Groningen Research Institute for Asthma and COPD. Annual Report 2013 UMCG

Transcription

1 Groningen Research Institute for Asthma and COPD Annual Report 2013 UMCG

2 CONTENT Mission statement... 3 Introduction... 4 Perspectives... 8 The year 2013 in review Highlights Prizes/Awards Visitors Special Topics Special Topic Special Topic Special Topic Special Topic Special Topic Members International collaboration Seminar program Research meetings Research projects Publications Dissertations Publications SCI journals Publications in Dutch Books / Book chapters Contributions to other research institutes

3 Mission statement The mission of GRIAC is the multidisciplinary translational study of obstructive airway and pulmonary diseases and healthy ageing Program leaders: Prof. dr. H.M. Boezen Prof. dr. G.H.Koppelman Visiting address: University Medical Center Groningen Hanzeplein 1 NL-9713 GZ Groningen Website: Webmaster: Prof.dr. W. Timens Secretariat: Ms. J. Blokzijl, Dept. of Pediatric Pulmonology and Pediatric Allergology Beatrix Children s Hospital University Medical Center Groningen Hanzeplein 1 P.O. Box NL-9700 RB Groningen Phone: Fax: h.m.boezen@umcg.nl and g.h.koppelman@umcg.nl 3

4 Introduction The Groningen Research Institute for Asthma and COPD (GRIAC) performs research on obstructive and pulmonary diseases on the edge between clinical and fundamental research, arising from a clinical-scientific background. GRIAC fits within the research of the University Medical Center Groningen, which has a central focus on healthy ageing. GRIAC is part of the governmentally accredited organization GUIDE (Groningen University Institute for Drug Exploration) which is embedded in the Groningen School of Medical Sciences (GSMS). Most research is funded by external support as given by NWO, Dutch Lung Foundation, the European Community and industry. The research conducted in Groningen results from internal discussions within the scientific forum of researchers on asthma and COPD in Groningen and somewhat broader in the Netherlands. It is also stimulated by new developments internationally. Members of the board of GRIAC from Groningen are internationally acknowledged experts in their research fields. In that sense, internationally GRIAC and GRONINGEN are used interchangeably, and we adapted our logo to reflect this. Participating departments There is an intensive collaboration between the researchers of GRIAC, consisting of our members from different disciplines. The disciplines involved are allergology, experimental pulmonology and inflammation research, epidemiology, general practice, molecular pharmacology, pathology, paediatric pulmonology and paediatric allergology, pulmonology and respiratory insufficiency. Collaboration is based on freedom, equivalence and consensus. There exists extensive collaboration with Departments of Dermatology, Gastroenterology, Genetics, Haematology, Medical oncology and Transplantation. Furthermore, collaboration exists with the Department of Analytical Biochemistry (University Center for Pharmacy). Epidemiology Allergology General Practice Respiratory insufficiency Molecular Pharmacology Pediatric Pulmonology & Pediatric Allergology Groningen Research Institute for Asthma and COPD Pathology Experimental pulmonology & inflammation research Pulmonology 4

5 How we collaborate Every two weeks GRIAC organises research meetings for the whole institute in which both internal and external speakers are invited to venture new ideas and to challenge the audience. This constitutes also the forum in which different types of research are being presented to all members of GRIAC. Members of GRIAC participate in very different aspects of asthma and COPD research, ranging from epidemiology, clinical allergology, pulmonology, pharmacology, and general practice to basic research in genetics, proteomics, tissue studies, cell cultures and animal models. Lively discussions always take place. To enhance collaboration and stimulate new areas of research, GRIAC organises twice yearly a research retreat and monthly brainstorm sessions on a specific topic. These brainstorm sessions are used to stimulate novel ideas for multidisciplinary research, and to discuss publication ideas for high impact journals. During the GRIAC retreat the members of the Board of directors, scientific staff and postdocs of GRIAC discuss future perspectives and new developments in research and explore potential new collaborations within their research, based on international developments in the field. During and after the research meeting investigators can discuss their grant proposals with the staff members, who are expert in a particular field. Every five years GRIAC organises an internationally well-received symposium aimed at understanding the differences and similarities between asthma and COPD. In 2009, the eighth symposium Bronchitis VIII was held and in 2014 Bronchitis IX will be organized from th of June in Groningen with again an excellent international faculty. The theme of Bronchitis IX is Lungs, on the edge of health and ageing ( At every occasion of the defence of a PhD thesis care is taken to also invite a top-researcher of a particular research field. He or she is asked to judge the thesis and participate in the PhD defence on site, and, in addition, to give a presentation. When these external visitors are present, workshops for exchange of ideas are organised for both senior and junior researchers. Finally, there are weekly meetings for junior researchers and staff members. At these meetings there is ample time for discussion on choosing the appropriate study design, the set-up of research protocols, analyses and interpretation of results of research, and for preparation and improvements in concepts of abstracts, and oral and poster presentations at international meetings. Introductory lectures are provided in lung function measurements, laboratory techniques, genetic research and so on. We aim to make our PhD students familiar with these research techniques. These weekly GRIAC meetings aim to teach the understanding of different aspects of the approach towards research on asthma and COPD in the various disciplines involved in GRIAC in order to improve the level of interdisciplinary research. PhD courses in epidemiology, statistics and genetic data analyses are being organised for members of GRIAC and others interested as well. Organisation Two program leaders lead the Institute. They have the following tasks: Representatives in GSMS and GUIDE Contacts with the UMCG Contacts with the University of Groningen Policy preparation for KNAW, FMW, UMCG and University of Groningen Preparing propositions for research development The coordinators are advised extensively by the Board of GRIAC, consisting of senior members of the participating departments, who all have their own specific expertise. This board advises in all aspects of research. The board meets once monthly to exchange ideas and prepare policies. 5

