Proteomic analysis of airway inflammation in murine asthma models

PROTEOMIC ANALYSIS OF AIRWAY INFLAMMATION IN MURINE ASTHMA MODELS ZHAO JING (B. MED., M. MED.) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF PHARMACOLOGY NATIONAL UNIVERSITY OF SINGAPORE 2006 ACKNOWLEDGEMENTS Four years may be nothing more than a numeric symbol to many people, but it means a lot to me. From a layman to a well-trained “skilled researcher”, I have learned many things which are invaluable to my future career. The project could not have been finished without Prof W.S. Fred Wong, who not only served as my supervisor but also encouraged and challenged me throughout my academic program. I will always remember the numerous discussions with him, his serious attitude to every even arbitrary ideas coming out of my mind, and his encouragement when the project got stuck (no matter how often it happens). Not only how to fish, but also how to weave the fishnet have I learned from him. Without help and support from my colleagues and friends, Chui Hong Wong, Hui Hwa Choo, Zhu Hua, Jasmine Chan, Amy Lin, Winston Liao, and Bao Zhang, I would never have finished these laborious works smoothly. Thanks also to Foo Tet Wei, Mok Lim Sum and Wang Xianhui for their earnest service and advice in proteomics practice. Last but not least, I would like to express sincere appreciation to the Research Scholarship support from the NUS, which gave me this precious chance of studying in Singapore. Zhao Jing Jun 2006 ii TABLE OF CONTENTS ACKNOWLEDGEMENTS ii TABLE OF CONTENTS iii SUMMARY vii LIST OF TABLES ix LIST OF FIGURES x LIST OF ABBREVIATIONS xii LIST OF PUBLICATIONS AND CONFERENCES ATTENDED xv 1. INTRODUCTION 1.1. Proteomics 1.1.1. Proteomics and genomics 1.1.2. Protein sample preparation 1.1.3. Protein separation 1.1.3.1 Two-dimensional electrophoresis 1.1.3.2 Chromatography 13 1.1.3.2.1 Two-dimensional approaches 14 1.1.3.2.2 Three-dimensional approaches 19 1.1.4. Mass spectrometry 19 1.1.4.1 Principles 20 1.1.4.2 Types of Mass spectrometry 23 1.1.5. Array-based proteomics 24 1.1.6. Applications of proteomics 26 iii 1.1.6.1 Expression proteomics 26 1.1.6.2 Functional proteomics 27 1.1.7. Proteomics in asthma 1.2. Asthma 1.2.1. Pathophysiology of asthma 1.2.1.1 Airway inflammation 29 33 33 33 1.2.1.1.1 Early response and late response 34 1.2.1.1.2 Chronic inflammation 35 1.2.1.2 Airway remodeling 39 1.2.1.2.1 Characteristics of airway remodeling 39 1.2.1.2.2 Mechanism of airway remodeling 44 1.2.2. Animal models 47 1.2.3. Treatment of asthma 51 1.2.3.1 Glucocorticoids 51 1.2.3.2 Other therapeutic agents 53 1.3. Rationale and objectives 57 2. MATERIALS AND METHODS 60 2.1. Mouse animal models 61 2.1.1. Acute asthma model 61 2.1.2. Chronic asthma model 61 2.2. Measurement of airway responsiveness 63 2.3. Immunoglobulin E measurement in serum 66 iv 2.4. Histology 66 2.5. Immunohistochemistry 67 2.6. Quantitative analysis of the airways 68 2.7. BAL fluid cell counts 69 2.8. Proteomic analysis 70 2.8.1. Sample collection and preparation 70 2.8.2. Two-dimensional electrophoresis 74 2.8.3. Silver staining 75 2.8.4. Image analysis 76 2.8.5. In-gel digestion and MS analysis 76 2.9. Western blotting 77 2.10. RT-PCR 78 2.11. Chitinase Activity Assay 78 2.12. Statistics 79 3. PROTEOMIC ANALYSIS OF ACUTE AIRWAY INFLAMMATION IN A MOUSE MODEL 81 3.1. Results 82 3.1.1. Acute mouse asthma model 82 3.1.2. 2-DE and image analysis of BAL fluid 85 3.1.3. Immunoblotting and RT-PCR 94 3.2. Discussion 97 v 4. PROTEOMICS OF AIRWAY INFLAMMATION AND REMODELING IN A CHRONIC MOUSE ASTHMA MODEL 104 4.1. Results 105 4.1.1. Serum IgE level and airway responsiveness 105 4.1.2. Airway inflammation and airway remodeling 108 4.1.3. Two-dimensional electrophoresis analysis 114 4.1.4. Gene Ontology Classification 123 4.2. Discussion 125 5. PHARMACOPROTEOMIC ANALYSIS OF DEXAMETHASONE IN AN ACUTE MOUSE ASTHMA MODEL 134 5.1. Results 135 5.1.1. Dexamethasone inhibits airway inflammation in mice 135 5.1.2. Dexamethasone alters mouse BAL fluid proteome 138 5.1.3. Immunoblotting and RT-PCR 144 5.1.4. Dexamethasone reduces BAL fluid chitinase activity 146 5.2. Discussion 149 6. CONCLUSIONS 158 7. REFERENCES 162 vi SUMMARY Proteomic techniques evaluate levels and post-translational