MicroRNAs and their epigenetic regulation as key factors in airway macrophage dysfunction in asthma.

Asthma affects 235 million people worldwide (according to WHO estimates) and is a great burden on the health economies of all nations. The disease is characterised by airflow obstruction that, over time, tends to become irreversible. The irreversibility, a consequence of airway remodelling and fibrosis, is associated both with treatment resistance and susceptibility to acute exacerbations usually induced by viral infections. Asthma exacerbations are a major unmet need and are estimated to cost £1.2 billion in lost productivity, £850 million in NHS health care provision and a further £161 million in social security costs within the UK. Understanding the basis for the increased susceptibility to disease exacerbation thus represents the primary need in asthma management especially when it is appreciated that there were just over 1,300 deaths (1,318) from asthma in the UK in 2005 (27 were children aged 14 years or under).
It is now appreciated that airway macrophages from asthmatics may be less able to deal with viral and bacterial infections. These cells form the first line of defence against pathogens, toxins and other environmental insults and hence, are critical to the airway response to the environment.
In this proposal we intend to determine the role of a novel family of short RNA molecules, microRNAs, which have been shown to participate in development, disease and many biological processes. We have found that, in asthma, these microRNAs are dysregulated, causing an imbalance in the functions of the immune system of the lung. Our preliminary results indicate that this deregulation is even more apparent when asthmatics have enhanced allergic inflammation with in the airways. The changes under these circumstances are those that would make asthmatics more prone to impaired viral responses and thus increase the likelihood of an asthmatic exacerbations. This "ties in" with our initial findings that, in macrophages, the deregulation in microRNAs seems to be an important factor in decreasing the secretion of protective interferon-beta (IFN-beta), increasing the over-expression of TNF-alpha, a potent pro-inflammatory cytokine, and reducing the pathogen binding ability of macrophages and thus their capacity to deal with pathogens. We intend to demonstrate that asthmatic macrophages show a deficiency in these three key functions and that environmental factors, such as viral infection and allergen challenge, further decrease their ability to appropriately deal with pathogens. MicroRNAs are relatively easy to manipulate with synthetic oligonucleotides. Thus, they make a good therapeutic target that could help reduce asthmatic exacerbations and the burden on asthma sufferers.
Finally, we will investigate the causes for this dysregulation in asthmatic macrophages both in microRNAs and the genes affected by them. We have preliminary evidence showing that microRNAs expression might be affected by a differential epigenetic control in asthmatic macrophages. Epigenetic control is an important mechanism for long-term and sustained regulation of gene expression and might explain the dysregulation of several genes associated with asthma in the onset and progression of the disease. Environmental factors can thus produce important epigenetic that will affect the functionality of macrophages in the lung. We will therefore study whether environmental or intrinsic factors affect the epigenetic control of crucial immune functions that may predispose to progression into a more severe asthmatic phenotype during life course.
The proposed study will thus address important issues relevant to asthma control and disease exacerbation and has significant potential to lead to the identification of realistic therapeutic targets as well as providing insight into the onset and progression of disease in this complex airway disorder.

1) We will determine the expression of a group of microRNAs (miR27a, miR-128, miR-135b, miR-141, miR-148a, miR-150, miR-152, miR-155, miR-340, miR-345, miR-374a, miR-375) and potential targets in peripheral blood monocytes and airway macrophages from asthmatic patients recovered by bronchoalveolar lavage (BAL) and compared to healthy donors. This will also be undertaken on BAL macrophages obtained from asthmatics before and after repeated experimental allergen challenge and on BAL macrophages exposed ex vivo to viral challenge. This will be done by RT-qPCR and Western Blot (the latter only for the genes involved in the defective pathways).
2) We will determine the functional role of the group of candidate microRNAs (as above) and miR-155 in regulating the pathways and cellular functions mentioned in the previous point. We will determine the effect of microRNAs on these pathways by employing synthetic oligonucleotides to manipulate microRNA expression, and the outcome will be measured by determining TNF-alpha and IFN secretion by ELISA. Pathogen binding ability to Haemophilus Influenzae and streptococcal sp, each labelled with propidium iodide, will be assessed by flow cytometry.
3) To determine whether this group of candidate microRNAs and their target genes are controlled by epigenetic marks in peripheral blood monocytes and asthmatic macrophages from patients in relationship to different levels of clinical severity (mild, severe asthmatics) or in relationship to the impact of environmental stimuli (in vivo experimental allergen challenge and in vitro exposure to rhinovirus). Comparison will be made to findings from healthy volunteer macrophages. The epigenetic studies will be done by Chromatin Immunoprecipitation determining the methylation of histones at the promoter of the relevant genes.
We will actively exploit and protect any IP at the University of Southampton through the Research and Innovation Services office.

The commercial private sector might benefit if, in the future, a therapy develops based on the findings of this work. MicroRNAs are relatively easy to manipulate and there are already products that deliver inhaled siRNA; the oligonucleotides used to manipulate microRNA expression can be delivered in the same way. This potential therapy would be more specific than the current treatments, since microRNA affect specific pathways, usually connected between them (like IL-13 and TGF-beta, for example). Such a therapy would suppose an extraordinary added value for the UK economy if commercialized. The idea behind our team is to direct our findings to make them translatable to a wider community of clinicians and, more importantly, the patients.
Beneficiaries of a successful outcome of this grant proposal may include NHS planning of how to deal with seasonal asthmatic exacerbation. As an example, if we prove that allergen challenges are increasing the risk of asthmatic exacerbations with cold virus, this could help establish the adequate protocols in periods where specific allergens are high and cold virus infections are stronger. Maybe by expecting more hospital admissions (passive) or maybe informing patients (the actual beneficiary of our research, hopefully) of what kind of measures they should take in order to avoid the risk. We also hope to establish a new diagnosis tool by determining whether there are epigenetic marks associated with asthma. This might actually allow stratification of different asthmatic phenotypes to allow more effective use of therapy and to personalise this to the individual according to a "epigenetic map".