Multicoloured bimodality biophotonic imaging for in vivo non-invasive analysis of IL-17 and IFN gamma immune effector programmes

显微术的最新进展为分析生物系统开辟了新的途径。过去通过试管实验或观察组织切片的“快照”推断出的体内细胞事件，现在可以直接在生活中实时可视化。其中一项技术，生物发光，特别吸引人，因为它可以被用来获得关于实验室老鼠细胞群的信息，具有敏感性和特异性，而不需要任何外科手术。由于该技术快速、无创、无压力且结果数量少，因此被认为是实验室动物福利的一大进步。这种方法基于这样一个事实：萤火虫、金龟子和一些细菌等物种会产生天然蛋白质，这些蛋白质会在正常化学反应中自然发光。这种光非常明亮，可以通过特殊的相机和数码图像拍摄和研究，在活老鼠的细胞内检测到。这些蛋白质以转基因的形式在老鼠细胞中表达，也就是说，蛋白质的DNA被整合到老鼠的基因组中。信息可以附着在基因上，指示它只在一种或另一种特定的细胞类型中被激活。这些“体内”报告者与过去的方法有着根本的不同，在过去的方法中，一个特定的基因被连接到一个荧光蛋白上，只有在老鼠被杀死和它的细胞被移除后才能被看到。这种新方法可以根据在活老鼠体内发现的特定细胞类型来观察图像。我们关注的具体生物学问题是，调节和协调免疫系统的白细胞，即CD4 T细胞，是如何选择它们属于哪种类型的CD4细胞的。这些效应细胞产生不同的化学物质，称为细胞因子，与不同类型的免疫反应有关。两种感兴趣的是干扰素- γ和IL-17。它们的主要产生者分别被称为Th1和Th17细胞。然而，关于这些细胞类型的大部分信息来自高度操纵的系统，通常在试管中。我们希望能够在不同免疫反应的背景下，直接在活体小鼠中实时观察细胞。我们在此应用程序中描述了实现此目标所需的许多技术步骤。主要目标是创建和优化一个系统，使我们能够同时研究两种不同的细胞因子反应，每种反应都用不同的颜色标记。我们将使用第三种颜色，这样我们就可以对免疫反应所针对的细菌进行成像。为了做到这一点，我们将制造不同的转基因小鼠系，其中不同的颜色被打开，从而报告不同的细胞因子。因为在精确决定需要哪些DNA序列以及需要多少DNA序列来获得准确的表示（例如，IL-17激活）方面存在一定的未知领域，因此我们通过建立我们的策略来降低风险，该策略是由德国的一个团队在分析荧光蛋白（而不是荧光素酶）检测时生产的，该团队已被证明是成功的、大型的构建体。我们描述了验证该系统所必需的一系列实验。关键是将这些小鼠应用于有争议的免疫方面，我们不了解干扰素- γ和基于IL-17的反应之间的关系。在某些情况下，人们认为Th17细胞可能转变为Th1细胞；利用这些老鼠，我们将能够确定这种情况发生的地点、时间和条件。一般来说，我们可以用这个系统来描述这些免疫亚群在免疫和感染中的细胞相互作用。这个实验系统的产生创造了一种可以被免疫学社区使用的资源，更广泛地说，它将加强多色成像的原则，作为生物学家观察多种细胞类型之间相互作用的一种方式。

Recent advances in microscopy have opened up new avenues for analysing biological systems. Cellular events in the body that used to be deduced from test-tube experiments or from 'snapshots' looking at tissue sections, can now be visualised directly in life in real-time. One such technique, bioluminescence, is particularly appealing because it can be used to gain information about cellular populations in a lab mouse with sensitivity and specificity, without any surgical procedure. Because the technique is rapid, non-invasive, stress-free and gives results with small numbers, it is considered an advance for laboratory animal welfare. The approach relies on the fact that there are natural proteins produced by species such as fireflies, click beetles, and some bacteria that naturally luminesce as part of a normal chemical reaction. This light is so bright, it can be detected within the cells of the living mouse, by a special camera and a digital image taken and studied. The proteins are expressed in mouse cells in the form of a transgene - that is, the DNA for a protein is incorporated into the mouse genome. Information can be attached to the gene directing it to be turned on only in one particular cell-type or another. These 'in-vivo' reporters are fundamentally different from past approaches in which a particular gene has been hooked onto a fluorescent protein that can only be seen once the mouse has been killed and its cells removed. The new approach allows images to be seen in terms of where particular cell-types are to be found in a live mouse. The specific biological question that concerns us is how the white blood cells central to regulating and orchestrating immunity, called CD4 T cells, make choices about which type of CD4 cell they will be. These effector cells make different chemicals called cytokines, associated with very different types of immune response. Two of interest are interferon-gamma and IL-17. Key producers of these are termed Th1 and Th17 cells, respectively. However, most of the information about those cell-types derives from highly manipulated systems, usually in the test tube. We would like to be able to visualise the cells directly in the live mouse in real time in the context of different immune responses. We describe in this application a number of technical steps required to achieve this goal. The major objective is to create and optimise a system that will allow us to study 2 different cytokine responses simultaneously, each labelled with a different colour. We will use a 3rd colour allowing us to image the bacteria to which the immune response is directed. In order to do this we will make different transgenic mouse lines in which the different colours are turned on, thus reporting the different cytokines. Because there is a certain amount of uncharted territory in deciding precisely which and how much DNA sequence would be needed to achieve an accurate representation of, for example, IL-17 activation, we have reduced the risk by building our strategy on the back of a proven, successful, large, construct produced by a group in Germany who were analyzing fluorescent protein (rather then luciferase) detection. We describe a series of experiments necessary to validate this system. The key point will be to apply these mice to debated aspects of immunity where we do not understand the relationship between interferon-gamma and IL-17 based responses. In some cases it is thought that Th17 cells may change into Th1 cells; using these mice we will be able to define where, when and under what conditions this occurs. In general, we can then use this system to describe cellular interactions of these immune subsets in immunity and infection. The generation of this experimental system creates a resource that can be used by the immunological community and, more widely, will reinforce the principle of multi-coloured imaging as a way for biologists to view interactions between multiple cell types.