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The role of epithelial-fibroblast communication in asthma and chronic obstructive pulmonary disease Osei, Emmanuel Twumasi 2017

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THE ROLE OF EPITHELIAL-FIBROBLAST COMMUNICATION IN ASTHMA AND CHRONIC OBSTRUCTIVE PULMONARY DISEASE by  Emmanuel Twumasi Osei  MSc, Cranfield University, 2011  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Pharmacology and Therapeutics)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)  September 2017  © Emmanuel Twumasi Osei, 2017   ii      The role of epithelial-fibroblast communication in asthma and COPD  PhD thesis  to obtain the degree of PhD at the University of Groningen on the authority of the Rector Magnificus Prof. E. Sterken and in accordance with the decision by the College of Deans.  and  Thesis submitted in partial fulfillment of the requirements for the degree of Doctor of philosophy in The Faculty of Graduate and Postdoctoral Studies The University Of British Columbia, Vancouver   This thesis will be defended in public on   Wednesday September 6 2017 at 16.15 hours  by  Emmanuel Twumasi Osei  born on 23 July 1986 in Sunyani, Ghana iii  Supervisor  Prof.  W. Timens Prof. T-L. Hackett Prof.  H.I. Heijink   Co-supervisor  Dr. C.A. Brandsma   Assessment committee  Prof. K.R. Bracke Prof. R. Gosens Prof. H.A.M. Kerstjens Prof. P. M. Lansdorp    iv  Abstract Asthma and COPD are incurable chronic inflammatory diseases of the lung that involve inflammation and remodeling in the airways. An abnormal interaction between the airway epithelium and fibroblasts in the epithelial mesenchymal trophic unit (EMTU) has been suggested to be a possible disease mechanism in asthma and COPD. The airway epithelium forms the first line of defense in the airways while fibroblasts are the structural cells responsible for producing extracellular matrix (ECM) proteins and maintaining the structural integrity of the lung.  In this thesis we developed a co-culture model to study the factors involved in airway epithelial cell and fibroblast interaction and how a dysregulated communication between these two cells contributes to asthma and COPD pathogenesis. We also used multiphoton imaging and 3-dimensional collagen contraction assays to assess the effect of abnormal ECM repair by airway fibroblasts, proposed to cause airway remodeling in asthma and COPD.  We found in the co-culture model that the airway epithelium through the release of IL-1α controls the inflammatory and ECM remodeling phenotype of lung fibroblasts. Further, exposing airway epithelial cells to cigarette smoke, the major risk factor for COPD, caused a higher release of IL-1α which subsequently led to the release of higher levels of inflammatory mediators. Again, due to the presence of a single nucleotide polymorphism, rs2910164 (GG allele), COPD-derived lung fibroblasts from our co-culture had a lower induction of miRNA-146a-5p, an anti-inflammatory miRNA that regulates IL-1 signaling, compared to controls.  Again, we found asthmatic-derived airway epithelial cells released higher levels of IL-1α and that IL-1 causes the release of inflammatory mediators, the down-regulation of ECM proteins in airway fibroblasts and affects their ability to organize gelatin into fibrillar collagen. In the airways of asthmatics patients, we found that there was disorganization of fibrillar collagen fibers v  in the lamina propria, due to a defect in the ability of asthmatic-derived airway fibroblasts to remodel collagen fibers compared to controls.  Taken together our work sheds new light on how abnormal epithelial-fibroblast communication may contribute to the chronic inflammation and airway remodeling described in asthma and COPD, further it opens new avenues for therapeutic research.      vi  Lay Summary  Asthma and Chronic Obstructive Pulmonary Disease (COPD) are incurable chronic inflammatory lung diseases that involve scaring of the airways. Abnormal communication between airway epithelial cells and fibroblasts has been suggested as a possible mechanism for scarring of the airways. We developed a model to study lung epithelial and fibroblast communication and found that Interleukin (IL-1α) is essential for epithelial regulation of fibroblast functions. Exposure to cigarette smoke, the major risk factor for COPD, caused a higher release of IL-1α from COPD-derived epithelial cells. Lung fibroblasts from COPD patients also had lower levels of the master regulatory molecule, miRNA-146a-5p, that normally controls excessive IL-1 signaling. Further, we found greater release of native IL-1α from asthmatic-derived airway epithelial cells and IL-1 caused disorganization of the matrix protein collagen Iα1 by airway fibroblasts. Our work sheds new light on the disease processes of asthma and COPD and opens new avenues for therapeutic research.      vii  Preface This dissertation is formatted in accordance with the regulations of the University of Groningen and submitted in partial fulfillment of the requirements for a PhD degree awarded jointly by the University of Groningen (UG) and the University of British Columbia (UBC). Versions of this dissertation will exist in the institutional repositories of both institutions.  All the research projects and the methods associated with this thesis undertaken in the University of Groningen was consistent with the Research Code of the University Medical Center Groningen (www.rug.nl/umcg/onderzoek/researchcode/ index), and the national ethical and professional guidelines (www.federa.org). In the University of British Columbia, all research and methods were approved by the Providence Health Care Research Ethics Board (H13-02173).    Chapter 1: Introduction  Chapter 1 was written by me after which it was proof-read and edited by Prof Tillie-Louise Hackett in UBC, Profs Wim Timens and Irene Heijink as well as Dr Corry-Anke Brandsma in UG.  Chapter 2: Unravelling the complexity of COPD by microRNAs it’s a small world after all I wrote Chapter 2 as the lead author jointly with Laura-Florez Sampedro who was a co-first author. I was the lead first author who was in charge of structuring the manuscript, writing 50% of the body and making all figures and tables. The manuscript was proof-read and edited by Profs Irene Heijink, Wim Timens Dirkje Postma as well as Dr Corry-Anke Brandsma.  This chapter has been published in the European Respiratory Journal   viii  Cite: Osei E.T., Florez-Sampedro L., Timens W., Postma S.D., Heijink I.H. and Brandsma C-A. (2015). Unravelling the complexity Of COPD by microRNAs; it’s a small world after all.  European Respiratory Journal 46:807–818.   Chapter 3: Interleukin-1α drives the dysfunctional cross-talk of the airway epithelium and lung fibroblasts in COPD I carried out approximately 70% of all experiments, carried out all data analysis, interpreted the data and wrote the manuscript in Chapter 3. I carried out co-culture experiments, ELISAs, Western blots and PCRs. Jacobien Noordhooek established the co-culture model and performed preliminary experiments. Anita Spanjer aided in some co-culture experiments. The study was conceived by Profs Wim Timens, Dirkje Postma and Irene Heijink, Tillie-Louise Hackett and Dr Corry-Anke Brandsma who all proof-read, edited and revised the manuscript. This chapter has been published in the European Respiratory Journal: Cite: Osei E.T., Noordhoek J.A., Hackett T.L., Spanjer A.I.R., Postma S.D., Timens, W., Brandsma C-A. and Heijink I.H. (2016). Interleukin-1α is an important driver of the disturbed cross-talk between airway epithelial cells and lung fibroblasts in COPD. European Respiratory Journal. 48 (2):359-369   Chapter 4: MiR-146a plays an essential role in the aberrant epithelial-fibroblast cross-talk in COPD I carried out all experiments in the manuscript for Chapter 4, analyzed the data and wrote the manuscript. Laura Florez Sampedro and Jacobien Noordhoek assisted with preliminary experiments of co-culture studies, miRNA PCRs and ELISAs. Dr Alen Faiz performed the SNP ix  analysis in the manuscript. I was involved in the study design and conceptualization of the project together with Laura Florez Sampedro, Profs Irene Heijink, Wim Timens and Dirkje Postma, Tillie-Louise Hackett and Dr Corry-Anke Brandsma who all also proof-read, edited and revised the manuscript. This chapter has been published in the European Respiratory Journal. Cite: Osei E.T., Florez-Sampedro L, Tasena H, Faiz A, Noordhoek JA, Timens W, Postma DS, Hackett TL, Heijink IH, Brandsma CA. (2017) miR-146a-5p plays an essential role in the aberrant epithelial-fibroblast cross-talk in COPD. Eur Respir J: 49(5).  Chapter 5: Interleukin-1 affects inflammatory mediator release and collagen I contraction by airway fibroblasts from asthmatic and non-asthmatic donors I carried out most experiments in Chapter 5, analyzed the data and wrote the manuscript. Dr Leila Mostaco-Guidolin assisted with all imaging and image analysis for the manuscript that included second harmonic generation non-linear optical microscopy and two-photon excitation fluorescence microscopy. May AL-Fouadi assisted with western blot experiments. Darren Cole and Dr Geoffrey N Maksym helped with optical magnetic twisting cytometry experiments. Drs Teal Hallstrand and Stephanie Warner helped to provide airway epithelial samples and assisted with air liquid interface cultures from which we obtained the primary epithelial cell data. The study was designed and conceptualized by myself and Prof Tillie-Louise Hackett who assisted in writing, proof-reading and editing of the manuscript. Profs Irene Heijink and Wim Timens as well as Dr Corry-Anke Brandsma also provided inputs throughout the study and helped in reviewing and editing the final manuscript.   x  Chapter 6: Answering a 90 year old question for asthma and airway fibrosis using multimodal nonlinear optical microscopy I performed approximately 30% of experiments in the manuscript for this chapter which included fibroblast seeded collagen gel experiments and western blots. Dr Leila Mostaco-Guidolin who is a shared co-first author with me, performed all imaging experiments and image analysis of the collagen gels as well as the airways of patients and controls. Soheil Hajimohammadi, Jari Ullah, , Vicky Li, Furquan Shaheen all helped in preliminary studies to establish the collagen gel contraction technique and imaging analysis of the collagen gels and airways. Darren J Cole and Geoffrey N Maksym assisted with optical magnetic twisting cytometry experiments. Xian Li, Fanny Chu and David Walker assisted with Transmission electron microscopy experiments. The study was designed and conceptualized by Prof Tillie-Louise Hackett who co-wrote the manuscript with Dr Leila Mostaco-Guidolin and myself. Prof Irene Heijink and Wim Timens as well as Dr Corry-Anke Brandsma proof-read, made contributions to the project, reviewed and edited the final manuscript.   Chapter 7: Summary, General Discussion and Future Perspectives I wrote Chapter 7 after which it was edited and reviewed by Profs Tillie-Louise Hackett, Wim Timens, Irene Heijink and Dr Corry-Anke Brandsma.  xi  Table of Contents  Abstract .......................................................................................................................................... ii Lay Summary ............................................................................................................................... vi Preface .......................................................................................................................................... vii Table of Contents ......................................................................................................................... xi List of Tables .............................................................................................................................. xiv List of Figures ...............................................................................................................................xv List of Abbreviations ............................................................................................................... xviii Acknowledgements ................................................................................................................... xxii Dedication ................................................................................................................................. xxiv Chapter 1: Introduction ............................................................................................................... 1 1.1 Definition and Epidemiology of Asthma and COPD ................................................. 1 1.2 Diagnosis of Asthma and COPD ................................................................................ 2 1.3 Pathophysiology of Asthma and COPD ..................................................................... 4 1.4 Exacerbations in Asthma and COPD ........................................................................ 11 1.5 Airflow Obstruction .................................................................................................. 11 1.6 Asthma and COPD-Overlap Syndrome .................................................................... 15 1.7 Management Strategies for COPD and Asthma ....................................................... 16 1.8 Role of Epithelial-Mesenchymal Trophic Unit in Lung Repair ............................... 21 1.9 Alterations in the EMTU in Asthma and COPD ...................................................... 24 1.10 IL-1 Signaling and Role in Asthma and COPD ........................................................ 32 1.11 MiRNAs in Asthma and COPD ................................................................................ 38 xii  1.12 Scope of Thesis ......................................................................................................... 39 Chapter 2: Unravelling The Complexity of COPD by MicroRNAs; It’s A Small World After All ................................................................................................................................... 42 2.1 Chapter Summary ..................................................................................................... 43 2.2 Introduction ............................................................................................................... 43 2.3 The Effects of Cigarette Smoke Exposure on miRNA Expression .......................... 49 2.4 Differential miRNA Expression in COPD ................................................................ 55 2.5 Involvement of miRNAs in Inflammatory Responses .............................................. 57 2.6 Involvement of miRNAs in Emphysematous Lesions .............................................. 59 2.7 Involvement of miRNAs in Tissue Repair ................................................................ 61 2.8 MiRNAs as Targets for Future Therapeutic Strategies in COPD ............................. 62 2.9 Conclusions ............................................................................................................... 65 Chapter 3: Interleukin-1α Drives the Dysfunctional Cross-Talk of the Airway Epithelium and Lung Fibroblasts in COPD ......................................................................... 66 3.1 Chapter Summary ..................................................................................................... 67 3.2 Introduction ............................................................................................................... 68 3.3 Methods and Materials .............................................................................................. 69 3.4 Results ....................................................................................................................... 74 3.5 Discussion ................................................................................................................. 84 Chapter 4: MiR-146a-5p Plays an Essential Role in the Aberrant Epithelial-Fibroblast Cross-Talk in COPD ............................................................................................................... 88 4.1 Chapter Summary ..................................................................................................... 89 4.2 Introduction ............................................................................................................... 90 xiii  4.3 Methods and Materials .............................................................................................. 91 4.4 Results ....................................................................................................................... 96 4.5 Discussion ............................................................................................................... 105 Chapter 5: Interleukin-1 Affects Inflammatory Mediator Release and Collagen I Contraction by Airway Fibroblasts from Asthmatic and Non-Asthmatic Donors ......... 109 5.1 Chapter Summary ................................................................................................... 110 5.2 Introduction ............................................................................................................. 111 5.3 Methods and Materials ............................................................................................ 113 5.4 Results ..................................................................................................................... 118 5.5 Discussion ............................................................................................................... 132 Chapter 6: Answering a 90 Year Old Question for Asthma and Airway Fibrosis Using Multimodal Nonlinear Optical Microscopy........................................................................ 137 6.1 Chapter Summary ................................................................................................... 138 6.2 Introduction ............................................................................................................. 138 6.3 Methods and Materials ............................................................................................ 140 6.4 Results ..................................................................................................................... 147 6.5 Discussion ............................................................................................................... 154 Chapter 7: General Summary, General Discussion and Future Perspectives ................ 161 7.1 General Summary ................................................................................................... 161 7.2 General Discussion ................................................................................................. 163 7.3 Future Perspectives ................................................................................................. 174 7.4 Conclusion .............................................................................................................. 179 References .............................................................................................................................. 181 xiv  List of Tables Table 2.1 MicroRNAs involved in different features of chronic obstructive pulmonary disease 49 Table 2.2 MicroRNAs involved in more than one feature of chronic obstructive pulmonary disease........................................................................................................................................................ 54 Table 3.1. Characteristics of severe chronic obstructive pulmonary disease patients from whom primary airway epithelial cells (AECs) were obtained. ................................................................ 71 Table 3.2 Characteristics of control donors and severe chronic obstructive pulmonary disease patients from whom primary human lung fibroblasts (PHLFs) were obtained. ........................... 72 Table 4.1Characteristics of COPD patients and non-COPD controls from whom primary human lung fibroblasts (PHLFs) were obtained. ...................................................................................... 93 Table 5.1 Characteristics of asthmatics and non-asthmatics from whom primary airway epithelial cell and primary airway fibroblasts were derived ....................................................................... 113 Table 6.1 Characteristics of asthmatics and non-asthmatics from whom airway biopsies and primary airway fibroblasts were derived. ................................................................................... 142   xv  List of Figures Figure 1.1 Mechanism of chronic airway inflammation in asthma. ............................................... 7 Figure 1.2 Mechanism of chronic airway inflammation in COPD. .............................................. 10 Figure 1.3 Features of airway remodeling in the large airways in asthma. .................................. 13 Figure 1.4 Features of small airway remodeling COPD. .............................................................. 15 Figure 1.5 The activation of the EMTU in lung disease. .............................................................. 23 Figure 1.6 Classical IL-1 signaling. .............................................................................................. 36 Figure 1.7 Regulation of IL-1 signaling. ...................................................................................... 38 Figure 2.1 Differential regulation of microRNAs in chronic obstructive pulmonary disease (COPD). ........................................................................................................................................ 47 Figure 3.1 Interleukin (IL)-8/CXCL8, heat shock protein (Hsp70) and IL-1β levels in co-culture of airway epithelial cells (AECs) and lung fibroblasts. ................................................................ 75 Figure 3.2 Decrease in the expression of extracellular matrix molecules and structural proteins in primary human lung fibroblasts (PHLFs) after co-culture with 16HBE14o- cells. ...................... 77 Figure 3.3 Interleukin (IL)-1α from 16HBE14o- cells is responsible for a pro-inflammatory phenotype switch in MRC-5 fibroblasts. ...................................................................................... 78 Figure 3.4 Interleukin (IL)-1α from primary airway epithelial cells (AECs) is responsible for a pro-inflammatory phenotype switch in MRC-5 fibroblasts. ............................................................... 80 Figure 3.5 Interleukin (IL)-1α is responsible for the decrease in expression of extracellular matrix molecules and structural proteins of lung fibroblasts after co-culture with epithelial cells. ........ 81 Figure 3.6 Interleukin (IL)-1α from cigarette smoke extract (CSE)-exposed airway epithelium causes a higher release of IL-8/CXCL8 in lung fibroblasts. ......................................................... 83 xvi  Figure 4.1 Epithelial-derived interleukin (IL)-1α is responsible for increased miR-146a-5p expression in lung fibroblasts. ...................................................................................................... 97 Figure 4.2 Expression of miR-146a-5p in primary human lung fibroblasts (PHLFs) upon co-culture with 16HBE14o- cells. .................................................................................................................. 98 Figure 4.3 Effect of RelB expression and rs2910164 polymorphism on miR-146a-5p expression in co-culture. ................................................................................................................................... 100 Figure 4.4 miR-146a-5p has anti-inflammatory effects in lung fibroblasts ................................ 102 Figure 4.5 miR-146a-5p reduces interleukin (IL)-1α-induced IL-8 release in lung fibroblasts. 103 Figure 4.6 Proposed role of miR-146a-5p in the cross-talk between airway epithelial cells (AECs) and lung fibroblasts [208, 243, 258, 260, 300]. .......................................................................... 104 Figure 5.1 Production of IL-1 & IL-33 in differentiated air-liquid interface (ALI) cultures of primary airway epithelial cells. ................................................................................................... 120 Figure 5.2 IL-1 but not IL-33 stimulates the release of inflammatory mediators from primary airway fibroblasts (PAFs). .......................................................................................................... 122 Figure 5.3 IL-1α and IL-1β induce decreased extracellular matrix protein expression in airway fibroblasts. ................................................................................................................................... 124 Figure 5.4 IL-1 but not IL-33 effects fibroblast collagen 1 gel contraction and fibrillar formation..................................................................................................................................................... 126 Figure 5.5 Interleukin-1 alters fibroblast interaction with collagen I. ........................................ 128 Figure 5.6 IL-1 down-regulates the expression of lysyl oxidase (LOX) in airway fibroblasts. . 130 Figure 5.7 Lysyl oxidase activity is essential for fibroblast contraction of collagen I gels ........ 131 Figure 6.1 (a) Schematic of the home-built nonlinear optical microscope. Light source is a Ti:Sapphire femtosecond oscillator (Tsunami, Spectra-Physics). .............................................. 144 xvii  Figure 6.2 Examples showing Verhoeff Van Giessen stained (a-b) tissue as well as label-free NLO images (c-e) acquired at the sub-epithelial region. ..................................................................... 144 Figure 6.3 Fibrillar collagen I is increased and disorganised in the lamina propria of asthmatic airways. ....................................................................................................................................... 149 Figure 6.4 Collagen fibrils are disorganized in biopsies from asthmatic airways. ..................... 150 Figure 6.5 Defective collagen I remodeling by asthma-derived airway fibroblasts. .................. 152 Figure 6.6 Asthma and non-asthma-derived airway fibroblasts are not different in cell stiffness...................................................................................................................................................... 153 Figure 6.7 Lower protein expression of decorin but not lysyl oxidase in asthma-derived airway fibroblasts .................................................................................................................................... 154   xviii  List of Abbreviations 16HBE14o- - Human Bronchial Epithelial cell line ACOS – Asthma-COPD Overlap Syndrome AECs – Airway Epithelial Cells AHR – Airway Hyperresponsiveness ALI – Air-Liquid Interface AP-1 – Activator Protein-1 BAL – Bronchoalveolar Lavage BAPN –β-aminopropionitrile BEBM –Bronchial Epithelial Basal Medium BEGM- Bronchial Epithelial Cell Growth Medium BMP-4 – Bone morphogenetic protein-4 BSA – Bovine Serum Albumin cAMP – Cyclic adenosine monophosphate CCL – Chemokine (C-C motif) ligand CD – Cluster of differentiation CM – Conditioned medium COPD – Chronic Obstructive Pulmonary Disease COX – Cyclooxygenase  CpG – 5’-C-phosphate-G-3’ CSE – Cigarette smoke extract CXCL- Chemokine (C-X-C motif) ligand DAMPs – Damage Associated Molecular Patterns DMEM – Dulbecco’s Modified Eagle Medium  ECM – Extracellular Matrix EGFR – Epidermal Growth Factor Receptor EMEM- Eagle’s Minimum Essential Medium EMT- Epithelial-mesenchymal transition EMTU – Epithelial-mesenchymal trophic unit EPac- Exchange Protein directly activated by cAMP xix  ERK- Extracellular signal-regulated kinases FCS – Fetal Calf Serum FEV1-Forced Expiratory Volume in 1 second FGF- Fibroblast Growth Factor FOS – First Order Statistics FPRL – Formyl peptide receptor-like  FVC – Forced Vital Capacity FZD – Frizzled receptor GAPDH – Glyceraldehyde 3-phosphate dehydrogenase GINA – Global Initiative for Asthma GLCM – Gray level coherence matrix GLI1- Glioma associated oncogene 1 GM-CSF – Granulocyte-monocyte colony stimulating factor GOLD – Global Initiative for Chronic Obstructive Lung Disease HCV – Hepatitis C virus HDM – House Dust Mite HSP – Heat Shock Protein ICS – Inhaled Corticosteroids IFN – Interferon  IL – Interleukin  IL-1RAcP – Interleukin-1 receptor accessory protein IRAK-1 – Interleukin 1 Receptor Associated Kinase 1 LABA – Long acting β agonists LAMA – Long acting muscarinic antagonists LOX – Lysyl oxidase LPS - Lipopolysaccharide MAPK – Mitogen-activated protein kinase MCP-1 – Monocyte Chemoattractant Protein-1 MiRNA – MicroRNA MMP – Matrix Metalloproteinase xx  MRC-5 – Fetal lung fibroblast cell line MUC5AC – Mucin 5AC MyD88 – Myeloid differentiation primary response gene 88 NAb – Neutralizing Antibody NF-κB – Nuclear factor kappa-light-chain enhancer of activated B cells NKx2.1 – NK2 homeobox 1 NLRP3 – Nod-like receptor P3 OMTC - Optical Magnetic Twisting Cytometry P21WAF – Cyclin-dependent kinase inhibitor 1 PAF – Primary Airway Fibroblasts PBS – Phosphate Buffered Saline PDGF – Platelet derived growth factor PGE2 – Prostaglandin E2 PHLF – Primary Human Lung Fibroblasts Poly I: C – Polyinosinic:polycytidylic acid ROS – Reactive Oxygen Species SABA - Short acting β agonists SAMA - Short acting muscarinic antagonists SCF – Stem Cell Factor SERPIN – Serine Protease Inhibitor SHG-NLOM – Second Harmonic Generation Non-linear Optical Microscopy SIGIRR – Single Ig IL-1 Related Receptor SiRNA – Small Interfering RNA SNP – Single Nucleotide Polymorphism ST2 – Interleukin 1 receptor-like 1 TEM – Transmission Electron Microscope TGF – Transforming Growth Factor TH2 – T helper 2 cell TIMP – Tissue Inhibitor of Metalloproteinase TLR – Toll-like receptor xxi  TNF – Tumor Necrosis Factor TPEF – Two-Photon Excitation Fluorescence TRAF-6 – TNF receptor associated factor - 6 Treg – Regulatory T cell TSLP – Thymic Stromal Lipoprotein TTF – Thyroid Transcription Factor UTR – Untranslated Region ZEB1 – Zinc Finger E-box-binding homeobox 1 ZO-1 – Zonula Occludens-1 α-SMA- α-Smooth Muscle Actin                 xxii  Acknowledgements A lot of help has gone into the successful completion of my PhD. Hence, I owe a lot of thanks to several people for all their contributions.   First and foremost I would like to acknowledge and thank God almighty the giver of all things who ordained for this to happen. Ebenezer! This is how far you have brought me Lord.  Next I will like to thank my team of supervisors in both UBC and the University of Groningen (RUG). Although my PhD was joint one and I had to spend time in different sites, we worked together in the time we had to achieve incredible success. I would first like to express my extreme gratitude to my Supervisor here in UBC, Dr Tillie Hackett. I thoroughly enjoyed working with you and the opportunity you gave me. You spent time to teach and learn new techniques with me and was very helpful in writing and completing my thesis on time. I truly appreciate this. Next I would like to thank my team of supervisors in RUG, Profs Wim Timens, Dirkje Postma and Irene Heijink as well as Dr Corry-Anke Brandsma. When I worked in Groningen for the first part of my PhD you all were instrumental in providing me a conducive atmosphere for my work. You were always on hand to help with whichever aspect of my work I had to do and you always reiterated your faith in me which really spurred me on. I appreciate you all very much and hope we can continue to work together in the future.  Next, I will also like to express my gratitude to the my PhD assessment committee, Prof Ken R. Bracke, Prof Peter Lansdorp, Prof Reinoud Gossens, and Prof Huib Kerstjens for their careful and critical reading as well as approval of my thesis  In the UBC Vancouver, I also had a PhD committee made up of Prof Peter Paré and Prof Chun Seow who have been instrumental in helping my degree program progress. I would like to express my thanks for all the contributions you made during my committee meeting that helped me to progress to my candidacy at UBC. I will also like to thank Dr Alasdair Barr for chairing my committee meeting and contributing to the progress of my degree program in UBC.  I will like to express my gratitude to other PI’s and researchers in the HLI who contributed in diverse ways to the success of my PhD including Prof Jim Hogg, Dr Dragos Vasilescu and Dr Lu Wang. I will also like to acknowledge all PI’s in Groningen including Dr Martijn Nawijn, Dr Machteld Hylkema and Dr Barbro Melgert and all the other GRIAC PIs. xxiii   I would like to thank the various technicians I have worked with both in RUG and UBC for the contributions to my PhD. In the EXPIRE laboratory in RUG, I would like to thank Jacobien Noordhoek for helping me to learn most of the techniques I needed at the start of my PhD and helping me to gain my footing in my research. I will like to also acknowledge Weird Kooistra for all the help he gave me with my molecular techniques in Groningen. Next I will like to thank Harold de Bruin, Uilke Brouwer, Marnix Jonker, Marjan Luinge, and Sharon Brouwer who were all technicians in RUG for their help in diverse ways with my PhD. At the HLI, I will like to thank Furquan Shaheen the former lab manager of Hackett Lab, Dimitri Pavlov formerly of the Bernatchez lab as well as Funny Chu and May AL-Fouadi our current lab manager for all their immense help with various aspects of my project.   I will also like to thank all collaborators who helped in doing specialized experiments and providing samples for some of my experiments. I will lie to acknowledge Dr Stephanie Warner and Dr Teal Hallstrand as well as Dr Geoffrey N Maksym and Darren Cole for all the help.  Next I will like to thank my colleague students and postdocs. In RUG, I will like to acknowledge, Dr Alen Faiz, Dr Daan S. Pouwels, Laura Hesse, Grissel Faura Tellez, Dr Maaike de Vries, Hataitip Tasena and Laura Florez Sampedro for all their contributions that made my stay comfortable as well made my projects successful. At the HLI, I would like to give special thanks to my fellow PhD student in the lab Steve Booth for all his help in contributions both to my PhD and outside work. I will also like to thank Dr Hyun-Kyoung Koo who was a former student in the lab for all her contributions and help with my projects. My thanks also goes to Dr Leila Mostaco-Guidolin the post-doc in the Hackett lab with whom I have collaborated on all my projects and for all her help.  Next, I will like to acknowledge all my friends in the UK, Netherlands and Ghana for all the contributions in diverse ways to my degree  Last but not the least, I will like to acknowledge my family and friends. For my parents Mr and Mrs F.K. Osei Jnr and my sisters, Irene Osei Wirekoaa and Marian Abrafi Wongnaa and brothers, Mr Gabriel Kumi and Mr Vincent Berko for all the support, physically emotionally and psychologically. I thank you guys a lot for being there for me. We made it!  xxiv  Dedication This thesis is dedicated to my Family . 1  Chapter 1:  Introduction  1.1 Definition and Epidemiology of Asthma and COPD The chronic inflammatory lung diseases asthma and chronic obstructive pulmonary disease (COPD) impose an enormous world-wide public health burden, with an estimated 300 million asthmatics [1] and 10% of the population over 40 years of age having moderate COPD [2]. The estimated global prevalence of COPD has grown over the last 30 years from 227.3 million people in 1990 to 384 million people in 2010 [3, 4]. The major risk factor for COPD is the inhalation of noxious gases, including cigarette smoke. The increased incidence of COPD has been associated with a rise in cigarette smoking, exposure to biomass fuels in non-developed countries and aging of the population [2]. The global prevalence of asthma is expected to increase to approximately 400 million people by 2025 [1]. This increasing prevalence is due to the fact that asthma symptoms are now more common in many low-and middle-income countries, with the prevalence of asthma remaining constant in high-income countries [5]. The factors responsible for increasing asthma rates in low-and middle-income countries, are not fully understood, but environment and life style changes are thought to play a key role [6]. The World Health Organization has estimated that asthma represents a 1.8% of the total global burden of disease with 346,000 deaths worldwide attributed to asthma each year [7], while  the mortality rates of COPD are approximately 5% per year [3].  Both diseases are defined as chronic inflammatory diseases, where the inflammatory reaction contributes to airway obstruction. Specifically, the Global Initiative for Obstructive lung 2  disease (GOLD) defines COPD as “a common preventable and treatable disease, characterized by airflow limitation and persistent respiratory symptoms resulting from abnormalities in the airway and alveoli, caused by exposure to noxious particles and gases” [2]. Asthma is defined by the Global Initiative for Asthma (GINA) as “a heterogeneous disease that is mainly characterized by chronic inflammation of the airways defined by a variable history of respiratory symptoms including dyspnea, cough, chest tightness and wheeze accompanied by a variable airflow obstruction” [1].   1.2 Diagnosis of Asthma and COPD Patients with asthma and COPD experience similar symptoms including shortness of breath, chest tightness, increased sputum production and chronic cough [2, 5, 8].  In the clinic, the functional abnormality used to diagnose asthma and COPD is the reduction in the ability of an individual to empty the lungs during a forced expiratory maneuver measured using spirometry. This emptying defect is measured by a reduction in the volume of air that can be forcibly expired in 1 second (FEV1) and its ratio to the forced vital capacity (FVC), the total volume of gas that can be forcibly expired when no time limit is applied.   1.2.1 COPD Diagnosis Based on these physiological measurements, COPD is diagnosed when the FEV1/FVC ratio falls below 0.7 following the administration of a bronchodilator [2]. COPD is further staged into 4 categories of increasing severity in the GOLD guidelines based on the predicted value of FEV1. Whereby, COPD patients with mild disease (GOLD stage 1) have an FEV1 ≥ 80%, patients with 3  moderate COPD (GOLD stage 2) have an FEV1 between 50 to 80%, patients with severe COPD (GOLD stage 3) have an FEV1  between 30 to 50%, and very severe COPD patients (GOLD stage 4) have an FEV1 below 30% [2, 9]. Although this is a widely used and accepted criterion for COPD diagnosis, some studies have shown that these guidelines may lead to a 5% false positive diagnosis in the elderly due to the natural decline in lung function and under diagnosis in individuals younger than 45 years, and therefore an additional equation to calculate the lower limit of normal (LLN) can be used [10].  1.2.2 Asthma Diagnosis As per the GINA guidelines asthma is diagnosed by an increase in FEV1 equal to or greater than 12% after the administration of a bronchodilator [1]. In addition, a change of more than 20% in the peak expiratory flow (PEF) which is the maximum expiration speed as measured by a peak flow meter can also be used as a diagnosis of asthma. In asthma, the reduction in FEV1/FVC is caused by airway hyperresponsiveness of the smooth muscle and airway remodeling, which both contribute to airway narrowing and airflow obstruction [1]. In cases where variability of airflow obstruction cannot be readily confirmed with spirometry, a bronchial provocation procedure may also be used to assess asthma [11]. In this case, inhalation of the choline ester methacholine, which activates specific muscarinic receptors in the airways, is used to evaluate the level of airway hyperresponsiveness (AHR). A positive test for AHR is confirmed when the concentration of methacholine needed to decrease an individual’s FEV1 by 20%, referred to as the provocative concentration (PC20), is less than 4mg/mL [11]. In the absence of metacholine, an inhaled histamine or mannitol test may also be used, although systemic side effects due to excess inflammation have been associated with the histamine challenge [12]. In addition, to allergic 4  asthma, a diagnosis of exercise-induced bronchoconstriction is established when there is a 15% reduction in FEV1 after exercising [1, 13]. For this test, patients exercise for six to eight minutes till a target ventilation or heart rate is reached (usually by the fourth minute) [13]   1.3 Pathophysiology of Asthma and COPD Both asthma and COPD are characterized by heterogeneous chronic airway inflammation and airflow obstruction [14]. As part of the pathophysiology of both diseases, genetic susceptibilities have been shown to exist that make certain individuals more vulnerable to the disease pathogenesis [15]. However, the importance of various genetic association studies has yet to be fully translated into understanding the genetic elements and how they interact with environmental triggers to cause both asthma and COPD [15]. In both of these conditions, airway inflammation affects the conducting and peripheral airways. However, different inflammatory cells are recruited and different mediators are produced due to abnormal responses to different environmental stimuli [16]. Consequently, asthma and COPD patients have different responses to therapy [16]. In COPD, direct smoking or second-hand (cigarette) smoke exposure is the major risk factor for the development of the disease in the western world. In fact, chronic exposure to any noxious particles and fumes from the combustion of biomass fuels or industrial agents has been shown to cause COPD in vulnerable individuals [9]. In allergic asthma, the cause of chronic airway inflammation is the exposure to allergens such as house dust mite or grasses [17]. Below, we review briefly the pathophysiology of asthma that results in primarily intermittent, and reversible airway obstruction, and the pathophysiology of COPD, which is associated with progressive and largely irreversible airway obstruction. 