Airway epithelial cell cilia transcriptomic dysregulation is associated with the inflammatory phenotype in asthma

Asthma is a heterogeneous disease in terms of phenotypes and endotypes, characterized by airway remodelling with epithelial dysfunction, including alterations of the mucociliary clearance.1 Previous studies identified motile cilia dysfunction in severe asthma.2, 3 Considering the importance of both motile and primary cilia in structural and functional epithelium integrity,4, 5 we investigated the associations between cilia dysregulation and asthma phenotypes and endotypes using a transcriptomic signature approach. We explored the transcriptomic signature of the 879 cilia-associated genes in the bronchial airway epithelial cells of 141 patients at a stable state from the U-BIOPRED study (61 severe asthma (SA), 36 mild/moderate asthma (MMA) and 44 healthy subjects). All participants in the UBIOPRED cohort gave written and signed informed consent.6 The clustering of patients according to the differentially expressed genes (DEGs; false discovery rate correction applied) identified two groups, one of which with high cilia dysregulation (Appendix S1) being enriched in asthmatics (51.4% SA, 34.3% MMA vs 40.6% SA, 22.6% MMA, p = .04), and characterized by high total IgE (p = .022), high eosinophil counts in blood (p = .001) and in sputum (p = .03), and low FEV1 (p = .02) (cilia dysregulated group) (Table S1, Figure S1). To further analyse the associations between cilia dysregulation and SA, we next focused on the SA population of both groups, comparing the 18 SA with high cilia dysregulation (SAd) with the other 43 SA. The SAd group was characterized by older age at diagnosis (p = .016), lower BMI (p = .010), less frequent GERD (p = .023), a more severe airway obstruction (p = .017) and a trend towards more frequent male gender (p = .059, Table 1). Exacerbation history and treatment did not differ between the groups. The SAd group was further characterized by high blood eosinophil (p = .023) and basophil counts (p = .011, Figure 1A); high sputum eosinophil count (p = .013, Table 1), less frequent paucigranulocytic sputum phenotype (0/7 vs 9/18, p = .024); higher concentrations of the T2-related cytokines IL13, CCL17 and MIP1b in the plasma (respectively 1.3 ± 1.2 pg/mL vs 0.8 ± 0.6 pg/mL, p = .038; 147 ± 108 pg/mL vs 76 ± 62 pg/mL, p = .001; 67 ± 20 pg/mL vs 54 ± 20 pg/mL, p = .011; Figure 1B), and more abundant macrophages in the bronchial submucosa (6.14 ± 6.05 cells mm−2 vs 3.40 ± 4.54 cells mm−2, p = .040) and in the epithelium (1.43 ± 2.42 cells mm−1 vs 0.12 ± 0.53 cells mm−1, p = .0026; Figure 1C). Epithelial remodelling features including mucus immunostaining did not significantly differ between groups. Thus, the alteration of the cilia-associated genes distinguished severe asthmatics featuring adult-onset, fixed airflow obstruction and T2-high eosinophilic asthma. We next investigated the global transcriptomic signature of the SAd compared with the other SA, to identify cellular mechanisms associated with cilia dysregulation. The SAd group exhibited 4117 DEGs including 315 cilia-associated DEGs compared with the SA group (Figure 1D). The top 100 DEGs, including three cilia-associated genes (Prokineticin 2, PROK2; Pleckstrin Homology Like Domain Family B Member 2, PHLDB2; and neuropilin-1, NRP1), were almost exclusively upregulated in SAd (Figure 1D,E). The analysis of the biological processes using Reactome database identified the immune system pathway (strength = 0.64, FDR = 1.61 × 10−14) (Figure S2). Protein–protein interaction network analysis identified cytokine–cytokine receptor interaction (strength = 0.93, FDR = 8.05 × 10−6) and NF-κB signalling pathway (strength = 1.14, FDR = 1.8 × 10−4) as principal KEGG pathways (Figure S3). Our original