6 Research program Research projects have to fit within the research program, describing the projects in their mutual cohesion. The tuning of projects and development into a program is the responsibility of the program leaders of GRIAC, in exchange with the Scientific Board of the Institute. In 2013, GRIAC defined obstructive airway and pulmonary disease in relation to healthy ageing, as its main topic, which is reflected in our mission statement. Program description Research is aimed to stretch from bench to bedside and back with feedback loops. Central to the research is the goal to translate fundamental findings into the clinical situation and vice versa, i.e. translational medicine (see figure below). Clinical research is conducted in different patient groups in comparison with normal control volunteers in order to unravel underlying mechanisms of the diseases (genetics, aetiology, pathogenesis, pathophysiology). Furthermore responses to intervention (mediated by either medical therapy, behavioural counselling, rehabilitation or other treatment modalities) as well as parameters of progression of disease are being assessed in relation to the underlying mechanisms of the disease. Questions that are generated, but unanswered by clinical research, are approached using in vitro cellular systems and in vivo animal models. The other way around, hypotheses generated from in vitro or in vivo research are translated to the general and clinical human situation. To this aim GRIAC focuses on the following main topics related to obstructive airway and pulmonary disease: Identification of risk factors for development, progression and remission of disease Identification of disease related genes and their functionality Unravelling the pathophysiology of allergen-, environment- and smoke- induced disease, in both humans and animal models Unravelling the effects of disease related inflammation on lung function, hyperresponsiveness and remodelling of large and small airways Defining new targets for intervention and evaluation of intervention strategies Development of non- or minor invasive tools to assess severity of disease and (side) effects of treatment. 6

7 Research area The focus of research is on asthma and COPD, which involves the sub-programs: 1. Epidemiology: Epidemiological studies on endogenous, environmental and lifestyle risk factors, both in general and patient-based populations, from prenatal onwards to old age. 2. Genomics: Studies on genes, epigenetics, gene expression and function, molecular mechanisms and gene-gene and gene-environment interactions in disease development, progression, remission, and severity, as well as disease intervention (pharmaco- genomics). 3. Pathophysiology and pathogenesis of allergen, smoking and other lifestyle factors, and environment-induced diseases: In vivo studies in humans and animal models using mice and unrestrained guinea pigs. Investigations include lung function techniques and studies of blood, tissues and/or cells derived from airways or lungs. Furthermore, in vitro studies assess cellular activation and interaction as well as signaling pathways in cells and tissue explants (e.g. lymphocyte subsets, epithelial cells, fibroblasts, intact airway, and smooth muscle preparations). Interactions of different cell types are studied in cells obtained by sputum induction as well as airway and lung tissue obtained by bronchoscopy, by surgical biopsy or autopsy. The Placebo controlled food provocation database provides an excellent method to investigate the allergic response to foods. 4. Assessment, modulation and intervention in disease severity, progression and remission: Disease outcome assessment is being studied with techniques such as exhaled breath analyses and small airway function. In addition, validated questionnaires on Quality of Life, drug side effects, hyperresponsiveness and symptoms are developed for diagnostic purposes as well as outcome assessment. Interventions can be at the level of cell cultures, animal models and clinical studies with targeted therapy. The main strategies to reach our goals are discussed below: Genomics The availability of genetic techniques and the collaboration with the Department of Genetics (Head: Prof. C. Wijmenga) have greatly extended the genetic sub-programs, allowing genome-wide association and methylation studies, high throughput genetic SNP detection, fine-mapping in relevant chromosomal regions and candidate gene studies. Deep sequencing techniques and analysis strategies are currently being developed. Since gene-environment interactions are important for understanding complex diseases like asthma and COPD, these have been explored in several sub-programs, in collaboration with multiple groups in the Netherlands and abroad. This has resulted in gene-environment interaction studies on atopy and asthma, and on COPD onset and progression. The GRIAC group collaborates internationally and takes the lead in some EC-funded FP7 projects (allergy and asthma: MeDALL; COPD: COPACETIC) on genetics and epigenetics of asthma and COPD, and in exploration of specific gene-environment interactions in these projects. Molecular biology techniques Molecular biology techniques are being used more effectively and widely, and are being introduced when not present (either in our own labs or as part of local facilities; for example, the recently developed custom micro-array development and accompanying data-mining). In vivo and in vitro silencing of genes are now established techniques that are operational at the University Medical Center and Pharmacy, including the development of knock out and transgenic mouse models. 7

