【作者】 孔小辉；

【导师】 田志刚；

【作者基本信息】 中国科学技术大学 ， 细胞生物学， 2014， 博士

【摘要】 肝脏作为重要的代谢器官,广泛参与糖代谢,蛋白质代谢以及脂类代谢。肝脏同时也是一个重要的免疫器官,富含大量天然样免疫细胞以及获得样免疫细胞,其在自免疫性炎症,微生物感染以及肿瘤的免疫监视过程中发挥着重要的作用。由于肝脏具备天然免疫优势状态,同时富含大量的调节性细胞以及独特的结构特征,容易在肝脏引发全身耐受,因而肝脏又是一个重要的耐受器官。嗜肝病毒感染,如乙型肝炎病毒(Hepatitis B virus, HBV)感染肝脏后,容易诱发肝脏耐受,形成慢性HBV感染(Chronic Hepatitis B,CHB)。病毒特异性T细胞免疫应答以及体液免疫应答对于病毒的清除至关重要,但是在CHB患者肝脏以及外周血中,病毒特异性T细胞经常发生耗竭甚至克隆丢失,从而失去抗病毒能力,使得病毒抗原在肝脏中持续性表达。天然免疫系统对调节获得性免疫应答具有重要的调节能力,在病毒感染过程中,天然免疫应答的强弱以及持续时间对于病毒特异性T细胞免疫应答或者抗体产生息息相关,比如树突状细胞(dendritic cell,DC), macrophage等呈递抗原的能力,又比如NK细胞,Thl细胞产生IL-2促进CD8+T细胞存活和扩增等。在本文中我们着重研究了γδT细胞—一类重要的天然样免疫细胞—在HBV耐受过程中的作用,我们的结果表明其似乎更像是发挥着旁观者效用。慢性HBV感染与肝细胞癌(Hepatocellular Carcinoma, HCC)的发生密切相关。我国目前每年约有30万人死于肝脏肿瘤,其中绝大部分为慢性HBV携带者。HBV感染肝细胞后,基因组发生的随机插入以及HBx蛋白的表达与肝细胞恶性化密切相关。同时HBV病理学进程表明,当肝脏中HBV病毒复制活跃,这时期可观察到显著的肝脏炎症,这类HBV携带者更容易发生肝细胞癌变,暗示肝脏慢性炎症对于HCC的发生密切相关,这也与目前广泛接受的慢炎诱癌学说不谋而合。因而研究HBV诱导的肝细胞癌为临床治疗提供了新的理论依据。本文将阐释在原发性肝脏肿瘤发生过程慢性炎症发挥着重要的作用。本文取得的主要结果从以下两个方面概述。I、γδT细胞调节肝脏免疫耐受的研究本研究采取pAAV/HBV1.2以及rAAV/HBV1.3病毒注射构建了HBV携带小鼠,WTHBV携带小鼠可检测到肝实质中病毒抗原的表达,并且分泌到胞外在血清中可以检测到高浓度的HBsAg, HBeAg, HBV-DNA等表达,同时肝脏几乎没有炎症,抗原特异性体液免疫应答以及T细胞免疫应答均受损；因而该模型模拟了人群中的慢性HBV携带者免疫耐受期的状态。首先我们检测到HBV耐受小鼠中γδT细胞的总数尤其是Vγ4细胞亚群发生了显著的上调。进一步我们采取TCRδ-/-小鼠观察到HBV耐受不能形成,血清中HBV抗原以及肝脏表达的HBc抗原明显下降直至消失。采用Anti-yδTCR清除HBV携带小鼠全身γδT细胞也获得了类似的结果；进一步我们分选出γδT细胞转输TCRδ-/-小鼠后,HBV耐受恢复,因此表明,γδT细胞对于HBV耐受的形成至关重要。接下来我们检测发现TCRδ-/-小鼠相比WT小鼠,肝脏HBV特异性体液免疫应答没有差异,调节性T细胞和Kupffer细胞比例也没有差异；非常有意思的是肝脏的HBV特异性CD8+T细胞比例以及功能均发生了显著的上升；表征CD8+T细胞的耗竭被逆转。进一步我们发现γδT细胞对于CD8+T细胞的耗竭发挥着旁观者效应。持续性表达的HBV抗原对于γδT细胞产生IL-17进而招募髓系来源抑制细胞(MDSC)进入肝脏至关重要.MDSC继而通过产生iNOS以及arginase-1直接抑制了CD8+T细胞的活化。在IL-17-/-小鼠中,我们也观察到MDSC显著减少,同时CD8+T细胞功能恢复,并且HBV耐受不能形成。体内抑制arginase-1亦显著改善CD8+T细胞功能并且加速HBV排斥。综上所述；γδT细胞作为一种天然样T细胞,在HBV耐受的形成过程中起始了MDSC的肝脏聚集,诱使CD8+T细胞发生功能耗竭而导致肝脏耐受。靶向这一过程能显著改善HBV特异性T细胞功能,促进HBV的排斥。Ⅱ、NK细胞抵抗CCl4诱导的肝细胞癌的研究本研究采取HBV转基因小鼠模型,由于该小鼠具备轻微自发炎症且自然发生肝脏肿瘤,很好的模拟了临床上HBV患者高发肝细胞癌(Hepatocellular Carcinoma, HCC)的现象。为了验证慢性炎症诱发肿瘤,我们采取连续CCl4注射诱导肝脏持续性炎症,观察到在HBV转基因小鼠中约6个月即发生肝脏肿瘤,表明慢性炎症确实促进了肝脏肿瘤的发生。进一步我们发现CCl4诱导HBV转基因小鼠肝脏炎症的同时,伴随着肝脏上皮间质转化(EMT)的发生,EMT被认为是肿瘤生长以及迁移的重要机制。同时我们注意到肝脏NK细胞活化性分子下调,抑制性分子上调,表明NK细胞受到抑制。进一步我们观察到清除NK细胞后,肝脏EMT标志蛋白vimentin表达显著上调。清除NK细胞后可观察到慢性炎症诱导的肝脏肿瘤显著增多；表明NK在该过程中发挥着免疫监视功能。采取Ployl:C活化NK细胞,可显著减少CCl4诱导的肝脏肿瘤的发生。此外,我们慢炎诱癌的模型还可以用于肝脏肿瘤的治疗和预防研究。比如由于免疫细胞可能介导了肝脏炎症,因而采取了免疫抑制剂处理,观察到抑制免疫细胞后,肝脏肿瘤生长更快；而采取非甾体类抗炎药处理,我们观察到一定的抑癌效果。非常有意思的是,PolyI:C处理HBV转基因小鼠后可以显著预防肝脏肿瘤的发生。综上所述,我们构建了一个非常实用的慢性炎症诱导肝脏肿瘤模型,并且观察到NK细胞通过靶向抑制EMT发挥免疫监视功能。采用非甾体类抗炎药饲喂HBV转基因小鼠可以一定程度上抑制肝脏肿瘤生长。更多还原

【Abstract】 The liver works as a metabolic organ and plays important roles in glucose, protein and lipid metabolism. While the liver is a unique immune organ, because of predominant innate immune cells, and actively involved in autoimmune diseases, microbial infection and tumor immune-surveillance. The liver is also recognized as immune-tolerant organ, since liver has enriched regulatory cells and the unique structural characteristics and therefore foster liver allo-grafts, blood and intestinal derived antigens and also chronic hepatotropic microbe infection.Hepatotropic virus infections, such as hepatitis B virus (HBV) infection