A 3-D perspective on neutrophil migration

Chemotaxis is the process by which cells can navigate towards sources of chemoattractant. It is crucial for development, immune function and the spread of cancer cells. It is based on the ability of cells to detect chemoattractants via highly specific receptors on their surface. Remarkably, when placed near to a source of chemottractant the cell can determine the direction towards its source. It achieves this by using both the fact that the attractant becomes more concentrated nearer the source and its ability to detect tiny differences in the concentration of chemoattractant around it. The cell then migrates in the direction of increasing concentration of chemottractant. Despite its importance our understanding of how chemotaxis works is very limited. It is clear that chemotaxis is made up of many component, or sub, responses. The ability to detect the chemottractant, the ability to sense changes in its concentration across very small distances and the ability to move in response to the chemoattractant (chemokinesis), which is in turn dependent on the ability of the cells to form reversible contacts with neighbouring cells or surfaces. A full understanding of chemotaxis will only emerge from an understanding of these elemental component responses. In this project we focus on the mechanisms underpinning chemokinesis (chemoattractant stimulated movement, in our assays the chemottractants are often uniform in concentration and the resulting migration is random and not directed as in chemotaxis) as a key event in chemotaxis. We aim to understand the chemical nature (the molecules that are acting as the signals) and spatial organisation of the processes inside cells that allow them to perform these responses. Neutrophils are a specialised form of white cell found in the blood. Their primary function is to ingest and kill bacterial and fungal pathogens. Neutrophils use the process of chemotaxis to migrate out of the blood stream towards areas of inflammation and also to home-in on pathogens. Thus chemotaxis is essential for neutrophils to perform their normal healthy function fighting disease. However, many long term inflammatory diseases are known to caused by neutrophils over-reacting. Too many neutrophils accumulate at the site of disease and actually contribute to host tissue damage, which in-turn leads to further accumulation of neutrophils. Hence a major objective in treating inflammatory disease such as arthritis is to reduce neutrophil migration into sites of inflammation. At the moment we have very few drugs that are able to effectively reduce neutrophil influx to sites of chronic inflammation with unwanted side effects because we do not know which steps in chemotaxis to best target. We aim to study neutrophil chemokinesis and chemotaxis. Recently there has been huge progress in understanding the events inside neutrophils that are involved in coordinating chemotaxis. This has been possible through the development of microscopes capable of seeing inside living neutrophils as they move and the use of genetic engineering techniques to make specific proteins we want to study inside cells fluorescent and hence possible to see with special illumination and detection systems in the microscopes. However, despite these advances our views of the inside of cells during chemotaxis has thus far been very 2-dimensional, that is as if we were looking down on them from above, and could not reveal any depth (the 'sides' of the cell). By using new imaging techniques we are able to begin to take many photos and combine them to give a sense of depth. What we have seen so far entirely changes our interpretation of the 2-D images and understanding of the spatial coordination of chemotaxis. In this project we hope to use mathematics and computers to improve our ability to create and interprete 3-D images of migrating neutrophils and to use this to find out the nature of the intracellular signals that regulate chemokinesis and chemotaxis.

