Research project P7/30 (Research action P7)
I. DOMAIN and STATE OF THE ART
Asthma and Chronic Obstructive Pulmonary Disease (COPD) are highly prevalent diseases of the lower airways, which cause worldwide significant morbidity and mortality and represent a substantial socio-economic burden 1,2. These lower airway diseases are linked with upper airway diseases such as chronic rhinosinusitis and nasal polyposis (“United Airways” concept). These airway diseases are heterogeneous and the underlying pathophysiological mechanisms contributing to specific phenotypes remain poorly characterized3-5. Major determinants in their development are environmental exposures and genetic predisposition, as evidenced by Genome Wide Association Studies (GWAS)6,7. Recently, culture-independent microbiological techniques demonstrated altered microbial communities (microbiome) in the lower airways of asthma and COPD patients 8,9.
II. FORMER ACCOMPLISHMENTS
In the last 5 years, our AIREWAY consortium has optimized several mouse models of airway inflammation (e.g. with ovalbumin and house dust mite as allergens; with toluene diisocyanate as chemical agent) and airborne pollutant exposure (e.g. cigarette smoke, diesel exhaust particles)10,11. We investigated the importance of environmental factors in the initiation and establishment of airway diseases in vivo12,13. Using a chronic COPD model, we addressed mechanisms involved in lymphoid neogenesis and disease persistence14,15. We also performed translational research using sputum samples (e.g. cytokine mRNA profiling in asthmatics and microRNA profiling in COPD patients), nasal polyps and lung tissues to understand which processes are implicated in chronic airway disease16-19.
III. MAIN OBJECTIVES
The acronym AIREWAY II (Importance and interaction of Allergy, Infections and Respiratory Environmental exposures in the development and chronicity of lower and upper airWAY diseases) describes the global objective of this project, and builds further on our P6/35 AIREWAY project that was initiated in 2007. With this interuniversity collaboration, we will further unravel the cellular and molecular mechanisms underlying lower (asthma and COPD) and upper (chronic rhinosinusitis and nasal polyposis) airway diseases, with a particular attention for the heterogeneity of these diseases, by combining molecular expertise, mouse models and human samples from the different partners of the consortium. Findings from basic research using experimental animal models will be validated by translational research on precious human samples. Vice versa, hypotheses generated from observations in man will be tested at a molecular level by using animal models in vivo and human cell cultures in vitro.
IV. METHODOLOGY
The strengths of the consortium are multiple. First, the partners have a long-lasting experience with a variety of in vivo animal models of airway diseases, mimicking the heterogeneity observed in human disease (e.g. eosinophilic vs. Neutrophilic asthma). Second, the different teams are complementary in expertise and equipment, which is shared within the consortium (e.g. measurement of bronchial hyperresponsiveness (BHR), neurogenic inflammation, flow cytometry, cellular and molecular in vitro studies on human primary tissue and cell cultures). Third, to assure clinical relevance, the research groups have built large biobanks of serum, sputum, nasal polyps and lung tissue from several well characterized patient groups (COPD, various asthma phenotypes, nasal polyposis, and chronic rhinosinusitis). Finally, two excellent international partners have joined the AIREWAY II consortium, adding their specific expertise on epigenetics and oxidative stress
(I. Adcock, London, UK) and on functional genomics and proteomics (U. Völker, Greifswald, Germany).
V. SPECIFIC AIMS
Investigate the mechanisms by which allergens, environmental pollutants and microbial agents initiate lower and upper airway diseases and modulate disease persistence
We hypothesize that the following mechanisms, modulated by environmental triggers, play a crucial role in the development of airway diseases:
1) Transient Receptor Potential (TRP) channels constitute a family of ion channels that function as molecular sensors of numerous physical and chemical stimuli. In the respiratory tract, inhaled noxious particles and compounds from cigarette smoke (e.g. nicotine, acrolein and oxidizing agents) are sensed by TRPA1 in airway sensory neurons and result in neurogenic inflammation and BHR20,21. We hypothesize that sensing of allergens and environmental pollutants by TRP channels affects the development of BHR and obstructive airway diseases.
2) Pathogen Associated Molecular Patterns (PAMPs) are molecules that are specifically related to microbes, whereas Damage Associated Molecular Patterns (DAMPs) are molecules that are released from injured cells or dying cells5. The recognition of PAMPs and DAMPs by pattern recognition receptors (PRR) is critical in mediating inflammatory responses to infection and sterile tissue damage. We hypothesize that exposure to allergens, microbial agents (e.g. bacterial superantigens, such as Staphylococcus aureus enterotoxins), or pollutants leads to activation of PRR, either directly (PAMPs) or indirectly by causing (epithelial) cell injury (DAMPs), thus affecting the development of obstructive airway diseases..
3) Exposure to environmental factors affects the phenotype of chronic airway diseases, explaining in part patient heterogeneity. We hypothesize that altered immunological and molecular mechanisms underlie the phenotypes in obstructive airways disease and want to focus on the role of defects in the induction of allergen-specific regulatory T-cells, on the role of B-lymphocytes and of regulatory molecules such as proteases in disease persistence. We also hypothesize that identification of molecular markers (e.g. proteomics of sputum and volatile organic compounds in exhaled breath) will reveal interesting phenotypes in obstructive airway diseases.
