Developing a model for the study of respiratory inflammation in the zebrafish
Lead Research Organisation:
Imperial College London
Department Name: Life Sciences
Abstract
Severe asthma is a disabling non-curable condition that can only be controlled by continuous administration of drugs. It has long been suggested that patients with a history of viral infection together with smoking have increased chances of developing severe asthma. Unfortunately, the pathophysiological mechanisms of this disease are not fully understood. More research is needed to shed light on how asthma develops in some people and to provide new targets for the pharmaceutical industry.
We propose to investigate whether and to what extent we could use zebrafish as a model to help us understand some of the basic mechanisms and pathway biology of severe asthma in humans. Although zebrafish do not have lungs, their gills serve the same function and have the same overall tissue structure and cell types as human lungs: an epithelial cell layer that facilitates gaseous exchange and a variety of immune cells that defend the tissue from infection. A major advantage is that gills are directly exposed to ambient water, which makes them easily accessible to drugs (addition to the water rather than by injection) or for collection of biopsies. Further, we can easily access the gill tissue under brief anaesthesia to take small biopsies instead of using invasive procedures like bronchoalveolar lavage as performed in mouse studies, where a small tube is inserted through the mouth into the lungs and fluid is injected and retrieved for analysis. In fact, we can take small biopsies a number of times from the same animal in longitudinal studies, reducing the number of animals otherwise used and providing us with higher quality data by avoiding the inherent variability that exists between individuals. In addition, we have a number of fish in which specific immune cells are labeled with fluorescence. Using microscopy we will visualise, in live fish, the behaviour of these cells under different conditions including exposure to smoke and/or virus which will enable study of the same animal over time, again reducing the total number of animals used.
This project will be performed in collaboration with our clinical colleagues at St Mary's Hospital who have already developed a platform which allows analysis of nasal samples in human volunteers (smokers and non-smokers) to study the effect of viral infection combined with smoking on lung function. Their experience will be invaluable in helping us develop non-invasive sampling in zebrafish and to allow the comparison of results across the species to work towards the identification of new therapeutic targets for respiratory disease. We will then be in a position to directly test the effects of intervention in these putative pathways and/or molecules on gill function in the fish.
Knowledge of fish husbandry, biology and behavior are essential for this project. Therefore, the work on zebrafish will be performed by one of our most experienced scientists who has been working in the field of zebrafish immunology for 5 years and who has already developed a number of the experimental procedures required for this proposal. This will minimise the time needed for training.
During this year-long project we aim to first develop methods for non-invasive sampling of gill fluids and tissues. We will apply these methods more than once on the same animal allowing studies over time on the same animal to obtain high quality data using fewer animals. During the second part of the project we will analyse our experimental samples and study many events from when, where and what type of cells are recruited to which genes/pathways play a role in inflammation. The results collected will eventually be compared with those obtained in the parallel study in humans to enable us to identify molecules or cells that play a critical role in the initiation of severe asthma. We hope to obtain a better understanding of the mechanisms of severe asthma in order to eventually develop successful treatments for this recalcitrant disease.
We propose to investigate whether and to what extent we could use zebrafish as a model to help us understand some of the basic mechanisms and pathway biology of severe asthma in humans. Although zebrafish do not have lungs, their gills serve the same function and have the same overall tissue structure and cell types as human lungs: an epithelial cell layer that facilitates gaseous exchange and a variety of immune cells that defend the tissue from infection. A major advantage is that gills are directly exposed to ambient water, which makes them easily accessible to drugs (addition to the water rather than by injection) or for collection of biopsies. Further, we can easily access the gill tissue under brief anaesthesia to take small biopsies instead of using invasive procedures like bronchoalveolar lavage as performed in mouse studies, where a small tube is inserted through the mouth into the lungs and fluid is injected and retrieved for analysis. In fact, we can take small biopsies a number of times from the same animal in longitudinal studies, reducing the number of animals otherwise used and providing us with higher quality data by avoiding the inherent variability that exists between individuals. In addition, we have a number of fish in which specific immune cells are labeled with fluorescence. Using microscopy we will visualise, in live fish, the behaviour of these cells under different conditions including exposure to smoke and/or virus which will enable study of the same animal over time, again reducing the total number of animals used.
This project will be performed in collaboration with our clinical colleagues at St Mary's Hospital who have already developed a platform which allows analysis of nasal samples in human volunteers (smokers and non-smokers) to study the effect of viral infection combined with smoking on lung function. Their experience will be invaluable in helping us develop non-invasive sampling in zebrafish and to allow the comparison of results across the species to work towards the identification of new therapeutic targets for respiratory disease. We will then be in a position to directly test the effects of intervention in these putative pathways and/or molecules on gill function in the fish.
