【作者】 殷文伟；

【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】 肝脏是一个独特的器官,它既是人体最大的代谢和解毒器官,同时也被认为是机体重要的免疫器官。与其它免疫器官主要诱导免疫应答不同,肝脏更倾向于诱导免疫耐受如口服免疫耐受,肝脏移植耐受等。一些病原菌如乙型肝炎病毒(Hepatitis B Virus, HBV)、丙型肝炎病毒(Hepatitis C Virus, HCV)和疟疾等利用肝脏诱导免疫耐受的特性在肝脏局部形成慢性感染,严重危害人类健康。HBV主要感染肝实质细胞,部分人群感染HBV后会导致终生携带,HBV携带者对HBV疫苗处于免疫耐受状态。长期以来,人们对于HBV诱导免疫耐受的机理研究主要基于HBV转基因鼠,但HBV转基因鼠免疫系统从胚胎期对HBV病毒就完全处于免疫耐受状态,与人类后天感染HBV状态有较大差别。本研究中我们采用pAAV/HBV1.2质粒高压注射的方式建立HBV慢性携带小鼠动物模型,研究了HBV慢性携带诱导外周免疫耐受的机理和HBV感染初期肝脏局部微环境改变对HBV免疫耐受的影响。一方面,我们发现Kupffer细胞在诱导HBV携带鼠外周免疫耐受过程中发挥重要作用；另一方面,肝脏过表达IL-15能够预防pAAV/HBV1.2质粒高压注射形成的免疫耐受状态。本研究中,我们采用对C57BL/6小鼠高压注射pAAV/HBV1.2质粒的方式建立HBV持续携带动物模型,模拟人类HBV慢性感染状态。运用放射免疫方法定量检测血清中HBsAg、HBeAg、Anti-HBs含量,免疫组化方法检测肝脏组织HBcAg表达,荧光定量PCR方法检测血清中HBV DNA含量,利用以上指标评价pAAV/HBV1.2质粒高压注射后HBV携带情况；通过细胞清除、细胞纯化和细胞转输实验观察特定细胞亚群在HBV诱导外周免疫耐受过程中的作用；采用构建重组表达质粒和高压注射的方式在小鼠体内内源性过表达IL-15；利用流式细胞术检测各细胞亚群的变化特征；利用ELISA方法检测血清中细胞因子含量的变化。采用上述研究方法,我们得到了以下结果：Kupffer细胞介导HBV免疫耐受。pAAV/HBV1.2质粒高压注射小鼠后血清中能检测到HBsAg、HBeAg持续表达,高压注射6周后肝脏组织仍可以检测到HBcAg蛋白,血清转氨酶在HBV质粒携带过程中一直维持在正常水平,这暗示HBV质粒携带鼠的免疫状态与处于免疫耐受状态的人类慢性HBV携带者的免疫状态相似。与人类HBV感染现象相一致,我们发现HBV疫苗预先免疫可以促进小鼠排斥HBV; HBV质粒携带鼠在HBV疫苗免疫后,不能产生Anti-HBs, HBV疫苗免疫也不能促进HBV质粒携带鼠排斥HBV,这表明肝脏表达HBV诱导外周对HBV疫苗处于免疫耐受状态。进一步我们对HBV质粒携带鼠免疫非HBV蛋白抗原OVA,发现HBV携带鼠可以对OVA产生免疫应答,这说明肝脏表达HBV诱导外周免疫耐受只针对HBV蛋白,是抗原特异性的。我们将HBV质粒携带鼠和对照鼠的脾脏淋巴细胞转输到获得性免疫系统缺陷的Rag1-/-鼠中,在Rag1-/-鼠上分别重建它们的外周免疫系统,观察重建的免疫系统对HBV疫苗的反应。实验结果表明,重建了对照鼠免疫系统的Rag1-/-鼠能对HBV疫苗产生应答,而重建了HBV携带鼠免疫系统的Rag1-/-鼠不能对HBV疫苗产生应答。接着,我们将HBV携带鼠脾脏淋巴细胞中的CD4+T细胞替换为对照鼠的CD4+T细胞,再转输给Rag1-/-,发现对照鼠的CD4+T细胞能辅助HBV携带鼠的获得性免疫系统对HBV疫苗产生免疫应答。这暗示HBV携带鼠免疫耐受状态可能是由于其CD4+T细胞功能缺陷造成的。肝脏抗原递呈细胞被证明在诱导肝脏免疫耐受过程中起重要作用。HBV携带鼠的非实质细胞转输到Naive鼠中能诱导受者鼠对HBV疫苗低免疫应答,进一步将肝脏非实质细胞分为Kupffer细胞和非Kupffer细胞再分别进行转输,我们发现Kupffer细胞具有很强的诱导受者鼠低免疫应答的能力,这表明Kupffer细胞介导了HBV免疫耐受。不过,检测Kupffer细胞在pAAV/HBV1.2质粒高压注射前后的变化情况,我们发现Kupffer细胞的状态并没有显著改变,这暗示肝脏Kupffer细胞一直处于诱导免疫耐受的状态。肝脏过表达IL-15诱导产生IFN-β预防HBV免疫耐受为了研究IL-15对HBV免疫耐受的影响,我们构建了含IL-15基因表达框的pLIVE-IL-15质粒,通过高压注射的方式使pLIVE-IL-15质粒在小鼠体内过表达IL-15。实验结果表明,pLIVE-IL-15质粒高压注射小鼠后能在体内长期表达。若将pLIVE-IL-15质粒或对照质粒pLIVE-EGFP与pAAV/HBV1.2质粒一起注射到小鼠体内,我们发现对照质粒组小鼠血清中长期表达HBsAg和HBeAg,而pLIVE-IL-15质粒注射组血清中低表达HBsAg和HBeAg并且HBV最终会被清除。这表明IL-15能有效预防HBV免疫耐受状态形成。IL-15是一种多能性细胞因子,对天然免疫系统细胞和获得性免疫系统细胞都具有调节作用。pLIVE-IL-15质粒在Rag1-/-鼠中仍能有效地抑制HBV表达,这表明IL-15发挥抗HBV功能不依赖于获得性免疫系统。尽管脾脏和肝脏NK细胞在IL-15质粒注射后数目有较大提升,但采用PK136抗体清除NK/NKT细胞并不影响IL-15抗HBV功能,采用IL-2Rγc-/-鼠实验也表明IL-15抗HBV能力不依赖于NK细胞。IFN-γ和IFN-α/β均可以抑制HBV表达。IL-15在IFN-γ缺陷鼠中仍具有抗HBV功能,这表明IL-15抗HBV能力不依赖于IFN-γ。研究发现,肝脏IFN-β mRNA和血清IFN-β蛋白水平在IL-15质粒注射后显著上调,而肝脏IFN-amRNA和血清IFN-α蛋白水平在IL-15质粒注射后变化不明显。进一步采用Anti-IFNAR1抗体阻断IFN-β功能可以完全逆转IL-15的抗HBV作用,这说明IL-15抗HBV功能依赖于IFN-β。Clodronate Lyposomes青除巨噬细胞不会改变IL-15的抗HBV作用,暗示IFN-β并不是由IL-15作用于巨噬细胞产生的。结论：pAAV/HBV1.2质粒高压注射模型很好地模拟了人类HBV慢性感染状况,可以用于研究HBV感染诱导外周免疫耐受的现象及机理。我们发现HBV免疫耐受状态与CD4+T细胞功能缺陷相关,Kupffer细胞在诱导HBV免疫耐受过程中发挥关键作用。细胞因子IL-15通过诱导产生IFN-β有效预防HBV免疫耐受状态的形成。本研究对于认识肝脏独特的免疫耐受特性,对于理解HBV慢性感染诱导外周免疫耐受的机理及HBV治疗都具有一定意义。更多还原