experimental approach, although hypothesis generating, and of cross-sectional design with uncertainty regarding the long-term stability of the endotype/phenotype we describe, provided clues of an intriguing, yet unreported link between cilia regulation and inflammation in asthma. Uncontrolled inflammatory mediators may initiate and extend airway remodelling.7 We hypothesized that an initial dysregulation of cilia could initiate and extend inflammation in a double whammy. Investigating the origin of altered cilia structure and function in light of the inflammatory response will pave the way towards novel therapeutic strategies in asthma. In conclusion, we identified a novel endotype of severe asthma centred on dysregulation of the cilia-associated gene signature and unveiled a panel of genes that may orchestrate or maintain epithelial remodelling in asthma via inflammatory modulation. Although our findings rely on a robust European cohort, additional experimental approaches are needed to elucidate the implications for asthma phenotyping. The U-BIOPRED consortium wishes to acknowledge the help and expertise of the following individuals and groups without whom the study would not have been possible: I.M. Adcock, National Heart and Lung Institute, Imperial College, London, UK; H. Ahmed, European Institute for Systems Biology and Medicine, CNRS-ENS-UCBL-INSERM, Lyon, France; C. Auffray, European Institute for Systems Biology and Medicine, CNRS-ENS-UCBL-INSERM, Lyon, France; P. Bakke, Dept of Clinical Science, University of Bergen, Bergen, Norway; A.T. Banssal, Acclarogen Ltd, St John’s Innovation Centre, Cambridge, UK; F. Baribaud, Janssen R&D, USA; S. Bates, Respiratory Therapeutic Unit, GSK, London, UK; E.H. Bel, Academic Medical Centre, University of Amsterdam, Amsterdam, The Netherlands; J. Bigler, previously Amgen Inc.; H. Bisgaard, COPSAC, Copenhagen Prospective Studies on Asthma in Childhood, Herlev and Gentofte Hospital, University of Copenhagen, Copenhagen, Denmark; M.J. Boedigheimer, Amgen Inc., Thousand Oaks, USA; K. Bønnelykke, COPSAC, Copenhagen Prospective Studies on Asthma in Childhood, Herlev and Gentofte Hospital, University of Copenhagen, Copenhagen, Denmark; J. Brandsma, University of Southampton, Southampton, UK; P. Brinkman, Academic Medical Centre, University of Amsterdam, Amsterdam, The Netherlands; E. Bucchioni, Chiesi Pharmaceuticals SPA, Parma, Italy; D. Burg, Centre for Proteomic Research, Institute for Life Sciences, University of Southampton, Southampton, UK; A. Bush, National Heart and Lung Institute, Imperial College, London, UK; Royal Brompton and Harefield NHS trust, UK; M. Caruso, Dept Clinical and Experimental Medicine, University of Catania, Catania, Italy; A. Chaiboonchoe, European Institute for Systems Biology and Medicine, CNRS-ENS-UCBL-INSERM, Lyon, France; P. Chanez, Assistance publique des Hôpitaux de Marseille - Clinique des bronches, allergies et sommeil, Aix Marseille Université, Marseille, France; K.F. Chung, National Heart and Lung Institute, Imperial College, London, UK; C.H. Compton, Respiratory Therapeutic Unit, GSK, London, UK; J. Corfield, Areteva R&D, Nottingham, UK; A. D’Amico, University of Rome ‘Tor Vergata’, Rome Italy; S.E. Dahlen, Centre for Allergy Research, Karolinska Institutet, Stockholm, Sweden; B. De Meulder, European Institute for Systems Biology and Medicine, CNRS-ENS-UCBL-INSERM, Lyon, France; R. Djukanovic, NIHR Southampton Respiratory Biomedical Research Unit and Clinical and Experimental Sciences, Southampton, UK; V.J. Erpenbeck, Translational Medicine, Respiratory Profiling, Novartis Institutes for Biomedical Research, Basel, Switzerland; D. Erzen, Boehringer Ingelheim Pharma GmbH & Co. KG, Biberach, Germany; K. Fichtner, Boehringer Ingelheim Pharma GmbH & Co. KG, Biberach, Germany; N. Fitch, BioSci