8 This has enabled the use of RNAi and pharmacological modulation of membrane and nuclear receptors and signaling proteins. Fundamental to this line of research is the exploration of intracellular pathways relevant for disease development. Proteomics Proteomic research has added important possibilities to develop disease susceptibility markers and disease progression and intervention tools. To enable clinical studies that require greater power, continuing and promoting collaboration with general hospitals in the region has expanded the recruitment population. To enhance the quality of the collaboration, local physicians in these hospitals are more involved in the research group and also propose their own studies for discussion in research meetings. Healthy ageing Healthy Ageing has been adopted as the main theme for research and clinical profile of the UMCG. An important long term project within this theme is LifeLines a planned 30-year survey on risk factors (obtained by questionnaire, objective physiological data and biological and genomic markers) for disease development, COPD being one of the leading themes. This fits very well with the research agenda of GRIAC, including co-morbidity and systemic manifestations of COPD. We are actively participating in the development and sustainment of this program within the UMCG. Perspectives Asthma and COPD research takes place in a lively and rapidly changing field. New developments will encompass the functional genomics (including proteomics) of asthma and COPD. We envisage that integration of the omics techniques will provide novel insight into the disease networks that lead to these obstructive airway diseases. The population for genetic analyses in asthma and COPD has been greatly expanded, and will be expanded even further, allowing replication and association studies. A number of international genome-wide association studies on asthma and COPD, including analysis on gene-environment interactions are ongoing, as well as gene methylation studies. This will lead to identification of novel genes and environmental factors playing a role in disease onset and progression. We will incorporate integrative genomic approaches in follow up studies. Functional studies on gene variations in asthmatic and healthy individuals have started, both in cells and in animal studies. Integration of longitudinal epidemiological data with genetics will provide insight into genetic variants as risk factors for the development, progression or remission of asthma and COPD. Finally, the integration of newly discovered genes with the results of gene expression in relevant tissues that are available and/or cell cultures allows further research into functional relevance and this can be integrated into systems medicine. Increasingly so, epigenetics (e.g. gene-by-environment interactions and differential (genome wide) methylation) is focus of our research. Within GRIAC, we translate the findings from these genomic approaches back to models of disease, such as animal models or cellular experiments. For both asthma and COPD, we will gain better insight into the intricate interplay between epithelial cells and fibroblasts on one hand and their interaction with different inflammatory cell types in the lung and airway smooth muscle cells on the other. With the recognition that the airway smooth muscle cell is a highly plastic cell governed by complex interactions between multiple receptor systems and environmental changes, research will remain focused on unraveling the interactive mechanisms that determine airway smooth muscle responsiveness and growth in chronic airways disease. Newly discovered genes will be incorporated into our studies on in vitro modification of epithelial, smooth muscle and fibroblast cell cultures. 8

9 A focus on the background question of why not all smokers develop COPD will remain a priority, in association with the consequences of smoking cessation and intervention in the progression of inflammation and remodeling. This knowledge is enhanced by studies regarding the effect of smoking (also during pregnancy) on allergy development, asthma progression and susceptibility to develop COPD as well as the effects on treatment response. The former topics will be investigated in animal models and in humans. Exacerbations are sometimes life-threatening occurrences in patients with asthma and COPD, which may affect activities of daily living, increase symptoms, reduce quality of life, and affect disease outcome. Research will focus on practical and minimal interventions to prevent these exacerbations, including research on the underlying mechanisms and the associated increase in symptoms. Finally, side effects of drugs will be assessed by questionnaires, which will help to further understand the optimal approach to asthma and COPD management. Novel techniques like bronchoscopic lung volume reduction in severe COPD patients are explored and evaluated in relation to their effects on e.g. daily physical activity. Physical inactivity, obesity, and a low grade systemic inflammation are increasingly recognized as important risk factors for the induction and clinical expression of asthma and COPD. The determinants and consequences of physical inactivity in COPD are systematically investigated in relation with co-morbid disorders. A physical activity enhancement strategy has been developed in collaboration with the faculty of human movement sciences, which may be used in the primary, secondary and tertiary echelons of our health care system. Research in the rehabilitation program has been recently reinforced with respect to asthma and COPD, and is expected to increase the input to and output of the GRIAC program. This has been expanded by novel invasive techniques such as applying stents in airway walls and chronic ventilatory support in COPD. As both improve exercise capacity in emphysema this might lead to a more effective rehabilitation. Notwithstanding the fact that understanding of a disease is of prime importance, the management of the disease as it exists in current patients is of importance. Thus, it is of great interest that transmural management of asthma and COPD is becoming more mature. Collaborative efforts of lung function departments, general practitioners and pulmonologists in addition to nurse practitioners help to provide better health care for individuals with respiratory symptoms that affect their daily life. This ultimately may improve the quality of life of individuals with asthma and COPD. Output, visibility and (external) funding Productivity of GRIAC is at present overall very good and the whole GRIAC institute was graded excellent in its most recent Mid Term Review. Results in internal medicine and basic science have been published in top peer reviewed journals and patents have also been filed. GRIAC members have been urged to focus on publication strategy and brainstorm sessions have been organized to even further improve the impact of scientific output. Asthma and COPD are highly prevalent in the general population, and thus focussing on these two syndromes is appropriate and has a high societal relevance. At current the priority of the institute is ranging from cellular models to the underlying disease models to the clinic (translational research) with transdisciplinarity as a major feature. The national and international academic reputation of the senior GRIAC members can be weighted at its merits judging the invitations to address international congresses and their prominent roles in various national and international research and professional societies and working groups in addition to their role in EU collaborations. In 2013, the Expertscape Website graded GRIAC amongst the top institutes in Europe in relation to asthma and COPD. Moreover, since several of these GRIAC members are relatively young and proven to develop their high potential in their specific research field (e.g. epidemiology, pediatric and 9