【Abstract】 The liver is the largest internal organ in body and plays an important role in maintaining normal metabolism and detoxifying the body. Also, the liver has been recognized as a unique immune organ which favours the induction of immune tolerance rather than immunity, such as oral tolerance, liver transplant tolerance. Some liver pathogens (HBV, HCV, and malaria) can establish persistent/chronic infections in the liver that lead to inflammatory liver disease.HBV infects the liver as its primary target. The immune system of HBV infected patient has become tolerant to HBV and shows no response to periphery HBV vaccine. Up to now, most in vivo studies on the mechanisms of HBV tolerance have been performed by using HBV transgenic mice; however, HBV transgenic mice are inherently tolerant to HBV virus and are not suitable for studying the mechanisms of HBV tolerance in HBV infected patient who is infected after birth and have limitations in addressing the factors at the onset of infection that influence the final outcomes of chronic HBV infection. Hydrodynamic injection of pAAV/HBV1.2leads to a persistent HBV gene expression in mice, which mimics human chronic HBV infections in the immune tolerant stage. So in the current study, we used this model to study the mechanism of HBV induced immune tolerance and examine the effects of IL-15on immune response to HBV.In this study, a mouse model for HBV tolerance was established by hydrodynamical injection of pAAV/HBV1.2plasmid into C57BL/6mice. The serum levels of HBsAg、HBeAg and Anti-HBs were measured by radioimmunoassay. Intrahepatic HBcAg expression was determined by immunohistochemistry. Serum HBV DNA was measured by quantitative PCR. In experiments of cell depletion, cell purification and cell transfer, we examined the exact role of specific cell population in HBV immune tolerance. With the aim to investigate the immunomodulatory effects of IL-15in vivo, we constructed a gene transfer vector to endogenously overexpress IL-15in mice. The status of lymphocyte subsets was analyzed with flow cytometer. Serum levels of cytokines were measured by ELISA. Our major findings are shown as follows: Involvement of Kupffer cells in HBV-induced immuntoleranceHydrodynamic delivery of the plasmid pAAV/HBV1.2into mouse liver allowed persistent HBV viremia and antigenemia in C57BL/6mice. Secretion of HBsAg and HBeAg into the serum remained high6weeks post pAAV/HBV1.2administration. Both cytoplasmic and nucleic HBcAg were detected in the livers of HBV-carrier mice at6weeks after injection. There were no detectable anti-HBs antibodies in the sera of these animals. The animal model mimics human chronic HBV infections in the immune tolerant stage. If mice were immunized with HBsAg vaccine ahead of tail vein hydrodynamic injection, all HBsAg-vaccinated mice could eliminated HBV virus by decreasing HBsAg and HBeAg in their sera and HBcAg in liver tissues. However, if HBV-carrier mice were immunized with HBsAg vaccine, serum anti-HBs can not be detected in HBV carrier mice and HBsAg vaccine can not cure HBV persistence in HBV carrier mice. Thus, HBV persistence in liver could induce peripheral immune tolerance. Meanwhile, the HBV-carrier mice preserved a strong immune response to other antigen such as OVA, so that peripheral tolerance toward HBsAg was antigen-specifically induced in HBV-carrier mice.Splenocytes from naive or HBV carrier mice were transferred to ragl-/-mice. After being challenged with HBV vaccine, ragl-/-mice with the immune system of naive mice developed protective levels of anti-HBs, whereas anti-HBs levels were nearly undetectable in ragl-/-mice with