Hepatocyte-hepatic stellate cell axis in potentiation of alcohol and HIV-induced liver injury

项目摘要 在高活性抗逆转录病毒治疗（ART）时代，肝病已成为肝硬化的常见原因。 住院治疗是艾滋病毒阳性者死亡的主要原因之一。的病因 HIV肝毒性是多方面的。HIV单一感染，HIV与嗜肝病毒（HBV）合并感染 和HCV）、HIV和酒精诱导的肝毒性以及ART诱导的肝毒性是肝损伤的已知原因。 艾滋病毒感染者的疾病。在肝毒性的所有原因中，酒精的机制- 在HIV感染细胞中诱导的肝毒性是未知的。鉴于所有艾滋病毒感染者都在接受抗逆转录病毒治疗， 由于ART的高可用性和可及性，很难评估酒精对 HIV感染者的肝脏。这就是为什么我在导师的帮助下采用体外系统 (Drs. Osna和Poluektova），以探索HIV感染肝细胞中酒精诱导的肝毒性。我 在我的导师的监督下，初步数据显示了以下发现：酒精代谢物 增强HIV在肝细胞中的积累，从而触发活性氧（ROS）， 肝细胞通过凋亡途径死亡。当肝星状细胞（HSC）暴露于HIV感染的 凋亡的肝细胞，促纤维化基因如Col 1A1，TIMP 1和TGF β的上调， 在暴露2小时后建立。这意味着在肝细胞和 HSC在HIV和酒精的前提下通过凋亡的肝细胞导致肝纤维化。这些 这些观察结果在暴露于HIV和酒精的人源化小鼠的肝脏中得到证实。用于治疗 目的了解酒精诱导的HIV肝毒性机制已成为必要， 被感染的细胞为了理解这一机制，我们面临着一些研究问题，这些问题试图 了解HIV进入肝细胞的受体和酒精代谢产物的影响，以增加 艾滋病毒进入。此外，酒精对肝细胞中HIV降解的影响还有待研究。不 已知肝细胞死亡是否是由于细胞凋亡或其他细胞死亡机制（例如坏死或坏死）引起的。 坏死性凋亡）。因此，了解细胞死亡机制的类型将有助于制定策略， 阻断/防止肝细胞死亡，肝细胞死亡介导肝纤维化。此外，我们将探索涉及的途径 在促纤维化途径的激活中，以了解阻断这些途径是否会阻止 纤维化基因所有的研究问题都将在体外和体内模型中进行测试。这些技能将 在致力于完成研究提案的3个目标的3年期间获得的成果是实验室 技术如蛋白质组学、流式细胞术、HIV的真实的实时聚合酶链反应（RT PCR RNA、mRNA、HIV DNA、数字液滴PCR（ddPCR）、流式细胞术、细胞培养和动物处理。期间 在这段时间里，我不仅将彻底配备赠款写作和演讲技巧，我将参与 作为一名实习生，我需要管理自己的资源，这是成为一名独立研究人员所需的技能。

Project Summary In the era of highly active antiretroviral therapy (ART), liver disease has become a common reason for hospitalization and one of the leading causes of mortality among HIV positive individuals. The etiologies of hepatotoxicity in HIV are multifaceted. HIV mono-infection, HIV co-infection with hepatotropic viruses (HBV and HCV), HIV and alcohol-induced hepatotoxicity and ART-induced hepatotoxicity are known causes of liver disease among HIV-infected individuals. Among all causes of hepatotoxicity, the mechanisms of alcohol- induced hepatotoxicity in HIV-infected cells are unknown. Given that all HIV-infected individuals are on ART due to high ART availability and accessibility, it becomes difficult to evaluate the single effects of alcohol on the liver of HIV-infected individuals. That is why I am adopting an invitro system with the help of my mentors (Drs. Osna and Poluektova) to explore alcohol-induced hepatotoxicity in HIV-infected hepatocytes. My preliminary data under the supervision of my mentors has revealed the following findings: Alcohol metabolites enhanced HIV accumulation in hepatocytes which triggers reactive oxygen species (ROS) that lead to hepatocyte death via apoptotic pathway. When Hepatic Stellate Cells (HSC) were exposed to HIV-infected apoptotic hepatocytes, an upregulation of profibrotic genes such as Col 1A1, TIMP 1 and TGFβ was established after 2 hours of exposure. This implies that there might be a cross talk between hepatocytes and HSC in the premises of HIV and alcohol via apoptotic hepatocytes which results in liver fibrosis. These observations were confirmed in the liver of humanized mice exposed to HIV and alcohol. For therapeutic purposes it has become necessary to understand the mechanisms of alcohol-induced hepatotoxicity in HIV- infected cells. To understand this mechanism, we are confronted with research questions which seek to understand the receptors for HIV entry into hepatocytes and the influence of alcohol metabolites to increase HIV entry. Moreover, the impact of alcohol on HIV degradation in hepatocytes needs to be explored. It is not known if hepatocyte death is due to apoptosis or other cell death mechanisms (such as necrosis or necroptosis) are involved. Hence understanding the type of cell death mechanism will help devise strategies to block/prevent the hepatocyte death, which mediates liver fibrosis. Also, we will explore the pathways involved in the activation of profibrotic pathways to understand if blocking these pathways will prevent activation of fibrotic genes. All the research questions will be tested in both in vitro and in vivo models. Skills that will be gained during the 3-year period dedicated to completing the 3 aims of the research proposal are laboratory techniques such as proteomics, flow cytometry, Real Time Polymerase Chain Reaction (RT PCR) for HIV RNA, mRNA, HIV DNA, digital droplet PCR (ddPCR), flow cytometry, cell culture, and animal handling. During this period, I will not only be thoroughly furnished with grant writing and presentation skills, I will be involved in the management of my resources as a trainee, a skill that is needed to become an independent researcher.