Use of Drosophila models to explore the function of asthma susceptibility genes

Aims

The aim of this project is to develop Drosophila models to better understand the role that asthma susceptibility genes play in epithelial barrier immunity and airways remodelling, and reduce the use of currently employed mammalian models for these studies.

Background

The incidence of asthma is increasing worldwide and there is no cure for the disease. For many sufferers, current therapies help manage the symptoms of asthma, but for approximately 10% of patients with the severest disease current therapies are ineffective. There is an urgent need to identify the underlying basis of asthma, and to develop appropriate new treatment strategies.

The causes of asthma are not fully understood. While exposure to environmental triggers can bring on an asthma attack, it is known that asthma tends to run in families and may be inherited. Over the last 10-15 years considerable progress has been made in identification of asthma susceptibility genes using a variety of genetic approaches. However, despite the advances at the genetic level, the functions of these genes and/or how their genetic polymorphism contributes to asthma remain poorly understood, especially for those genes involved in local tissue susceptibilities such as barrier immunity and tissue remodelling. Traditionally, approaches for understanding gene function rely heavily on the use of murine models which are costly, time consuming and may not result in a quantifiable phenotype. Therefore, there is a need to develop alternative approaches that reliably offer the potential for introducing several disease-genes into the same organism to allow assessment of gene-gene, as well as gene-environment effects.

Research details and methods

Drosophila lack an adaptive immune system, possess a simple airway structure which shares many features with the human airways, and benefit from a comprehensive toolbox of methods for directly manipulating genes of interest. These properties make the Drosophila an exciting new model system for assessing the effect of asthma susceptibility genes on airway structure and function.

Transgenic flies overexpressing the asthma susceptibility genes, ADAM33 or CDHR3 will be generated to study their function in epithelial barrier immunity and airway remodelling. High resolution light-sheet and confocal microscopy will be used to explore if similar phenotypes are observed in the transgenic flies as compared to human and murine models. Effects on epithelial barrier, immune responses and oxygen sensitivity will be assessed at baseline and after challenge with cigarette smoke or bacteria. If tractable, such a system will facilitate studies of remodelling and barrier immunity in asthma and provide a tool for high-throughput screening of compounds that modify the function of the susceptibility genes which may lead to development of novel therapeutic interventions that act close to the origin of the disease.

We wish to test the hypothesis that Drosophila models can replace, augment and inform vertebrate asthma models by allowing first-pass elucidation of key asthma genes, as well as providing an in vivo model for rapid high through-put drug screening. We will demonstrate proof-of-principle by targeting two susceptibility genes, ADAM33 and CDHR3, where we have prior experience in mammalian systems, so enabling direct comparison with the invertebrate system.

We will use the GAL4/UAS (driver/effector) system to analyse gene function in Drosophila.  Novel transgenic flies will be generated which express human sADAM33 or human sADAM33 (E346A) (inactive control). These lines will be crossed with relevant driver lines to allow organ-specific conditional expression of ADAM33 in third instar larvae. The remodelling effect of sADAM33 will be assessed using high resolution light-sheet and confocal microscopy in anaesthetized living intact larvae. Other readouts will include assessment of epithelial barrier immunity, collagen deposition and oxygen sensitivity.  

To study CDHR3, we will use two approaches: the first will focus on the effects of the CDHR3 asthma-related point mutation, on the function of the natural Drosophila DE-cadherin. We will also create transgenic lines expressing human CDHR3 or its allelic variant in the larval trachea, as described for ADAM33. Effects on epithelial barrier, immune responses and oxygen sensitivity will be assessed at baseline and after challenge with cigarette smoke or bacteria.

The use of Drosophila for such studies could provide rapid and novel insights into genetic mechanisms of airways disease and allow screening of novel compounds for alternative therapeutic interventions in the context of these susceptibility mutations.

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Strategic grant

Grant reference number

NC/M001482/1

Award date:

Apr 2015 - Mar 2017

Grant amount

£89,116