Consulting, Maasmechelen, Belgium; L.J. Fleming, National Heart and Lung Institute, Imperial College, London, UK; Royal Brompton and Harefield NHS trust, UK; E. Formaggio, previously CROMSOURCE, Verona, Italy; S.J. Fowler, Centre for Respiratory Medicine and Allergy, Institute of Inflammation and Repair, University of Manchester and University Hospital of South Manchester, Manchester Academic Health Sciences Centre, Manchester, UK; U. Frey, University Children’s Hospital, Basel, Switzerland; M. Gahlemann, Boehringer Ingelheim (Schweiz) GmbH, Basel, Switzerland; T. Geiser, Dept of Respiratory Medicine, University Hospital Bern, Switzerland; Y. Guo, Data Science Institute, Imperial College, London, UK; S. Hashimoto, Academic Medical Centre, University of Amsterdam, Amsterdam, The Netherlands; J. Haughney, International Primary Care Respiratory Group, Aberdeen, UK; G. Hedlin, Dept Women’s and Children’s Health & Centre for Allergy Research, Karolinska Institutet, Stockholm, Sweden; P.W. Hekking, Academic Medical Centre, University of Amsterdam, Amsterdam, The Netherlands; T. Higenbottam, Allergy Therapeutics, West Sussex, UK; J.M. Hohlfeld, Fraunhofer Institute for Toxicology and Experimental Medicine, Hannover, Germany; C. Holweg, Respiratory and Allergy Diseases, Genentech, San Francisco, USA; I. Horváth, Semmelweis University, Budapest, Hungary; P. Howarth, NIHR Southampton Respiratory Biomedical Research Unit, Clinical and Experimental Sciences and Human Development and Health, Southampton, UK; A.J. James, Centre for Allergy Research, Karolinska Institutet, Stockholm, Sweden; R. Knowles, Arachos Pharma, Stevenge, UK; A.J. Knox, Respiratory Research Unit, University of Nottingham, Nottingham, UK; N. Krug, Fraunhofer Institute for Toxicology and Experimental Medicine, Hannover, Germany; D. Lefaudeux, European Institute for Systems Biology and Medicine, CNRS-ENS-UCBL-INSERM, Lyon, France; M.J. Loza, Janssen R&D, USA; R. Lutter, Academic Medical Centre, University of Amsterdam, Amsterdam, The Netherlands; A. Manta, Roche Diagnostics GmbH, Mannheim, Germany; S. Masefield, European Lung Foundation, Sheffield, UK; J.G. Matthews, Respiratory and Allergy Diseases, Genentech, San Francisco, USA; A. Mazein, European Institute for Systems Biology and Medicine, CNRS-ENS-UCBL-INSERM, Lyon, France; A. Meiser, Data Science Institute, Imperial College, London, UK; R.J.M. Middelveld, Centre for Allergy Research, Karolinska Institutet, Stockholm, Sweden; M. Miralpeix, Almirall, Barcelona, Spain; P. Montuschi, Università Cattolica del Sacro Cuore, Milan, Italy; N. Mores, Università Cattolica del Sacro Cuore, Milan, Italy; C.S. Murray, Centre for Respiratory Medicine and Allergy, Institute of Inflammation and Repair, University of Manchester and University Hospital of South Manchester, Manchester Academic Health Sciences Centre, Manchester, UK; J. Musial, Dept of Medicine, Jagiellonian University Medical College, Krakow, Poland; D. Myles, Respiratory Therapeutic Unit, GSK, London, UK; L. Pahus, Assistance publique des Hôpitaux de Marseille, Clinique des bronches, allergies et sommeil, Espace Éthique Méditerranéen, Aix-Marseille Université, Marseille, France; I. Pandis, Data Science Institute, Imperial College, London, UK; S. Pavlidis, National Heart and Lung Institute, Imperial College, London, UK; P. Powell, European Lung Foundation, Sheffield, UK; G. Praticò, CROMSOURCE, Verona, Italy; M. Puig Valls, CROMSOURCE, Barcelona, Spain; N. Rao, Janssen R&D, USA; J. Riley, Respiratory Therapeutic Unit, GSK, London, UK; A. Roberts, Asthma UK, London, UK; G. Roberts, NIHR Southampton Respiratory Biomedical Research Unit, Clinical and Experimental Sciences and Human Development and Health, Southampton, UK; A. Rowe, Janssen R&D, UK; T. Sandström, Dept of Public Health and Clinical