Knowledge of fish husbandry, biology and behavior are essential for this project. Therefore, the work on zebrafish will be performed by one of our most experienced scientists who has been working in the field of zebrafish immunology for 5 years and who has already developed a number of the experimental procedures required for this proposal. This will minimise the time needed for training.
During this year-long project we aim to first develop methods for non-invasive sampling of gill fluids and tissues. We will apply these methods more than once on the same animal allowing studies over time on the same animal to obtain high quality data using fewer animals. During the second part of the project we will analyse our experimental samples and study many events from when, where and what type of cells are recruited to which genes/pathways play a role in inflammation. The results collected will eventually be compared with those obtained in the parallel study in humans to enable us to identify molecules or cells that play a critical role in the initiation of severe asthma. We hope to obtain a better understanding of the mechanisms of severe asthma in order to eventually develop successful treatments for this recalcitrant disease.
Technical Summary
Aim 1: Tool development for non-invasive sampling.
We will take advantage of the techniques developed by TH and colleagues for clinical use which uses swabs generated from bioengineered substrates to collect nasal tissue and fluids from human volunteers. We will optimize these techniques for sampling zebrafish gill tissue and fluids to allow non-invasive, longitudinal studies in live fish with only short exposures to anaesthetic (and analgesia).
Aim 2: Cell and pathways analysis.
We will analyse the profile of innate immune markers with our collection of TaqMan Q-PCR assays on cDNA samples of gills exposed to cigarette smoke (CS) using previously optimised protocols. We will optimise exposure of fish to Poly:IC (as a viral mimetic) directly added to the water. Poly:IC and CS will then be combined and their inflammatory effects studied using live imaging over time with our (various) transgenic zebrafish bearing fluorescently tagged neutrophils and macrophages. We will apply findings from aim 1 to sample tissue for longitudinal analysis. Fish will be killed at the end of the study and all tissues collected for histological and end point gene expression analysis. We will also assess mast cell presence and pending our initial findings Ketotifen which targets this cell type will be used to assess the role of mast cells in tissue inflammation and remodeling. We will further assess whether epithelial inflammation mediated by CS and TLR induces smooth muscle impairment and breathing difficulties by monitoring opercular rate with video and by the key histological features (muscle thickness) of asthma.
Aim 3: Data will be compared with human data obtained by others (including TH's team) to generate hypotheses regarding the involvement of innate immune pathways in human respiratory disease. If time allows we will target pathways identified in our comparative study using drugs or genetic approaches.
We will take advantage of the techniques developed by TH and colleagues for clinical use which uses swabs generated from bioengineered substrates to collect nasal tissue and fluids from human volunteers. We will optimize these techniques for sampling zebrafish gill tissue and fluids to allow non-invasive, longitudinal studies in live fish with only short exposures to anaesthetic (and analgesia).
Aim 2: Cell and pathways analysis.
We will analyse the profile of innate immune markers with our collection of TaqMan Q-PCR assays on cDNA samples of gills exposed to cigarette smoke (CS) using previously optimised protocols. We will optimise exposure of fish to Poly:IC (as a viral mimetic) directly added to the water. Poly:IC and CS will then be combined and their inflammatory effects studied using live imaging over time with our (various) transgenic zebrafish bearing fluorescently tagged neutrophils and macrophages. We will apply findings from aim 1 to sample tissue for longitudinal analysis. Fish will be killed at the end of the study and all tissues collected for histological and end point gene expression analysis. We will also assess mast cell presence and pending our initial findings Ketotifen which targets this cell type will be used to assess the role of mast cells in tissue inflammation and remodeling. We will further assess whether epithelial inflammation mediated by CS and TLR induces smooth muscle impairment and breathing difficulties by monitoring opercular rate with video and by the key histological features (muscle thickness) of asthma.
Aim 3: Data will be compared with human data obtained by others (including TH's team) to generate hypotheses regarding the involvement of innate immune pathways in human respiratory disease. If time allows we will target pathways identified in our comparative study using drugs or genetic approaches.
Planned Impact
In this project we will use Danio rerio (zebrafish) as model organism to study asthma pathway biology, primarily refining procedures and reducing animal numbers and for a small part of the project potentially replacing the use of animals.
Studying gill biology in fish rather than lungs in mammals offers one obvious advantage: the gill is accessible to ambient water and therefore drugs but is also accessible for live imaging and biopsy. Therefore this project will contribute to the 3Rs in the following ways:
A - By using non-invasive delivery methods to access the zebrafish respiratory system with inflammatory compounds (cigarette smoke and viral TLR agonists) and therefore refining protocols otherwise used in rodents such as intra-tracheal or intra-nasal delivery of inflammatory compounds.