5  1.3.1 Chronic airway inflammation in asthma In allergic asthma, exposure of the airways to various allergens in pollen, house dust mite, animal dander etc. leads to chronic airway inflammation [18]. When inhaled, allergens first encounter the airway epithelium, and trigger an inflammatory response that causes damage with disruption of cell-cell contacts [19]. In allergic asthmatics, epithelial disruption leads to the release a variety of cytokines and damage associated molecular patterns (DAMPs), including thymic stromal lipoprotein (TSLP) that conditions dendritic cells (DCs) to recognize antigens from various allergens and present these to naïve T cells leading to T helper 2 (TH2) differentiation [20]. The disruption of the epithelial barrier also facilitates sampling of antigens in the airway lumen by DCs [20]. In addition to DCs, macrophages may also play a role as antigen presenting cells in asthma [21]. The release of chemokines including CC-chemokine ligand 11 (CCL11) or eotaxin-1 and granulocyte-monocyte colony stimulating factor (GM-CSF), after allergen encounter with DCs is vital for eosinophil influx and maintenance in the airways [22]. There is also the release of CCL17 (TARC) and CCL22 (MDC) from the airway epithelium [22] which causes the influx of TH2-type cells in the airways [17, 23]. TH2 cells produce a variety of cytokines, including interleukin (IL)-4, IL-9, IL-13 and GM-CSF, that drive immunoglobulin (Ig) E  switching from B cells as well as IL-5 that is a chemo-attractant for eosinophils [23]. IL-4 and IL-13 also act on the epithelial layer to induce goblet cell hyperplasia [17]. In addition, mast cells in the airway mucosa maintained by epithelium-derived stem cell factor (SCF) are activated through antigen-specific IgE cross-linking of surface Fc receptors. This encounter and complexing of mast cell receptors by allergens leads to degranulation and the release of various potent bronchoconstrictors such as histamine, prostaglandin D2, and leukotrienes C4, D4 and E4 [14, 24, 25]. Recruited basophils which may also be precursors of mast cells, have been shown to release histamine through IgE cross-linking  [26]. 6  Normally, tolerance towards allergens is induced, in which a T cell subset called regulatory T (Treg) cell plays a role. Tregs control TH1 and TH2 responses, e.g. by suppressing cytokine production [27]. However, the development of this T cell subset may be impaired in asthma, as reduced Treg numbers have been observed in broncho-alveolar lavage (BAL) fluid in asthmatic children compared to controls, and reduced Treg numbers correlated positively with poor lung function [28] (figure 1.1).    7                 Figure 1.1 Mechanism of chronic airway inflammation in asthma. The airway epithelium is damaged with goblet cell hyperplasia and chronic mucus hypersecretion. When damaged the airway epithelium releases cytokines such as TSLP that conditions dendritic cells (DCs). DCs then prime naïve T cells into TH2 cells through the release of cytokines such as CCL17 and CCL22. TH2 cells release a variety of cytokines including IL-4 and IL-13 that cause IgE production from B cells. IL-5 also causes eosinophilic infiltration. The damaged epithelium is also a source of chemokines and growth factors such as CCL11 or Eotaxin-1 that causes eosinophil influx. In non-allergic asthma, non-specific triggers lead to mast IgE cross-linking on mast cells as well as basophils and the release of bronchochonstrictors such as prostaglandins and leukotrienes. Mast cells are maintained in the airways by epithelial-derived SCF. TH2 cells also release IL-9 that stimulates the proliferation of mast cells. Treg cells control TH2 responses by lowering cytokine production. Further, the airway epithelium is a source of fibrogenic factors such as TGF-β, PDGF, and FGF that stimulates fibroblasts to produce ECM proteins in excess that leads to airway remodeling [14].   TSLPMast CellBasophilIgETreg cellB cellIL-9IL-13IL-4IL-5Eosinophilic inflammationIncreased ECM production byFibroblasts TGF-β, PDGF, FGFProstaglandins,Cysteinyl Leukotrienes,HistamineSmooth muscleBronchoconstrictionCCL17,CCL22CCL11/Eotaxin-1, SCF, GM-CSF, IL-1Dendritic CellTH2 cellInhaled AllergensAirway epitheliumBasement membrane thickeningCCR4Goblet cell hyperplasia8  1.3.2 Chronic airway inflammation in COPD Traditionally, the underlying chronic airway inflammation in COPD has been shown to include neutrophilic influx driven by the airway epithelium, which produces chemoattractants for neutrophils, such as CXCL8 (IL-8). At present it is clear that COPD involves a complex inflammatory response with the involvement of macrophages and CD8 T cells in the airway wall and neutrophils acting at the epithelial surface and in the airway lumen [23, 29, 30]. Specifically in subjects susceptible to develop COPD, inhaled, noxious particles cause damage to the airway epithelium, which forms the first line of the innate immune defense in the lungs, [10], leading to the release of DAMPs [31] and innate immune cytokines (tumor necrosis factor α, IL-1, IL-6 and IL-8/CXCL8) [32]. These factors induce the recruitment of innate immune cells, i.e. neutrophils, and macrophages [33, 34]. Macrophages release CXCL11, CXCL9 and CXCL10 which in concert with antigen-presenting DCs cause an influx of TH1 and TC1 cells to activate the adaptive immunity [35]. In the parenchyma, the release of proteases (e.g. metalloproteases (MMPs), elastase) and reactive oxidative species (ROS) from recruited neutrophils and macrophages alters the protease, anti-protease balance in favor of proteases leading to tissue destruction resulting in emphysema [10, 36]. It is well established that a deficiency in the α1-antitrypsin enzyme, which neutralizes neutrophil elastase, is responsible for causing emphysematous disease in a subset of COPD patients [37]. This deficiency leads to diffuse parenchymal tissue destruction referred to as pan-lobular emphysema, as compared to centrilobular emphysema caused by cigarette smoke exposure [38].  Chronic airway inflammation is perpetuated by activation of the adaptive immune defense in the airway. The adaptive immune response is activated upon the priming of naive T cells by dendritic cells (DC), presenting antigens from inhaled foreign particles and damaged cells [9]. 9  Activated antigen-specific CD4 and CD8 positive T cells as well as antibody-producing plasma cells have been found in lymphoid aggregates around the small airways and in parenchymal lung tissue and add to the repetitive inflammatory cycle that is propagated even years after smoke cessation [9, 39] (figure 1.2).   10                     Figure 1.2 Mechanism of chronic airway inflammation in COPD. Inhaled cigarette smoke particles cause damage to the airway epithelium leading to the release of damage associated molecular patterns (DAMPs) and innate immune cytokines including; interleukin (IL)-1, tumor necrosis factor (TNF)-α, IL-6 and IL-8/ CXCL8. These factors induce the recruitment of neutrophils, and macrophages. Neutrophils add to small airway disease and emphysema by producing reactive oxygen species and proteases such as neutrophil elastase and matrix metalloproteinase (MMP) 9. Macrophages produce CXCL11, CXCL9 and CXCL10 which together with dendritic cells that present inhaled antigenic smoke particles cause an influx of TH1 and TC1 cells to orchestrate the adaptive immunity. Increased release of elastase and MMPs cause parenchymal tissue destruction that leads to emphysema. Activated antigen-specific CD4 and CD8 positive T cells in the small airways add to the repetitive inflammatory cycle that is propagated even years after smoke cessation. The increased production of mucus as a result of chronic mucus hypersecretion causes mucus plugs that occlude the small airways. The airway epithelium is also a potent source of fibrogenic growth factors such as TGF-β and FGF that acts on fibroblasts to cause the deposition of excess ECM proteins and cause fibrosis of the small airways [14].   Fibroblasts produce excessECMTGF-β, PDGF, FGFIL-1, TNF-α, CXCL8/IL-8, IL-6CXCR2IL-1, CXCL8/IL-8CXCL11, CXCL9, CXCL10TH1 Cell TC1 CellNeutrophilMacrophageFibrosis of small airwaysChronicMucus secretionSmall airways diseaseAlveolar destruction,EmphysemaDendritic cellCXCR3Airway EpitheliumGoblet cellHyperplasiaROS, Inflammatory mediators11  1.4 Exacerbations in Asthma and COPD In addition to the chronic inflammation present with disease, acute episodes that aggravate the inflammatory conditions, termed exacerbations, can occur in asthma and COPD patients [40]. "An exacerbation of COPD is defined as an event in the natural course of the disease characterized by a change in the patient's baseline dyspnea, cough, and/or sputum that is beyond normal day-to-day variations; is acute in onset; and may warrant a change in regular medication" [40]. Exacerbations are often triggered by respiratory infections and are associated with increased inflammation in the lower airways [41]. Several studies have shown that frequent severe exacerbations are associated with increased morbidity and mortality, poor health status, and a faster decline in lung function in both asthma and COPD [42]. Therefore, prevention and optimal treatment of exacerbations is a global priority.  1.5 Airflow Obstruction The movement of gas in and out of the lung involves 1) bulk transport of air along a pressure gradient developed through the action of respiratory muscles and 2) diffusion of gases due to a concentration gradient due to the uptake of oxygen and diffusion of carbon dioxide within the alveoli [43]. Airway obstruction based on spirometric values of FEV1/FVC can result from bronchospasm, mucosal edema and inflammation, mucus hypersecretion and the formation of mucus plugs, as well as structural changes that can lead to fibrosis (airway remodeling) or loss of elastic recoil by tissue destruction (emphysema) [9, 17].  Histopathological studies of patients with asthma and COPD have established that airway inflammation and remodeling occurs in parallel both in the proximal airways (>2 mm in diameter), and distal airways (<2 mm in diameter) [43, 12  44]. Below we describe the specific pathological features that lead to airflow obstruction in COPD and asthma.   1.5.1 Airflow obstruction in asthma  Airway hyperresponsiveness (AHR) describes the ability of the airways to narrow following exposure to a bronchoconstrictor agonist. The initial observation that bronchoconstriction occurs more frequently in asthmatic patients compared to non-asthmatic patients was made in 1921[45]. AHR is a prominent feature of almost all symptomatic asthma patients and correlates with the severity of asthma [45]. Asthma used to be considered an entirely reversible disease in terms of airway hyperresponsiveness. However, a number of longitudinal cohort studies have demonstrated that asthmatics, as a group, experience an accelerated rate of respiratory functional deterioration [46-49]. It is therefore now well understood that patients with severe asthma also present with persistent airflow limitation, which has been attributed to structural changes within the airways termed airway remodeling [44, 50, 51]. These structural changes have been studied in bronchial biopsies, which have shown that asthmatic airways are remodeled with the following features: epithelial metaplasia and damage, thickening of the basement membrane (laminar reticularis), hypertrophy and hyperplasia of airway smooth muscle, mucus-gland hyperplasia, angiogenesis and fibrosis with an altered deposition and composition of the extracellular matrix [17] (figure 1.3). Importantly, investigations of the airways of asthmatic children have shown that airway remodeling can occur even prior to the onset of symptoms and diagnosis [50-52]. These findings highlight the significance of airway remodeling in asthma, and suggest that it may occur concurrently or prior to inflammation [44].  13    Figure 1.3 Features of airway remodeling in the large airways in asthma. Airway sections from formalin-fixed paraffin embedded tissue stained with Masson’s trichrome stain for collagen (blue-green), cytoplasm and intercellular space (light purple) and keratin and muscle (red)  of a) A large airway from a normal control individual with no observable remodeling of the airways and b) An age and gender matched large airway of an asthmatic individual with arrows showing airway remodeling including i; increased smooth muscle mass, ii; damaged airway epithelium, iii; basement membrane thickening, iv; chronic mucus hypersecretion to occlude the airway lumen and v; fibrosis in the sub-epithelial space. Image by kind courtesy of F. Shaheen and T.L Hackett (Vancouver, Canada)   1.5.2 Airflow obstruction in COPD The diagnostic hallmark of COPD is irreversible expiratory airflow limitation caused by a combination of large and small airways disease and emphysema. The precise role of each of these pathological features remains poorly understood, especially in the mild to moderate stages of COPD [43]. It has been known since 1968 that the small conducting airways <2mm in internal diameter become the major site of airflow obstruction in COPD [53-55]. Airflow obstruction, which leads to an irreversible decline in lung function, partly results from chronic airway 14  remodeling that involves chronic mucus hypersecretion [43] and activation of fibroblasts with the production of excess ECM proteins, leading to thickening of the small wall (figure 1.4). In addition to fixed airflow obstruction, most patients with COPD have been shown to also present with AHR when studied over long periods which demonstrates an over-lap with the pathogenesis of asthma [8]. Using micro-CT in 2011, McDonough et al, demonstrated that in end-stage disease there can be up to 72% and 89% reduction in the number of last generation of conducting airways, the terminal bronchioles, in very severe panlobular and centrilobular emphysema patients respectively [56]. In addition, this study also showed that loss of terminal bronchioles can occur in regions of lung where there was no emphysema detected using micro-CT to measure the airspace size (mean linear intercept (Lm)), suggesting that bronchiolar obliteration may be independent from and/or precede emphysematous tissue destruction [56].  Emphysema is defined pathologically as the presence of permanent enlargement of the airspaces distal to the terminal bronchioles, accompanied by destruction of their walls and capillary networks without obvious fibrosis, resulting in non-functional airspaces [57]. This results in the reduction of driving pressure and obstructive collapse of the peripheral airways causing a reduction in FEV1 [57, 58]. Acinar wall destruction can be characterized as two specific types. In centrilobular emphysema, by far the most common form associated with smoke exposure, tissue destruction is limited to the central part of the acinar lobule. In contrast, panacinar emphysema is characterized by widespread destruction of the entire lobule, and is associated with alpha-1-anti-trypsin deficiency [59].    15              Figure 1.4 Features of small airway remodeling COPD. Airway sections from formalin-fixed paraffin embedded tissue stained with a haemtoxylin and eosin for the cell nuclei (purple) and cytoplasm (pink) counter-stained with alcian blue for acidic mucopolysacharides (blue) a) An airway from a normal control individual with no occlusion or obstruction for comparison, b) A bland mucus plug occluding a small airway due to mucus hyper-secretion, c) A small airway occluded with inflammatory exudates showing an active inflammatory process, d) A narrowed small airway due to airway remodeling resulting from excess extracellular matrix deposition in the peribronchiolar space. Image taken from [9] Hogg and Timens, 2009, Annu. Rev. Pathol. Mech. Dis. 4:435-59    1.6 Asthma and COPD-Overlap Syndrome As described above, there are fundamental differences in the presentation and pathogenesis of asthma and COPD. Asthma is primarily an allergic disease associated with AHR that often develops in childhood, while COPD is associated with progressive, irreversible lung function decline that is typically linked to smoke exposure and usually presents later in life, >40 years of 16  age [42]. However, it has become more apparent through several epidemiological studies that asthma and COPD may coexist, or at least one condition may evolve into the other mostly after smoke exposure, which has led to the term Asthma and COPD Overlap Syndrome (ACOS) [42, 60]. As an example, AHR is present in almost all patients with asthma, at least when they are experiencing symptoms, and in up to two thirds of patients with COPD, especially when reassessed overtime [61]. In asthma, the presence of AHR and neutrophilia is associated with a decreased lung function and enhanced mucus hypersecretion in the airways [8]. Further, the patterns of airway inflammation associated with asthma and COPD are heterogeneous, although significant overlap can be present. For example eosinophilic airway inflammation has been observed in some COPD patients, and has been associated with greater reversibility of airflow obstruction when treated with steroids [14]. Further, neutrophilic inflammation has been shown to occur in a subset of severe asthma patients, and has been hypothesized to confer resistance to steroids [42]. Therefore the overlap between asthma and COPD should be considered especially in smokers and elderly people and careful history taking is important to detect asthmatic features in earlier life [42].   1.7 Management Strategies for COPD and Asthma The current treatment strategies defined by GOLD and GINA for both COPD and asthma are outlined below. The problem is that these treatment strategies only serve to manage symptoms of both diseases and do not alter the course of airway remodeling [6, 62-67]. Due to the common mechanisms and presentations of airway inflammation and airflow limitation in both diseases, there is significant overlap in the types of therapies used for disease management.   17  1.7.1 COPD Treatment Smoking cessation as well as the avoidance of polluting agents has been shown to reduce FEV1 decline by almost 35 mL per year although it cannot completely halt disease progression in COPD patients [63, 68]. The first line of therapy is therefore the use of nicotine replacement products to increase long-term smoking abstinence rates [69]. The use of anti-depressants bupropion, nortriptyline and varenicline have been shown to have long-term efficacy for maintained smoking cessation as part of an intervention program involving nicotine replacement and or counseling programs [70-72]. While many smokers are using E-cigarettes, these products are not regulated and it is currently not understood if these products also cause harm [71, 72].  In COPD, the aim of pharmacotherapy is to reduce symptoms, exacerbation rates, prevent further lung damage and enhance exercise tolerance of patients [2]. For all COPD patients (GOLD 1-4), it is recommended to be vaccinated each year to prevent seasonal infections with influenza and pneumococcal viruses, and this preventative measure is associated with reduced exacerbations and mortality rates [73, 74]. Proper education on the use of inhaler devices, proper recognition and attention to symptoms as well as strict adherence to self-management and integrated care programs have shown a lot of benefits in the management of COPD [2]. COPD patients are also prescribed a short acting inhaled bronchodilator including β2-adrenergic receptor agonists (SABA) and short-acting muscarinic antagonists (SAMA) to be taken when required to offer relief in acute attacks of symptoms [75]. These drugs are effective for <12 hours and help to reduce symptoms, improve FEV1 and reduce dynamic hyperinflation at rest and during daily activities [76]. In addition to the SABA and SAMA combination therapy, GOLD stage 2 to 4 patients are provided a regular treatment of long acting bronchodilator therapy including long acting β2-agonists (LABA) and long-acting muscarinic antagonists (LAMA). These drugs act between 12 and 24 hours to offer 18  lasting relief from chronic inflammatory symptoms [77]. In severe and very-severe (GOLD 3 to 4) GOLD patients with frequent exacerbations the use of inhaled corticosteroids (ICS) in combination with LABA and/or LAMA therapy has been reported to greatly improve lung function and reduce exacerbation rates [2]. The combination therapy of LABA/LAMA/ICS is essential in individuals with severe disease since COPD patients do not respond as well to corticosteroids as asthmatics [8, 78, 79].  When patients with moderate to severe COPD experience acute exacerbations the administration of antibiotics such as azithromycin and moxifloxacin as well as antioxidants and mucolytic agents have been shown to reduce exacerbation rates [80, 81]. In severe and very severe COPD patients, phosphodiesterase-4 inhibitors such as Roflumilast can be used to control severe exacerbations when all other combinations of therapies have been used [82, 83]. The effects of other medications such as leukotriene modifiers, infliximab which is an anti-TNF-α antibody and simvastatin are still being investigated [2]. Augmenting α-1 antitrypsin in COPD patients with this deficiency has been shown to present lung function improvements with preservation of lung parenchyma [84-86]. For patients with severe disease and respiratory failure, oxygen therapy and ventilator support can be provided. In addition surgical interventions including lung volume reduction surgery, bullectomy or bronchoscopic procedures to decrease hyperinflation can be used but these surgeries are now less common [87-89]. For very-severe COPD disease lung transplantation is often the only option [90], but surgical procedures such as lung volume reduction surgery as well as new non-surgical procedures such as the one-way endobronchial valve therapy [91], trans-bronchial airway bypass [92] nitinol coil treatment [93] and parasympathetic lung denervation [93] among others have shown promising effects on lung function and quality of life scores.   19  1.7.2 Asthma Treatment In asthma, it is essential to control exposure to environmental triggers [6]. Reduction in the rate of exposure of asthmatic children to indoor allergens has been shown to reduce adverse outcome associated with asthma [94]. However, in asthmatic adults where allergen-avoidance measures such as the removal of causative allergen in occupational asthma were instituted, desirable outcomes were seen in only a third of subjects [95]. This suggests allergen-avoidance measures alone are not effective in all patients and therefore adhering to pharmacological therapy is required to control disease symptoms [96]. In step 1 of asthma disease management, the GINA guidelines recommend SABAs such as albuterol, levalbuterol and pirbuterol be prescribed and used as needed to relax constricted airway smooth muscles and provide a quick relief from disease symptoms [1, 6]. In addition to SABA, a regular low dose ICS is recommended to help lower the risk of exacerbations and improve quality of life [97, 98]. In patients with increasing rates of exacerbations, therapy can be increased to step 2 which recommends the use of combination low dose LABA with ICS therapy, in addition to regular SABA use [97]. As disease symptoms increase, step 3 includes a fixed combination of low dose ICS and LABA together with the use of SABA when needed [99]. The dose of ICS/LABA being administered is increased in step 4 to a medium dose in patients whose treatment options are still not effective. In step 4, the addition of LAMA to the ICS/LABA combination can also be considered in addition to regular SABA usage [100]. In patients who are unresponsive to either ICS or LABA treatments, cysteinyl leukotriene-receptor antagonist therapy with pranlukast, montelukast and zafirlukast may be helpful [6]. This causes bronchodilation within the first few hours of administration and can last multiple days by blocking leukotriene C4, D4 and E4 which are major bronchoconstrictors in asthma [101]. Montelukast treatment has been considered a safe and side effect-free alternative to ICS, and 20  recommended for use in infants as young as 1 year old [6]. Although leukotriene modifiers are generally used in patients unresponsive to ICS, the addition of this medication to low dose ICS has proven helpful [102, 103]. In severe refractory asthma where there is no response to ICS, LABA or leukotriene modifiers, Omalizumab, an anti-IgE monoclonal antibody is used as an add-on therapy [104]. Omalizumab binds to the high affinity FcεR1 receptor on basophil and mast cell surfaces and prevents IgE cross-linking and subsequent degranulation [104, 105]. Circulating levels of IgE are reduced to less than 95% after subcutaneous administration of this drug and has been shown to decrease exacerbation frequency in severe asthmatics [105]. The use of antibodies against IL-4 and IL-5 as add-on therapies to LABA/ICS combinations have shown some benefit to sub-groups of patients [1, 6]. After asthma symptoms are adequately controlled in a 3 to 6 month period, a step down management is recommended where therapy is reduced from high to lower doses to help minimize adverse side-effects [6].   1.7.3 The need for new therapeutic targets in asthma and COPD Taken together, current therapy for both asthma and COPD targets the chronic inflammatory processes and the bronchoconstriction that have been implicated in the pathogenesis of both diseases. Although these treatments can help alleviate symptoms and improve the quality of life of some patients, there is still no cure for both asthma and COPD and the structural changes in the airways are unaffected [22, 44]. Hence, a closer look at the underlying mechanisms of airway remodeling is essential. In line with this, it has been proposed that an abnormal interaction of the epithelium and fibroblasts in the lung epithelial-mesenchymal trophic unit (EMTU) acts in concert with chronic inflammatory processes and leads to aberrant ECM repair and remodeling that drives 21  airflow obstruction [106]. Thus, a closer look at this aberrant communication may provide new therapeutic targets for both asthma and COPD.     1.8 Role of Epithelial-Mesenchymal Trophic Unit in Lung Repair The role of the lungs in gas exchange places them in direct contact with the inhaled external environment which contains airborne allergens, particles and pathogens [107]. The first chemical, structural, and immunological barrier to the inhaled environment is the airway epithelium, which normally via the mucocilliary elevator and secreted airway/alveolar-lining-fluid, removes inhaled-foreign bodies from the lung [108]. Within the lung, the large conducting airways are lined with a pseudostratified epithelium consisting of ciliated, basal and goblet cells, which transitions to a more simple columnar epithelium in the small conducting airways, and lastly to a simple squamous alveolar epithelium for gas exchange [109]. The airway epithelium forms a tightly regulated barrier, which is held together by the formation of cell-cell contacts adherens (comprised of e.g. E-cadherin) and tight junctions (comprised of e.g. the interacting molecules zona occludens (ZO)-1, claudins, and occludins) that is attached to the basement membrane by desmosome junctions [110]. However, when damaged, the airway epithelium becomes immunologically activated to protect and maintain tissue homeostasis. The result is an acute inflammatory response followed by epithelial repair, which involves induction of cell migration to form a temporary barrier in response to growth factors (e.g. transforming growth factor (TGF)-β, fibroblast growth factor (FGF)) released into the underlying mesenchyme to form a provisional secreted fibrin extracellular matrix [108]. Furthermore, the injured epithelium releases fibroproliferative and fibrogenic growth factors (e.g.  fibroblast growth factor (FGF)2, and platelet derived growth factor (PDGF)), which 22  stimulate the underlying mesenchyme to proliferate and secrete ECM [111]. Such communication between the epithelium and underlying mesenchyme is reminiscent of the processes that drive branching morphogenesis during lung development, where the endoderm and mesoderm act as a trophic unit [112]. Specifically, as the lung develops, the reciprocal interactions between the endoderm and mesoderm occur in a temporal, spatial and cell-type specific manner through the expression of intrinsic factors, such as transcription factors (e.g. thyroid transcription factor (TTF)1/ NKx2.1), signaling molecules (e.g. FGF, bone morphogenetic protein (BMP)-4) and extracellular matrix proteins (proteoglycans and various collagens) [113]. Plopper and Evans first introduced the concept of the epithelial-mesenchymal trophic unit (EMTU) in 1999, and its role in lung development, repair and homeostasis [114]. Since then re-activation of the EMTU in response to chronic mucosal injury has been proposed to play a role in airway inflammation and remodeling [44, 106] (figure 1.5). 23                    Figure 1.5 The activation of the EMTU in lung disease. After damage to the airway epithelium as a result of epithelial fragility, the exposure to allergens and noxious particles as in asthma and COPD, the airway epithelium releases are variety of inflammatory mediators such as interleukin-1 (IL-1), tumor necrosis factor (TNF)-α, IL-8 and IL-6. These cytokines act on airway fibroblasts in the epithelial-mesenchymal trophic unit to cause the release of inflammatory mediators such as IL-8 and IL-6 that for example, can cause inflammation through neutrophil chemotaxis. The epithelium is also a source of fibrogenic factors such as TGF-β, PDGF, FGF and VEGF that act on fibroblasts to induce ECM deposition and remodeling. In addition to this, fibroblasts can also produce growth factors such as EGF and TGF-α that act on the airway epithelium to cause airway remodeling in a feedback cycle in the lung EMTU.    Intact Epithelium Repaired EpitheliumEpithelial injuryInflammatory mediators & DAMPSIL-1, TNF-α, IL-8, IL-6, Fibroblasts release growth factorsTGF-β, EGFFibrogenicmediatorsTGF-β, PDGF, FGF, VEGF IL-8, IL-6, HSP70Fibroblast-derived inflammationNeutrophil chemotaxisIncreased ECM deposition by FibroblastsFibroblastsAirway Smooth muscleEpithelial Fragility24  1.9 Alterations in the EMTU in Asthma and COPD In both allergic asthma and COPD, repetitive insult of the lung by environmental triggers is thought to cause chronic activation and aberrant regulation of the repair processes within the EMTU described above. An impaired capacity of the lung epithelium to repair itself has been closely linked to asthma and COPD disease progression [115, 116]. Below we describe the alterations in the airway epithelium and mesenchymal cells that have been reported both in asthma and COPD, with specific focus on the alterations in the intrinsic factors, transcription factors, growth factors and ECM that are important in repair of the EMTU.  1.9.1 Alterations in the airway epithelium in asthma and COPD In COPD, muco-ciliary clearance has been shown to decline along with chronic mucus hypersecretion, due to goblet cell hyperplasia as well as disturbed ciliary movement, and squamous metaplasia of the epithelium. This together, with an increased production of a more viscous mucus altogether leads to increased airflow obstruction after cigarette smoking [39, 117]. The airway epithelium from COPD patients has been shown to display lower expression of junctional proteins [118] and loss of cell-cell contacts with a disrupted expression of ZO-1 compared to non-COPD patients [119-121]. Further, exposure of the airway epithelium to cigarette smoke extract has been shown to disrupt airway epithelial cell-cell contacts due to an effect on epidermal growth factor receptor (EGFR)-dependent formation of tight junctions, which delays epithelial recovery upon wounding [119]. The 4700 chemicals found in a single inhalation of cigarette smoke have been shown to produce close to 1014 reactive oxygen species [122]. The resultant high oxidant concentration leads to cellular damage, apoptosis and necrosis of the airway epithelium, leading to the release of DAMPs that bind to various pattern recognition receptors (PRR) to initiate an 25  innate immune response. [31, 122]. Whereby, DAMPs can induce pro-inflammatory responses in neighboring epithelial cells and fibroblasts, and also directly act on innate immune cells to cause their recruitment [31, 122]. Cigarette smoke exposure has also been shown to directly induce the release of various cytokines and growth factors from the airway epithelium thought to play a role in COPD. Specifically, cigarette smoke induces robust release of TNF-α, IL-1, CXCL8 (IL-8) [123] and chemokines ligand 2 (CCL2) or MCP-1 (monocyte chemoattractant) [124]. Further, upon exposure to various noxious particles, the airway epithelium is also a source of growth factors such as TGF-β, vascular endothelial growth factor (VEGF) and PDGF, which are pivotal in causing the fibrotic lesions seen in COPD [9]. An increased TGF-β expression in the lungs of COPD patients has been associated with myofibroblast transformation from resident fibroblasts and an increased ECM production [125]. The expression of growth factor receptors, such as epidermal growth factor receptor (EGFR), have been shown to be upregulated in response to cigarette smoke and is thought to cause the increase in MUC5AC expression leading to the chronic mucus hypersecretion in COPD [126].   In asthma, abnormal airway epithelial barrier function has also been shown to be an integral part of disease progression. It has been shown in several studies that there is goblet cell hyperplasia [127-129] as well as airway epithelial fragility with decreased expression of adherens and tight junctions in asthma [130-135]. Furthermore, the asthmatic epithelium has been shown to express features of aberrant repair, with increased expansion potential of the basal cell population [130, 132] and increased expression of repair markers p21WAF, TGF-β and EGFR [136, 137]. In addition, the fragile airway epithelium in asthmatics has been shown to be a robust source of SCF and TSLP, which are vital for mast recruitment and maintenance as well as TH2 polarization in the airways respectively [23]. In line with this, airway epithelial knockdown of E-cadherin expression using 26  siRNA resulted in increased expression of TSLP and TH2 attracting chemokine CCL17 [138]. Other important cytokines released after damage to the airway epithelium due to allergen exposure include IL-1, IL-6, CXCL8 (IL-8), CCL5, IL-5, CCL22, granulocyte macrophage colony-stimulating factor (GM-CSF) and TGF-α, which all contribute to eosinophilia, mesenchymal cell activation, TH2 cell chemotaxis and/or TH2 cell polarization [23, 139-141]. The airway epithelium in asthmatics has been shown to have an increased production of growth factors TGF-β2, PGE2 [142-144], EGF, FGF, amphiregulin and heparin-binding EGF-like growth factor, which have all been shown to be of importance to the remodeling seen in the airways [145]. These growth factors are important for airway hyper-responsiveness and myofibroblast proliferation [22, 44]. In addition, the deposition of various ECM proteins and proteoglycans (e.g. collagen I, III, and IV, fibronectin, versican and lumican) from fibroblasts after exposure to these pro-fibrotic factors released from the airway epithelium has been documented and has been proposed to be responsible for the increased airway wall thickness in asthma [146, 147].   In both asthma and COPD, it has been suggested that the epithelium may contribute to aberrant repair and fibrosis in the airway wall through the process of epithelial-to-mesenchymal transition (EMT), a process involved in tissue repair and remodeling [148-150]. Disruption of epithelial junctions is an important hallmark of EMT, where loss of epithelial characteristics is accompanied by transition into a more mesenchymal phenotype and expression of remodeling markers. While the down-regulation of epithelial junctional proteins and expression of mesenchymal markers has been described in airway epithelium of both asthma and COPD patients [120, 121], the role of this epithelial plasticity and EMT in pathogenesis and disease progression is still in dispute [150, 151]. 27  1.9.2 Alterations in lung fibroblasts in asthma and COPD The various cells found in the lung mesenchyme including fibroblasts, fibrocytes, and smooth muscle cells which are essential structural cells involved in the maintenance of tissue homeostasis [152]. Of these cells, fibroblasts in particular are the producers of the rich glycoprotein and proteoglycan ECM proteins [152], which provide cells with the structural and biochemical support essential for cell type determination and maintenance [153]. Under normal repair conditions, fibroblasts are activated to differentiate into α-smooth muscle actin (SMA) expressing myofibroblasts, which are highly contractile and producers of new ECM proteins [154]. Once tissue repair is complete, myofibroblasts undergo apoptosis and are cleared by immune cells [154, 155]. However, under disease conditions, these cells persist and cause fibrotic lesions due to the damaged and activated epithelium, causing attraction and activation of immune cells. Aside from their role in ECM production, mesenchymal cells have been shown to be a source of pro-inflammatory cytokines such as CXCL8 (IL-8), TNF-α and IL-6 as well as pro-fibrotic mediators, including TGF-β and MMPs that interact to promote the further release of more growth factors and chemokines to perpetuate remodeling and inflammatory processes [156].    The fibrotic lesions that result in the narrowing of small airways in COPD have been shown to be associated with dysfunctional activity of fibroblasts. α-SMA, vimentin and tenascin- C expressing myofibroblasts increase and cluster in the bronchi of smokers compared to controls, which correlates with lung function decline in COPD patients [157]. Parenchymal lung fibroblasts from severe (GOLD 4) COPD patients produced higher levels of collagen I in vitro and lower levels of decorin upon stimulation with TGF-β1 and FGF compared to mild (GOLD 1) patients [158]. This may have implications for small airway disease, because decorin is an important proteoglycan involved in the cross-linking and spacing of collagen fibrils[158]. Hence, decreased 28  decorin by parenchymal lung fibroblasts points to loosening of collagen and surrounding alveolar attachments that is implicated in loss of elastic recoil and collapsibility of the airways [9, 158]. In addition to increased ECM production, fibroblasts derived from COPD patients have been shown to be more senescent, proliferate slower and secrete increased levels of the pro-inflammatory cytokines IL-6, CXCL8 (IL-8) and PGE2 as well as fibrogenic cytokines such as TGF-β1. This inflammatory phenotype of COPD-derived fibroblasts was shown to correlate with a poor lung function in severe COPD patients compared to mild (GOLD 1) patients or non-COPD controls [159, 160]. TGF-β1 as well as EGF and IL-1β induce higher expression of WNT-5B and WNT ligand receptors frizzled-6 (FZD6) and frizzled-8 (FZD8) in COPD-derived fibroblasts compared to controls [125, 161]. Since WNT signaling is an essential part of EMTU in lung morphogenesis, this suggests a reactivation of the EMTU in COPD. The increase in WNT5B and receptors may cause higher production of ECM proteins, MMPs, growth factors, enzymes and pro-inflammatory cytokines involved in small airway remodeling and chronic bronchitis in COPD [125, 161]. Interestingly, an increased expression of the FZD8 receptor on COPD-derived fibroblasts is associated with a single nucleotide polymorphism (SNP) rs663700 that may contribute to chronic mucus hyper-secretion in COPD. Since this increased expression of FZD8 is also involved in the release of pro-inflammatory cytokines, this points to an important role of fibroblast function in COPD pathogenesis [162].  In asthma, the enhanced proliferation and hypercontractility of the ASM in the airway has been implicated in AHR and the pathogenesis of asthma, and is a main target of current drug therapy. However, the fact that chronic remodeling in the airways is not affected by most asthma medication calls for a closer look at the role of other mesenchymal cells in the pathogenesis of the disease [106]. Although the source of activated fibroblasts in asthma is unclear at the moment 29  [163], it has been suggested that this could be due to an increased proliferation of resident fibroblasts and chemotaxis of circulating fibrocytes [156]. Of interest, an increased ECM deposition by these fibroblasts apposing to the reticular basement membrane of the asthmatic airway is a hallmark of asthma [164]. In line with this, the increased mesenchymal cell population in asthmatics may be due to increased production of growth factors in the airways of asthmatic patients, such as TGF-β, PDGF (pro-fibrotic cytokines) and trans-retinoic acid [165]. Further, the chronic inflammatory milieu in which mesenchymal cells reside within the asthmatic airways has been shown to provide a source of a variety of mediators that serve to promote excessive repair and remodeling processes [156]. Increased levels of inflammatory mediators such as the TH2 cytokine IL-13, [166], IL-4  (mast cell cytokine) [167] as well as IL-6 and IL-23 (Th17 differentiation cytokines) have been found, and these cytokines can  interact with TGF-β and MMP activity to increase the expression of ECM proteins as well as the potential migration and invasiveness of fibroblasts from asthmatics compared to controls as shown in vitro [168]. In line with the interaction between inflammatory mediators and MMPs, an imbalance between the production of tissue inhibitor of metalloproteinases (TIMP)-1 and MMP-1 activity correlates with an increased procollagen synthesis in fibroblasts in the asthmatic airway [169]. In addition to a higher rate of collagen synthesis, there is increased production of the proteoglycans versican and biglycan in asthma-derived airway fibroblasts [170]. This imbalance has been proposed to lead to fibrotic lesions in the airways of asthmatic patients [170].   Taken together, these studies demonstrate important alterations in the epithelium and mesenchymal cells in the airways of both asthmatic and COPD patients. What is unclear at this time is how these alterations affect the EMTU and what their role in disease pathogenesis is.  30  1.9.3 Aberrant epithelial-fibroblast communication in asthma and COPD In vivo data related to the EMTU in asthma and COPD has been primarily focused on imaging the structure of airway biopsies. In asthma, thickening of the basement membrane in remodeled airways has long been proposed to result in disturbed communication between epithelial cells and fibroblasts, leading to aberrant activation of the EMTU [106]. Using transmission electron microscopy Behzad et al, demonstrated that the cytoplasmic extensions between alveolar epithelial cells and parenchymal fibroblasts are significantly reduced in COPD patients compared to smokers without COPD, due to excessive collagen deposition [171]. On the other hand, biopsies of the conducting airways from current smokers and COPD patients compared to controls have been shown to have reticular basement membrane fragmentation (RBM), which may cause an increased exposure of resident fibroblasts to the release of mediators from the epithelium seen during cigarette smoke exposure [172]. This may indicate that the communication between cells within the EMTU in the conducting airways and parenchymal tissue may be affected in different ways.  