10 adult pulmonology, and molecular pharmacology), GRIAC can face its future with confidence. We will continue to invest in the training of young scientists in the field of obstructive airways and pulmonary disease, with a focus on multidisciplinary translational research. Given the true interdisciplinary nature of the institute, we feel confident that ongoing close collaboration of GRIAC members who share their in-depth knowledge of specific research fields in asthma and COPD will keep the institute at the internationally acknowledged level of excellence in the future, and that they will be able to generate sufficient resources to finance this research. We have shifted our focus from smaller (University Medical Centers) towards larger (inter)national and interdisciplinary research grants (Lung foundation consortium grants, NWO TOP grants, European funding) as well as personal grants (VENI, VIDI, VICI and ERC grants). Within the U4 collaboration of the Universities of Groningen, Ghent, Göttingen and Uppsala, international collaborations are ongoing to prepare PhD students to stay at 2 or 3 of these universities for an international PhD project. 10

11 The year 2013 in review All contributions to the scientific work in GRIAC are important and highly appreciated. It cannot be stressed enough that all the scientific output and results obtained are only possible due to the contribution of every single person who works within our research institute. Nevertheless, without disrespect to the work of members who are not specifically mentioned, we like to highlight some topics that drew particular attention in Highlights Prof. Dr. D.S. Postma was granted an Honorary Degree at the University of Lund, Sweden (May 2013) Prof. Dr. D.S. Postma was granted the Turner Warwick award from the National Heart and Lung Institute and the Royal Brompton and Harefield NHS Foundation Trust, UK (June 2013) Dr. R. Gosens, Dr. H. Maarsingh, and Prof. Dr. H. Meurs organized the 8th International Young Investigators Meeting on Airway Smooth Muscle and Fibroblast Biology Groningen, Sept. 4th-6th, 2013 Prof. Dr. M.G. Belvisi and Prof. Dr. H. Meurs organized an ERS Research Seminar: Translational Animal Models of Asthma Barcelona, Nov. 29th-30th, Prof. Dr. H.M. Boezen and Prof. Dr. D.S. Postma obtained a Lung Foundation Consortium grant. Genes and exposures underlying COPD onset ( euro).( Co-investigators Prof. Dr. C.M. van Duijn, Dr. C.C. van Diemen). Prof. Dr. H. M. Boezen was appointed certified instructor Epidemiology on behalf of the Netherlands Epidemiological Society and the SMBWO. Prof. Dr. M. Schmidt was appointed as Science without Borders Visiting Professor. Prof. Dr. H. Meurs was appointed as member of the Board of the Dutch Pharmacological Society. Dr. R. Gosens was appointed as a member of the Editorial Board of the European Journal of Pharmacology Dr. J.W.H. Kocks was appointed as assistant professor and was granted a Research Fellowship with prof Richard Beasley, Medical Research Institute of New Zealand Dr. J.W.H. Kocks and Dr. B. Flokstra-de Blok were appointed as associate editors of the Primary Care Respiratory Journal, which is the 2nd highest journal in ISI Family Medicine category Prizes/Awards C.D. van Ginkel, MSc won a Poster Award at the Pediatric Allergy and Asthma Meeting Dr. I.H. Heijink won an ERS Travel Grant for best abstracts on COPD L.E.M. Kistemaker, MSc won an International Trainee Award from the American Thoracic Society 11

12 E.I. Metting, MSc won the public award of The Longdagen 2013 meeting of the Netherlands Respiratory Society A. Oldenburger, MSc won a Young Investigators Award from the British Pharmacological Society and a Travel Award from the Netherlands Respiratory Society W. Poppinga, MSc won a Travel Award from the Netherlands Respiratory Society S.D. Pouwels, MSc won the NRS Young Investigators award P. Robbe, MSc won a travel award from the American Thoracic society P. Robbe. MSc won the ATS Val Vallyathan Junior award for outstanding contribution to basic and translational science in occupational medicine Visitors Prof. Dr. A. Agusti, Institut Clínic del Tòrax, Hospital Clínic, University of Barcelona. February 28, 2013 Dr. R. Rottier, ErasmusMC, Rotterdam. Protein complexes in lung development. August 20, Dr. M.J. Schuliga, Department of Pharmacology, University of Melbourne, Australia, The plasminogen activation system: new targets in lung inflammation and remodeling. September 3, Dr. R. Krishnan, Harvard Medical School, Boston, USA, Mechanosensing in the lung: implications for ARDS and asthma. September 24, Prof. Dr. P. Pare, St. Paul s hospital, Vancouver, Canada, Genetic screening for respiratory disease: are we there yet?. September 27, Prof. Dr. G. Folkerts, Department of Pharmacology, University of Utrecht, Nutrition in respiratory diseases. October 4, Prof. D. Jarvis, Respiratory Epidemiology, Occupational Medicine and Public Health, National Heart and Lung Institute, London. October 9, Prof. Dr. L. Bjermer, Department of Respiratory Medicine and Allergology, Lund University, Lund, Sweden. October 10, Prof. Dr. P. Sterk, Department of Respiratory Medicine, Academic Medical Center, Amsterdam. October 10,

