【作者】 刘海波；

【导师】 曹雪涛；

【作者基本信息】 第二军医大学 ， 免疫学， 2010， 博士

【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。更多还原

【Abstract】 Fas (CD95/Apo-1), a representative of tumor necrosis factor (TNF) receptor superfamily, triggers cell apoptosis following engagement by Fas ligand (FasL, CD95 ligand, TNFSF6) or by agonistic anti-Fas antibodies. It is generally accepted that Fas-mediated apoptosis is essential for the maintenance of immune homeostasis and immune privilege in brain, eye and testis. Fas-mediated apoptosis is considered to be a non-inflammatory process, leading to the resolution of immune and inflammatory responses. However, accumulating evidence indicates that Fas also initiates proliferative and activating signals, contributing to inflammatory responses. It is now well established that Fas signal can trigger the secretion of chemotactic factors and proinflammatory cytokines. Fas ligation has been reported to stimulate chemokine production by dendritic cells (DCs), early passage fetal human astrocytes and human vascular smooth muscle cells. Moreover, Fas also triggers the production of inflammatory cytokines such as TNFa, IL-6 and IL-8 in macrophages, fibroblasts, epithelial cells and synoviocytes. In addition, activation of CD95 also triggers the secretion of key inflammatory cytokine IL-1β. As yet the mechanisms how Fas ligation triggers the secretion of IL-1βremain elusive.In contrast to other inflammatory cytokines, IL-1βis produced as inactive cytoplasmic precursors (pro-IL-1β), which is cleaved by caspase-1 (IL-1β-converting enzyme [ICE]) to its mature active form. IL-18 and IL-33 also need to be processed by caspase-1 to become their active forms as IL-1β. It is well documented that caspase-1 activation requires a molecular complex termed the "inflammasome". Inflammasome recognizes specific ligands such as muramyl dipeptide, ATP or uric acid, leading to conformational change of the complex and activation of caspase-1. ATP, a well known danger signal, binds purinergic P2X7 receptor (P2X7R) to trigger NALP3 inflammasome activation. NOD-like receptor family members such as NALP3, NALP1 or IPAF is responsible for the detection of the danger signals or PAMPs (pathogen-associated molecular patterns).Dendritic cells (DCs) are professional antigen-presenting cells (APCs), which are crucial for initiating immune responses. Several studies have demonstrated that DCs, irrespective of their maturation state, are resistant to Fas-mediated apoptosis even though the existence of Fas on the surfaces of DCs. Increased expression of the anti-apoptotic protein FLICE-inhibitory protein (FLIP) may account for the resistance of DCs to Fas-mediated apoptosis. Our previous studies also showed that Fas ligation induces DCs maturation and survival instead of apoptosis. Thus, Fas signals an alternative pathway rather than the classical apoptotic signaling pathway in DCs, which may benefit DCs to fulfill antigen presentation in initiating immune response in vivo. However, Fas-triggered non-apoptotic signaling pathway in DCs remains unclear.Our previous studies showed that Fas ligation on DCs triggers the secretion of IL-1p, and this encourages us to explore whether Fas signal induces inflammasome assembly. In the present study, we demonstrate that Fas ligation by the agonistic anti-Fas monoclonal antibody (mAb) Jo-2 causes the release of endogenous ATP from DCs. Then the released ATP activates P2X7R in DCs via autocrine loop and subsequently triggers a series of signaling pathway that induces NALP3 inflammasome activation, leading to IL-1βsecretion and maturation of DCs.1. Fas signal induces IL-1βsecretion from DCs by activating NALP3 inflammasomeTo investigate the role of Fas signal in DCs functions, we first tested whether Fas signal induces apoptosis in DCs. In our experimental context, we used the agonistic anti-Fas monoclonal antibody (mAb) Jo-2 to stimulate CD11c positive DCs derived from bone marrow. In accordance with previous studies, we found that Jo-2, even in a high concentration with 24 hours incubation, failed to induce apoptosis in both immature DCs (imDCs) and mature DCs (mDCs). In contrast, the thymocytes, as positive control, were sensitive to Jo-2-induced cell apoptosis, but not thymocytes from Fas deficient mice. These results suggest that DCs, irrespective their maturation state, were not sensitive to Fas-mediated apoptosis.We next examined the effects of Jo-2 on the cytokine production from DCs. We showed that Jo-2 specifically elicited IL-1βproduction but not TNF-a or IL-6 from DCs. The control antibody showed no any effects on cytokine production from DCs. FasL showed similar effects on cytokine production from DCs as Jo-2. Interestingly, IL-1βproduction induced by Jo-2 stimulation could be inhibited by Caspase-1 specific inhibitor YVAD. We confirmed the Caspase-1 activation induced by Jo-2 stimulation using Western blot assay. Since the Caspase-1 activation is a hallmark of inflammasome activation, we presumed a link between Fas signal and inflammasome in DCs. More interestingly, we found that Jo-2 stimulation induced a striking increase in IL-1βproduction from LPS-primed DCs, and the increase in IL-1βproduction could be inhibited by Caspase-1 specific inhibitor YVAD or ATP channel antagonists. It is well known that LPS turns on the gene expression of components of NALP3 inflammasome. Jo-2 induced large amounts of IL-1βproduction from LPS-primed DCs may be associated with inflammasome activation and ATP channel P2X7R.To elucidate the association of Fas signal and inflammasome, we performed Real-time PCR analysis to measure the effects of Jo-2 on inflammasome expression. Real-time PCR analysis showed that Jo-2 increased the expression of NALP3 and IL-1β, but did not affect ASC and Caspase-1 mRNA expression. NF-κB specific inhibitor PDTC blocked the increase in NALP3 and IL-1βmRNA expression induced by Jo-2 stimulation. Therefore, Jo-2 