Due to the complexity of cellular cross-talk in vivo, in vitro co-culture systems have primarily been used to study cellular communication within the EMTU. From early work on the normal EMTU, it was shown in a co-culture model that proliferation and differentiation of the airway epithelium is affected by fibroblast mediator release, while in turn the proliferative capacity of fibroblast was regulated by the airway epithelium [173]. A later study of the airway EMTU by Thompson and colleagues in 2006 showed that the release of active TGF-β2 following airway epithelial damage caused by a scrape wound elevated the expression of α-smooth muscle actin and tenascin-C, and increased the expression of fibrillar collagen in lung fibroblasts when embedded in collagen I gels [174]. Further, mechanical stress comparable to those generated during broncho-constriction, applied to the airway epithelium was found to induce increased concentrations of 31  Endothelin 1 and 2 as well as TGF-β2 which causes fibroblast activation and profibrotic changes [175].    With regard to COPD, Sun and colleagues found by the use of a co-culture model that involved cigarette smoke extract (CSE) exposure that an increased release of the LL-37, a member of the human cathelicidin anti-microbial peptide family from the bronchial epithelium, upregulated collagen production in human lung fibroblasts [176]. This effect was mediated by the formyl peptide receptor-like 1 (FPRL)-dependent extracellular signal-regulated kinase (ERK) pathway and was due to an aberrant epithelial-fibroblast interaction, since direct stimulation of lung fibroblasts with CSE did not affect the expression of collagen [176]. In terms of alterations in inflammatory mediators within the COPD EMTU, Suwara and colleagues showed that epithelial damage via thapsigargin to simulate endoplasmic reticulum (ER) stress and H2O2 to induce reactive oxidative species (ROS) caused an increased release of the alarmin IL-1α. Exposing lung fibroblasts to conditioned medium from these damaged bronchial epithelial cells induced IL-1α-dependent release of pro-inflammatory mediators CXCL8 (IL-8) and IL-6 [177]. The use of polycytidylic acid (poly I:C) as a viral mimic has also been shown to amplify bronchial epithelial-derived IL-1α-mediated release of CXCL8 (IL-8) and IL-6 from lung fibroblasts, indicating that multiple stimuli can alter EMTU communication [177].   To assess the contribution of epithelial-fibroblast interaction in the pathogenesis of asthma, Reeves and colleagues used differentiated airway epithelium and lung fibroblast co-cultures [178]. Here, they demonstrated that the airway epithelium suppresses the production of ECM proteins and profibrotic mediators such as collagen Iα1, collagen 3α1 and hyaluron synthase 2 by lung fibroblasts. Asthma-derived airway epithelium was less able to suppress the expression of these pro-fibrotic molecules [178]. It was further shown that this defect may be due to a decreased 32  expression of prostaglandin E2 synthase in parallel with a higher TGF-β2 release in the asthma-derived airway epithelium [178]. In line with this, Hostettler et al, used conditioned medium to show that airway epithelial cell-derived PGE2 acting in synergy with TGF-β reduced the proliferation of fibroblasts by almost 50% [179]. Further to this, Reeves et al, again used co-cultures to demonstrate that compared to controls the airway epithelium from asthmatic children is less able to suppress the expression of fibroblast to myofibroblast transition (FMT) markers, such as α-smooth muscle actin and tropomysin-I in lung fibroblasts [180]. This defect in epithelial regulation was again due to an increased production of TGF-β2, which emphasizes the importance of the TGF-β family in the aberrant communication that drives airway remodeling in the asthmatic EMTU [180]. TGF-β has also been shown to interact with other pro-inflammatory mediators [181]. A cytokine implicated in this interaction is IL-1, which is a master regulator with both inflammatory and fibrogenic effects [181, 182].   1.10 IL-1 Signaling and Role in Asthma and COPD The role of various mediators in driving aberrant repair and remodeling processes in the lung EMTU has been studied in various models as described above. One important regulatory cytokine that has been shown to have important roles in asthma and COPD inflammation and remodeling is interleukin (IL)-1[183-189]. In the lungs, IL-1 plays an essential role in the normal immune defense against inhaled particles and infections [190]. There is great overlap in the activation of the IL-1 pathway after IL-1/IL-1 receptor (IL-1R) coupling and the lipopolysaccharide (LPS)/Toll-like receptor (TLR) 4 signaling pathway, which is important for immune defense against infections in the lung [191]. The airway epithelium constitutively produces and stores IL-1α at the plasma 33  membrane, primarily as a membrane-associated form [190, 192]. However, when stimulated IL-1α can be rapidly shuttled into the nucleus of the cell where it binds strongly to DNA [182]. In apoptotic conditions, IL-1α in the cytosol moves into the nucleus and remains bound to chromatin. In necrotic conditions, however, IL-1α is released after being made available in the cytosol to act as a bona fide ‘alarmin’, which induces sterile inflammation by activating the synthesis and release of a wide variety of chemokines and growth factors [191]. The receptors for IL-1 can be found on most immune and structural cells in the lung and acts in concert with other innate cytokines such as TNF-α to activate NF-B which has been shown to be critical also for the activation of the adaptive immunity.   While mesenchymal cells such as lung fibroblast may not be the major source of IL-1 in the EMTU, IL-1 acts on these cells in a robust way to affect airway inflammation and remodeling in both asthma and COPD. Indeed, IL-1 stimulates fibroblasts in the mesenchyme to secrete cytokines such as CXCL8 (IL-8), TGF-β, PDGF and MCP-1, which directly affects ECM production and repair in the lungs [193]. IL-1β has been demonstrated as a major inducer of cyclooxygenase-2 (COX-2) and PGE2 in human lung fibroblasts, which can stimulate or inhibit the proliferation of fibroblasts at specific concentrations, demonstrating a biphasic role of IL-1β-induced PGE2 release from fibroblasts [194]. The effects of IL-1 have also been demonstrated to be important for the regulation of wound repair and fibrosis by interacting with TGF-β, which is a hallmark growth-factor in fibrosis and airway remodeling. Here, TGF-β has been shown to increase the survival of myofibroblasts by inhibiting IL-1β induced apoptosis [155] while IL-1β also inhibits TGF-β1 -dependent myofibroblast transformation [181]. This point to an important balance between IL-1 and TGF-β signaling in the lungs during repair processes which could be dysregulated in diseases such COPD and asthma. In asthma, an increased production of IL-1 has 34  been associated with airway hyperresponsiveness, smooth muscle hyper-contractility and TH2 activation [187-189]. In COPD an increased release of IL-1 is involved in the release of chemo-attractants that drives neutrophilic inflammation and small airway disease [183-186].   1.10.1 IL-1 signaling The IL1 gene cluster is found in a loci on chromosome 2q12-13 spanning a 360 kb region [195]. There are currently 11 known members of the IL-1 superfamily which include 7 agonists; IL-1α, IL-1β, IL-18, IL-33, IL-36α, IL-36β and IL-36γ, 3 receptor antagonists; IL-1 receptor antagonist (IL-1Ra), IL-36Ra and IL-38 and IL-37 which is an anti-inflammatory cytokine. IL-1 family members exert their biological functions by binding to a variety of IL-1 (R) receptors including IL-1R1, the decoy IL-1R2, IL-1R accessory protein (IL-1RAcP), ST2 (IL-1R4), IL-1R5, IL-1R6, IL-1R7, single Ig IL-1-related receptor (SIGIRR) also referred to as IL-R8, IL-R9 and IL-1R10 [182].  Although both IL-1α and IL-1β are distinctly encoded by different genes, IL1A and IL1B, both activate the same pathway by binding to IL-1R1 and are synthesized as 31-kDa precursor molecules missing leader sequences [192]. Pro-IL-1α is biologically active but can undergo cleavage by calpain, a cysteine protease activated by calcium to generate an 18KDa mature form which is also biologically active. On the other hand, pro-IL-1β requires cleavage by caspase-1 (also termed IL-1β converting enzyme) which is derived after processing by the NLRP3- inflammasome complex to generate the mature 17kDa biologically active IL-1β [192]. Of the other IL-1 family members important to asthma and COPD pathogenesis, IL-33 is synthesized as a 30kDa protein which is constitutively expressed in several structural cells including the airway epithelium and signals through the ST2 receptor found on structural cells such as fibroblasts and 35  immune cells such as mast cells, basophils and T cells [182]. IL-33 partly resides in the nucleus but can be released during necrosis and cell damage to act as an alarmin [196]. Another IL-1 family member, IL-18, is also expressed in its pro-form in most mesenchymal cells. IL-18 is membrane-associated in monocytes and signals through the IL-18R [196]. With regard to biological signaling of IL-1 and its family members, 4 receptor complexes are formed; IL-1R (IL-1R1/IL-1RAcp), IL-33 receptor (ST2/IL-1RAc), IL-18 receptor complex (IL-18Rα/IL-18Rβ) and IL-36 receptor complex (IL-36R/IL-1RAcp). These receptor complexes signal through a similar IL-1 pathway involving intracellular toll interleukin receptor (TIR) domains and the recruitment of the adaptor MyD88 [196].  Specifically for classical IL-1 signaling through IL-1R, a conformational change in IL-1R after it binds to IL-1 allows for a heterodimer complex with the IL-1 receptor accessory protein (IL-1RAcP) [197]. This leads to the accumulation of TIR domains adjacent to the complex that causes the recruitment of adaptor proteins including MyD88, IL-1R associated kinase (IRAK)-1 and tumour necrosis factor (TNF) receptor associated factor (TRAF)-6. Activated signal transduction pathways leads to the nuclear translocation of transcription factors such as p38 mitogen-associated protein kinase (MAPK), c-Jun N-terminal kinase (JNK), activator protein (AP)-1 and nuclear factor (NF)-B that promotes the transcription of a wide variety of inflammatory cytokines and growth factors [198, 199] (figure 1.6). Interestingly, IL-1α can also act as a transcription factor itself and has been labeled as “dual function cytokine” [182, 200]. Another mediator that also found to belong to this dual function family is fellow IL-1-family member IL-33 [200].   36   Figure 1.6 Classical IL-1 signaling. IL-1α and IL-1β both share the same receptor IL-1 receptor (IL-1R)-1. IL-33 binds to the ST2 receptor while IL-18 binds to the IL-18 receptor (IL-18R)-β. The binding of these receptors by their respective ligands cause a formation of receptor complexes with the IL-1R accessory protein (IL-1RAcp). The formation of these receptor complexes leads to the recruitment of the adaptor Myeloid differentiation primary response gene 88 (MYD88) which leads to the further recruitment of IL-1R associated kinase (IRAK)-1 and tumour necrosis factor (TNF) receptor associated factor (TRAF)-6. This leads to the phosphorylation of inhibitory- B kinase (IKK) -α, IKK-β and IKK-γ that leads to the degradation of inhibitory- B kinase, the translocation of the nuclear factor- (NF-) B subunits p65 and p60 into the nucleus and the transcription of various inflammatory genes. Other nuclear transcription factors that may be activated include activator protein-1 (AP-1), c-June N-terminal kinase (JNK) and p38 mitogen-associated protein kinase (MAPK).   1.10.2 Regulation of IL-1 signaling The signaling and activity of the IL-1 family members is regulated at various levels by other family members and receptors as well as by other epigenetic factors (figure 1.7). In line with this, the 37  discovery of IL-1 receptor (IL-1R) 2 as a decoy biologically inert receptor which binds to and prevents IL-1α and IL-1β signaling was a shift in the classical definition of a receptor put forward by John N. Langley in 1906 [182, 201]. Likewise, the IL-18 binding protein (IL-18BP) is similar in structure and function to the IL-1R2 which inhibits the activity of IL-1 family member, IL-18 which is important for the production of IFN-γ [182]. Again, the inflammatory effects of IL-1 family member IL-33, is controlled by the soluble ST2 (sST2) and IL-1RAcP (sIL-1RAcP) as well as Toll/IL-1R domain (TIR) 8 that bind to and inhibits the activity of IL-33 [202]. The IL-1 family also has 2 receptor antagonists that compete for binding and regulate the biological function of family members. Here, the IL-1 receptor antagonist (IL-1) Ra further regulates IL-1 signaling by competing with IL-1 for the IL-1R1 binding site. IL-1Ra binds to IL-1R1 with comparable avidity as IL-1 and prevents activation of the signaling pathway [192]. The IL-36 receptor antagonist (IL-36) Ra is the other receptor antagonist in the IL-1 family which has been shown to bind to the IL-1Rrp2 and inhibits the effects of IL-36 [203, 204].   In addition to self-regulation by IL-1 family members, the function of IL-1 can also be regulated by various epigenetic factors. Here, it has been shown that methylation of specific CpG sites on the IL-1β promoter leads to a suppression of its transcription in some mesenchymal cells [205]. Further, an increased methylation of the IL1R2 gene loci in asthma has been associated with a lower expression of IL-1R2 which may add to increased IL-1 activity in patients [206]. As part of other epigenetic regulatory mechanisms, IL-1 expression and production has also been shown to be a target of various miRNAs, including miR-149 and miR-146a in chronic inflammatory diseases including COPD [207, 208]. These miRNAs have been shown to down-regulate the expression and induction of IL-1 and have been suggested as possible targets for new therapy to control aberrant IL-1 signaling in disease. 38    Figure 1.7 Regulation of IL-1 signaling. The regulation of IL-1 signaling includes anti-inflammatory family members and epigenetic mechanisms such as miRNA regulation. The IL-1 receptor antagonist (IL-1RA) competes with IL-1α and IL-1β and binds to the IL-1R1 receptor to prevent IL-1 signaling. The decoy IL-1 receptor (IL-1R) II which can be membrane bound or in a soluble form also binds IL-1α and IL-1β but lacks a cytoplasmic domain that can initiate signaling. The soluble ST2 receptor binds to IL-33 and prevents the stimulation of the membrane bound ST2 receptor while the IL-18 binding protein (IL-18Bp) binds and prevents IL-18 from binding to IL-18Rβ. IL-1 signaling can also be controlled by epigenetic mechanisms such as the increased expression of miR-146a which binds and down-regulates IRAK-1 and TRAF-6 to prevent biological signaling.   1.11 MiRNAs in Asthma and COPD MiRNAs are part of an endogenous mechanism that controls various aspects of cellular behavior including cellular interaction [209]. They are small non-coding RNA molecules that range from 21 to 23 nucleotides in length and form part of the epigenetic regulation in cells [209]. MiRNAs regulate target genes through post-transcriptional modification by binding to the 3’ untranslated region (UTR) of mRNA and causing degradation through the RNA-Induced Silencing Complex 39  (RISC) as well as inhibition of protein translation [210]. A dysregulation of various miRNAs has been suggested to be involved in the pathogenesis of both asthma and COPD. In various asthma models miRNAs have been shown to be involved in disease pathogenesis. As an example, there is a decreased expression of miR-570-3p in asthmatics compared to controls which correlate with lung function decline [211] while there is lower induction of let 7g in the airway epithelium of HDM sensitized mice [212]. Further, increasing the expression of let 7g in the lung epithelium led to an attenuation of the HDM-induced increased in α-SMA expression in the lung mesenchyme, demonstrating the importance of let-7g in airway remodeling in asthma [212]. On the other hand there was an elevated expression miR-146a, miR-146b, miR-150, miR-181a and miR-155 in CD4+ T cells [213] in an OVA-sensitized mouse model of asthma compared to controls. Taken together, this indicates a possible role of miRNA regulation in the pathogenesis of asthma.  As in asthma, a dysregulated miRNA control has been shown to also contribute to various aspects of COPD pathogenesis including the role in driving cigarette responses, chronic inflammation, emphysema and airway remodeling. As we focused on understanding the involvement of miRNAs in the cross-talk between the airway epithelium and fibroblasts in COPD, we have reviewed relevant literature in this field in Chapter 2.    1.12 Scope of Thesis An aberrant epithelial-fibroblast communication has been suggested to drive the chronic inflammation and remodeling seen in both asthma and COPD. However, over the years studies have focused on the individual roles of epithelial and fibroblast dysregulation in disease pathogenesis rather than understanding how these defects within the lung EMTU will affect 40  epithelial-fibroblast communication and how this could contribute to disease pathogenesis. Thus in this thesis we hypothesize that a “dysregulated epithelial-fibroblast cross-talk, drives chronic inflammation and airway remodeling in the lung EMTU and contributes to the pathogenesis of asthma and COPD”.  As part of the possible factors that could have great impact on epithelial-fibroblast communication in a chronic inflammatory disease such as COPD, microRNAs could serve as major player in regulating the activity of various cytokines. Hence in chapter 2, we provide a detailed assessment of the role of miRNAs in COPD pathogenesis and link the expression of miRNAs to various features of the disease. In chapter 3 we developed a co-culture model of airway epithelial cells and lung fibroblasts to assess the specific mediators through which the cells communicate in the normal lung EMTU and found epithelial-derived IL-1α drives fibroblast responses. We assessed the effect of the release of epithelial-derived IL-1α on fibroblast-derived inflammation and the expression of ECM molecules. Next we determined the effect of cigarette smoke exposure, the major risk factor of COPD on this communication to determine the relevance for disease pathogenesis of COPD. A failure of regulatory mechanisms of the IL-1 pathway has been implicated in the increased activity of IL-1 in some chronic inflammatory diseases such as COPD. In chapter 4 we assessed the effect of miR-146a-5p a known epigenetic regulator of the IL-1 pathway, on the aberrant epithelial-fibroblast crosstalk in COPD. We assessed the expression of miR-146a-5p in lung fibroblasts in response to epithelium derived IL-1α and through functional assays, determined the mechanism of regulation of fibroblast-derived inflammation comparing COPD to control individuals.  41  In chapter 5 we assessed the possible effect of airway epithelial repair on the release of IL-1 family members in the lung EMTU of asthmatics. Further, we examined the possible role of IL-1 in driving an abnormal repair phenotype of airway fibroblasts in asthma compared to control individuals. This was assessed by the use of functional collagen contraction assays as well as high powered multimodal non-linear optical microscopy (NLOM) imaging.  In chapter 6, we then examined the role of dysregulated collagen fiber repair by airway fibroblasts in driving excessive remodeling in the lung EMTU of asthma patients. We used high powered multimodal non-linear optical microscopy (NLOM) imaging which allows for visualization of fibrillar collagen (second harmonic generation) and elastic (two-photon excited auto fluorescence) fibers to study for the first time, the morphological changes in fiber organization and repair in the EMTU of asthmatics compared to normal individuals. We further used functional collagen contraction assays to assess the potential contribution of fibroblasts to this defective repair and remodeling processes in asthma.  In chapter 7 a general discussion, summary and implications of findings in chapter 3  to chapter 6 together with future perspectives are provided. These data provide new dimensions for understanding of pathogenesis and possible future targets for therapeutic research in asthma and COPD.  42  Chapter 2:  Unravelling The Complexity of COPD by MicroRNAs; It’s A Small World After All  Emmanuel T. Osei1,2,4, Laura Florez-Sampedro1,2,4, Wim Timens1,2, Dirkje S. Postma2,3, Irene H. Heijink1,2,3,5 and Corry-Anke Brandsma1,2,5  Affiliations:  1University of Groningen, University Medical Center Groningen, Dept of Pathology and Medical Biology, Groningen, The Netherlands. 2University of Groningen, Groningen Research Institute for Asthma and COPD (GRIAC), University Medical Center Groningen, Groningen, The Netherlands. 3University of Groningen, University Medical Center Groningen, Dept of Pulmonology, Groningen, The Netherlands. 4Both authors contributed equally as first authors. 5Both authors contributed equally as last authors.   43  2.1 Chapter Summary Chronic obstructive pulmonary disease (COPD) is a progressive lung disease and is currently the fourth leading cause of death worldwide. Chronic inflammation and repair processes in the small airways are characteristic of COPD. Despite extensive efforts from researchers and industry, there is still no cure for COPD, hence an urgent need for new therapeutic alternatives. MicroRNAs are such an option; they are small noncoding RNAs involved in gene regulation. Their importance has been shown with respect to maintaining the balance between health and disease. Although previous reviews have discussed the expression of microRNAs related to lung disease, a detailed discussion regarding the function of differential miRNA expression in the pathogenesis of COPD is lacking.  In this review we link the expression of microRNAs to different features of COPD and explain their importance in the pathogenesis of this disease. We further discuss their potential to contribute to the development of future therapeutic strategies.   2.2 Introduction Chronic obstructive pulmonary disease (COPD) is a chronic heterogeneous disease of the lungs characterized by persistent and excessive inflammation, leading to tissue remodeling, alveolar lesions, airflow limitation and accelerated lung function decline[10]. Over 300 million people suffer from COPD. It is currently the fourth leading cause of death worldwide and predicted by the World Health Organization to become the third leading cause by 2030 [214]. Cigarette smoking is the predominant risk factor for COPD, while exposure to other noxious gases has also been identified as a risk factor. Although smoking cessation can delay disease progression, there is no cure for COPD and current medication cannot reverse the long-term decline in lung function.  44  Inhaled cigarette smoke first encounters the airway epithelium, which forms a continuous and highly regulated barrier. The exposure of airway epithelium to noxious particles like cigarette smoke causes the release of pro-inflammatory mediators, including damage-associated molecular patterns and cytokines like interleukin (IL)-1, IL-8 and tumor necrosis factor (TNF)-α [10, 31, 215]. Inflammatory cytokines cause lung infiltration by inflammatory cells that further amplify the inflammation and release proteases and reactive oxidative species that damage the parenchymal lung tissue, contributing to emphysema development in COPD patients [9]. Repeated injury leads to fibroblast activation, which causes excessive extracellular matrix deposition and remodeling of the small airways in COPD [216]. However, fibroblasts appear unable to provide adequate tissue repair after smoke-induced damage in the parenchyma of COPD lungs [9].  Besides the inflammatory and structural cells in the lung and pro-inflammatory mediators that contribute to the pathogenesis of COPD, microRNAs (miRNAs) have recently been implicated in COPD pathogenesis [217]. MiRNAs are small noncoding RNAs of ~19–25 nucleotides. MiRNAs can cause post-transcriptional gene repression either by increasing mRNA degradation or by inhibiting protein translation of specific mRNA targets.  Currently, over 2400 mammalian miRNAs (listed in miRBase release 21 [218, 219]) are known to be expressed in a wide variety of cell types and have been predicted to target about 60% of mammalian mRNAs [216].  There is large overlap in gene regulation by miRNAs, as several miRNAs can regulate a single gene, and multiple genes can be affected by a single miRNA [217]. Potential miRNA targets can be predicted with bioinformatics tools that recognize complementary elements in the 3' untranslated region (UTR) of genes and in the seed sequence of the miRNAs [220]. However, evidence suggests that this sequence pairing is not necessarily a reliable predictor of a miRNA 45  function, since it only compares the sequences; it does not consider other variables and interactions that are present in vivo [220]. Therefore, experimental validation of miRNA targets is crucial to understand mRNA function. The most direct way of miRNA target validation is by luciferase assays, where the 3'UTR of the mRNA of interest is cloned into the 3' region of a luciferase reporter plasmid for further evaluation of target gene expression in cells that are transfected with these constructs [220]. Another, indirect, way of validating miRNA targets is by assessing the effect of miRNA up- or downregulation on protein expression of predicted targets. In the present review, differences between validated and predicted targets are indicated.  MiRNA expression levels can be determined by different techniques such as small RNA sequencing, microarray hybridization and quantitative reverse transcriptase PCR (qRT-PCR). The first two techniques are unbiased screening approaches that provide a large number of results at once, giving an estimate of the overall changes in miRNA expression profile between different samples. In general, the key findings of these unbiased approaches are validated using a miRNA-specific qRT-PCR approach, which is a more sensitive and quantitative method.  Regulation by miRNAs is a form of epigenetic regulation that involves inherited alterations in gene expression, which cannot be attributed to modifications in the DNA sequence. Aside from the regulation by noncoding RNAs of which miRNAs are part, epigenetic mechanisms include histone modification and DNA methylation [221]. These mechanisms do not only individually affect gene expression, but can also interact to affect disease pathogenesis. For example, the methylation of CpG islands in the promoter regions of miRNAs can inhibit miRNA transcription, while miRNAs can target and inhibit the activity of DNA methyltransferases [222, 223].  The presence and stable expression of miRNAs in serum, plasma, sputum, urine and several organ tissues and the role of their dysregulation in disease indicate their importance, 46  allowing further exploration of their use as biomarkers and therapeutic targets [224]. The first successful trials using miRNAs as therapeutic targets in liver disease have been reported [225].  Although some recent reviews have discussed the role of miRNAs in lung diseases, a detailed review focusing on the specific role of miRNAs in COPD pathogenesis is lacking. Hence, in this review we will provide an overview of the studies published on differential miRNA expression in COPD and healthy individuals, and we will discuss the role of specific miRNAs involved in different aspects of COPD pathogenesis. We will cover the following topics: 1) the effects of cigarette smoke exposure on miRNA expression; 2) the potential roles of miRNAs in the excessive inflammatory response, the presence of emphysema and the tissue repair response in COPD; 3) the association of miRNAs with lung function; and 4) the potential of miRNAs as therapeutic targets in COPD. Overviews of the discussed miRNAs can be found in tables 1 and 2 and a schematic overview is shown in figure 1.  47     Figure 2.1 Differential regulation of microRNAs in chronic obstructive pulmonary disease (COPD). A schematic representation of the pathophysiology of COPD with the various differentially regulated microRNAs highlighted. ROS: reactive oxidative species; TGF: transforming growth factor.    48  Feature MicroRNA Finding Ref. Smoke exposure miR-101 ↑ in bronchial epithelium from COPD patients versus controls and in 16HBE cells with CSE [226] miR-144 ↑ in 16HBE cells with CSE [226] miR-200c ↓ in CSE-exposed human bronchial epithelial cells [227] miR-7# ↑ in CSE-exposed airway smooth muscle cells [228] let-7c#, miR-34c# and miR-222 ↓ with CS exposure in rat lungs [229] miR-135b ↑ with CS exposure in mouse lungs [230] miR-146a, miR-92a-2*, miR-147, miR-21 and miR-20 ↑ with CS exposure in rat lungs [231] miR-21 ↑ with CS exposure in rat lungs and in serum of COPD patients and control smokers [231] miR-181a ↓ with CS exposure in rat lungs and in serum of COPD patients and control smokers [231] miR-340 ↓ in induced sputum of control smokers versus control nonsmokers [232] let-7c# and miR-125b ↓ in induced sputum of COPD smokers versus control nonsmokers [232]  miR-210, miR-150, miR-146b-3p and miR-452 ↓ in BAL macrophages of smokers versus nonsmokers [233] miR-218#, miR-128b and miR-500 ↓ in bronchial airway epithelial cells from current smokers versus never-smokers [234] miR-181d ↑ in bronchial airway epithelial cells from current smokers versus never-smokers [231] miR-34b#, miR-345, miR-421, miR-450b, miR-466 and miR-469 ↓ with CS exposure in mouse lungs and not reversed after 1 week CS cessation [235] COPD miR-20a, miR-28-3p, miR-34c-5p and miR-100 ↓ in serum of COPD patients [224] miR-7# ↑ in serum of COPD patients [224] miR-29b, miR-483-5p, miR-152, miR-629, miR-26b, miR-101, miR-106b, miR-532-5p and miR-133b ↓ in plasma of COPD patients [236] miR-518b ↑ in in whole blood samples of COPD patients [237] miR-15b#, miR-132, miR-145, miR-212, miR-223, miR-342-5p, miR-422a, miR-423-5p, miR-425 and miR-486-3p ↑ in BAL cell fraction of COPD patients [238]     49   Table 2.1MicroRNAs involved in different features of chronic obstructive pulmonary disease CSE: cigarette smoke extract; CS: cigarette smoke; BAL: bronchoalveolar lavage; Tregs: regulatory T-cells; FEV1: forced expiratory volume in 1 s; IL: interleukin; TNF: tumour necrosis factor. #: microRNAs that have more than one association with different traits of COPD.   2.3 The Effects of Cigarette Smoke Exposure on miRNA Expression As cigarette smoking is the major risk factor for COPD, we first describe the effects of cigarette smoke exposure on miRNA expression profiles. Several studies using different models have shown that direct exposure to cigarette smoke or exposure to cigarette smoke extract (CSE) affects the miRNA expression in the lungs.  Feature MicroRNA Finding Ref. COPD miR-29, miR-34a#, miR-98, miR-146b-5p, miR-193a-5p, miR-218#, miR-324-5p, miR-342-3p and miR-365 ↓ in BAL cell fraction of COPD patients [238]  miR-15b#, miR-223, miR-1274a and miR-424 ↑ in lung tissue samples of COPD patients [239]  miR-199a-5p# ↓ in Tregs from COPD patients [240]  miR-1# ↓ in quadriceps of COPD patients [241]  miR-199a-5p# and miR-34a# ↑ in lung tissue samples of COPD patients compared with nonsmoking controls [242] Lung function let-7c#, miR-34b#, miR-34c#, miR-125a-5p, miR-30e-3p and miR-30a-3p Positively correlated with FEV1 in sputum [232] miR-1# Positively correlated with FEV1 in skeletal muscle cells [241] miR-199a-5p# and miR-34a# Negatively correlated with FEV1 [242] Inflammation miR-146a ↓ in IL-1β/TNF-α-stimulated primary lung fibroblasts from smokers with COPD compared with smokers without COPD [243] Emphysema miR-520e, miR-302d, miR-92a, miR-638, miR-211 and miR-150 ↑ with decreased mean linear intercept (thus more emphysema) in lung tissue samples [244] let-7c#, let-7d, let-7e, let-7f, miR-181c, miR-181d, miR-30a-3p, miR-30c, miR-30e-5p and miR-30e-3p ↓ with decreased mean linear intercept (thus more emphysema) in lung tissue samples [244] miR-15b# Localizes in emphysematous areas in the lung [239] miR-34c#, miR-34b#, miR-149, miR-133a and miR-133b ↓ in lung tissue samples of COPD patients with moderate compared with mild emphysema [245] Tissue repair miR-15b# Localizes in fibrotic areas in the lung [239] 50  An in vitro study by Hassan et al. [226] evaluated the effect of CSE on the human bronchial epithelial cell line 16HBE and showed that 24-h CSE exposure induces upregulation of miR-101 and miR- 144 expression. These two miRNAs are predicted to target the cystic fibrosis transmembrane conductance regulator [226], which has previously been shown to have a reduced function upon cigarette smoke exposure [246]. Interestingly, they also found a higher miR-101 expression in slides of lung tissue samples from COPD patients compared with controls, suggesting that this miRNA may also play a role in COPD pathogenesis [226]. Another in vitro study showed that CSE exposure of human bronchial epithelial cells leads to a decrease in miR-200c expression [227]. This miRNA was suggested to maintain the epithelial phenotype as it was shown to target the expression of the E-cadherin transcriptional repressors ZEB1 and ZEB2 [247, 248]. Therefore, a decrease in miR-200c expression may contribute to an increase in epithelial to mesenchymal transition (EMT). EMT represents the loss of epithelial properties and the acquisition of mesenchymal cell characteristics, associated with pathological processes and tissue remodeling. EMT has previously been linked to COPD and cigarette smoke exposure [120, 249]. The expression of miR-7 was also found to be altered by CSE. MiR-7 expression was significantly increased in CSE-exposed human airway smooth muscle cells, while this was not observed for 16HBE cells and MRC5 fibroblasts [228]. Overexpression of miR-7 in human airway smooth muscle cells led to decreased protein levels of exchange protein directly activated by cAMP (Epac)1, a predicted target for miR-7 that has previously been shown to be decreased upon CSE exposure and to have a low expression in COPD lung tissue samples [250].  In addition to in vitro studies, several animal models have evaluated the effect of cigarette smoke exposure on the miRNA profile. A microarray analysis of a rat model of 4 weeks’ cigarette smoke exposure showed downregulation of 24 miRNAs and upregulation of one miRNA (miR-51  294) in lung homogenates [229]. From the downregulated miRNAs, the differential expression of three (let- 7c, miR-34c and miR-222) was validated by qRT-PCR. These miRNAs are predicted to regulate genes involved in cell proliferation and p53 regulation. It is noteworthy that these miRNAs have human homologues and thus may be involved in the response to cigarette smoke exposure in humans as well. In a mouse model of 4 days’ cigarette smoke exposure, miR-135b was shown by qRT-PCR to be increased in lung tissue samples [230]. The authors suggest that the upregulation of miR-135b may act as a counter-regulatory mechanism for cigarette smoke-induced inflammation, by regulating the IL-1 receptor (IL-1R1) expression. Xie et al. [231] studied the effects of 4 and 15 weeks’ cigarette smoke exposure in a rat model and demonstrated differential expression of 30 and 37 miRNAs, respectively, by miRNA array in lung homogenates. Upon validation by qRT-PCR, miR-146a, miR- 92a-2*, miR-147, miR-21 and miR-20 were shown to significantly increase at 4 and 15 weeks, while miR-181a significantly decreased upon cigarette smoke exposure. Of interest, both miR-146a and miR-181a have been linked to “inflammaging”, an ageing-related state characterized by systemic chronic inflammation [251]. Since the authors also found significantly increased serum levels of miR-21 and decreased miR-181a expression in heavy smokers and COPD patients compared with healthy controls [231], they suggested that a high ratio of serum miR-21 to miR-181a expression may be a risk factor for the development of COPD and can be associated with the risk of developing COPD in heavy smokers.  Studies that have analyzed samples from human smokers have also contributed to understanding the effect of cigarette smoke exposure on the miRNA expression profile. Van Pottelberge et al. [232] analyzed the miRNA expression profile in induced sputum of nonsmoking and smoking controls and smoking COPD patients; their analysis was based on comparing the miRNA expression profile of these groups in a screening cohort versus a validation cohort. When 52  comparing smoking and nonsmoking controls in the screening cohort, they found 34 differentially expressed miRNAs, most being decreased in smokers. In their validation cohort, only one of these miRNAs, miR-340, could be validated. When comparing smoking COPD patients versus nonsmoking controls, they initially found eight differentially expressed miRNAs of which two, let-7c and miR-125b, were validated. Another human ex vivo study performed miRNA array analysis on human alveolar macrophages from bronchoalveolar lavage (BAL) of smoking and nonsmoking individuals. Their analysis consisted of three subject cohorts, two of which were used for the screening and validation of the array results. This study showed an overall decrease in miRNA expression in smokers [233], with a decrease of 43 miRNAs and an increase of 11 miRNAs at a two-fold cut-off, in the first cohort. Three of the downregulated miRNAs (miR-210, miR-150 and miR-146b-3p) were validated by qRT-PCR in two of the cohorts. The most downregulated miRNA, miR-452, was followed up in vitro, where its expression was shown to be inversely related to the expression of its predicted target, matrix metalloproteinase (MMP)-12. This is of interest, since MMP-12 has been found to be increased in sputum of COPD patients [252] and contributes to the development of emphysema [253]. In addition to these studies, miRNA profiling on bronchial airway epithelial cells of current and never-smokers revealed differential expression of 28 miRNAs, most of which were downregulated in the current smokers [234]. Of these, miR-218, miR-128b, miR-500 and miR-181d were validated using qRT-PCR, and all were downregulated with smoking except miR-181d, which was upregulated. MiR-218 was found to be most strongly correlated with genes that were strongly upregulated by cigarette smoke exposure. Within the negatively correlated genes, there was an overrepresentation of miR- 218 predicted target genes involved in cell structure, cell–cell adhesion, and cell signaling and ion transport pathways [234].  53  Considering all the evidence from these studies, we propose that the cumulative effects of cigarette smoke on lung tissue damage and inflammation are reflected by an effect on the miRNA profile, and that smokers with and without COPD develop differential miRNA profiles. Interestingly, Izzotti et al. [235] demonstrated that cigarette smoke-induced miRNA changes in mouse lungs were dose dependent and only partly reversible, as the expression levels (by miRNA microarray) of several miRNAs, including miR-34b, miR-345, miR-421, miR-450b, miR-466 and miR-469, did not revert to basal levels after 1 week of smoking cessation. These findings suggest that the stability of alterations in some miRNAs after smoking cessation might be dependent on the cigarette smoke dose. In particular, irreversible changes could contribute to the development of COPD, although it is important to take into consideration that, due to the high variability of the genetic background that exists between humans, a high-dose long-term cigarette smoke exposure will not always lead to COPD. Again, it is also important to note that effects of smoking cessation in an animal model might not be comparable to those observed in humans. In line with this study, in collaboration with Bossé et al. [254], we have previously shown that smoking deregulates the levels of many genes in human lung tissue and that for most of the genes, expression levels are reverted to control levels upon smoking cessation. However, some genes, including serpin peptidase inhibitor clade D, member 1 (SERPIND1), showed no or a very slow reversal to basal levels.  Although most of the results from these studies do not point at the same miRNAs, some miRNAs have been associated with multiple features of COPD (table 2), and may thus be relevant for the pathogenesis of COPD.   54   Table 2.2 MicroRNAs involved in more than one feature of chronic obstructive pulmonary disease. CS: cigarette smoke; FEV1: forced expiratory volume in 1 s; CSE: cigarette smoke extract; GOLD: Global Initiative for Chronic Obstructive Lung Disease; BAL: bronchoalveolar lavage; Tregs: regulatory T-cells.  MicroRNA Feature Finding Ref. let-7c Smoke exposure ↓ with CS exposure in rat lungs [229] Lung function ↓ in COPD sputum and positively correlated with FEV1 [232] Emphysema ↓ with decreased mean linear intercept (thus more emphysema) in lung tissue samples [244] miR-1 COPD ↓ in quadriceps of COPD patients [241] Lung function Positively correlated with FEV1 in skeletal muscle cells [241] miR-7 Smoke exposure ↑ in CSE-exposed airway smooth muscle cells [217] COPD ↑ in serum of COPD patients [224] miR-15b COPD ↑ in lung tissue samples of COPD patients; higher in GOLD stage IV [239] Emphysema Localized in emphysematous areas in the lung [239] Tissue repair Localized in fibrotic areas in the lung [239] miR-34a COPD ↓ in BAL cell fraction of COPD patients [238] Lung function Negatively correlated with FEV1 [242] ↑ in lung tissue samples of COPD patients compared with nonsmoking controls miR-34b Smoke exposure ↓ with CS exposure in mouse lungs and not reversed after 1 week CS cessation [235] Lung function Positively correlated with FEV1 in sputum [232] Emphysema ↓ in lung tissue samples of COPD patients with moderate compared with mild emphysema [245] miR-34c Smoke exposure ↓ with CS exposure in rats lungs [229] COPD ↓ in serum of COPD patients [224] Lung function Positively correlated with FEV1 in sputum [232] Emphysema ↓ in lung tissue samples of COPD patients with moderate compared with mild emphysema [245] miR-199a-5p COPD ↓ in Tregs from COPD patients [240] Lung function Negatively correlated with FEV1 [242] ↑ in lung tissue samples of COPD patients compared with nonsmoking controls  miR-218 Smoke exposure ↓ in bronchial airway epithelial cells from current smokers versus never-smokers [234] COPD ↓ in BAL cell fraction of COPD patients [238] 55  2.4 Differential miRNA Expression in COPD To date, seven studies have reported differential miRNA expression in COPD patients in comparison with (smoking) control subjects. Overlap between these studies is rather limited and this is mainly due to different types of samples (i.e. serum, plasma, BAL, whole blood or lung tissue), small sample sizes, differences in (statistical) methods and inclusion criteria used. Using a qRT-PCR miRNA array approach, Akbas et al.