Characterization of lung dendritic cells in a novel murine model of asthma

CHARACTERIZATION OF LUNG DENDRITIC CELLS IN A NOVEL MURINE MODEL OF ASTHMA ZHOU QIAN A THESIS SUBMITTED FOR THE DEGREE OF PHD DEPARTMENT OF MICROBIOLOGY NATIONAL UNIVERSITY OF SINGAPORE 2013 DECLARATION I hereby declare that the thesis is my original work and it has been written by me in its entirety. I have duly acknowledged all the sources of information which have been used in the thesis. This thesis has also not been submitted for any degree in any university previously. ZHOU QIAN December 2013 Acknowledgements Pursing a PhD is a tough journey. For me, however, it is never a lonely one. Many people have put energy in helping me along the way. My gratitude goes out to everyone who devoted time, knowledge and heart to my work. I not have room to name all of you here, but I could not have written this thesis without you. First and foremost, I owe endless thanks to Prof Kemeny. I feel grateful beyond measure to him and I must say, to the fates that allow my path to cross his. I am not sure why he accepted my application -I am the first student in his lab who obtained bachelor degree in mainland China. I probably had the least experience in the lab when I joined it, so I felt a little out of place at the beginning. Regardless how busy he was, Prof Kemeny was always helpful and encouraging. He has a genius for reading people, engaging us in a way that brings out the best in us. He helped me to build my confidence, pushed me to achieve goals, to be the best I can be. I thank for Tang Yafang for her invaluable mentorship in teaching me skills in lab techniques and experimental designs. Beyond this, her spirit, her determination and unbelievable strength in pursuing her dream was an inspiration that motivated me along the way. This thesis would not have been possible without the generous help from Adrian, whose sharp mind, tireless patience and enthusiasm were invaluable in helping to build my work block by block. Word is powerless in expressing my gratitude to his unwavering support. I feel lucky to have Dr Florent Ginhoux as my collaborator and scientific advisor. Besides offering his scientific expertise and many useful transgenic mice crucial for this thesis, he encouraged me with his enthusiasm constantly. I am in debt to many others as well: Shu Zhen, Pey Yng, Nayana, Kenneth, Fei Chuin, Richard, Isaac, Guo Hui, LC and Fiona. I owe tremendous gratitude to these people who not only have been great teachers for me in the lab, but most importantly, for being my “family” in Singapore, who rendered comfort and support when things are not all right, who shared my bitter-sweet experiences in the journey. I also thank Prof Chew Fook Tim, Prof Paul A MacAry, Dr Veronique Angeli for providing important technical support and facility access, as well as scientific guidance in my PhD project. I am endlessly thankful to my mother, who has never faltered in her belief in me. She has kept me sane through pep talks and reality checks. She taught me to see the world with never-ending curiosity, to strive for what I believed. Her drive, her determination and courage have enormous impact on me. She is not only my mother but my friend, my comrade in the battlefield of life. For that I am grateful and lucky beyond measure. In the end, I owe everything to the Lord who gives me the world full of wonder and this life with countless blessings. What I have done and what I have accomplished would not be possible without Him. Table of Contents CHAPTER 1: Introduction 15 1.1 Asthma 16 1.1.1 Asthma as a major public asthma problem 16 1.1.2 Asthma as a complex disease caused by multiple factors 17 1.1.3 Asthma as a chronic inflammatory disease 18 1.1. 4Clinical symptoms of asthma . 19 1.2 Animal model of asthma . 19 1.2.1 Chronic asthma model vs acute asthma model 21 1.2.2 Ovalbumin asthma model vs dust mite asthma model . 22 1.3 Overview of allergic asthma: the classic paradigm 24 1.4 Innate immune cells and asthma . 26 1.5 Innate immune cytokines and asthma . 31 1.6 Innate effector cells and asthma 35 1.7 Dendritic cells . 37 1.7.1 Origin of peripheral tissue DCs . 38 1.7.2 Heterogeneity of peripheral tissue DCs . 39 1.8 Lung dendritic cells . 40 1.8.1 Lung DC subsets 40 1.8.2 Lung DC function 41 1.8.3 Lung DCs in asthma . 42 1.8.4 Epithelial cells and DCs . 45 1.9 Adaptive immune system in asthma . 