bREATH-EASy: UNDERSTANDING THE ROLE OF INFECTION & EXTRACELLULAR MATRIX IN IDIOPATHIC PULMONARY FIBROSIS USING STEM CELL DERIVED ALVEOLAR CELLS

In the UK 1 in 4 people die from respiratory disease. Poor access to lung samples, an inability to culture lung cells and a lack of animal models that reproduce human lung disease has left us with an inadequate understanding of how to diagnose and treat lung disease. These issues are exemplified by idiopathic pulmonary fibrosis (IPF), a disease where rapid scarring reduces lung function leading to death within an average of 3 years. The cause of IPF is unknown and prognosis is difficult to predict. A greater knowledge of lung disease including IPF is needed so diagnosis, prognosis and treatment can be improved.

Human pluripotent stem cells are providing new ways to model and understand disease. Until recently however, models for lung diseases were not available. Our recently-published work has addressed this need and we can now produce large numbers of lung alveoli cells, one of the cell types affected by IPF. We can now make 3D lung organoids allowing modelling of complex biological functions and interactions, and can utilise these technologies to understand IPF.

To this end we have assembled an international team to address 3 interlinked objectives:

1) Generate a hIPSC model of IPF to understand how SFTPC mutations affect cellular phenotype and gene expression in AE2 cells. There are no known mutations that cause of IPF, however mutations in surfactant protein C (SFTPC) can cause inherited IPF also known as familial IPF, the mechanism however remains elusive. We will generate a stem cell model of familial IPF by taking a skin biopsy from patients with a SFTPC mutation and then turning their skin cells into lung cells using our established protocol. We will then use the cells to study how the mutation causes lung cell damage, fibrosis and to identify new therapeutic targets to treat IPF.

2) Model respiratory infection in-vitro to understand the effect of infection on AE2 cells carrying SFTPC mutation. IPF patients have more frequent respiratory infections and >90% mortality rate per infection, how bacterial infection exacerbates IPF is not well understood. Using the SFTPC mutant cells created in Obj. 1 we will use our 3D culture platform to grow lung organoids and then infect them with bacteria or viruses. We will create a profile of changes in gene expression in response to infection and compare this to uninfected cells. This will provide insight into how respiratory infection affects normal lung cell function and importantly deliver insight into the mechanism by which infection exacerbates IPF.

3) Model change in ECM composition to understand how ECM impacts cell phenotype. As IPF progresses, the scaffold that holds the lungs together, the extracellular matrix (ECM), dramatically changes, causing the lungs to become stiff and less elastic as well as reducing oxygen absorption. How the changes in ECM affect lung cell function, and whether lung cells actively contribute to further ECM changes has not been well characterised. We will develop our understanding of this process by growing cells created in Obj. 1 in simple 3D gels that will allow us to model how the ECM changes during early and late stages of IPF. We will then assess how these changes affect cell functionality and gene expression. This will provide insight into how ECM influences lung cell function and importantly deliver potential therapeutic targets that aim to prevent or reverse changes to ECM in IPF.

These objectives will address poor access to human IPF disease models, and provide insight to the pathogenesis of lung fibrosis and infection in IPF, offering the opportunity to develop exciting new drug targets as well as diagnostic and prognostic markers for the disease. Importantly, this platform could be modified to model other fibrotic lung diseases, and other diseases that have a fibrotic component.

In the UK 1 in 4 people die from respiratory disease, costing the NHS more than any other disease area. Poor access to lung samples, an inability to culture lung cells and a lack of animal models that faithfully reproduce human lung disease has left us with an inadequate understanding of how to diagnose and treat lung disease. These issues are exemplified by idiopathic pulmonary fibrosis (IPF), a disease where rapid scarring reduces lung function leading to death within an average of 3 years.

