【作者】 侯昕；

【导师】 田志刚；

【作者基本信息】 中国科学技术大学 ， 细胞生物学， 2009， 博士

【摘要】 肝脏作为一个天然免疫器官,包含了众多的天然免疫细胞,如巨噬细胞、NK细胞和NKT细胞。这些细胞大量聚集在肝脏中,表明它们在肝脏生物学中具有重要的地位,能够为机体提供抵御肿瘤和外来抗原入侵的免疫监视及保护。但是近年来的研究表明过度的免疫细胞活化有时可能对肝细胞进行意想不到的自身攻击,引起自身免疫性的肝脏损伤。Kupffer细胞被细菌的外毒素(LPS)活化后能分泌大量的炎性细胞因子介导肝脏损伤;NKT细胞被刀豆蛋白A(ConA)和α-半乳糖神经酰胺(α-Galcer)激活后通过分泌细胞因子和直接杀伤肝实质细胞导致肝脏严重的损伤。NK细胞同样是肝脏中重要的天然免疫细胞,占肝脏淋巴细胞总数的30%-40%。很多研究提示NK细胞参与了人类肝炎发病的过程,在小鼠模型的研究中也发现NK细胞过度活化会导致肝脏损伤,但是NK细胞通过哪些机制介导肝脏损伤尚未研究清楚。本研究通过给小鼠同时注射poly I:C(1ug/只)和D-GalN(10mg/只),建立了一种比较新的爆发性肝炎的模型,有利于深入探讨肝脏天然免疫细胞在肝脏免疫损伤中的作用及机理。我们采用赖氏法检测血清转氨酶ALT水平、组织H-E染色观察肝脏病理学变化,并利用上述指标综合评估肝细胞损伤程度;通过细胞清除研究特定淋巴细胞群在肝脏损伤过程中的作用;采用流式细胞术测定各淋巴细胞亚群的表型,活化状态以及细胞因子分泌功能;用ELISA法和实时定量PCR法检测肝脏中、血清中以及细胞培养上清中细胞因子的表达水平;我们用MACS磁珠分离方法分离纯化肝脏中的NK细胞和Kupffer细胞,并且在体外poly I:C刺激下共同培养,同时在体内和体外运用特异抗体中和实验观察NK细胞和Kupffer细胞间相互作用的分子机制。通过上述研究方法取得的结果如下:1.poly I:C能够在D-GalN鼠中引起严重的肝脏损伤D-GalN致敏的小鼠接受poly I:C注射后爆发严重的肝脏损伤。联合注射18小时后肝脏转氨酶水平可以达到14560±2910 IU/L,直到48小时才基本恢复到正常水平。通过HE染色可以看到poly I:C和D-GalN联合注射鼠肝脏有局部充血和肝细胞的坏死。这种严重的肝损伤现象只在poly I:C和D-GalN联合注射鼠中可见,单独注射poly I:C或者D-GalN都不会引起小鼠转氨酶的升高和肝脏的坏死。而且poly I:C/D-GalN诱导的损伤是肝脏特异性的,小鼠的肺脏、结肠和小肠都没有明显的损伤。2.poly I:C和D-GalN诱导的爆发性肝损伤是NK细胞介导的流式细胞检测结果表明,poly I:C/D-GalN能够诱导大量的NK细胞在肝脏局部聚集,而且这些NK细胞活化程度增强,表现在细胞表面CD69分子表达水平升高。体内清除NK细胞后,poly I:C/D-GalN诱导的肝脏损伤明显降低。Poly I:C/D-GalN同样能够在SCID鼠中引发严重的肝脏损伤,这一结果进一步证明了NK细胞是该模型中导致肝脏损伤的重要效应细胞,而且其发挥作用时不依赖T细胞和B细胞的协助。3.在poly I:C和D-GalN联合注射小鼠中NK细胞的活化依赖Kupffer细胞的存在由脂质体介导的巨噬细胞清除实验表明,poly I:C和D-GalN联合注射诱导的肝脏损伤是依赖Kupffer细胞的。Kupffer细胞被清除后,poly I:C/D-GalN不能够引起血清中转氨酶水平的急剧升高。和Kupffer细胞未清除小鼠相比,polyI:C/D-GalN注射后,肝脏中NK细胞的比例降低,分泌IFN-γ的NK细胞也明显减少。4.NK细胞分泌的IFN-γ和Kupffer细胞分泌的TNF-α协同诱导肝脏损伤ELISA检测结果显示,poly I:C/D-GalN注射后,血清中IFN-γ和TNF-α的量显著增多,在18小时达到顶峰,48小时回复到正常水平,这和转氨酶的变化是一致的。而定量PCR的结果表明,在肝脏局部,IFN-γ的RNA转录水平2小时就开始升高,4小时到峰值,24小时回复到正常水平;TNF-αRNA的转录水平则在2小时就可以达到峰值,48小时回落到正常值。中和体内的TNF-α后,poly I:C/D-GalN诱导的肝脏损伤明显减弱。给IFN-γ缺陷鼠注射poly I:C/D-GalN后,血清中转氨酶的水平较野生型鼠也显著降低。表明IFN-γ和TNF-α在该模型中参与了肝脏损伤的形成。给予外源性重组IFN-γ的实验表明,D-GalN能够使肝脏对IFN-γ造成的损伤更加敏感。而且给D-GalN致敏鼠注射IFN-γ和TNF-α时,两种细胞因子同时注射诱导的肝脏损伤远大于单独注射其中一种细胞因子引发的损伤,表明IFN-γ和TNF-α在介导肝脏损伤时有协同效应。另外,胞内细胞因子染色实验证明,IFN-γ主要是由肝脏中的NK细胞分泌的,和T细胞、NKT细胞无关。当用抗体将NK细胞清除后,poly I:C/D-GalN诱导的IFN-γ水平就显著降低;而免疫荧光染色实验则证明TNF-α主要是由肝脏中的Kupffer细胞分泌的,清除Kupffer细胞后可以明显降低血清中TNF-α的含量。5.活化后的NK细胞和Kupffer细胞通过NKG2D/Rae 1相互作用通过RT-PCR方法对poly I:C/D-GalN注射后肝脏组织的检测,我们发现NKG2D的配体Rae 1转录水平升高,另一个配体Mult 1的RNA转录水平基本没有变化。进一步的流式结果证明了poly I:C/D-GalN注射后,肝脏中Kupffer细胞表达Rae1,NK细胞表面的NKG2D表达没有变化。阻断小鼠体内NKG2D通路后,poly I:C/D-GalN诱导的肝脏损伤明显减弱,血清中IFN-γ的含量也显著降低。体外纯化的NK细胞和Kupffer细胞共同培养实验进一步证明了Kupffer细胞依赖NKG2D和Rae 1的识别促进NK细胞分泌IFN-γ。6.IL-12和IL-18也参与了NK细胞和Kupffer细胞之间的相互作用ELISA实验显示poly I:C/D-GalN能够诱导肝脏局部和血清中IL-12和IL-18的表达水平升高,在注射后12小时和18小时明显高于0小时,提示IL-12和IL-18可能参与了肝脏损伤的过程。体外NK细胞和Kupffer细胞共培养实验进一步证明,分别中和IL-12和IL-18后,poly I:C刺激产生的IFN-γ明显降低,预示IL-12和IL-18在NK细胞和Kupffer细胞的相互作用中起到了积极的作用。结论:本文描述了一个NK细胞介导的小鼠爆发性肝炎模型,这对于已经存在的巨噬细胞介导的肝炎模型和NKT细胞介导的肝炎模型是个很好的补充。同时,本文首次描述了肝脏中的NK细胞和巨噬细胞通过Rae1和NKG2D的识别协同诱导肝脏损伤。该发现对于研究天然免疫识别在肝脏损伤中的作用有一定的提示,对于控制、治疗天然免疫细胞诱发的肝脏疾病有指导意义。更多还原

