PI3K Signaling and Aberrant Neutrophil Function in COPD; Implications for Disease Susceptibility, Prognosis and Therapeutic Targeting

Chronic Obstructive Pulmonary Disease (COPD) is a term that describes conditions such as emphysema and chronic bronchitis which are characterised by narrowing of the breathing tubes (airways), and are associated with lung tissue destruction. It is debilitating, affects 3 million people in the UK, and is the 5th leading cause of death (25,000 deaths in the UK/year). It is the only chronic disease with a rising death rate. UK COPD costs are estimated at £3.2 billion per annum. Annual NHS costs are £900M, 50% of which are due to unscheduled hospital admissions due to deteriorating symptoms. Better disease management would reduce this disease burden, but currently no treatments prevent disease onset or halt progression.

COPD is a chronic inflammatory condition, where the continued presence of excessive inflammatory proteins and cells in the lung cause damage. Smoking cigarettes is the most important risk factor, but only 15% of smokers develop COPD, and the disease runs in families, suggesting there are genetic factors that predispose towards COPD. Inflammation and lung destruction continue even after smoking cessation. This is poorly understood, but we believe that COPD patients have abnormal immune responses that drive disease even after noxious stimuli (cigarette smoke) have gone. Presently we cannot predict who will develop COPD or if all disease features are caused by the same underlying immune problem. It is vital to understand this, as it will help direct new treatments.

Neutrophils are white blood cells crucial for fighting infection. They leave the blood, moving (migrating) accurately to areas of infection, where they ingest bacteria, killing them with an arsenal of proteins contained within the cell. These proteins cause damage and inflammation if they are released in the body's tissues. Neutrophils contain genes that control the cell by dictating what proteins are expressed. These proteins could be enzymes, controlling chemical reactions within the neutrophil, or structural proteins, controlling how they move.

Neutrophils are central to COPD. COPD patients have many neutrophils in their lungs and the chemicals neutrophils release can cause the characteristic lung damage. Our data show that neutrophils from COPD patients are defective, migrating less accurately than cells from healthy subjects or people with other lung diseases and ingest less bacteria in models of infection. This is important, as inaccurate migration and reduced bacterial clearance could lead to more lung damage and poorer outcomes during infections; both of these are thought central in COPD. We have shown this harmful cell behaviour is due to increased activity of a protein enzyme called phophosinositide-3 kinase (PI3K). Abnormal PI3K signalling may be the cause of altered COPD neutrophil behaviour and correcting this may offer a new treatment in COPD.

We wish to investigate the molecular signals and identify the relevance of injurious neutrophil behaviour in COPD, using neutrophils isolated from blood and lung secretions of COPD patients, those at risk of developing COPD (family members of COPD patients) and healthy volunteers. This will determine how prevalent the defective neutrophil behaviour is across all the differing features of COPD, and whether you can predict who might develop the disease by assessing their neutrophils. We will then identify the cause of the defective neutrophil behaviour, by studying the activity of structural proteins and enzymes (and the genes that control them) within COPD neutrophils, using our extensive preliminary data and knowledge of cell signaling to focus our experiments.

Identifying the specific cause of defective neutrophil behaviour will allow us to form new targeted treatments for COPD, improving the health of patients with the disease. It may also allow us to predict who is most at risk of the COPD, developing a screening tool, helping to inform people about their lifestyle choices.

COPD is a major cause of mortality and morbidity. Data supports a genetic basis but screening (limited to Alpha-1-antitrypsin deficiency) can detect <2% of cases. We cannot predict who else is at risk. COPD encompasses different clinical phenotypes, however it is unclear if the same disease mechanism underpins all disease pathologies. Neutrophils (PMNs) are central to the inflammation seen in all clinical phenotypes and we have demonstrated that circulating COPD PMNs have defective chemotactic and phagocytic responses. This is seen in all disease severities, including early disease, is not present in other pulmonary conditions and is associated with abnormal Phosphoinositide3 kinase (PI3K) signaling. We hypothesise that aberrant PI3K signaling causes chemotactic and phagocytic defects which contribute to COPD pathogenesis.

