A non-mammalian model to study innate immune modulation of airways remodelling in asthma.
Lead Research Organisation:
University of Sheffield
Department Name: Infection Immunity & Cardiovasc Disease
Abstract
In asthma, patients are very familiar with the day to day variability in their symptoms, and this can cause hospitalisation and sudden death. However, over the long-term, it is scarring of the airways that causes breathlessness and ultimately disability. The process of scarring in the airways is not well understood and no treatments prevent it. Scarring is often called fibrosis, and scar tissue in the airways is made by fibrotic cells in the lungs. These fibrotic cells come from circulating cells called fibrocytes. We think fibrocytes are programmed to be pro-scarring by white blood cells of the immune system.
One of the reasons we don't understand airway fibrosis is that it is that no single model, in either the test tube or in animals, has shown the ability to mimic the human disease. Despite this many mice are used each year, sometimes in experiments lasting many weeks. We would like to reduce these mouse experiments and make sure that any experiments that are done are focussed on the best research question and shaped by work in other models.
We therefore propose to take circulating cells from patients with asthma and to graft these into transparent zebrafish embryos. These cells are not rejected and become the cells that make fibrous tissue in the developing zebrafish. In the first days after fertilisation of a zebrafish egg, experiments on the developing embryos are not legally considered to be animal experiments; we believe this is more humane than using fully developed mice. This approach will establish a model where we can see how fibrocytes interact with white blood cells and test whether this interaction is important in driving fibrosis.
In the longer term this will allow us to better understand the process of fibrosis and to develop treatments for it, without impacting on animal welfare.
One of the reasons we don't understand airway fibrosis is that it is that no single model, in either the test tube or in animals, has shown the ability to mimic the human disease. Despite this many mice are used each year, sometimes in experiments lasting many weeks. We would like to reduce these mouse experiments and make sure that any experiments that are done are focussed on the best research question and shaped by work in other models.
We therefore propose to take circulating cells from patients with asthma and to graft these into transparent zebrafish embryos. These cells are not rejected and become the cells that make fibrous tissue in the developing zebrafish. In the first days after fertilisation of a zebrafish egg, experiments on the developing embryos are not legally considered to be animal experiments; we believe this is more humane than using fully developed mice. This approach will establish a model where we can see how fibrocytes interact with white blood cells and test whether this interaction is important in driving fibrosis.
In the longer term this will allow us to better understand the process of fibrosis and to develop treatments for it, without impacting on animal welfare.
Technical Summary
A substantial part of the impact of asthma relates not to acute exacerbations, but to chronic breathlessness resulting from airways remodelling. We have no clear way to prevent this remodelling, which can occur in all immune phenotypes of asthma. Current, widely exploited mouse models cannot fully recapitulate this chronic pathway.
We hypothesise that airway remodelling in asthma is driven by direct communication between innate immune cells and fibroblasts derived from incoming fibrocytes. Furthermore, we predict that pharmacological interruption of this communication will provide a new approach for long-term drug discovery in asthma.
We therefore propose to develop a non-mammalian in vivo model for the study of the interactions between fibrocytes, tissues, and other innate immune cells. Zebrafish larvae are transparent and genetically tractable, with transgenically labelled neutrophils and macrophages enabling detailed study of cell behaviours in vivo. In addition, xenotransplants into developing larvae are tolerated since they are present before immune maturation. For example, xenotransplanted human lung mesenchymal stem cells integrate into the tissues of the developing zebrafish giving a unique model for the real-time study of disease fibroblast function in vivo. By combining these approaches in a single model, we will be able to offer a unique system for the study of a range of hypotheses relating to immune cell interaction with fibrotic cells isolated from human asthmatics.
In this one year pilot grant we will optimise parameters for isolation and simulation of fibrocytes from human asthmatic and control subjects, establish a transplant protocol into larval zebrafish and observe the interaction with native innate immune cells.
We anticipate a major impact on the 3Rs: reducing the number of mice used for chronic remodelling studies of asthma, informing and refining those experiments that are performed.
We hypothesise that airway remodelling in asthma is driven by direct communication between innate immune cells and fibroblasts derived from incoming fibrocytes. Furthermore, we predict that pharmacological interruption of this communication will provide a new approach for long-term drug discovery in asthma.
We therefore propose to develop a non-mammalian in vivo model for the study of the interactions between fibrocytes, tissues, and other innate immune cells. Zebrafish larvae are transparent and genetically tractable, with transgenically labelled neutrophils and macrophages enabling detailed study of cell behaviours in vivo. In addition, xenotransplants into developing larvae are tolerated since they are present before immune maturation. For example, xenotransplanted human lung mesenchymal stem cells integrate into the tissues of the developing zebrafish giving a unique model for the real-time study of disease fibroblast function in vivo. By combining these approaches in a single model, we will be able to offer a unique system for the study of a range of hypotheses relating to immune cell interaction with fibrotic cells isolated from human asthmatics.
In this one year pilot grant we will optimise parameters for isolation and simulation of fibrocytes from human asthmatic and control subjects, establish a transplant protocol into larval zebrafish and observe the interaction with native innate immune cells.
We anticipate a major impact on the 3Rs: reducing the number of mice used for chronic remodelling studies of asthma, informing and refining those experiments that are performed.