【Abstract】 The role of liver as a major organ of the innate immune system has been increasingly recognized.The liver lymphocyte population is enriched in macrophages（Kupffer cells）,natural killer and natural killer T cells,which constitute the innate immune system and play a critical role in immune defense against tumors and microbial pathogen.More and more evidences also suggest the contribution of these innate immune cells in the pathogenesis of hepatitis.Kupffer cells can produce various inflammatory mediators that may damage hepatocytes upon activation by bacterial products,such as endotoxin（lipopolysaccharide,LPS） NKT cells are also pivotal in Con A- and alpha-galactosylceramide（α-Galcer）-mediated liver damage via cytokineds and direct cytotoxicity against hepatocytes.NK cells are another population of innate immune cells in the liver,comprising 30-40%of total intrahepatic lymphocytes in human,and 10-20%in mice.The contribution of NK cells in the pathogenesis of human hepatitis or animal models has been reported,but the role and mechanisms remain to be further investigated.In this study,to induce liver injury,we injected mice intravenously with poly I:C （1ug/mice） and intraperitoneally with D-GalN（10 mg/mice）.Then we examined the liver injury by determining serum transaminase ALT levels and liver pathologic changes.By cell depletion,effects of the specific lymphocyte population and cellular interactions in the process of liver injury were investigated.The FACS analysis provided us with the precise information about lymphocyte phenotype,activation and cytokine production.Hepatic NK cells and Kupffer cells were isolated by positive magnetic cell sorting（MACS） according to the manufacyure’s protocol,and were coculture in vitro with poly I:C stimulation.Moreover,specific neutralization with antibody was applied in vivo and in vitro to explore the potential mechanisms involved in cell interaction.We used ELISA and real time PCR to examine cytokine levels.Our major findings are shown as followed:1.Treatment with poly I:C induces severe liver injury in D-GalN-sensitized miceIn this study,we found that poly I:C could induce severe liver injury in D-GalN sensitized mice,which differed from the mild liver injury of our previous study, where poly I:C alone was injected.Co-administration with poly I:C and D-GalN induced significant elevation of serum ALT and histological necrosis in the liver,but injection with poly I:C or D-GalN alone did not cause any liver injury.We also found that the same amount of poly I:C/D-GalN did not induce any injury in other organs,such as the lungs,colon,and small intestine.2.Poly I:C/D-GalN-induced