Medicine, Umeå University, Umeå, Sweden; W. Seibold, Boehringer Ingelheim Pharma GmbH, Biberach, Germany; A. Selby, NIHR Southampton Respiratory Biomedical Research Unit, Clinical and Experimental Sciences and Human Development and Health, Southampton, UK; D.E. Shaw, Respiratory Research Unit, University of Nottingham, UK; R. Sigmund, Boehringer Ingelheim Pharma GmbH & Co. KG, Biberach, Germany; F. Singer, University Children’s Hospital, Zurich, Switzerland; P.J. Skipp, Centre for Proteomic Research, Institute for Life Sciences, University of Southampton, Southampton, UK; A.R. Sousa, Respiratory Therapeutic Unit, GSK, London, UK; P.J. Sterk, Academic Medical Centre, University of Amsterdam, Amsterdam, The Netherlands; K. Sun, Data Science Institute, Imperial College, London, UK; B. Thornton, MSD, USA; W.M. van Aalderen, Academic Medical Centre, University of Amsterdam, Amsterdam, The Netherlands; M. van Geest, AstraZeneca, Mölndal, Sweden; J. Vestbo, Centre for Respiratory Medicine and Allergy, Institute of Inflammation and Repair, University of Manchester and University Hospital of South Manchester, Manchester Academic Health Sciences Centre, Manchester, UK; N.H. Vissing, COPSAC, Copenhagen Prospective Studies on Asthma in Childhood, Herlev and Gentofte Hospital, University of Copenhagen, Copenhagen, Denmark; A.H. Wagener, Academic Medical Center Amsterdam, Amsterdam, The Netherlands; S.S. Wagers, BioSci Consulting, Maasmechelen, Belgium; Z. Weiszhart, Semmelweis University, Budapest, Hungary; C.E. Wheelock, Centre for Allergy Research, Karolinska Institutet, Stockholm, Sweden; S.J. Wilson, Histochemistry Research Unit, Faculty of Medicine, University of Southampton, Southampton, UK. Additional contributors: Antonios Aliprantis, Merck Research Laboratories, Boston, USA; David Allen, North West Severe Asthma Network, Pennine Acute Hospital NHS Trust, UK; Kjell Alving, Dept Women’s & Children’s Health, Uppsala University, Uppsala, Sweden; P. Badorrek, Fraunhofer ITEM, Hannover, Germany; David Balgoma, Centre for Allergy Research, Karolinska Institutet, Stockholm, Sweden; S. Ballereau, European institute for Systems Biology and Medicine, University of Lyon, France; Clair Barber, NIHR Southampton Respiratory Biomedical Research Unit and Clinical and Experimental Sciences, Southampton, UK; Manohara Kanangana Batuwitage, Data Science Institute, Imperial College, London, UK; An Bautmans, MSD, Brussels, Belgium; A. Bedding, Roche Diagnostics GmbH, Mannheim, Germany; A.F. Behndig, Umeå University, Umea, Sweden; Jorge Beleta, Almirall S.A., Barcelona, Spain; A. Berglind, MSD, Brussels, Belgium; A. Berton, AstraZeneca, Mölndal, Sweden; Grazyna Bochenek, II Dept of Internal Medicine, Jagiellonian University Medical College, Krakow, Poland; Armin Braun, Fraunhofer Institute for Toxicology and Experimental Medicine, Hannover, Germany; D. Campagna, Dept of Clinical and Experimental Medicine, University of Catania, Catania, Italy; Leon Carayannopoulos, previously at MSD, USA; C. Casaulta, University Children’s Hospital of Bern, Switzerland; Romanas Chaleckis, Centre of Allergy Research, Karolinska Institutet, Stockholm, Sweden; B. Dahlén, Karolinska University Hospital & Centre for Allergy Research, Karolinska Institutet, Stockholm, Sweden; Timothy Davison, Janssen R&D, USA; Jorge De Alba, Almirall S.A., Barcelona, Spain; Inge De Lepeleire, MSD, Brussels, Belgium; Tamara Dekker, Academic Medical Centre, University of Amsterdam, Amsterdam, The Netherlands; Ingrid Delin, Centre for Allergy Research, Karolinska Institutet, Stockholm, Sweden; P. Dennison, NIHR Southampton Respiratory Biomedical Research Unit, Clinical and Experimental Sciences, NIHR-Wellcome Trust Clinical Research Facility, Faculty of Medicine, University of Southampton, Southampton, UK; Annemiek Dijkhuis, Academic