46 1.9.1 B cells in asthma 47 1.9.2 Th2 cells in asthma 47 1.9.3 Th1 cells in asthma 48 1.9.4 Th17 cells in asthma 49 1.9.5 CD8 T cells in asthma 51 1.10 Aims of the study 52 CHAPTER 2: Materials and Methods 54 2.1 Media and buffers . 54 2.2 Mice 57 2.3 Cell isolation . 60 2.3.1 Isolation of CD4 T cells by magnetic separation . 60 2.3.2 Adoptive transfer of CD4 T cells . 61 2.3.3 Sorting of CD4 T cells . 61 2.3.4 CFSE labeling of CD4 T cells . 62 2.3.5 Isolation of dendritic cells from lung Parenchymal . 62 2.3.6 Sorting of dendritic cells from lung draining lymph node . 64 2.4 Asthma model: sensitization and airway challenges 65 2.4.1 Allergen extraction . 65 2.4.2 Precipitation of Blo t-alum . 65 2.4.3 Sensitization and challenge protocol (intraperitoneal sensitization) . 65 2.4.4 Sensitization and challenge protocol (intransal sensitization) . 66 2.5 Bronchoalveolar lavage (BAL) analysis . 67 2.6 Ex vivo assays of mediastinal lymph node (MLN) . 68 2.6.1 Ex vivo restimulation of MLN cells . 68 2.6.2 Intracellular cytokine staining of re-stimulated MLN cells . 69 2.6.3 Ex vivo analysis of IL-4-producing innate and adaptive cells in MLN . 70 2.6.4 Ex vivo antigen presentation assay of MLN dendritic cells . 70 2.6.5 Ex vivo analysis of MLN dendritic cells 71 2.7 Ex vivo assays for lung parenchymal 72 2.7.1 Cytokine assay for lung homogenate . 72 2.7.2 Cytokine assay for sorted lung cells 72 2.7.3 GM-CSF receptor expression measurement of lung dendritic cells 73 2.8 Lung histology 74 2.8.1 Preparation of lung tissue . 74 2.8.2 Processing and sectioning of lung tissue . 75 2.8.3 Tissue mounting . 75 2.8.4 Staining 76 2.9 Lung immunofluorescence histology 79 2.10 Assessment of lung function . 79 2.11 Measurement of cytokines 81 2.12 Measurement of serum immunoglobulins 83 2.12.2 ELISA for Blo t-specific IgE . 85 2.12.3 Dotblots for Blo t-specific IgE and IgG . 86 2.13 Gel Filtration of Blo t extracts 86 2.14 In vivo and in vitro assays for bone marrow-derived dendritic cells 87 2.14.1 Generation of GM-CSF-derived bone marrow dendritic cell (BMDCs) . 87 2.14.2 Immunization of mice with BMDCs . 88 2.14.3 Stimulation of BMDCs with lipopolysaccharides (LPS) 89 2.14.4 Co-culture of BMDCs and CD4 T cells 89 2.15 Statistical analysis . 90 CHAPTER 3: Murine Model of Asthma Induced by Blo t allergen 91 3.1 Introduction . 91 3.2 Blo t induces a profound airway allergy phenotype through systemic sensitization. 91 3.2.1 Immunization protocol (systemic sensitization) 91 3.2.2 Increased serum immunoglobulin E production 92 3.2.3 Increased eosinophil and lymphocyte infiltration in the airway 93 3.3 Blo t induces a robust airway allergy phenotype through respiratory sensitization. 97 3.3.1 Immunization protocol (respiratory sensitization) . 97 3.3.2 Increased serum immunoglobulin E production 98 3.3.3 Increased eosinophils and lymphocytes in the airway . 99 3.3.4 Elevated Th2 cytokine production in mediastinal lymph node . 100 3.3.5 Mucus hyper-secretion in the lung . 101 3.3.6 Airway hyper-responsiveness (AHR) 102 3.4 Discussion . 103 CHAPTER 4: Characterization of Innate and Adaptive Immunity to Inhaled Blo t allergen 106 4.1 Introduction . 106 4.2 Examination of IL-4-eGFP+ cells in Blo t-immunized 4get mice 107 4.3 Th2 adjuvant-like property of Blo t 110 4.4 Discussion . 112 CHAPTER 5: Lung Dendritic Cells in Th2 Immunity Induced by Inhaled Blo t allergen 115 5.1 Introduction . 115 5.2 Ex vivo characterization of lung dendritic cells 116 5.3 Inhibited dendritic cell migration greatly diminishes Th2 immunity induced by inhaled Blo t . 121 5.4 In vivo depletion of lung CD103+ DCs does not alter the development of allergic responses to Blo t. 124 5.5 Lung CD11b+ DC deficiency abrogates the development of allergic responses to Blo t 128 5.6 CCR2-dependent monocyte-derived CD11b+ DCs are not required for the development of allergic responses to Blo t. . 134 5.7 Discussion . 137 CHAPTER 6: Granulocyte-Macrophage Colony-Stimulating Factor in the Development of Allergic Response Induced by Inhaled Blo t . 139 6.1 Introduction . 139 6.2 In vivo source of GM-CSF responding to inhaled Blo t . 140 6.3 Lung epithelium-derived GM-CSF mediates allergic responses induced by Blo t 144 6.4 Lung epithelium-derived GM-CSF is a critical regulator of CD11b+ DC Th2 cell priming . 148 6.5 Discussion . 154 CHAPTER 7: Direct Instrumental effect on Dendritic Cells Exerted by Blo t allergen . 157 7.1 Introduction . 157 7.2 Generation of GM-CSF-derived BMDCs . 159 7.3 Blo t-conditioned BMDCs could promote Th2 immunity in vivo 160 7.4 BMDCs conditioned by Blo t allergen fail to display TLR signaling activation 162 7.5 Discussion . 