A mouse model of rhinovirus induced asthma exacerbation

A MOUSE MODEL OF RHINOVIRUS - INDUCED ASTHMA EXACERBATION ZHANG XUEYU (B.Sc. ZHEJIANG UNIVERSITY) A THESIS SUBMITTED FOR THE DEGREE OF MASTER OF SCIENCE DEPARTMENT OF PHARMACOLOGY 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 that have been used in the thesis. This thesis has also not been submitted for any degree in any university previously. _________________ ZHANG XUEYU 01 August 2013 ACKNOWLEDGEMENTS First and foremost, I would like to thank my supervisor Professor Fred Wong Wai-Shiu for his guidance and assistance through my M.Sc. studies. Without his help, I could not overcome the obstacles in the project. I would also like to thank Professor Vincent Chow and Mrs. Phoon Meng Chee for their invaluable advice on my research works. I am grateful to Cheng Chang, Tao Lin, Eugene, Shou Ping, Fera Goh, Tze Khee, Alan and all lab members for their constant guidance and support. Without them, this project would not have been completed smoothly. Finally, I would like to thank my parents and all my friends for their encouragement and support during these years.   iii   SUMMARY Asthma is a chronic airway disease that affects millions of people around the world. The exacerbation of asthma symptoms is tightly associated with the mortality of this disease and brings a heavy healthcare and economic burden. Respiratory virus infection is long regarded as a crucial factor that induces asthma exacerbation. With the development of PCR techniques, rhinovirus, the major cause of common cold, was detected in most wheezing cases and considered to be the major trigger of asthma exacerbation. To date, the immune reactivity in response to infections is considered as a great contributor to the pathogenesis of rhinovirus-induced asthma exacerbation. The development of a mouse model of asthma exacerbation would greatly contribute to the further understanding of the relationship between rhinovirus infection and asthma exacerbation. We modified the protocol of a model recently published by Bartlett et al. (2008). Allergen challenge followed by rhinovirus infection induced inflammatory cells infiltration in airways, mucus hyper-secretion and increasing trend of airway hyper-responsiveness (AHR) in mice. The amplification of three characteristic features of asthma indicates that our mouse model successfully imitated the rhinovirus-induced allergic asthma exacerbation. Analysis of the expression of several inflammatory genes showed that rhinovirus infection increased the expression of Th2 cytokines such as   iv   IL-4 and IL-13, along with the expression of eotaxin-1, the classic chemokine attracts eosinophils. These inflammatory factors are typically expressed in allergic asthma, particularly during acute attack period, and contribute to the pathophysiology of this process. Rhinovirus infection also induced and enhanced the expression of other inflammatory chemokines including CXCL10 and MCP-1, two chemoattractants for T lymphocytes and monocytes. CXCL10 acts as a chemokine relating Th1 response, and recently considered as a novel biomarker of rhinovirus-induced asthma exacerbation. Besides the cytokines and chemokines, rhinovirus infection also increased the level of serum IgE, especially in the later days post challenge. In addition to the increase of IgE levels, mucus overproduction also showed an increased trend along with time. Thus, considering the time course of rhinovirus-induced inflammatory factors, virus infection may also contribute a possible role to the development and aggravation of allergic airway disease. Taken together, the establishment of mouse model of rhinovirusinduced asthma exacerbation in this project may further contribute to the investigation of virus-induced asthma exacerbation and facilitate the discovery of effective therapy for this disease.   v   TABLE OF CONTENTS ACKNOWLEDGEMENTS ............................................................................... iii   SUMMARY ...................................................................................................... iv   TABLE OF CONTENTS .................................................................................. vi   LIST OF TABLES............................................................................................ ix   LIST OF FIGURES ........................................................................................... x   LIST OF ABBREVIATION ............................................................................... xi   Chapter 1 Introduction ................................................................................... 1   1.1   Asthma ........................................................................................... 2   1.1.1   Epidemiology of asthma........................................................... 2   1.1.2   Development of asthma ........................................................... 3   1.1.3   Pathophysiology of asthma ...................................................... 5   1.2   Asthma exacerbation ...................................................................... 