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

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.

The aim is to develop, optimize and validate a methodology using multimodality in-vivo gene reporters for simultaneous analysis of immune activation with respect to different cytokines. Transgenic reporter constructs utilizing luciferases emitting at different wavelengths for detection in vivo will be expressed alongside fluorescent proteins of different wavelengths, detectable by confocal microscopy and flow cytometry. This will be applied to in vivo imaging of the immunological choice to activate either IL-17 or IFNgamma effector functions. Knowledge of the relationship between these cell types derives largely from reductionist, in-vitro experiments and we lack the tools to track their interactions in vivo. Several labs reported IL-17 reporters hooked to fluorescent proteins, but this technology is fundamentally different, since it does not allow in vivo imaging, the key goal of our study. We will generate reporter transgene constructs in which luciferases (that emit at different wavelengths) are driven by promoters for IFNgamma and IL-17 using a BACs approach successfully applied by our collaborator (A Waisman). Constructs also carry fluorescent proteins of different colours and are codon-optimised, delivering an enhanced signal. Basic studies will validate the behaviour of each cytokine reporter with respect to actual cytokine release and the effector cell-types responsible. Studies will progress to analysis of the two cell populations simultaneously following immunization and then in 'complex' settings such as bacterial infection. In the latter case, the bacteria will be tagged in a third colour, allowing comprehensive in vivo imaging of the host-pathogen interaction. The project aims to supply an imaging modality for in vivo, longitudinal imaging of multiple cell types simultaneously, with specific advances to understanding of the in vivo interactions of Th17 and Th1 cells during an immune response including analysis of plasticity between these cell-types.

The short to medium-term impact of this research is primarily for the T cell immunology, biological research community and, more widely, for researchers with applications requiring multi-colour BPI. Thus, the ability to image IL-17 and IFN gamma choices in immunological contexts in real time in vivo should have a major impact on current debates in the field, offering a very new perspective on the problem. More generally, BPI is one of the fastest moving areas with respect to advances in technical capacity. The approach that we describe here is not simply a matter of buying a machine and applying an off-the-shelf luciferase strategy. We will be contributing considerable refinements to the current status of such experiments in terms of: codon-optimized reporters for better detection, dual fluorescent protein combinations and novel methods for separation of distinct bandwidths. These advances should impact on biologists wanting to use BPI in different areas. One extremely important outcome of this work is that it will have a considerable benefit in terms of cost, by significantly reducing the numbers of animals used in immune experiments, and welfare by utilising non-invasive imaging techniques without the need for restraint. Health: While this is not a proposal aimed at specific human, medical research issues, there will be substantial long-term benefits accruing from experimental systems that allow in vivo imaging of cytokine activation profiles and thus the testing of therapeutics, across a wide range of inflammatory, infectious and autoimmune diseases. Wealth: These mice and the technology would potentially constitute a major advance in immune imaging. Earlier similar advances have led to development of the mouse models as a commercial resource for licensing to Biopharma and other users. We anticipate that progress as described above would filter rapidly into the research community through presentations of data at meetings and submissions to high-profile journals. Findings will be freely and openly communicated to other researchers. We have an excellent track-record for making our transgenic models available to researchers across the world.