Effect of exposure of children to particulate matter air pollution on airway macrophages and dendritic cells.

Microscopic particles of soot (particulate matter, PM) from petrol and diesel engine emissions are linked to a wide range of adverse health effects in children. The most consistent findings in survey studies are that long-term exposure of children to air pollution increases their risk of developing allergic asthma and reduces the function of their airways. Thus more cases of asthma occur in high soot-exposed communities, and children living in these communities take longer to forcibly breathe out air (lung function). To date, how soot damages children's lungs in unknown. In part this is because sampling cells from deep inside children's lungs has been very difficult, and in part, because measuring long-term exposure of individual children to sooty particles has been impossible.

We recently developed a new method that both measures long-term exposure to soot and samples cells from deep inside the airways of children. This method involves children coughing up plugs of cells after breathing a salty mist. Within these plugs are large cells called airway macrophages whose normal function is to take up soot that is breathed into the lung. Measuring the amount of soot in macrophages is a very good marker of an individual child's exposure to soot over the preceding months. In addition to removing soot, animal studies strongly suggest that airway macrophages, and another cell type called dendritic cells, are involved in the development of asthma and the regulation of normal lung growth. It is therefore very likely that subtle changes in airway macrophages and dendritic cells are key to the adverse health effects of inhaled soot. We speculate that breathing in soot affects airway macrophages and dendritic cells so that they release more tissue damaging substances, and stimulates these airway cells to support more allergic reactions in the lung.

In this proposed study, we will measure the amount of soot in macrophages of healthy children, and for each child, compare the amount of macrophage soot to the number and function of airway macrophages and dendritic cells. We will focus on cellular changes that are known from animal models to enhance risk of developing asthma and reduce the growth of the airways. Airway macrophages and dendritic cells will be analysed using flow cytometry - a technique that suited to the analysis of small numbers of cells. We will also use portable soot monitoring to assess where children are exposed most when they move around their local environment. In order to control for other factors that may affect lung cells (e.g. gender and age), and that some children will not be able to produce sufficient lower airway cells for detailed analysis, this study needs to recruit a large number of subjects. In total, 400 children will be studied, 250 more than once, over a 36 month period. We will recruit from healthy children attending the Centre of the Cell experience, a hands-on multimedia resource that is embedded within our Institute at Whitechapel. To date, over 30,000 children have visited the Centre. Children attending our Centre mainly live in the East End of London - one of the worst areas for air pollution in Europe. We will engage with children by developing an multi-media educational presentation on air pollution and lung cells. Using this resource, we are maximising the study's potential to impact on the lives of deprived urban children.

This study will be the first that focuses on the mechanisms for the long-term effects of air pollution in children. All the techniques are ethically acceptable in children, and we have preliminary results that show we can identify both airway macrophages and dendritic cells from the small number of cells in induced sputum samples from young children. We envisage that the study's results will strengthen the argument that soot causes lung diseases in childhood, and will provide new insights into how the environmental affects asthma development and normal lung growth.

We will determine the association between chronic exposure of children to PM air pollution and; i) the proportion of small airway macrophages (AM), ii) the number, proportion and activation status of dendritic cells with a mature phenotype, and iii) the capacity of dendritic cells to stimulate naïve CD4+ T cells in vivo. Using AM carbon as a biomarker of individual exposure to PM, we will test the hypothesis that PM, by stimulating mediator release by lung cells, enhances the recruitment of the myeloid precursors of AM and dendritic cells, and directly stimulating expression of co-stimulatory receptors on dendritic cells, results in an airway milieu with an increased proportion of smaller "pro-inflammatory' AM, and a dendritic cell phenotype with increased capacity to stimulate CD4+ T cell proliferation and generate Th2 responses. To assess this, we will recruit 400 children via the Centre of the Cell resource - a multimedia teaching experience embedded in our Institute. Lung function, atopic status, exhaled breath condensate pro-inflammatory mediators, and glutathione gene polymorphism status (GSTP, GSTM) will be done in all children. The local sources of PM exposure will be assessed in all children using personal black carbon monitoring on a typical school day. Lower airway cells will be sampled using induced sputum. AM carbon will be determined using our established image analysis method. Markers for recruitment of myeloid precursors and co-stimulatory molecule expression will be assessed by flow cytometry. In functional studies, we will assess the capacity of induced sputum cells to stimulate proliferation of naïve CD4+ T cells proliferation. The study is powered to detect a 0.6 SD in the primary outcome variables between children in the upper and lower tertiles of airway macrophage carbon. All the sampling procedures to be used have been ethically approved in children, and our flow cytometry analysis standard operating procedure is based on robust pilot data.

Primary beneficiaries of research
The main beneficiary of this research will be children living in Tower Hamlets - one of the most deprived areas of the UK. Children living in our borough are disproportionally exposed to environmental threats such as air pollution. Strengthening the argument for causality for asthma and reduced lung function will have a major impact on the cost-benefit analysis for local air pollution reduction initiatives, such as further tightening of the low emission zone, aimed at protecting their long-term health. Since exposure to traffic-derived particulate matter air pollution is ubiquitous, our results will also be generalisable to all children living in urban areas - both nationally and internationally.

Secondary beneficiaries
Data on dendritic cells and inflammatory mediators will be of significant relevance to researchers focusing on asthma pathogenesis and normal lung function growth. Furthermore, the extensive alveolar macrophage carbon data set will impact on researchers intending to assess personal exposure of children in developing world settings. For example, members of the WHO children's environmental health network (CI; member). There will be unrestricted access to anonymised raw data.

Communications and Engagement.
The public engagement programme, that will run throughout the 36 month study period, will include i) development of family and school workshops to facilitate recruitment of children and their parents for the study and raise local understanding or air pollution issues, and ii) narratives of children who/or have been participating in the research project. All aspects of this combined educational/research initiative will undergo front-end evaluation starting with Centre of the Cell's "Youth Forum" and followed by regular evaluation of impact.

Capacity and involvement
The CI and co-applicants will be responsible for communicating the programme's findings to the public. To date, this group has an outstanding track record in communicating our studies via our direct links to the national and international press oranisations e.g.
http://news.bbc.co.uk/1/hi/7837649.stm
http://news.bbc.co.uk/1/hi/world/africa/8092182.stm
http://www.guardian.co.uk/society/2010/jan/24/traffic-pollution-child-pneumonia
http://www.bbc.co.uk/news/uk-england-london-15053299
http://articles.latimes.com/2011/sep/26/news/la-heb-cyclists-pollution-20110926
http://www.thehindu.com/health/medicine-and-research/article2486953.ece

Data on personal exposure of children will be disseminated to the new multi-disciplinary "Healthy Cities" Initiative at Queen Mary University of London (CI; member )
http://www.qmul.ac.uk/research/cities/index.html , with the view to directly influencing urban planning to reduce children's exposure to traffic-derived air pollution. Resources specific to impacts are the costs for the Centre of the Cell activities described in the justification section; one full time Centre of the Cell outreach officer, workshop materials, travel, marketing materials, website interactive development, curation and development of objects, and 'Nucleus' Interactive development and running costs total; £10,000.