One of the key features of asthma is chronic breathlessness, which develops over time as a result of airway remodelling. Immune responses drive fibrosis of the airway wall, where circulating fibrocytes are recruited to the airway and produce matrix, leading to a non-reversible fixed airway obstruction. The link between airway inflammation and remodelling is not well understood and currently airway remodelling cannot be prevented therapeutically. Direct communication between immune cells and the fibroblasts derived from fibrocytes may be responsible for airway remodelling and, if this could be interrupted pharmacologically, could provide an avenue for drug discovery in asthma. Current in vivo systems for the study of interactions between immune and fibrotic cells require whole animal models, most commonly mice, and use sensitisation/challenge protocols to induce airway inflammation resembling some aspects of asthma.
Why we funded it
This Strategic Grant aims to develop a zebrafish larval model1 as a replacement for mice used to study airway remodelling in asthma. While zebrafish do not have lungs, the disease mechanism being investigated in this pilot study involves two cell types that are present in zebrafish larvae, making them amenable models for these types of asthma studies.
The development of chronic asthma models has significant implications for mouse welfare and is classified as a moderate procedure under the Animals (Scientific Procedures) Act. A literature search showed in 2013 approximately 800 studies were published where murine asthma models were used. A typical study investigating asthma using mice requires a minimum of six mice per experimental group, which increases to twelve animals per group in repeated studies. This results in an estimated 10,000 mice required globally for asthma studies per annum.
To investigate the interactions between the immune cells and fibrocytes, human fibrocytes will be isolated from normal and asthmatic patients before being xenotransplanted into zebrafish larvae. Fibrocytes will be fluorescently labelled and either inoculated after isolation or treated to induce proliferating and/or differentiating populations. Zebrafish larvae are transparent and genetically tractable, so lines will be used with transgenically labelled neutrophils and macrophages to enable visualisation of direct interaction of the engrafted fibrocytes and the immune cells in vivo. Development of the labelled fibrotic cells will be followed using in vivo Z-stack confocal microscopy and the extent of fibrosis analysed by measuring the total volume of transplanted material, the surface area to volume ratio of fibrosis and the extent of branching.
1 The Animals (Scientific Procedures) Act 1986 defines protected larval forms of vertebrate animals as independently feeding. Zebrafish larvae begin independently feeding 120 hours post fertilisation so are not considered protected until this time.
Hamilton et al. (2018). A method for transplantation of human HSCs into zebrafish, to replace humanised murine transplantation models F1000Research 7:594. doi: 10.12688/f1000research.14507.1
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