【作者】 徐龙；

【导师】 田志刚；

【作者基本信息】 中国科学技术大学 ， 细胞生物学， 2011， 博士

【摘要】 肝脏是一个天然免疫耐受器官。很早就观察到,肝脏移植后易诱导受体免疫耐受；一些嗜肝病原体如：乙型肝炎病毒(Hepatitis B Virus, HBV).丙型肝炎病毒(Hepatitis C Virus, HCV)和疟疾易导致机体形成长期慢性感染,并诱导全身免疫耐受。如：与正常人相比,外周免疫乙肝疫苗后HBV携带者并不能产生保护性抗体。但是,目前我们对肝脏免疫耐受形成的机理,以及肝脏免疫耐受如何进一步诱导全身免疫耐受的细胞机制均知之甚少,探索其机制机理对慢性肝炎的临床治疗具有一定的指导意义。本研究通过高压注射HBV质粒pAAV/HBV1.2,建立了HBV慢性感染成年小鼠模型。利用该模型深入探索了肝脏免疫耐受的形成以及如何诱导外周免疫耐受的机理。我们采用免疫放射法(IRMA)定量检测小鼠血清中乙肝表面抗原、表面抗体,定量PCR检测血清中HBV DNA拷贝数等指标来评价小鼠是否处于慢性感染HBV状态；采用赖氏法检测血清转氨酶ALT水平、H-E染色观察肝脏组织病理学变化来判断肝细胞损伤程度：并利用上述指标综合评估小鼠慢性感染HBV模型是否成功,并将高压注射HBV质粒4周后血清中HBsAg较高的小鼠定义为HBV携带鼠(HBV carrier mice)(>100ng/m);通过外周脾脏免疫HBsAg/CFA检测血清中anti-HBs说明HBV鼠对HBsAg处于免疫耐受状态,进一步细胞转输来证明耐受具有可传递性。细胞清除实验研究各细胞亚群在肝脏免疫耐受中的相互作用；采用ELISA和多细胞因子检测的方法检测体外细胞培养上清中IL-10、TGF-β、IFN-γ和IL-4等细胞因子的浓度；采用流式细胞术分析在HBV鼠中负调细胞抑制HBsAg外周免疫应答的过程。通过上述一系列实验方法取得的实验结果如下：1.成年小鼠HBV慢性感染模型的建立有文献报道通过尾静脉高压注射pAAV/HBV1.2质粒的方法能建立成年C57BL/6小鼠慢性感染HBV的模型。我们通过摸索不同剂量的注射质粒发现6gg质粒能使小鼠血清中HBsAg持续保持较高浓度,5周后小鼠HBV阳性率100%,肝脏组织免疫组化的结果同样证实5周后,HBV鼠肝脏中存在HBcAg阳性细胞；通过ALT检测以及H-E染色发现,HBV鼠肝脏组织并没有淋巴细胞浸润、肝脏损伤等出现；将血清中HBsAg含量与HBV DNA拷贝数进行比较,发现二者之间呈明显的相关性。与6μg质粒结果相反,小鼠高压注射20μg pAAV/HBV1.2质粒后,在4周左右,小鼠血清中HBsAg急剧下降,第5周时,HBsAg阳性率为0。因此,6μg pAAV/HBV1.2高压注射可以获得稳定、可重复、标准的HBV慢性感染模型。为了方便观察高压注射pAAV/HBV1.2质粒后,小鼠有哪些器官组织表达抗原。我们将GFP表达框插入pAAV/HBV1.2质粒,使得质粒进入细胞后能表达很强的GFP蛋白。通过免疫荧光以及免疫组化方法,我们发现只有肝脏组织在高压注射后表达GFP蛋白,而心脏、肺脏和肾脏都不表达GFP蛋白,因此高压注射质粒后只能靶向进入肝脏,并启动表达蛋白。2.HBV携带鼠诱导HBsAg外周免疫耐受高压注射pAAV/HBV1.2质粒后形成HBV携带小鼠,通过检测总淋巴细胞比例以及表面分子,发现HBV携带鼠淋巴细胞中CD4+T细胞或CD8+T细胞等比例没有明显变化,NK细胞比例以及表面分子(NKG2D、CD69以及NKG2A)均无明显变化。提示小鼠HBV肝脏感染并未引起全身免疫应答能力整体变化。通过小鼠手术进行脾内注射1μg HBsAg/CFA,发现正常小鼠均能产生anti-HBs; HBV携带鼠(携带时间为1周或8周)在HBsAg免疫后,血清中检测不到anti-HBs。表明肝脏中持续表达HBV导致小鼠外周免疫系统对HBsAg刺激无应答,表现为免疫耐受状态。如果pAAV/HBV1.2质粒高压注射1天后进行HBsAg疫苗免疫,小鼠能产生较高滴度的anti-HBs,并能清除小鼠自身的HBV提示针对HBsAg的特异性免疫耐受的形成需要肝脏中HBV的持续存在,大约需要1周以上时间。在免疫耐受形成前,外周的免疫刺激所形成的免疫应答可以清除尚未诱发免疫耐受或后续的肝脏HBV感染。同时导致耐受一旦形成,机体将出现全身性HBV无应答状态。3.HBV携带鼠可将免疫耐受特征转移至正常小鼠将HBV鼠或对照鼠脾脏细胞转输至正常C57BL/6小鼠中,1天后免疫HBV疫苗,再过1周检测血清中anti-HBs的滴度。发现小鼠转输了HBV鼠脾细胞后产生anti-HBs水平明显低于对照组。并且随着转输的细胞数量增多,anti-HBs水平逐渐降低。转输对照鼠或HBV鼠脾脏细胞后,检测受体鼠OVA免疫以后血清中anti-OVA IgG并没有区别,说明HBV鼠抑制HBsAg的免疫应答具有抗原特异性。4.HBV携带鼠中产生具有负调功能的HBsAg特异性CD4+T细胞转输对HBV形成免疫应答的anti-HBs阳性小鼠脾脏细胞至HBV携带Rag1-/-鼠或HBV携带鼠,发现2周内供体细胞能明显清除HBV携带Ragl-/-鼠中的HBV,但是无法清除HBV携带鼠的HBV, HBsAg几乎不受影响。进一步将HBV携带鼠脾脏细胞与anti-HBs阳性小鼠脾脏细胞1：1混合后转输至HBV携带Rag1-/-鼠,发现HBV携带鼠来源的脾脏细胞能在一定程度上抑制anti-HBs阳性鼠脾脏细胞对HBV的清除能力,但HBV携带Rag1-/-鼠脾脏细胞则没有此抑制作用,提示这种具负调功能的细胞归属淋巴细胞。通过MACS分选HBV携带鼠脾脏中CD4+T细胞进行免疫细胞转输实验,证明所转输得CD4+T细胞与所转输的脾脏总免疫细胞具有相同的抑制效果,提示抑制HBsAg免疫应答的负调细胞是CD4+T细胞。5.HBV携带鼠具负调功能的CD4+T细胞为Trl样细胞流式细胞检测结果表明,在HBV鼠肝脏和脾脏淋巴细胞中CD4+CD25+Foxp3+Treg细胞比例没有明显变化,推测在HBV免疫耐受中Treg细胞不参与重要作用。将分离的小鼠肝脏单个核细胞在体外抗CD3、CD28抗体刺激并培养4天,ELISA检测发现HBV携带鼠的肝脏单个核细胞产生IL-10和IFN-γ显著上升,而TGF-β和IL-4则没有明显变化,基本符合Trl细胞的细胞因子表型,提示HBV鼠肝脏淋巴细胞中产生了Trl样细胞。6. Kupffer细胞在HBV携带鼠免疫耐受形成中起关键作用采用尾静脉注射Clodronate/liposomes清除Kupffer细胞的实验表明,Kupffer细胞在HBV形成免疫耐受过程中起着关键作用。