modifications of a large number of proteins simultaneously. Currently, proteomics has been used for studying human diseases in a wide variety of biomedical areas including cardiovascular diseases, cancer, neurological disorders, and respiratory diseases. The proteomic approach allows us to search for new bio-markers and explore the pathogenesis of allergic airway inflammation based on the analysis of protein expression differences between healthy state and diseased state. The purpose of this study was firstly to analyse and quantify the alterations in global protein expression in bronchoalveolar lavage (BAL) fluid from mice with acute allergic airway inflammation compared with normal mice by employing a proteomic technology. A typical 2-dimensional map of BAL fluid of mouse was constructed. Secondly, the protein profiles from both BAL fluid and lung tissue from mice with chronic allergic airway inflammation were compared with the samples from normal mice. A typical 2-dimensional map of lung tissue of mouse was also constructed. Finally, we investigated pharmacoproteomics of a glucocorticoid drug, dexamethasone, the most effective class of drug for treating asthma, in an acute allergic mouse asthma model. To achieve these objectives, representative animal models are required. No single animal asthma model can simulate all features of human asthma. vii Therefore, we established two types of asthma model, the acute and the chronic, to demonstrate different stages of asthma conditions in human. The acute asthma model focuses on early asthmatic responses and airway acute inflammation with mild structural changes, while the chronic asthma model illustrates more features of airway structural alterations but the acute airway inflammation is attenuated. We have identified many classes of proteins which were for the first time shown to be related to the pathophysiology of asthma. Our findings shed new light on the exploration of new mechanisms of the development of asthma. The identified proteins may be considered as potential biomarkers for monitoring the progression of asthma or potential therapeutic targets for novel drug development. Moreover, the pharmacoproteomics study further broadens our understanding of the spectrum of gene target regulation by steroids and may be useful for the new drug development. viii LIST OF TABLES Table Title Page List of various chromatographic methods 13 Allergic responses in different mouse strains 50 Glucocorticoid-regulated genes 53 Running protocols for IEF 74 Primers for RT-PCR analysis 80 Proteins identified in acute asthma model 90 Proteins identified in chronic asthma model 116 Proteins identified from dexamethasone-treated mice 140 ix LIST OF FIGURES Figure Title Page Schematic summary of steps in the LCM Workflow for DIGE system 11 ICAT strategy for quantifying differential protein expression 17 Mass spectrometry technology for proteomics 22 Scheme of fluorescence resonance energy transfer (FRET) 25 Pathogenesis of asthma 30 Aerosol delivery system 62 The Buxco system 64 Computation of the airway responsiveness 65 10 Type of cells in BAL fluid 72 11 Scheme of proteomics workflow and instrumentation 73 12 Development of a murine acute asthma model 83 13 Representative 2-D gels of BAL fluid in acute asthma model 87 14 A representive searching result (part) 88 15 Differential BAL fluid protein expression in acute asthma model 91 16 Graphical representation of 24 identified proteins 92 17 A representative MALDI TOF-TOF MS spectrum 93 18 Western blot analysis of BAL fluid in acute asthma model 95 19 RT-PCR analysis in acute asthma model 96 20 Serum level of OVA-specific IgE in chronic asthma model 106 x Biochem 2001;70: 649-676. 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Chemokines in asthma: cooperative interaction between chemokines and IL-13. J Allergy Clin Immunol 2003;111:227-42 189 [...]...21 Airway responsiveness in chronic asthma model 107 22 H&E staining for airway inflammation in chronic asthma mode 108 23 PAS staining for mucus production in chronic asthma model 110 24 Smooth muscle thickness in chronic asthma model 111 25 Masson Trichrome staining for collagen deposition 112 26 Immunohistochemistry staining for fibronectin 113 27 Representative 2-D gels in chronic asthma model... 