Multiphoton imaging in human liver tissues: validation of a new tool for drug discovery.

人体肝脏组织中的多光子成像：药物发现新工具的验证。

• 批准号: NC/R002061/1
• 负责人: Scott Davies
• 金额: $ 13.94万
• 依托单位: University of Birmingham
• 依托单位国家: 英国
• 项目类别: Fellowship
• 财政年份: 2018
• 资助国家: 英国
• 起止时间: 2018 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=NC%2FR002061%2F1
• 关键词: Multiphoton; imaging; human; liver; tissues

Chronic liver disease (CLD) is one of the world's leading causes of death. Rates of mortality due to CLD have steadily increased over the past 50 years and CLD is now the 5th most prevalent killer in the UK. There are over 100 known forms of liver disease, each of which requires a tailored course of treatment. Therapies are often unavailable due to delays in diagnosis or lack of insight into disease pathogenesis, and liver transplantation is often the only curative treatment. This is creating a huge burden on healthcare services for available and suitable donors. To avoid the need for transplantation, a greater understanding is needed on how the immune system responds throughout the course of liver disease. Herein I propose to develop and validate new methodology for testing therapeutics in authentic CLD models derived from human tissue. Experiments in mice which receive putative treatments or genetic alterations to mimic human liver disease are a valuable tool for various stages of drug discovery. Immune responses can be monitored in vivo liver injury progresses; unfortunately, although we have access to representative mouse models of liver injury induced by toxicity and we can measure the induction and resolution of fibrosis in vivo, there are no representative models for immune-mediate liver damage. Autoimmune liver diseases and chronic inflammatory liver diseases lack representative mouse models of comparable inflammation. Additionally, available models do not always display most symptoms associated with human liver disease, in part because the mouse immune system differs significantly to that of humans. As such, a broader and more relevant strategy for drug-discovery is required. Our laboratory has identified that the major cell type of the liver, the hepatocyte, is able to phagocytose and clear dead cells effectively (apoptotic and necrotic cells are abundant in liver injury and hepatocytes make up 80% of the liver cell composition). I showed that both mouse and human hepatocytes clear dead cells in a similar manner. Moreover, I pioneered experiments whereby perfused donor human liver tissue was treated with a drug which prevented hepatocytes from clearing dead cells in the same manner as it did in mice. My experiments demonstrated that it may prove more informative to use human liver tissue for the testing of therapeutics in place of mice to study resolution of liver injury with a readout of phagocytosis and dead cell clearance. I propose to adapt the models that I developed using static microscopy techniques, to real time imaging using our new multiphoton microscopes (most advanced of their kind in Europe). Multiphoton microscopy allows high resolution imaging deep into tissues in real time and is performed in living anaesthetised mice. This fellowship aims to refine and validate the use of this technology for monitoring immune cells in samples of mouse and human liver explants in comparison to live imaging of mice. Our team recently observed that hepatocytes can selectively delete live immune cells which normally dampen inflammation, known as T-regulatory cells (Treg). This cell subtype has conserved properties in mice and men. We also have a compound that perturbs Treg capture by hepatocytes, and I intend to apply the same principles as for the aforementioned dead cell clearance assays developed in my PhD to adapt the human liver imaging technologies to live measurements by multiphoton microscopy. These experiments will reveal an unappreciated role for hepatocytes in immune regulation in mice and men. I have letters from translational researchers stating that successful comparison of T cell-hepatocyte interactions in perfused mouse and human tissue compared to mouse intravital experiments would encourage them to reduce their use of mice and increase the use of human liver tissue in basic biology and drug discovery research.

慢性肝病(CLD)是世界主要的死亡原因之一。在过去的50年里，慢性阻塞性肺病的死亡率一直在稳步上升，目前在英国，慢性阻塞性肺病是第五大最常见的杀手。已知的肝病有100多种，每一种都需要一个量身定制的疗程。由于延误诊断或缺乏对疾病发病机制的了解，治疗方法往往不可用，而肝移植往往是唯一的根治方法。这给可用的和合适的捐赠者的医疗服务造成了巨大的负担。为了避免移植的需要，需要更多地了解免疫系统在整个肝病过程中的反应。在此，我建议开发和验证新的方法学，用于在源自人类组织的真实CLD模型中测试治疗方法。在老鼠身上进行的实验，接受了假定的治疗或基因改变，以模拟人类肝病，对于药物发现的不同阶段来说，是一个有价值的工具。免疫反应可以在体内监测肝损伤的进展；不幸的是，尽管我们可以获得有代表性的毒性肝损伤小鼠模型，我们可以在体内测量肝纤维化的诱导和消退，但还没有具有代表性的免疫介导肝损伤模型。自身免疫性肝病和慢性炎症性肝病缺乏具有代表性的类似炎症的小鼠模型。此外，现有的模型并不总是显示与人类肝病相关的大多数症状，部分原因是小鼠的免疫系统与人类的免疫系统显著不同。因此，需要一种更广泛和更相关的药物发现战略。本实验室已鉴定出肝脏的主要细胞类型--肝细胞能够有效地吞噬和清除死亡细胞(肝损伤中大量的凋亡和坏死细胞，肝细胞占肝细胞组成的80%)。我发现，小鼠和人类的肝细胞都以类似的方式清除死亡细胞。此外，我开创了一项实验，用一种药物治疗灌流的供体人类肝组织，这种药物可以防止肝细胞像在小鼠身上那样清除死亡细胞。我的实验证明，用人肝组织代替小鼠测试治疗药物，通过读取吞噬功能和死亡细胞清除的读数来研究肝损伤的解决方案，可能会被证明是更有意义的。我建议将我开发的使用静态显微镜技术的模型调整为使用我们的新多光子显微镜(欧洲同类中最先进的)进行实时成像。多光子显微镜允许实时深入组织的高分辨率成像，并在活体麻醉的小鼠身上进行。这项奖学金的目的是改进和验证这项技术用于监测小鼠和人类肝脏移植样本中的免疫细胞，并与小鼠的活体成像进行比较。我们的团队最近观察到，肝细胞可以选择性地删除通常抑制炎症的活免疫细胞，称为T调节细胞(Treg)。这种细胞亚型在小鼠和人身上具有保守的特性。我们还有一种化合物可以干扰肝细胞对Treg的捕获，我打算应用与我在博士论文中开发的前述死亡细胞清除分析相同的原理，使人类肝脏成像技术适用于多光子显微镜的实时测量。这些实验将揭示肝细胞在小鼠和人类免疫调节中的未被认识的作用。我收到了翻译研究人员的来信，他们表示，与小鼠体内实验相比，在灌流的小鼠和人类组织中成功地比较T细胞和肝细胞的相互作用，将鼓励他们减少对小鼠的使用，并在基础生物学和药物发现研究中增加对人类肝组织的使用。