Groningen Research Institute for Asthma and COPD. Annual Report 2014 UMCG

Transcription

1 Groningen Research Institute for Asthma and COPD Annual Report 2014 UMCG

2 Content Mission statement... 3 Introduction... 4 Research area... 7 Perspectives... 8 The year 2014 in review Highlights Prizes/Awards Visitors Special Topics Special Topic Special Topic Special Topic Special Topic Special Topic Departments Members International collaboration Seminar program Research meetings presentations Genome-wide nasal gene expression in COPD Brainstorm sessions Research projects in 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. Pediatric Pulmonology and Pediatric Allergy 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) is dedicated to research on obstructive and pulmonary diseases on the edge of clinical and fundamental research, arising from a clinical-scientific background. GRIAC operates within the research framework of the University Medical Center Groningen, which has a central focus on healthy ageing and the Faculty of Science of the University of Groningen, which has a focus on molecular life and health. 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 from consortia with researchers from collaborating institutes inside the Netherlands and abroad. Participating departments The multidisciplinary and translational research of GRIAC is the result of 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. GRIAC recently added new members from the department of Clinical Pharmacy and from ERIBA, the European Research Institute for the Biology of Aging. Collaboration is based on freedom, equivalence and consensus. Extensive collaboration exists with Departments of Dermatology, Gastroenterology, Genetics, Haematology, Medical oncology and Transplantation. Furthermore, collaboration exists with the Department of Analytical Biochemistry (University Center for Pharmacy). 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 2014 Bronchitis IX has been organized from th of June in Groningen with again an excellent international faculty. The theme of Bronchitis IX was 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 GRIAC defines obstructive airway and pulmonary disease in relation to healthy ageing, as its main topic, which is reflected in our mission statement. Research projects have to fit within this research topic, 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. Program description Our 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: Omics Genomics/transcriptomics/exposomics 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. The exposome concept refers to the totality of environmental exposures from conception onwards, and is a novel approach to studying the role of the environment in the development of lung disease in the Lifelines cohort. Furthermore, GRIAC is leading studies on gene expression profiling (transcriptomics) and collaborates internationally (University of British Columbia, Laval University) on studies linking gene expression to (epi)genetic variants (eqtl analysis). 7

8 Proteomics/lipidomics Proteomic and lipidomic 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. Molecular Medicine GRIAC is actively engaged in studies linking clinical outcomes to (molecular) pathophysiology. Often inspired by outcomes from omics studies, the functionality of genes and proteins in disease is studied using molecular approaches in cells and tissues from patients, in cell lines and in animal models. Molecular 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.this has enabled the use of RNAi and pharmacological modulation of membrane and nuclear receptors and signaling proteins in cells and tissue slices. Fundamental to this line of research is the exploration of intracellular pathways relevant for disease development. Clinical Medicine Patient centered research is at the heart of GRIAC. Our translational research approach included clinical and intervention studies in asthma and COPD. Moreover, several cohort studies, both regional as well as national, are lead by GRIAC investigators. The LifeLines cohort study is a unique asset for large scale epidemiological research into healthy ageing. Healthy ageing Healthy Aging 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 exposomics, lipidomics and 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. 8

9 Functional studies on gene variations in asthmatic and healthy individuals are ongoing, 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. The molecular basis of pathophysiology including airway (hyper)responsiveness, inflammation and remodeling in asthma and COPD is increasingly on the forefront of GRIAC research. For both asthma and COPD, we will gain better insight into the intricate interplay between tissue resident celss (epithelial cells, fibroblasts, smooth muscle cells) on one hand and their interaction with the extracellular matrix and different inflammatory cell types in the lung on the other. With the recognition that most tissue resident cells are highly plastic governed by complex interactions between multiple receptor systems and environmental changes, research will remain focused on unraveling the interactive mechanisms that determine inflammation and remodeling 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.. In addition, a new area of interest in this research line is tissue repair in COPD and the role of resident cells (including progenitor cells and structural cells of the alveoli) in this response. 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. 9

10 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 2014, 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 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 for PhD students to stay at 2 or 3 of these universities for an international PhD project. 10

