Screening for, and characterisation of, novel immune cell extravasation genes in Drosophila, mice and man
Lead Research Organisation:
University of Bristol
Department Name: Biochemistry
Abstract
An inflammatory response is pivotal in many normal physiological responses, for example wherever there is a wound or site of infection. In such scenarios, if inflammation is delayed or insufficient then the consequences to health can be catastrophic. Conversely, if the inflammatory response is excessive or slow to resolve, then 'chronic' inflammation can also lead to several pathologies, ranging from tissue scarring through to atherosclerosis or even cancer. A better understanding of how inflammatory cells are recruited to their target tissues will help guide us how the inflammatory response might be modulated therapeutically to treat these conditions.
One of the key 'rate-limiting' steps in an inflammatory response is the movement of leukocytes (white blood cells) from the circulation across blood vessel walls (known as 'extravasation') to sites of damage or infection. However, despite decades of research in this field, many aspects of this step in leukocyte recruitment remain poorly understood and there is still much to learn; this is in part because breaching of vessel walls is a highly complex process to dissect at the molecular level and it is incredibly difficult to image in opaque tissues of traditional mammalian models (e.g. mouse).
In Bristol we have recently developed a new model in which to study immune cell 'extravasation' using the fruitfly, Drosophila. Fruitflies offer many advantages for this research, being genetically 'tractable' (easy to mutate and study individual genes) and also translucent (enabling us to watch these movements live using microscopy). Since fruitflies largely have an 'open circulation' without discrete blood vessels, they were not previously considered a useful model for this field. However, we have identified a period in pupal life when immune cells circulate through the wing "veins" and a laser-induced wound triggers immune cells to leave the veins and move towards the damage. We have already used this model to identify one novel receptor protein (Tre1) that is essential for immune cell extravasation, and excitingly, the equivalent gene in mammals (GPR84) has proven to be required for neutrophil extravasation in mice (our pilot studies in QMUL), and is associated with several inflammatory conditions in human patients.
Our findings suggest that Drosophila may provide a powerful fast-track approach to identify more genes required in immune cells (or vessel cells) for this pivotal step in inflammation. Here we propose to use our fly model to initiate such a 'screen' and develop an even faster screening process in newly hatched adults to ultimately search through the whole genome. Studies in the fly will also provide new detail on how proteins like Tre1 function at the molecular level. At QMUL, we will complement this work by exploring how the equivalent genes function in mammals during extravasation and investigate how these genes might impact on important physiological and pathological processes, such as healing of a skin wound and during peritonitis.
Ultimately, we want to identify which of the genes highlighted by our studies in fly and mouse are most promising as targets for modulation of inflammation in the clinic. To extend our findings towards translation, we will undertake studies using human cells where we knockdown candidate genes/signalling pathways in human vessel or immune cells to determine how this impacts leukocyte extravasation. Alongside this, we will use "population health" approaches to search human genetics data to identify which of our candidate genes are associated with human disease.
In summary, we propose to employ a multi-disciplinary, cross-institutional and multi-organism approach to uncover more genes with important functions in immune cell extravasation, that will provide potential therapeutic targets for inflammatory disorders in the clinic. We envision this multi-modal approach will be far more insightful than studies in a single model alone.
One of the key 'rate-limiting' steps in an inflammatory response is the movement of leukocytes (white blood cells) from the circulation across blood vessel walls (known as 'extravasation') to sites of damage or infection. However, despite decades of research in this field, many aspects of this step in leukocyte recruitment remain poorly understood and there is still much to learn; this is in part because breaching of vessel walls is a highly complex process to dissect at the molecular level and it is incredibly difficult to image in opaque tissues of traditional mammalian models (e.g. mouse).
In Bristol we have recently developed a new model in which to study immune cell 'extravasation' using the fruitfly, Drosophila. Fruitflies offer many advantages for this research, being genetically 'tractable' (easy to mutate and study individual genes) and also translucent (enabling us to watch these movements live using microscopy). Since fruitflies largely have an 'open circulation' without discrete blood vessels, they were not previously considered a useful model for this field. However, we have identified a period in pupal life when immune cells circulate through the wing "veins" and a laser-induced wound triggers immune cells to leave the veins and move towards the damage. We have already used this model to identify one novel receptor protein (Tre1) that is essential for immune cell extravasation, and excitingly, the equivalent gene in mammals (GPR84) has proven to be required for neutrophil extravasation in mice (our pilot studies in QMUL), and is associated with several inflammatory conditions in human patients.