13 Special Topics Special Topic 1 Muscarinic M 3 receptors mediate airway inflammation and remodeling Loes Kistemaker & Reinoud Gosens; Dept. Molecular Pharmacology Acetylcholine is the primary parasympathetic neurotransmitter in the airways, and is traditionally associated with bronchoconstriction and mucus secretion. Parasympathetic activity is increased in COPD and asthma, which is the basis for the use of anticholinergics as bronchodilator therapy in COPD, and in acute exacerbations of asthma. The lungs express muscarinic M 1, M 2 and M 3 receptor subtypes, each with a specific physiological role and relationship with disease. M 1 receptors facilitate neurotransmission in the parasympathetic ganglia and regulate electrolyte and water secretion by mucus producing cells. The M 2 receptor is an auto-inhibitory prejunctional receptor on vagal nerves inhibiting acetylcholine release. M 3 receptors on airway smooth muscle and glands mediate bronchoconstriction and mucus secretion, the primary roles of muscarinic receptors in lung diseases. Because of the specific involvement of the M 3 receptor in airway smooth muscle contraction, and because anticholinergics are primarily used as bronchodilators, M 3 receptor selective anticholinergics have been advocated for. However, there is a growing body of evidence from in vitro and in vivo studies indicating that acetylcholine also contributes to airway inflammation and remodeling (see (1) for review) and it is not known which muscarinic receptor subtype(s) mediate these effects. Therefore, our studies aimed to gain insight into the functional roles of muscarinic M 1, M 2 and M 3 receptors in airway inflammation and remodeling using muscarinic receptor subtype knock-out mice. First, we used an acute cigarette smoke exposure model to investigate the role of the individual muscarinic receptors in cigarette smoke-induced inflammation. Cigarette smoke induced an increase in macrophages, lymphocytes and neutrophils in bronchoalveolar lavage fluid of wild-type mice. Neutrophilic inflammation was lower in M 3 R -/- mice, but higher in M 1 R -/- and M 2 R -/- mice compared to wild-type mice (figure 1). Accordingly, the release of KC, MCP-1 and IL-6 was lower in M 3 R -/- mice, and higher in M 1 R -/- and M 2 R -/- mice. Markers of remodeling were not increased after four days of cigarette smoke exposure. However, M 3 R -/- mice had reduced expression of TGF-β1 and matrix proteins. Figure 1: Effect of cigarette smoke exposure on neutrophil numbers in the BAL fluid of WT mice and M 1, M 2 and M 3 receptor knock-out mice. In subsequent studies, wild-type animals were pretreated with the M 3 receptor antagonist 4- DAMP, to investigate whether we could confirm the pro-inflammatory role of the M 3 receptor 13

14 observed in knock-out mice by pharmacological inhibition. Indeed, cigarette smoke-induced inflammatory cell recruitment and KC release were also prevented by pretreatment with the M 3 antagonist. Collectively, these data demonstrated a pro-inflammatory role for the M 3 receptor in cigarette smoke-induced neutrophilia and cytokine release, yet an antiinflammatory role for M 1 and M 2 receptors. This is the first demonstration of the differential regulation of inflammation by muscarinic receptors in vivo, and suggests an important role for the M 3 receptor (2). Second, we used a chronic ovalbumin model to investigate the role of the individual muscarinic receptors in asthma. The results provide an equally convincing case for the M 3 receptor, but also show that not all aspects of the allergic response can be prevented by M 3 receptor knock-out. Allergen exposure induced goblet cell metaplasia, airway smooth muscle thickening, pulmonary vascular smooth muscle remodeling and deposition of collagen I and fibronectin in the airway wall of wild-type mice. These effects were absent or markedly lower in M 3 R -/- mice (30-100%), as shown for airway smooth muscle thickening in figure 2. M 1 R -/- and M 2 R -/- mice responded similar to wild-type mice with respect to remodeling. Interestingly, allergen-induced airway inflammation, assessed as infiltrated eosinophils and Th2-cytokine expression, was similar or even enhanced in M 3 R -/- mice. Again, M 1 R -/- and M 2 R -/- mice responded similar to wild-type mice. These data indicate that acetylcholine contributes to allergen-induced remodeling and smooth muscle mass via the M 3 receptor, and not via M 1 or M 2 receptors. No stimulatory role for M 3 receptors in allergic inflammation was observed, suggesting that the role of acetylcholine in remodeling is independent of the allergic inflammatory response (3). We hypothesize that bronchoconstriction by itself, without an inflammatory response, can induce remodeling. This will be the subject of further studies. Figure 2: Effect of ovalbumin challenge on airway smooth muscle thickening in WT and M 3R -/- mice. Taken together, these data underscore the important role for the M 3 receptor, suggesting that kinetic M 3 subtype selectivity of anticholinergics is a desired property. Whether anticholinergic therapy also affects inflammation and remodeling in patients with COPD and asthma still needs to be confirmed. References: (1) Kistemaker LE, Oenema TA, Meurs H, Gosens R. Regulation of airway inflammation and remodeling by muscarinic receptors: perspectives on anticholinergic therapy in asthma and COPD. Life Sci 2012;91: (2) Kistemaker LE, Bos IS, Hylkema MN, Nawijn MC, Hiemstra PS, Wess J, Meurs H, Kerstjens HA, Gosens R. Muscarinic receptor subtype-specific effects on cigarette smoke-induced inflammation in mice. Eur Respir J 2013;42: (3) Kistemaker LE, Bos ST, Mudde WM, Hylkema MN, Hiemstra PS, Wess J, Meurs H, Kerstjens HA, Gosens R. Muscarinic M Receptors Contribute to Allergen-Induced Airway Remodeling in Mice. Am J Respir Cell Mol Biol 2013, doi: /rcmb OC. 14