8   1.2.1   Epidemiology of asthma exacerbation ..................................... 8   1.2.2   Factors inducing asthma exacerbation .................................. 10   1.2.2.1 Rhinovirus .......................................................................... 16   1.2.3   Pathophysiology of asthma exacerbation .............................. 19   1.2.3.1 Epithelial cells .................................................................... 19   1.2.3.2 Eosinophils......................................................................... 20   1.2.3.3 Neutrophils ......................................................................... 25   1.2.3.4 Macrophages ..................................................................... 27   1.2.3.5 Mucus hyper-secretion ....................................................... 29   1.2.3.6 Airway Hyper-responsiveness (AHR) ................................ 31   1.3   Animal models for asthma exacerbation ...................................... 32   Chapter 2 Rational ........................................................................................ 38     vi   Chapter 3 Materials and methods ............................................................... 40   3.1   Materials ....................................................................................... 41   3.2   Virus culture and purification ........................................................ 42   3.3   Virus titration ................................................................................. 43   3.4   Mouse model of asthma exacerbation .......................................... 44   3.5   Bronchoalveolar lavage (BAL) fluid collection .............................. 44   3.6   Total and differential BAL fluid cell counts .................................... 45   3.7   Histological examination ............................................................... 46   3.8   Immunoglobulin E levels in serum ................................................ 48   3.9   Reverse transcription-polymerase chain reaction (RT-PCR)........ 49   3.10   Measurement of airway hyper-responsiveness (AHR) ............... 50   3.11   Statistical analysis ...................................................................... 52   Chapter 4 Results ......................................................................................... 53   4.1   Rhinovirus-induced BAL fluid inflammatory cell increases in experimental allergic asthma murine model .......................................... 54   4.2   Rhinovirus-induced lung tissue inflammatory cell infiltration in experimental allergic asthma murine model .......................................... 57   4.3   Rhinovirus-induced airway mucus hyper-secretion in experimental allergic asthma murine model ............................................................... 60   4.4   Rhinovirus-induced serum IgE production in experimental allergic asthma murine model ............................................................................ 63   4.5   Rhinovirus-induced expression of other inflammatory markers in experimental allergic asthma murine model .......................................... 65   4.6   Rhinovirus-induced AHR in experimental allergic asthma murine model .................................................................................................... 69   Chapter 5 Discussion ................................................................................... 71   5.1   Development of rhinovirus-induced asthma exacerbation mouse model .................................................................................................... 72   5.2   Increased chemokine expression in rhinovirus-induced allergic airway disease ...................................................................................... 77     vii   5.3   Immune response in virus-induced allergic asthma exacerbation 81   5.4   Further direction and limitations ................................................... 85   Chapter 6 Conclusion................................................................................... 89   Chapter 7 References .................................................................................. 91   APPENDIX ................................................................................................... 113       viii   LIST OF TABLES Table 3.1 Primer sequences of targets for RT-PCR ....................................... 51     ix   LIST OF FIGURES Figure 1.1 The annual cycle of asthma exacerbation. ............................. 11   Figure 1.2 Variation in the frequency of rhinovirus isolation in sampled illnesses and rates of rhinoviral respiratory illness. .................................. 13   Figure 1.3 The structure of rhinovirus. ..................................................... 18   Figure 1.4 The immunological role of epithelial cells in virus-indcued asthma exacerbations............................................................................... 21   Figure 1.5 Eosinophils modulate the function of other leukocytes. .......... 24   Figure63.1 Cytopathic effect in Rhinovirus 14 infected HeLa cells. .......... 