severe liver injury is NK cell-dependentSince we previously found that NK cells could be activated by poly I:C in vivo and played important roles in poly I:C-induced mild liver injury,we examined the role of NK cells in poly I:C/D-GalN co-administration model.The results showed that treatment with poly I:C/D-GalN induced the accumulation and activation of NK cells in the liver,and depletion of NK cells before poly I:C/D-GalN administration could significantly prevent liver injury.Further,the severe liver injury in SCID mice triggered by poly I:C/D-GalN proved that NK cells could mediate poly I:C/D-GalN-induced liver injury without the help of B cells and T cells.3.NK cell activation depends on the presence of Kupffer cells in poly I:C/D-GalN-treated miceMacrophages express abundant TLRs that enable them to sense the presence of pathogens,and are pivotal in several murine models of hepatitis.To examine the role of Kupffer cells in this model,we depleted Kupffer cells with clodronate-liposomes and verified the effective depletion using immunofluorescent staining with macrophage-specific mAb anti-F4/80.Poly I:C/D-GalN-induced liver injury was markedly reduced in Kupffer cell-depleted mice,indicating that Kupffer cells were indispensable in this model.Further,depletion of Kupffer cells significantly inhibited the accumulation of NK cells in the liver,and suppressed production of IFN-γby NK cells.These results suggested that Kupffer cells were involved in the accumulation and activation of NK cells induced by poly I:C/D-GalN.4.Poly I:C/D-GalN-induced liver injury results from the synergic effects of NK cell-derived IFN-γand Kupffer cell-derived TNF-αIn accordance to the time course of poly I:C/D-GalN-induced liver injury, cytokine levels in the serum and liver up to 48 hours after co-administration were measured by ELISA and real-time RT-PCR,respectively.The levels of TNF-αand IFN-γin the serum increased significantly at 18 hours post poly I:C/D-GalN administration.IFN-γand TNF-αmRNA levels in liver tissue preceded the serum peak by approximately 14 hours and 16 hours,respectively.To evaluate the roles of increased TNF-αand IFN-γin liver injury,we injected poly I:C/D-GalN into IFN-γ^-/- or TNF-α-neutralized mice.The results showed that both TNF-αand IFN-γwere critical in this injury.Further,as a supplement to previous conclusion that D-GalN-treated mice were more sensitive to TNF-α-mediated liver injury,we proved that D-GalN-treated mice were also very sensitive to IFN-γ-mediated liver injury. Co-administration of IFN-γand TNF-αcould cause much more severe liver injury than either cytokine alone,suggesting that TNF-αand IFN-γhad a synergic effect on liver injury.The study demonstrated that after poly I:C/D-GalN injection,IFN-γwas mainly produced by liver