A live model to study mucociliary clearance in health and disease
Lead Research Organisation:
University of Manchester
Department Name: School of Biological Sciences
Abstract
The human lungs are the sites of gaseous exchange where air, containing oxygen, is breathed in and carbon dioxide is breathed out. This direct interaction with the outside world makes the lungs particularly vulnerable to the entry of harmful foreign agents such as pollutants and bacteria, viruses and fungi. In order to prevent their entry into the body, a protective barrier shields the exposed surfaces of the lungs. This barrier is composed of mucus, a gel-like material that acts like a 'molecular sieve' to trap inhaled infectious agents and pollutants. Once trapped, these foreign bodies need to be removed from the lung before they cause damage. Specific lung cells, called ciliated cells, project tiny hair-like structures (cilia) into the overlying mucus layer. The cilia move together in a coordinated manner so as to generate a directional flow that takes the mucus, and any trapped material, away from the lungs. This process is known as mucociliary clearance and if it is defective this can lead to disease. Examples of diseases with defective mucociliary clearance includes cystic fibrosis, where mucus is thick and sticky and cannot be moved; asthma, where an overproduction of mucus can cause airway obstruction; and primary ciliary dyskinesia where cilia movement is impaired and so mucus cannot be cleared. These diseases, particularly cystic fibrosis and primary ciliary dyskinesia, lead to increase vulnerability to infection, which can prove fatal.
In order to effectively treat these diseases, we need to understand precisely how mucociliary clearance works so that we can design drugs to improve outcomes for patients. Scientists often employ model organisms to understand the details of a biological process that is difficult, or ethically improper, to study in humans. With mucociliary clearance, mammalian models, particularly mice, are commonly used to either mimic diseases or to understand the role of particular genes in the process. However, the anatomical location of the lungs within the body means that it is difficult to study the dynamics of mucociliary clearance and often involves invasive techniques that can be harmful to the mice. In this research proposal, we introduce an alternative model, the tadpole of the frog species, Xenopus tropicalis, to replace the use of mice in these studies. Importantly, their skin surface produces mucus and has ciliated cells that move the mucus, just like in the human lungs. Details about the function of particular genes in mucociliary clearance and how mucus interacts with cilia and with other agents (e.g. bacteria) can be elucidated with this model, extending our understanding of mucociliary clearance in humans. Crucially, the tadpoles that we use are at an early developmental stage and are considered to be non-sentient. Our aim for this project is to develop and validate this live model of mucociliary clearance using the tadpole skin in order to gain new scientific insight at the same time as replacing, and reducing the reliance on, mice.
In order to effectively treat these diseases, we need to understand precisely how mucociliary clearance works so that we can design drugs to improve outcomes for patients. Scientists often employ model organisms to understand the details of a biological process that is difficult, or ethically improper, to study in humans. With mucociliary clearance, mammalian models, particularly mice, are commonly used to either mimic diseases or to understand the role of particular genes in the process. However, the anatomical location of the lungs within the body means that it is difficult to study the dynamics of mucociliary clearance and often involves invasive techniques that can be harmful to the mice. In this research proposal, we introduce an alternative model, the tadpole of the frog species, Xenopus tropicalis, to replace the use of mice in these studies. Importantly, their skin surface produces mucus and has ciliated cells that move the mucus, just like in the human lungs. Details about the function of particular genes in mucociliary clearance and how mucus interacts with cilia and with other agents (e.g. bacteria) can be elucidated with this model, extending our understanding of mucociliary clearance in humans. Crucially, the tadpoles that we use are at an early developmental stage and are considered to be non-sentient. Our aim for this project is to develop and validate this live model of mucociliary clearance using the tadpole skin in order to gain new scientific insight at the same time as replacing, and reducing the reliance on, mice.
Technical Summary
A critical defence mechanism in the respiratory tract against inhaled particulates and pathogens is mucociliary clearance (MCC) in which mucus secreted by specialist cells is moved by ciliated epithelial cells. Defective MCC occurs in asthma, cystic fibrosis, chronic obstructive pulmonary disease and in primary ciliary dyskinesia leading to infection. It is important to study the respiratory epithelium to understand its function in both health and in disease with the aim of developing new therapeutic strategies for treatment. Mammalian models are widely used in respiratory research. However, studying the respiratory tract in these systems has inherent difficulties, particularly observing events in real time. This is because the lungs are located internally and studying live events requires harvesting and re-constituting the tissue in vitro, taking the tissue out of its normal context and resulting in the death of many animals. In this project, we propose the skin of the Xenopus tadpole as a model for the replacement of mammals to study mucociliary epithelia. The tadpole skin has a mucociliary surface, with both mucin-secreting cells and ciliated cells, making it ideal for live imaging of its surface and for challenge through changes in the local environment. We will use transgenic tadpoles that secrete fluorescently tagged mucins onto the skin and cilia that are also fluorescently tagged allowing study of the dynamics of MCC. The model will be challenged with agonists/antagonists of mucin secretion and cilia beating, and pathogen products. Such a live fluorescent model for MCC has the potential to replace mammalian models, particularly mice. We anticipate a diverse use of the model in fundamental research, in the testing of novel therapeutics (as well as measuring secondary impacts of other drugs on MCC) and in mimicking human disease, including those due to genetic mutations.
