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.

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.

Publications

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