Investigation into the role of ceramide in the pathogenesis of cystic fibrosis lung disease

Cystic fibrosis (CF) is the most common life-limiting inherited disease in the United Kingdom (UK). Over 9,000 people have CF and it affects around 1 in 2,500 newborn babies. CF is a chronic condition that is associated with significant medical problems that occur from an early age and may present at birth. Living with CF necessitates a daily burden of treatment, including oral medications, nebulisers, physiotherapy and a special diet, plus frequent clinic visits and prolonged hospital admissions.

Progressive lung disease is the most important problem in CF and regularly leads to premature death in young adulthood. The median life expectancy for people with CF in the UK is 34 years. Although we know that CF is a condition that is inherited and involves problems with an ion channel in cells the exact cause of the lung disease is not fully understood. Current treatments do not cure patients, all of whom get progressive complications, most notably recurrent lung infections and inflammation leading to gradual loss of lung function and ultimately respiratory failure. Many patients require assessment for lung transplantation although this is not a viable option for everyone and there is a severe shortage of donor organs.

A major problem with CF research is that it has traditionally been difficult to obtain highly relevant cells from inside the lungs of people with CF. My previous work performed during my PhD funded by a Medical Research Council (MRC) and CF Trust Training Fellowship identified a reliable way to do this by growing the cells directly from the diseased lungs removed when people with CF undergo lung transplantation at the Institute of Transplantation, Freeman Hospital, Newcastle upon Tyne. I was also able to collect lung tissue for a research archive during this work.

Recently researchers have found increased levels of a fat called 'ceramide' in lung tissue from a CF mouse model. This accumulation was associated with lung infection and inflammation - as we see in people with CF. In this current project I aim to use the valuable human CF cells and tissue to measure the levels of ceramide present. I will then investigate why ceramide may accumulate in CF cells, how it may cause lung inflammation and importantly a potential novel treatment to try and reduce ceramide levels.

I will visit and collaborate with centres of excellence in Cambridge, Paris, Essen and New York to perform this work and will bring back knowledge and experience gained in these centres to further develop research in Newcastle.

Cystic fibrosis (CF) is the most common genetically acquired life-limiting disorder in the UK. Lung disease is responsible for over 95% of morbidity and mortality, the exact pathogenesis of which remains poorly understood. There is an acute need for the development of novel therapeutic approaches.

Raised levels of the sphingolipid ceramide have been described in a CF mouse model that are associated with lung inflammation, a key feature of human CF lung disease. In this Clinician Scientist Fellowship proposal I will combine primary bronchial epithelial cells and human CF lung tissue with the latest mass spectrometry techniques to accurately measure and image ceramide in the airway epithelium. I will then examine a potential mechanism whereby ceramide may accumulate in the plasma membrane due to reduced acid ceramidase activity and cause inflammation. I hypothesise that ceramide accumulates in microdomains of the plasma membrane in CF airway epithelial cells that causes NADPH oxidase to cluster resulting in raised production of reactive oxygen species, increased production of inflammatory mediators and airway inflammation. I will also examine whether the altered ceramide metabolism is associated specifically with defective CF transmembrane regulator function (CFTR). Finally I will investigate the potential therapeutic use of recombinant acid ceramidase in models of CF lung disease to reverse the accumulation of ceramide and reduce airway inflammation.

This fellowship will advance knowledge, improve methodology and answer key questions that may support ceramide metabolism as a potential novel therapeutic avenue for CF lung disease including the use of recombinant acid ceramidase.

BACKGROUND
Cystic fibrosis (CF) is the most common life-limiting genetically acquired disorder in the UK. Lung disease is responsible for over 95% of morbidity and mortality, the exact pathogenesis of which remains poorly understood, and ultimately leads to respiratory failure in young adulthood. Living with CF necessitates a heavy and life-long burden of care. It follows that there is an urgent need to improve understanding of pathogenesis and develop novel therapeutic strategies.

BENEFITS TO HEALTH
Patients, carers and families: The work in this fellowship will increase understanding of the mechanisms involved in CF lung disease and provide information on ceramide metabolism as a potential new therapeutic avenue. I will specifically explore if treatment with recombinant acid ceramidase may reverse the accumulation of ceramide in models of CF.
Wider benefits to the NHS and healthcare research: Data generated in this fellowship will inform future research directions including refinement of approaches to pharmacological manipulation of sphingolipid metabolism. The development of technologies to accurately measure sphingolipids in primary airway epithelial cells and to image lipids in human lung tissue will represent an important improvement in the methodology currently used to study sphingolipid biology. Abnormalities in sphingolipid metabolism have been identified in a number of other lung diseases and these approaches are likely to be of benefit beyond CF including emphysema and asthma. The fellowship will also provide support for the ongoing utilisation of explanted CF lung tissue at one of the largest lung transplantation centres in Europe. The importance and scarcity of primary tissue and cells is widely recognised by the CF research community and there is a clear intention to continue active collaboration with researchers nationally and internationally along with ongoing dissemination of the culture techniques. The value of such a resource is exemplified by the recent development of ivacaftor, which was validated in primary cells prior to successful translation.

ECONOMIC BENEFIT
In addition to potential economic benefits to the NHS the development of novel techniques to accurately measure and image ceramde in primary cells and lung tissue are likely to generate further research and drug development income to the UK and European Union. The post-doctoral research assistant employed as part of the fellowship will acquire transferable skills equally relevant to a future career in academia or the pharmaceutical industry.

CHANGE IN POLICY
Through prompt communication to a broad range of researchers, clinicians and health care professionals at large national and international meetings and in open access peer- reviewed journals the results of the research will be widely disseminated to key decision and policy makers. This will be complemented by a strong policy of engagement with
influential patient groups such as the CF Trust.

Reference:
[1] Ramsey et al. A CFTR potentiator in patients with cystic fibrosis and the G551D mutation. N Engl J Med. 2011;365:1663-1672