11 The year 2014 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 The Bronchitis IX symposium entitled: Lungs: on the edge of health and aging, organized by GRIAC, was held from June in the UMCG in Groningen. There was an outstanding program with leading researchers in the field of obstructive pulmonary diseases. The presentations were highly interesting and followed by lively discussions. The organizing committee consisted of Dr. M. van de Berge, Prof. G.H. Koppelman, Dr. J.N.G. Oude Elberink, Prof. W. Timens and C. Verver. Following the Bronchitis symposium, the COST congress entitled Early origins of chronic lung disease: a Birds eye view on the ageing lung was organized on June 26 and 27, by Dr. R. Gosens and Dr. M.N. Hylkema. 102 registrants from 19 countries presented their work in scientific lectures or the poster session. The COST conference was preceded by the 2nd COST training school on Molecular Mechanisms of Ageing, also organized by Dr. R. Gosens and Dr. M.N. Hylkema. Thirteen participants originating from 6 countries registered for the summer school that consisted of workshops and scientific sessions. For some of the scientific sessions, participants benefited from the parallel Bronchitis conference. This not only ensured high quality speakers from all around the world, but also enabled close interaction with these researchers during breaks and at the poster session. In honor of the retirements of Prof. E.J. Duiverman and Prof. J.G.R. de Monchy farewell symposia were organized and in relation to the thesis defences of Dr. O.E.M. Savenije, Dr. D. Ierodiakonou, Dr. A. Oldenburger, and Dr. N.J. Goossens minisymposia were organized. Speakers at these symposia were all national and international leading researchers. Prof. H.M. Boezen organized, in collaboration with Dr. V. Schlünssen from the Aarhus University in Denmark, a hot topic symposium at the annual ERS-conference in Munich entitled: Determine personalized exposure and gene-environment interactions underlying chronic airway disease; the exposome and the epigenome. In March and April two information evenings for the general public entitled: the hereditary causes of asthma and COPD, were organized by Prof. D.S. Postma and Prof. G.H. Koppelman. These evenings were very well attended. In March, Dr. H. Oude Elberink organized a patient information day for patients with Mastocytosis. Prof. H.M. Boezen was elected chair of the ERS working group 6.4 of the ERS Epidemiology assembly genes and environment. Prof. H. Meurs and Prof. D.S. Postma were awarded as ERS honorary fellow (FERS). Prof. M. Schmidt was appointed as Teaching Director of the Groningen Research Institute of Pharmacy. 11

12 Prizes/Awards C.E. Boorsma, MSc won the NRS Chiesi Young Investigators award Dr. O.E.M. Savenije won the NRS Young Investigator SAB award for her thesis J. Saleh-Langenberg, MSc won an abstract award at the Food Allergy and Anaphylaxis meeting in Dublin L. Hesse, MSc won an award for the best poster presentation at the EAACI summer school E. van Dijk, MSc won the NRS Young Investigators Presentation award A.I.R. Spanjer, MSc obtained the Presentation Award of the Pharmacy Day, GRIP, Visitors Dr. C. Grainge, University of Southampton, Southampton, UK. January 9, Prof. Dr. M. Kabesch, University Hospital Regensburg, Regensburg, Germany. January 14, Prof. Dr. A. Bush, Imperial College, London, UK. January 14, Dr. S. Krauss-Etschmann, Helmholtz Center, Munich, Germany. February 4, Prof. Dr. S.T. Weiss, Channing lab, Harvard University, Boston, USA. March 3, Prof. Dr. J. Vestbo, University of Manchester, Manchester, UK. June 17, Prof. Dr. I. Sabroe, University of Sheffield, Sheffield, UK. September 2, Prof. Dr. T. Sigsgaard, Aarhus University, Denmark. September 23, Dr. G.A. Lockett, University of Southampton, Southampton, UK. October 7, Prof. Dr. M.C. Michel, University of Mainz, Germany. October 24, Prof. Dr. E. Klussmann, Max Delbrück Centrum für Molekulare Medizin (MDC), Berlin, Germany. October 24, Prof. Dr. W. Windisch, University of Witten/Herdecke, Köln, Germany, October 24, Prof. Dr. H. Maarsingh, Palm Beach Atlantic University, USA. October 30, Prof. Dr. L.C. Porto, Rio de Janeiro State University, Brazil. October 30, Prof. Dr. M. Peters-Golden, University of Michigan, Michigan, USA. October 30, Dr. A. Kruis, Leiden University Medical Center, Leiden, NL. November 18, Prof. Dr. D. Heederick, University of Utrecht, Utrecht, NL. December 16, Dr. J. Burgess, University of Sydney, Sydney, Australia. December 19,