15 Special Topic 2 Small airway disease in asthma Erica van der Wiel, Ilse M. Boudewijn, Nick H.T. ten Hacken, Dirkje S. Postma, Maarten van den Berge - Department of Pulmonology Small airways have long been disregarded in the pathophysiology of asthma. However, the small airways are an important site of inflammation and remodeling in asthma (1,2). The small airways are usually defined as airways with an internal diameter < 2mm, usually deeper than generation 8 of the bronchial tree (3,4). The last generations, 17-23, contribute for 95% to the total airway surface area. The small airways are difficult to investigate directly, because they are relatively inaccessible. There are several tests available assessing the patency of small airways and they measure different aspects of dysfunction as summarized in Table 1 (5,6). So far, there is no gold standard available to diagnose small airway dysfunction. Recent studies suggest that abnormalities in the small airways can contribute to the clinical expression of asthma (7). For example, Farah et al showed that improvement of ventilation heterogeneity after treatment with high dose inhaled corticosteroids was independently associated with improvement in asthma control (8). In our group, Telenga et al recently showed that a lower FEF 50% was an independent predictor of more severe bronchial hyperresponsiveness (9). In addition to this, we have recently investigated subjects without any respiratory symptoms, but with bronchial hyperresponsiveness (BHR), and measured small airway dysfunction before and during a methacholine provocation test by Impulse Oscillometry (IOS) (10). We showed that these subjects with so-called asymptomatic BHR have less small airway resistance than asthma patients, both at baseline and after a methacholine test (Figure 1). This might explain their absence of respiratory symptoms. So far, we have no reliable instruments to identify asthmatic subjects with small airway dysfunction. For this reason we are studying new tools that can help to assess the presence of small airway dysfunction in asthma. First, we started with the development of a questionnaire (a collaboration of the departments of General Practice and Pulmonology in GRIAC). To this end, asthma patients with and without small airway dysfunction were interviewed and items that differed were collected, leading to a 63-item small airway dysfunction tool (SADT) (11). The items identified were related to several asthma signs and symptoms e.g. bronchial hyperresponsiveness, response to allergens or physical exercise. In the near future, we will test and validate this 63-item tool retaining the most important items. Next, we have built further on the research of Cohen et al who studied the response to a small particle provocation test, aiming to identify which asthma patients would most likely benefit from small particle treatment (12). To this end, we developed a new provocation test in collaboration with the department of Pharmaceutical Technology and Biopharmacy, during which small particle adenosine is administered with a dry powder inhaler instead of the regular wet nebulized adenosine 5 -monophosphate (13). After validation of the provocation test in 5 asthmatic subjects, we started a study comparing small (2.7µm MMAD) and large (6µm MMAD) particle dry powder adenosine inhaled with different flows. In this way, we wish to select the optimal provocation test to assess small airway dysfunction in patients with asthma. Since small airway dysfunction clearly contributes to asthma symptoms, treatment of small airways is of interest. Cohen et al from our group showed that treatment with a small particle inhaled corticosteroid (ICS), ciclesonide, improves bronchial hyperresponsiveness measured with small particle adenosine 5 -monophosphate (Figure 2) (12). In another study it was shown that small airway resistance in mild-to-moderate asthma patients improved after treatment with small particles ICS but not after treatment with coarse particle ICS (14). It is, however, not yet clear whether all asthma patients benefit from small particle treatment or 15

16 whether this comprises only a subset of asthma patients with small airway dysfunction. Such a subset may be asthma patients who smoke, since smoking is related to small airway dysfunction (15). Moreover, it has been suggested that smokers with asthma respond less to treatment with coarse particle ICS. Therefore, we hypothesize that smokers with asthma will benefit from small particle ICS treatment. We are currently performing a clinical study involving smoking and ex-smoking asthma patients which are treated with small- and large particle ICS. In conclusion, asthma severity and response to treatment varies widely among asthma patients. Identifying sub-phenotypes of asthma is needed to develop patient-specific treatment strategies and small airway dysfunction is such a sub-phenotype of asthma. Developing better diagnostic tools to identify patients with small airway disease, and subsequently develop treatment regimes targeted at the small airways, is important as it may lead to improved asthma care. References (1) Hamid Q, Song Y, Kotsimbos TC, Minshall E, Bai TR, Hegele RG, et al. Inflammation of small airways in asthma. J Allergy Clin Immunol 1997 Jul;100(1): (2) Bergeron C, Hauber HP, Gotfried M, Newman K, Dhanda R, Servi RJ, et al. Evidence of remodeling in peripheral airways of patients with mild to moderate asthma: effect of hydrofluoroalkane-flunisolide. J Allergy Clin Immunol ;116(5): (3) Macklem PT, Mead J. Resistance of central and peripheral airways measured by a retrograde catheter. J Appl Physiol ;22(3): (4) Weibel ER. Morphometry of the Human Lung. Berlin-Göttingen-Heidelberg: Springer Verlag; (5) Contoli M, Bousquet J, Fabbri LM, Magnussen H, Rabe KF, Siafakas NM, et al. The small airways and distal lung compartment in asthma and COPD: a time for reappraisal. Allergy 2010 Feb;65(2): (6) van den Berge M, ten Hacken NH, Cohen J, Douma WR, Postma DS. Small airway disease in asthma and COPD: clinical implications. Chest ;139(2): (7) van der Wiel E, ten Hacken NH, Postma DS, van den Berge M. Small-airways dysfunction associates with respiratory symptoms and clinical features of asthma: a systematic review. J Allergy Clin Immunol 2013 Mar;131(3): (8) Farah CS, King GG, Brown NJ, Downie SR, Kermode JA, Hardaker KM, et al. The role of the small airways in the clinical expression of asthma in adults. J Allergy Clin Immunol 2012 Feb;129(2):381-7, 387.e1. (9) Telenga ED, van den Berge M, Ten Hacken NH, Riemersma RA, van der Molen T, Postma DS. Small airways in asthma: their independent contribution to the severity of hyperresponsiveness. Eur Respir J 2013 Mar;41(3): (10) Boudewijn IM, Telenga ED, van der Wiel E, van der Molen T, Schiphof L, Ten Hacken NH, et al. Less small airway dysfunction in asymptomatic bronchial hyperresponsiveness than in asthma. Allergy 2013 Nov;68(11): (11) Schiphof L, van der Wiel E, ten Hacken NH, van den Berge M, Postma DS, van der Molen T. Development of a tool to recognize small airways dysfunction in asthma (SADT). C42. Creating the evidence: generating tools and developing methodologies for pulmonary and critical care research. May 1, 2013, A4220-A4220. (12) Cohen J, Postma DS, Douma WR, Vonk JM, De Boer AH, ten Hacken NH. Particle size matters: diagnostics and treatment of small airways involvement in asthma. Eur Respir J 2011 Mar;37(3): (13) Lexmond AJ, Hagedoorn P, van der Wiel E, Ten Hacken NHT, Frijlink HW, de Boer AH. Adenosine dry powder inhalation for bronchial challenge testing, part 1: Inhaler and formulation development and in vitro performance testing. Eur J Pharm Biopharm. In press. doi: /j.ejpb (14) Yamaguchi M, Niimi A, Ueda T, Takemura M, Matsuoka H, Jinnai M, et al. Effect of inhaled corticosteroids on small airways in asthma: investigation using impulse oscillometry. Pulm Pharmacol Ther 2009 Aug;22(4): (15) Verbanck S, Schuermans D, Meysman M, Paiva M, Vincken W. Noninvasive assessment of airway alterations in smokers: the small airways revisited. Am J Respir Crit Care Med 2004 Aug 15;170(4):

