CF, which causes premature death by progressive respiratory failure, is a genetic disorder resulting from mutations in the cystic fibrosis transmembrane conductance regulator (CFTR). The CFTR plays an important role in homeostatic balance of fluid composition. In CF, impaired CFTR-mediated chloride ion efflux increases sodium and, consequently, water resorption from the airway surface. This leads to the accumulation of thick mucus and a collapse of mucociliary clearance, that promotes chronic infection and excessive inflammation, which conspire to fatally damage the lungs. In addition to known mucociliary defects, several lines of evidence suggest that primary alterations in immune responses contribute to pulmonary pathology in CF. As principal researcher Audrey Bernut explains: “Currently our understanding is limited by the lack of suitable animal models that recapitulate the immune abnormalities found in CF patients.” Available CF models (e.g. patient-derived cells or mammalian models) suffer from several limitations, predominantly the evaluation of phenomena in a pre-existing inflammatory environment. Consequently, the mechanisms by which the CFTR directly regulates host immunity, and how CF mutations contribute to infectious and inflammatory pathogenesis in CF, have remained obscure. To get a better understanding of the links between a dysfunctional CFTR and the deleterious immune responses in CF, Bernut’s research project, CFZEBRA, developed zebrafish larvae as a tractable animal model. Young zebrafish are transparent, allowing for the non-invasive, real-time monitoring of the behaviour of immune cells during inflammatory processes in the whole organism. The zebrafish CFTR retains close sequence identity with the human one (56.24 % identity). Like mammals, the zebrafish CFTR is expressed in epithelial surfaces and immune cells and plays an important role in homeostatic balance of fluid composition. “While the absence of lungs might, at first sight, appear to reduce the relevance of this model, my data suggest that changes to CFTR function in epithelial and innate immune cells are conserved across tissues and species,” says Bernut, whose project was undertaken with the support of the Marie Skłodowska-Curie programme and supervised by Stephen Renshaw, professor at the University of Sheffield, UK. Using CRISPR-Cas9 technology, a simple yet powerful tool for editing genomes, Bernut was able to ‘cut’ DNA sequences and alter the CFTR function by generating mutations. The result was the generation of zebrafish with CF, the creation of which allowed Bernut to assess the role of CFTR in directly regulating host inflammatory and immune potential in vivo. Bernut uncovered a number of altered immunity and inflammatory processes which are critical mechanisms underlying infection and inflammatory disease in CF. These could be addressed therapeutically to prevent inflammatory lung damage in CF patients, leading to potential improvements in disease outcomes. “I have already identified interesting compounds that could be therapeutic, and which are currently used for the treatment of other diseases. Experimental studies in patients will now be necessary to evaluate the efficacy of these novel, immune-targeted therapies for CF. If all goes well, these could then lead to clinical trials,” Bernut concludes.
CFZEBRA, Cystic fibrosis, CFTR, zebrafish, inflammation, infection, immune-targeted therapies, pulmonary pathology, animal models, CRISPR-CAS9