13 Special Topics Special Topic 1 First results of the GENEVA study: Research on the genetics of food allergy in children C. Dorien van Ginkel, Gerard H. Koppelman, Ewoud A. Dubois. Departments of pediatric pulmonology and pediatric allergy. Food allergy is a potentially lethal disease and its prevalence is increasing in the Western world 1. Furthermore, food allergy reduces quality of life in patients to a greater extent than diabetes 2 and is strongly heritable 3. One study reported a 5-fold increase for the risk of peanut allergy for a child with a peanut allergic sibling or parents 4. Remarkably, only 1 in 2 children suspected to be food allergic and sensitised to food has a positive outcome of a double blind placebo-controlled food challenge (DBPCFC) and is thereby diagnosed as clinically reactive 5. We hypothesize that the genetic makeup of a child influences the difference between sensitisation with and without clinical reactivity to food. Specifically, we hypothesize that genes that contribute to skin epithelial integrity are associated with clinical food allergy. To study this hypothesis we use the database of the Food Challenge Unit of the Beatrix Children s Hospital of the UMCG which contains phenotypic information on food allergy as well as other atopic morbidities on over one thousand children who have been diagnosed by the gold standard test, the Double-Blind Placebo Controlled Food Challenge. This will be coupled to a DNA collection which will be expanded to include samples from 800 children and their parents. This will be achieved by collecting DNA from blood- and saliva samples. We will focus on candidate genes involved in epithelial integrity, and are aiming to perform a genome-wide association study in the future. The first results of the GENEVA study examined the association between food allergy and the filaggrin gene (FLG). The FLG gene is located on chromosome 1q21 and is part of the epidermal differentiation complex. These genes play an important role in skin barrier function since the filaggrin protein helps aggregate the epidermal cytoskeleton to form a protein-lipid barrier 6. An impaired barrier function may permit intact proteins to pass the barrier and to elicit an immune response. Loss of function (LOF) variants of the FLG gene result in a defective form of the filaggrin protein and have a prevalence of about 10% in Western populations 7. FLG LOF variants are associated with ichtyosis vulgaris, characterized by palmar hyperlinearity, keratosis pilaris and a fine white scale on the extremities. Ichtyosis vulgaris is strongly associated with atopy 8. Furthermore, the FLG LOF variants are strong risk factors for atopic dermatitis 9 and are associated with allergic rhinitis and sensitization 10. In children with atopic dermatitis, these variants are also associated with asthma 11. Other studies suggest a role of FLG in sensitization to foods and clinical food allergy 12. We showed that in high risk children suspected of being food allergic, those carrying one or more loss of function variants of the FLG gene are 1.5 times more likely to be clinically allergic as diagnosed by the DBPCFC than children carrying wild type alleles. This strong association is replicated using a family based design, confirming the robustness of this observation 13. We furthermore showed that genetic markers may be useful as an addition to clinical assessment in the diagnosis of food allergy

14 Figure 1. Prevalence of food allergy in subjects with and without FLG risk alleles. The genetic findings of FLG being an important gene for food allergy have led to the dualallergen exposure hypothesis for the pathogenesis of food allergy. This proposes that lowdose cutaneous exposure to food allergens triggers Th2 responses and IgE production by B cells while early oral exposure induces tolerance by stimulating regulatory T cells and Th1 cells 14. This hypothesis is supported by a study showing that epicutaneous exposure to peanut protein causes Th2-type immunity with high levels of peanut specific IgE and prevents the development of oral tolerance to peanut 15. A recent study showed that early life environmental exposure to peanuts, as measured by peanut in household dust, is indeed associated with peanut sensitization and allergy as confirmed by open food challenges, but only in children carrying FLG mutations 16. This percutaneous priming is also proposed for the role of FLG in asthma and rhinitis 10. In conclusion, we recently showed that in high risk children, loss of function variants of the filaggrin gene are associated with clinical food allergy, as diagnosed by the double blind placebo-controlled food challenges. The FLG gene is therefore likely to be important in the mechanism driving the difference between asymptomatic sensitisation and food allergy, most likely through an impaired skin barrier. Since children carrying one or more loss of function variants of the FLG gene are 1.5 times more likely to be food allergic, such genetic markers may be useful as an addition to clinical assessment in the diagnosis of food allergy. After completing the DNA collection, we hope to replicate the results of the filaggrin gene in a larger study population and study the effect and diagnostic role of the other candidate genes. Furthermore we will use the genome wide data of the LifeLines cohort in the search for new genetic variants associated with food allergy. The GENEVA study is supported by grants of the Nutricia Research Foundation and the JK de Cock stichting. References 1. Branum, A. M. & Lukacs, S. L. Food allergy among children in the United States. Pediatrics 124, (2009). 2. Flokstra-de Blok, B. M. J. et al. Health-related quality of life of food allergic patients: comparison with the general population and other diseases. Allergy 65, (2010). 3. Tsai, H.-J. et al. Familial aggregation of food allergy and sensitization to food allergens: a family-based study. Clin. Exp. Allergy 39, (2009). 14