Kupffer细胞清除后再高压注射HBV质粒, KC清除组早期(d3)血清中HBsAg明显高于末清除组,提示正常情况下Kupffer细胞可能参与HBV的清除；随着时间推移(3-4w), Kupffer细胞清除组血清HBsAg急剧下降,HBV被清除,HBV免疫耐受不能形成,提示Kupffer细胞在免疫耐受形成中起重要作用；Rag1-/-小鼠在清除Kupffer细胞后血清中HBsAg值长期(8w)保持较高的水平,提示Kupffer细胞诱发机体形成免疫耐受需要获得性免疫细胞的存在；通过细胞转输实验,证明KC清除后HBV携带鼠脾脏淋巴细胞不能抑制受者小鼠对HBsAg的免疫应答,进一步提示KC与获得免疫细胞(T细胞和B细胞)的相互作用是HBV携带小鼠免疫耐受形成的基础。7.HBV携带鼠中Tfh和GC B细胞的活化受阻是全身免疫耐受形成的关键环节采用流式细胞术对HBV疫苗免疫后小鼠胭窝淋巴结免疫细胞进行检测,发现HBV携带鼠对HBV疫苗没有免疫应答,表现为anti-HBs不能产生,生发中心不能形成,GC B细胞和Tfh细胞不能活化。这种免疫耐受现象与DC没有明显关系,表现为HBV疫苗免疫后HBV鼠中DC细胞比例与活化均无变化。这些结果提示在HBV携带鼠诱导外周免疫耐受过程中,抗原特异性Trl细胞可能对Tfh细胞或/和GC B细胞进行了负相调节,从而导致针对HBV疫苗的免疫耐受而无法产生anti-HBs。结论：本文通过建立HBV慢性感染小鼠模型,发现肝脏持续表达HBV能诱导外周淋巴器官对HBsAg失去免疫应答能力。在肝脏免疫耐受诱导外周免疫耐受的机制中,我们发现Kupffer细胞在诱导HBV特异性的Trl样负调细胞的产生中起着非常关键的作用。负调细胞参与血液循环至全身淋巴结和脾脏,通过抑制Tfh细胞和GC B细胞的活化而介导全身免疫耐受的形成。首次阐述了Kupffer细胞、Trl样细胞以及Tfh细胞三者的相互作用及其免疫耐受形成的机制。更多还原

【Abstract】 The liver exhitits a distinctive form of immune tolerance, in which orthotopic liver transplantation are predisposed to induce tolerance and foreign antigenic material or other liver targeted pathogens (such as:HBV, HCV and malarial parasite) could not induce an immune response. Futhermore, liver virus persistence results in systemic immune tolerance. For example, HBV carrier could not produce protective levels of anti-HBs when compare with normal person after HBV vaccination. However, the precise mechanism as to how liver participates in induction of systemic immune tolerance without immune resoponse remains poorly understood.In this study, we established adult mouse model for HBV chronic infection by hydrodynamical injection with6μg of pAAV/HBV1.2palsmid to explore the mechanism about the liver immune tolerance and systemic tolerance. Then we evaluated the state of liver tolerance by determining serum levels of HBsAg or anti-HBs using IRMA and serum HBV DNA copies using real-time PCR. Serum transaminase ALT levels and liver pathologic changes were observed to examine the liver injury. These data together helped to determine whether the mouse HBV model was available and the mice whose serum HBsAg surpassed100ng/ml after4weeks HBV plasmid injection were considered to be HBV carrier mice. We found HBV carrier mice induced peripheral tolerance after1μg of CFA/HBsAg immunization and tolerance could be transferred by adptive splenocytes transfer. By cells depletion, effects of the specific lymphocyte interactions in the process of liver tolerance were investigated. We used ELISA to measure cytokine levels after ex vivo activation including IL-10, TGF-P, IFN-y and IL-4. Flow cytometry analysis provided us with the precise information about lymphocyte phenotype and activation. Our major results are shown as followed:1. Establishment of a mouse model for HBV persistenceHuang and colleagues have established a mouse mode of persistent HBV infection by hydrodynamic injection of the plasmid pAAV/HBV1.2into