28 Protein grouping using Gene Ontology Annotation 124 29 Interactions between actin binding proteins and actin 129 30 Anti-inflammatory effects of dexamethasone 136 31 Histological examinations of effects of dexamethasone 137 32 Representative 2-D gels in dexamethasone experiment 139 33 Locations of identified proteins in dexamethasone experiment 141 34 Optical intensities of identified proteins 142... (2005) Increased lungkine and chitinase levels in allergic airway inflammation: a proteomics approach Proteomics 5(11):2799-807 2 Zhao J, Yeong LH, Wong WSF (2007) Dexamethasone alters bronchoalveolar lavage fluid proteome in a mouse asthma model International Archives of Allergy and Immunology 142(3):219-229 [Epub ahead of print] 3 Zhao J, Lin YZ, Yeong LH, Wong WSF (2006) Proteomics of airway remodelling... Congress, Nov 3-5, 2003, Singapore 3 Zhao J, Zhu H, Wong CH, Leung KY, Wong WSF Increased lungkine xv and chitinase levels in allergic airway inflammation: a proteomics approach 9th Congress of the Asia Pacific Society of Respirology, Dec 10-13, 2004, Hong Kong 4 Zhao J, Zhu H, Wong CH, Leung KY, Wong WSF Increased lungkine and chitinase levels in allergic airway inflammation: a proteomics approach American... it has a higher loading capacity and allows increased spatial resolution Different staining methods have been developed for the protein detection, such as Coomassie Blue staining, silver staining, radioactive labeling, and fluorescence staining (Hirsch et al., 2004; Gorg et al., 2004) The detection sensitivity varies from 100 ng of proteins for Coomassie staining to 200 fg of proteins for radiography... proteins analysis, ranging from a single protein to thousands in one experiment Proteomics thus has replaced the phrase ‘protein science’ (Baak et al., 2005) The growth of proteomics is a direct result of rapid advances made in genome study The first complete genome of an organism, Hemophilus influenzae, was sequenced in 1995, 42 years after the landmark description of the DNA double helix structure in. .. proteome in a mouse asthma model Combined Scientific Meeting 2005, Nov 4-6, 2005, Singapore xvi 1 INTRODUCTION 1 1.1 Proteomics 1.1.1 Proteomics and genomics The word ‘proteome’ was first coined in 1994 to describe all proteins content present in a cell, tissue, or body fluid at a given time (Wilkins et al., 1996) The study of the proteome, called proteomics, was proposed in 1995 and was defined as... proteins for radiography Due to the shortcomings of the organic dyes, radiolabelling and silver staining for visualization and quantitation of proteins, fluorescent detection has increasingly gained popularity for proteomic analysis Two major approaches for the fluorescent detection, pre-electrophoretic staining (Urwin and Jackson, 1993) and post-electrophoretic staining (Berggren et al., 2002), are currently... be characterized and the targets of drugs be identified 1.1.2 Protein sample preparation A typical proteomics experiment (such as protein expression profiling) can be broken down into the following steps: (i) the separation and isolation of proteins from a cell line, tissue, or organism; (ii) the acquisition of protein structural information for the purposes of protein identification and characterization;... proteins in a dynamic range of approximately 4 orders of magnitude (Hirsch et al., 2004) Affinity purification is a powerful approach to reduce the complexity of a sample by specifically isolating individual proteins or “protein complexes” (Bauer and Kuster, 2003) These preparation steps are often more time consuming than the subsequent analysis steps and influence the sensitivity 5 and discriminative . PROTEOMIC ANALYSIS OF AIRWAY INFLAMMATION IN MURINE ASTHMA MODELS ZHAO JING (B. MED., M. MED.) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF. PROTEOMICS OF AIRWAY INFLAMMATION AND REMODELING IN A CHRONIC MOUSE ASTHMA MODEL 104 4.1. Results 105 4.1.1. Serum IgE level and airway responsiveness 105 4.1.2. Airway inflammation and airway. chronic asthma model 107 22 H&E staining for airway inflammation in chronic asthma mode 108 23 PAS staining for mucus production in chronic asthma model 110 24 Smooth muscle thickness in chronic