166 CHAPTER 8: Identification of Major Component in Blo t extracts Responsible for Inducing Th2 Immunity in vivo . 169 8.1 Introduction . 169 8.2 The activity of Blo t extracts in inducing asthmatic responses is protease sensitive 170 8.3 Biochemical characterization of airway allergy inducing activity in Blo t extracts. 173 8.4 Discussion . 175 CHAPTER 9: Final Discussion 178 9.1 Brief summary of our study 178 9.2 Final discussion of our study 179 9.3 Limitation and future direction of our study . 183 Reference . 185 Summary The Blomia tropicalis (Blo t) dust mite is prevalent in tropical and sub-tropical regions of the world. Although it is a leading cause of asthma, little is known how it induces allergy. Using a novel murine asthma model induced by intra nasal exposure to Blo t, we observed that a single intranasal sensitization to Blo t extract induces strong Th2 priming in the lung draining lymph node. Resident CD11b+ DCs preferentially transport antigen from the lung to the draining lymph node and are crucial for the initiation of Th2 CD4+ T cell responses. As a consequence, mice selectively deficient in CD11b+ DCs exhibited attenuated Th2 responses and more importantly did not develop any allergic inflammation. Conversely, mice deficient in CD103+ DCs and CCR2-dependent monocyte-derived DCs exhibited similar allergic inflammation compared to their wildtype counterparts. We also show that CD11b+ DCs constitutively express higher levels of GM-CSF receptor compared to CD103+ DCs and are thus selectively licensed by lung epithelial-derived GM-CSF to induce Th2 immunity. Taken together, our study identifies GM-CSF licensed CD11b+ lung DCs as a key component for induction of Th2 responses and represents a potential target for therapeutic intervention in allergy. List of figures Figure 3.2.1 Immunization protocol (systemic sensitization) 92 Figure 3.2.2 Immunoglobulin E (IgE) in the serum of immunized mice. . 93 Figure 3.2.3 Infiltration of cells into the airways in response to asthmatic. 94 Figure 3.2.4 Images of sorted BAL fluid cells (H&E staining). 95 Figure 3.2.5 Cell infiltration in BAL fluid of Blo t-treated mice. . 95 Figure 3.2.6 Cytokine production from lung draining lymph node (MLN) of Blo t-treated mice. 96 Figure 3.3.1 Immunization protocol (respiratory sensitization). . 97 Figure 3.3.2 Immunoglobulins in the serum of Blo t immunized mice. 99 Figure 3.3.3 Cell infiltration in BAL fluid of Blo t-treated mice. . 100 Figure 3.3.4 Cytokine production from lung draining lymph node (MLN) of Blo t-treated mice. 101 Figure 3.3.5 Representative histological images of PAS staining of the lung . 102 Fig 3.3.6 Airway hyper-responsiveness following methacholine inhalation. 103 Figure 4.2.1 Time points for MLN analysis in 4get mice after intranasal immunization. 108 Figure 4.2.2 Identification of IL-4-producing cells of MLN in 4get mice. . 108 Figure 4.2.3 Kinetics of IL-4-eGFP+ CD4 T cells in MLN of 4get mice. . 110 Figure 4.2.4 Kinetics of IL-4-eGFP+ innate cells in MLN of 4get mice. 110 Figure 4.3.1 Experimental design of adoptive transfer from DO11.10 × 4get mice mice. . 111 Figure 4.3.2 Percentage of OVA-specific-IL-4-eGFP+ CD4 T cells of total CD4 T cells in MLN. . 112 Figure 5.2.1 Kinetics of lung dendritic cells in draining lymph node. 117 Figure 5.2.2 Identification of lung dendritic cells and their recruitment in MLN. 118 Figure 5.2.3 OVA up-taking by lung dendritic cells in MLN. 120 Figure 5.2.4 Antigen presentation capacity of lung dendritic cells. 121 Figure 5.3 Inhibited dendritic cell migration greatly diminishes Th2 immunity induced by inhaled Blo t. . 124 Figure 5.4 In vivo depletion of lung CD103+ DCs does not alter the development of allergic responses to Blo t. . 128 Figure 5.5 Lung CD11b+ DC deficiency abrogates the development of allergic responses to Blo t. 133 Figure 5.6 CCR2-dependent monocyte-derived CD11b+ DCs are not required for the development of allergic responses to Blo t. . 136 Figure 6.2 In vivo source of GM-CSF upon Blo t immunization. . 144 Figure 6.3 Lung epithelium-derived GM-CSF mediates allergic responses induced by Blo t 148 10 Fogg, D.K., C. 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Clin Exp Allergy 37:973-988. 205 [...]