From Department of Medicine Solna Translational Immunology Unit Karolinska Institutet, Stockholm, Sweden Epigenetic Mechanisms of Asthma and Allergy Nathalie Acevedo Stockholm 2015 All previously published papers were reproduced under the Creative Commons Attribution license (CC-BY) and (CC-BY-NC) Published by Karolinska Institutet Printed by E-print © Nathalie Acevedo, 2015 ISBN 978-91-7549-830-0 Epigenetic Mechanisms of Asthma and Allergy THESIS FOR DOCTORAL DEGREE (Ph.D.) By Nathalie Acevedo Caballero Principal Supervisor: Opponent: Professor Annika Scheynius Professor John Holloway Karolinska Institutet University of Southampton, UK Department of Medicine Solna Faculty of Medicine Division of Translational Immunology Co-supervisor(s): Examination Board: Professor Juha Kere Associate Professor Maja Jagodic Karolinska Institutet Karolinska Institutet Department of Biosciences and Nutrition Department of Clinical Neuroscience Division of Molecular Genetics Associate Professor Cilla Söderhäll Professor Claes Wadelius Karolinska Institutet Uppsala University Department of Biosciences and Nutrition Department of Immunology, Division of Molecular Genetics Genetics and Pathology Associate Professor Lennart Nilsson Linköping University Department of Clinical and Experimental Medicine 1 2 A Luis Caraballo, mentor y amigo cuyo ejemplo y enseñanzas fueron esenciales para iniciar mis estudios de doctorado "Whether men will be able to survive the changes of environment that their own skill has brought about is an open question. If the answer is the affirmative, it will be known some day; if not, not" - B. Russell (1955) 3 4 ABSTRACT Allergic diseases such as atopic eczema (AE) and asthma are chronic inflammatory disorders in which the regulation of the immune system is altered, in concert with disease-specific defects in target organs. Their inception requires gene-environment interactions but the molecular mechanisms contributing to the pathogenesis remain unclear. During the last decades there was a dramatic increase in the prevalence of allergic diseases worldwide and several environmental conditions have been implicated (e.g. pollution, changes in lifestyle, reduced biodiversity). These exposures are supposed to affect leukocyte function, possibly through epigenetic mechanisms, which are the ultimate connection between the cell and the environment. The overall aim of this thesis was to elucidate the DNA methylation signatures as an epigenetic marker associated to cell lineage (I) and increasing age (IV) in primary blood leukocytes, and to compare DNA methylation (II, III) and miRNA expression (II) between healthy controls (HC) and patients with AE and asthma in connection with changes in mRNA expression. In study I, we found a particular DNA methylation landscape for each population of blood leukocytes that discriminates their lineage and that lymphoid cells are more methylated than myeloid cells. At the DNA methylation level, CD19+ B cells are the most different among all leukocytes. This study revealed that differences in cell composition may bias DNA methylation analyses conducted in DNA extracted from whole blood and provided a catalog of lineage-specific CpG sites. In study II, we compared DNA methylation and miRNA levels between AE patients and HC in four T cell populations isolated from peripheral blood. We discovered that circulating skin-homing memory CD4+CLA+ T cells isolated from AE patients have significant DNA methylation differences in 49 CpG sites and differential expression of 16 miRNAs compared to HC. Integrative data analyses with the differentially expressed mRNAs revealed that epigenetic signatures in AE patients are connected to the IL-10/IL-13 pathway. In study III, we analyzed the relation between genotype and epigenotype in the GSDMB/ORMDL3 asthma-susceptibility locus. We found significant differences in DNA methylation between children with controlled persistent asthma and HC. Methylation levels in three of these differentially methylated CpG sites correlate with the mRNA levels of ORMDL3. Causal inference tests revealed that both, single nucleotide polymorphisms and non-polymorphic CpG sites have independent effects on ORMDL3 mRNA expression. We also discovered that CD8+ T cells contain a differentially methylated region in