the tail veins of C57BL/6mice. We found that HBV persistence in mice was determined by the doses of the plasmid in the model. C57BL/6mice were injected hydrodynamically with6μg and 20μg of pAAV/HBV1.2plasmid, respectively. In the6μg group, the mice kept high serum levels of HBsAg, and almost all mice were still HBsAg positive at5weeks post injection. Cytoplasmic and nucleic HBcAg were detected in the livers of HBsAg positive mice at5weeks post injection of pAAV/HBV1.2. ALT and Histological analysis of the liver sections from HBsAg positive mice showed that there was no inflammatory response or injury in the liver. On the contrary, the HBsAg level dropped quickly during3to5weeks after pAAV/HBV1.2injection and all mice were negative for HBsAg at5weeks post injection in the20μg group. So, in the subsequent experiments, we chose the6μg of pAAV/HBV1.2to establish the mouse model for HBV persistence.To easily test which organs expressed the HBsAg after hydrodynamic injection of pAAV/HBV1.2, we digested GFP expression cassettes and inserted into the plasmid. By the achievment of GPF and HBV coexpression, we can observe the infected cell directly by fluorscence or immunohistochemistry. The GFP expression was found only in liver, but not in heart, lung or kindny. Therefore, the hepatocytes were targeted by pAAV/HBV1.2and initiated to express HBV related proteins by hydrodynamic injection.2. Induction of peripheral tolerance toward HBsAg in HBV carrier mice.To investigate the immune response toward HBsAg in HBV carrier mice, we carried out the flow cytometry analyses of the NK, T and B cells isolated from liver and spleen. We found the presentage of these cells and surface maker of NK cells (such as:NKG2D, CD69and NKG2A) were unaffected in HBV carrier mice when compared with control mice, indicating the weak immune response in HBV carrier mice and NK cell did not play a key role in the process.To determine whether the sustained HBV expression in liver could induce peripheral tolerance, control or HBV carrier mice were immunized intrasplenically with1μg of CFA/HBsAg. Protective levels of anti-HBs can be detected in control mice after the vaccination, but not in HBV carrier mice. When the mice were immunized at HBV persistence1day, HBsAg immunization could overcome tolerance and all mice produced the high levels of anti-HBs. These data suggested that it was difficult to reverse the tolerance which was induced by liver HBV persistence within about1week by peripheral immunization of HBsAg. 