17 Table 1. Small airway function measurements Test Airway obstruction Parameters of small airway dysfunction Spirometry FEF 25-75% ; FEF 50%, FVC/SVC Resistance Impulse oscillometry (IOS) R5-R20; AX; X5, Fres R20 Air trapping Body plethysmography Ventilation heterogeneity Single breath nitrogen washout test (SBNT) Multiple breath nitrogen washout test (MBNW) Imaging HRCT scan H 3 HeMRI scan Inflammation FRC, RV, RV/TLC CV, CV/VC; slope phase III Sacin, Scond Air trapping Regional ventilation defects Parameters of large airway dysfunction FEV 1,FEV 1 /FVC, PEF Bronchoscopic biopsy Transbronchial biopsy Central bronchial biopsy Bronchoscopy Bronchoalveolar lavage (BAL) Exhaled nitric oxide (eno) Alveolar eno Bronchial eno Table is adapted from previously published tables from reference 5 and 6. AX: Reactance area, BAL: Bronchoalveolar lavage, CC: Closing capacity, CV: Closing volume, FEF 25-75% : Forced expiratory flow at 25% to 75% of the FVC, FEF 50% : Forced expiratory flow at 50% of the FVC, FEV 1 : Forced expiratory flow in one second, FRC: Functional residual capacity, Fres: Resonant frequency of reactance, FVC: Forced vital capacity, HRCT: High resolution computed tomography, IOS: Impulse oscillometry, MBNW: Multiple breath nitrogen washout test, eno: exhaled nitric oxide, PEF: Peak expiratory flow, R5: Resistance of the respiratory system at 5 Hertz, R20: Resistance of the respiratory system at 20 Hertz, R5-R20: Difference of R5 and R20, RV: Residual volume, Sacin: Ventilation heterogeneity generated in the acinar lung zone, SBNT: Single breath nitrogen test, Scond: Ventilation heterogeneity generated in the conductive lung zone, TLC: Total lung capacity, X5: Reactance of the respiratory system at 5 Hertz 17

18 Figure 1. Small airway resistance (R5-R20, measured with Impulse Oscillometry) at a provocative concentration of methacholine causing a 20% fall in FEV 1 in patients with asymptomatic BHR and asthma (10). Figure 2. Provocative concentration of small particle AMP causing a 20% fall in FEV 1 before and after treatment of (a) Ciclesonide, a small particle steroid and (b) Fluticasone, a coarse particle steroid (12). 18