[224] showed that four miRNAs (miR-20a, miR- 28-3p, miR-34c-5p and miR-100) were downregulated and one miRNA (miR-7) was upregulated in serum of COPD patients compared with smokers without COPD. Another study, evaluating miRNA expression levels in the plasma of healthy smokers and current smoking COPD patients, found nine differentially expressed miRNAs in COPD (miR-29b, miR-483-5p, miR-152, miR-629, miR-26b, miR-101, miR-106b, miR-532-5p and miR-133b) [236]. Using qRT-PCR, this study also showed that in ex-smoking COPD patients, miR-106b expression negatively correlates with the duration of COPD since diagnosis, whereas in current smoking COPD patients, miR-106b expression negatively correlates with years of smoking. Therefore, the authors suggest that plasma levels of miR-106b may reflect persistent and systemic changes even after smoking cessation in COPD.  A study by Leidinger et al.[237] focused on comparing miRNA profiles from whole blood of COPD patients, lung cancer patients and healthy volunteers. Here, 140 miRNAs were found to be differentially expressed between COPD and control, of which miR-518b was most significantly upregulated; this result, however, was not confirmed by qRT-PCR. Another study compared miRNA profiles by miRNA array in the cell fraction of BAL fluid of 23 COPD patients and 15 smokers and one ex-smoker without COPD. qRT-PCR validation was performed for a group of random miRNAs among those that were differentially expressed. The study found 10 miRNAs to 56  be upregulated (miR-15b, miR-132, miR-145, miR-212, miR-223, miR-342-5p, miR-422a, miR-423-5p, miR-425 and miR-486- 3p) and nine to be downregulated (miR-29, miR-34a, miR-98, miR-146b-5p, miR-193a-5p, miR-218, miR-324-5p, miR-342-3p and miR-365) in the COPD group when compared with the control group without COPD [237].  A miRNA profile analysis of lung tissue samples from COPD patients and smokers without airflow limitation showed differential expression of 70 miRNAs; 13 were downregulated and 57 were upregulated in COPD [239]. Four miRNAs (miR-15b, miR-223, miR-1274a and miR-424) were validated by qRT-PCR. Of those, miR-15b was highly expressed in lung tissue samples from COPD patients, with the highest expression in stage IV COPD patients compared with other stages of COPD and with control smokers. SMAD7, a protein of the transforming growth factor (TGF)-β pathway, which inhibits the production of collagen and other matrix proteins, was found to be one of the predicted targets for miR-15b that decreased upon miR-15b overexpression in the human bronchial epithelial cell line BEAS-2B. These results correlate with previous findings from our group, showing that SMAD7 protein expression is significantly lower in lung epithelium from stage II and IV COPD patients compared with control subjects, while other SMAD proteins did not significantly differ [255].  Another miRNA profiling study that analyzed regulatory T-cells (Tregs) showed that miR-199a-5p is downregulated in Tregs of COPD patients compared with control smokers and healthy nonsmokers; this result was confirmed with qRT-PCR [240]. Several of the predicted targets of this miRNA are strongly related to the TGF-β pathway. Additional in vitro experiments with a miR-199-5p inhibitor showed that this miRNA has potential targets belonging to bone morphogenetic protein signaling, which promotes TGF-β-induced Treg differentiation [256]. 57  Thus, the altered miR-199-5p expression might contribute to the dysregulation of pro-inflammatory T-cell-mediated inflammation in COPD.  The ultimate effects of the changes in the miRNome that differentiate COPD patients from smokers with normal lung function are possibly accumulating over time and may be related to the onset of disease and disease severity. With respect to the relationship of changes in miRNome to lung function, the expression of let-7c, miR-34b, miR-34c, miR-125a-5p, miR-30e-3p and miR-30a-3p has been positively correlated with forced expiratory volume in 1s (FEV1) [232]. Furthermore, Lewis et al. [241] evaluated the role of miR-1 in the skeletal muscle dysfunction that accompanies COPD. MiR-1 expression, evaluated by qRT-PCR, was decreased in skeletal muscle of COPD patients compared with controls and positively correlated to FEV1. Mizuno et al.[242] evaluated the expression of miR-199-5p and miR-34a in COPD, since these miRNAs were thought to be playing a role in regulation of hypoxia-inducible factor-1α, previously shown to be altered in COPD. This study found by qRT-PCR that miR-199-5p and miR-34a were upregulated in lung tissue samples from COPD patients with moderate to severe COPD compared with nonsmoking individuals with normal lung function, and this increased expression was clearly associated with impaired lung function. This suggests that these changes in miRNA profile might not only be related to cigarette smoke exposure, but are in fact changes associated with pathogenesis of disease and reflected by the reduced lung function present [242].   2.5 Involvement of miRNAs in Inflammatory Responses Although different miRNAs have been implicated in different aspects of the regulation of the inflammatory response, there is one miRNA in particular that has been directly linked to COPD 58  pathogenesis, miR-146a. Sato et al. [243] found that lung fibroblasts from COPD patients have a lower induction of miR-146a expression upon IL-1β/TNF-α stimulation in comparison with fibroblasts from healthy controls. In addition, they showed that downregulation of miR-146a expression increases the levels of its target, cyclooxygenase 2, subsequently leading to higher prostaglandin (PG)E2 production in lung fibroblasts. These findings are in line with the high levels of PGE2 in sputum of COPD patients and the association of higher PGE2 levels with more severe airflow limitation during progression of COPD [257]. Of note, cigarette smoke exposure has been shown to increase miR- 146a expression in murine pulmonary fibroblasts by a mechanism involving the nuclear factor- κB member RelB [258]. Various other studies have linked miR-146a to the regulation of inflammatory responses, showing that miR-146a targets IRAK1 and TRAF6, two adapter proteins involved in cytokine receptor signaling [259], and IL-1β-induced IL-8 production in human lung epithelial cell lines [260]. Furthermore, miR-146a negatively modulates the inflammatory mediators IL-6 and IL-8 in human skin fibroblasts [261].  There are other miRNAs that, although not directly related to the inflammatory response in COPD, have been linked to inflammation and to COPD separately. Among the most downregulated miRNAs in COPD found by van Pottelberge et al. [232] there are several miRNAs that are known to regulate inflammatory responses, including miR-125b, miR-150 and miR-203. MiR-125b was found to significantly reduce lipopolysaccharide (LPS)-induced pulmonary inflammation in a mouse model, as reflected by reductions in total and neutrophil cell counts [262]. Furthermore, higher miR-150 expression correlated with lower CXCL1 expression in mice, while miR-203 was found to target MyD88 in LPS-stimulated monocytes [263]. In addition, miR-181a and miR-20a, which are also downregulated in COPD [231], have been linked to inflammatory processes. MiR-181a regulates inflammatory responses associated with IL-1β levels in LPS-59  stimulated monocytes [264] and IL-8 levels in human fibroblasts [265]. Transfection of miR-20a mimics in human monocytes decreased the release of IL-1β and TNF-α, and injection of miR-20a mimics in mice led to a global decrease in IL-6 secretion in macrophages that were stimulated with LPS in vitro [266]. Considering that miR-125b, miR-150, miR-203, miR-181a and miR-20a are downregulated in COPD patients and that they all regulate inflammatory processes, it is possible that their alteration is involved in the pro-inflammatory phenotype observed in COPD.   2.6 Involvement of miRNAs in Emphysematous Lesions Emphysema is induced by chronic inflammation upon cigarette smoking and characterized by loss of alveolar tissue and a decrease in lung elastic recoil. In collaboration with the groups of Spira and Hogg, we were involved in the first study investigating the direct relationship between mRNA [267] and miRNA expression changes, both measured by microarrays, and emphysema severity as measured by micro-computed tomography scans [244]. Increased emphysema severity was associated with increased expression of 35 miRNAs, including miR-520e, miR-302d, miR-92a, miR-638, miR- 211 and miR-150. Increased emphysema severity was associated with decreased expression of 28 miRNAs, including let-7c, let-7d, let-7e, let-7f, miR-181c, miR-181d, miR-30a- 3p, miR-30c, miR-30e- 5p and miR-30e-3p. The expression of five miRNAs (miR-638, miR-18a-3p, miR-483-3p, miR-181d and miR-30c) was significantly correlated with more than 50 of their predicted target genes. Of those, miR-638, miR-30c and miR-181d had the most anti-correlated predicted target genes, and these target genes were enriched in pathways associated with emphysema, suggesting that these miRNAs may be important regulators of the gene expression changes associated with emphysema severity. Subsequent inhibition of miR-638 in primary lung 60  fibroblasts led to the modulation of pathways that were previously implicated in oxidative stress and accelerated lung ageing responses.  In a study by Ezzie et al.[239], in situ hybridization showed miR-15b localization in areas of emphysema as well as in areas of fibrosis, with increased expression in COPD patients compared with controls. Although the authors did not further investigate the role of miR-15b in emphysema, they found that miR-15b overexpression in bronchial epithelial BEAS-2B cells resulted in decreased SMAD7, decorin and SMURF2 protein expression. Our group has shown that stimulation with TGF-β1 and basic fibroblast growth factor induces a significantly more pronounced downregulation of decorin in cultured fibroblasts from patients with severe emphysema than from those with mild emphysema [158], and miR-15b may thus be related to the severity of emphysema in COPD.  Savarimuthu Francis et al.[245] evaluated miRNA expression by miRNA microarray in lung tissue samples of COPD patients with moderate and mild emphysema defined by gas transfer measurements. They found five miRNAs that were significantly downregulated in lung tissue from patients with moderate emphysema compared with mild emphysema: miR-34c, miR-34b, miR- 149, miR-133a and miR-133b. MiR-34c exhibited the largest difference. Although it is not clear if and how miR-34c may contribute to the severity of emphysema, the authors found that higher miR-34c expression correlated with lower mRNA expression of five of its predicted targets: MAP4K4, SERPINE1, ALDOA, HNF4A and ZNF3. Among these, SERPINE1 had the strongest correlation with the miR-34c expression. Since SERPINE1 is a protease inhibitor, it is possible that miR-34c expression in emphysema is involved in the disturbed protease–antiprotease imbalance that is characteristic of the pathogenesis of emphysema [245]. 61  2.7 Involvement of miRNAs in Tissue Repair There are several miRNAs that have been linked to the development of pulmonary fibrosis, but not many have been described as regulators of hampered tissue repair, airway remodeling and fibrosis in COPD. As mentioned, Ezzie et al.[239] reported increased expression of miR-15b in areas of fibrosis in lung tissue samples from COPD patients and found that miR-15b targets several genes from the TGF-β superfamily, including SMAD7.  Since tissue repair and the fibrotic response have common initiating events, it is possible that the same miRNAs are involved in their regulation. Therefore, it would be of interest to investigate whether miRNAs that have been associated with fibrosis are also involved in tissue repair and airway remodeling in COPD. Potential candidates are miR-21 [231] and let-7 [229, 232, 244] since these were found to be involved in repair of damaged lungs after influenza infection [268], while miR-21 also mediates the fibrogenic activation of pulmonary fibroblasts [269]. Huleihel et al. [270] showed that the overexpression of let-7d in lung fibroblasts induced a delay in tissue repair and reduced motility and proliferation of fibroblasts. Furthermore, let-7d attenuated the expression of TGF-β downstream targets such as the high-mobility group AT-hook 2 (HMGA2) and α-smooth muscle actin (α-SMA) [270]. Several members of the let-7 family, including let-7d and let-7c, were associated with emphysema severity in lung tissue samples [244], and let-7c was found to be decreased in sputum of COPD patients and correlated positively with FEV1 [232].  Other miRNAs that could be of importance for tissue repair in COPD are miR-29b (downregulated in COPD) and miR-145 (upregulated in COPD) [238, 271]. MiR-29b has been found to mediate TGF-β1- induced extracellular matrix synthesis through activation of the PI3K-AKT pathway in human lung fibroblasts [272]. MiR-145 has been shown to regulate myofibroblast 62  differentiation and lung fibrosis [271]. Yang et al.[271] found that miR-145 expression is upregulated in TGF-β1-treated lung fibroblasts and that its expression is also higher in the lungs of patients with idiopathic pulmonary fibrosis compared with normal human lungs. Overexpression of miR-145 in lung fibroblasts increased α-SMA expression, enhanced contractility and promoted formation of focal and fibrillar adhesions [271]. One of the most interesting results of this study is the protection of miR-145−/− mice from bleomycin-induced pulmonary fibrosis, suggesting that miR-145 may be a potential target in the development of novel therapies to treat pathological fibrotic processes, as observed in the airway wall in COPD.   2.8 MiRNAs as Targets for Future Therapeutic Strategies in COPD The first successful miRNA-based therapy involved treating hepatitis C virus (HCV) infections. Miravirsen is a locked nucleic acid-modified DNA phosphorothioate antisense oligonucleotide that sequesters mature miR-122 in a highly stable heteroduplex. This inhibits the function of miR-122, which is required for the propagation of HCV in the liver [225]. The success of the phase 1 and 2 clinical trials of this new drug represented a major breakthrough for miRNA therapy in humans.  Depending on how a particular miRNA is dysregulated in disease, two different therapeutic approaches can be used. The first approach involves the use of miRNA antagonists, including anti-miRNAs, locked nucleic acids and antagomiRs, to inhibit the activity of miRNAs. These molecules are complementary oligonucleotides that either degrade the endogenous miRNA or trap it in a specific configuration to prevent its processing by the RNA-induced silencing complex (RISC) [273]. In the second approach, oligonucleotide miRNA mimics or gene vectors are used to 63  increase the expression levels of miRNAs. This approach has been studied extensively in various cancers, where miRNA mimics are introduced into cells to restore miRNA function where they are downregulated [216, 273, 274]. An important advantage of miRNA therapy is the potential to affect and regulate several pathways that are dysregulated in disease [273].  The various miRNAs that are deregulated in COPD pathogenesis could be candidates for miRNA-based therapy. We have described studies in this review that have highlighted this dysregulation and may serve as the basis for miRNA therapy studies. Although there is little overlap in the miRNAs reported, common miRNAs to various features of COPD, such as let-7c, miR-15b, miR-21 and miR- 34b (table 2), could serve as a basis for future therapeutic investigations.  As described, we have also shown that inhibiting miR-638 in lung fibroblasts modulates genes that may be important in emphysema [244]. This could serve as the beginning of studies that investigate the use of miRNA therapy to reverse the cellular damage and aberrant repair in emphysema. Another example is the use of a miRNA antagonist to inhibit miR-15b, which has been shown to be upregulated in lung tissue from COPD patients [239]. Studies may also focus on the use of mimics to increase the expression of miR-146a, which has been shown to have anti-inflammatory effects on both pulmonary fibroblasts [243] and bronchial epithelium [234]. Due to the fact that remodeling processes are opposite in COPD airways (fibrosis) compared with parenchyma (emphysema), when considering these approaches, attention must also be paid to targeting strategies to direct the miRNA modulators to the correct lung compartment.  MiRNA antagonists and mimics have been studied in experimental models, providing promising outcomes. Although these studies were mainly performed with other lung disease models, they can serve as good examples for investigating miRNA therapy in animal models of 64  COPD. For example, in a mouse model of lung cancer, let-7 mimics were successfully used to inhibit tumor growth after intravenous administration of a neutral lipid emulsion that contained the mimic [275]. A synthetic miR- 34a mimic delivered to another mouse model of lung cancer inhibited lung tumor growth and suppressed known cancer-promoting genes [276]. The knock-down of miR-155 in a mouse model of allergic asthma led to an attenuated T-helper cell (TH)2 response, with a reduction in TH2 cytokines compared with wildtype mice [277]. These studies show the potential of bringing in vitro miRNA studies in COPD to animal models and, in due course, to clinical trials.  It is important to realize that there may also be limitations to a therapeutic approach involving miRNAs. There is the possibility of toxicity as a result of off-target miRNAs or unintended effects of miRNA mimics or antagonists, which could be due to nonspecific delivery. Furthermore, the pathways affected by miRNAs could be adversely deregulated into hyper- or hypo-activation, which may have deleterious consequences for normal cells [273]. In this respect, it is worth noting that most manuscripts describing the use of miRNA mimics and antagonists have not adequately addressed the quantitative effects and physiological relevance of the increased or decreased miRNA levels. These concerns especially apply to in vitro studies, where miRNA levels may be increased far beyond levels of physiological relevance. When making the translation of experimental in vitro and in vivo models to clinical trials in humans, as was done for Miravirsen, dose-finding experiments are mandatory. In the case of Miravirsen, no dose-limiting toxic effects or binding-site escape mutations were observed after delivery of 3, 5 or 7 mg·kg−1 of body weight for 5 weeks [225]. Notwithstanding this, the toxicity of exogenous miRNAs still remains an important area to investigate in miRNA therapeutics, in which specific cell or tissue compartment targeting may also play an important role [273]. 65  2.9 Conclusions In this review, we have provided an up-to-date overview of the current knowledge on differential miRNA expression in COPD and discussed how the dysregulation of miRNAs may contribute to different aspects of COPD pathogenesis. Increasing evidence supports a role for miRNA deregulation in persistent inflammation, tissue repair and tissue remodeling, leading to large and small airways pathology and emphysema in COPD. Although there are several publications on miRNAs and their dysregulation in COPD, there is relatively limited overlap in the observed miRNAs and few studies have directly related the observed dysregulation in miRNAs to their biological function in COPD. This limited overlap might be due to the fact that miRNAs are cell type specific and the discrepancy between studies in terms of the use of different samples (e.g. cell type, type of tissue, serum versus plasma) and different experimental approaches. However, several miRNAs have been shown to be involved in more than one feature of COPD (table 2) and those could serve as a starting point for the functional studies that are needed to make the translation to new therapeutics in COPD that are directed towards inflammation and remodeling in COPD. This will improve our insights into the various roles of miRNAs and their potential as novel therapeutic targets in COPD. 66  Chapter 3:  Interleukin-1α Drives the Dysfunctional Cross-Talk of the Airway Epithelium and Lung Fibroblasts in COPD Emmanuel T. Osei1,2,3, Jacobien A. Noordhoek1,2,4, Tillie L. Hackett3, Anita I.R. Spanjer2,5, Dirkje S. Postma2,4, Wim Timens1,2, Corry-Anke Brandsma1,2,6 and Irene H. Heijink1,2,4,6   Affiliations:  1University of Groningen, University Medical Center Groningen, Dept of Pathology and Medical Biology, Groningen, The Netherlands.  2University of Groningen, University Medical Center Groningen, GRIAC Research Institute, Groningen, The Netherlands. 3University of British Columbia, Centre for Heart Lung Innovation, Dept of Anesthesiology, Pharmacology and Therapeutics, Vancouver, BC, Canada.  4University of Groningen, University Medical Center Groningen, Dept of Pulmonology, Groningen, The Netherlands. 5University of Groningen, Dept of Molecular Pharmacology, Groningen, The Netherlands. 6These two authors contributed equally to this work    67  3.1 Chapter Summary Chronic obstructive pulmonary disease (COPD) has been associated with aberrant epithelial–mesenchymal interactions resulting in inflammatory and remodeling processes. We developed a co-culture model using COPD and control-derived airway epithelial cells (AECs) and lung fibroblasts to understand the mediators that are involved in remodeling and inflammation in COPD.  AECs and fibroblasts obtained from COPD and control lung tissue were grown in co-culture with fetal lung fibroblast or human bronchial epithelial cell lines. mRNA and protein expression of inflammatory mediators, pro-fibrotic molecules and extracellular matrix (ECM) proteins were assessed.  Co-culture resulted in the release of pro-inflammatory mediators interleukin (IL)-8/CXCL8 and heat shock protein (Hsp70) from lung fibroblasts, and decreased expression of ECM molecules (e.g. collagen, decorin) that was not different between control and COPD-derived primary cells. This pro-inflammatory effect was mediated by epithelial-derived IL-1α and increased upon epithelial exposure to cigarette smoke extract (CSE). When exposed to CSE, COPD-derived AECs elicited a stronger IL-1α response compared with control-derived airway epithelium and this corresponded with a significantly enhanced IL-8 release from lung fibroblasts.  We demonstrate that, through IL-1α production, AECs induce a pro-inflammatory lung fibroblast phenotype that is further enhanced with CSE exposure in COPD, suggesting an aberrant epithelial– fibroblast interaction in COPD.   68  3.2 Introduction Chronic obstructive pulmonary disease (COPD) is mainly caused by exposure to noxious particles, of which cigarette smoking is the major risk factor [9]. Presently, no cure exists for COPD, and current pharmacological treatments can only partly suppress symptoms and exacerbations [278]. The disease is characterized by chronic inflammation and defective tissue repair leading to irreversible chronic airflow limitation as a result of destruction of the gas-exchanging surface of the lung (emphysema), remodeling and narrowing of the small airways [278].  When inhaled, cigarette smoke first encounters the airway epithelium, which normally forms a continuous and highly regulated structural barrier that is part of the innate immune defense [279]. We have previously shown that cigarette smoke inhibits epithelial barrier function [119]. This damage can also cause the release of pro-inflammatory mediators (interleukin (IL)-8/CXCL8, IL-6) and danger signals known as damage-associated molecular patterns (DAMPS), such as IL-1α and heat shock protein (Hsp70) [31]. Additionally, airway epithelium is a source of growth factors (e.g. transforming growth factor (TGF)-β1) that can act on the underlying mesenchymal cells in the lamina propria to induce repair [280]. Mesenchymal fibrocytes, fibroblasts and smooth muscle cells are essential structural cells that produce various extracellular matrix (ECM) proteins within the lung, including collagens and decorin [152].  We have previously demonstrated decreased decorin production by primary lung fibroblasts from severe (Global Initiative for Chronic Obstructive Lung Disease (GOLD) 4) COPD patients compared with mild (GOLD 1) COPD patients [158]. Other groups have also demonstrated that lung fibroblasts respond to IL-1β, IL-1α [177] and prostaglandin PGE2 [281] stimulation by releasing IL-8/CXCL8 and IL-6, and specifically in the case of IL-1β, by also downregulating their ECM protein production [181]. Epithelial–fibroblast communication has 69  been shown to be involved in the pathogenesis of asthma [180, 282] and has also been proposed to contribute to idiopathic pulmonary fibrosis [283]. However, detailed knowledge on the direct interaction between airway epithelial cells (AECs) and the underlying pulmonary fibroblasts in COPD is limited, also with respect to the effects of cigarette smoke.  We hypothesize that dysfunctional epithelium–fibroblast communication through the release of mediators plays a key role in the chronic inflammation and remodeling processes in COPD, and that cigarette smoke exposure contributes to this aberrant process. The objective of the study was to develop a co-culture cell model to investigate the effect of cross-talk between AECs and lung fibroblasts from severe COPD patients and control subjects on pro-inflammatory mediator release and ECM expression. Furthermore, we investigated the role of cigarette smoke exposure on the cross-talk between AECs and fibroblasts in COPD.   3.3 Methods and Materials 3.3.1 Human airway epithelial and lung fibroblasts Human bronchial epithelial 16HBE14o- cells (kindly donated by Dr D.C. Gruenert, University of California, San Francisco, CA, USA) were cultured in Eagle’s minimal essential medium (EMEM)/10% fetal calf serum (FCS) as described previously [138]. Primary AECs were isolated as described previously [284] from tracheobronchial tissue of 13 COPD patients with severe disease undergoing lung transplantation and from leftover tracheobronchial tissue of 16 non-COPD control donor lungs, for whom no further information was available. Subject characteristics of the donors are given in table 3.1. Primary AECs were cultured in hormonally supplemented bronchial epithelium growth medium (Lonza, Basel, Switzerland) and used at passage 3 as 70  described previously [138]. Fetal lung fibroblast cells (MRC-5; BioWhittaker, Walkersville, MD, USA) were cultured in EMEM/10% FCS. Primary human lung fibroblasts (PHLFs) were derived from nine COPD patients with severe disease undergoing lung transplantation and five non-COPD controls undergoing tumour resection surgery. Fibroblasts were isolated from lung parenchyma using the explant technique as described previously [158, 285] grown in Ham’s F12 medium/10% FCS (Lonza, Basel, Switzerland) and used for experiments at passage 5. The full protocol of PHLF isolation can be found in the online supplementary material. Subject characteristics of PHLF donors are available in table 3.2. The study protocol was consistent with the Research Code of the University Medical Center Groningen (www.rug.nl/umcg/onderzoek/researchcode/ index), and national ethical and professional guidelines (www.federa.org).   71   Table 3.1. Characteristics of severe chronic obstructive pulmonary disease patients from whom primary airway epithelial cells (AECs) were obtained. FEV1: forced expiratory volume in 1s; FVC: forced vital capacity. Primary AECs were either used for co-culture experiments with MRC-5 or conditioned medium experiments           Patient Age years Sex Smoking status Pack-years FEV1 % pred FEV1/FVC % Experiment 1 53 Male Ex 40 25 25 Co-culture and conditioned medium 2 58 Female Ex 38 60 46 Co-culture 3 57 Male Ex 30 11 31 Co-culture 4 64 Male Never 0 39 53 Co-culture 5 44 Male Ex 25 60 50 Co-culture 6 60 Male Ex 23 16 29 Conditioned medium 7 48 Male Ex 25 17 21 Conditioned medium 8 57 Female Ex 45 23 24 Conditioned medium 9 57 Female Ex 40 18 25 Conditioned medium 10 61 Female Ex 35 19 23 Conditioned medium 11 49 Male Ex 11 20 22 Conditioned medium 12 55 Female Ex 72 14 29 Conditioned medium 13 62 Male Ex 44 22 19 Conditioned medium 72  Patient Age years Sex Smoking status Pack-years FEV1 % pred FEV1/FVC % Control-derived PHLFs  1 67 Female Never 0 101 81  2 65 Female Current 38 98 76  3 74 Male Ex 50 100 71  4 65 Male Ex 40 97 76  5 50 Male Ex 31 97 78 COPD-derived PHLFs  6 59 Male Ex 38 19 25  7 58 Female Ex 30 18 28  8 60 Male Ex 30 37 49  9 66 Male Ex NA 24 31  10 62 Male Ex 44 22 19  11 57 Female Ex 40 18 25  12 48 Male Ex 27 12 23  13 57 Female Ex 33 25 33  14 44 Male Ex 25 60 50  Table 3.2 Characteristics of control donors and severe chronic obstructive pulmonary disease patients from whom primary human lung fibroblasts (PHLFs) were obtained. FEV1: forced expiratory volume in 1s; FVC: forced vital capacity; NA: not available   3.3.2 Co-culture model 16HBE14o- and MRC-5 cells were initially used to develop the model. Significant observations were replicated using: 1) primary AECs from severe COPD patients or controls with MRC-5 fibroblasts and 2) 16HBE14o- cells with PHLFs from COPD patients or controls to assess disease-specific effects in each cell type separately. Briefly, AECs were plated on 0.4-μM pore 6.5-mm transwell membranes (Costar; Corning, New York, NY, USA) and fibroblasts were seeded on 24-73  well plates. When both cell layers were confluent, the transwell insert with AECs was placed in co-culture with the fibroblasts and left for 72 h in the appropriate medium (see online supplementary material).  3.3.3 Conditioned Medium experiments and neutralizing antibody experiments 16HBE14o- cells or primary AECs when confluent were serum/hormone-deprived overnight and stimulated with or without 20% cigarette smoke extract (CSE) for 6 h. The CSE was thoroughly washed off and cells were incubated for another 24 h prior to the CSE-free conditioned medium being collected. Fibroblasts that had been serum-deprived overnight were then treated for 24 h with the CSE-free conditioned medium that had been pre-incubated for 1 h with or without 4 μg·mL−1 IL-1α neutralizing antibody (AB-200-NA) or IL-1β neutralizing antibody (MAB601) (R&D Systems, Europe, Abingdon, UK). Cell-free supernatants were collected and analyzed by ELISA, and cell lysates were harvested for RNA and protein examination. Refer to the online supplementary material for full protocols of experiments.  3.3.4 Statistics Data were analyzed using SPSS (IBM, Armonk, NY, USA). The Mann–Whitney U-test was used for comparison between subject groups and the Wilcoxon signed-rank test for paired comparisons within groups of primary cells. We tested for normal distribution on the outcomes of the experiments with cell lines and used the t-test for paired differences accordingly. p<0.05 was considered to be statistically significant.   74  3.4 Results  3.4.1 Increased inflammatory mediator release in lung fibroblasts when in co-culture with airway epithelial cells.   When 16HBE14o- and MRC-5 cells were placed in co-culture, we found a significant increase in basolateral IL-8/CXCL8 (figure 3.1a) and Hsp70 (figure 3.1b) secretion compared with epithelial and fibroblast mono-cultures. IL-1β protein levels were undetectable (data not shown). Subsequent mRNA analyses on the epithelial and fibroblast cell fractions demonstrated that fibroblasts are the main source of secreted IL-8/CXCL8 (figure 3.1c). Similarly, IL-1β mRNA levels were increased in fibroblasts, but not in epithelial cells, when placed in co-culture (figure 3.1d). To determine if the results obtained from the cell lines in co-culture were representative of primary cells, we paired the 16HBE14o- cells with PHLFs derived from COPD and control subjects, and also paired primary AECs from COPD and control donors with MRC-5 cells. As in the co-culture cell line model, we found that co-culture of PHLFs with 16HBE14o- cells also induced a significant increase in basolateral IL-8/CXCL8 (figure 3.1e) and Hsp70 (figure 3.1f), without differences in the IL-8/CXCL8 response of PHLFs from ex-smokers, the never-smoker and the current smoker. Furthermore, we confirmed that the mRNA for IL-8/CXCL8 (figure 3.1g) and IL-1β (figure 3.1h) was only upregulated in the PHLFs with co-culture. Additionally, combining primary AECs with MRC-5 fibroblasts resulted in increased basolateral IL-8/CXCL8 and Hsp70 levels as well as a trend towards an increase of IL-6 protein levels (supplementary figure A.8), while levels of granulocyte-macrophage colony-stimulating factor and IL-33 were undetectable. Hsp70 and IL-6 levels correlated strongly with IL-8/CXCL8 release in co-culture (supplementary figure A.9). 75  We found no significant difference in the release of mediators between control and COPD-derived AECs and PHLFs.  Figure 3.1 Interleukin (IL)-8/CXCL8, heat shock protein (Hsp70) and IL-1β levels in co-culture of airway epithelial cells (AECs) and lung fibroblasts.  16HBE14o- (16HBE) cells were cultured alone and with MRC-5 cells or with primary human lung fibroblasts (PHLFs) derived from control (open triangles) and chronic obstructive pulmonary disease patients (filled triangles). a) IL-8/CXCL8 concentration (with median) and b) Hsp70 concentration (with median) in cell-free supernatants (24 h) of the basolateral compartment of co-culture system. c) IL-8/CXCL8 and d) IL-1β mRNA expression levels (with median) in epithelial cells and fibroblasts (6 h) harvested separately comparing co-culture and mono-cultures of 16HBE14o- and MRC-5 cells. e) IL-8/CXCL8 concentration (with median) and f) Hsp70 concentration (with median) in cell-free supernatants (24 h) of the basolateral compartment. g) IL-8/CXCL8 and h) IL-1β mRNA expression levels (with median) in PHLFs (6 h) harvested separately comparing co-culture and mono-cultures of 16HBE14o-cells and PHLFs. mRNA levels were related to the housekeeping genes β2-microglobulin and protein phosphatase 1α, and expressed as 2−ΔCt. *: p<0.05; **: p<0.01; ***: p<0.001 between the indicated values. 76  3.4.2 Decreased expression of ECM molecules and pro-fibrotic proteins in lung fibroblasts in co-culture with airway epithelial cells In contrast to the increased pro-inflammatory response, co-culture of PHLFs from COPD and control subjects with 16HBE14o- cells resulted in a significant downregulation in the mRNA expression of α-smooth muscle actin (α-SMA) (figure 3.2a), TGF-β1 (figure 3.2b), and the ECM molecules decorin (figure 3.2c), fibulin-5 (figure 3.2d), collagen-Iα1 (figure 3.2e) and fibronectin (figure 3.2f) compared with mono-culture conditions. The downregulation of fibronectin and α-SMA was confirmed on the protein level using Western blotting (figure 3.2g and 3.2h). Neither the baseline expression nor the decrease of these ECM and structural proteins upon co-culture was significantly different between COPD and control-derived PHLFs. 77    Figure 3.2 Decrease in the expression of extracellular matrix molecules and structural proteins in primary human lung fibroblasts (PHLFs) after co-culture with 16HBE14o- cells. mRNA expression levels (6 h) (with median) of a) α-smooth muscle actin (α-SMA), b) transforming growth factor (TGF)-β1, c) decorin, d) fibulin-5, e) collagen-Iα1 and f) fibronectin in PHLFs from control donors (open triangles) and chronic obstructive pulmonary disease (COPD) patients (filled triangles) comparing co-culture with 16HBE14o- and mono-cultures. mRNA levels were related to the housekeeping genes β2-microglobulin and protein phosphatase 1α, and expressed as 2−ΔCt. g, h) Relative protein expression levels (with median) and representative blots for g) fibronectin and h) α-SMA in PHLFs from control donors (open triangles) and COPD patients (filled triangles) comparing co-culture with 16HBE14o- and mono-cultures. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as loading control. *: p<0.05; **: p<0.01; ***: p<0.001 between the indicated value  78  3.4.3 Epithelium-derived IL-1α is responsible for pro-inflammatory phenotype switch in lung fibroblasts Next, we used conditioned medium from 16HBE14o- cells to investigate whether the observed effects could be due to a soluble factor. Indeed, we observed that epithelial conditioned medium also induced an increase in IL-8/CXCL8 secretion by MRC-5 fibroblasts (figure 3.3a). Since IL-1β and PGE2 have been shown to induce similar phenotype switches in fibroblasts [177, 181, 281], we first studied the effect of an IL-1β neutralizing antibody (figure 3.3b) and inhibition of the downstream effect of PGE2 by the use of an adenylate cyclase inhibitor, MDL-12,330A hydrochloride (supplementary figure A.11). Both did not prevent the IL-8/CXCL8 release from MRC-5 cells. The other agonist of the IL-1R1 receptor is IL-1α. Interestingly, we found that the use of a neutralizing antibody against IL-1α completely abrogated IL-8/CXCL8 secretion by MRC-5 fibroblasts (figure 3.3c).   Figure 3.3 Interleukin (IL)-1α from 16HBE14o- cells is responsible for a pro-inflammatory phenotype switch in MRC-5 fibroblasts. IL-8/CXCL8 concentration (with median) released from confluent MRC-5 cells incubated a) without (MRC-5 basal) or with conditioned medium from confluent 16HBE14o- cells (16HBE CM), b) with 16HBE14o- conditioned medium in the presence and absence of 4 µg·mL−1 IL-1β neutralising antibody (NAb) and c) with 16HBE14o- conditioned medium in the presence and absence of 4 µg·mL−1 IL-1α NAb. *: p<0.05; **: p<0.01 between the indicated values.  79  To determine if the results obtained using 16HBE14o- conditioned medium were representative of primary cells, we conducted the same experiments using conditioned medium from primary AECs from COPD and control donors on MRC-5 fibroblasts. As shown in figure 4, neutralization of IL-1α also abrogated primary AEC conditioned medium-induced IL-8/CXCL8 secretion (figure 3.4a) as well as IL-8/CXCL8 and IL-1β mRNA expression (figure 3.4b and 3.4c) in MRC-5 fibroblasts. Furthermore, we assessed the effect of the neutralizing antibody in the co-culture model itself. As shown in figure 3.4d, the addition of IL-1α neutralizing antibody in the basal compartment also inhibited the basolateral release of IL-8/CXCL8 upon co-culture of primary AECs from COPD and control subjects with MRC-5. There was also a strong trend towards the downregulation of basolateral IL-6 release (supplementary figure A.9). Moreover, IL-1α neutralization in the co-culture also prevented the downregulation of TGF-β1, decorin, fibulin-5 and collagen-Iα1 mRNA expression in MRC-5 cells when co-cultured with primary AECs (figure 3.5). 80      Figure 3.4 Interleukin (IL)-1α from primary airway epithelial cells (AECs) is responsible for a pro-inflammatory phenotype switch in MRC-5 fibroblasts. MRC-5 cells were grown to confluence, serum-deprived overnight and subsequently incubated without (MRC-5 basal) or with conditioned medium from primary AECs (PAEC CM) of control donors (open triangles) and chronic obstructive pulmonary disease (COPD) patients (filled triangles) in the presence or absence of 4 μg·mL−1 IL-1α neutralising antibody (NAb). a) IL-8/CXCL8 concentration (with median), and b) IL-8/CXCL8 and c) IL-1β mRNA expression (with median) in MRC-5 cells. d) IL-8/CXCL8 concentration (with median) in cell-free supernatants (24 h) of the basolateral compartment comparing co-culture and mono-cultures of primary AECs from control donors (open triangles) and COPD patients (filled triangles) and MRC-5 cells in the presence or absence of 4 µg·mL−1 IL-1α NAb. mRNA levels were related to the housekeeping genes β2-microglobulin and protein phosphatase 1α, and expressed as 2−ΔCt. **: p<0.01; ***: p<0.001 between the indicated values.   81   Figure 3.5 Interleukin (IL)-1α is responsible for the decrease in expression of extracellular matrix molecules and structural proteins of lung fibroblasts after co-culture with epithelial cells. mRNA expression levels (with median) of a) transforming growth factor (TGF)-β1, b) decorin, c) fibulin-5 and d) collagen-Iα1 in MRC-5 cells comparing mono-culture and co-culture with primary airway epithelial cells from control donors (open triangles) and chronic obstructive pulmonary disease patients (filled triangles) in the presence and absence of 4 µg·mL−1 IL-1α neutralising antibody (NAb). mRNA levels were related to the housekeeping genes β2-microglobulin and protein phosphatase 1α, and expressed as 2−ΔCt. *: p<0.05 between the indicated values.      82  3.4.4 Cigarette smoke exposure increases IL-1α expression in airway epithelial cells and subsequent IL-8/CXCL8 production in lung fibroblasts To evaluate if epithelial exposure to cigarette smoke alters communication with lung fibroblasts, we exposed fibroblasts to conditioned medium from 16HBE14o- cells pre-treated with CSE. Exposure of epithelial cells to CSE significantly increased the release of IL-1α protein (figure 3.6a) and mRNA (figure 3.6b) compared with basal levels. Subsequent exposure of MRC-5 fibroblasts to conditioned medium from CSE-treated 16HBE14o- cells induced a significantly stronger increase in IL-8/CXCL8 production than stimulation with basal 16HBE14o- conditioned medium (figure 3.6c). Similarly, CSE exposure significantly increased IL-1α mRNA expression in our primary AECs (figure 3.6d). Interestingly, COPD-derived primary AECs showed a stronger increase in IL-1α mRNA expression after CSE exposure than control-derived primary AECs (figure 3.6d). In line with this increase in IL-1α expression, conditioned medium from CSE-exposed COPD-derived primary AECs caused a significantly stronger increase in IL-8/CXCL8 release from MRC-5 fibroblasts than conditioned medium from CSE-exposed control primary AECs (figure 3.6e).         83    Figure 3.6 Interleukin (IL)-1α from cigarette smoke extract (CSE)-exposed airway epithelium causes a higher release of IL-8/CXCL8 in lung fibroblasts. a–c) 16HBE14o- cells were pre-stimulated without or with 20% CSE. a) IL-1α concentration (with median) in conditioned medium (16HBE CM) and b) IL-1α mRNA expression (with median) in 16HBE14o- cells. c) IL-8/CXCL8 concentration (with median) released from MRC-5 cells after incubation with 16HBE14o- conditioned medium. d, e) Primary airway epithelial cells (AECs) from control (Ctrl) donors (open triangles) and chronic obstructive pulmonary disease (COPD) patients (filled triangles) were pre-stimulated with 20% CSE or not. d) Fold expression of IL-1α in primary AECs. e) IL-8/CXCL8 concentration released from MRC-5 cells after incubation with conditioned medium from primary AECs. mRNA levels were related to the housekeeping genes β2-microglobulin and protein phosphatase 1α, and expressed as 2−ΔCt.*: p<0.05; **: p<0.01 for indicated values between groups; ##: p<0.01 for indicated values within groups.   