15 4. Hourihane, J. O., Dean, T. P. & Warner, J. O. Peanut allergy in relation to heredity, maternal diet, and other atopic diseases: results of a questionnaire survey, skin prick testing, and food challenges. BMJ 313, (1996). 5. Van den Berg, M. E. et al. Parental eczema increases the risk of double-blind, placebo-controlled reactions to milk but not to egg, peanut or hazelnut. Int. Arch. Allergy Immunol. 158, (2012). 6. Sandilands, A., Sutherland, C., Irvine, A. D. & McLean, W. H. I. Filaggrin in the frontline: role in skin barrier function and disease. J. Cell Sci. 122, (2009). 7. Van den Oord, R. a H. M. & Sheikh, a. Filaggrin gene defects and risk of developing allergic sensitisation and allergic disorders: systematic review and meta-analysis. Bmj 339, b2433 b2433 (2009). 8. Smith, F. J. D. et al. Loss-of-function mutations in the gene encoding filaggrin cause ichthyosis vulgaris. Nat. Genet. 38, (2006). 9. Sandilands, A. et al. Comprehensive analysis of the gene encoding filaggrin uncovers prevalent and rare mutations in ichthyosis vulgaris and atopic eczema. Nat. Genet. 39, (2007). 10. Weidinger, S. et al. Filaggrin mutations, atopic eczema, hay fever, and asthma in children. J. Allergy Clin. Immunol. 121, e1 (2008). 11. Rodríguez, E. et al. Meta-analysis of filaggrin polymorphisms in eczema and asthma: robust risk factors in atopic disease. J. Allergy Clin. Immunol. 123, e7 (2009). 12. Brown, S. J. et al. Loss-of-function variants in the filaggrin gene are a significant risk factor for peanut allergy. J. Allergy Clin. Immunol. 127, (2011). 13. Van Ginkel, C. D. et al. Loss-of-function variants of the filaggrin gene are associated with clinical reactivity to foods. Allergy (2015). 14. Lack, G. Update on risk factors for food allergy. J. Allergy Clin. Immunol. 129, (2012). 15. Strid, J., Hourihane, J., Kimber, I., Callard, R. & Strobel, S. Epicutaneous exposure to peanut protein prevents oral tolerance and enhances allergic sensitization. Clin. Exp. Allergy 35, (2005). 16. Brough, H. a et al. Peanut allergy: Effect of environmental peanut exposure in children with filaggrin lossof-function mutations. J. Allergy Clin. Immunol. 134, e1 (2014). 15

