Development of an in vitro model to dissect the mechanism of fungal persistence in the cystic fibrosis lung

Cystic fibrosis (CF) is the most common fatal genetically inherited disease in Caucasian populations. This disease is caused by mutations in the cystic fibrosis transmembrane regulator gene (CFTR) which produces defective ion fluxes and calcium homeostasis in the epithelia. Patients with cystic fibrosis are therefore susceptible to recurrent and lethal lung infections including those caused by the fungal pathogen Aspergillus fumigatus.

Animal models play a central role in the study of cystic fibrosis. Researchers have used mice, ferrets, pigs and sheep to model the inflammatory response seen in human patients with this disease. Although such models generate useful knowledge on the pathophysiology of this disease, animal models are limited for a number of reasons. One is the ethical concerns about the numbers of mice that needed to generate gene or tissue specific knock-outs which likely die early after birth. Additionally, mice, the most common vertebrate animal used, do not completely recapitulate the disease phenotypes seen in the human lung during infection and larger vertebrate such as sheep, pigs and ferrets are often used instead. The use of larger animals intrinsically has a number of limitations including housing space, high cost of the experimentations and the need to use very high number of animals to get enough statistical significance. In vitro cell culture systems using immortalised cystic fibrosis epithelial cells from donors have been developed to overcome those problems. However, genetic variability between healthy immortalised and cystic fibrosis cell lines prevents precise and statically useful comparison of events in CF and control cells which has limited them to be widely adopted by the community.

Here we propose to establish a new in vitro model of isogenic cystic fibrosis and healthy controls bronchial epithelial cell lines carrying different CF mutations in the CFTR gene using CRISPR/Cas9 mutagenesis of 16HBE bronchial cells. Using an established methodological approach developed in our laboratory including in vitro infection, confocal microscopy, expression analysis, cytokine measurements and competitive fitness analysis the student will be able to define critical stages of the interaction between the cystic fibrosis epithelia and A. fumigatus compared to healthy cells and the fungal factors governing this process.

The successful completion of this project will achieve a number of scientific objectives. Firstly, the development of an isogenic cell culture system of epithelial cell lines will be of great value for researchers focused on the study of epithelia-pathogen interactions in cystic fibrosis without using any mice. Secondly, screening of A. fumigatus null libraries on CF epithelia will increase our knowledge on the pathogen factors leading persistence in the context of disease. Additionally, the student will be enrolled in a 3Rs focused programme that will give the student the opportunity to enrol with industrial partners in Manchester and our fungal community to promote the implementation on this resource in other laboratories at an international level. As a PhD studentship we will also provide extensive training opportunities for a student, and the wider research community, on the importance of alternatives to replace animal testing.

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PhD Studentship

Status:

Active

Principal investigator

Dr Paul Bowyer

Institution

University of Manchester

Co-Investigator

Dr Sara Gago
Dr Michael Bromley

Grant reference number

NC/T001798/1

Award date:

Oct 2020 - Sep 2022

Grant amount

£90,000