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

慢性肝病（CLD）是世界上主要的死亡原因之一。在过去的50年里，CLD的死亡率稳步上升，现在CLD是英国第五大最常见的杀手。已知的肝病有100多种，每一种都需要量身定制的治疗方案。由于诊断延误或对疾病发病机制缺乏了解，通常无法获得治疗，肝移植通常是唯一的治疗方法。这给现有和合适的捐助者的医疗保健服务造成了巨大负担。为了避免移植的需要，需要对免疫系统在整个肝脏疾病过程中的反应有更深入的了解。在此，我建议开发和验证新的方法，以测试来自人体组织的真实CLD模型的治疗方法。在小鼠身上进行实验，接受假定的治疗或基因改变来模拟人类肝脏疾病，是药物发现各个阶段的宝贵工具。免疫反应可以监测体内肝损伤的进展；遗憾的是，尽管我们已经获得了具有代表性的毒性肝损伤小鼠模型，并且我们可以测量体内纤维化的诱导和消退，但目前还没有具有代表性的免疫介导性肝损伤模型。自身免疫性肝病和慢性炎症性肝病缺乏具有代表性的小鼠模型。此外，现有的模型并不总是显示与人类肝脏疾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有很大不同。因此，需要一个更广泛和更相关的药物发现战略。我们实验室发现肝脏的主要细胞类型肝细胞能够有效地吞噬和清除死细胞（肝损伤中凋亡和坏死细胞丰富，肝细胞占肝细胞组成的80%）。我发现老鼠和人类的肝细胞清除死细胞的方式是相似的。此外，我还开创了一项实验，用一种药物治疗灌注的供体人类肝脏组织，这种药物可以阻止肝细胞清除死细胞，就像在老鼠身上一样。我的实验表明，通过吞噬和死细胞清除的读数来研究肝损伤的解决方案，用人类肝组织代替小鼠来测试治疗方法可能会提供更多信息。我建议将我使用静态显微镜技术开发的模型，用于使用我们新的多光子显微镜（欧洲同类中最先进的）进行实时成像。多光子显微镜允许高分辨率成像深入组织的实时，并在活体麻醉小鼠进行。该研究旨在完善和验证该技术在小鼠和人类肝脏移植样本中监测免疫细胞的使用，并与小鼠的实时成像进行比较。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，即t调节细胞（Treg）。这种细胞亚型在小鼠和男性中具有保守的特性。我们还有一种化合物会干扰肝细胞对Treg的捕获，我打算应用与我博士期间开发的上述死细胞清除测定相同的原理，使人类肝脏成像技术适应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男性免疫调节中的作用。我收到一些转译研究人员的来信，他们说，将灌注小鼠和人体组织中的T细胞-肝细胞相互作用与小鼠活体实验成功比较，将鼓励他们在基础生物学和药物发现研究中减少对小鼠的使用，增加对人类肝脏组织的使用。

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.