usually results in Chronic Hepatitis B (CHB) infection, accompanying with persistent expression of HBV antigens and impaired virus-specific cellular and humoral immune response though they are crucial to eliminate virus. Innate immune response, shortage of viral specific property, is also important for viral control and can promote the adaptive immune response. For example, NK cells and type I helper T cell derived IL-2, Dendritic cells and kupffer cells presented antigens are critical for adaptive immune response. However, they maybe harbor regulatory function. In this study we focus on an innate like T cells-γδT cells-and the roles in the process of HBV induced liver tolerance, which act more like bystander.Moreover, CHB has been considered closely related to HCC (Hepatocellular Carcinoma, HCC). Because about300thousand people die from liver tumors in China each year and HBV carriers take up the majority. HBV infection induced liver tumor has been explained with the viral genome insertion induced and HBx protein induced liver cells malignancy. Meanwhile persistent liver inflammation is always observed in the chronic HBV carrier, suggesting chronic inflammation can promote the development of HCC. Thus, targeting liver inflammation maybe provides a theoretical basis for clinical treatment. Our study here support persistent liver inflammation promote liver tumor development, and NK cells play an important function in this process.I, γδT cells modulate HBV-induced toleranceWe constructed HBV-carrier mice by using pAAV/HBV1.2plasmid and recombinant AAV/HBV1.3virus, wild type C57BL/6mice carry persistent expression of viral antigens in the liver hepatocytes and secret outside the cell in the serum with high levels of expression of HBsAg, while almost no liver inflammation, accompanying insufficient antigen-specific humoral immune response and T cell immune response; which can mimic immune tolerance phase during the chronic HBV infection.Next, we detected the number of yδT cells-Vy4subset in particular, is significant upregulation in the HBV-carrier mice. While we injected TCR-/-mice with pAAV/HBV1.2, were observed HBV-tolerance could be not established with quickly disappeared HBV antigen expression in the serum and liver. Similar results obtained after y8T cells depletion by anti-yδTCR antibody. Further we adoptively transferred yδT cells to TCRδ-/-mice, serum high concentration of HBV antigen maintained. Thus, y8T cell is crucial for HBV-induced tolerance.Later, we did not detect elevated HBV-specific humoral immune response between HBV-carrier wild type and TCRδ-/-mice; in addition, hepatic regulatory T cells and Kupffer cells were also not alter between them. Interestingly, HBV-specific CD8+T cell number and activity were notable increased as there were exhausted in the wild type HBV-carrier mice, HBV tolerance was reserved if depletion of CD8+T cells in TCRδ-/-HBV-carrier mice.Furthermore, we found that y8T cells play a bystander effect to CD8+T cells exhaustion. Persistent HBV antigen expression induced y8T cells to produce IL-17, which