19 Special Topic 3 How can studying eqtls and pqtls be of help in understanding the genetics of asthma and COPD? M van den Berge, CA Brandsma, GH Koppelman During the last decades, genome-wide association studies (GWASs) have been performed aiming to uncover the genetic basis of asthma and COPD. GWAS usually provide information about 300,000 or more single nucleotide polymorphisms (SNPs) throughout the genome. Several new susceptibility genes for asthma and COPD have been identified during the last decade. Although GWAS studies have gained important new insights in biological mechanisms underlying asthma and COPD, it is important to consider that both are complex genetic diseases. Thus, the change in relative risk caused by any single genetic variant, even when highly statistically significant, will be only small. Rather, a combination of multiple genetic variants working together may explain a clinically significant increase the explained variance associated with the risk of having the disease. A limitation of GWAS studies has been that is often difficult to identify which genes are regulated by the genetic variants 1. This is partly due to the small effect size of each individual genetic variants. In addition, SNPs are often in near-perfect linkage disequilibrium with nearby SNPs, meaning that they occur together in the population. Finally, the majority of disease associated SNPs are located in non-coding regions of the genome, e.g. promotor regions, and do not actually change the gene or protein structure. Integrative genomics is a new approach to identify true causal genes and variants. By using mrna expression as a phenotype and examining which genetic variants contribute to both gene expression (eqtls) and disease phenotype, true causal genetic variants may be discovered. eqtls can be divided into those that have local effects (cis-eqtls) where the SNP is located near the gene (e.g. within 1 megabase within the transcription start site (TSS) of that gene) and those with distant effects (trans-eqtls), e.g. more than 5 megabases away from the TSS of the gene or even on a completely different chromosome. We have recently shown how studying eqtls can be used to unravel how the genetic variants identified by earlier GWAS studies regulate the molecular events leading to asthma 2. As part of an eqtl consortium, GWAS as well as genome-wide gene-expression data were available from 1,111 patients undergoing lung surgery at one of the three university hospitals: The University Medical Center Groningen (Groningen, The Netherlands), Laval University (Quebec, Canada) and the University of British-Columbia (Vancouver, Canada). First, we idenitified cis-eqtls according to standard methods 3. Association between SNPs within 1 megabase up- or downstream the transcription probeset with an FDR-corrected q-value below 0.1, were defined as cis-eqtls. When more than one SNP was associated to the expression of a gene, the most significant SNP was referred to as the cis-esnp. A total of 5,655, 10,630, and 7,953 cis-eqtls were found in the Laval, Groningen, and UBC cohorts respectively. As a next step, we investigated the consistency of our findings between the three different cohorts, Groningen, Laval, and UBC. A successful replication was defined as the presence of an eqtl with the same direction of the effect and a nominal p-value below in at least one of the two other cohorts. Using this criterion, more than 70% of the Groningen cis-eqtls could be replicated indicating that the data are robust. Next, we related our findings to the previously published genome wide association study of asthma (the Gabriel study) 4;5. In contrast to most other GWAS studies, the Gabriel study published the asthma association results for all SNPs investigated rather than only reporting the top signals allowing us to perform an in-depth analysis. 19

20 Of the 567,589 GABRIEL SNPs, 60,530 were esnps in lung tissue. Interestingly, we found that these 60,530 esnps were enriched for a significant association with asthma in the GABRIEL study (Figure 1). Figure 1. Q-Q plot for the Gabriel meta-analysis among esnps found in the lung eqtl study. Among all SNPs surveyed by the Gabriel study, 60,530 were esnps. The graph shows that esnps are significantly enriched for lower p- values. This is compatible with previous studies showing that SNPs associated with complex diseases are more likely to be eqtls. In the Gabriel, the rs t allele in the 17q21 asthma susceptibility locus was identified as the genetic variant related to highest increase in relative risk of asthma with an odds ratio of Originally, this SNP was suggested to regulate the expression of ORMLD3 or GSDMB as the causal genes. However, when exploring the lung tissue eqtl dataset, the rs t allele was found to be most strongly associated with a lower expression of GSDMA (Figure 2). Figure 2. Boxplot for lung gene expression levels of GSDMA according to genotype for the SNP rs

21 This genetic variant was also associated with expression of ORMDL3 and GSDMB, but the effect was much weaker. In addition, several other SNPs in the 17q21 asthma susceptibility locus were found to be associated with lower GSDMA expression. The strongest effect was found for the rs Further support for GSDMA as an asthma susceptibility gene was derived from the observation that it is abundantly expressed in human bronchial epithelium. Additionally, a network analysis revealed that genes in the network neighborhood of GSDMA were enriched for immune response genes. In conclusion, these data show the strength of studying eqtls. First, they can identify functional genetic variants which are more likely to play a role in complex genetic diseases. In addition, it enables a better interpretation of GWAS studies by identifying which are causal genes regulated by asthma susceptibility SNPs. Whereas QTL mapping is mostly restricted to the study of regulating mrna, we recently reported one of the first examples of protein (p)qtl mapping in respiratory disease 6. This is a collaboration between the University of Nottingham (group led by dr I. Sayers) and GRIAC. In contrast to eqtls, pqtl mapping has the potential to uncover post translational mechanism of SNPs that change protein, but do not alter transcriptional events. In this study, we investigated the genetic regulation of soluble levels of serum upar. In a previous study, upar was found to be associated with astma, BHR and lung function decline in three different family studies 7. Serum upar levels were higher in asthma and COPD compared to healthy controls (figure 3). Figure 3. Serum upar levels in healthy controls, patients with asthma, and patients with COPD. Next, we performed a GWAS on serum UPAR levels in Dutch Asthma Patients that were part of the Dutch Asthma Genetics Consortium. A remarkable finding was that we did not observe a strong pqtl on chromosome 19 in the UPAR gene, but a genome wide significant trans p QTL on chromosome 4 in the Kallikrein1 gene. Further work showed that serum KLKB1 enzymatic activity was driven by this SNP (rs ) and is inverse to scupar levels. Biochemical analysis identified that KLKB1 cleaves scupar and negates scupar's effects on primary human bronchial epithelial cells (HBECs) in vitro. Importantly, this effect would not have been found when only eqtl mapping was performed, since this mechanism is based on proteins, not on mrna. 21