84  3.5 Discussion We demonstrate that lung fibroblasts are directly regulated by AECs to release pro-inflammatory mediators and downregulate ECM synthesis and pro-fibrotic responses. Our data indicate that this regulation is driven by epithelium-derived IL-1α, as neutralizing IL-1α in epithelial conditioned media completely reversed the release of inflammatory mediators by lung fibroblasts. Additionally, we demonstrate that cigarette smoke exposure induces higher IL-1α levels in AECs, particularly in epithelial cells from severe COPD patients. Moreover, cigarette smoke exposure may contribute to an aberrant cross-talk between epithelial cells and the underlying fibroblasts in COPD, as we observed that CSE-exposed epithelium derived from COPD patients induces a stronger increase in IL-8/CXCL8 secretion by lung fibroblasts than CSE-exposed control-derived epithelium.  In the lung, fibroblasts are located within the interstitium in close proximity to the airway epithelium, and hence can be easily influenced by the release of several factors by the epithelium in normal repair and disease states [152]. In COPD, however, the interaction between AECs and the underlying fibroblasts may be increased, and cells may be in closer contact because of the observed fragmentation of the basement membrane in the mucosa [121, 172]. Thus, the epithelium may exert stronger pro-inflammatory effects on fibroblasts in COPD. Fibroblasts in the lung have been shown to not only contribute to repair processes through contraction, synthesis and remodeling of granulation tissue, but also through the production of cytokines to aid the normal immune defense mechanisms within the lung [286]. IL-8/CXCL8 is a chemoattractant for neutrophils in the lungs [190, 287], and chronic neutrophilic inflammation may contribute to abnormal tissue repair, remodeling and destruction in COPD [9]. Increased IL-8/CXCL8 release in the lungs has been associated with the pathogenesis of COPD [288]. Thus, higher IL-8/CXCL8 85  secretion by lung fibroblasts upon epithelial exposure to cigarette smoke may play a role in the pathogenesis of COPD [9]. Similarly, the increased release of Hsp70 and IL-6, which are both inflammatory mediators, has been implicated in chronic inflammatory processes in COPD [31, 289]. The airway epithelium has been reported to be the source of mediators that drive chronic inflammation and structural changes in COPD. Among others, IL-1β, IL-1α, PGE2, tumor necrosis factor-α, IL-6 and various matrix metalloproteinases are increased in pulmonary epithelial cell supernatants from COPD patients compared with healthy controls [106]. PGE2 and IL-1β as well as IL-1α have been shown to induce an increase in IL-8/CXCL8 secretion from fibroblasts [177, 281]. Our results highlight that neutralization of IL-1α completely blocked the production of IL-8/CXCL8 by lung fibroblasts upon stimulation with epithelial conditioned media. Thus, our data provide strong support for a role of epithelium-derived IL-1α in the cross-talk between epithelial cells and fibroblasts during both normal immune defense in the lungs and aberrant repair in COPD. IL-1α is a member of the IL-1 superfamily of inflammatory cytokines. It plays a crucial role in normal immune responses in vivo and is constitutively expressed in lung epithelium [182]. IL-1α and its agonist IL-1β bind to the IL-1R1 receptor and illicit a similar downstream response with the subsequent activation of transcription factors such as nuclear factor-κB and activator protein-1 [182]. While the activity of IL-1β is dependent on the activation of the NLRP3 inflammasome and subsequent cleavage by caspase-1, IL-1α is both active in its pro-form and upon cleavage by caspases [183].  Our findings suggest that cigarette smoke may increase the release of IL-1α from the airway epithelium in severe COPD, which subsequently causes an additional increase in IL-8/CXCL8 release from fibroblasts, contributing to neutrophilic inflammation in vivo. Relatively little work has been done on the role of IL-1α in COPD and our data suggest the need for a closer 86  look at the role of IL-1α in the pathogenesis of COPD. In line with our findings, PAUWELS et al. [183] showed that cigarette smoke-induced inflammation in mice is dependent on the IL-1R1 receptor, and both IL-1α and its agonist IL-1β are involved in neutrophilic inflammation. Furthermore, BOTELHO et al. [184] showed that smoke-induced neutrophilic inflammation in mice was dependent on IL-1α, but not IL-1β. They also measured IL-1α protein in bronchial biopsies and sputum, and showed an increased expression in the inflammatory infiltrate and epithelium in biopsies from COPD patients compared with controls [184]. SUWARA et al. [177] used another model of COPD where human AECs were injured in vitro with hydrogen peroxide and thapsigargin to induce reactive oxidative species injury and endoplasmic reticulum stress, respectively. They found increased levels of IL-1α and IL-1β in the conditioned medium from these cells, which induced the release of IL-8/CXCL8 and IL-6 in MRC-5 fibroblasts [177]. This effect was completely blocked using IL-1α neutralizing antibody and only partially by using the IL-1β neutralizing antibody. This is in line with our study, demonstrating a crucial role for epithelium-derived IL-1α in regulating fibroblasts to become pro-inflammatory in a co-culture model representative of in vivo conditions. Our data further indicate that COPD-derived epithelial cells are more prone to release IL-1α upon CSE exposure. It will be worthwhile to further study the mechanism of IL-1α release in order to understand why COPD epithelial cells express more IL-1α upon CSE exposure, e.g. whether this involves endoplasmic reticulum stress or an oxidant/ antioxidant imbalance as shown previously [177].  In addition to the production of inflammatory mediators in our co-culture model, AECs via IL-1α also reduced the expression of the pro-fibrotic cytokine TGF-β1, the structural molecule α-SMA and various ECM molecules, including decorin, fibulin-5 and collagen-Iα1. The exact meaning of this finding needs to be investigated further, as the lack of demonstrating differences 87  between COPD and control fibroblasts could also be due to power limitation. Nevertheless, our current findings support a mechanism whereby small airways may be lost due to defective tissue repair, which would be in line with the findings of McDonough et al, [290] who demonstrated up to 90% loss of small airways in end-stage (GOLD 4) COPD patients. Although our epithelium–fibroblast model reflects the in vivo situation more closely than mono-cultures, our model may not fully reflect the in vivo situation, where other cell types are present as well. Also in our model, we compared control-derived primary AECs from first-generation tracheobronchial tissue to COPD-derived primary AECs from the third to fifth generation of the bronchial tree. Although these cells are not from the exact same site in the lung, various studies have shown similar genomic and epigenomic similarities in AECs from the tracheal and bronchial origin [291, 292]. We used cells derived from lung tissue of COPD patients undergoing transplantation for end-stage disease. Further experiments will be required to assess if the observed aberrant cross-talk between epithelial cells and fibroblasts is also present in early, mild and moderate stages of the disease. Future work will need to assess if the increased IL-8/CXCL8 secretion observed in our model additionally promotes neutrophil chemotaxis.  In conclusion, our data show that lung fibroblasts are regulated by AECs to become pro-inflammatory in their function. This regulation is driven by epithelial-derived IL-1α, further enhanced by CSE exposure and stronger in epithelial cells from severe COPD patients than from healthy individuals. Our study offers novel insights into the role of epithelial cells and fibroblasts in the pathogenesis of chronic remodeling and inflammation seen in COPD.    88  Chapter 4:  MiR-146a-5p Plays an Essential Role in the Aberrant Epithelial-Fibroblast Cross-Talk in COPD  Emmanuel T. Osei1,2,3 , Laura Florez-Sampedro2,4, Hataitip Tasena1,2, Alen Faiz1,2, Jacobien A.Noordhoek1,2,5, Wim Timens1,2, Dirkje S. Postma2,5, Tillie L. Hackett3, Irene H. Heijink1,2,5,6 and Corry-Anke Brandsma1,2,6   Affiliations:  1Dept of Pathology and Medical Biology, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands.  2GRIAC Research Institute, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands.  3Centre for Heart and Lung Innovation, University of British Columbia, Vancouver, BC, Canada.  4Dept of Pulmonology, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands.  5Dept of Pharmacokinetics, Toxicology and Targeting, University of Groningen, Groningen, The Netherlands 6These two authors contributed equally to this work.     89  4.1 Chapter Summary We previously reported that epithelial-derived interleukin (IL)-1α drives fibroblast-derived inflammation in the lung epithelial–mesenchymal trophic unit. Since miR-146a-5p has been shown to negatively regulate IL-1 signaling, we investigated the role of miR-146a-5p in the regulation of IL-1α-driven inflammation in chronic obstructive pulmonary disease (COPD). Human bronchial epithelial (16HBE14o-) cells were co-cultured with control and COPD-derived primary human lung fibroblasts (PHLFs), and miR-146a-5p expression was assessed with and without IL-1α neutralizing antibody. Genomic DNA was assessed for the presence of the single nucleotide polymorphism (SNP) rs2910164. miR-146a-5p mimics were used for overexpression studies to assess IL-1α-induced signaling and IL-8 production by PHLFs.  Co-culture of PHLFs with airway epithelial cells significantly increased the expression of miR-146a-5p and this induction was dependent on epithelial-derived IL-1α. miR-146a-5p overexpression decreased IL-1α-induced IL-8 secretion in PHLFs via downregulation of IL-1 receptor-associated kinase-1. In COPD PHLFs, the induction of miR-146a-5p is significantly less compared with controls and was associated with the SNP rs2910164 (GG allele) in the miR-146a-5p gene.  Our results suggest that induction of miR-146a-5p is involved in epithelial–fibroblast communication in the lungs and negatively regulates epithelial-derived IL-1α induction of IL-8 by fibroblasts. The decreased levels of miR-146a-5p in COPD fibroblasts may induce a more pro-inflammatory phenotype, contributing to chronic inflammation in COPD.  90  4.2 Introduction Chronic obstructive pulmonary disease (COPD) is a progressive disease in which chronic neutrophilic inflammation is associated with destruction of the lung parenchyma (emphysema) and small airway disease, which both contribute to airflow limitation and lung function decline [10]. The inhalation of noxious particles, specifically from cigarette smoke, is the most common risk factor for COPD and smoking is known to induce the recruitment of neutrophils [9, 293]. However, not all smokers develop COPD, indicating a genetic susceptibility to the disease process [293]. The inconsistency in replicating target genes related to COPD in different study populations points to an important role of epigenetic regulation [294]. Epigenetic mediators such as microRNAs (miRNAs) can regulate the transcriptional activity of various genes involved in lung function and inflammation that are thought to be involved in the pathogenesis of COPD [295].  miRNAs are small noncoding RNAs of approximately 19–25 nucleotides that cause post-transcriptional gene repression by increasing mRNA degradation or by inhibiting protein translation of specific mRNA targets [296]. They are involved in various biological processes and alterations in their expression can result in pathological conditions, including pulmonary diseases [239]. We recently provided an up-to-date review of studies showing the role of miRNA dysregulation in COPD and how it is associated with the various features of COPD [295]. In particular, it has been shown that exposure to cigarette smoke alone can change miRNA expression in the lungs [233]. Several studies have shown that various miRNAs are differentially expressed in whole lung tissue, serum and/or sputum of smoking COPD patients compared with smokers without COPD [224, 232, 254]. Of these, miR-146a-5p has been shown to regulate the release of interleukin (IL)-1-induced inflammatory mediators from pulmonary epithelial cells, including the neutrophil chemoattractant IL-8 [260]. We recently showed in a co-culture model that cigarette 91  smoke extract (CSE)-induced IL-1α expression is higher in airway epithelial cells (AECs) from COPD patients compared with control-derived epithelial cells [297]. Furthermore, the higher levels of epithelial IL-1α induce a stronger release of the pro-inflammatory cytokines including IL-8 from lung fibroblasts [297]. Interestingly, others have shown that when lung fibroblasts are stimulated with IL-1β and tumor necrosis factor (TNF)-α, miR-146a-5p expression is induced to a lesser extent in lung fibroblasts from COPD patients when compared with control fibroblasts [243].  In this study, we hypothesized that a failure to upregulate miR-146a-5p in lung fibroblasts contributes to the disturbed communication within the epithelial–mesenchymal trophic unit in COPD, leading to increased inflammation in the disease. Therefore, we studied the expression of miR-146a-5p in COPD and non-COPD control-derived primary human lung fibroblasts (PHLFs) in our co-culture model, and conducted functional assays to investigate the mechanism of regulation of pro-inflammatory activity by miR-146a-5p in pulmonary fibroblasts.   4.3 Methods and Materials  4.3.1 Subjects and cell culture conditions The human bronchial epithelial cell line 16HBE14o- was kindly donated by Dr D.C. Gruenert (University of California, San Francisco, CA, USA) and cultured in Eagle’s minimal essential medium (EMEM)/10% fetal calf serum (FCS; Lonza/BioWhittaker, Verviers, Belgium) on collagen/bovine serum albumin-coated flasks as previously described [138]. Fetal lung fibroblast cells (MRC-5; BioWhittaker, Walkersville, MD, USA) were cultured in EMEM/10% FCS on 24-92  well plates before experiments. PHLFs were isolated from peripheral parenchymal lung tissue of eight non-COPD control donors undergoing tumor resection surgery and 12 COPD patients with severe disease undergoing lung transplantation using the explant technique as previously described [158, 285]. PHLFs from controls were isolated from histologically normal tissue taken as far away as possible from the tumor. The tissue was checked for cancer or other pathology by an experienced pathologist, and found to be cancer-free and showing no other pathology. Clinical information on the COPD patients and non-COPD control subjects is presented in table 4.1. The study protocol for this project was consistent with the Research Code of the University Medical Center Groningen (www.rug.nl/umcg/onderzoek/researchcode/index) and national ethical and professional guidelines (www.federa.org). PHLFs were cultured in Ham’s-F12 medium/10% FCS (Lonza) on 24-well culture plates before experiments.   93   Table 4.1Characteristics of COPD patients and non-COPD controls from whom primary human lung fibroblasts (PHLFs) were obtained.  FEV1= Forced expiratory volume in 1 second, FVC= Forced Vital Capacity, F=female M= male CS= current smoker, ES= ex-smoker, NS= never smoker. #FEV1% Predicted not available, $Pack years and FEV1/FVC not available. CM= conditioned medium. All COPD donors were on inhaled or oral steroids before transplantation. There was a significant difference as expected, in FEV1%predicted (p=0.04) and FEV1/FVC% (p=0.04), as well as in age between the COPD patients and controls, with the COPD patients being slightly younger (p=0.03), while there was no significant difference in pack-years. Patient Age years Sex Smoking status Pack-years FEV1 % pred FEV1/FVC % Experiment Control-derived PHLFs  1 74 Male Ex 50 100 71 Co-culture  2 50 Male Ex 31 97 78 Co-culture  3 65 Male Ex 40 97 76 Co-culture and conditioned medium  4# 68 Male Ex 51  74 Co-culture and conditioned medium  5 67 Female Never 0 101 81 Co-culture and conditioned medium  6 65 Female Current 38 98 76 Co-culture and conditioned medium  7 46 Male Ex 32 97 82 Conditioned medium  8¶ 60 Male Ex  85  Conditioned medium COPD-derived PHLFs  7 58 Female Ex 30 18 28 Co-culture  8 60 Male Ex 30 37 49 Co-culture  9 62 Male Ex 44 22 19 Co-culture  10 48 Male Ex 27 12 23 Co-culture  11 57 Female Ex 33 25 33 Co-culture  12 44 Male Ex 25 60 50 Co-culture  13 44 Male Ex 27 14 28 Conditioned medium  14 56 Male Ex 38 23 35 Conditioned medium  15 53 Female Ex 40 24 26 Conditioned medium  16 52 Male Ex 20 18 62 Conditioned medium  17 51 Male Ex 42 28 29 Conditioned medium  18 59 Female Ex 37 21 21 Conditioned medium 94  4.3.2 Co-culture model We used 16HBE14o- and MRC-5 co-cultures for the mechanistic studies. 16HBE14o- cells were co-cultured with COPD and control-derived PHLFs to determine the disease-specific effects. Briefly, 16HBE14o- cells were plated on coated 0.4-μM pore 6.5-mm transwell membranes (Costar; Corning, New York, NY, USA), while fibroblasts were cultured separately on a 24-well plate. After a confluent layer was obtained for both cell types, the transwell with 16HBE14o- cells (upper compartment) was placed in co-culture with lung fibroblasts in the 24-well plate (lower compartment) and left for 72 h in EMEM/10% FCS or Ham’s-F12 medium/10% FCS (Lonza) for co-culture with MRC-5 cells or PHLFs, respectively [297]. Before experimentation, cells were serum-deprived overnight.  4.3.3 Conditioned medium and neutralizing antibody experiments 16HBE14o- cells were serum-deprived overnight and stimulated for 6 h with or without 20% CSE, which we have previously reported did not affect cell viability [297]. After stimulation, the CSE was thoroughly washed off and CSE-free conditioned medium was collected after a further 24 h incubation. CSE-free conditioned medium was pre-incubated for 1 h with or without 4 μg·mL−1 IL-1α neutralizing antibody (AB-200-NA, MAB601; R&D Systems, Abingdon, UK) and was used to stimulate serum-deprived fibroblasts for 24 h. For experiments with recombinant human (rh) IL-1α (R&D Systems), a 1 ng·mL−1 concentration was chosen as this is comparable to levels previously reported in sputum of COPD patients [19]. Cell-free supernatants were collected and analyzed by ELISA, and cell lysates were harvested with TRI Reagent (Molecular Research Center, Inc. Cincinnati, OH, USA) for RNA isolation.  95  4.3.4 MiR-146a-5p mimic transfection MRC-5 fibroblasts were transfected with the miR-146a-5p mimic at 25 nM (MirVana miRNA mimic, assay ID: MC10722; Applied Biosystems, Carlsbad, CA, USA) and scrambled small RNA at 25 nM (AllStars Negative Control siRNA; Qiagen, Hilden, Germany) as a non-targeting control to assess the effects of miR-146a-5p induction on IL-1 signaling in lung fibroblasts. miR-146a-5p expression was analyzed by quantitative PCR, protein lysates were assessed by Western blot and IL-8 concentrations were determined by ELISA (R&D Systems) in the cell-free supernatant as described in the supplementary material.  4.3.5 Single nucleotide polymorphism genotyping To investigate if the lower induction of miR-146a-5p by COPD-derived primary human lung fibroblasts (PHLFs) in co-culture was caused by a common G>C SNP rs2910164, genotyping for this SNP was performed. A subset of samples were previously genotyped on Illumina Human1M-Duo BeadChip array and imputed using MACH program for genotype imputation using HapMap release 22 template, as previously described [298]. Five samples which were not genotypes on arrays had their DNA extracted from lung tissues of the same subjects from which PHLFs were obtained, according to the standard salt-chloroform extraction method. The rs2910164 polymorphism was identified using TaqMan® SNP Genotyping assay (C_15946974_10) according to the manufacturer’s protocol. Each PCR was done in duplicates with 10 ng DNA template and 40 cycles of amplification was used. The PCR was carried out on a ABI7800HT machine (Applied Biosystems).  96  4.3.6 Statistical analysis SPSS statistics version 23 (IBM, Armonk, NY, USA) was used for data analyses. Differences between COPD patients and non-COPD control subjects were analyzed with a Mann–Whitney U-test. Differences between treatments within a group were analyzed using paired t-tests for the cell lines and Wilcoxon signed-rank tests for the primary cells. p<0.05 was considered statistically significant.   4.4 Results 4.4.1 Epithelium-derived IL-1α is responsible for the increased miR-146a-5p expression in lung fibroblasts As our previous study showed that IL-1α derived from the airway epithelium (primary and 16HBE14o- cells) is an important regulator of pulmonary fibroblast-derived inflammation [297], we first determined whether epithelial-derived IL-1α causes an induction of miR-146a-5p in lung fibroblasts. We stimulated PHLFs from non-COPD controls and COPD patients with 16HBE14o- conditioned medium in which IL-1α levels had been previously determined [297] in the presence or absence of the IL-1α neutralizing antibody. We found that the expression of miR-146a-5p was increased in PHLFs treated with conditioned media from 16HBE14o- cells and this was further enhanced by pre-stimulation of the 16HBE14o- cells with CSE (figure 4.1a). Furthermore, the induction of miR-146a-5p was completely abrogated by the addition of IL-1α neutralizing antibody (figure 4.1a). We also assessed the direct effects of rhIL-1α stimulation on miR-146a-5p expression in PHLFs and found, as with the 16HBE14o- cell conditioned media, a significant increase in miR-146a-5p expression in PHLFs (figure 4.1b). Lastly, stimulation with 1 ng·mL−1 97  (figure 4.1c) or 0.01 ng·mL−1 (supplementary figure B.5) rhIL-1α led to a significant release of IL-8 concentration from PHLFs (figure 4.1c), which significantly correlated with the increased expression of miR-146a-5p in PHLFs (figure 4.1d). Neither the expression level of miR-146a-5p nor the release of IL-8 was significantly different between COPD and control-derived PHLFs after 16HBE14o- conditioned medium or rhIL-1α stimulations.             Figure 4.1 Epithelial-derived interleukin (IL)-1α is responsible for increased miR-146a-5p expression in lung fibroblasts. Primary human lung fibroblasts (PHLFs) from control donors (n=6) and chronic obstructive pulmonary disease (COPD) patients (n=6) were grown to confluence and serum-deprived overnight. a) miR-146a-5p expression in PHLFs after stimulation with conditioned medium (CM) from 16HBE14o- (16HBE) cells pre-treated with or without cigarette smoke extract (CSE) in the presence or absence of 4 µg·mL−1 IL-1α neutralising antibody (NAb). b) miR-146a-5p expression in serum-deprived PHLFs after stimulation with/without 1 ng·mL−1 recombinant human IL-1α for 24 h. miR-146a-5p expression levels were related to the housekeeping noncoding RNU48, expressed as 2–ΔCt. c) IL-8 concentration released from PHLF. Medians are indicated. ***: p<0.001, between the indicated values. d) Correlation of IL-8 concentration released from PHLFs to miR-146a-5p expression in PHLFs after IL-1α stimulation. Data are shown on a log scale. 98  4.4.2 MiR-146a-5p expression is decreased in PHLFs from COPD patients in co-culture with 16HBE14o- cells We have previously shown in our co-culture model that COPD-derived AECs, through higher induction of IL-1α, induce lung fibroblasts to be more pro-inflammatory [297]. Hence, we were interested in the regulation of miR-146a-5p expression in control and COPD-derived PHLFs. In our co-culture model, 16HBE14o- cells significantly upregulated miR-146a-5p expression in PHLFs from both control and COPD donors. Interestingly, there was a significantly lower induction of miR-146a-5p in COPD-derived PHLFs compared with those from control donors upon co-culture with 16HBE14o- cells (figure 4.2).                   Figure 4.2 Expression of miR-146a-5p in primary human lung fibroblasts (PHLFs) upon co-culture with 16HBE14o- cells. PHLFs from control donors or chronic obstructive pulmonary disease (COPD) patients were cultured alone or co-cultured with 16HBE14o- cells for 72 h after which they were serum-deprived. miR-146a-5p expression in PHLFs from control donors and COPD patients in mono-culture and co-culture with 16HBE14o- cells was related to the housekeeping noncoding RNU48, expressed as 2–ΔCt. Medians are indicated. *: p<0.05; **: p<0.01, between the indicated values  99  4.4.3 Low induction of miR-146a-5p in COPD fibroblasts is associated with the single nucleotide polymorphism rs2910164 To explain the difference in miR-146a-5p induction between COPD and control fibroblasts, we considered two possibilities. i) A difference in expression of RelB, a member of the NF-κB family of transcription factors that has been shown to regulate miR-146a-5p in mouse lung fibroblasts [258]. ii) A difference in the presence of the SNP rs2910164 in the primary miR-146a-5p sequence as this SNP has been shown to cause a reduction in the expression of mature miR-146a-5p [299]. i) We examined the expression of RelB in our PHLFs from the co-culture model, and we found no significant difference in mRNA expression of RelB between control and COPD-derived PHLFs (figure 4.3a). ii) We compared the genotypes for the rs2910164 SNP of the PHLFs used in our co-culture experiments and found that donors with the GG genotype had a lower miR-146a-5p induction after co-culture than those with the CG genotype (figure 4.3b). Importantly the donors with the GG genotype were all, except one, COPD patients.     100   Figure 4.3 Effect of RelB expression and rs2910164 polymorphism on miR-146a-5p expression in co-culture. Primary human lung fibroblasts (PHLFs) from control donors or chronic obstructive pulmonary disease (COPD) patients were cultured alone or co-cultured with 16HBE14o- cells for 72 h after which they were serum-deprived. a) RelB expression in PHLFs from control donors and COPD patients in mono-culture and co-culture with 16HBE14o- cells. b) Effect of the single nucleotide polymorphism rs2910164 on miR-146a-5p expression in PHLFs before and after co-culture with 16HBE14o- cells. mRNA levels were normalised to the housekeeping genes β2-microglobulin and protein phosphatase 1α, while miR-146a levels were related to the housekeeping noncoding RNU48, both expressed as 2–ΔCt. Medians are indicated. *: p<0.05; **: p<0.01, between the indicated values.   4.4.4 MiR-146a-5p overexpression has anti-inflammatory effects on lung fibroblasts miR-146a-5p has been reported to exert anti-inflammatory properties by targeting the proteins of IL-1 receptor-associated kinase (IRAK)-1 and TNF receptor-associated factor (TRAF)-6, which are key downstream mediators in the cellular response to IL-1 [208]. Hence, we were interested in examining the anti-inflammatory mechanism of miR-146a-5p in human lung fibroblasts. For overexpression of miR-146a-5p experiments we used a human lung fetal fibroblast cell line (MRC-5). First, we examined the expression of miR-146a-5p in MRC-5 fibroblasts co-cultured with 16HBE14o- cells and found a similar induction of miR-146a-5p in MRC-5 fibroblasts upon co-culture (figure 3.4a) as in the PHLFs. Next, we successfully overexpressed miR-146a-5p levels in MRC-5 fibroblasts by treatment with the miR-146a-5p mimic compared with the scrambled non-101  targeting control mimic (figure 3.4b). We then assessed the protein expression levels of IRAK-1 and TRAF-6 in MRC-5 fibroblasts after overexpressing miR-146a-5p. We found the protein expression of IRAK-1 was significantly reduced after miR-146a-5p overexpression compared with scrambled control, but TRAF-6 was unaffected (figure 3.4c and d). This indicates that miR-146a-5p indeed regulates IL-1 receptor downstream signaling in human lung fibroblasts. To determine the role of miR-146a-5p in the suppression of IL-1α-induced-IL-8 release, we treated MRC-5 fibroblasts with rhIL-1α after miR-146a-5p overexpression (figure 3.5a). Here, we found a significant decrease of IL-8 release from MRC-5 fibroblasts when miR-146a-5p was overexpressed compared with cells treated with the scrambled control (figure 3.5b). Taken together, our data show that miR-146a-5p regulates IL-1α-induced pro-inflammatory responses in lung fibroblasts (figure 3.6).  102                  Figure 4.4 miR-146a-5p has anti-inflammatory effects in lung fibroblasts a) MRC-5 fibroblasts were cultured alone or co-cultured with 16HBE14o- cells. miR-146a-5p expression in the fibroblasts was related to the housekeeping noncoding RNU48, expressed as 2–ΔCt. Medians are indicated. b) MRC-5 fibroblasts were seeded and immediately transfected with 25 nM miR-146a-5p mimic or the scrambled control for 48 h. miR-146a-5p expression in the fibroblasts was related to the housekeeping noncoding RNU48, expressed as 2–ΔCt. Data are presented as mean±sem (n=3 or 4 independent experiments). c) Tumour necrosis factor receptor-associated factor (TRAF)-6 and d) interleukin-1 receptor-associated kinase (IRAK)-1 protein expression with representative blots and densitometry in MRC-5 fibroblasts after transfection with miR-146a-5p mimic (Mm) or scrambled control (Sc). Bas: basal. β-Actin was used as the loading control for protein expression. Data are presented as mean±sem (n=3 or 4 independent experiments). *: p<0.05; **: p<0.01, between the indicated values.  103   Figure 4.5 miR-146a-5p reduces interleukin (IL)-1α-induced IL-8 release in lung fibroblasts. MRC-5 fibroblasts were seeded and immediately transfected with 25 nM miR-146a-5p mimic or the scrambled control for 48 h. MRC-5 fibroblasts were then serum-deprived overnight and stimulated with/without 1 ng·mL−1 recombinant human IL-1α for 24 h. a) miR-146a-5p mRNA expression in MRC-5 fibroblasts was related to the housekeeping noncoding RNU48, expressed as 2–ΔCt. Data are presented as mean±sem. b) IL-8 release from MRC-5 fibroblasts. Data are presented as mean±sem (n=3 or 4 independent experiments). *: p<0.05, between the indicated values.            104                  Figure 4.6 Proposed role of miR-146a-5p in the cross-talk between airway epithelial cells (AECs) and lung fibroblasts [208, 243, 258, 260, 300]. a) In controls, epithelial-derived interleukin (IL)-1α causes an induction of miR-146a-5p expression as well as release of IL-8 from lung fibroblasts. miR-146a-5p then binds to and downregulates the expression of IL-1 receptor (IL-1R)-associated kinase (IRAK)-1 downstream of the IL-1 pathway in a feedback loop to dampen the NF-κB activation and the inflammatory effects of the epithelial-derived IL-1α on pulmonary fibroblasts. b) In chronic obstructive pulmonary disease (COPD), cigarette smoke exposure causes an increased IL-1α release from AECs which further increases IL-8 release from fibroblasts. However, in COPD-derived fibroblasts, the IL-1α-induced increase in miR-146a-5p expression is lower compared with control-derived fibroblasts, which then contributes to lower feedback inhibition of the NF-κB activation and an exaggerated pro-inflammatory response. MyD88: myeloid differentiation primary response gene 88; TRAF: tumour necrosis factor receptor-associated factor; IKK: IκB kinase.  105  4.5 Discussion We investigated the role of miR-146a-5p in aberrant IL-1α signaling between the airway epithelium and lung fibroblasts in COPD. We found that co-culture of PHLFs with AECs significantly increases the expression of miR-146a-5p, which is completely dependent on epithelial-derived IL-1α. We demonstrate that miR-146a-5p expression has an anti-inflammatory role, by downregulating the expression of IRAK-1, which is downstream of the IL-1 pathway, and subsequently reduces IL-8 release from lung fibroblasts. Furthermore, we show that the induction of miR-146a-5p is significantly less in COPD fibroblasts and this was associated with the SNP rs2910164 (GG allele) in the miR-146a-5p gene. miR-146a-5p has been well studied as a regulator of cellular function in both innate and adaptive immunity [301]. Specifically, miR-146a-5p has been suggested to target various inflammatory pathways including Toll-like receptor and IL-1 receptor signaling [300, 301]. Overexpressing miR-146a-5p in the liver prevents the release of pro-inflammatory cytokines and protects mice from ischemia–reperfusion injury by targeting and reducing the protein expression of IRAK-1 and TRAF-6, which are downstream of the IL-1 pathway [208]. Perry et al. [260] showed that miR-146a-5p overexpression reduces IL-1β-induced IL-8 production in mono-cultures of alveolar epithelial cells. Additionally, Bhaumik et al. [261] found a high expression of miR-146a-5p in senescent human neonatal foreskin fibroblasts compared with quiescent cells. This high expression was shown to reflect a negative feedback mechanism that modulates the secretion of IL-1α-induced IL-6 and IL-8 release due to a robust senescent-associated secretory phenotype activity in fibroblasts [261]. In line with our present study, this effect was linked to the inhibition of IRAK-1, but not TRAF-6 [261]. This finding is particularly important since senescence of 106  various cell types such as epithelial cells and fibroblasts in the lung has been shown to contribute to COPD pathogenesis [302].  IL-1α is an important driver of innate immune responses [182]. This cytokine is constitutively present in the lung epithelium as part of the immune defense against inhaled particles and is responsible for the release of chemokines, such as IL-8, responsible for neutrophilic recruitment [182, 190]. In COPD, there is an increased release of IL-1α as indicated by the increased levels in sputum and bronchoalveolar lavage fluid compared with control subjects [183]. We have previously shown that exposure to CSE induces a stronger expression of IL-1α in airway epithelium from COPD patients compared with controls, leading to enhanced release of pro-inflammatory mediators such as IL-8 and IL-6 from lung fibroblasts upon their co-culture [297]. In addition, we showed that IL-1α was secreted at baseline and was responsible for a pro-inflammatory switch in lung fibroblasts [297].  In the present study, overexpression of miR-146a-5p reduced the IL-1α-induced IL-8 secretion from lung fibroblasts. Several regulatory mechanisms to modulate the effects of IL-1α are present in vivo, such as the secretion of the naturally occurring IL-1 receptor antagonist (IL-1Ra) and the decoy IL-1R2 receptor [182, 192]. Apart from these mechanisms, miR-146a-5p has emerged as a crucial regulator of the IL-1 pathway [261]. In line with previous studies [208, 261], we show that the induction of miR-146a-5p is dependent on IL-1α stimulation and also causes a downregulation in IRAK-1 protein expression. IRAK-1 is an important serine/threonine kinase that associates with the IL-1R1 receptor complex upon stimulation [303]. This eventually leads to the activation of transcription factors such as activator protein AP-1 and NF-κB, which subsequently leads to the induction and release of several inflammatory mediators, including IL-8 [303]. Thus, we hypothesize that the IL-1-induced increase in miR-146a-5p acts in a negative 107  feedback loop to regulate the observed lung fibroblast pro-inflammatory activity upon co-culture with AECs. The induction of miR-146a-5p was further enhanced when epithelial cells were pre-stimulated with CSE. This indicates an increased demand for miR-146a-5p induction as a negative feedback mechanism to counteract the enhanced release of IL-1α from the airway epithelium in smokers. Of interest, the observed increase in miR-146a-5p expression upon co-culture with epithelial cells was smaller in COPD-derived lung fibroblasts compared with control-derived lung fibroblasts. In line with our previous study [297], this reduced induction of miR-146a-5p may lead to an impaired feedback inhibition of the fibroblast-derived inflammation resulting from a higher production of IL-1α from COPD-derived epithelium exposed to CSE (figure 6). Sato et al. [243] additionally found less induction of miR-146a-5p in fibroblasts from COPD patients compared with healthy subjects after IL-1β/TNF-α stimulation. This downregulation was associated with increased expression of the cyclooxygenase-2 enzyme and an increased production of the inflammatory mediator prostaglandin E2 in the sputum of COPD patients [243]. The difference in the induction of miR-146a-5p in PHLFs from our model was only seen after 72 hours of co-culture, but not after the 24-h stimulation of PHLFs with epithelial conditioned medium. This suggests that prolonged periods of exposure to IL-1α are required to induce differential miR-146a-5p induction between COPD and control-derived fibroblasts which may be representative of the lung epithelial–mesenchymal trophic unit in COPD where there is a chronic exposure to cigarette smoke and the resultant epithelial-derived IL-1α [177, 184, 297]. This is also in line with Perry et al. [260], who suggested a time- and concentration-dependent effect of IL-1 on miR-146a-5p expression. To elucidate the underlying mechanism responsible for the lower induction of miR-146a-5p by COPD-derived PHLFs upon co-culture with epithelial cells, we investigated the possible 108  involvement of RelB, a member of the NF-κB family, which has been shown to regulate miR-146a-5p expression [258]. Although Sheridan et al. [304] reported a lower expression of RelB in PHLFs from smokers with and without COPD compared with those from nonsmokers, we did not find a difference in expression in RelB mRNA expression between non-COPD and COPD-derived PHLFs in our co-culture model. A common G>C SNP rs2910164 in the primary miR-146a-5p sequence has been associated with a reduction in the expression of mature miR-146a-5p [299]. Of interest, we found that PHLFs from donors homozygous for the GG allele of SNP rs2910164, which were all but one from COPD patients, had a lower miR-146a-5p induction after co-culture than fibroblasts from donors heterozygous for this allele. This indicates that the expression of this SNP is associated with a lower miR-146a-5p induction in COPD-derived lung fibroblasts in our co-culture model. Of interest, Wang et al. [305] showed that this particular SNP is also associated with a lower miR-146a-5p expression in relation to COPD. In conclusion, this study demonstrates that the pro-inflammatory phenotype of COPD lung fibroblasts resulting from a dysregulated epithelial–fibroblast interaction in our co-culture model may, at least in part, be due to the reduced ability of COPD-derived fibroblasts to upregulate miR-146a-5p to counter-regulate pro-inflammatory activity. miRNAs are likely to have therapeutic potential [295] with miRNA therapies recently making it through to clinical trials [225]. Hence, our finding could provide a basis for further investigations to target chronic inflammation in COPD   109  Chapter 5:  Interleukin-1 Affects Inflammatory Mediator Release and Collagen I Contraction by Airway Fibroblasts from Asthmatic and Non-Asthmatic Donors Emmanuel T. Osei1,2,3,4, Leila. Mostaco-Guidolin1, Stephanie. Warner1, May. AL-Fouadi1, Darren J. Cole7, Geoffrey N. Maksym7, Wim Timens3,4, Teal Hallstrand6,  Corry-Anke Brandsma,3,4, Irene H. Heijink3,4,5 and Tillie-Louise. Hackett1,2.     Affiliations:  1University of British Columbia (UBC), Centre for Heart Lung Innovation, Vancouver, B.C., Canada 2Department of Anesthesiology, Pharmacology and Therapeutics, University of British Columbia, Vancouver, BC, Canada 3University of Groningen, University Medical Center Groningen, Department of Pathology and Medical Biology; Groningen, the Netherlands 4University of Groningen, GRIAC (Groningen Research Institute of Asthma and COPD), University Medical Center Groningen; Groningen, the Netherlands 5University of Groningen, University Medical Center Groningen, Department of Pulmonology; Groningen, the Netherlands 6University of Washington Medical Center, Washington, USA 7Dalhousie University, School of Biomedical Engineering, Halifax, Nova Scotia, Canada    110  5.1 Chapter Summary Asthma is a chronic inflammatory disease associated with airway remodeling, which is regulated by interaction between the epithelium, mesenchymal cells and extracellular matrix (ECM) within the epithelial mesenchymal trophic unit (EMTU). Our previous work has shown that airway epithelial cells, through the production of IL-1α, regulate the phenotype of fibroblasts within the lung EMTU. The objective of this study was to assess the role of epithelial-derived IL-1 in fibroblast inflammatory and remodeling responses in asthma.  Primary airway epithelial cells (PAECs) and primary airway fibroblasts (PAFs) were obtained from asthmatic and non-asthmatic donor lungs deemed not suitable for transplantation (n=10). PAECs at passage 2 were cultured in an air-liquid interface (ALI) and the expression and release of IL-1α and other IL-1 family members were determined by PCR and ELISA respectively. PAFs were stimulated with 1ng/mL IL-1α, IL-1β and IL-33. The release of pro-inflammatory mediators was measured using ELISA and contraction of collagen I gels over time was quantified. Collagen I fiber formation was assessed using second harmonic generation-nonlinear optical microscopy (SHG-NLOM).  We found higher mRNA and protein expression of IL-1α, IL-1β and IL-33 in PAECs derived from asthma patients compared to control-derived PAECs. Exogenous stimulation of fibroblasts with IL- 1α and IL-1β but not IL-33 significantly increased the release of pro-inflammatory cytokines IL-6, IL-8, TSLP and GM-CSF and inhibited collagen I, fibronectin, and periostin expression in asthma and control PAFs. Additionally, we found that fibroblasts stimulated with IL-1α and IL-1β were inhibited in their ability to contract collagen I gels. This effect was due to downregulation of lysyl oxidase, which affected microtubule formation, as was confirmed by inhibition of LOX with β-aminoproprionitrile.  111  IL-1 production is increased in asthmatic-derived PAECs, and IL-1α and -β regulate the remodeling and pro-inflammatory phenotype of PAFs. This has important implications for IL-1 release during epithelial damage in asthma, and may help in understanding abnormal collagen deposition and remodeling in asthma, creating potential opportunities for therapeutic intervention.   5.2 Introduction Asthma is defined as a chronic inflammatory disease of the airways associated with airway hyperresponsiveness (AHR) and airway remodeling [96]. Longitudinal studies of children to adulthood have shown that current pharmacologic treatments only manage symptoms and do not change airflow obstruction caused by airway remodeling [46-49]. Thus, there is a need to understand the mechanisms of airway remodeling to identify new therapeutic targets. There is growing evidence that aberrant repair and remodeling within the asthmatic airway epithelial mesenchymal trophic unit (EMTU) plays an important role in airway remodeling [22, 44, 114].  