Planned Impact
We propose that pre-feeding stage Xenopus tropicalis tadpoles, which are considered non-sentient, can be used to replace mice in the study of lung mucociliary clearance, including in diseases such as asthma, cystic fibrosis (CF), chronic obstructive pulmonary disease (COPD) and primary ciliary dyskinesia (PCD). Specifically, this would come under the NC3Rs term of 'Partial Replacement', since it replaces a mammalian model with an immature form of a vertebrate species. The tadpole skin is a mucociliary epithelium easily accessible to manipulation, unlike the airways of mice (and other mammalian models), which require highly invasive procedures to access the tissues of interest. Whilst recognising that this model is not applicable to studying human lung function per se, it will provide a platform for asking fundamental questions about mucociliary clearance and even to mimic disease states. Currently, many mouse models are used to answer these questions. Using similar mouse models employed in our own laboratories we estimate that a typical publication uses 300 mice and this correlates with taking averages from a selection of publications in this research area. We have taken all this into account when estimating the potential levels of replacement possible.
To accurately reflect the replacement potential, we identified studies using mice investigating the function of a gene or effect of a treatment on mucociliary clearance including in the diseases PCD, CF, COPD and asthma. These types of study are most readily replaceable with the tadpole model and this formed the basic criterion for our analysis of the literature. We first searched the literature for 'mucociliary clearance' in the three years from 2015-2017, filtered for 'mice'. By assessing each publication, we identified 16 primary research articles meeting our criteria for replacement. We next analysed the literature for 'PCD' and 'CF', identifying 13 publications suitable for replacement. Asthma and COPD are multifactorial diseases, some aspects of which are not appropriate to study with our tadpole model. However, these diseases all have alterations in mucin production, secretion or expression and this is where the tadpole model has the potential to replace current mouse models. Using the same criteria as before, we searched each disease together with the term 'mucin' and analysed search results for potential replacement which identified a further 14 publications. This gives a total of 43 publications over three years. Using our local estimate of 300 mice per publication, this represents a potential replacement of 12,900 mice (i.e. 43 x 300 mice) in three years or 4,300 mice per year. In the longer term, we also envisage the tadpole as being a simple screening tool to analyse the effects of drugs (e.g. to reduce mucin secretion or improve cilia beating), pathogens and particulates on mucociliary clearance. This would naturally lead to a further replacement in the numbers of animals, including mice, currently used for such purposes in industry.
These replacement estimates rely on uptake of the model by other researchers. As our letters of support from world leaders in muco-obstructive diseases and PCD demonstrate, we are already well integrated in the respiratory research community and can identify specific research groups that may adopt our model. To increase the uptake of the model we will deliver a hands-on workshop in collaboration with the NC3Rs Regional Programme Manager (Dr. Kamar Ameen-Ali) demonstrating the basics of using the model, as well as techniques specific for mucociliary research. As the workshop will be targeted more locally in the UK, we shall support this initiative through generating a series of professionally produced video tutorials that we will make publicly available as well as sending to targeted research groups globally. In addition, we will use the North West Regional AWERB hub meetings as a forum to describe the applicability of the model.
To accurately reflect the replacement potential, we identified studies using mice investigating the function of a gene or effect of a treatment on mucociliary clearance including in the diseases PCD, CF, COPD and asthma. These types of study are most readily replaceable with the tadpole model and this formed the basic criterion for our analysis of the literature. We first searched the literature for 'mucociliary clearance' in the three years from 2015-2017, filtered for 'mice'. By assessing each publication, we identified 16 primary research articles meeting our criteria for replacement. We next analysed the literature for 'PCD' and 'CF', identifying 13 publications suitable for replacement. Asthma and COPD are multifactorial diseases, some aspects of which are not appropriate to study with our tadpole model. However, these diseases all have alterations in mucin production, secretion or expression and this is where the tadpole model has the potential to replace current mouse models. Using the same criteria as before, we searched each disease together with the term 'mucin' and analysed search results for potential replacement which identified a further 14 publications. This gives a total of 43 publications over three years. Using our local estimate of 300 mice per publication, this represents a potential replacement of 12,900 mice (i.e. 43 x 300 mice) in three years or 4,300 mice per year. In the longer term, we also envisage the tadpole as being a simple screening tool to analyse the effects of drugs (e.g. to reduce mucin secretion or improve cilia beating), pathogens and particulates on mucociliary clearance. This would naturally lead to a further replacement in the numbers of animals, including mice, currently used for such purposes in industry.
These replacement estimates rely on uptake of the model by other researchers. As our letters of support from world leaders in muco-obstructive diseases and PCD demonstrate, we are already well integrated in the respiratory research community and can identify specific research groups that may adopt our model. To increase the uptake of the model we will deliver a hands-on workshop in collaboration with the NC3Rs Regional Programme Manager (Dr. Kamar Ameen-Ali) demonstrating the basics of using the model, as well as techniques specific for mucociliary research. As the workshop will be targeted more locally in the UK, we shall support this initiative through generating a series of professionally produced video tutorials that we will make publicly available as well as sending to targeted research groups globally. In addition, we will use the North West Regional AWERB hub meetings as a forum to describe the applicability of the model.