Medical Centre, University of Amsterdam, Amsterdam, The Netherlands; Paul Dodson, AstraZeneca, Mölndal, Sweden; Aleksandra Draper, BioSci Consulting, Maasmechelen, Belgium; K. Dyson, CROMSOURCE, Stirling, UK; Jessica Edwards, Asthma UK, London, UK; L. El Hadjam, European Institute for Systems Biology and Medicine, University of Lyon, France; Rosalia Emma, Dept of Clinical and Experimental Medicine, University of Catania, Catania, Italy; Magnus Ericsson, Karolinska University Hospital, Stockholm, Sweden; C. Faulenbach, Fraunhofer ITEM, Hannover, Germany; Breda Flood, European Federation of Allergy and Airways Diseases Patient’s Associations, Brussels, Belgium; G. Galffy, Semmelweis University, Budapest, Hungary; Hector Gallart, Centre for Allergy Research, Karolinska Institutet, Stockholm, Sweden; D. Garissi, Global Head Clinical Research Division, CROMSOURCE, Italy; J. Gent, Royal Brompton and Harefield NHS Foundation Trust, London, UK; M. Gerhardsson de Verdier, AstraZeneca; Mölndal, Sweden; D. Gibeon, National Heart and Lung Institute, Imperial College, London, UK; Cristina Gomez, Centre for Allergy Research, Karolinska Institutet, Stockholm, Sweden; Kerry Gove, NIHR Southampton Respiratory Biomedical Research Unit and Clinical and Experimental Sciences, Southampton, UK; Neil Gozzard, UCB, Slough, UK; E. Guillmant-Farry, Royal Brompton Hospital, London, UK; E. Henriksson, Karolinska University Hospital & Karolinska Institutet, Stockholm, Sweden; Lorraine Hewitt, NIHR Southampton Respiratory Biomedical Research Unit, Southampton, UK; U. Hoda, Imperial College, London, UK; Richard Hu, Amgen Inc. Thousand Oaks, USA; Sile Hu, National Heart and Lung Institute, Imperial College, London, UK; X. Hu, Amgen Inc., Thousand Oaks, USA; E. Jeyasingham, UK Clinical Operations, GSK, Stockley Park, UK; K. Johnson, Centre for Respiratory Medicine and Allergy, Institute of Inflammation and repair, University Hospital of South Manchester, NHS Foundation Trust, Manchester, UK; N. Jullian, European Institute for Systems Biology and Medicine, University of Lyon, France; Juliette Kamphuis, Longfonds, Amersfoort, The Netherlands; Erika J. Kennington, Asthma UK, London, UK; Dyson Kerry, CromSource, Stirling, UK; G. Kerry, Centre for Respiratory Medicine and Allergy, Institute of Inflammation and Repair, University Hospital of South Manchester, NHS Foundation Trust, Manchester, UK; M. Klüglich, Boehringer Ingelheim Pharma GmbH & Co. KG, Biberach, Germany; Hugo Knobel, Philips Research Laboratories, Eindhoven, The Netherlands; Johan Kolmert, Centre for Allergy Research, Karolinska Institutet, Stockholm, Sweden; J.R. Konradsen, Dept Women’s and Children’s Health & Centre for Allergy Research, Karolinska Institutet, Stockholm, Sweden; Maxim Kots, Chiesi Pharmaceuticals, SPA, Parma, Italy; Kosmas Kretsos, UCB, Slough, UK; L. Krueger, University Children’s Hospital Bern, Switzerland; Scott Kuo, National Heart and Lung Institute, Imperial College, London, UK; Maciej Kupczyk, Centre for Allergy Research, Karolinska Institutet, Stockholm, Sweden; Bart Lambrecht, University of Gent, Gent, Belgium; A-S. Lantz, Karolinska University Hospital & Centre for Allergy Research, Karolinska Institutet, Stockholm, Sweden; Christopher Larminie, GSK, London, UK; L.X. Larsson, AstraZeneca, Mölndal, Sweden; P. Latzin, University Children’s Hospital of Bern, Bern, Switzerland; N. Lazarinis, Karolinska University Hospital & Karolinska Institutet, Stockholm, Sweden; N. Lemonnier, European Institute for Systems Biology and Medicine, CNRS-ENS-UCBL-INSERM, Lyon, France; Saeeda Lone-Latif, Academic Medical Centre, University of Amsterdam, Amsterdam, The Netherlands; L.A. Lowe, Centre for Respiratory Medicine and Allergy, Institute of Inflammation and Repair, University Hospital of South Manchester, NHS