The airway epithelium is the first chemical, structural, and immunological barrier to the inhaled environment. It removes inhaled-foreign bodies from the lung via mucocilliary function and secretion of mucus [108, 306]. When damaged, the airway epithelium becomes immunologically activated to alarm immune cells and eventually maintain tissue homeostasis [150]. Furthermore, the injured epithelium releases fibroproliferative and fibrogenic growth factors (e.g. FGF2, PDGF) that stimulate the underlying mesenchymal cells to proliferate and secrete ECM [111].  In asthma, repetitive insult by environmental triggers is thought to cause a chronic activation and aberrant regulation of the repair processes within the EMTU [106]. There is an impaired capacity of the airway epithelium to repair itself, which has been closely linked to disease 112  progression [115, 116]. It has been shown in several studies that the airway epithelium in asthma is fragile with decreased expression of adherens and tight junctions [127-135]. The asthmatic epithelium has also been shown to express features of aberrant repair, with increased expansion of the basal cell population [130, 132] and increased expression of repair markers p21WAF, TGF-β and EGFR [136, 137]. Cytokines released following damage to the airway epithelium due to allergen exposure include IL-1, IL-6, CXCL8/IL-8, thymic stromal lipoprotein (TSLP), CCL5, IL-5, CCL17, CCL22, granulocyte macrophage colony-stimulating factor (GM-CSF) and TGF-α, which contribute to eosinophilia, mesenchymal cell activation, TH2 cell chemotaxis and/or TH2 cell polarization [23, 139-141]. We recently demonstrated that airway epithelial cells through the release of IL-1α are able to stimulate the release of inflammatory mediators such as CXCL8/IL-8 and IL-6 as well as modulate extracellular matrix production by lung fibroblasts using a co-culture model [297]. IL-1α is a master regulatory cytokine involved in innate immune regulation in the lungs [182]. It has been shown that IL-1α and other family members, IL-1β, IL-33 and IL-18, are vital in driving various aspects of asthma pathogenesis including; eosinophil recruitment, TH2 activation and airway hyperresponsiveness [187-189, 307-309]. Thus, in the present study we hypothesized that IL-1 is essential for driving inflammation and remodeling in the asthmatic EMTU. We assessed the expression and release of IL-1 and its family members in the airway epithelium of asthmatics and non-asthmatics using submerged and air-liquid interface (ALI) cultures. Further, we examined the effects of the IL-1 family on airway fibroblast-derived inflammation, and on the ability of fibroblasts to remodel collagen I using collagen contraction assays.    113  5.3 Methods and Materials  5.3.1 Sample collection Primary Airway epithelial cells (PAECs) were obtained via endobronchial airway brushings from asthmatic and healthy control subjects as previously described [310]. Primary airway fibroblasts (PAFs) were isolated via outgrowth technique [311] from human lungs not suitable for transplantation from asthmatic and healthy control donors who donated their lungs for medical research to the International Institute for the Advancement of Medicine (Edison, NJ). A lung was identified as healthy if the donor had no history of respiratory disease or other co-morbidities, while asthmatic individuals had a history of physician diagnosed asthma. The donor demographics are provided in Table 5.1.          Table 5.1 Characteristics of asthmatics and non-asthmatics from whom primary airway epithelial cell and primary airway fibroblasts were derived   Epithelial Donors    Non-Asthma Asthma Subjects  5 10 Age, mean (range)  31 (22-58) 30 (21-44) Female/Male  3/2 7/3 Fibroblast Donors    Non-Asthma Asthma Subjects  9 9 Age, mean (range)  17 (5-42) 21 (21-44) Female/Male  3/6 5/4 114  5.3.2 Cell culture conditions PAECs were cultured in bronchial epithelial growth medium (BEGM, Lonza, Walkersville, MD) containing 100 U/mL penicillin and 100 ug/mL streptomycin. Fibroblasts were grown in Dulbecco’s Modified Eagle’s medium (DMEM; Invitrogen, Burlington, ON, Canada) supplemented with 10% fetal calf serum, 2 mM L-glutamine, and 1% antibiotic/antimycotic solution in standard conditions (37˚C in humidified 5% CO2 atmosphere).    5.3.2.1 Air-liquid interface Cultures At passage 2 or 3, PAECs were grown in an air-liquid interface (ALI) on 0.4μm polyester transwell inserts (Corning, New York). Cells were exposed to air when confluent and cultured for 20 subsequent days based on the methods of Fulcher et al. [312] in a 1:1 mix of BEBM and DMEM with added bovine pituitary extract (52 μg/ml), transferrin (10 μg/ml), epidermal growth factor (EGF) (0.5 ng/ml), hydrocortisone (0.5 μg/ml), epinephrine (0.5 ng/ml) (all Cambrex), 100 μg/ml streptomycin, 0.1 μM All-trans retinoic acid, insulin (5.0 μg/ml), 100 U/ml penicillin and (all from Sigma, St. Louis, MO). RNA and cell culture supernatant was harvested at day 0, 5, 11 and 20 of ALI.  5.3.2.2 Recombinant protein stimulations  Six well tissue culture plates were coated overnight with 50μg/mL of rat tail collagen I (Corning, New York) in DMEM. Following washing with PBS, PAFs at passage 3-5 were seeded at a density of 50,000 cells per well onto the plates and allowed to grow to confluence. Cells were then serum-deprived overnight and stimulated with either control media or 1 ng/mL recombinant IL-1α, IL-115  1β or IL-33 for 24 hours. Cell culture supernatant, protein and RNA was harvested for subsequent analysis.  5.3.3 Collagen I gel contraction assay Collagen I gels were made according to a previously described method [267]. Briefly, 12 well tissue-culture plates were coated with 1% BSA (Sigma) in DMEM (Lonza) for 2 h. The medium was then removed and 1 ml of 0.4 mg/ml type I Rat tail collagen (Corning) in DMEM was added and allowed to polymerize for 16 h at 37°C. Collagen gels were carefully detached from the sides of the plate before PAFs were trypsinized and seeded at a density of 40,000 cells per well. Immediately after seeding, PAFs were treated with either control media, recombinant human 1 ng/ml IL-1α, IL-1β or IL-33 (R&D systems). Fibroblast contraction of collagen I gels after 24 hours was quantified by imaging gels before and after the experiment and extent of gel contraction was analyzed using Image J software, and measuring semi-dry weight of gels after the experiment. Lastly, to understand the remodeling of collagen fibers during gel contraction, gels were then fixed in 4% paraformaldehyde for 20 minutes then stained with 4',6-diamidino-2-phenylindole (DAPI) to identify nuclei and Phalloidin (Thermo Fisher Scientific, Waltham, USA) to stain for F-actin. To assess the effects of lysyl oxidase (LOX) activity on collagen I gel contraction, seeded gels were stimulated with 10 mg/ml of β-aminopropionitrile (BAPN) fumarate salt (Sigma) which is a broad inhibitor of LOX activity.  5.3.4 Non-Linear Optical Microscopy and Texture analysis of Collagen I gels Second Harmonic Generation Non-linear optical microscopy (SHG-NLOM) was carried out on fixed collagen I gels using a multi-photon microscope as described previously [313]. The peak 116  intensity of fibrillar collagen was expressed as arbitrary unit (au). Texture analysis using a gray level co-occurrence matrix (GLCM) was used to calculate the probability of pixels within the image occurring with a particular gray-tone, in a predetermined direction and separated by a pre-defined distance as described by Haralick and colleagues [314]. This enables us to calculate the Entropy (∑ 𝑃𝑖,𝑗 log𝑖,𝑗N-1IJ=0 ) of fibriliar collagen to determine the organization of collagen fibrils. Textural features were extracted by using a Matlab custom-built texture analysis toolkit. Some functions were based on the Matlab image processing toolbox [313] as well as ImageJ’s histogram analysis toolbox.  5.3.5 Optical magnetic twisting cytometry (OMTC) To assess the effect of IL-1 on cell stress, cell stiffness was measured using OMTC as previously described [315, 316]. Briefly, primary airway fibroblasts at passage 3 were seeded on a 96 well plate with surface bound magnetic beads and grown to confluency. Cells were then serum starved and treated with control media or recombinant IL-1α for 24 hours. The 96 well plate was then placed on the stage on an inverted microscope (DM-IRB, Leica Microsystems) equipped with an electro-magnetic twisting device and a charge-coupled device camera (1,280 × 1,024 pixels, 12-bit gray scale, SensiCam, The Cooke, Auburn Hills, MI). The Beads were twisted with a specific torque of 56 Pa at 0.5 Hz, and the camera imaged the beads continuously (~200 beads at a time) at 16 frames per twisting cycle. Using an intensity centroid algorithm, the bead positions in each recorded image were automatically determined [315] and then Fourier transformation was used to extract the displacement of each bead in response to the applied torque [315-317]. Beads with erratic, irreproducible motions were not analyzed. Thus, for a given specific torque (T̃) applied to a bead and the resultant bead displacement (D̃), as described above, a complex stiffness (G̃) of the 117  cell was defined as the ratio of the torque to the displacement, i.e., G̃ = (T̃/D̃) = G′ + iG”, where G′ is the in-phase component or elastic stiffness, which we hereafter refer to as cell stiffness (in Pascal/nm)  5.3.6 ELISA Concentrations of IL-1α, IL-1β, IL-33, released from PAECs and concentrations of IL-8, IL-6, thymic stromal lipoprotein (TSLP) and granulocyte-monocyte colony stimulating factor (GM-CSF) released from PAFs were measured by ELISA (R&D Systems, Minneapolis, USA) according to the manufacturer’s instructions.  . 5.3.7 Gene expression analysis Total RNA was harvested from PAECs and PAFs using the miRNEasy and RNEasy (Qiagen) respectively. For targeted RNA sequencing of PAECs, the Illumina HiSeq 2000 (Illumina, San Diego, USA) was used. TopHat2 (v2.0.6) was used to align the RNA reads of IL-1α, IL-1β and IL-33 to the human genome (build hg19) [318]. The BEDTools’ coverage utility was then used to specify the number of RNA reads that aligned to every metagen locus after a BED file of Ensembl Gene (ENSG) loci was constructed from the Ensembl build 69 [319]. RNA from PAFs was converted to cDNA with the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA) as per the instructions of the manufacturer. qRT-PCR was then performed for specific gene expression assays (all from Life Technologies) including collagen Iα1 (Hs00264051_m1), fibronectin (Hs00365052_m1), periostin (Hs01566734_m1), glioma-associated oncogene homolog 1 (GLI-1) (Hs00942480_m1) and Lysyl oxidase (Hs00942480_m1) on the VIIA7 with Protein phosphatase 1 catalytic subunit, alpha isoenzyme (PP1A) 118  (Hs00267568_m1), β-2 microglobulin (B2M) (Hs00984230_m1) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Hs02786624_g1) as the housekeeping genes according to the manufacturer’s instructions.  5.3.8 Statistics  Statistics were done with the Graphpad Prism software version 6. Differences between disease groups and conditions were assessed using a 2-way ANOVA and Bonferroni post-hoc correction while differences between multiple stimulations were assessed with the non-parametric Friedman’s test and the post hoc Dunn’s test. Further, direct comparison between diseased and control groups were assessed with the Mann-Whitney U test and paired observations were analyzed with the Wilcoxon signed rank test as well as t tests. P<0.05 was considered statistically significant.   5.4 Results  5.4.1 Asthmatic-derived undifferentiated basal cells release increased levels of IL-1 We assessed the expression of the IL-1 family members during epithelial differentiation using the ALI model to mimic the process of epithelial re-differentiation in vivo. As shown in figure 1, we found that PAECs from asthmatics express higher mRNA levels of IL-1α (figure 5.1a), IL-1β (figure 5.1b) and IL-33 (figure 5.1c) than those from controls on day 0 of the ALI culture, of the confluent monolayer. However, by day 5 of ALI culture, as the cells polarize and increase barrier function to differentiate into a pseudostratified epithelium, the expression of these IL-1 family 119  members in asthmatic-derived cells was downregulated to the levels of control-derived cells (figure 5.1a-c), and remained low for at least 20 days. The expression of other IL-1 family members (IL-18 and IL-1 receptor antagonist) that may be involved in the pathogenesis of asthma did not change during epithelial differentiation (data not shown). Importantly, at the protein level, asthmatic-derived undifferentiated epithelial cultures also secreted significantly higher levels of IL-1α compared to non-asthmatic samples, as measured in the basolateral compartment (figure 5.1d). This further supports a potential role of secreted IL-1 in epithelial repair and these levels again decreased upon epithelial differentiation. IL-1β secretion by asthmatic-derived PAEC also tended to be higher at day 0 compared to healthy-derived cultures (figure 5.1e), but this failed to reach statistical significance. IL-33 levels were below the detection limit of the assay.     120   Figure 5.1 Production of IL-1 & IL-33 in differentiated air-liquid interface (ALI) cultures of primary airway epithelial cells. Primary airway epithelial cells (PAECs) from non-asthmatic (n=5) and asthmatics (n=10) were cultured at an air-liquid interface, RNA and supernatants were collected at Days (D) 0, 5, 11 and 20. a) IL-1α b) IL-1β and c) IL-33 expression from PAECs are expressed as normalized to base pair reads. The concentration of d) IL-1α & e) IL-1β released from PAECs was measured by ELISA. Medians ± IQR are shown,*=P<0.05 and **=P<0.01 between the indicated values.     121  5.4.2 IL-1 stimulates an innate inflammatory response in airway fibroblasts To ensure the responses observed in fibroblasts cultures were specific to each IL-1 family member, we used commercially available recombinant proteins in our experiments. We chose to study the effects of both IL-1α, IL-1β and IL-33 using recombinant proteins to ensure we were capturing the response of airway fibroblasts to specific IL-1 family members. As shown in figure 5.2, stimulation with IL-1α and IL-1β, but not IL-33, induce a significant increase in IL-6 (figure 5.2a), CXCL8/IL-8 (figure 5.2b), GM-CSF (figure 5.2c) and TSLP (figure 5.2d) release from PAFs compared to basal conditions (P<0.01). Of interest, there was no difference in the response of PAFs derived from non-asthmatic and asthmatic donors. As our in vitro experiments demonstrated that airway epithelial cells release ~30 pg/ml of IL-1 during differentiation, we performed dose response experiments with IL-1 ranging from 0.001 to 1 ng/ml, and all doses demonstrated significant effects on CXCL8/IL-8 release from airway fibroblasts (supplemental figure C.2). We also assessed the viability of PAFs after cytokine stimulations and found no effect on cell death (supplemental figure C.3).           122   Figure 5.2 IL-1 but not IL-33 stimulates the release of inflammatory mediators from primary airway fibroblasts (PAFs). Primary airway fibroblasts from non-asthmatics and asthmatics were grown to confluence on collagen I coated plates and stimulated with or without 1ng/ml recombinant human IL-1α, IL-1β or IL-33 for 24hours. Concentration of a) IL-6, b) CXCL8/IL-8, c) granulocyte-monocyte colony stimulating factor (GM-CSF) & d) thymic stromal lipoprotein (TSLP) released from primary airway fibroblasts after 24 hours. ****=P<0.0001 between the indicated values.     123  5.4.3 IL-1 induces down-regulation of extracellular matrix proteins in airway fibroblasts Next, we assessed the effects of cytokine stimulation on the expression of ECM proteins in the fibroblasts and found that both IL-1α and IL-1β, but not IL-33, caused significant down-regulation of the mRNA expression of collagen Iα1 (figures 5.3a, b and c), periostin (figures 5.3e, f and g) and fibronectin (figure 5.3h, i and j).  There were no differences in the mRNA expression of collagen Iα1, periostin or fibronectin comparing asthmatics and non-asthmatics with or without IL-1 stimulation. When we assessed the protein expression of collagen Iα1, unlike the mRNA, we found no changes in protein levels following 24 hours exposure to IL-1α and IL-1β (supplementary figure C.4). However, changes in collagen I protein expression have been shown to take up to 48 hours to stimuli such as TGF-β and therefore at the 24 hour time point used in this study we may not capture the subsequent change in protein expression [311]. As previous work has shown that IL-1 can down-regulate collagen expression via down-regulation of the sonic Hedgehog (SHH) transcription factor glioma-associated oncogene homolog 1 (GLI-1) [181], we assessed the expression of GLI-1 in our fibroblast cultures. We found that IL-1α (figure 5.3l) and IL-1β (figure 5.3m), but not IL-33 (figure 5.3n), stimulation caused a down-regulation of GLI-1 in fibroblasts independent of disease.        124                                          Figure 5.3 IL-1α and IL-1β induce decreased extracellular matrix protein expression in airway fibroblasts. Primary airway fibroblasts from non-asthmatics and asthmatics were grown to confluence on collagen I coated plates and stimulated with or without 1ng/ml recombinant human IL-1α, IL-1β or IL-33 for 24hours. The mRNA expression of Collagen Iα1 (a-c), Periostin (d-f) Fibronectin (g-i) and glioma-associated oncogene homolog 1 (GLI-1) (j-l) was assessed at 24 hours. *=P<0.05, **=P<0.01 and ***=p<0.001 between the indicated values. 125  5.4.4 Collagen I fiber formation and contraction by airway fibroblasts is inhibited by IL-1 It is well established that airway fibroblasts are important for the production, repair and contraction of fibrillar collagens during wound healing [150, 156, 320]. Figure 5.4a, shows representative images of collagen I gels seeded with PAFs and incubated with medium control, IL-1α, IL-1β or IL-33 for 24 hours. We first compared the basal rates of contraction, and found asthmatic PAFs were less able to contract collagen gels compared to non-asthma-derived PAFs (figure 5.4b). In comparison to the basal rate of contraction of collagen I gels by PAFs after 24 hours, treatment with IL-1α and IL-1β but not IL-33, inhibited the contraction of collagen I gels (figure 5.4c). Due to the fact that collagen gel contraction in this model is due to matrix compaction and extrusion of water from the gel, gel weight can also be used to determine gel contraction [321]. As shown in figure 5.4d, our data were further validated by IL-1α and IL-1β treated collagen gels having a significantly greater weight compared to control and IL-33 conditions.  Free floating 3-dimensional collagen gels confer a mechanically compliant environment which enables fibroblasts to compact the collagen matrix and transmit mechanical force into the surrounding matrix inducing local and global matrix remodeling [322]. In the collagen I gelatin gel this leads to fibrillar collagen formation and arrangement [322, 323]. When we analyzed the expression of fibrillar collagen I using SHG-NLOM (figure 5.4e) we found that IL-1α and IL-1β but not IL-33, inhibited the ability of airway fibroblasts to remodel gelatin collagen I into fibrillar collagen I compared to control conditions shown by a decreased peak intensity of fibrillar collagen (figure 5.4f). Using texture analysis, we assessed the orientation of fibrillar collagen within the collagen I gels. We found that in collagen I gels treated with IL-1α and IL-1β, the fibrillar collagen had a higher measure of entropy indicating more disorganization of collagen fibers compared to 126  control and IL-33-treated samples (Figure 5.4g). We found no difference in the response of non-asthmatic and asthmatic-derived PAFs to the stimulation of IL-1α, IL-1β and IL-33.    Figure 5.4 IL-1 but not IL-33 effects fibroblast collagen 1 gel contraction and fibrillar formation Primary airway fibroblasts (PAFs) from non-asthmatics and asthmatics were grown to confluence and seeded in collagen I gels in the presence or absence of 1ng/ml IL-1α, IL-1β or IL-33 and allowed to contract for 24 hours. a) Representative gel contraction images, b) Percentage gel contraction comparing basal rates of non-asthma and asthma derived PAFs c) Percentage gel contraction after stimulation with IL-1α, IL-1β and IL-33 d) semi-dry weight of contracted gels after stimulation with IL-1α, IL-1β and IL-33. e) Representative images of fibrillar collagen 1 taken with second harmonic generation (SHG) assessing non-linear optical microscopy (NLOM) in gels, f) SHG peak intensity of fibrillar collagen I g) Entropy score for collagen I fiber orientation derived using textural analysis of SHG-NLOM images.  *=P<0.05, **=P<0.01 and ****=p<0.0001 between the indicated values.  127  5.4.5 IL-1 alters the interaction of airway fibroblasts with collagen I but not cell stiffness To assess if IL-1 influenced the interaction of PAFs with collagen fibers, the collagen I gels were stained with DAPI to localize cell nuclei and with phalloidin to stain F-actin in the cytoskeleton. As shown by the representative images in figure 5.5a, treatment with IL-1α and IL-1β but not IL-33 inhibited the formation of dendritic extensions by airway fibroblasts compared to basal conditions, where fibroblasts clearly formed dendritic extensions. We quantified this by assessment of cell morphology and found that IL-1α and IL-1β exposure, but not IL-33, reduced the total cell area of airway fibroblasts (figure 5.5b) indicating a lack of dendritic extensions. These effects were not different between asthmatic and control-derived fibroblasts. To further investigate if IL-1α affects fibrillar collagen formation through effects on cell stiffness, we assessed the effect of IL-1α on the rheology of airway fibroblasts using Optical Magnetic Twisting Cytometry, enabling the measurement of cell stiffness in live cells [315, 316]. As shown in figure 5.5c, treatment with IL-1α did not affect the cell stiffness of airway fibroblasts (P=0.31).                   128     Figure 5.5 Interleukin-1 alters fibroblast interaction with collagen I. Primary airway fibroblasts from non-asthmatics and asthmatics were grown to confluence and seeded in collagen I gels in the presence or absence of 1ng/ml IL-1α, IL-1β or IL-33 and allowed to contract for 24 hours. Collagen I gels were then stained with DAPI and Phalloidin 594 for F-actin in the seeded fibroblasts a) Representative of composite images of fibroblasts and fibrillar collagen I taken with the confocal microscope and second harmonic generation (SHG) assessing non-linear optical microscopy (NLOM) in gels, b) Cell area measured as pixels2 of fibroblasts seeded in collagen I gels after contraction. c) Fibroblasts were seeded on 96 well plates in the presence of 1ng/ml IL-1α. Optical magnetic twisting was then used to measure cell stiffness presented as G1 (Pa/nm).  *=P<0.05 between the indicated values.     129  5.4.6 IL-1 controls fibroblast remodeling via lysyl oxidase regulation of microtubule formation It has been suggested the IL-1 affects collagen remodeling by fibroblasts through the  regulation of the collagen cross-linking enzyme lysyl oxidase (LOX) [181]. Fibroblasts in free-floating collagen gels have been shown to make little new ECM, while LOX has recently been shown to be an important microtubule formation [324]. Hence, we proposed that IL-1 via LOX may play a role in collagen I remodeling through modulation of microtubule formation. When we examined the expression of LOX in airway fibroblasts, we found that IL-1α and IL-1β, but not IL-33, induce down-regulation of LOX (figures 5.6a, b and c). To test if LOX was important for collagen I compaction by airway fibroblasts, we treated cells with the LOX inhibitor β-aminopropionitrile (BAPN). With BAPN inhibition we observed loss of dendritic extensions and rounding of airway fibroblasts (figure 5.7a), leading to a decreased cell area (figure 5.7b). This was similar to the rounding phenotype observed with IL-1α and β treatment, and accompanied by an almost complete inhibition of collagen I gel contraction by airway fibroblasts (figure 5.7c & d) compared to basal conditions. Cell death analysis indicated BAPN stimulation did not affect cell viability (figure 5.7e).         130   Figure 5.6 IL-1 down-regulates the expression of lysyl oxidase (LOX) in airway fibroblasts. Primary airway fibroblasts (PAFs) from non-asthmatics and asthmatics were grown to confluence on collagen I coated plates. mRNA expression of LOX after stimulating with or without 1ng/ml recombinant a) IL-1α, b) IL-1β & c) IL-33 for 24hours., ***=P<0.001 between indicated values.              131                    Figure 5.7 Lysyl oxidase activity is essential for fibroblast contraction of collagen I gels Primary airway fibroblasts from non-asthmatics and asthmatics were grown to confluence and seeded in collagen I gels and allowed to contract for 24 hours in the presence or absence of 10mg/ml β-aminopropionitrile which inhibits lysyl oxidase activity. Collagen I gels were then stained with DAPI and Phalloidin 594 for F-actin in the seeded fibroblasts a) Representative of composite images of fibroblasts and fibrillar collagen I taken with the confocal microscope and second harmonic generation (SHG) assessing non-linear optical microscopy (NLOM) in gels, b) Cell area measured as pixels2 of fibroblasts seeded in collagen I gels after contraction. c) Percentage gel contraction of collagen I gels d) semi-dry weight of contracted gels. e) Primary airway fibroblasts (PAFs) were grown to confluence on collagen I coated plates and stimulated with or without 10mg/ml BAPN. Percentage lactate dehydrogenase (LDH) released from cells after 24 hours. **=P<0.01 and ***=P<0.001 between indicated values.  132  5.5 Discussion We report that the production of IL-1α is elevated in undifferentiated asthmatic airway epithelium compared to that of healthy controls. Furthermore, we show that IL-1 is important for regulating fibroblast pro-inflammatory responses (IL-8, IL-6, TSLP, GM-CSF), ECM production (collagen, fibronectin, periostin) and fibroblast interactions with fibrillar collagen I via LOX activity. These data support a role for IL-1 in the asthmatic EMTU and a potential role in disease pathogenesis.   In the lung, IL-1 plays an essential role in the normal immune defense against inhaled particles and infections [190]. The airway epithelium is a constitutive source of IL-1, storing it primarily as a plasma membrane-associated pro-form [182]. After damage to the airway epithelium by inhaled allergens, as seen in asthma, IL-1 can be released as an alarmin to help initiate immune responses [31, 325]. In this study we used air-liquid interface cultures to model the process of epithelial re-differentiation. We found higher IL-1α mRNA and protein expression in undifferentiated cells forming a simple cuboidal epithelial monolayer, compared to differentiated, pseudostratified cells. Importantly, we also found that asthmatic-derived epithelial cells express significantly higher mRNA and protein levels of IL-1α than control-derived epithelial cells.  While there is strong evidence to support a role for IL-1 release by airway epithelial cells following infection or damage by inhaled particles or stimuli [188, 189, 308, 309], this is the first report to demonstrate IL-1 release in undifferentiated, leaky airway epithelium, especially in asthmatic-derived cultures. This undifferentiated airway epithelial phase parallels the first stages of epithelial wound repair/re-epithelization models, where epithelial cells display a similar phenotype during the initial “inflammatory phase” [326]. In line with this, acute exposure of a mouse model to silica induced an early release of IL-1α, which peaked between the first 12 to 24 hours of exposure and was then switched off [327]. In the present study, we also found IL-1 was switched off following 133  5 days of epithelial polarization and differentiation. In the human airways, the epithelium is exposed daily to inhaled allergens [130, 328] causing epithelial barrier dysfunction and an inflammatory environment in asthma. We propose this could lead to an environment of persistent and exaggerated IL-1α release from the asthmatic airway epithelium. Airway epithelial damage in asthmatics has been suggested to have direct effects on the underlying fibroblasts in the EMTU, leading to structural changes [106, 114]. By the use of co-cultures and conditioned medium exposure studies, we and others have previously shown that epithelial-derived IL-1α is important for the regulation of lung fibroblast-mediated inflammation and ECM production [177, 183, 184, 297]. When IL-1 is released into the airway EMTU, it has been shown to have a strong effect on fibroblast pro- inflammatory and repair responses [177, 297, 329]. We demonstrated that IL-1α is the most prominent IL-1 family member released from undifferentiated epithelium and that this release is exaggerated in asthmatic epithelium, while IL-1β levels were lower and IL-33 was undetectable. For all of the read-outs in the current study, only IL-1α and IL-1β, but not IL-33, affected fibroblast function. This could be due the fact that IL-1α and IL-1β both bind and signal through the same IL-1 receptor (IL-1R) 1 with the activation of a similar down-stream pathway, while IL-33 signals through a different receptor, ST2. This receptor is also expressed on mesenchymal cells, but IL-33 has been shown to primarily affect immune cell types such as mast cells, basophils, T and B cells instead of airway fibroblasts in asthma [330, 331]. In this study, we demonstrate that in response to exogenous recombinant IL-1α and IL-1β, airway fibroblasts release the pro-inflammatory cytokines IL-6, CXCL8/IL-8, TSLP and GM-CSF, which are vital for eosinophilia, mast cell activation and TH2 driven inflammation in asthma [23]. Increased levels of these cytokines has been linked to release of IL-4 and IL-5, all of which 134  has been shown to be involved in allergic sensitization, hypercontractility of smooth muscle cells and airway hyperresponsiveness in asthma [23, 44]. In addition to the effect on inflammatory mediator release, IL-1 stimulation caused a down-regulation of airway fibroblast expression of ECM proteins collagen Iα1, periostin and fibronectin. This supports the role of IL-1 in counteracting an increased ECM expression by fibroblasts that we and others have previously demonstrated [297, 332, 333]. There was no difference in the ECM response of airway fibroblasts from asthmatics and non-asthmatics to exogenous IL-1 stimulation in the present study. This notwithstanding, Reeves et al demonstrated with epithelial-fibroblast co-cultures that an aberrant ECM response by asthma-derived airway fibroblasts is due to a defective ability of the asthma-derived airway epithelium to downregulate fibroblast ECM expression [178]. This was the result of increased production of TGF-β2 by asthma-derived epithelial cells compared to controls [178]. IL-1 and TGF-β have been shown to influence each other’s biological activity in lung fibroblasts [155, 181]. IL-1 can inhibit TGF-β-dependent myofibroblast transformation [181], while TGF-β enhances myofibroblast survival by suppressing IL-1-induced apoptosis [155]. Hence a dysregulated balance between the production of these regulatory cytokines may cause fibroblasts to be more pro-fibrotic in later stages of disease. This defect may add to the pro-fibrotic phenotype of fibroblasts in asthmatic airways that may lead to airway remodeling [178]. Therefore, it will be of interest to study the combined effect of TGF-β and IL-1 in future studies. Although IL-1 is a classical inflammatory mediator, it has been suggested to have regulatory effects on fibroblast activation and ECM molecule expression via the downregulation of the SHH pathway [334]. In agreement with this, we demonstrate in the present study that IL-1 down-regulates the SHH transcription factor GLI1, which has been shown to be involved in fibroblast activation and the expression of ECM molecules, including collagen I [334].   135  In addition to fibroblast ECM expression, we also examined the potential effect of epithelial-derived IL-1 release on the functional repair phenotype of airway fibroblasts in the asthmatic EMTU. It is well known that cell morphology is highly dependent on the surrounding matrix environment [335]. Fibroblasts in connective tissues under resting conditions are normally organized in to a dendritic network [336]. It has previously been shown that in relaxed collagen gels, fibroblasts form dendritic extensions which have a neuronal-like appearance, with microtubule cores and actin-rich tips, that are reminiscent of fibroblasts under resting conditions in vivo [337]. These dendritic extensions enable the cell to spread and become entangled with collagen fibrils, resulting in integrin-dependent mechanical interactions [337]. Further, it has been shown in low-density fibroblast seeded collagen gels, that microtubule formation is essential for the first 24 hours of collagen I compaction by lung fibroblasts. However, after 48 hours fibroblasts in compacted collagen gels can develop stress fibers and lamellipodia, which resemble fibroblasts during wound repair [338]. In our experiments, we found asthmatic airway fibroblasts have a basal abnormal fibrillar collagen remodeling phenotype. In addition to this, IL-1α and IL-1β further inhibited collagen I contraction, fiber formation and formation of dendritic extensions in treated fibroblasts while causing a disordered fibrillar collagen matrix (Chapter 6). Disorganization of fibrillar collagen may potentially be vital to chronic airway remodeling in asthma, since disorganization and fragmentation of collagen may serve as a signal for enhanced ECM production by fibroblasts [16]. In addition, small modifications in the structure and organization of ECM has been shown to modify its bioactive regulation on various cells through processes including increased oxidative stress, which may be implicated in structural changes in the asthmatic EMTU [16, 339]. 136  As to the mechanism of how IL-1 can affect microtubule formation and subsequent contraction of collagen I, it has been shown that LOX is recruited to mitotic spindles during mitosis and that it can regulate the G2/M checkpoints in cancer cells [324]. Further, LOX is important for the cross-linking collagen by fibroblasts during repair [340, 341], however, it has been previously shown that in relaxed collagen gels fibroblasts initiate little ECM production. We demonstrated that inhibition of LOX resulted in inhibition of collagen contraction and loss of dendritic cell extensions. Interestingly, we further found that IL-1 stimulation caused a down-regulation of LOX expression in airway fibroblasts compared to basal conditions and IL-33 stimulation. This indicates that IL-1 regulates the fibroblast repair phenotype by targeting the expression of LOX, which is line with work by Mia and colleagues, who demonstrated that IL-1 inhibits TGF-β induced myofibroblast differentiation in lung fibroblasts by down-regulating LOX expression [181].  In conclusion, this study demonstrates that the excess production of IL-1α and IL-1β from undifferentiated asthmatic airway epithelium may contribute to increased inflammation as well as abnormal remodeling of airway fibroblasts in the asthmatic EMTU. This study offers important new insights into epithelial regulation of fibroblast responses in the asthmatic EMTU, whereby IL-1 released in the early stages of epithelial differentiation acts in the initial phase of injury to promote fibroblast-mediated inflammation for cell recruitment while counteracting profibrotic actions of lung fibroblasts through the down-regulation of ECM expression. Furthermore, this early effect of IL-1 on fibroblast ECM production promotes a phenotype that leads to fibrillar collagen disorganization. The IL-1R1 and IL-1 pathway have been suggested as possible therapeutic targets in asthma. Hence our findings may create potential opportunities for therapies that do not only target airway inflammation but also provide ways of modulating fibrillar collagen I remodeling in asthma. 137  Chapter 6:  Answering a 90 Year Old Question for Asthma and Airway Fibrosis Using Multimodal Nonlinear Optical Microscopy *Leila B. Mostaço-Guidolin1,2 , *Emmanuel T. Osei1,2,3,4, Soheil Hajimohammadi 1,2, Jari Ullah1,2, Xian Li1,2, Vicky Li1,2, Furquan Shaheen1, Fanny Chu1, Darren J. Cole6, Wim Timens3,4, Corry-Anke Brandsma3,4, Irene H. Heijink3,4,5, Geoffrey N. Maxsym6, David Walker1, Tillie-Louise Hackett1,2  *Co-first author  Affiliations: 1Centre for Heart Lung Innovation, St. Paul’s Hospital, Vancouver, BC, Canada 2Department of Anesthesiology, Pharmacology and Therapeutics, University of British Columbia, Vancouver, BC, Canada 3University of Groningen, University Medical Center Groningen, Department of Pathology and Medical Biology; Groningen, the Netherlands 4University of Groningen, GRIAC (Groningen Research Institute of Asthma and COPD), University Medical Center Groningen; Groningen, the Netherlands 5University of Groningen, University Medical Center Groningen, Department of Pulmonology; Groningen, the Netherlands 6Dalhousie University, School of Biomedical Engineering, Halifax, Nova Scotia, Canada    138  6.1 Chapter Summary Asthma is characterized as a chronic inflammatory airway disease associated with airway fibrosis that leads to thickening of the airway wall and a reduction in lung function. Using histological stains for over 90 years has led to the understanding that the fibrosis in asthmatic airways is mainly the result of increased collagen deposition within the basement membrane and lamina propria of the airway mucosa. However, histological stains are not able to resolve the biochemical and structural composition of specific extracellular matrix (ECM) proteins such as fibrillar collagen formation, which is found in fibrotic scar tissue. Here, we used nonlinear optical microscopy (NLOM) for label-free visualization of fibrillar collagen (second harmonic generation) and elastin (two-photon excitation fluorescence) fibers within airway tissue. By applying textural analysis algorithms, we demonstrate for the first time that fibrillar collagen (scar tissue) is highly disorganized and fragmented within asthmatic airways, which can affect the structural composition and cellular responses within the airway tissue. Further, we demonstrate that asthmatic-derived fibroblasts are deficient in their ability to remodel fibrillar collagen I compared to healthy controls due to defective decorin production. This imaging approach has the potential to enhance the clinical interpretation of airway remodeling in asthma and direct new avenues of research for therapeutic strategies to prevent tissue fibrosis.    6.2 Introduction An estimated 300 million people worldwide suffer from asthma, with 250,000 annual deaths attributed to the disease [1]. Treating life-long sufferers with asthma in the US alone cost over $56 billion, in the most recent assessment in 2007 [7, 342]. While reliever therapies exist for asthma 139  symptoms, there is still no cure for the disease [343]. The disease is characterized as a chronic inflammatory airway disease associated with airway remodeling that involves all the tissues of the airway wall, including the epithelium, basement membrane, lamina propria [344], smooth muscle and vascular structures [17, 343, 345]. To analyze airway remodeling, airway tissue biopsies are taken and several histological chemical stains such as Masson’s Trichrome or pentachrome stain are used to visualize tissue components [346, 347]. Using such stains since 1927, it has been shown that collagen is greatly accumulated within asthmatic airways compared to controls [348]. However, such histological stains are not able to distinguish between non-fibrillar and fibrillar collagen, the latter being the predominant collagen found in scar tissue [163]. Additionally, the histological stains provide no information on the biochemical and structural characteristics of collagen fibers.  Collagen is the most abundant protein in the animal kingdom and is found in all connective tissues, providing structural integrity of all internal organs [349]. Type I fibrillar collagen is the predominant collagen in the lung, skin, cornea, liver, tendon and bones and the concentration and distribution of collagen fibers is essential for the size, shape, density and strength of tissues [350]. Fibrillar collagens have a triple helix structure (known as a Madras helix), which imparts tremendous strength on the protein’s structure enabling it to act like “rebar” (reinforcement rods used in concrete construction) within the extracellular matrix [351-353]. Models of wound healing have shown that type III fibrillar collagen is the major collagen deposited following injury, however once the granulation tissue is remodeled to form a scar, it is primarily composed of type I fibrillar collagen [326]. During wound repair the ECM molecule elastin, which normally provides the ability of tissues to undergo repetitive strain and relaxation, such as during breathing, is lost [353, 354]. While type I collagen is needed to repair the structure and function of damaged tissues, 140  excessive collagen within a wound results in loss of anatomical structure, compromised function, and tissue fibrosis [355, 356]. Conversely, insufficient collagen deposition results in weak scar tissue [357]. Therefore understanding collagen and elastin fiber formation is vital to understanding normal wound healing and how this process is dysregulated in disease pathologies such as airway remodeling in asthma. Here we report on the use of multimodal nonlinear optical microscopy (NLOM) imaging, which has the ability to visualize collagen (second-harmonic-generation) and elastin (two-photon excited auto fluorescence (TPEF)) fibers without chemical stains [313, 358]. We provide new insight into the organization of fibrillar collagen fibers in the remodeled airways of asthmatic donors compared to healthy controls using texture analysis. Further, we confirm our findings using transmission electron microscopy. Lastly, our data are supported by in vitro data, demonstrating that airway fibroblasts from matched asthmatic patients are unable to effectively remodel and contract collagen fibers compared to healthy controls. This study helps in expanding our understanding of collagen deposition and remodeling in asthma and creates potential opportunities for therapeutic intervention.   