We will address the following questions: Does the aberrant PMN phenotype predict the onset or progression of COPD? Is the chemotactic defect due to aberrant PI3K signaling? What is the molecular basis of the defect?

We will study PMNs from patients with COPD, across disease phenotypes, compared to healthy smoking controls to determine if the cell phenotype bridges all clinical phenotypes or is limited to specific pathologies. We will study circulating and bronchoalveolar lavage PMNs to assess if migration to the tissues modulates the chemotactic phenotype. Crucially, we will study family members of index cases to assess if the PMN phenotype is preserved across families, providing evidence of a genetic association for both COPD and cell behavior. We will study PI3K signaling using immunohistology, phase, DIC and confocal microscopy, cell sorting, mass spectrometry, selective inhibitors and PCR to identify the protein signals involved, their role in cell functions, the sites of their activities and their individual molecular signatures. Identified targets will be tested across COPD families to determine if they are a signal of risk.

Who will benefit from this research?
We believe that this research will lead to new therapeutic strategies in COPD and in time, new treatments that are likely to reduce prevalence, improve COPD outcomes, reduce hospital admissions and slow disease progression. Identification of molecular causes could also lead to the development of screening tools to identify people most at risk of disease. The non-academic beneficiaries of this research will be patients, their family, carers and Pharma, but may also include employers and potentially the UK economy

How will they benefit?
COPD is progressive, debilitating and associated with considerable mortality. Recent research suggests there are 3.7million people with COPD in the UK and the Health and Safety Executive report that 40% of COPD patients are below retirement age and one quarter of these people cannot work due to their symptoms. Direct COPD-associated health costs in the UK have been estimated at £900M per annum, with 50% of this being hospital admissions during exacerbations. In my hospital, we estimate that reducing COPD re-admissions by 10% or COPD-exacerbation length of stay by one day on average would save >£500,000 per year. However, despite the high mortality, morbidity, prevalence and cost of the disease, a survey by the British Lung Foundation suggested that most people within the UK have not heard of COPD. Treatments are limited, none prevent disease or halt decline. COPD needs to be better understood, have more treatments for early disease and its profile needs to be raised.

The proposed research will enhance understanding of the mechanisms of disease and identify novel therapeutic targets. Poor neutrophil migratory accuracy and bacterial phagocytosis are compatible with increased extracellular proteinase release, inflammation and tissue damage and reduced bacterial clearance and increased susceptibility to invasive infection. We have identified a potential mechanism for the aberrant behaviour that is correctable in vitro. This is an exciting development in COPD pharmacologics and could be of significant benefit to patients and their families. Improved health, reduced hospitisations and lower COPD-related unemployment would have substantive public-health and economic benefits, and will be of interest to the media (raising COPD awareness), private commercial sector and policy makers.

Identifying the molecular mechanisms of COPD neutrophil behaviour will support the development of a COPD animal model based on a mechanism proven to be present in COPD patients. This would facilitate the development and assessment of emerging treatments in COPD and would be a considerable resource for the academic community and Pharma.

This project includes development opportunities for the applicants through collaborative exposure to well characterised patients and new assays. These skills will be shared among colleagues to support academic development in Birmingham and Cambridge. The project will also benefit researchers of other neutrophil-based inflammatory diseases. Methodologies and resources will be shared, to support UK academic excellence, raising the profile of UK-based research internationally.

Within 3 years, we will identify the mechanism of aberrant neutrophil function, the patients groups that demonstrate this abnormality (supporting either a focused phenotype or generic disease treatment strategy which will help with trial development) and will have identified candidate screening signals for people at risk of disease. Based upon this work, and on-going links with Pharma, we will test emerging tool compounds and will develop a murine model of COPD within 4 years, to be used in putative studies of new therapies. With emerging tool compounds being developed by Pharma, it is likely that human trials of medicants would require a further 5 years for development, including stage III clinical trials.