Role of microRNAs in the innate immune response to viral infection in asthma

Asthma affects over 5 million people in the UK, costs the NHS #900 million each year in direct health care costs and is responsible for 12.7 million lost working days each year. Despite therapy designed to control asthma, many continue to have disease exacerbations, most of which are caused by simple viral infections (colds). Such exacerbations have significant impact, affecting ability to work, quality of life, and rarely even leading to death. Understanding the basis for disease exacerbation is thus an important goal.

My preliminary research has suggested that the genetic regulation in an important airway cell called the alveolar macrophage is different in asthma from that in healthy non-asthmatic individuals and that this may lessen its ability to fight viral infection. My study will thus focus on assessing this in greater detail. I will compare macrophages from individuals who have asthma with those who do not, to evaluate their genetic make-up, how this is regulated and how it influences the response to viral infection. I hope that by improving our understanding of how these airway cells respond to viral infection in asthma that my work will ultimately lead to new treatments that prevent exacerbations in this disease.

Respiratory viral infections are a common cause of asthma exacerbations. A deficient epithelial innate immune response to viral infections has been described in asthma by colleagues in Southampton and Imperial College. MicroRNAs are non-coding RNA molecules that control gene expression by inhibiting protein translation. My preliminary study of microRNA expression in alveolar macrophages (AM), using microRNA microarrays, have suggested that an innate immune defect against airway pathogens may also be present in these key effector cells of the airway immune defence in asthma.

I hypothesise that the differential expression of certain microRNAs in patients with asthma leads to reduced expression of TLR-7 by their AM and an altered interferon response, leading to a defective innate immune response against viral pathogens.

Specific aims of the study:

1. Demonstrate that the altered expression of specific microRNAs in asthmatic AM is linked to reduced expression of TLR-7. I will use RT-qPCR to quantify levels of the miRNAs highlighted by my preliminary work and then use flow-cytometry, western blotting and immunohistochemistry to assess the expression of the TLR-7 by AM. I aim to recruit patients with mild, moderate and severe asthma and healthy volunteers.

2. Investigate the molecular mechanism of action underlying microRNA regulation of TLR-7 expression. I will initially work with a cell line and techniques including gene cloning and Luciferase assays will be used to link the identified microRNAs with the TLR-7 mRNA. I will also transfect AM with specific anti-microRNAs to experimentally inhibit the identified microRNAs to try and demonstrate a return of TLR-7 levels to normal range. Successful transfection will be assessed by RT-qPCR and levels of TLR-7 will be assessed as described in Aim1.

3. Determine whether microRNA deregulation affects viral detection by macrophages. AM from asthmatic patients and healthy controls will be exposed to TLR-7 agonists and levels of inflammatory cytokines and chemokines (type I interferons, IP-10, TNF-?, IL-6, IL-8 and RANTES) released will be measured by flow-cytometry and ELISA. I will also transfect asthmatic AM with specific anti-microRNAs to explore whether TLR-7 function can be restored. Success in this aim would support the use of anti-miRs as potential therapy in asthma.

My goal is to deepen our understanding of the role of microRNAs in the pathogenesis of viral exacerbations in asthma, improve our characterisation of the defects in the innate immune response to viral exacerbations and aid in the identification of new therapy for the disease.