Systems Biology analysis of biological timers and inflammation

As we get older our immune system tends to get weaker and it becomes increasingly difficult to shake off diseases. At the same time we tend to develop arthritis and other auto-immune diseases which are localised instances of uncontrolled inflammation. In this grant application our multidisciplinary team of scientists consisting of physiologists, biologists, mathematicians and computer scientists aim to look at a very important signalling system NF-kappaB. This system plays an important role in stress and the immune responses and determines the fate of cells in the body, which is essential for general health and wellbeing. We will use a combined experimental and mathematical approach to analyse integrated systems that control NF-kappaB signalling in normal cells and tissues.
We found that NF-kappaB carries signal information in the timing of its movements between the nucleus and cytoplasm. The timing of these movements determines which genes are switched on. We now wish to understand how this key process of NFkappaB signalling is controlled through normal life. How is it controlled by the cell division cycle, and how is it controlled by the sleep-wake cycle of the 24 h circadian clock.
Recently, we have found that the speed of the oscillations in the NF-kappaB signalling system are very temperature sensitive in the physiological and fever range between 35 and 40 degrees C. This also appears to markedly change the pattern of which genes are switched on or off. Our work so far has been in cell lines grown in the laboratory and we now wish to investigate NF-kappaB signalling in normal cells taken from transgenic mice that have fluorescently labelled NF-kappaB proteins. We will use these cells to determine the NF-kappaB response to temperature, a range of physiological stimuli including glucocorticoids (which are often used in inflammatory treatment), to screen small molecule drugs to find ones that modulate the response and to study the quantitative relationships with the cell cycle and the circadian clock. The data from these experiments and others published in the literature will be used to build integrated mathematical models that can predict important aspects of cell, tissue and animal physiology relevant to understanding the maintenance of a healthy organism and how this may change with age.
Key aims will be to understand how the clock and cell cycle together affect the timing and level of NF-kappaB signalling and which target genes are switched on. One of the NF-kappaB family of proteins, p105 encoded by the NFkB1 gene, has been found to cause faster ageing in mice when this gene is missing (Mann and von Ziglnicki, personal communication). We wish to investigate whether changes in NF-kappaB dynamics are involved in this ageing condition. Therefore, we will make a BAC reporter for p105 with fluorescent fusions at either end of the protein.
We are an ideal team to perform this work, because we have complementary interdisciplinary skills in cell imaging, image analysis, molecular cell biology, physiology, genomics, bioinformatics and mathematical modelling. A core part of the team has an excellent track record of working together to analyse the NF-kappaB signalling system. In addition, this new project brings in new team members with considerable expertise in animal physiology, circadian clocks and endocrinology.

We will develop a new quantitative understanding of coordinated inflammatory signalling in primary cells & tissues. We previously showed novel single-cell dynamics & function of the key inflammatory NF-kappaB signaling system in cell lines. This work has established a central hypothesis that coupling between multiple dynamic systems may allow different signals to be coordinated to ensure accurate & appropriate cell & tissue inflammatory responses. A key aim of this project is to understand process coordination between the NF-kappaB, circadian clock & cell cycle systems. Work will include 1) exploitation of transgenic models for NF-kappaB analysis system (using RelA & IkappaBalpha fluorescent BAC reporter mice) & the coupling with the circadian clock (cross with the Per2 luciferase reporter mice). 2) Analysis of the coupling between NF-kappaB, circadian clock & cell cycle in cell lines, primary cells & tissues using dynamic cell & single molecule imaging, quantitative proteomic & gene expression assays. 3) Perturbation analyses of system coordination using physiological stimuli, temperature, stimulation with glucocorticoids, disruption of clock (knockout & fast clock mutants) & a chemical screen to identify new agents that modulate NF-kappaB dynamics. 4) A key collaboration will be initiated with D. Mann & T. von Ziglnicki (Newcastle) to analyse NF-kappaB dynamics in NFKB1 KO that show an ageing phenotype. The theoretical work will produce new multi-scale deterministic & stochastic models to enable accurate predictions of temporal gene responses for a given input, thus providing new understanding of process coordination during inflammation. Modelling & experimental approaches will be used iteratively to direct experimental design. Our key aim is to provide new insights for understanding coupled biological systems that underlie the control of inflammation in cells & tissues in healthy & ageing organisms.

The application of systems biology offers great potential for a better understanding of cell signalling and decision-making pathways. This in turn creates the opportunity for the identification of better drug targets and more efficacious modes of treatment of disease. This application is relevant to important processes in human and animal disease. In inflammation and innate immunity, NF-kappaB is a critically important regulator. The clock and cell cycle are important regulatory timing systems that control fundamental aspects of biology. This project is important for applied research and is of relevance to healthcare and to the pharmaceutical industry.
In this respect we have strong support for this project from GlaxoSmithKline (GSK, see letter from S. Farrow). They will assist us in conducting a new screen for small molecules that modulate the dynamics of NF-kappaB signalling (providing £20k pa of in kind support). GSK are also substantially supporting other work in the labs of A. Loudon and D. Ray. Until recently, M. White had an established collaboration with AstraZeneca (AZ) through Dr J. Unitt, who was a co-author on our major NF-kappaB papers. With the closure of AstraZeneca Charnwood this collaboration was put on hold. Recently AZ and GSK have each jointly funded the new £15m Manchester Collaborative Centre for Inflammation Research (MCCIR). It has been agreed by all involved that the present project would be affiliated to MCCIR. Following the likely appointment of a new Director in the next few weeks, it is anticipated that details of available in-kind support for the current project from the MCCIR will be formally agreed.
The major technologies being used in this project are based on bio-imaging. This is an important and currently growing area. We have close relationships with instrumentation companies and in particular with Carl Zeiss and Hamamatsu Photonics with whom MW has collaborated for 16 and 20 years respectively. This involves the loan and testing of equipment and the exchange of ideas, for new developments in microscopy and detection. Both companies will sponsor the project through support for the microscopy training courses in years 1,3 and 5 (the first course will be joint with the last SABR course thus saving on costs) The companies will provide speakers for the course from Germany and Japan, the loan of demonstration equipment and financial support. While the course is focused on providing training for the staff on the SABR project, places have been made available to staff on other systems biology projects from around the country. Representatives of each of the instrumentation companies are on the scientific advisory board for the BBSRC SABR project.
The use of microscopy generates movies and images that are colourful and visual. They represent an excellent resource for the development of public understanding of science. MW has given lectures at Public Understanding of Science meetings (e.g. ASE lecture in 2008). In 2006 a group from the CCI led by MW and DS, presented an exhibit entitled "The Language of Cells" at the Royal Society exhibitions in London, Glasgow and at Science Day at Buckingham Palace. The whole group are very keen apply to exhibit at future Royal Society exhibitions and this project may well give us a useful theme from which to develop a new exhibit. This offers a specific opportunity and we will seek others through talking to schools and other groups. When publicity of outcomes from this project are important, we will engage with the University of Manchester Press Office to coordinate this. We have good experience of media publicity and have previously worked with the BBSRC Press office in publicising high impact publications.