NK cells,and NK cell depletion could significantly inhibit IFN-γproduction in the serum.The immunofluorescent staining of liver tissues showed that TNF-αwas produced mainly by Kupffer cells.Similarly,Kupffer cell depletion could markedly inhibit TNF-αproduction in the serum.5.Cross talk between NK cells and Kupffer cells occurs via NKG2D-Rae 1 recognition after being triggered by TLR3 activationWe attempted to determine whether NKG2D-ligand interactions were involved in the cooperation between NK cells and Kupffer cells.Expression of Rae 1 and Mult 1,two important ligands of NKG2D,in the liver was investigated by RT-PCR. Rae 1 expression was markedly increased after poly I:C/D-GalN injection,while Mult 1 expression was not changed.We further found that Rae 1 expression was significantly up-regulated on the surface of Kupffer cells but not on hepatocytes. Poly I:C/D-GalN injection did not significantly affect NKG2D expression on NK cells.To investigate whether the interaction between Rae 1 and NKG2D contributed to the liver damage,we injected mice with NKG2D blocking mAb before poly I:C/D-GalN treatment.Blockade of NKG2D recognition alleviated poly I:C/D-GalN-induced liver injury,and reduced levels of IFN-γin the serum.The in vitro data also showed that blockade of NKG2D recognition reduced IFN-γsecretion by NK cells when co-incubated with poly I:C-stimulated Kupffer cells. 6.IL-12 and IL-18 are also involved in the cross talk between NK cells and Kupffer cellsThe critical roles of IL-12 in poly I:C-induced NK cell-mediated liver mild injury have been demonstrated in our previous study.Here,we observed that both IL-12 and IL-18 were expressed constitutively in the liver,but markedly increased 12 to 18 hours after injection.Modest induction of IL-18 and IL-12 in the serum was observed 18 hours and 12 to18 hours after injection,respectively.Moreover, blockade of either of the two cytokines in vitro reduced IFN-γproduction by NK cells if co-incubated with poly I:C-stimulated Kupffer cells.Conclusion:the present work describes an NK cell-mediated murine model of fulminant hepatitis which is a valuable supplement to established Kupffer cell- or NKT cell-mediated hepatitis（e.g.,LPS/D-GalN model and Con A model）.We also demonstrate that the reciprocal activation of hepatic NK cells and Kupffer cells initiates the liver injury in poly I:C/D-GalN-induced hepatitis.To our knowledge, this is the first report depicting cellular cross talk between hepatic NK cells and Kupffer cells mediated by NKG2D-Rae1 recognition.Taken together,our findings may provide insight into innate immune recognition,which is pivotal in acute liver injury;further,our results may aid in investigating potential therapeutic strategies against innate immune-mediated injury in disease settings.更多还原