3. Deliver of HBV-immune tolerance to naive miceTo investigate whether immune tolerance of HBV-carrier mice can be transferred to naive mice in vivo, splenocytes from control or HBV carrier mice were transferred into wild type (WT) mice. The serum levels of anti-HBs were determined after being challenged with HBV vaccine. We found the transferred splenocytes from HBV-carrier mice significantly inhibited the production of anti-HBs against HBsAg in WT mice. The degree of inhibition was positively correlated with the amount of transferred splenocytes from HBV carrier mice to WT mice prior to HBV vaccination. Next, using the same experimental procedure, mice transferred with splenocytes were challenged with OVA in order to determine whether the observed suppression was specific to HBsAg. As expectation, mice transferred with splenocytes from HBV carrier mice produced the same serum levels of anti-OVA IgG as that of WT mice, suggesting that the transferred splenocytes from HBV-carrier mice could specifically inhibit anti-HBV immunity in WT mice.4. HBV carrier mice produce regulatory HBsAg-specific CD4+T cellsAfter the adoptive splenocytes transfer, we found Rag1-/-HBV carrier mice, but not WT HBV carrier mice, transferred with anti-HBs (+) splenocytes, but not control splenocytes, could eliminate HBV totally within two weeks. To further observe the direct suppressed effect of regulatory cells from HBV carrier mice, the recipient Rag1-/-AHBV+mice were transferred intravenously with splenocytes isolated from either HBV carrier mice, anti-HBs (+) mice, or mixture from HBV carrier mice and anti-HBs (+) mice (ratio1:1), and then serum HBsAg levels were measured at2weeks after cells transfer. We found HBsAg levels of recipient transferred mixture of splenocytes were obviously higher than recipient transferred anti-HBs (+) splenocytes only. The interpretation of these findings was that HBV carrier mice generated some regulatory cells which were absent in Ragl"7" HBV carrier mice to inhibit anti-HBV immunity.We further confirmed that Ragl^HBV carrier mice were absence of HBsAg-specific regulatory cells via adoptive splenocytes transfer. Suppression of anti-HBs production was lost by transferring splenocytes from Ragl-/-HBV carrier mice instead of HBV carrier mice. Furthermore, anti-HBs production was found to be unaffected after transferring of CD4+T cells with the purity of92%compared with total splenocytes from HBV carrier mice, indicating the regulatory cells in HBV carrier mice were CD4+T cells.5. The regulatory HBsAg-specific CD4+T cells might be Trl-Iike cellsThe immunotolerance induced by HBV was not due to development of CD4+CD25+Foxp3+Treg cells, as we found no significant increase in Treg cells in HBV carrier mice. To explore the possible role Trl like cells play in the induction of tolerance to the HBsAg, at week2post pAAV/HBV1.2injection, liver MNCs and splenocytes were isolated and activated ex vivo to determine the IL-10, IFN-y, TGF-β and IL-4production by ELISA. We found IL-10and IFN-y production by liver MNCs were strongly elevated in HBV carrier mice when compared with control mice, but production