Planned Impact
The mechanisms regulating innate immune cell-fibrocyte interaction are of fundamental importance in airway remodelling in asthma. Very few targets with critical roles in airway remodelling have been identified to date. The discoveries from this project will therefore significantly enhance the knowledge economy with new scientific advancement, as described in 'academic beneficiaries".
1. Animal Welfare. The focus on developing a new non-mammalian model of airway remodelling will have important impacts on both reduction and refinement of mouse experiments. Initially, we expect a reduction in the region of 500 mice per year used for long-term asthma studies (approx 5% of all experiments). As this model becomes successful and is more widely adopted, we would expect this to increase - perhaps dramatically. In addition, the new understanding arising from our models will inform experimental design and allow the experiments that are done on mice to be more focussed on the organ specific questions that cannot be addressed directly in zebrafish. This will allow better experiments and better data from mouse studies.
2. The pharmaceutical industry has considerable interest in discovering regulators of and treatments for airway remodelling. We anticipate that the unique nature of the mechanisms investigated in this project will make them attractive targets for anti-fibrotic therapeutics. Protection of IP for these targets as they are discovered will bring significant economic gains to UK plc. Integration with the Pharma industry will allow rapid drug development, building on existing collaborations such as Renshaw's Fellowship-Partnership award with GlaxoSmithKline.
3. Patient care. It is our ultimate aim that drugs identified in my research programme find their way into our clinical practice to treat the patients that we currently cannot treat. This programme will deliver successful identification of drug targets and identification of lead candidates which I hope will ultimately have an impact on patient care. Experimental Medicine approaches are in development to take forward such advances towards clinical use. Via existing and new links, we will encourage Pharma investment in this programme, and develop IP sharing arrangements to ensure mutual benefit from emerging knowledge and know how. The advances in knowledge, and potential for driving drug development will ultimately impact on quality of life, health and well-being for asthma patients.
4. Interdisciplinary working. The project uses cross-disciplinary approaches from mammalian cell biology, zebrafish models and molecular biology. These methodologies will be used to develop and make use of innovative approaches to the study of inflammation and fibrosis in vivo. The project will contribute to new expertise in developing these unique tools to address biological questions by a systematic, and ultimately, high throughput approach. The project will strengthen links between different disciplines and forge a greater understanding of how we can engage, complement and enhance research for the future.
5. Innovative training opportunities. This proposal will deliver highly trained researchers offering unique skills. The PDRA will combine skills in fish models and state of the art in vivo microscopy, in parallel with mammalian cell based fibrocyte biology. They will develop distinctive skills in generating new datasets and new approaches to understanding biological problems. This expertise will provide transferable skills to other non-academic beneficiaries, but will also be used to train researchers from other groups in our methodologies. The PDRA and PIs will be actively involved in public engagement and broader dissemination, with regular school visits and high-level involvement with public exhibitions such as Royal Society Summer Science exhibition, and the University of Sheffield Festival of the Mind.
1. Animal Welfare. The focus on developing a new non-mammalian model of airway remodelling will have important impacts on both reduction and refinement of mouse experiments. Initially, we expect a reduction in the region of 500 mice per year used for long-term asthma studies (approx 5% of all experiments). As this model becomes successful and is more widely adopted, we would expect this to increase - perhaps dramatically. In addition, the new understanding arising from our models will inform experimental design and allow the experiments that are done on mice to be more focussed on the organ specific questions that cannot be addressed directly in zebrafish. This will allow better experiments and better data from mouse studies.
2. The pharmaceutical industry has considerable interest in discovering regulators of and treatments for airway remodelling. We anticipate that the unique nature of the mechanisms investigated in this project will make them attractive targets for anti-fibrotic therapeutics. Protection of IP for these targets as they are discovered will bring significant economic gains to UK plc. Integration with the Pharma industry will allow rapid drug development, building on existing collaborations such as Renshaw's Fellowship-Partnership award with GlaxoSmithKline.
3. Patient care. It is our ultimate aim that drugs identified in my research programme find their way into our clinical practice to treat the patients that we currently cannot treat. This programme will deliver successful identification of drug targets and identification of lead candidates which I hope will ultimately have an impact on patient care. Experimental Medicine approaches are in development to take forward such advances towards clinical use. Via existing and new links, we will encourage Pharma investment in this programme, and develop IP sharing arrangements to ensure mutual benefit from emerging knowledge and know how. The advances in knowledge, and potential for driving drug development will ultimately impact on quality of life, health and well-being for asthma patients.
4. Interdisciplinary working. The project uses cross-disciplinary approaches from mammalian cell biology, zebrafish models and molecular biology. These methodologies will be used to develop and make use of innovative approaches to the study of inflammation and fibrosis in vivo. The project will contribute to new expertise in developing these unique tools to address biological questions by a systematic, and ultimately, high throughput approach. The project will strengthen links between different disciplines and forge a greater understanding of how we can engage, complement and enhance research for the future.
5. Innovative training opportunities. This proposal will deliver highly trained researchers offering unique skills. The PDRA will combine skills in fish models and state of the art in vivo microscopy, in parallel with mammalian cell based fibrocyte biology. They will develop distinctive skills in generating new datasets and new approaches to understanding biological problems. This expertise will provide transferable skills to other non-academic beneficiaries, but will also be used to train researchers from other groups in our methodologies. The PDRA and PIs will be actively involved in public engagement and broader dissemination, with regular school visits and high-level involvement with public exhibitions such as Royal Society Summer Science exhibition, and the University of Sheffield Festival of the Mind.