stimulates the expression of NALP3 inflammasome components via NF-κB signaling pathway. We confirmed Jo-2 induced NF-κB activation as evidenced by the phosphorylation and degradation of NF-κB inhibitor protein IκB. To further investigate the relationship of Fas signal and NALP3 inflammasome activation, we measured Jo-2 stimulation induced IL-1βproduction after knockdown of components of NALP3 inflammasome. We found that IL-1βproduction induced by both LPS and Jo-2 was significantly inhibited after knockdown of NALP3 inflammasome components. These results suggest that Fas-signal induced IL-1βproduction was dependent on NALP3 inflammasome.2. Fas signal triggers the release of endogenous ATP to activate NALP3 inflammasomeSince our results indicate that Jo-2 induced IL-1 p secretion was correlated with ATP, we next examined the effects of Fas signal on ATP release using reporter gene assay. We found that LPS elicited ATP release from DCs, whereas FasL or Jo-2 induced large amounts of ATP release that could not be inhibited by pan Caspase inhibitor zVAD. Ecto-ATPase inhibitor ARL increased ATP concentration about two folds. These results suggest that Jo-2 elicited endogenous ATP release which was not correlated with cell death. Accordingly, LPS and Jo-2 induced IL-1βproduction that could be inhibited by pan Caspase inhibitor zVAD, this may associate with Caspase-1 inhibition. ARL increased IL-1βproduction from DCs induced by LPS and Jo-2 stimulation. LDH release assay showed that both LPS and Jo-2 failed to trigger LDH release from DCs. These results demonstrate that Jo-2 specifically elicits endogenous ATP release from DCs without inducing cell death.Extracellular ATP engages on P2X7R on cell surface to initiate intracellular signal transduction, leading to NALP3 inflammasome activation. We next focused on the role of P2X7R in Fas signal-induced IL-1βsecretion. Pretreatment DCs with P2X7R specific inhibitors compromised Jo-2-induced IL-1βsecretion and DCs maturation as indicated by lab and CD86 staining. We confirmed the prerequisite role of IL-1βduring Jo-2-induced DCs maturation. Actually, the antagonists of P2X7R inhibited Jo-2-induced DCs maturation via inhibition of IL-1βsecretion.We next explored signal transduction pathways in DCs elicited by ATP. Western blot assay showed that Jo-2 stimulation induced potent ERK activation after engagement on DCs. Whereas P2X7R antagonists could reduce Jo-2-induced both ERK and Caspase-1 activation dose dependently, indicating that ATP acted upstream of ERK and Caspase-1. In addition, ERK specific inhibitor PD98059 also inhibited Jo-2-induced both ERK and Caspase-1 activation dose dependently. Therefore, ERK signaled upstream of Caspase-1. Accordingly, both PD98059 and Caspase-1 inhibitor YVAD inhibited IL-1βproduction and DCs maturation. In sum, we showed that the signaling axis ATP-P2X7R-ERK-Caspase-1 was required for Fas signal-induced IL-1βsecretion and DCs maturation.3. Fas signal promotes DCs to prime T cells by inducing endogenous ATP release and IL-1βsecretionThe source of ATP in vivo remains elusive since ATP is quickly degraded by ATPase once secreted into extracelluar compartment. The concentration of ATP is tightly controlled in vivo. Since our results suggested that Fas signal triggered the release of endogenous ATP, we presumed that FasL-Fas cross-linking may play important role in DC-T interaction by inducing ATP release.We examined the role of ATP in DC-T interaction by detecting peptide-specific T cell proliferation triggered by peptide-pulsed DCs. We introduced P2X7R antagonist KN-62 and ATPase (apyrase) into the DC-T coculture system. ELISA assay showed that both KN-62 and apyrase significantly inhibited IL-1βproduction during DC-T cognate interaction. In addition, KN-62 or apyrase also impaired the production of IL-2 and IFN-γ, and the inhibitory effects on cytokine production by KN-62 could be partially restored by recombinant IL-1β. More importantly, both KN-62 and apyrase also inhibited peptide specific T cell proliferation, and the inhibition of T cell proliferation by KN-62 could be restored by recombinant IL-1β. These results suggest that ATP involves in the initiation of antigen-specific T cell proliferation via regulation of IL-1βor other cytokine production during DC-T cognate interaction. To directly document the presence of ATP during the priming of T cells by DCs, we measured the ATP level in the supernatants of DC-T co-culture system. We found that ATP was released quickly into the DC-T co-culture supernatant and peaked (approximately 100nM) at four hours in the presence of antigen specific peptide, then decreased and maintained at almost 50nM level for more than 40 hours. Fas signal in DCs was not required for the peak of ATP release but was crucial in the maintenance of ATP concentration in the later period. In addition, we did not found evident LDH release into the DC-T cu-culture supernatant. Therefore, our data showed the presence of ATP during the priming T cells by DCs, and Fas signal in DCs is crucial for ATP release, and consequently Fas signal-induced ATP release may contribute to the priming of T cells by DCs.In conclusion, Fas signal triggers the release of endogenous ATP from DCs, which functions in an autocrine manner to initiate the axis ATP-P2X7R-ERK-Caspase-1-IL-1βwith resultant IL-1βsecretion. IL-1βalso acts through an autocrine manner to promote the maturation of DCs, leading to the enhanced proliferation of antigen specific T cells. Our study provides the direct evidence of Fas signal in inflammation and raises new view of DCs in immune regulation. This will contribute to a better understanding of the relationship between FasL-Fas and inflammation, and provide insight into the pathogenesis of inflammation-associated diseases. Moreover, our study might also identify potential targets for therapeutic intervention of inflammation-associated diseases.更多还原