of TGF-β and IL-4kept unchanged in two groups, which strongly suggesting the involvement of Trl like cells in HBV-induced immunotolerance.6. Kupffer cells are critical in induction of systemic toleranceTo explore the role of KCs in induction of tolerance, mice were depleted transiently of KCs by a single intravenous administration of clodronate liposome on day-2prior to HBV plasmids injection. Strikingly, this treatment resulted in total elimination of HBV during3to4weeks. However, HBsAg found to be unaffected at8weeks in Rag1-/-HBV carrier mice after depletion of KCs. The performance of adoptive splenocytes transfer indicated depletion of KCs, but not NK/NKT, resulted in functional deficiency of regulatory cells in HBV positive mice. It suggested strongly that KCs contribute to induction of regulatory HBsAg-specific Trl like cells, which was responsible for suppression of HBsAg-activated immune response and maintenance systemic tolerance.7. Inactivation of Tfh cells to HBV vaccination in peripheral lymph nodes in HBV-carrier miceIt was well known there were many definite steps between antigen immunization and antibody production, including DCs activation, percentage of Tfh cells and GC B cells increase, generation of germinal center, and then secretion of antibody. To further explore the mechanism how Trl like cells maintained the tolerance and inhibited the production of anti-HBs after HBV vaccination in HBV carrier mice, we measured the percentage of GC B cells, Tfh cells, and DC cells using flow cytometry. After HBV vaccination, the frequencies of GC B cells and Tfh cells increased significantly in control mice, but not in HBV carrier mice. However, a dramatic relative increase in DC numbers and expression of costimulatory molecular CD86was evident at4days following HBV vaccination both in control and HBV carrier mice. These data suggested that the exact step between DCs and Tfh cells was inhibited in HBV carrier mice, leading to suppression of anti-HBs production after HBsAg immunization.These data suggested that DCs activation and expansion were not influenced but Tfh cells were inhibited in HBV carrier mice, leading to the dysformation of GC and subsequent reduction in anti-HBs synthesis after HBsAg immunization.Conclusion:The present work described an immunotolerance model whereby liver persistent expression of HBV could cause peripheral immune tolerance toward HBsAg immunization. We found KCs in liver played key roles in inducing the generation of regulatory HBsAg-specific Trl like cells which mediated systemic tolerance via migrating to lymph nod and spleen and inhibiting the Tfh and GC B cells activation after HBsAg-vaccination. Our study firstly elaborated a KCs-Trl like cells-follicular Th cells interplay mediated the systemic immunotolerance Induced by liver HBV persistence.更多还原