以被外源性的重组IL-1β部分逆转。更为重要的是,KN-62和apyrase也能够明显抑制抗原肽特异性的T细胞增殖,而外源性的重组IL-1β可以部分逆转KN-62的抑制效应。这些结果提示ATP参与调控了DC-T相互作用时IL-1β的产生,并间接性的影响其它细胞因子的分泌,最终影响了抗原肽特异性的T细胞的增殖。为了更直接的证明在DC刺激T细胞增殖时有ATP存在与参与,我们用报告基因的方法检测了DC-T培养上清中ATP的分泌情况。结果发现,在抗原肽存在的条件下,一旦DCs和T细胞共孵育,上清中ATP的浓度迅速增高,在第四个小时达到高峰(约100nM),然后迅速回落,维持在50nM左右的水平达40个小时以上。DCs的Fas缺陷对ATP分泌的高峰没有影响,但在维持后期ATP的浓度时发挥重要作用。另外,培养上清中的LDH没有观察到明显的释放。可见,在DCs刺激T细胞增殖时有高浓度的ATP存在并发挥重要作用,而DCs的Fas信号在ATP的释放中也起关键作用。综上所述,Fas信号可以通过促进DCs内源性ATP的释放从而触发ATP-P2X7R-ERK-Caspase-1-IL-1β通路导致IL-1β的成熟、释放,释放后的IL-1β可以以自分泌的方式作用于DCs本身促进DCs的成熟与活化,最终促进抗原特异性T细胞的增殖。本研究发现了Fas信号参与炎症反应的直接证据,对于DCs参与免疫调控提出了新的学术观点,将有助于更好地理解FasL-Fas系统与炎症之间错综复杂的关系以及为某些炎症性临床疾病发病机制的研究提供了理论基础,也为相关疾病的治疗提供了潜在的靶标。
[Abstract]:Fas ( CD95 / Apo - 1 ) belongs to the superfamily of tumor necrosis factor ( TNF ) receptors and can trigger apoptosis of cells after interaction with its ligand FasL or activated antibody . Fas - mediated apoptosis plays a crucial role in maintaining immune homeostasis and immune immunity of brain , eye and testis organs . However , it has been shown that the Fas signal can trigger inflammatory cytokines such as tumor necrosis factor 伪 ( TNF - 伪 ) , IL - 6 and IL - 8 . The Fas signal can also induce the secretion of inflammatory cytokines such as tumor necrosis factor 伪 ( TNF - 伪 ) , IL - 6 and IL - 8 .

IL - 18 and IL - 33 are involved in the activation of caspase - 1 . IL - 18 and IL - 33 also need to be processed by Caspase - 1 . IL - 18 and IL - 33 also need to be involved in the activation of the active mature form . The inflammatory complex can recognize that the activation of Caspase - 1 requires the activation of a molecule complex known as an inflammatory complex . A large number of studies have shown that the activation of Caspase - 1 requires the activation of a molecule complex known as an inflammatory complex . A large number of studies in recent years have shown that activation of the NALP3 inflammatory complex is required by the activation of an NALP3 , NALP1 or IPAF , and the NALP3 , NALP1 or IPAF is involved in the identification of the risk signal and the PAMP ( pathogen - associated molecular pattern ) .

DCs are the most potent antigen - presenting cells known in vivo and play a crucial role in the initiation of immune responses . Several studies have shown that the expression of Fas on DCs induces apoptosis in DCs and does not depend on their maturation .

In our study , we found that the expression of Fas signal can induce the secretion of IL - 1尾in DCs , which suggests that the Fas signal may be associated with inflammatory complex . In this study , we find that the activation antibody Jo - 2 of Fas acts on DCs , which causes endogenous ATP release . The released ATP acts on the surface of DCs in self - secretion , triggering intracellular signal transduction to induce the activation of NALP3 inflammatory complex , which ultimately leads to the activation of the NALP3 inflammatory complex .

The activation of Caspase - 1 , the processing of IL - 1尾 and the secretion of IL - 1尾 result in maturation and activation of DCs .

1 . The Fas signal induces DCs to secrete IL - 1beta by activating the NALP3 inflammatory complex .

In order to investigate the effect of Fas signal on the function of DCs in detail , we first examined whether the Fas signal induced apoptosis of DCs . In our trial system , we found that both imDCs and mDCs were insensitive to Fas - mediated apoptosis .

The results showed that Jo - 2 could not induce the secretion of TNF - 伪 and IL - 6 , but it could induce the secretion of IL - 1尾 . The effect of Jo - 2 on the secretion of IL - 1尾could be enhanced by the activation of Caspase - 1 . The effect of Jo - 2 on the secretion of IL - 1尾could be significantly enhanced by the activation of Caspase - 2 . The effect of Jo - 2 on IL - 1尾 secretion was obviously related to activation of inflammatory complex and was closely associated with ATP .

In order to further clarify the relationship between the expression of NALP3 and IL - 1尾in NALP3 , the expression of NALP3 and IL - 1尾in NALP3 was significantly promoted by the method of real - time quantitative PCR .

2 . Fas signal activates NALP3 inflammatory complex by promoting release of endogenous ATP

We also found that LPS and Jo - 2 were able to induce ATP release .

We found that the secretion of IL - 1尾induced by Jo - 2 was significantly inhibited and the maturation of IL - 1尾was inhibited by inhibiting IL - 1尾secretion .

In addition , the ERK signal pathway inhibitor PD98059 can inhibit the activation of ERK signal pathway and the activation of Caspase - 1 . It is suggested that the ERK signal pathway inhibitor PD98059 can inhibit the secretion of IL - 1尾and the maturation of DCs .

3 . Fas signal is involved in DC - activated T cells by inducing endogenous ATP release and IL - 1尾 production

The source of ATP in vivo has not been clear since ATP is rapidly degraded by extracellular ATP once secreted outside of the cell , and its concentration is tightly controlled . As we find that the Fas signal can promote the release of a large amount of ATP , and the interaction between FasL - Fas in the DC - T interaction , we suggest that a large amount of ATP can be released and involved in the DC - T interaction at the time of the DC - T interaction .

It was found that the presence and absence of ATP involved in the production of IL - 1尾in DC - T co - culture system . The results suggested that KN - 62 and apytosis could significantly inhibit the secretion of IL - 1尾 . The results suggested that KN - 62 and apytosis could significantly inhibit the secretion of IL - 1尾 . In addition , KN - 62 and apytosis could significantly inhibit the secretion of IL - 1尾. In addition , KN - 62 and apytosis could significantly inhibit the secretion of IL - 1尾 .

In conclusion , the expression of Fas signal can trigger the maturation , release and release of IL - 1beta by promoting the release of endogenous ATP in DCs . The direct evidence of the involvement of the Fas signal in DCs per se can promote the maturation and activation of DCs . It is helpful to understand the complex relationship between FasL - Fas system and inflammation and provide a theoretical basis for the study of pathogenesis of certain inflammatory clinical diseases , and also provides a potential target for the treatment of related diseases .
【学位授予单位】：第二军医大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R392

【共引文献】