the 5’UTR of ORMDL3 that is less methylated compared to other leukocyte subtypes, suggesting that methylation differences in ORMDL3 might promote lymphocyte driven inflammation. In study IV, we performed a pilot genome-wide DNA methylation analysis in whole blood, aiming to elucidate which genes had longitudinal changes in DNA methylation between 3 and 60 months after birth in healthy children. After filtering by lineage-CpG sites, we found 330 age-methylated CpG sites and 464 age-demethylated CpG sites that reflect the immunological window and are located on genes encoding for cytokines and chromatin remodelers. In summary, we discovered DNA methylation signatures in blood leukocytes associated to terminal differentiation and development, and demonstrated that compared to HC, patients suffering of AE and asthma have differences in DNA methylation and miRNA levels that might have functional impact on mRNA expression. The findings of this thesis provide new insights on genomic loci containing epigenetic differences between allergic patients and HC, that may be studied in the future as candidates for the development of preventive strategies, disease biomarkers or novel pharmacological therapies. 5 LIST OF SCIENTIFIC PAPERS I. Reinius LE, Acevedo N, Joerink M, Pershagen G, Dahlén SE, Greco D, Söderhäll C, Scheynius A, Kere J. Differential DNA methylation in purified human blood cells: implications for cell lineage and studies on disease susceptibility. PLoS One. 2012;7(7):e41361. II. Acevedo N, Bruhn S, Andersson A, Katayama S, Krjutshkov K, Wickberg G, Lundeberg L, Söderhäll C, Kere J, Greco D, Scheynius A. Differential DNA methylation and miRNA expression in skin-homing CD4+CLA+ T cells of atopic eczema patients. Manuscript. III. Acevedo N, Reinius LE, Greco D, Gref A, Orsmark-Pietras C, Persson H, Pershagen G, Hedlin G, Melén E, Scheynius A, Kere J, Söderhäll C. Risk of childhood asthma is associated with CpG site polymorphisms, regional DNA methylation and mRNA levels at the GSDMB/ORMDL3 locus. Human Molecular Genetics. 2015; 24(3):875-890. IV. Acevedo N, Reinius LE, Vitezic M, Fortino V, Söderhäll C, Honkanen H, Veijola R, Simell O, Toppari J, Ilonen J, Knip M, Scheynius A, Hyöty H, Greco D, Kere J. Age-associated DNA methylation changes in immune genes, histone modifiers and chromatin remodeling factors within 5 years after birth in human blood leukocytes. Under revision in Clinical Epigenetics. 6 Related publications not included in this thesis Liu Y, Aryee MJ, Padyukov L, Fallin MD, Hesselberg E, Runarsson A, Reinius L, Acevedo N, Taub M, Ronninger M, Shchetynsky K, Scheynius A, Kere J, Alfredsson L, Klareskog L, Ekström TJ, Feinberg AP. Epigenome-wide association data implicate DNA methylation as an intermediary of genetic risk in rheumatoid arthritis. Nat Biotechnol. 2013;31(2):142-7. Reinius LE, Gref A, Sääf A, Acevedo N, Joerink M, Kupczyk M, D'Amato M, Bergström A, Melén E, Scheynius A, Dahlén SE; BIOAIR Study Group, Pershagen G, Söderhäll C, Kere J. DNA methylation in the Neuropeptide S Receptor 1 (NPSR1) promoter in relation to asthma and environmental factors. PLoS One. 2013;8(1):e53877. Acevedo N, Sääf A, Söderhäll C, Melén E, Mandelin J, Pietras CO, Ezer S, Karisola P, Vendelin J, Gennäs GB, Yli-Kauhaluoma J, Alenius H, von Mutius E, Doekes G, Braun- Fahrländer C, Riedler J, van Hage M, D'Amato M, Scheynius A, Pershagen G, Kere J, Pulkkinen V. Interaction between retinoid acid receptor-related orphan receptor alpha (RORA) and neuropeptide S receptor 1 (NPSR1) in asthma. PLoS One. 2013;8(4):e60111. Bornelöv S, Sääf A, Melén E, Bergström A, Torabi Moghadam B, Pulkkinen V, Acevedo N, Orsmark Pietras C, Ege M, Braun-Fahrländer C, Riedler J, Doekes G, Kabesch M, van Hage M, Kere J, Scheynius A, Söderhäll C, Pershagen G, Komorowski J. Rule-based models of the interplay between genetic and environmental factors in childhood allergy. PLoS One. 2013;8(11):e80080. Fuertes E, Söderhäll C, Acevedo N, Beckere A, Brauer M, Chan-Yeung M, Dijk N, Heinrich J, de Jongste J, Koppelman G, Postma D, Kere J, Kozyrskyj A, Pershagen G, Sandford A, Standl M, Tiesler C, Waldenberger M, Westman M, Carlsten C, Melén E. Associations between the 17q21 region and allergic rhinitis in five birth cohorts. J Allergy Clin Immunol. Epub pii: S0091-6749(14)01188-9. 7 8