Modeling of wound repair and inflammation in the Drosophila embryo

Wound healing is the body's process of repairing damaged tissue and takes place for all wounds, be they a nick to the finger or the repair of internal organs after abdominal surgery. There are many occasions when tissue repair fails, leading to chronic non-healing wounds such as venous leg ulcers which are a huge clinical burden for elderly patients and suffered by about 500,000 people in the UK. Equally, the process can be too exuberant leading to fibrosis and scarring as a consequence of excessive contraction, healing and inflammation. In order to understand how tissue repair goes awry and how it might be improved, we need to better understand the process, and one way to do this is by turning to a very simple model, the fruitfly, Drosophila. Using the fly it is possible to make movies of healing wounds in living animals and observe precisely how individual cells are involved at every stage. Moreover, Drosophila also have hugely simpler genetics so we can relatively easily determine which genes are pivotal in each component of each stage of the repair process. We have already shown that for the wound closure step and for the associated inflammatory step where white blood cells are recruited to the wound, much of what we find in flies holds true for mice and man. Here we propose using Drosophila to gain a fast track understanding of:
1. How molecular switches in skin cells (both those at the wound edge and the ones further back), respond to the wound signals to assemble the actomyosin contractile cables (just like in muscle) that move the cells forward to heal the wound. These signals will not only be chemical but we think also mechanical, like stretch, and our studies in fly will let us investigate this too. We also want to know which of the genes that are switched on in the wound edge cells are most important, and what the steps are that enable them to be switched on. This fundamental knowledge will lend clues when designing potential therapeutics to "kick start" healing in patients suffering from chronic, non-healing wounds.
2. What signals draw white blood cells to the wound and how do they sense these signals? Since the white blood cells are there to deal with wound infection we want to watch how they do this and also to determine what it is that forces them to leave the wound site when healing is complete because many human pathologies are a consequence of inflammation failing to resolve. Being able to artificially modulate the inflammatory response in patients would enable us to prevent some of the negative consequences of inflammation at wound sites including fibrosis. The fly offers a chance to take the first steps in achieving this goal.

Because we are doing all these experiments in flies which have a very short lifecycle and very powerful genetics, we can find answers to these questions much faster than would be possible in any other model organism, but it will be important to take what we discover in flies and apply it to more clinically relevant models. This important step is made considerably easier for us since one of our labs also works on vertebrate wound healing models in zebrafish and mouse and has clinical collaborations too with a group in Cardiff that have a wound healing clinic dealing with human patient samples.

Drosophila embryos provide a model of wound healing and the associated inflammatory response that is both genetically tractable, and amenable to live, high resolution imaging of these very dynamic processes. This combination of genetics and live imaging is very powerful for uncovering the mechanisms that underpin repair and is not available in other model organisms. We have established a laser wound model that allows us to observe both the wound re-epithelialisation response and the associated recruitment and resolution of inflammatory cells to and from the wound.

We will use a variety of well tested Drosophila genetic techniques, in particular the GAL4 UAS system to drive various fluorescent gfp and/or mCherry fusion proteins in either epithelial cells or hemocytes (Drosophila macrophages) to track the dynamic behaviours of actin, or microtubules or junctional proteins during the healing process. The same system will be used to drive reporters (eg of Calcium or H202) or RNAi constructs to knock down genes in specific tissues. We also propose several microarray experiments using the FlyChip facility in Cambridge to analyse the transcriptional changes following wounding and/or bacterial infection as well as genetic screens which will take advantage of publically available p-element insertion lines. Our standard tool for imaging the wound response in wild type embryos or in embryos where a component has been genetically or pharmacologically modified, will be live confocal microscopy using both conventional confocal microscopes together with spinning disk confocal microscopy. Complementary TEM studies will also be used where understanding of ultrastructure is important also. Our work will also take advantage of a bead implantation assay developed in our labs which allows local application of pharmacological reagents to wounds and subsequent live imaging. This technique opens up a wealth of experimental possibilities as outlined in the case for support.

It is essential that all living organisms possess a robust ability to repair damaged structures if they are to survive. However, this healing machinery can fail catastrophically, leading to chronic non-healing wounds such as venous leg ulcers which are currently a huge clinical burden for elderly patients and suffered by more than 500,000 people in the UK. By contrast the repair process can also be overactivated, for example after burn or scald wounds, triggering excessive wound bed contraction and inflammation. Such an over-exhuberent response leads to fibrosis and scarring which can cause great discomfort and be severely debilitating, and often requires further surgery to remedy, again adding to the clinical burden. There are very few science-driven therapies to improve wound repair in the clinic, as highlighted by the current approaches used which include pressure bandaging for leg ulcers and massage therapy for reducing scarring. The clear need for research-driven therapies in the clinic is obvious. One way to increase our understanding of how tissue repair goes awry and how it might be improved is to turn to a simple model, the fruitfly, Drosophila whose powerful genetics allow us to dissect out the genes required for each step of the repair process and to live image healing at single cell level within a living animal . Our previous studies have already shown that much of what we find in Drosophila holds true for vertebrate repair also. In this application we propose using Drosophila to gain a fast track understanding of two key aspects of the wound healing response:
1. Re-epithelialisation. We want to understand how molecular switches in epithelial cells respond to wound cues to assemble the actomyosin machinery that moves front row cells and those further back to heal the wound. We believe that these signals are likely to be a combination of mechanical signals such as tension changes, together with chemical cues. We also want to study the role of the microtubule cytoskeleton in the process of re-epithelialisation and to investigate how genes switched on in the wound edge cells influence the wound closure process, and how epigenetic changes in these cells might 'unlock' key repair genes.
2. Inflammation. We want to investigate what signals attract immune cells to the wound and how these cells sense the early inflammatory signals, and how they integrate these early signals with other later inflammatory cues and how bacterial infection impacts on their inflammatory migrations. Since many human pathologies are a consequence of inflammation failing to resolve we also want to determine what drives immune cells to leave the wound site when healing is complete.
The powerful genetics, short lifecycle and amenability for live imaging of Drosophila means that we can much more rapidly achieve these goals in Drosophila than in other model organisms. An additional benefit of studying tissue repair in Drosophila is that it is consistent with the 3R's. Indeed, PM is plenary speaker at the 3Rs roadshow highlighting how Drosophila can, in part, substitute for and complement in vivo studies of wound repair in mice. Drosophila embryos are also a powerful in vivo system for testing drugs and exploring their pharmacology in the healing wound.
Our long term goal is to take what we learn in flies to higher model organisms and finally to man in order to develop therapies that enhance healing. This will be facilitated both within the lab of PM where wound healing is also studied in mouse, and with other colleagues working in mouse, and with clinical collaborators, in particular Prof Harding who has a wound healing clinic in Cardiff. While our studies will focus on skin repair they will also have implications for repair of other tissues and organs, where chronic non-healing wounds, and inflammation driven fibrosis are major clinical problems also (eg colitis and adhesions in the gut and fibrosis in lung and kidney and liver).