16 Special Topic 2 New avenues for Epac in inflammation and tissue remodeling in COPD Anouk Oldenburger, Wim Timens, Herman Meurs, Harm Maarsingh, Martina Schmidt Departments of molecular pharmacology and pathology. Chronic obstructive pulmonary disease (COPD) is a chronic inflammatory disease of the airways and the lung parenchyma, further characterized by airway obstruction and remodeling (1). Symptoms are treated with glucocorticosteroids, anticholinergics, β 2 -agonists or phosphodiesterase (PDE)-4 inhibitors (2,3). Both β 2 -agonists and PDE4 inhibitors elevate the second messenger cyclic AMP (camp), although by distinct mechanisms (4,5). The two main effectors of camp are protein kinase A (PKA) and the exchange protein directly activated by camp (Epac), which consists of the two isoforms Epac1 and Epac2. Epac1 and Epac2: Inflammation An important cytokine that is increased in COPD is interleukin-8 (IL-8). Our data indicate that Epac and PKA decrease cigarette smoke extract (CSE)-induced IL-8 release by airway smooth muscle (ASM) cells, via inhibition of NF-κB and ERK signalling, respectively. Importantly, we also observed reduced Epac1 expression in lung tissue from COPD patients (6,7). To investigate the potential cause for the downregulation of Epac1, we studied micrornas (mirnas). MiRNAs are epigenetic regulators involved in fine-tuning of cellular activities by posttranscriptional repression of mrna. In COPD patients, mirna-7 is increased in serum (8). We investigated the potential interaction of Epac1 and mirna-7. CSE induced mirna-7 specifically in human ASM cells. In line, mirna-7 was increased in bronchial smooth muscle of COPD stage II patients isolated by laser dissection. Importantly, Epac1 expression is reduced in mirna-7 overexpressing human ASM cells (Figure 1). Our data implicate that upregulation of mirna-7 by cigarette smoke correlates with the downregulation of Epac1 in COPD (8). Both Epac1 and Epac2 seem to be implicated in the reduction of CSE-induced IL-8 release from human ASM cells. However, phospholipase Cε (PLCε), a direct effector of Epac, acts pro-inflammatory. To translate our findings on inflammation in vitro to the in vivo situation, Epac1-/-, Epac2-/- and PLCε-/- mice were exposed to cigarette smoke for 5 days. We demonstrated that compared to wild-type (WT) mice exposed to cigarette smoke, the number of total inflammatory cells, macrophages, and neutrophils as well as IL-6 release were lower in Epac2-/- mice, which was also the case for neutrophils and IL-6 in PLCε-/- mice (Figure 2, ref 9). Compared to WT mice exposed to cigarette smoke, the number of macrophages was reduced in Epac1-/- mice. Whereas, the numbers of lymphocytes, only present in low numbers in BALF of air-exposed WT mice, were increased in Epac1-/- mice. Together our data indicated that particularly Epac2 acts pro-inflammatory in vivo (9). Aberrant epithelial repair is also regarded as a pathophysiological feature of COPD. It is of interest that the A-kinase anchoring protein (AKAP) family member AKAP9 enhanced the endothelial barrier function in concert with Epac1. AKAPs compartmentalize cellular camp upon generation of multiprotein complexes. We found that CSE reduced the barrier function in human bronchial epithelial cells, a process accompanied by a reduced membrane expression of E-cadherin and AKAP9 (Figure 1). Silencing of AKAP9 reduced the functional epithelial barrier and prevented the ability of st-ht31, an inhibitor of AKAP-PKA interactions, to restore membrane localization of E-cadherin. Our data indicate that AKAP9 maintains the bronchial epithelial barrier function and may be important in the pathophysiology of COPD (10). 16

17 Altogether, Epac1 and Epac2 seem to be involved in cigarette smoke induced inflammation, although with a clear distinction regarding their relative contribution. Compartmentalization of camp driven by AKAP family members, may be responsible for the distinct biological effects of camp. Remodeling: Epac1 and Epac2 Figure 1: (A) In human bronchial epithelial cells, cigarette smoke extract reduces the barrier function (resistance)which is prevented by St-Ht31, a inhibitory peptide of AKAP-PKA interactions. In addition, the expression of both AKAP9 and E- cadherin is reduced by cigarette smoke extract (further details see ref 9). (B) In airway smooth muscle cells, cigarette smoke extract enhances mirna-7 levels. Overexpression of mirna-7 inhibits the protein expression of Epac1. In bronchial smooth muscle of COPD patients a upregulation of mirna-7 is observed. (C) Epac: implications for structural lung cell functioning(further details see ref 6). 17