INTRO The capacity of neutrophils to fight bacterial and fungal infections is dependent on their ability to chemotax into zones of inflammation. Neutrophil's, like other cells, chemotaxis is built upon both their abilities to polarize and move in response to uniform stimulation (chemokinesis) and to sense, and navigate in accord with the sense of, gradients of chemottractants. Our, and others, work has shown that PIP3 signals induced by chemottractants are thought to become polarized, high at the 'leading edge', and important for polarization and chemokinesis. SUPPORTING DATA We argue, and show preliminary data indicating, that the dominant features of this polarization, both in terms of PIP3 and F-actin accumulation, are created by a combination of chemoattractant AND adhesion-generated signals. This creates a situation where, in contrast to the '2-D, leading edge dogma', the PIP3 is highest in the zone of attachment UNDER new protusions and is low under the uropod and the F-actin, although initially in a crescent at the front of the cell, becomes associated with substrate-fixed focal complexes in the centre of the cell. HYPOTHESIS The above is due to cycle started by GPCR/PI3Kgamma/PIP3-mediated activation of integrin adhesion. This leads to integrin activation of Class IA PI3Ks, further PIP3 accumulation, the evolution of focal complexes and ultimately the recruitment of suppressors such as SHIPI, creating a gradient of both PIP3 and adhesion/de-adhesion. QUESTIONS & AIMS What are the key regulators that govern the 3-D distribution of PIP3 signals during chemokinesis? Within this we consider how PIP3 signals can both direct, and get shaped by, the cycle of integrin and focal complex-dependent adhesion and de-adhesion. To answer these questions convincingly will require the development of analytical tools capable of interrogating and assimilating data from movies capturing the 3-D distribution of signalling molecules, we also aim to create such tools.

A) Who will benefit (outside of our immediate academic community, see beneficiaries)? 1) The pharmaceutical sector, specifically, those interested in targetting inflammatory disease. The work is directly relevant to an understanding of the intracellular signalling underpinning neutrophil migration. This process is a key step in the development of inflammatory disease. Furthermor, our owrk focuses on PI3Ks, which are already accepted to be validated anti-inflammatory targets with a number of international pharmaceutical organisations taking PI3K inhibitors through clinical trials as anti-inflammatories. We have collaborations with a number of companies in this domain including Karus pharmaceuticals and in the recent past PIraMed (now Genentech). 2) Researchers within the NHS. We have substantial, long term collaborations with NHS, clinical researchers who are interested in human neutrophil migration. These clinicians co-supervise some students in our lab and attend our lab meetings. This helps us ensure our work is therapeutically and commercially relevant. 3) The medical charities. Aspects of our work on neutrophil signalling have been and are funded by the medical charities, specifically, the Wellcome trust, The Arthritis research council and the British Lung Foundation. We have a number of long term contacts in all of these organisations and attend some of their meetings. 4) PDRAs employed on the grant. B) How will they benefit? 1) In the context of the benefits to the pharmaceutical sector the benefits will be improved target selection for focused medicinal chemistry, better understanding of the mechanism of action of PI3K-selective drugs. These effects will be in both the short and medium term in the context of the fact PI3K-selective drugs are already in trials. Through any longer term successes in these trials there are potential benefits to health and, in the setting that our commercial collaborators are based in the UK, the competitiveness of the UK economy and jobs. The PDRAs will gain skills and experience that are directly relevant to the pharmaceutical sector. 2) Clinical colleagues in the NHS will gain from our work with mouse genetic models (that cannot be usedin human studies) and apply its conclusions to their work with clinical or human material. 3) The medical charities that have funded out work have gained through delivery of their strategic objectives. C) What will we do to ensure we engage our users and maximise the impact of this work? 1) We are already regularly invited to present our work in the commercial and NHS sectors, this would continue during this project. We already take time at internaional meetings to discuss our work with commercial colleagues that would continue. Clinicians already attend our lab meetings on a regular basis, that would continue. We would continue to seek out opportunties to obtain funding in this area from the commercial sector by listening and finding out their priorities. The Babraham Institute organises forums with pharmaceuticalc companies, we always try to attend and present our work at these events. 2) We will continue to collaborate with commercial and clinical colleagues as described above. We currently have active collaborations with Horizon Discovery, AstraZeneca and Karus pharmaceuticals, this is evidence of the effectiveness of our approach in this area. 3) If we obtain date with clear commercial implications we will discuss potential ways ahead with the commercial arm of BI, BB. However, in this project it unlikely this will be in the form of a patentable chemical entity. We have experience in translating our research into potentially valuable patents both in our lab (eg, we are named on patents with Onyx pharmaceuticals protecting the principle of targetting PI3Kgamma for anti-inflammatories) and in BBT. We also have a track record of transferring knowledge to the commercial sector through consultancies and other routes described above.