6.3 Methods and Materials  6.3.1 Human lung tissue & cell preparations The lungs of 10 asthmatic and 10 non-asthmatic subjects, not suitable for transplantation, were donated for research through the International Institute for the Advancement of Medicine (IIAM, Edison, NJ, USA; www.iiam.org) and biobanked within the James Hogg Lung Registry (Ethics 141  protocol number H00-50110). The demographics of the subject lungs assessed are shown in Table 6.1. This study was approved by the Providence Health Care Research Ethics Board (H13-02173). There was no statistical difference between the sex and age of the asthmatic and non-asthmatic group of subjects. Airway and parenchymal tissue was dissected via blunt dissection and airway and parenchymal fibroblasts were derived using the outgrowth technique as previously described [311, 359]. Donor lungs were then inflated with Tissue-Tek (Miles, Inc., Elkhart, IN) and frozen as per the James Hogg Lung Registry protocol previously described elsewhere [360]. Once frozen, lungs were band sawed into 2 cm lung slices; from each slice, a 15mm x 20 mm tissue sample was taken in an unbiased uniform fashion, fixed with formalin and paraffin embedded. Tissue cores were sectioned with a microtome at 5 µm to 20 µm thickness. A sequential section for all tissue blocks was used to identify all airways contained within each section and then segregated into small (< 2mm diameter) and large (> 2 mm diameter) airways. There was no statistical difference in the mean small airway diameter for non-asthmatics (704.1 µm ±83.1) versus asthmatics (753.1 µm ±117.2). This was also the case for the mean size of large airways in non-asthmatics (5.81 ± 0.98 mm) and asthmatics (5.3 ±1.27 mm).   Airway samples were also fixed with 2.5% glutaraldehyde for 2 hours, and then washed with cacodylate buffer, a solution of 1% osmium and 1% potassium ferrocyanide for 1 hour, and distilled water for transmission electron microscopy. Next, the samples were dehydrated through a graded acetone series and embedded in epoxy resin. The resin blocks were sectioned with a diamond knife on a Leica EM UC7 ultramicrotome, placed on Formvar-coated grids and stained with lead citrate and uranyl acetate for transmission electron microscopy.    142      Table 6.1 Characteristics of asthmatics and non-asthmatics from whom airway biopsies and primary airway fibroblasts were derived.   6.3.2 Non-Linear Optical Microscopy Samples were investigated using an in-house built multimodal femtosecond nonlinear optical microscope which is capable of acquiring co-localized two-photon excited fluorescence (TPEF) and second harmonic generation (SHG) images of a sample simultaneously, either in the forward- or in the epi-direction. Its general setup is shown in figure 6.1a. In summary, the laser source is a Ti:Sapphire oscillator. The output fs pulses from the Ti:sapphire oscillator are split into the reflected pulses and the transmitted pulses using a beam splitter. The reflected pulse is transmitted through various optical components, including a delay line stage, and reaches the sample through an objective lens. Two non-descanned PMT detectors are mounted on the microscope for simultaneous detection of TPEF and SHG signals through arrays of dichroic and color filters.  Typically 25 mW of pump and 8 mW of Stokes (measured after the objective) were used for imaging. We used ScanImage (ver. 3.5) software for sample stage control and image acquisition. Post image processing and image viewing were carried out in ImageJ (ver 1.42b) and Matlab. We used a 20×, 0.75-NA objective lens in order to combine a large field of view and a good spatial resolution.  Non-Asthma Asthma Subjects 10 10 Age, mean (range) 17 (5-42) 21 (21-44) Female/Male 3/7 5/5 143   Developed by Denk et al. [361], TPEF microscopy measures the simultaneous absorption of two photons, where the total energy is equivalent to that of a single photon at half the wavelength, leading to electron excitation of fluorescent molecules in the sample (figure 6.1b). Due to this excitation process, TPEF microscopy possesses some interesting features: enhanced depth of penetration owing to the use of a near infrared excitation, good optical sectioning, good axial resolution, and reduced photo-bleaching and photo-toxicity [361-363].  The second nonlinear optical process detected is the SHG [364]. It is a coherent elastic process where two excitation photons are combined in an optically nonlinear medium, to create a SHG photon with a wavelength exactly half of the excitation wavelength [364]. This process is summarized in the energy level diagram shown in figure 6.1c. As the SHG signal arises from samples lacking inversion symmetry, SHG microscopy has been proven to be an ideal tool to analyze the spatial arrangement of collagen fibers in tissue and quantification. Representative images showing the sub-epithelial region (SER) and the BM under the NLO microscope as well as images acquired from histology are presented in figure 6.2   144     Figure 6.1 (a) Schematic of the home-built nonlinear optical microscope. Light source is a Ti:Sapphire femtosecond oscillator (Tsunami, Spectra-Physics). F-ISO: Faraday isolator; CM: chirp laser mirrors; M1, M2: mirrors; GS: salvo scanner; DM: dichroic mirror; BF: bandpass filter; PMT: photomultiplier tube. Schematic description of two nonlinear optical techniques used in this study: (b) two-photon excited auto-fluorescence (TPEF), used to image elastic fibers; (c) second harmonic generation (SHG), which was used to image collagen type-I. Examples showing H&E and Van Giesson stained tissue as well as label-free NLO images acquired at the (d) sub-epithelial region and at the (e) smooth-muscle cells region from an asthmatics donor. Elastic fibers are color-coded in green (TPEF), collagen fibrils are shown in blue (SHG).    Figure 6.2 Examples showing Verhoeff Van Giessen stained (a-b) tissue as well as label-free NLO images (c-e) acquired at the sub-epithelial region. Elastic fibers are shown in black and collagen is shown in brown-yellowish colour (a-b). Elastic fibers imaged with TPEF are shown in (c), and color-coded in white (e); collagen fibers, imaged by SHG are shown in (d) and colour coded in blue (e); epithelial cells are shown in magenta (e). BM: basement membrane; EP: epithelium; SER: sub-epithelial region.    145  6.3.3 Image analysis  SHG images from the lamina propria regions of interest were acquired for each donor. Post image processing was performed in ImageJ software. Image background correction, intensity normalization and calculation of various image texture parameters were carried out using Matlab7.5. A custom-built texture analysis toolkit based on some of the texture analysis functions available in the Matlab image processing toolbox was used to calculate gray level co-occurrence matrix (GLCM) parameters. First order statistics (FOS) parameters were calculated using ImageJ’s analysis toolbox and plugins [313]. A general overview of all processing and data extracted from each SHG image is presented in supplementary figure D.1.  6.3.4 Texture analysis Texture analysis with the use of a GLCM was employed to calculate the probability of pixels within the image occurring with a particular gray-tone, in a predetermined direction and separated by a pre-defined distance as described by Haralick and colleagues [314]. This enabled us to compute the Entropy (∑ 𝑃𝑖,𝑗 log𝑖,𝑗N-1IJ=0 ) of fibrillar collagen which assessed the organization of collagen I fibrils. Textural features were extracted by using a Matlab custom-built texture analysis toolkit. Some functions were based on the Matlab image processing toolbox [313] as well as ImageJ’s histogram analysis The co-occurrence matrix was calculated in four orientations: horizontal, vertical, and the two diagonals (directions defined by four angles: 0, 45, 90, and 135), and then the average value was obtained. A window size with 14 pixels was adopted to extract features from 8 bit images.   146  6.3.5 Collagen gel contraction assays and analysis Collagen gels were made as previously described [267]. Briefly, 1 ml of 0.4 mg/ml type I Rat tail collagen (Corning) in DMEM was added to 12 well tissue-culture plates coated with 1% BSA (Sigma) in DMEM (Lonza) and allowed to polymerize for 16 h at 37°C. Collagen I gels were carefully detached from the sides of the plate before airway fibroblasts were trypsinized and seeded at a density of 40,000 cells per gel in the presence or absence of 10 ng/ml TGF-β. Fibroblast contraction of collagen I gels after 72 hours was quantified by imaging gels before and after the experiment and extent of gel contraction was analyzed using Image J software. Gels were then fixed in 4% paraformaldehyde for SHG-NLOM as described above. The peak intensity of fibrillar collagen was expressed as arbitrary unit (au) and textural analysis to calculate entropy using a GLCM, was done as described above.  6.3.6 Optical magnetic twisting cytometry (OMTC) Cell stiffness was measured using OMTC as previously described [315, 316]. Briefly, cells were seeded at passage 3 and grown to confluency on a 96 well plate with surface bound magnetic beads. Cells were then serum deprived and treated with control media or recombinant transforming growth factor (TGF)-β for 24 hours. The 96 well plate was then placed on the stage on an inverted microscope (DM-IRB, Leica Microsystems) equipped with an electro-magnetic twisting device and a charge-coupled device camera (1,280 × 1,024 pixels, 12-bit gray scale, SensiCam, The Cooke, Auburn Hills, MI). Beads were twisted with a specific torque of 56 Pa at 0.5 Hz, and the camera imaged the beads continuously (~200 beads at a time) at 16 frames per twisting cycle. Using an intensity centroid algorithm, the bead positions in each recorded image were automatically determined [315] and Fourier transformation was used to extract the displacement 147  of each bead in response to the applied torque [315-317]. Beads with erratic, irreproducible motions were not analyzed. Thus, for a given specific torque (T̃) applied to a bead and the resultant bead displacement (D̃), as described above, a complex stiffness (G̃) of the cell was defined as the ratio of the torque to the displacement, i.e., G̃ = (T̃/D̃) = G′ + iG”, where G′ is the in-phase component or elastic stiffness, which we hereafter refer to.  6.3.7 Statistical analysis The Graphpad prism software version 6 was used to perform all statistical analysis. A Shapiro-Wilk normality test was used and the data were found to be not normally distributed. The Mann-Whitney U test was used to determine significant differences while comparing non-asthmatics and asthmatics. The Wilcoxon signed rank test as well as t tests was used to assess significant differences between paired observations p-value lower than 0.05 was considered to be statistically significant.    6.4 Results  6.4.1 Fibrillar collagen is disorganized in the lamina propria of both small and large airways of asthmatic subjects Representative NLOM images of a cross-section through a large airway are shown for a non-asthmatic and asthmatic donor (figure 6.3a). First order statistics analysis demonstrated increased levels of fibrillar collagen (figure 6.3b) but not elastin (figure 6.3c) in the laminar propria of large airways from asthmatics compared to non-asthmatics. Using texture analysis, we found 148  significantly increased entropy of fibrillar collagen I in asthmatics compared to non-asthmatics (figure 6.3d), indicating that collagen fibers are more disorganized in asthmatic airways while the entropy of elastin was unchanged (figure 6.3e). When we assessed the small airways, we found a similar increase in the signal intensity of fibrillar collagen (figure 6.3f) but not elastin (figure 6.3g) in the lamina propria of asthmatics compared to non-asthmatics. Using texture analysis, we also found significantly increased entropy of fibrillar collagen fibers in the lamina propria of the small airways of asthmatics compared to non-asthmatic donors (figure 6.3h), while elastin was unaffected (figure 6.3i).        149                  Figure 6.3 Fibrillar collagen I is increased and disorganized in the lamina propria of asthmatic airways. a) Representative SHG-NLOM and TPEF images showing the lamina propria of large airways of a non-asthmatic compared to an asthmatic showing fibrillar collagen I signal in blue and elastin in white. SHG-NLOM and TPEF imaging was done in the large airways comparing non-asthmatic and non-asthmatic individuals. b) Mean intensity levels of fibrillar collagen I c) Mean intensity levels of elastin d) Entropy of fibrillar collagen I after textural analysis e) Entropy of elastin after textural analysis. SHG-NLOM and TPEF imaging was done in the small airways comparing non-asthmatic and non-asthmatic individuals, f) Mean intensity levels of fibrillar collagen I g) Mean intensity levels of elastin h) Entropy of fibrillar collagen I after textural analysis i) Entropy of elastin after textural analysis. Graphics with blue bars represent results obtained from collagen images and graphics with gray bars represent results obtained from elastin images. A: asthmatics; NA: non-asthmatics; L: lumen. Differences were considered statistically significant for p<0.05.  150  6.4.2 Collagen fibers are disorganized at the fibril level Using the resolution of transmission electron microscopy, we were able to assess the organization of collagen fibers at the fibrillar level. As shown in figure 6.4a, when we assessed collagen fibers within the laminar propria in cross-section, we found that asthmatic fibers have a disorganized structure of collagen fibrils compared to collagen fibers from non-asthmatics. We quantified the fibrils using a circularity score, where 0 would be a line and 1.0 a perfect circle. We found asthmatic donors had significantly more variation in object size compared to non-asthmatic donors (figure 6.4b).             Figure 6.4 Collagen fibrils are disorganized in biopsies from asthmatic airways. a) Representative transmission electron microscopy (TEM) images comparing a cross-section of a collagen fiber from the lamina propria of a non-asthmatic to an asthmatic airway. b) A histogram of the collagen fibrils Circularity score distribution is shown. The mean score for non-asthmatics was 0.48 and for asthmatics was 0.82, and was statistically distinct (p<0.05).  Non-Asthmatics Asthmatics 151  6.4.3 Airway fibroblasts derived from asthmatic donors are defective in collagen 1 contraction Using a free-floating collagen I gel assay, we assessed if airway fibroblasts that are normally present within the lamina propria are responsible for the defective packing of collagen I in asthma. Figure 6.5a shows representative images of collagen I gel contraction 72 hours after seeding with fibroblasts derived from both the large conducting airways or lung parenchyma of asthmatic and non-asthmatic donors. We found that airway fibroblasts derived from asthmatic patients are unable to contract collagen I gels as well as non-asthmatic derived airway fibroblasts when quantified by gel area (figure 6.5b). However, this abnormal response is specific to asthmatic airway derived fibroblasts as there was no difference in collagen I contraction by parenchymal lung fibroblasts derived from the same asthmatic and non-asthmatic donors (figure 6.5c). The pro-fibrotic cytokine TGF-β is a well-known activator of fibroblasts during repair; however treatment with 10 ng/ml TGF-β for 72 hours did not alter the response of asthmatic-derived airway fibroblasts nor non-asthmatic-derived airway fibroblasts (figure 6.5d).  To determine if defective collagen I gel contraction by asthmatic-derived airway fibroblasts was due to abnormal collagen fiber formation, we analyzed the expression of fibrillar collagen I using SHG-NLOM. Figure 6.5e is a representative image of SHG-NLOM imaging of collagen I gels after contraction comparing non-asthmatic and asthmatic–derived airway fibroblasts. As shown in figure 6.5f, collagen I gels seeded with asthma-derived airway fibroblasts had a significantly lower SHG peak intensity signal for fibrillar collagen compared to non-asthmatic fibroblasts. Using texture analysis we also assessed the orientation of fibrillar collagen within the collagen I gels and found that the fibrillar collagen present in the collagen I gels seeded with asthma-derived airway fibroblasts had a greater measure of entropy, indicating more 152  disorganization of collagen fibers compared to non-asthmatic airway fibroblast seeded gels (figure 5g).   Figure 6.5 Defective collagen I remodeling by asthma-derived airway fibroblasts. Primary parenchymal and airway fibroblasts from non-asthmatics and asthmatics were grown to confluence, seeded in collagen I gels and allowed to contract for 72 hours. a) Representative gel contraction images, b) Percentage gel contraction of airway fibroblasts c) Percentage gel contraction of parenchymal lung fibroblasts d) Percentage gel contraction of airway fibroblasts after stimulating with and without 10 ng/ml TGF-β, e) Representative images of fibrillar collagen 1 taken using second harmonic generation (SHG) assessed using non-linear optical microscopy (NLOM) in gels, f) SHG peak intensity of fibrillar collagen I g) Entropy score for collagen I fiber orientation derived using textural analysis of SHG-NLOM images.  *=P<0.05, **=P<0.01 and ****=p<0.0001 between the indicated values.   6.4.4 Fibroblast cytoskeletal functions are not altered in asthmatic-derived airway fibroblasts  To understand the mechanism involved in the defective repair of fibrillar collagen I by asthma-derived airway fibroblasts, we first assessed the rheology of airway fibroblasts using Optical 153  Magnetic Twisting Cytometry, which enables the measurement of cell stiffness in live cells [315, 316]. As shown in figure 6.6a, there was no difference in the basal cell stiffness of airway fibroblasts derived from asthmatic and non-asthmatics. To assess if a difference in cell stiffness exists in response to fibrogenic factors, we stimulated fibroblasts with TGF-β and again found no difference in cell stiffness (figure 6.6b).   Figure 6.6 Asthma and non-asthma-derived airway fibroblasts are not different in cell stiffness. Primary airway fibroblasts from non-asthmatics and asthmatics were seeded on 96 well plates in the presence for optical magnetic twisting experiments to measure cell stiffness presented as G1 (Pa/nm). a) Basal cell stiffness comparing non-asthma and asthma-derived fibroblasts, b) Cell stiffness of non-asthma and asthma-derived fibroblasts after stimulation with TGF-β.   6.4.5 Asthmatic-derived airway fibroblasts produce less decorin, essential for normal collagen packaging First, to ensure defective collagen I contraction was not due to a difference in the expression of collagen I, we examined protein expression and found no difference in the expression of collagen I protein between asthmatic-derived and non-asthmatic-derived airway fibroblasts (figure 6.7a). 154  Lysyl oxidase (LOX) is an essential extracellular enzyme produced by fibroblasts for the cross-linking of collagen and other ECM molecules. When we assessed the expression of LOX protein in airway fibroblasts from asthmatic and non-asthmatic donors seeded in collagen I gels, we also found no statistical differences (figure 6.7b). Decorin is an important proteoglycan vital for cross-linking as well as the spacing of collagen fibers. When we assessed airway fibroblasts seeded in collagen I gels for the expression of decorin, we found that asthmatic-derived airway fibroblasts had significantly lower expression of decorin protein compared to airway fibroblasts from non-asthmatics (figure 6.7c).        Figure 6.7 Lower protein expression of decorin but not lysyl oxidase in asthma-derived airway fibroblasts Primary airway fibroblasts non-asthmatics and asthmatics were grown to confluence, seeded in collagen I gels in and allowed to contract for 72 hours. a) Relative protein expression of collagen Iα1 normalized to total protein b) Relative protein expression of lysyl oxidase normalized to total protein c) relative protein of Decorin normalized to β2-microglobulin. *=P<0.05 between the indicated values.   6.5 Discussion The presented NLOM analysis demonstrates essential new insights into the disorganization of fibrillar collagen fibers in the remodeled airways of asthmatic donors. For nearly a century, histological stains have been used as the gold standard to visualize morphological changes in 155  asthmatic airways. The importance of NLOM in addition to these histological stains is that important information is obtained on the biochemical and structural characteristics of ECM fibers. Further, we demonstrate that airway fibroblasts from asthmatic patients are unable to effectively remodel and contract collagen fibers compared to healthy controls, which provides a potential mechanism for collagen fiber disorganization in asthma. All components of the airway wall (adventitia, submucosa and smooth muscle) have been shown to be increased by 50 – 300 % in cases of fatal asthma and 10-100% in cases of non-fatal asthma compared with non-asthmatic controls [365-367]. Many elements contribute to this response, including an increase in airway smooth muscle; edema; inflammatory cell infiltration; glandular hypertrophy; and connective tissue deposition [367]. Regarding connective tissue and specifically collagen deposition, the majority of studies have focused on changes in the basement membrane (lamina reticularis). While the relationship of asthma severity with the degree of basement membrane thickening remains uncertain, the majority of studies have demonstrated that the basement membrane is thickened in all asthmatics compared to controls [368]. Furthermore, these changes in the basement membrane can be observed after 1 year of birth [369]. As yet, there are limited data on the collagen composition in the lamina propria of asthmatic subjects. Evaluation of the lamina propria is important as it has a larger area than the basement membrane, and therefore has the potential to exert a greater effect on airway narrowing [366]. Also, inflammatory cells are prominent in the lamina propria, and its anatomical location places the lamina propria adjacent to the airway smooth muscle, which is thickened in asthma [116]. Patients with moderate to severe asthma have been shown to have more collagen III and V in the lamina propria compared to healthy controls and mild asthmatics [368] whereas other studies did not show any differences in collagen deposition in the lamina propria between asthmatics and non-asthmatics [163, 370]. A potential 156  explanation for the conflicting results is that the subjects in these studies were on varying steroid treatments for severe asthma and that these studies used endo-bronchial biopsies, for which it is difficult to sample the lamina propria consistently between subjects.  In this study, using tissue sections from non-transplantable donor lungs and NLOM, we demonstrate for the first time that the lamina propria in asthmatic airways contains more disorganized fibrillar collagen. Further, we demonstrate that texture analysis (GLCM) enables us to understand more about the tissue environment and biochemical properties of collagen in the asthmatic airways, whereby we report that fibrillar collagen is disorganized and fragmented in the lamina propria. We have previously validated the use of GLCM parameters to effectively quantify the biochemical properties of fibrillar collagen in a myocardium infarct rat model [313]. With regard to the structural properties of collagen, it has been shown that the arrangement of tropocollagen molecules into a staggered assembly of collagen fibrils enables collagen to withstand tensile strains of up to 50% before they break [371]. Thus, the structural formation of fibrillar collagens is essential for the biochemical properties of tissues under load or strain [371]. In this study, we demonstrate that fibrillar collagen within the lamina propria of the asthmatic airways is disorganized both at the fiber and fibril level. No studies have focused on the relationship of collagen cross-linking (collagen organization) and the tensile properties of the airway tissue. However, it has been shown in lung parenchymal tissue that the total amount of collagen fibers correlates with the resistance and elasticity of the tissue [372]. There is also significant literature from surgical repair models of the skin showing that the tensile strength of the repairing tissue increases as collagen-cross linking occurs [373, 374]. With regard to elastin, we found no difference in the signal intensity or the organization of elastin fibers in the lamina propria between asthmatic and non-asthmatic donors. This is contradictory to Mauad et al 1999., 157  who demonstrated elastosis and fragmentation of elastic fibers when assessing the whole airway mucosa of fatal asthmatics compared to non-asthmatic airways [375]. The only difference in these studies is that the latter used histological stains and morphometric quantification in the entire airway compared to NLOM and texture analysis in the lamina propria in the present study. Changes in individual ECM components or networks as in the case of collagen and elastin could contribute to changes in airway mechanics through changes in tissue biomechanics and geometric effects [376]. The incorporation of morphologic and physiologic measurements on asthmatic and non-asthmatic tissues into mathematical models has demonstrated that the magnitude of airway wall thickening is sufficient to contribute substantially to asthmatic airway hyperresponsiveness (AHR) [377]. Furthermore, the “perturbed equilibrium hypothesis” suggests that airway wall thickening can destabilize the dynamic forces that control airway caliber, causing airway collapse [378]. Despite this, pulmonary measurements in asthmatics have shown the airways to be less distensible [344]. Future studies have to show how modeling disorganized fibrillar collagen in the asthmatic airways would affect airway closure and AHR.  From the late stages of gestation to adult life, the collagen content in the lung increases 5-fold [379]. Specifically in the rabbit, it has been shown in the maturing lung, rapid growth after birth, leads to a 15% increase in collagen synthesis relative to the rate of total protein synthesis which declines after two months [379, 380]. However, no such data exist on human lungs or on how collagen deposition may differ in the lungs of asthmatics. The current study consisted of donor lungs from both children (4 – 15 years of age) and young adults (18 -25 years of age). Despite the young age of the donors in this study, we demonstrate significant collagen deposition and remodeling consistent with previous autopsy studies on fatal asthmatics with subjects greater than 40 years of age [366]. Therefore, these data further support the notion that airway remodeling 158  occurs early in the pathogenesis of asthma. Whether the collagen deposition in the maturing lung is affected by early life events that contribute to asthma pathogenesis is still to be determined.   While collagen I is capable of spontaneously forming fibrils in vitro in the absence of cells [351], assembly in vivo requires the presence of fibronectin, integrins, lysyl oxidase, and decorin, thus enabling cellular control over assembly and organization [351]. The primary cell responsible for collagen formation are fibroblasts which in the airway are mainly situated in lamina propria [156]. In the present study we demonstrated that compared to controls, airways fibroblasts from asthma patients are abnormal in their ability to remodel denatured fibrillar collagen I compared to patient-matched parenchymal derived fibroblasts. The free floating 3D collagen I gels used in the present study enabled us to mimic the tissue micro-environment enabling fibroblasts to interact with collagen I through dendritic extensions and subsequently actin stress fibers once gels are contracted [336]. During tissue damage and repair, activated fibroblasts secrete ECM components such as collagen while interacting with and remodeling them [320]. In the sputum of asthmatics, collagen synthesis by measurement of the procollagen type I C-terminal peptide has been shown to be elevated, and significantly increase upon exacerbations [381]. In our in vitro model, there was no difference in collagen I protein production by airway fibroblasts between asthmatics and non-asthmatics. Again further stimulation of airway fibroblasts seeded on collagen I gels with TGF-β caused no significant alterations in their rate of contraction. These data indicate that fibrillar collagen I disorganization was due to an intrinsic abnormal remodeling by asthmatic-derived fibroblasts. Further, this defect was only observed in airway-derived fibroblasts and not parenchymal-derived fibroblasts from the same donor, indicating that this defect is specific to the airways. 159  To determine the underling defect that led to the abnormal remodeling phenotype of airway fibroblasts in asthmatics, we assessed if there were differences in the cytoskeletal and contractile filament activity as determined by cell stiffness [316]. We found no difference in basal or TGF-β-stimulated stiffness of airway fibroblasts comparing asthmatics to non-asthmatics. Fibroblast synthesis and remodeling of collagen involves a complex series of intra and extracellular events that involves synthesis of procollagen α chains, packaging and exporting by secretory vesicles into the extracellular matrix where fibrils are assembled [382]. Assembled fibrils are then stabilized by covalent cross-linking which is highly dependent on the activity of the lysyl oxidase enzyme [383]. However, we found no significant difference in the expression of lysyl oxidase comparing airway fibroblasts from controls and asthmatics. Additionally, collagen synthesis is highly influenced by small leucine rich proteoglycans (SLRP), such as decorin, which is essential for coating the surface and spacing of fibrils [384]. We found a lower protein expression of decorin in the asthmatic-derived airway fibroblasts compared to controls. This is line with studies demonstrating that, in addition to the spacing and coating of collagen fibers, decorin is involved in the modulation of fibroblast collagen I gel contraction by increasing the mechanical properties of contracting gels [385, 386]. As further support of the findings of the current study, decreased expression of decorin has been associated with defective repair and collagen disorganization in other chronic lung diseases [158, 387].  A limitation of the present study is that NLOM does not enable us to distinguish between types of fibrillar collagens. As fibrillar collagen types I and III have been shown to be the major fibrillar collagens in both wounded and unwounded lung tissue, [388] we can infer that the fibrillar collagen signal detected is composed of collagen I and III. Further research will be required to develop standards to differentiate which fibrillar collagen is altered in asthma. 160   In conclusion, using NLOM and texture analysis we demonstrate that fibrillar collagen in the lamina propria of asthmatic airways is increased and most importantly is disorganized. In aging skin it has been shown that fragmented collagen fibers impairs wound healing, vascularization [389], and cannot support normal cell morphology and mechanical tension in fibroblasts [390, 391]. As we demonstrate that airway, but not parenchymal derived fibroblasts contribute to abnormal organization of fibrillar collagen I fibers, our in vitro data suggest that abnormal collagen packaging by airway fibroblasts may play a significant role in the development of airway remodeling. Because of the abnormal fibril organization, this does not only imply simple thickening of the airway wall, but also effects on ECM  signaling to inflammatory and resident cells in the airways.  161  Chapter 7:  General Summary, General Discussion and Future Perspectives  7.1 General Summary The prevalence, morbidity and mortality of both asthma and COPD are expected to rise in the next 10 to 30 years and present a serious economic burden [1, 3, 4]. This trend points to the need for more effective therapeutics that will not only manage the chronic inflammatory symptoms, but also aid in resolving the remodeling of the airways that is unaffected by current therapy. Hence, a closer look at the mechanisms that drive structural changes in both the small and large airways in COPD and asthma is vital. One of the mechanisms that has been proposed to drive these structural changes and implicated in the pathogenesis of both asthma and COPD is a dysregulated communication between the epithelium and lung fibroblasts [44, 106]. The airway epithelium is the first barrier to inhaled particles, forms the first part of innate immunity and has been shown to be damaged by cigarette smoking, the major risk factor for COPD, and various environmental triggers of allergic asthma [22, 115, 116, 306]. Increased epithelial fragility and epithelial damage causes the release of a wide variety of inflammatory mediators including interleukin -1 (IL-1) α, that affect underlying fibroblasts in the mesenchyme to promote their activation and increased matrix deposition [44, 106, 392, 393]. In turn, activated fibroblasts also produce growth factors and cytokines that act on the airway epithelium, resembling the process of epithelial-mesenchymal trophic unit (EMTU) activation during lung development [114, 392].   Thus, we hypothesized that an aberrant interaction between the airway epithelium and lung fibroblasts drives chronic airway inflammation and remodeling in the lung epithelial-mesenchymal 162  trophic unit (EMTU) and contributes to the pathogenesis of asthma and COPD. By the use of an in vitro co-culture system in chapter 3, we discovered epithelial-derived IL-1α to be a vital mediator that regulates fibroblast-mediated inflammation and ECM expression. We showed that IL-1α levels are increased after cigarette smoke exposure, which stimulated an increased fibroblast-mediated inflammatory response. In chapter 4, we then assessed if a failure of miR-146a-5p regulation, a known epigenetic regulator of IL-1 signaling, is involved in the aberrant epithelial-fibroblast interaction in COPD. Here, we found a lower induction of miR-146a-5p in COPD-derived lung fibroblasts compared to controls upon co-culture with epithelial cells. This lower induction of miR-146a-5p expression, which is associated with a SNP, rs2910164 (GG allele) may contribute to the increased inflammatory response by lung fibroblasts in COPD. In chapter 5, we studied the potential role of IL-1 in the dysregulated epithelial-fibroblast communication in asthma and observed that IL-1 release is higher in airway epithelial cells from asthmatics than from non-asthmatics and reduced upon differentiation. We assessed the effect of IL-1 stimulation on fibroblast phenotype and found IL-1 stimulation leads to fibroblast-dependent inflammatory response and a down-regulation of ECM expression, which was independent of whether the fibroblasts were asthma or control-derived. In addition, we found that IL-1 inhibits contraction of collagen I gels through the down-regulation of lysyl oxidase (LOX) and glioma-associated oncogene homolog 1 (GLI-1). Further in chapter 6, we studied lung tissue from asthmatics and non-asthmatics with SHG-NLOM and two-photon excitation fluorescence microscopy and found an increased deposition as well as highly disorganized levels of fibrillar collagen I both in the large and small airways of asthmatics compared to controls. We propose that this increased disorganization of collagen I fibers may be due to an aberrant repair phenotype of 163  airway fibroblasts derived from asthmatics compared to non-asthmatics, which supported our in vitro findings.  Taken together, work done in this thesis sheds new light on the role of epithelial-fibroblast interactions and microRNA regulation in the chronic inflammatory and airway remodeling processes that occur in the pathogenesis of asthma and COPD. This provides an important background for future therapeutic studies and interventions that will not only focus on chronic inflammation but also target airway remodeling in asthma and COPD.    7.2 General Discussion  7.2.1 The airway epithelium regulates fibroblast inflammatory release and ECM expression in the lungs. The airway epithelium plays a vital role as the first line of defense, which is part of the innate immune response in the lungs [108]. This protection is achieved through a physical barrier of epithelial tight junctions, the secretion of broncho-alveolar lining fluid, and the activity of muco-ciliary clearance, which clears inhaled particles in the lung [9]. Importantly, the airway epithelium also serves as the source of immune mediators and forms part of a non-specific immune defense to inhaled particles and infectious agents in the airways [394]. Pattern recognition receptors (PRRs), such as Toll-like receptors (TLR) and NOD-like receptors, on the airway epithelium recognize and bind to pathogen-associated molecular patterns (PAMPs) and damage associated molecular patterns (DAMPs) [9, 31]. This causes the release of inflammatory mediators such as tumor necrosis factor (TNF)-α, IL-1α, IL-6, CXCL8 (IL-8), prostaglandin E2, as well as fibrogenic 164  mediators such as TGF-β and epidermal growth factor (EGF) among others [108, 190]. The release of these mediators has been demonstrated to affect various immune cells as well as structural cells in the lung mesenchyme, such as fibroblasts [106]. Although fibroblasts were traditionally seen as mainly structural cells, it is now well known that fibroblasts are highly metabolically active [156]. Lung fibroblasts have been shown to respond to the activity of fibrogenic cytokines by increasing their expression of ECM proteins while also responding to a variety of inflammatory mediators such as PGE2, IL-1 and TNF-α by releasing inflammatory mediators such as CXCL8 (IL-8) as well as IL-6 [156].      To find out how the epithelium interacts with fibroblasts to regulate pro-inflammatory responses in the airways, we studied in chapter 3, through the use of an in vitro co-culture model to assess how epithelial cells and fibroblasts interact, and demonstrated that IL-1α released from airway epithelial cells regulates the lung fibroblast phenotype. Here, we found epithelial-derived IL-1α stimulates pro-inflammatory responses of fibroblasts, which potentially plays a role in inflammatory responses in the lungs. This basal expression of IL-1α could derive from an already available pool of IL-1α in the cytoplasm or bound to the cell membrane [182] and is in line with the measureable cytokine levels in healthy airways [9]. Importantly we found in chapter 5 that IL-1α is produced in the initial stages of re-differentiation by the airway epithelial cells and may play important roles in regulating fibroblast-derived inflammation and ECM responses. We demonstrated through the use of the air-liquid interface (ALI) culture, that IL-1α as well as its family members IL-1β and IL-33 are expressed, and in the case of IL-1α, also released when epithelial cells form a simple columnar epithelial monolayer. However, as the cells start to differentiate into a pseudo-stratified epithelial layer by day 5, the expression of IL-1α and family members and release of IL-1α is lost. Interestingly, this time frame matches the initial 165  inflammatory phase observed in days 0 to 5 in several epithelial wound repair models [326]. Thus, our findings support the view that IL-1α is an acute phase mediator that is involved in initial responses to damage and injury in the airways [395].  Further to the release of IL-1α from the airway epithelium, we demonstrated with the in vitro co-culture model in chapter 3 that epithelial-derived IL-1α stimulates lung fibroblasts to increase their expression and release of pro-inflammatory mediators including CXCL8 (IL-8) and IL-6 and the DAMP, heat shock protein (HSP) 70. This finding was further corroborated in chapter 5 where we also found an IL-1α and IL-1β -induced release of CXCL8 (IL-8) and IL-6 as well as TSLP and GM-CSF by airway fibroblasts. These cytokines are vital for the influx of neutrophils, the maturation of immune cells and regulation of T cell responses, demonstrating the role of epithelial-derived IL-1 in driving fibroblast inflammatory responses [23, 44, 396, 397]. In recent years, lung fibroblasts have been proven to act not only as structural cells, but to be also important in modulating inflammatory responses [286]. In line with this, fibroblasts respond to a wide variety of cytokines including IL-1α, IL-1β, and TNF-α [152]. Our findings support a vital role of epithelial-derived IL-1 in driving fibroblast pro-inflammatory responses that may contribute to the non-specific immune response to inhaled particles in the lung. Lung fibroblasts are the main structural cells in the mesenchyme and responsible for the production of extracellular matrix (ECM) proteins such as collagen, fibronectin and decorin [156]. Hence, apart from the effect of epithelial regulation on fibroblast-derived inflammation, we were interested in assessing the effects of epithelial-fibroblast interactions on the ECM repair functions of lung fibroblasts. It is well known that there is an increased remodeling of the airways that leads to airway narrowing due to fibrosis in COPD and asthma [43, 163]. This increased remodeling has been shown to be as a result of the increased production of profibrotic mediators including TGF-166  β by structural cells such as the airway epithelium [16]. In chapter 3, when we assessed the expression of ECM molecules in lung fibroblasts after co-culture with airway epithelial cells, we found a down-regulation of ECM proteins including collagen Iα1, fibronectin, fibulin-5 and decorin as well as the cell cytoskeleton molecule α-smooth muscle actin (SMA), for which expression was epithelial-IL-1α dependent. This decreased expression of ECM was corroborated by our findings in chapter 5, where exogenous stimulation of airway fibroblasts with IL-1α and IL-1β both caused a down-regulation in the expression of collagen Iα1 and periostin. Although these findings seem to be counter-intuitive to the pathology of the disease in vivo, our findings are in agreement with previous work by Reeves and colleagues who also showed that the airway epithelium suppresses the expression of ECM proteins by lung fibroblasts in a co-culture model [178]. This effect on fibroblast ECM expression has also been demonstrated in cardiac fibroblasts, where both IL-1α and IL-1β decreased the expression and synthesis of collagen, while promoting the release of matrix metalloproteinases (MMPs) that cause the degradation of ECM proteins [332, 333] Although these models may help in understanding aspects of the complex cellular interactions in vivo, they do not totally recapitulate the full complexity of cellular communications in the tissue micro-environment. This notwithstanding, work done by Mia et al. showed that IL-1β inhibits TGF-β-induced expression of collagen in lung fibroblasts [181].This helps to understand how epithelial-derived IL-1 signaling may interact with TGF-β activity to modulate fibroblast ECM expression. Taken together, these findings support an emerging concept that highlights the airway epithelium as a modulator of ECM production by fibroblasts. We propose that a dysregulation in this mechanism may contribute to abnormal tissue repair, airway remodeling and tissue destruction in COPD and asthma.  