18 Tissue remodeling is another feature of COPD and covers different characteristics of the disease such as mucus hypersecretion, airway fibrosis and emphysema. The majority of extracellular matrix (ECM) proteins, including collagens and fibronectin, are produced by fibroblasts. Alterations in the tightly controlled balance of production and degradation of ECM proteins causes structural changes in the lung such as emphysema, characterized by excessive degradation of parenchymal ECM, and (small) airway fibrosis characterized by excessive deposition of ECM proteins (1). Although a role for camp in the regulation of remodeling has been shown (4,5), the exact role of the camp effectors Epac and PKA on the different aspects of remodeling is not completely known. Interestingly, it has been reported that camp-elevating drugs reduce collagen I synthesis in human lung fibroblasts. A PDE inhibitor and a camp analog have been shown to reduce MMP9 and TIMP1 gene expression and activity in different cell types. CSE exposure of the human bronchial epithelial cell line 16HBE14o- leads to increase of MMP9 mrna and thereby of the MMP9/TIMP1 ratio. Induction of MMP9 mrna was reduced by specific PKA activation. Pro-MMP9 levels induced by CSE were reduced by the fenoterol, an effect specifically mimicked by pharmacological activation of PKA. PKA inhibition may enhance the MMP9/TIMP1 ratio and thereby reduce emphysema. In contrast, activation of PKA may reduce emphysema by a decrease of the MMP9/TIMP1 ratio (further details see ref 6). In addition, we tried to identify the role of Epac1 and Epac2 in remodeling processes in the lung by exposure of Epac1-/- and Epac2-/- mice to cigarette smoke. We demonstrated that Epac1-/- mice expressed higher levels of TGF-β1, collagen I and fibronectin. We propose that Epac1, but not Epac2, acts anti-fibrotic. Concerning mucus hypersecretion, Epac1-/- and Epac2-/- were characterized by a constitutively higher expression of MUC5AC mrna at basal level (Figure 2). We observed that goblet cells tended to be increased in Epac2-/- and PLCε-/- mice, whereas primarily Epac1-/- mice tended to stain positive for the inducer of goblet cell differentiation SPDEF (9). Overall, we identified distinct roles of Epac1 and Epac2 in remodeling processes. This suggest that Epac1 alone controls remodeling. In contrast, both Epac1 and Epac2 seem to control MUC5AC (9). Similar as for inflammation, compartmentalization of Epac by AKAPs will most likely define a potential role of AKAP-dependent compartmentalization of camp in remodeling processes. 18

19 Figure 2: (A) In an acute mouse model of cigarette smoke exposure, Epac2, possibly via PLCε, enhances neutrophils (left panel). Epac1 bears the capacity to reduce the deposition of collagen I in lung tissue (right panel). (B) Both Epac1 and Epac2 regulate mucus hypersecretion in mice. (C) Epac1 and Epac2: implications in inflammation and remodelling in vivo. Epac2 contributes to cigarette smoke induced-increase in macrophages and IL-1β release. Possibly via PLCε, Epac2 also enhances neutrophils and IL-6 release. Epac1 inhibits collagen I and fibronectin deposition. Both Epac1 and Epac2 play an inhibitory role in mucus hypersecretion. For further details see refs 6, 9. 19

