CTL Effector Mechanisms in Adenoviral Hepatitis

描述(申请人提供)：这个项目的广泛目标是阐明导致病毒感染的肝细胞被杀死的免疫机制。作为该项目的一部分进行的研究发现，肝细胞对穿孔素和颗粒酶依赖的细胞毒性T淋巴细胞(CTL)杀伤机制具有抵抗力，并且病毒感染的肝细胞从肝脏清除主要是通过替代的CTL效应机制来介导的，这些机制利用Fas/FasL、TNF/TNFR1和/或其他死亡受体介导的途径。这些结果使我们推测，肝细胞对穿孔素和颗粒酶介导的CTL效应通路的细胞毒作用的抵抗在限制肝内免疫反应中的肝损伤程度方面具有重要意义。为了解决这一假说，并探索在肝脏抗病毒免疫过程中使用的独特的CTL效应器机制的机制，我们建议探索病毒感染的肝细胞对CTL效应器功能的颗粒胞吐途径产生抵抗的机制。初步研究结果表明，在抗病毒免疫反应中产生的细胞因子可分别诱导人和小鼠肝细胞表达颗粒酶B特异性抑制物--蛋白酶抑制物9(PI-9)和丝氨酸蛋白酶抑制物6(SPI-6)。在拟议的研究中，我们将确定在正常和病毒感染的小鼠肝细胞中表达的丝氨酸蛋白酶抑制物(丝氨酸酶)的谱系，并确定细胞因子在体外和体内调节这种丝氨酸蛋白酶表达的作用。然后，我们将分别探讨蛇毒和/或组织蛋白酶B在介导肝细胞对颗粒酶和穿孔素的抵抗中所起的作用。最后，我们将确定体内沉默丝氨酸和/或组织蛋白酶B基因是否会使病毒感染的肝细胞对穿孔素和颗粒酶依赖的细胞毒作用敏感，从而加速病毒感染的肝细胞的免疫清除，从而加重病毒性肝炎。

DESCRIPTION (provided by applicant): The broad objective of this project is to elucidate the immune mechanisms responsible for killing of virally infected hepatocytes. Studies conducted as part of this project have found that hepatocytes are resistant to perforin and granzyme dependent mechanisms of cytotoxic T lymphocyte (CTL) killing and that clearance of virally infected hepatocytes from the liver is mediated predominately by alternative CTL effector mechanisms that utilize Fas/FasL, TNF/TNFR1 and/or other death receptor mediated pathways. These results lead us to hypothesize that hepatocyte resistance to the cytotoxic effects of the perforin and granzyme mediated CTL effector pathway is important in limiting the degree of liver injury during intrahepatic immune responses. To address this hypothesis and to explore the mechanisms responsible for the unique repertoire of CTL effector mechanisms employed during anti-viral immunity in the liver; we propose to explore mechanisms responsible for resistance of virally infected hepatocytes to the granule exocytosis pathway of CTL effector function. Preliminary findings indicate that cytokines produced during antiviral immune responses induce expression of the granzyme B specific inhibitors proteinase inhibitor 9 (PI-9) and serine proteinase inhibitor 6 (SPI-6) in human and mouse hepatocytes respectively. In proposed studies, we will determine the repertoire of serine proteinase inhibitors (serpins) expressed in normal and virally infected murine hepatocytes and determine the role of cytokines in regulating such serpin expression in vitro and in vivo. We will then explore the role that serpins and/or cathepsin B play in mediating hepatocyte resistance to granzymes and perforin, respectively. Finally, we will determine whether silencing of serpin and/or cathepsin B genes in vivo renders virally infected hepatocytes susceptible to perforin and granzyme dependent cytotoxicity and thereby accelerates immune clearance of virally infected hepatocytes and exacerbates viral hepatitis.