recruited myeloid suppressor cells (MDSC) into the liver. And MDSC directly inhibited CD8+T cells activation in a dependent manner of iNOS and arginase-1. We also observed a significant decrease MDSC in HBV-carrier IL-17-/-mice, while CD8+T cell function recovered. Treatment HBV-carrier mice with arginase-1in vivo could restore CD8+T cell function and promote elimination of HBV.In summary; γδT cells as an innate-like T cells, play important role in the formation of HBV-induced tolerance by inducing hepatic accumulation of MDSC mediated viral specific CD8+T cell exhaustion. Targeting this process can significantly improve HBV-specific T cells response and promote HBV clearance.II, NK cells resist to CC14-induced hepatocellular carcinoma.We use HBV-transgenic mice in this study as these mice can spontaneous develop liver tumors with sustained liver inflammation at about17month-old, which greatly mimic HBV-induced hepatocellular carcinoma in clinical patients. To strengthen the hypothesis that chronic inflammation may promote tumor development, we continuously injected CC14to the HBV-transgenic mice, and observed liver tumor after six months treatment, suggesting that chronic inflammation is indeed fostering liver tumorigenesis.Furthermore, we found aggravating epithelial-mesenchymal transition between (EMT) occurred in HBV transgenic mice during chronic liver inflammation. EMT is considered as an important mechanism for tumor growth and migration. We noted that NK cells were suppressed during chronic inflammation induced liver tumor with downregulated activation molecules and upregulated inhibitory molecules. We later observed that enhanced vimentin expression-EMT marker-in the liver after NK cells depletion. And a significant increase in liver tumors developed during chronic inflammation in the HBV-transgenic mice after clearing NK cells, thus NK cells are critical for liver tumor immunosurveillance.Moreover, our model of chronic inflammation induced liver tumor may also be useful for studying the prevention and therapy of liver cancer. Non-steroidal anti-inflammatory drug (NSAIDs) and immune-suppressive agents were used to investigate the effect in prevention persistent inflammation induced liver tumor. We found here that immune-suppressive agents-rapamycin and cyclosporine treatment promoted liver tumor development. We also examined two pieces of NSAIDs-diclofenac and aspirin; diclofenac could quickly downregulated CC14-induced liver damage but had aggravated side effect during long-time oral administration. However, oral aspirin seems benefit for control tumor development and body weight. Besides, PolyI:C-a short RNA double strand mimics used for hepatic NK cell expansion and activation-treatment may significantly resist liver cancer development.In summary, we have built a practical model of chronic inflammation-induced liver tumors, and observed NK cells were important to resist liver tumor by inhibition of EMT. The usage of NSAIDs aspirin but not immunosuppressive agents can suppress tumor development in the HBV-transgenic mice.更多还原