167  7.2.2 Abnormal epithelial regulation of fibroblast-pro-inflammatory responses in COPD and asthma In addition to determining that epithelial-derived IL-1α is the main specific mediator involved in the epithelial regulation of fibroblast inflammatory responses, we assessed how a possible abnormality in this interaction could contribute to disease pathogenesis. Hence in chapter 3, we exposed primary airway epithelial cells derived from COPD patients and controls to cigarette smoke extract (CSE) to simulate the major environmental risk factor for COPD. We observed an increased expression of IL-1α in COPD-derived epithelium compared to controls. Interestingly, exposing lung fibroblasts to conditioned medium (CM) from CSE-exposed COPD-derived airway epithelium led to a higher release of the neutrophil chemo-attractant CXCL8 (IL-8) compared to CM from control-derived cells. Apart from the exposure to CSE, IL-1α as well as family member IL-1β have been shown to be released via reactive oxygen species (ROS) and endoplasmic reticulum (ER) stress, which have been also implicated in the pathogenesis of COPD [13]. Of importance to our findings in chapter 3, others found that release of CXCL8 (IL-8) by lung fibroblasts was dependent on epithelial-derived IL-1α and not IL-1β after ROS and ER stress injury [13]. Again, our findings support work previously done in mouse models of COPD, demonstrating that IL-1α signaling is indispensable in driving neutrophilic influx after cigarette smoke exposure [36, 37]. Neutrophilic influx has been demonstrated to be a vital part of the disease pathogenesis in COPD. Increased neutrophil numbers have been demonstrated in the bronchoalveolar lavage (BAL) fluid [38-40], sputum [41-43] and the airway wall [44, 45] of COPD patients compared to controls. This increased neutrophil influx forms a part of chronic airway inflammation and the production of neutrophil elastase is involved in the emphysematous destruction of the lung parenchyma [46]. 168   In the asthmatic EMTU, damage to the airway epithelium leaves the epithelium fragile and in a chronic state of activation [7]. Hence, through the use of an ALI culture in chapter 5 we compared, for the first time, the difference in the release of IL-1 between the differentiating airway epithelium of asthmatics and non-asthmatics. Of importance we observed a higher expression of IL-1α, IL-β and family member IL-33 as well as a corresponding higher release in the case of IL-1α in asthma-derived airway epithelium compared to controls when cells were in a mono-layer. However upon differentiation, this increased expression was reduced. This finding points to a higher IL-1 release from airway epithelium when being undifferentiated and not forming a tight barrier as in asthma, which could potentially enhance the response of airway fibroblasts in the lung EMTU. In line with this finding, we demonstrated by the use of exogenous cytokine stimulation that IL-1α as well as IL-1β caused the release of cytokines IL-6 and CXCL8 (IL-8) in airway fibroblasts. This supported our findings in chapter 3 where we found in our co-culture model that epithelial-derived IL-1α stimulates inflammatory mediator release from fibroblasts. In addition to the release of these cytokines, IL-1 stimulation in chapter 5 also caused the release of GM-CSF and TSLP, which are vital for eosinophilia, mast cell activation, immune cell maturation and TH2 cell responses in the pathogenesis of asthma [24, 47-49]. In our studies, stimulating fibroblasts with IL-33 showed no effects on airway fibroblasts. This indicates that although IL-33 expression is increased in the damaged airway epithelium in asthma [50], airway fibroblasts in the lung EMTU may not be its main effector cell. Indeed, IL-33 has been demonstrated to affect the responses of other cells including immune cells such as basophils, mast cells, T and B cells [51, 52]. Thus increased IL-1 release from the damaged epithelium in COPD and asthma may contribute to increased inflammatory responses.   169  7.2.3 Defective miRNA regulation of IL-1 signaling involved in epithelial-fibroblast interaction in COPD Since IL-1 signaling is vital for immune defense, there are several regulatory mechanisms available to control the effects of this important cytokine [12, 53]. In addition to the anti-inflammatory effects of IL-1 family members, several epigenetic mechanisms have been shown to be involved in regulating IL-1 signaling. As an example, the methylation of specific CpG sites of the IL-1β promoter suppresses its transcription in mesenchymal cells such as fibroblasts [54]. Further to this, several miRNAs such as miR-149 and miR-146a-5p have been shown to target and regulate IL-1 signaling [55, 56]. Of these, the anti-inflammatory miR-146a-5p has been widely studied in IL-1-mediated inflammation involved in other chronic inflammatory diseases in the body [57]. Hence in chapter 4, we studied if a dysregulated expression of miR-146a-5p may be involved in the abnormal epithelial regulation of fibroblast responses in COPD as demonstrated in chapter 3. Here, we found an epithelial-derived IL-1α –dependent induction of miR-146a-5p in lung fibroblasts. Interestingly, this induction of miR-146a-5p was significantly less in COPD-derived lung fibroblasts compared to controls. This lower induction was associated with a single nucleotide polymorphism (SNP) rs2910164 (GG allele) in the COPD-derived lung fibroblasts which has been shown to cause a lower expression of mature miR-146a-5p [58]. We further confirmed the anti-inflammatory effects of miR-146a-5p induction on the IL-1 pathway by demonstrating that it targets and down-regulates the expression of the IL-1 receptor (IL-1R)-associated kinase (IRAK)-1 and lowers the IL-1α-induced CXCL8 (IL-8) release by lung fibroblasts [56]. Thus, the defective induction of miR-146a-5p could add to chronic inflammation in the COPD lungs. 170  Although we did not study if there are regulatory defects in the IL-1 pathway in the asthmatic airways, previous studies have assessed this. Gagné-Ouellet and colleagues demonstrated increased methylation of IL-R2, which was associated with a lower expression of the receptor in asthmatic patients [206]. Since IL-1R2 is a decoy receptor that binds to and inhibits IL-1 signaling, a decreased expression of this receptor is suggested to cause an increased activity of IL-1 in asthmatic patients compared to controls [206]. With reference to miRNA regulation, decreased expression of miR-570-3p upon stimulating airway epithelial cells with TNF-α was shown to be accompanied by increased epithelial-derived inflammation including the release of CXCL8/IL-8, CCL2, TNF, IL-6 and CCL4. miR-570-3p has been found to have a down-regulated expression in the serum and exhaled breath condensates of asthmatic patients compared to controls [398]. Hence, it may be worthwhile to examine if this decrease can be seen in the airway epithelium and how this impacts epithelial-fibroblast communication in the lungs of asthmatic patients.   7.2.4 Epithelial regulation of fibroblast ECM expression and remodeling phenotype in asthma and COPD The structural changes that occur in asthma and COPD, including the airway wall deposition of ECM proteins such as fibrillar collagen, lead to airflow obstruction and lung function decline [4]. Although the resulting increase in airway remodeling is a well-documented feature of both asthma and COPD, current therapeutics are unable to reverse or manage this [4, 8]. In chapter 5, we demonstrate that IL-1 stimulation causes a down-regulation of ECM proteins such as collagen Iα1, periostin and fibronectin through the regulation of the sonic hedgehog (SHH) transcription factor, glioma-associated oncogene homolog 1 (GLI-1) in healthy/asthmatic-derived fibroblasts. This IL-1α-dependent down-regulation of ECM expression confirmed our findings in chapter 3, where we 171  also found an epithelial-derived IL-1α-dependent decrease in the expression of ECM proteins in lung fibroblasts including collagen Iα1, fibronectin, decorin and fibulin-5. The potential effects of IL-1 released from the airway epithelium in both asthmatics and COPD patients after epithelial damage may form part of an initial response to suppress exaggerated ECM production, while switching fibroblasts to a more pro-inflammatory phenotype.  A decreased expression of ECM proteins, including decorin, in lung fibroblasts after an increased exposure to IL-1α due to CSE stimulation of the airway epithelium as shown in chapter 3, may be a mechanism to regulate the increased activity of fibrogenic mediators such as TGF-β1 [35, 61, 62]. Indeed, different studies have shown an interaction between TGF-β and IL-1 activity in lung fibroblasts in vitro, whereby TGF-β increases myofibroblast survival by inhibiting IL-1β induced apoptosis [61], while IL-1β also inhibits TGF-β1 -dependent myofibroblast transformation [35]. This IL-1/TGF-β interaction will be of importance in COPD where there is an increased expression of TGF-β1 and its receptors TGF-β receptor type I and II [62]. The increased expression of TGF-β in COPD has been linked to small airway remodeling and shown to be involved in the influx of mast cells and macrophages [63]. However, an increased expression of IL-1α may also contribute to a mechanism whereby small airways are lost in some COPD patients [29]. Although there is fibrosis of the airway wall due to ECM deposition in COPD, a proportion of the small airways may be lost which precedes emphysematous destruction in very severe disease [64]. This was shown by McDonough and colleagues, who found small airway loss of up to 90% in very severe (GOLD 4) COPD patients compared to controls [64]. How small airway fibrosis, as well as small airway loss and emphysema may occur adjacent to each other in COPD is still largely unknown. However, we have demonstrated that an excess expression of epithelial-derived IL-1α that causes the down-regulation of ECM molecules such as decorin may 172  contribute to this mechanism in vivo. In line with this, work from our group previously found a lower expression of decorin in the lungs of patients with severe COPD compared to controls [65]. This finding was further attributed to an abnormal phenotype of parenchymal lung fibroblasts of severe COPD compared to patients with mild disease where TGF-β stimulation led to lower expression of decorin [66]. Since decorin is an essential proteoglycan involved in collagen cross-linking and spacing, the loss of this protein in end-stage COPD would not only contribute to tissue destruction, but may cause a loosening of alveolar attachments, loss of elastic recoil and collapsibility of the lungs [17]. Again, loss of decorin could potentially add to an increased TGF-β activity since decorin has been shown to be a natural inhibitor of TGF-β activity [67] The effect of IL-1 on lung fibroblast ECM expression was recapitulated in chapter 5 where 24 hours of IL-1 stimulation also caused a down-regulation of ECM proteins collagen Iα1, periostin and fibronectin in airway fibroblasts from asthmatics and non-asthmatics. We did not find differences between asthmatics and non-asthmatics in fibroblast ECM protein expression after IL-1 stimulation. This notwithstanding, Reeves et al, demonstrated with epithelial-fibroblast co-cultures that an aberrant ECM response by asthma-derived airway fibroblasts is due to an abnormal ability of the asthma-derived airway epithelium to downregulate fibroblast ECM expression [32]. This was the result of increased production of TGF-β2 by asthma-derived epithelial cells compared to controls after 96 hour of co-culture [32]. The discrepancy between our findings and the findings reported by Reeves and colleagues may be due to different experimental set-ups where we used a 24 hour time-point with exogenous cytokine stimulation while they performed a co-culture experiment for 96 hours [32]. However, the two studies taken together further points to the possibility of a dysregulated balance between the production of regulatory cytokines IL-1 and TGF-β in vivo being involved in ECM regulation and fibrotic response in asthma and COPD  173  We further asked in chapter 5 if epithelial-derived IL-1 regulation has an impact on fibroblast functional repair phenotype. Here, we studied the effects of IL-1 at 24 hours since IL-1α is an acute-phase mediator that is released within the first 12 to 24 hours of lung injury [68]. Of interest, we observed that even at 24 hours, there was a basal abnormal phenotype of airway fibroblasts from asthmatics compared to non-asthmatics in their ability to contract collagen gels. In addition, exogenous stimulation of fibroblast-seeded collagen I gels with IL-1α and IL-1β led to the loss of dendritic extensions in airway fibroblasts, a defective interaction of fibroblasts with fibrillar collagen and an increased disorganization of collagen fibrils. The loss of dendritic extensions in fibroblasts has been associated with defects in microtubules and the actin cytoskeleton and linked to the activity of the collagen I cross-linking enzyme, lysyl oxidase [69]. In line with this, we demonstrated that IL-1 stimulation caused a decrease in the expression of lysyl oxidase in fibroblasts. This is a novel finding that shows that IL-1 does not only affect fibroblast repair phenotype through regulating the cross-linking of collagen I, but also affects fibroblast morphology. Our findings in chapter 5 were confirmed by data in chapter 6, where we studied for the first time, lung tissue from asthmatics and non-asthmatics with high powered multimodal non-linear optical microscopy (NLOM) imaging. Here, we demonstrated novel findings showing that similar to what has been shown in COPD [70], collagen fibers within asthmatic airways are more disorganized in their structure compared to non-asthmatic airways. Further, we showed this disorganization of fibrillar collagen in asthmatic airways could be due to an inherent abnormality in the ability of asthma-derived airway fibroblasts to remodel fibrillar collagen I. This finding affirmed our data from chapter 5 where we also demonstrated a basal abnormal phenotype of asthma-derived fibroblasts compared to controls even at 24 hours.  174  Combining the findings from all four chapters, we show a clear picture in this thesis that an increased release of IL-1 by the damaged epithelium may be a major contributor to not only inflammation but also defective repair responses by fibroblasts that may drive airway remodeling in asthma. Disorganized or fragmented collagen I has been demonstrated to stimulate an excess production of ECM [71]. Hence, excess production of IL-1 that causes fibrillar collagen disorganization and fragmentation could potentially be a vital mechanism that links inflammation and airway remodeling in both asthma and COPD.   7.3 Future Perspectives In this thesis we report findings which demonstrate that an abnormal interaction between the airway epithelium and lung fibroblasts may contribute to the pathogenesis of asthma and COPD. We show that IL-1α may play a crucial role in this interaction which suggests the activity and signaling of this cytokine may be an interesting target for future therapeutic strategies.   In our in vitro co-culture model for COPD, we found a CSE-dependent increase in epithelial-derived IL-1α that caused higher release of the chemo-attractant IL-8/CXCL8 from lung fibroblasts which plays an essential role in the innate immunity. Future studies could determine if increased fibroblast-derived inflammation could also play a role in driving the adaptive immunity through the release of cytokines to influence T and B cells. Airway epithelium and fibroblasts used for our studies in chapter 3 were derived from patients with very severe (GOLD 4) COPD. Further experiments to assess if this dysregulated communication is also essential for mild to moderate disease could throw more light on the role of this interaction during the development of COPD. We also demonstrated in our experiments that epithelial-derived IL-1α down-regulates fibroblast 175  ECM expression in lung fibroblasts. Here, we proposed that this could serve to counteract the effects of fibrogenic mediators such as TGF-β on one hand while potentially causing the loss of small airways in severe COPD patients. Future studies could determine the balance between IL-1 and TGF-β activity in COPD patients and how this affects the pathogenesis of the disease. In addition the expression of IL-1α in biopsies of small airways from COPD patients with different disease stages could also be further examined to determine the involvement of IL-1 in small airway loss in COPD patients. Again, through the use of collagen contraction assays and high powered multiphoton microscopy as used in chapter 5 and 6, it will be of interest to study the effects of epithelial-derived IL-1 and TGF-β interaction on the functional phenotype of COPD-derived fibroblasts. This will enable us understand how the interaction of IL-1α as well as observed TGF-β release by the airway epithelium may affect the functional phenotype of fibroblasts and contribute to small airway loss in COPD. Of interest, senescent lung fibroblasts acquire the senescence-associated secretory phenotype (SASP) and have been shown to be a robust source of IL-1α which then could drive fibroblast-derived inflammation in an autocrine way [72]. Since aging and senescence has been suggested to be main pathogenic mechanisms of COPD, further studies could assess how this impacts epithelial-fibroblast communication.   We assessed potential effects of IL-1 release from asthmatic airway epithelium on airway fibroblast phenotype at 24 hours, since IL-1α has been shown to be an acute-phase mediator that acts in the early stages of immune response [68]. In line with this, the increased release of IL-1α by asthmatic compared to normal airway epithelium was seen in the early stages of differentiation in the ALI culture. However, since it has been demonstrated that due to TGF-β2 activity the asthmatic airway epithelium may differentially regulate fibroblast phenotype in 96 hours compared to non-asthmatics [32], it will be worthwhile to conduct future studies to assess IL-1 effects on the 176  fibroblast phenotype and TGF-β interaction at different time points. This will give a better idea of the dynamics of IL-1 release with respect to time and how this could differentially regulate lung fibroblasts in disease. Although some studies have demonstrated an increased expression of IL-1 in mouse models of asthma [73-75], there is a lack of studies that have compared the expression of IL-1 in the airways of asthmatic patients to those of healthy controls. After demonstrating an increased release and expression of IL-1α in ALIs of asthmatic airway epithelium, future studies should assess the expression of IL-1α in biopsies as well as concentrations in bronchoalveolar lavage and sputum from asthmatic patients compared to controls. This will aid in providing a clear picture and corroborate findings to find further support for a role of epithelial-derived IL-1 in the pathogenesis of asthma.  In this thesis we assessed the effects of miR-146a-5p regulation on the IL-1-dependent defective epithelial regulation of fibroblast responses in our COPD in vitro model. This demonstrated how a defective regulatory mechanism of the IL-1 pathway may add to chronic inflammation in COPD. As we reviewed in chapter 2 of this thesis, several other miRNAs may have an effect on various aspects of COPD. As an example, an up-regulation of miR-135b in lung biopsies after smoke exposure in a mouse model has been shown to regulate the IL-1R1 receptor and serve as an anti-inflammatory modulator. Hence it might be worth-while to assess if miR-135b has a role in regulating epithelial-derived IL-1 modulation of fibroblast phenotype in COPD. In the same light, several miRNAs, including miR-146a-5p as well as miR-223-3p, miR-629-3p, miR-570-3p, miR-155 and miR-126 have been associated with airway inflammation in asthma [60, 76-78]. Future studies could assess the impact of the differential expression of these miRNAs on the abnormal epithelial-fibroblast interaction in the asthma EMTU.  177   We discovered IL-1α as a vital mediator driving epithelial regulation of fibroblast responses. However, other IL-1 family members have also been shown to be involved in the pathogenesis of both asthma and COPD. In chapter 5 we found an increased expression of IL-1β and IL-33 in addition to IL-1α in undifferentiated asthmatic airway epithelium. IL-18 and IL-38 are additional IL-1 family members which have been shown to be involved in the pathogenesis of asthma [51, 79-81]. Thus it will be worthwhile to determine if differential release of these cytokines compared to normal conditions could have an effect on the epithelial-fibroblast interaction we demonstrated. IL-18 and IL-33 have also been shown to be involved in the pathogenesis of COPD and further studies could assess their contribution to inflammation and remodeling in COPD [82-87]. Several members of the IL-1 family have anti-inflammatory properties that control the signaling activities of the IL-1 family members. These include the IL-1 receptor antagonist, the decoy IL-1R2 and IL-18Rα [12]. While some studies have reported increased expression of these anti-inflammatory factors in asthma and COPD [88], some have reported no changes [89] or decreased expression [59]. Future studies could assess the expression and activity of these anti-inflammatory members of the IL-1 family and their contribution to the abnormal epithelial-fibroblast crosstalk demonstrated in this thesis. In our in vitro model we discovered cellular interaction was mainly through epithelial-derived IL-1α regulation of fibroblast responses. However, it has been suggested that lung fibroblasts may also produce mediators such as fibroblast growth factor (FGF) that could also influence airway epithelial phenotype and function and thus play a role airway remodeling [11, 90]. Future studies should therefore assess how and to what extent the release of these mediators from lung fibroblasts could also affect the airway epithelium and how this might play a role in the EMTU of asthma and COPD patients. 178  There have been a few studies in animal models assessing IL-1 signaling as a target for therapy directed against chronic airway inflammation in both asthma and COPD. However, none of these have examined the role of IL-1 in airway remodeling and repair. Hence future studies using animal models of asthma and COPD could assess the effects of targeting and modulating components of the IL-1 signaling to ascertain the possible therapeutic benefits on both inflammation and remodeling of the airways in asthma and COPD. This will aid in translating the findings in this thesis for potential therapeutic research.  7.3.1 Regulation of IL-1 signaling, current and potential therapeutics in COPD and asthma Taken together, the findings presented in this thesis shows IL-1 as a master regulator which contributes to an abnormal epithelial-fibroblast interaction in the lungs and may play an important role in the disease pathogenesis in COPD and asthma. Apart from the contribution of IL-1 to an abnormal interaction in the EMTU, it has been shown to contribute to most hallmarks of both asthma and COPD. Hence, more studies are needed to take a closer look at how the IL-1 signaling pathway could be targeted for new therapeutics in both asthma and COPD. To date a few trials have looked at the possibility of treating COPD and asthma with the commercially available IL-1β antibody (Canakinumab), which has proven successful in treating auto-inflammatory disorders [91]. In a randomized double-blind placebo controlled trial that was the first of its kind, patients with mild asthma were given 10 mg/kg of Canakinumab twice with a 15 day interval between the first and second shot and an allergen challenge test was performed on the start date and day 28 [92]. Although the dosage used was based on a computed model and no actual data, there was a significant reduction of circulating levels of IL-1β and on the late phase asthma response compared to pre-treatment [91, 92]. A recent phase 1/2 clinical trial has looked at the pharmacokinetic 179  properties of Canakinumab in COPD patients and findings have been inconclusive [93]. However, based on studies done in mouse models as well as current data presented in this thesis, blocking the effects of only IL-1β may not be effective, since IL-1α is an acute phase mediator that is pivotal in the initiation of inflammation and can serve to be the initial inducer of IL-1β release. Hence studies or trials that are directed towards a concerted approach that would potentially target the activity of IL-1α and IL-1β as well as other family members such as IL-18 and IL-33 might provide a great new potential targets for both COPD and asthma. However moving forward, it is also important to carefully understand the differences in IL-1 activity and disease phenotypes of patients involved and trials directed towards the right patient. As an example, taking our findings and data already published into account [35], an increased activity of IL-1 may be beneficial to patients with a corresponding increased TGF-β activity. Again, in these trials targeting IL-1 signaling in the various compartments of the lung through localized administration of neutralizing antibodies could aid in determining the differential effects of IL-1 signaling in different parts of the lung.    7.4 Conclusion  In conclusion, we demonstrate in this thesis that epithelial-derived IL-1α is vital for the regulation of lung fibroblast responses by increasing the release of inflammatory cytokines while down-regulating their remodeling and repair phenotype. This interaction is aberrant in COPD, where CSE stimulation leads to an increased expression in epithelial-derived IL-1α that causes a heightened fibroblast inflammatory phenotype. In asthma, abnormal repair, keeping the epithelial 180  cells in a basal and frail state may cause an increased release of IL-1α, which may drive fibroblast-derived inflammation and an abnormal remodeling phenotype. 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MicroRNA-570-3p regulates HuR and cytokine expression in airway epithelial cells. Am J Clin Exp Immunol 2014: 3(2): 68-83. 206    Appendix A  : Supplementary information and Data for Chapter 3 Interleukin-1α drives the dysfunctional cross-talk of the airway epithelium and lung fibroblasts in COPD  A.1 Isolation and culture of primary human lung fibroblasts Fibroblasts were isolated from lung parenchyma using the explant technique as previously described [158, 285]. Lung tissue was cut into 1 mm3 26 cubes and put in a 12-well culture plate (Corning Costar Europe Ltd., Badhoevedorp, the Netherlands). The tissue was left for 10 minutes at 20˚C and allowed to adhere after which 1.5ml of Ham’s-F12 medium/ 10% FCS (Lonza) was added. This was placed in the incubator under standard conditions and medium was replaced once a week. Tissue explants were usually removed after four weeks when primary fibroblast outgrowth contained enough cells. Primary fibroblasts were then trypsinized (0.05% trypsin/0.02% EDTA; BioWhittaker) and cells were transferred into a 25cm2 culture flask. Primary fibroblasts were grown till confluence, frozen in liquid nitrogen and used for experiments at passage 5 [158, 285].  A.2 Full protocol of co-culture model Primary airway epithelial cells (AECs) and 16HBE14o- cells were plated at 20,000 cells hormonally-supplemented bronchial epithelium growth medium (BEGM, Lonza) or Eagle’s minimal essential medium (EMEM)/10% FCS (Lonza) respectively on coated 0.4μm polyester transwell membrane inserts (upper compartment). Primary human lung fibroblasts (PHLFs) or MRC-5 cells were plated at 20,000 cells in EMEM medium/10% FCS in a 24 well culture plate. Confluent layers of epithelial cells and fibroblasts were obtained after one week of culturing in standard conditions. After this, MRC-5 cells 1 and PHLFs were placed on EMEM/10% FCS 207  (Lonza) when co-cultured with 16HBE14o- cells or BEGM (Lonza) when co cultured with primary AECs. Mono-culture controls consisted of either epithelial cells on inserts placed in wells without fibroblasts, or confluent fibroblast wells with empty inserts on top in the same medium as used for co-culture. The cell inserts were carefully placed in fibroblast wells and left in co-culture for 72 hours in standard conditions. The cells were placed on serum/hormone free conditions before experimentation. For IL-1α neutralization experiments, the cells were placed in co-culture with or without 4μg/ml IL-1α neutralizing antibody (AB-200-NA, R&D Systems) for 72 hours and the antibody was present until cells were harvested. Epithelial cells and fibroblasts were harvested separately with TRIreagent for RNA isolation, Laemmli buffer for cell lysate preparation and cell-free supernatants were collected for ELISA.  A.3 Conditioned medium (CM) experiments 16HBE14o- cells or primary AECs were plated in their appropriate medium on coated 24 well plates. When confluent, cells were serum/hormone-deprived overnight and subsequently stimulated with 20% cigarette smoke extract (CSE) for 6 hours. CSE was thoroughly washed off and cells were incubated for another 24 hours. The epithelial cells and CSE-free conditioned medium (CM) were collected and stored at -80°C until use. CSE was made as previously described [121]. Smoke of 2 Kentucky 3R4F research21 reference cigarettes without filter (The Tobacco Research Institute, Lexington, Kentucky, USA) were bubbled through 25ml BEBM (Lonza) to make 100% cigarette smoke extract. For each experiment fresh CSE was prepared and diluted in serum/hormone-free medium to 20%. Viability experiments showed that a concentration of 20% CSE did not cause epithelial cell death (supplement figure A.10).  208  A.4 Neutralizing antibody experiments Fibroblasts were serum-deprived overnight after which they were stimulated with CM for 24 hours. Epithelial CM was pre-incubated for 1 hour with or without 4μg/ml IL-1α neutralizing (AB-200-NA) or IL-1β neutralizing antibody (MAB601, R&D Systems, Europe, Abingdon, UK). Cell-free supernatants were collected and fibroblasts were harvested for RNA isolation or cell lysate preparation. In the co-culture model, cells were placed together for 72 hours with or without 4μg/ml IL-1α neutralizing antibody (R&D Systems) and the antibody was present until the cells were harvested. Epithelial cells and fibroblasts were harvested separately with TRIreagent for RNA isolation and subsequent qRT-PCR, Laemmli buffer for cell lysates preparation for western blot and cell-free supernatants were collected for ELISA. An LDH assay, (G7890, Promega, Southampton, UK) was done to assess cell viability after co-culture and in the presence of the IL-1α neutralizing antibody. This showed no significant changes in cell viability (data not shown).  A.5 Measurement of protein levels by ELISA IL-8/CXCL8, IL-1α, Hsp70 (R&D Systems) and IL-6 (Sanquin, Pelikine, Amsterdam) were measured with sandwich ELISAs in cell-free culture supernatants and performed according to the manufacturer’s instructions.  A.6 RNA Isolation and qRT-PCR for structural proteins RNA was harvested using the standard TRIreagent method. cDNA synthesis was done with iScript cDNA synthesis kit (BioRad, Herts, UK) according to the manufacturer’s instructions. Taqman® Gene expression assays were used for IL-8/CXCL8 (Hs00174103_m1), IL-1α (Hs00174092_m1), IL-1β (Hs01555410_m1), decorin (Hs00754870_s1), fibulin-5 (Hs00197064_m1), collagen-Iα1 209  (Hs00264051_m1), fibronectin (Hs00365052_m1), α- smooth muscle actin (Hs00426835_g1),  periostin (Hs01566734_m1), MMP-2 (Hs01548727_m1) and TGF-β (Hs00998133_m1) according to  the manufacturer’s instructions. qRT-PCR was performed on the Taqman® or LightCycler 480 II  (Roche). Expression of the genes of interest was normalized to expression of the housekeeping genes protein phosphatase 1, catalytic subunit, alpha isoenzyme (Hs00267568_m1) and β2- microglobulin (Hs00984230_m1) with approximately equal amplification efficiency.  A.7 Western blotting for ECM molecules Total cell lysates were obtained by harvesting epithelial cells and fibroblasts separately in 1X Laemmli buffer (containing 2% SDS, 10% glycerol, 2% β-mercaptol, 60mM Tris-Hcl (pH 6.8) and bromophenol blue) and boiled for 5 minutes. Samples were subjected to SDS-PAGE and blotted on a nitrocellulose membrane (Schleider and Schuell GmbH, Einbeck, Germany). Expression of fibronectin and α-smooth muscle actin was analyzed using mouse anti-human fibronectin (Sigma, F6140) and mouse anti-human α smooth muscle actin (Sigma, A5228) respectively, with anti-GAPDH (Santa Cruz Biotechnology, Santa Cruz, CA) as loading control as previously described,[138] after which visualization was done with the ODYSSEY® infra-red imaging system (LICOR®, Lincoln, NE).     210   A.8  IL-8/CXCL8, Hsp70 and IL-6 in co-culture of airway epithelial cells and lung fibroblasts.  Primary airway epithelial cells (AECs) derived from control (open triangles) and COPD patients (filled triangles) were cultured alone or with MRC-5 cells. A) IL-8/CXCL8 concentration (with median) in cell-free supernatants (24 hours) of the baso-lateral compartment comparing co-culture and mono-cultures. B) IL-8/CXCL8 mRNA expression levels (with median) in MRC-5 cells (6 hours) harvested separately comparing co-culture and mono-cultures C) Hsp70 concentration (with median) in cell-free supernatants (24 hours) of the baso-lateral compartment comparing co-culture and mono-cultures. D) IL-6 concentration (with median) in cell-free supernatants (24 hours) of the baso-lateral compartment comparing co-culture and mono-cultures cells in the presence or absence of 4 μg/ml IL-1α Nab mRNA levels were related to the housekeeping genes (β2μG and PP1α) and expressed as 2-∆Ct. **=p<0.01 and *=p<0.05 between the indicated values.  211   A.9 Correlation of IL-8/CXCL8 secretion to Hsp70 and IL-6 secretion in co-culture. IL-8/CXCL8 concentration from baso-lateral part of co-cultures of primary airway epithelial cells (AECs) and MRC-5 cells was correlated with A) Hsp70 and B) IL-6 concentrations measured in the baso-lateral part of the same experiments. Concentrations are shown in a Log scale.           212   A.10  Assessment of epithelial cell death after cigarette smoke extract stimulation. 16HBE14o- cells were grown to 90% confluence and then serum-deprived overnight before being stimulated with 0-60% CSE for 6 hours. Cell death was analyzed using the Annexin-V-PI staining for flow cytometry as described previously [32]. Percentage cell death of 16HBE14o- cells after CSE stimulation. Data is presented as mean±SEM of 3 independent experiments. *=p<0.05 & **=p<0.01 between the indicated values.          213               A.11  The adenlylate cyclase inhibitor (ACI) MDL-12330A hydrochloride does not block the epithelial conditioned medium-induced IL-8 release. Conditioned medium was harvested from 16HBE14o- cells grown to confluence. IL-8 concentration released from confluent MRC-5 fibroblasts incubated with 16HBE14o- CM in the presence or absence of 10μM adenylate cyclase inhibitor MDL-12330A hydrochloride. Data is presented as mean±SEM of 3 independent experiments *=p<0.05 between the indicated values  214  Appendix B  : Supplementary Information and Data for Chapter 4 MiR-146a plays an essential role in the aberrant epithelial-fibroblast cross-talk in COPD  B.1 MiR-146a-5p mimic transfection MRC-5 fibroblasts were seeded at a density of 1x105 cells per well in 24-well plates in EMEM/10% FCS and transfected shortly after seeding, using the HiPerFect reagent (QIAGEN, Venlo, Netherlands) and the miR-146a-5p mimic at 25nM (MirVana miRNA mimic, assay ID= MC10722; Life Technologies). A scrambled small RNA at 25nM (AllStars Negative Control siRNA; QIAGEN) was used as a control. After 48 hours, cells were washed and placed in serum-free medium, followed by 24 hour stimulation with or without 1ng/ml IL-1α (R&D Systems) and harvested with TriReagent for RNA isolation or Laemmli buffer for protein lysates preparation and cell-free supernatants were collected for IL-8 measurement.  B.2 rs2910164 Genotyping To investigate if the lower induction of miR-146a-5p by COPD-derived PHLFs in co-culture was caused by a common G>C SNP rs2910164, genotyping for this SNP was performed. A subset of samples were previously genotyped on Illumina Human1M-Duo BeadChip  array and imputed using MACH program for genotype imputation using HapMap release 22 template, as previously described [298]. Five samples which were not genotypes on arrays had their DNA extracted from lung tissues of the same subjects from which PHLFs were obtained, according to the standard salt-chloroform extraction method. The rs2910164 polymorphism was identified using TaqMan® SNP Genotyping assay (C_15946974_10) according to the manufacturer’s protocol. Each PCR was 215  done in duplicates with 10 ng DNA template and 40 cycles of amplification was used. The PCR was carried out on a ABI7800HT machine (Applied Biosystems).  B.3 RNA isolation and qRT-PCR RNA was harvested from cells with the standard TRIreagent method. For miRNA expression analysis, miRNA-specific cDNA synthesis was done using the TaqMan microRNA reverse transcription kit (Life Technologies, Bleiswijk, Netherlands) together with reverse transcription primers (Life Technologies) for miR-146a (000468). qRT-PCR was performed on the LightCycler 480 II (Roche, Almere, Netherlands) and expression of the miRNAs of interest was normalized to the expression the small nuclear RNA, RNU48 (001006) as an endogenous control with approximately equal amplification efficiency. For expression analysis of the NF-B family member RelB, the iScript cDNA kit (Biorad, Herts, UK) was used for cDNA synthesis, after which qRT-PCR was performed using the TaqMan® Gene expression assay RelB (Hs00232399_m1) with the protein phosphatase 1, catalytic subunit, alpha isoenzyme (Hs00267568_m1) and β2- microglobulin (Hs00984230_m1) as the housekeeping genes. The reaction was followed for 40 cycles and the Ct value calculated using the LightCycler software. DeltaCt (∆Ct) values were calculated (Average triplicate Gene of Interest – average triplicate Housekeeping gene) and the data were represented as 2^-∆Ct.  B.4 IL-8 ELISA and Western blotting Protein levels of IL-8 were measured with a sandwich ELISA (R&D Systems) according to the manufacturer’s instructions. Total cell lysates of fibroblasts were obtained by harvesting cells in 1X Laemmli buffer (containing 2% SDS, 10% glycerol, 2% β-mercaptol, 60mM Tris-Hcl (pH 6.8) 216  and bromophenol blue) and boiling for 5 minutes after transfection. Samples were then blotted on a nitrocellulose membrane (Schleider and Schuell GmbH, Einbeck, Germany) after being subjected to SDS-PAGE.  Expression of the interleukin 1 receptor associated kinase (IRAK)-1 and TNF receptor-associated factor (TRAF)-6 was analyzed using mouse anti-human IRAK-1 (Sc-5288) and mouse anti-human TRAF-6 (Sc-8409) with goat anti-human β-Actin (all antibodies from, Santa Cruz Biotechnology, Santa Cruz, CA) as loading control as previously described [305]. Detection of bands were done by enhanced chemilumuniscence according to the manufacturer’s instruction (ECL, Amersham) and imaged with the ChemiDocTM MP system (Biorad, Veenendaal, Netherlands).          B.5  Recombinant human IL-1α caused IL-8 from PHLFs. PHLFs from control donors (open triangles, n=6) and COPD patients (closed triangles, n=6) were grown to confluence and serum deprived overnight. IL-8 concentration (with median) released from PHLFs IL-1α stimulation. *** & ### = p<0.001     217  Appendix C  : Supplementary Information and Data for Chapter 5 Interleukin-1 affects inflammatory mediator release and collagen I contraction by airway fibroblasts from asthmatic and non-asthmatic donors  C.1 Western blot Cell lysates were prepared by harvesting with protein extraction buffer (PEB) containing phosphatase and protease inhibitor cocktails (Sigma) as previously described [82]. Samples harvested with PEB were then subjected to SDS-PAGE with standard molecular weight ladder as a control after which they were blotted on a nitrocellulose membrane. Expression of collagen Iα1 was analyzed using a rabbit anti-human collagen I antibody (Abcam, ab34710) with a goat anti-rabbit IRDye 800 CW (LICOR®, Lincoln, NE) as the secondary antibody after which visualization was done with the ODYSSEY® infrared imaging system (LICOR®). Signal from detected bands were normalized to total protein using the REVERT™ total protein stain (LI-COR®, Lincoln, NE, USA) according the manufacturer’s instruction.   218          C.2  Recombinant human IL-1α stimulates the release of CXCL8/IL-8 release from primary lung fibroblasts. Primary lung fibroblasts from control individuals were seeded on culture plates, grown to confluence and serum deprived overnight. CXCL8/IL-8 concentration released after fibroblasts after stimulation with IL-1α at concentrations 0.001, 0.01, 0.5 and 1ng/ml for 24 hours. **=<p=0.01 and **** = p<0.0001 between the indicated values.         C.3  Assessment of cell death of primary airway fibroblasts after cytokine stimulation. Primary airway fibroblasts from non-asthmatics and asthmatics were grown to confluence on collagen I coated plates and stimulated with or without 1ng/ml recombinant human IL-1α, IL-1β or IL-33 for 24hours. Percentage lactate dehydrogenase (LDH) released from cells after 24 hours was then assessed.  B a s a l 0 .0 0 1 n g /m l 0 .0 1 p g /m l 0 .5 n g /m l 1 n g /m l 02 0 0 0 04 0 0 0 06 0 0 0 0IL-8  (pg/ml)**************219         C.4  Effect of Interleukin-1 stimulation collagen Iα1 protein expression in primary airway fibroblasts (PAFs).  Primary airway fibroblasts from non-asthmatics and asthmatics were grown to confluence on collagen I coated plates. Relative protein expression normalized to total protein stimulating with or without 1ng/ml recombinant a) IL-1α, b) IL-1β & c) IL-33 for 24hours.   220  Appendix D  : Supplementary Information and Data for Chapter 6 Answering a 90 year old question for asthma and airway fibrosis using multimodal nonlinear optical microscopy            D.1  An overview of data processing and of SHG images.  (a) SHG images from collagen present within asthmatics and non-asthmatics airways were acquired; (b) each images was then pre-processed in order to correct background and intensity normalization; (c) the number of objects present in each images as well as the fractal dimension of collagen fibers and bundles was calculated; (d) the preferred direction of accumulated fibers in each sample was also recorded; finally, (e) several textural parameter were extracted, from both first and second order statistics (FOS and GLCM, respectively).    

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