A non-mammalian model to study innate immune modulation of airways remodelling in asthma

Aims

This project aims to reduce the number of murine experiments using long-term remodelling protocols by generating a novel zebrafish model to study the interaction of innate immune cells and fibrocytes in driving fibrosis of the airway wall in asthma.

Background

A substantial part of the impact of asthma relates not to acute exacerbations, but to chronic breathlessness resulting from airways remodelling and persistent fixed airways obstruction. Such non-reversible obstruction can occur in all phenotypes of asthma and there is no clear way to prevent it therapeutically. The links between key pathways of airways inflammation and remodelling remain unclear and potential for therapeutic intervention is underdeveloped. Direct communication between innate immune cells and fibroblasts derived from incoming fibrocytes may be responsible for driving airway remodelling in asthma and pharmacological interruption of this communication may provide a new approach for long-term drug discovery in asthma.

Current in vivo systems examining immune/fibrotic interaction require whole animal models and invasive allergen challenges, which do not model the human disease well and which have important implications for the 3Rs. This project will develop a zebrafish larvae model to examine the interaction between immune cells and fibrocytes in vivo, replacing the need for mammalian models.

Research details and methods

Zebrafish larvae are transparent and genetically tractable. Transgenically labelled neutrophils and macrophages have been created which enable cell behaviours to be studied in detail in vivo. In addition, xenotransplants of human cells into developing larvae are tolerated since they are present before immune maturation. In this project, fibrocytes from normal and asthmatic subjects will be fluorescently-labelled and xenotransplanted into embryos from zebrafish lines expressing fluorescent proteins in neutrophils and macrophages. The interactions of the engrafted fibrocytes with innate immune cell populations (number of cell-cell interactions per unit time, macrophage or neutrophil interaction, duration of interaction and frequency of interaction) will be recorded using time-lapse 4D spinning disc confocal microscopy. Differences between normal controls and fibrocytes from patients with different asthma phenotypes will be determined. Combining these approaches in a single model offers a unique system for the study of a range of hypotheses relating to immune cell interaction with fibrotic cells isolated from human asthmatics.

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.

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Strategic grant

Status:

Active

Principal investigator

Professor Stephen Renshaw

Institution

University of Sheffield

Co-Investigator

Professor Ian Sabroe

Grant reference number

NC/M001490/1

Award date:

Apr 2015 - Dec 2016

Grant amount

£98,320