Here we propose to apply our recently published stem-cell-based lung differentiation platform to model IPF and address 3 interlinked objectives:

1) Generate a hIPSC model of IPF to understand how SFTPC mutations affect cellular phenotype and gene expression in AE2 cells; no mutations have been described that cause IPF, however mutations in surfactant protein-C (SFTPC) lead to familial IPF. There are currently few robust human specific models to study familial IPF and the mechanism causing fibrosis remains elusive.

2) Model respiratory infection in-vitro to understand the effect of infection on AE2 cells carrying SFTPC mutation; Familial IPF patients have more frequent respiratory infections and have a 90% mortality rate per infection, how bacterial infection impacts and exacerbates the SFTPC phenotype is not well understood.

3) Model change in ECM composition to understand how ECM impacts cell phenotype; as IPF progresses the ECM changes dramatically, negatively impacting respiratory function. How these changes influence cellular behaviour and a potential pro-fibrotic feed-forward loop is not well characterised.

Together, these objectives will address the poorly understood issues related to genetic risk factors, respiratory infection and ECM and how they individually and collectively impact the prognosis of IPF. This will provide opportunities for identification of novel diagnostic and prognostic markers as well as development of new therapies for IPF.

Social impact:
Currently, treatment options for lung diseases including IPF are very limited and diagnosis and management of lung disease within the UK is one of the most expensive undertakings for the NHS. This project will generate a robust in-vitro model of lung fibrosis and infection and will identify potential diagnostic, prognostic and therapeutic markers. This in turn will build on our knowledge of respiratory fibrosis and create a new understanding of how IPF initiates, progresses and is complicated by infection and ECM remodelling. This will lead to new, faster and cheaper approaches to diagnosis, advance management and treatment, improve prognosis and enhance quality of life for patients. New approaches to managing and treating IPF will reduce costs to the NHS and the government and free-up NHS recourses that can be invested into other areas of healthcare. Additionally, the insight delivered from this project will inform onto public service providers and will improve government policy in dealing with respiratory diseases and ensure patients are receiving care based on the most relevant and up-to-date knowledge.

Economic impact:
Treating lung diseases causes significant economic pressure. The EU spends 280 billion annually and in the USA the cost is nearly $450 billion annually. This project will identify new therapeutic targets to treat IPF which will reduce the severity of the condition and the cost to treat it. Identification of new diagnostic, prognostic and therapeutic targets will generate new wealth for the UK through the creation of intellectual property, and the opportunity to commercialise and create new health products and devices for respiratory disease. This will stimulate further UK investment opportunities to create new UK companies, jobs and infrastructure for the management of respiratory disease. The combined effect of reducing economic burden while creating new technology and wealth could have a positive economic impact generating billions of pounds annually.

Knowledge:
This project focuses on advancing the field of regenerative medicine by developing our understanding of the basic mechanism of lung disease and infection. Currently, most respiratory disease modelling is performed using animal models that don't accurately reproduce the disease phenotype and are inaccurate predictors of drug efficacy and safety. We will advance our knowledge of respiratory disease by creating a human specific platform that will generate human specific data and provide a more accurate assessment of drug targets, and the efficacy and safety of novel therapeutic agents. In addition to improving modelling accuracy, our platform will lift the burden placed on use of animal models for respiratory research and improve our knowledge on how in-vito models can be constructed and exploited to gain accurate information. We will also generate several unique techniques relating to manipulation of the platform for infection modelling which will be unique to the UK stem cell research community. Lastly, this proposal will develop a novel disease model for lung infection and fibrosis both of which fall within the remit of the MRC and have been identified as priority targets in the MRC Strategic Plan "Research Changes Lives" 2014-2019 priority theme one "Resilience, repair and replacement".

People:
Currently there are no other research groups in the UK, and very few in the world that have the knowledge and expertise to propose this type of work. This proposal will provide a unique opportunity to train UK based researchers, both directly and indirectly involved with this project and expand this area of expertise within the UK. This will ensure a continued production of researchers with the necessary skills to further utilise, expand and adapt these platforms for their own use within the respiratory research field or in other areas of research that lack appropriate in-vitro models.