... and airway hyper-responsiveness As a model of asthma, however, experimental acute allergic inflammation of the lung parenchyma has significant limitations Characteristic features airway remodelling of human asthma such as intraepithelial recruitment of eosinophils, lamina propria inflammation and subepithelial fibrosis are virtually absent in these models 1.2.2 Ovalbumin asthma model vs dust mite asthma. .. and acute asthma models, acute ovalbumin asthma model (ovalbumin as antigen) and acute dust mite model (dust mite allergen as antigen), which is the most relevant for this study 1.2.1 Chronic asthma model vs acute asthma model For years, attempts have been made to induce chronic asthma in mice (Kumar and Foster, 2001) These chronic asthma models have involved either repeated intratracheal/intranasal... mimicking of the cause and progression of human asthma Although different laboratory animals such as guinea pigs, dogs, sheep, rats have been implemented in asthma research, mice are the most commonly used in experimental models because of advantages such as the availability of various transgenic animals, the wide array of reagents available for analysis, the low cost of maintenance and the 20 CHAPTER... the nature of the allergen and the route of sensitization can significantly influence the overall innate and adaptive Furthermore, not only does respiratory exposures to OVA lead to inhalation tolerance , but continuous challenge with OVA via intranasal route in already sensitized animal results in an attenuation and even complete abrogation instead of an increase or maintenance of airway inflammation... prevalence of asthma keeps increasing and currently affects millions of people worldwide Apart from imposing a social burden in terms of quality of life, the cost of asthma patient health care also places a significant burden on public health and thus represents a major public health concern 1.1.1 Asthma as a major public asthma problem Approximately, 300 million people suffer from asthma and its prevalence... prevalence has been increasing over the past few decades (Braman, 2006) The increase in asthma and atopic diseases has been described as an epidemic event According to statistics released by American Academy of Allergy Asthma and Immunology, the prevalence of asthma is particularly high in developed countries with highest prevalence in the United Kingdom and New Zealand (Masoli et al., 2004) In developing... (Th2) cells are found in the lungs of almost all patients asthma, in particularly, patients with allergic asthma (Robinson et al., 1992) It is well established that Th2 cells are key players in the pathogenesis of asthma through the cytokines they produce (IL-4, IL-5 and IL-13) they mediate several hallmarks of asthma/ allergy, namely, eosinophilia, goblet cell hyperplasia, airway 24 CHAPTER 1: Introduction... allergic asthma, patients are usually interviewed about the incidence of symptoms, their day/night time occurrence and history of allergy and so on to facilitate the diagnosis of asthma 1.2 Animal model of asthma Clinical observations on asthmatic patients have laid the foundation knowledge for scientific studies on the pathogenesis of asthma At the very beginning, association 19 CHAPTER 1: Introduction... countries, asthma prevalence is increasing sharply with the progress of urbanization The increase in China and India will lead to a dramatic increase in the economic burden due to their giant populations (Masoli et al., 2004) Specifically, the high prevalence 16 CHAPTER 1: Introduction rate has markedly increased the cost of this disease as measured in health care dollars, time away from work and school, and... of symptoms, forced expiratory volume and peak expiratory flow rate In asthma research, nevertheless, it is more commonly classified according to the origin and cause of the disease, namely allergic asthma and non-allergic asthma as described earlier (Romanet-Manent et al., 2002) Allergic asthma, the most common type of asthma experienced by approximately 80% of asthmatic patients, is more widely investigated . Asthma as a chronic inflammatory disease 18 1.1. 4Clinical symptoms of asthma 19 1.2 Animal model of asthma 19 1.2.1 Chronic asthma model vs acute asthma model 21 1.2.2 Ovalbumin asthma model. mite asthma model 22 1.3 Overview of allergic asthma: the classic paradigm 24 1.4 Innate immune cells and asthma 26 1.5 Innate immune cytokines and asthma 31 1.6 Innate effector cells and asthma. promote asthma. Over the past 20 years, asthma research has therefore almost exclusively focused on inflammation as a cause of disease. 1.1.4 Clinical symptoms of asthma Allergic asthmatic patients