Our findings suggest that Drosophila may provide a powerful fast-track approach to identify more genes required in immune cells (or vessel cells) for this pivotal step in inflammation. Here we propose to use our fly model to initiate such a 'screen' and develop an even faster screening process in newly hatched adults to ultimately search through the whole genome. Studies in the fly will also provide new detail on how proteins like Tre1 function at the molecular level. At QMUL, we will complement this work by exploring how the equivalent genes function in mammals during extravasation and investigate how these genes might impact on important physiological and pathological processes, such as healing of a skin wound and during peritonitis.
Ultimately, we want to identify which of the genes highlighted by our studies in fly and mouse are most promising as targets for modulation of inflammation in the clinic. To extend our findings towards translation, we will undertake studies using human cells where we knockdown candidate genes/signalling pathways in human vessel or immune cells to determine how this impacts leukocyte extravasation. Alongside this, we will use "population health" approaches to search human genetics data to identify which of our candidate genes are associated with human disease.
In summary, we propose to employ a multi-disciplinary, cross-institutional and multi-organism approach to uncover more genes with important functions in immune cell extravasation, that will provide potential therapeutic targets for inflammatory disorders in the clinic. We envision this multi-modal approach will be far more insightful than studies in a single model alone.
Technical Summary
We have developed a new in vivo model to search for novel genes that might drive immune cell extravasation from vessels to wounds, which allows us to utilise the live-imaging opportunities of translucent Drosophila pupae, together with their genetic tractability. We have already identified one novel "extravasation" gene (Tre1) which is essential for this process in flies. We now have pilot data showing that murine neutrophils lacking the mammalian ortholog of Tre1, GPR84, exhibit a clear inability to breach vessel walls, indicating our screening approaches in the fly offer a powerful approach to reveal novel vertebrate extravasation genes.
Here we propose a multi-disciplinary investigation of immune cell extravasation in fly and mouse models and extension towards human studies, through human cell co-culture and population health approaches. We will perform an in-depth characterisation of the role of Tre1/GPR84, with in vivo studies providing mechanistic detail and human assays to explore clinical relevance. We will exploit the unique genetic tractability of Drosophila to perform large-scale screens in pupae and subsequently adult flies (using tissue-specific RNAis) to identify novel genes required for extravasation in either immune or vessel cells. We will be guided by RNA-seq studies revealing those genes upregulated at the time of extravasation.
Building on our Tre1/GPR84 findings, we aim to establish a pipeline to validate novel extravasation targets from our Drosophila screens in our established murine intravital imaging assays (using lineage-specific knockouts) as well as testing how these genes impact physiological responses such as peritonitis and wound-healing. To complement our in vivo studies, we will examine whether the human orthologs of these genes are required for extravasation in flow assays with human leukocytes and endothelial cells, and search for associations with human inflammation-related pathologies using genetic epidemiology techniques.
Here we propose a multi-disciplinary investigation of immune cell extravasation in fly and mouse models and extension towards human studies, through human cell co-culture and population health approaches. We will perform an in-depth characterisation of the role of Tre1/GPR84, with in vivo studies providing mechanistic detail and human assays to explore clinical relevance. We will exploit the unique genetic tractability of Drosophila to perform large-scale screens in pupae and subsequently adult flies (using tissue-specific RNAis) to identify novel genes required for extravasation in either immune or vessel cells. We will be guided by RNA-seq studies revealing those genes upregulated at the time of extravasation.
Building on our Tre1/GPR84 findings, we aim to establish a pipeline to validate novel extravasation targets from our Drosophila screens in our established murine intravital imaging assays (using lineage-specific knockouts) as well as testing how these genes impact physiological responses such as peritonitis and wound-healing. To complement our in vivo studies, we will examine whether the human orthologs of these genes are required for extravasation in flow assays with human leukocytes and endothelial cells, and search for associations with human inflammation-related pathologies using genetic epidemiology techniques.