Foundation Trust, Manchester, UK; Alexander Manta, Roche Diagnostics GmbH, Mannheim, Germany; Lisa Marouzet, NIHR Southampton Respiratory Biomedical Research Unit, Southampton, UK; Jane Martin, NIHR Southampton Respiratory Biomedical Research Unit, Southampton, UK; Caroline Mathon, Centre of Allergy Research, Karolinska Institutet, Stockholm, Sweden; L. McEvoy, University Hospital, Dept of Pulmonary Medicine, Bern, Switzerland; Sally Meah, National Heart and Lung Institute, Imperial College, London, UK; A. Menzies-Gow, Royal Brompton and Harefield NHS Foundation Trust, London, UK; Leanne Metcalf, previously at Asthma UK, London, UK; Maria Mikus, Science for Life Laboratory & The Royal Institute of Technology, Stockholm, Sweden; Philip Research Ltd, Southampton, UK; Centre for Allergy Research, Karolinska Institutet, Stockholm, Sweden; K. Boehringer Ingelheim Pharma GmbH & Co. KG, Biberach, Germany; University of Southampton, Southampton, UK; U. previously at AstraZeneca, Mölndal, Sweden; Science for Life Laboratory & The Royal Institute of Technology, Stockholm, Sweden; R. North West Severe Asthma Network, University Hospital South Manchester, UK; B. Dept Women’s and Children’s Health & Centre for Allergy Research, Karolinska Institutet, Stockholm, Sweden; S. Royal Brompton Hospital, London, UK; AstraZeneca, Mölndal, Sweden; Catania, Italy; European Federation of Allergy and Airways Diseases Patient’s Associations, Brussels, Belgium; J. European Institute for Systems Biology and Medicine, CNRS-ENS-UCBL-INSERM, Lyon, France; University of Rome ‘Tor Vergata’, Rome Italy; Centre for Allergy Research, Karolinska Institutet, Stockholm, Sweden; NIHR Southampton Respiratory Biomedical Research Unit, Southampton, UK; C. European Institute for Systems Biology and Medicine, CNRS-ENS-UCBL-INSERM, Lyon, France; University of Southampton, UK; previously at Asthma UK, London, UK; Academic Medical Centre, University of Amsterdam, Amsterdam, The Netherlands; NIHR Southampton Respiratory Biomedical Research Unit, Southampton, UK; Centre for Allergy Research, Karolinska Institutet, Stockholm, Sweden; Leanne previously at Asthma UK, London, UK; K. Boehringer Ingelheim Pharma GmbH & Co. KG, Biberach, Germany; MSD, Brussels, Belgium; Royal Brompton and Harefield NHS Foundation Trust, UK; C. National Heart and Lung Institute, Imperial College, London, UK; National Heart and Lung Institute, Imperial College, London, UK; Longfonds, Amersfoort, The Netherlands; G. Università Cattolica del Sacro Cuore, Milan, Italy; University of Rome ‘Tor Vergata’, Rome Italy; M. European Institute for Systems Biology and Medicine, CNRS-ENS-UCBL-INSERM, Lyon, France; Boehringer Ingelheim Pharma GmbH & Co. KG, Biberach, Germany; Centre for Proteomic Research, Institute for Life Sciences, University of Southampton, Southampton, UK; S. North West Severe Asthma Network, of Hospital, UK; N. North West Severe Asthma Network, Pennine Acute Hospital NHS Centre for Allergy Research, Karolinska Institutet, Stockholm, Sweden; Academic Medical Centre, University of Amsterdam, Amsterdam, The Netherlands; Caroline NIHR Southampton Respiratory Biomedical Research Unit, Southampton, UK; Jessica Asthma UK, London, UK; M. University of Nottingham, UK; P. Dept Women’s and Children’s Health, Karolinska Institutet, Stockholm, Sweden; A. Royal Brompton and Harefield NHS Foundation Trust, London, UK; F. University Hospital Dept of Pulmonary Medicine, Bern, Switzerland; University of Southampton, Southampton, UK; S. Centre for Respiratory Medicine and Allergy, Institute of Inflammation and Repair, University Hospital of South Manchester, NHS Foundation Trust, Manchester, UK; J. University of Copenhagen and Asthma Centre Denmark; K. Karolinska University Hospital & Karolinska Institutet, Stockholm, Sweden; M. Centre for