20 Conclusions These studies implicate an important role for camp in COPD. We defined distinct roles of Epac1 and Epac2 in inflammation and remodeling. In an acute model of cigarette smoke exposure in mice, we unraveled pro-inflammatory actions of Epac2. We showed that Epac1 alone is able to reduce remodeling, whereas for the reduction of MUC5AC a concerted action with Epac2 seemed to be required. Based on our findings, the development of selective Epac1 and Epac2 activators and/or inhibitors could be of additive value to alleviate symptoms of COPD. Targeting of these selective Epac activators or inhibitors to specific areas in the lung represents a future goal. Research into compartmentalization of camp by AKAPs should be another subject of future research. Acknowledgement This work was supported by a Long Fonds (grant ) and a Rosalind Franklin Fellowship from the University of Groningen to Martina Schmidt. Harm Maarsingh was supported by the Schering-Plough Research Institute (Oss, The Netherlands). References 1. Hogg JC, Timens W: The pathology of chronic obstructive pulmonary disease. Annual Rev Pathol 2009, 4, Hoonhorst SJ, Ten Hacken NH, Vonk JM, Timens W, Hiemstra PS, Lapperre TS, Sterk PJ, Postma DS: Steroid resistance in COPD? overlap and differential anti-inflammatory effects in smokers and ex-smokers. PloS One 2014, 9, e Meurs H, Oenema TA, Kistemaker LE, Gosens R: A new perspective on muscarinic receptor antagonism in obstructive airways diseases. Curr Opin Pharmacol 2013, 13, Oldenburger A., Maarsingh H, Schmidt M: Multiple facets of camp signalling and physiological impact: CAMP compartmentalization in the lung. Pharmaceuticals 2012, 5, Dekkers BG, Racke K, Schmidt M: Distinct PKA and epac compartmentalization in airway function and plasticity. Pharmacol Therap 2013, 137, Oldenburger A: New avenues for Epac in inflammation and tissue remodelling in COPD. Thesis 2014, University Groningen. 7. Oldenburger A, Roscioni SS, Jansen E, Menzen MH, Halayko AJ, Timens W, Meurs H, Maarsingh H, Schmidt M: Potential anti-inflammatory role of the camp effectors Epac and PKA: implications in chronic obstructive pulmonary disease. PLoS ONE 2012, 7, e Oldenburger A, van Basten B, Kooistra W, Meurs H, Maarsingh H, Krenning G, Timens W, Schmidt M: Interaction between Epac1 and mirna-7 in airway smooth muscle cells. Naunyn Schmiedebergs Arch Pharmacol 2014, 387, Oldenburger A, Timens W, Bos S, Smit M, Smrcka AV, Laurent A, Cao J, Hylkema M, Meurs H, Maarsingh H, Lezoualc'h F, Schmidt M: Epac1 and Epac2 are differentially involved in inflammatory and remodeling processes induced by cigarette smoke. FASEB J 2014, 28, Oldenburger A, Poppinga WJ, Kos F, de Bruin HG, Rijks WJ, Heijink IH, Timens W, Meurs H, Maarsingh H, Schmidt M: A-kinase anchoring proteins contribute to the loss of E-cadherin and bronchial epithelial barrier by cigarette smoke. Am J Physiol 2014, 15, 306, C

21 Special Topic 3 Occupational pesticide exposure increases risk for COPD Kim de Jong, Judith M. Vonk, Dirkje S. Postma, H. Marike Boezen Departments of epidemiology and pulmonology Chronic obstructive pulmonary disease (COPD) is characterized by persistent and often progressive airflow obstruction caused by and abnormal inflammatory response to noxious particles and gases. Tobacco smoking is considered to be the main risk factor for COPD, yet a substantial proportion of 15 20% of all cases has been attributed to occupational exposures (1), with proportions up to 30% in never smokers (2). Because occupational exposures are common, yet also potentially modifiable contributors to the global burden of COPD, it is important to determine which occupational factors drive the development of this disease. Within two Dutch general populations based cohorts, i.e. the LifeLines cohort study and the Vlagtwedde-Vlaardingen study, we estimated occupational exposure to amongst others pesticides using a job exposure matrix (JEM). The JEM classified subjects based on ISCO- 88 job codes into no, low and high exposure categories (0/1/2). First, we showed cross-sectional associations between pesticide exposure and lower levels of FEV 1 and FEV 1 /FVC in 11,851 individuals from the LifeLines cohort study and 2,364 subjects included in the last survey (1989/1990) of the Vlagtwedde-Vlaardingen cohort (see figure 1 for associations in LifeLines). Subjects with high levels of exposure to pesticides had also an increased risk for COPD gold stage 2 and higher (OR = 1.95 ( ) in LifeLines and OR = 1.78 ( ) in Vlagtwedde-Vlaardingen) (3). There were no consistent associations between exposure to pesticides and the FEF 25-75%, a marker of small airways obstruction (4). Figure 1. Cross-sectional associations between occupational pesticide exposure and the level of FEV 1 (A) and FEV 1/FVC (B), for he whole group and stratified according to smoking status (never/ever smoker) in the LifeLines cohort study. Associations with FEV 1 were significantly stronger (p<0.05) for ever compared to never smokers. Additionally within 12,772 observations from 2,527 subjects assessed between 1965 and 1990 in the Vlagtwedde-Vlaardingen cohort we found that subjects with high pesticide exposure had an accelerated decline in FEV 1 and FEV 1 %IVC (figure 2) (5). Especially within smokers these implied substantial losses of lung function, for example smokers with high 21