4) Epigenetic changes are alterations in gene function, not -coded within the DNA (e.g. DNA methylation, histone acetylation and microRNAs), that are evoked by the environment (e.g. air pollution, cigarette smoking)22. We hypothesize that epigenetic changes are induced in lower and upper airway disease and affect disease pathogenesis.
Read outs: We will determine the role of the above mentioned mechanisms in different disease phenotypes using their respective models and using patient samples. In the murine models, we will measure pulmonary function (BHR), determine TRP functionality and evaluate inflammation (flow cytometry, immunohistochemistry) and remodeling. At a molecular level, we will measure release of DAMPs, cytokines and chemokines (ELISA and RT-PCR) in murine and human samples. At a cellular level, we will examine the role of antigen presenting cells (dendritic cells), macrophages and the epithelium in initiation of the disease, as well as the importance of T- and B-cells in orchestrating the inflammatory processes. In mechanistic studies we will block proteins or gene transcripts of interest (e.g. receptors, epithelial derived-mediators, and proinflammatory cytokines) with specific monoclonal antibodies, antagonists or siRNA. This will be addressed by both in vitro and in vivo experiments. In addition, we will use genetically modified mice with selective gene over-expression or (constitutive or conditional) suppression. In our epigenetics approach we will examine DNA methylation profiles, histone acetylation and perform microRNA profiling and determine the role of differentially expressed microRNAs in vitro and in vivo (e.g. by administering antagomirs or microRNAs).
VI. REFERENCES ( * with authors from the AIREWAY II consortium)
1. Pauwels, R. A. & Rabe, K. F. Burden and clinical features of chronic obstructive pulmonary disease (COPD). Lancet 364, 613-620 (2004).*
2. Global Initiative for Asthma. Www.ginasthma.org.
3. Wenzel, S. E. Asthma: defining of the persistent adult phenotypes. Lancet 368, 804-813 (2006).
4. Moore, W. C. et al. Identification of asthma phenotypes using cluster analysis in the Severe Asthma Research Program. Am. J. Respir. Crit Care Med. 181, 315-323 (2010).
5. Brusselle, G. G., Joos, G. F. & Bracke, K. R. New insights into the immunology of chronic obstructive pulmonary disease. Lancet 378, 1015-1026 (2011).*
6. Hancock, D. B. et al. Meta-analyses of genome-wide association studies identify multiple loci associated with pulmonary function. Nat. Genet. 42, 45-52 (2010).*
7. Moffatt, M. F. et al. A large-scale, consortium-based genomewide association study of asthma. N. Engl. J. Med. 363, 1211-1221 (2010).
8. Hilty, M. et al. Disordered microbial communities in asthmatic airways. PLoS. One. 5, e8578 (2010).
9. Erb-Downward, J. R. et al. Analysis of the lung microbiome in the "healthy" smoker and in COPD. PLoS. One. 6, e16384 (2011).
10. Vanoirbeek, J. A. et al. How long do the systemic and ventilatory responses to toluene diisocyanate persist in dermally sensitized mice? J. Allergy Clin. Immunol. 121, 456-463 (2008).*
11. Provoost, S. et al. Diesel exhaust particles stimulate adaptive immunity by acting on pulmonary dendritic cells. J. Immunol. 184, 426-432 (2010).*
12. Robays, L. J. et al. Concomitant inhalation of cigarette smoke and aerosolized protein activates airway dendritic cells and induces allergic airway inflammation in a TLR-independent way. J. Immunol. 183, 2758-2766 (2009).*
13. Provoost, S., Maes, T., Joos, G. F. & Tournoy, K. G. Monocyte-derived dendritic cell recruitment and allergic T(H)2 responses after exposure to diesel particles are CCR2 dependent. J. Allergy Clin. Immunol. (2011). Epub *
14. Demoor, T. et al. Role of lymphotoxin-alpha in cigarette smoke-induced inflammation and lymphoid neogenesis. Eur. Respir. J. 34, 405-416 (2009).*
15. Demoor, T. et al. The role of ChemR23 in the induction and resolution of cigarette smoke-induced inflammation. J. Immunol. 186, 5457-5467 (2011).*
16. Manise, M. et al. Cytokine production from sputum cells and blood leukocytes in asthmatics according to disease severity. Allergy 65, 889-896 (2010).*
17. Bachert, C. et al. Presence of IL-5 protein and IgE antibodies to staphylococcal enterotoxins in nasal polyps is associated with comorbid asthma. J. Allergy Clin. Immunol. 126, 962-8, 968 (2010).*
18. Provoost, S. et al. Decreased FOXP3 protein expression in patients with asthma. Allergy 64, 1539-1546 (2009).*
19. Pottelberge, G. R. et al. MicroRNA expression in induced sputum of smokers and patients with chronic obstructive pulmonary disease. Am. J. Respir. Crit Care Med. 183, 898-906 (2011).*
20. Andre, E. et al. Cigarette smoke-induced neurogenic inflammation is mediated by alpha,beta-unsaturated aldehydes and the TRPA1 receptor in rodents. J. Clin. Invest 118, 2574-2582 (2008).
21. Talavera, K. et al. Nicotine activates the chemosensory cation channel TRPA1. Nat. Neurosci. 12, 1293-1299 (2009).*
22. Adcock, I. M., Tsaprouni, L., Bhavsar, P. & Ito, K. Epigenetic regulation of airway inflammation. Curr. Opin. Immunol. 19, 694-700 (2007).*