Respiratory Medicine and Allergy, Institute of Inflammation and Repair, University Hospital of South Manchester, NHS Foundation Trust, Manchester, UK; M. Semmelweis University, Budapest, Hungary; L. Semmelweis University, Budapest, Hungary; K. NIHR Southampton Respiratory Biomedical Research Unit, Clinical and Experimental Sciences, NIHR-Wellcome Trust Clinical Research Facility, Faculty of Medicine, University of Southampton, Southampton, UK; Karolinska University Hospital, Stockholm, Sweden; COPSAC, Copenhagen Prospective Studies on Asthma in Childhood, Herlev and Gentofte Hospital, University of Copenhagen, Copenhagen, Denmark; S. Università Cattolica del Sacro Cuore, Milan, Italy; van de Academic Medical Centre, University of Amsterdam, Amsterdam, The Netherlands; van Academic Medical Centre, University of Amsterdam, Amsterdam, The Netherlands; UCB, Slough, UK; previously at Asthma UK, London, UK; Philips Research Laboratories, Eindhoven, The Netherlands; C. University Hospital Bern, Switzerland; A. North West Severe Asthma Network, NHS Trust, UK; Boehringer Ingelheim Pharma GmbH & Co. KG, Biberach, Germany; Asthma UK, London, UK; Histochemistry Research Unit, Faculty of Medicine, University of Southampton, Southampton, UK; Boehringer Ingelheim Pharma GmbH, Biberach, Germany; National Heart and Lung Institute, Imperial College, London, UK; International Primary Care Respiratory Group, Aberdeen, UK; Data Science Institute, Imperial College, London, UK; UK Clinical Operations, GSK, Stockley Park, UK; W. Amgen Inc., Thousand Oaks, USA; W. Dept Women’s and Children’s Health & Centre for Allergy Research, Karolinska Institutet, Stockholm, Sweden; Z. NIHR Southampton Respiratory Biomedical Research Unit, Clinical and Experimental Sciences and Human Development and Health, Southampton, UK; A.H. Academic Medical Centre, University of Amsterdam, The Novartis Pharma University of Southampton, Southampton, UK; Academic Medical Centre, University of Amsterdam, Amsterdam, The Netherlands; Imperial London, London, UK; University of Catania, Catania, Italy; University of Rome ‘Tor Vergata’, Italy; Hospital, Denmark; Jagiellonian College, Krakow, Poland; University Hospital, Bern, Switzerland; Semmelweis University, Budapest, Hungary; University of Manchester, Manchester, UK; Marseille, France; Fraunhofer Institute, Hannover, Germany; University Hospital, Umea, Sweden; University, Belgium; Lyon, France; Università Cattolica del Sacro Cuore, Italy; University Hospital, Copenhagen, Denmark; Karolinska Institutet, Stockholm, Sweden; University Hospital, Nottingham, UK; University of Bergen, Bergen, Norway; Asthma Foundation, The Netherlands; European Lung Foundation, Sheffield, UK; Asthma UK, London, UK; of Allergy and Airways Diseases Associations, Brussels, Belgium; Catania, Italy; International Primary Care Respiratory Group, Aberdeen, UK; Philips Research Laboratories, Eindhoven, The Netherlands; Research Ltd, Southampton, UK; Stockholm, Sweden; BioSci Consulting, Maasmechelen, Belgium; AstraZeneca; Boehringer Janssen Amgen Merck & of the The Netherlands; Gahlemann, Germany; Italy; Southampton, UK; The Netherlands; Asthma UK; Asthma UK; de and The Netherlands; Sigmund, and The Roberts, UK; David UK; The Netherlands; UK; Kamphuis, The Netherlands; UK; Italy; de The Netherlands; The of the and de and Gahlemann, and Sigmund, was by University of and the National Institute for Health and Medical Research and J.M. study A. S.J. and J.M. analysis and A. G. R. and J.M. of M. G. R. and J.M. and J.M. G. from and the R. for at by and for and Novartis as of and in a asthma by is a and and in a University of Southampton from Chiesi and AstraZeneca; the J.M. from and for from and the are further to The that the findings of study are from the The is not for the or of by the to the for the