B - We will explore the possibility that inflammation induced by cigarette smoke-infused water leads to impaired muscle contraction and oxygenation that will be readily visible by simple visual observation of accelerated ventilation behavior (i.e. increased gill opercular rate) recorded by filming thereby refining procedures otherwise performed in mammals which employ metabolic chambers.
C - By performing live-imaging, rather than immunohistology for analysis of immune cell infiltration using transgenic animals with fluorescence tagged immune cells, thereby reducing the number of animals used.
D - By developing new non-invasive tissue sampling and biopsies on zebrafish gills by applying small swabs made of bioengineered materials previously tested in human to refine invasive procedures otherwise performed in rodents such as bronchio-alveolar lavages where tubes are inserted inside the lung to collect samples and animals killed.
E - By enabling longitudinal studies through sampling at different time points for a kinetic analysis on the same animal. This procedure will be combined with a live-imaging for analysis of immune cell infiltration. Altogether, this will provide more information per animal and therefore reduce the number of animals used per experiment. The data will also be of higher quality than that obtained from different mice at different time points. For a kinetic analysis over a period of 3 weeks different mouse groups would be needed for gene expression and cell recruitment analysis following initiation of lung inflammation (e.g. 4 time points in the first week) plus further separate groups to assess the effect of long term inflammation (3 time points for longer term analysis). Therefore with 6 mice per group multiplied by each time point this would equal 42 mice per condition and per experiment, rather than 6 fish per condition for our proposed longitudinal study, a reduction of 85%.
Future work that will result from this project is likely to contribute to refinement and replacement in the following way:
In order to characterise signalling pathways and genes involved in inflammation induced by irritants (e.g. smoke) we will aim to target specific pathways. In mouse drugs would be administered by systemic injection (IV, IP) or locally (intranasal and intratracheal) administration. In zebrafish drugs and especially those targeting the gills, are added directly to the water therefore refining the procedure. New drugs that have not been used previously in zebrafish will be first tested for toxicity on embryos (<5 days post fertilization) for survival therefore replacing the use of animals. We do not, however, anticipate that this will be a major part of the current project.
Studying gill biology in fish rather than lungs in mammals offers one obvious advantage: the gill is accessible to ambient water and therefore drugs but is also accessible for live imaging and biopsy. Therefore this project will contribute to the 3Rs in the following ways:
A - By using non-invasive delivery methods to access the zebrafish respiratory system with inflammatory compounds (cigarette smoke and viral TLR agonists) and therefore refining protocols otherwise used in rodents such as intra-tracheal or intra-nasal delivery of inflammatory compounds.
B - We will explore the possibility that inflammation induced by cigarette smoke-infused water leads to impaired muscle contraction and oxygenation that will be readily visible by simple visual observation of accelerated ventilation behavior (i.e. increased gill opercular rate) recorded by filming thereby refining procedures otherwise performed in mammals which employ metabolic chambers.
C - By performing live-imaging, rather than immunohistology for analysis of immune cell infiltration using transgenic animals with fluorescence tagged immune cells, thereby reducing the number of animals used.
D - By developing new non-invasive tissue sampling and biopsies on zebrafish gills by applying small swabs made of bioengineered materials previously tested in human to refine invasive procedures otherwise performed in rodents such as bronchio-alveolar lavages where tubes are inserted inside the lung to collect samples and animals killed.
E - By enabling longitudinal studies through sampling at different time points for a kinetic analysis on the same animal. This procedure will be combined with a live-imaging for analysis of immune cell infiltration. Altogether, this will provide more information per animal and therefore reduce the number of animals used per experiment. The data will also be of higher quality than that obtained from different mice at different time points. For a kinetic analysis over a period of 3 weeks different mouse groups would be needed for gene expression and cell recruitment analysis following initiation of lung inflammation (e.g. 4 time points in the first week) plus further separate groups to assess the effect of long term inflammation (3 time points for longer term analysis). Therefore with 6 mice per group multiplied by each time point this would equal 42 mice per condition and per experiment, rather than 6 fish per condition for our proposed longitudinal study, a reduction of 85%.
Future work that will result from this project is likely to contribute to refinement and replacement in the following way:
In order to characterise signalling pathways and genes involved in inflammation induced by irritants (e.g. smoke) we will aim to target specific pathways. In mouse drugs would be administered by systemic injection (IV, IP) or locally (intranasal and intratracheal) administration. In zebrafish drugs and especially those targeting the gills, are added directly to the water therefore refining the procedure. New drugs that have not been used previously in zebrafish will be first tested for toxicity on embryos (<5 days post fertilization) for survival therefore replacing the use of animals. We do not, however, anticipate that this will be a major part of the current project.