the immune system of HBV carrier mice. If we replaced CD4+T cells of splenocytes from HBV carrier mice with CD4+T cells from naive mice and transferred the reconstructive splenocytes to ragl-/-mice and vaccined with HBsAg, we found the reconstructive immune system could response to HBsAg, indicating that the immune tolerant status of HBV carrier mice was associated with the dysfunction of CD4+T cells.The inherent tolerogenicity of the liver are mediated by tolerogenic antigen-presenting cells. Although phenotype of kupffer cells has no obvious change after pAAV/HBV1.2injection, the mice received NPCs or only KCs from HBV carrier mice secreted obvious low anti-HBs levels after HBsAg immunization, suggesting that kupffer cells are critical in induction of immune tolerance.Prevention of hepatitis B virus-induced immunotolerance by liver over-expression of interleukin-15via promoting IFN-beta productionWith the aim to investigate the immunomodulatory effects of IL-15in vivo, we constructed a gene transfer vector to endogenously overexpress IL-15in mice. We found that hydrodynamic injection of the plasmid pLIVE-IL-15resulted in a sustained concentration of IL-15in serum. Liver IL-15over-expression resulted in great suppression of HBsAg and HBeAg expression early after pAAV/HBV1.2injection and even eliminated HBV virus at five weeks post injection. So, IL-15liver gene transfer induced enhanced immune responses to HBV.IL-15is a pleiotropic cytokine known to modulate both innate and adaptive immune cells. Over-expression of IL-15similarly decreased HBsAg and HBeAg levels in Ragl-/-mice, suggesting that IL-15exerted anti-HBV activity in a T-cell and B-cell independent manner. Interestingly, despite an increase in NK cell numbers in both spleen and liver of IL-15-expression mice, the anti-HBV effect of IL-15was neither dependent on presence of NK cells in mAb depletion experiment and by using NK-deficient mice (IL-2Rγc-/-).Type Ⅰ IFNs (IFN-α/β) and IFN-y are known to inhibit HBV gene expression noncytopathically. IL-15inhibits HBV gene expression in IFN-γ-/-mice, suggesting that IFN-y did not play a critical role in IL-15-mediated anti-HBV activity. IL-15treatment increased intrahepatic IFN-β mRNA expression as well as circulating serum IFN-β levels, whereas IFN-a mRNA and protein levels were unchanged. Blockade of IFN-β function led to a significant increase in expression of HBsAg and HBeAg in IL-15treated mice, indicating the anti-HBV activity of IL-15was probably mediated by IFN-β. Depletion of macrophages with clodronate lyposomes did not block the ability of IL-15to inhibit HBV expression in our study, suggesting that IFN-β was not produced by macrophages.Conclusion:The characteristics of the mouse model for HBV-induced immuntolerance are analogous to those of human chronic HBV infections. The status of HBV induced immuntolerance is associated with dysfunction of CD4+T cells. Kupffer cells play an important role in inducing periphery immune tolerance. Liver over-expression of IL-15prevents HBV-induced immune tolerance in an IFN-β-dependent manner. So, the study on the mechanism of HBV-induced immuntolerance and a novel function of IL-15in anti-HBV immunity might provide an implication in clinical practice.更多还原