43   Figure74.1 Rhinovirus-induced increase in BAL inflammatory cell counts in experimental allergic asthma murine model. ............................................ 56   Figure84.2 Rhinovirus-induced lung tissue inflammatory cell infiltration in experimental allergic asthma murine model. ............................................ 59   Figure94.3 Rhinovirus-induced airway mucus hyper-secretion in experimental allergic asthma murine model. ............................................ 62   Figure14.4 Rhinovirus-induced serum IgE productions in experimental allergic asthma murine model. .................................................................. 64   Figure14.5 Rhinovirus-induced inflammatory marker expression changes in experimental asthmatic lungs. .............................................................. 68   Figure14.6 Rhinovirus-induced AHR in experimental allergic mice. ......... 70   x   LIST OF ABBREVIATION AHR airway hyper-responsiveness Al(OH)3 aluminium hydroxide AMV avian myeloblastosis virus ASM airway smooth muscle APC antigen present cells BAL bronchoalveolar lavage BCA bicinchonic acid BSA bovine serum albumin CCR CC-chemokine receptor Cdyn dynamic compliance CPE cytopathic effect CXCR CXC-chemokine receptor DALY disability-adjusted life year DC dendritic cell ECP eosinophil cationic protein EDN eosinophil derived neurotoxin EGFR epidermal growth factor receptor Eos eosinophil EPO eosinophil peroxidase FBS fetal bovine serum GM-CSF macrophage-colony stimulation factors HDM house dust mite   xi   HRP horseradish peroxidase ICAM-1 intercellular adhesion molecule-1 IL interleukin LDL low-density lipoprotein LT leukotriene Lym lymphocyte Mac macrophage MCP-1 monocyte chemoattractant protein-1 MBP major basic protein Neu neutrophil NF-κB Nuclear factor-κB NH4Cl ammonium chloride OVA ovalbumin PBS phosphate buffered saline PCR polymerase chain reaction RI airway resistance RSV respiratory syncytial virus STAT signal transducer and activator of transcription Th2 T helper 2 TLR Toll-like receptor TNF-α tumor necrosis factor-alpha TSLP thymic stromal lymphopoietin UTR untranslated region VCAM-1 vascular cell adhesion molecule-1   xii   Chapter 1 Introduction   1   1.1 Asthma 1.1.1 Epidemiology of asthma Asthma is a chronic airway disease with typical symptoms including cough, wheezing, chest tightness and shortness of breath. To date, the development of asthma has been linked to genetic and environmental components, but the full-spectrum of pathogenesis is still not clear. According to the Global strategy for asthma management and prevention, one description of asthma is: a chronic inflammatory disorder of airways in which many cells and cellular elements play a role. The chronic inflammation is associated with airway hyperresponsiveness that leads to recurrent episodes of wheezing, breathlessness, chest tightness, and coughing, particularly at night or in the early morning. These episodes are usually associated with widespread, but variable, airflow obstruction within the lung that is often reversible either spontaneously or with treatment (Global Initiative for Asthma (GINA), 2011). Asthma affects nearly 300 million people of all ages and all ethnic background (Masoli et al., 2004). In different countries, the prevalence of asthma ranges from 1% to 18% of the population. Higher prevalence (>10%) is found in urbanized countries, such as New Zealand (15.9%), Australia (14.7%) and the United States (10.9%). With the projected increase in urban population from 45% to 59%, there may be an estimated additional 100 million asthmatic persons by   2   the year 2025 (Masoli et al., 2004). The morbidity and mortality among asthma sufferers are significant. The number of hospital admissions for asthma has increased worldwide. From 1960s to 1980s, a 200% increase in rates of hospitalization of asthmatic adults and a 50% rise for children were reported in the United States (DeMeo and Weiss, 2009). The mortality rate of asthma ranges from 0 to 2.5 per 100,000 people around the world, and most of the preventable deaths are associated with poor asthma care (Masoli et al., 2004). In addition to direct costs of health care and indirect costs of lost productivity, the social and economic burden of asthma cannot be ignored. The number of disability-adjusted life years (DALYs) lost associated with asthma is estimated to be 15 million per year, making asthma the 25th leading cause of DALYs lost worldwide in 2001 (Masoli et al., 2004). The annual economic cost of asthma in the United States from 2002 to 2007 was $56.0 billion, with $50.1 billion direct health care costs and $5.9 billion indirect costs (lost productivity) (American Lung Association, 2012). 1.1.2 Development of asthma Asthma is a phenotypically heterogeneous disease involving complex interactions of multiple factors. Besides the common category of allergic asthma and non-allergic asthma, phenotypes can also be clarified   by asthma triggers (virus, environmental allergens, 3   occupational irritants, etc.), by inflammatory pathology (eosinophilic, neutrophilic, and paucigranulocytic), or by clinical and physiological categories (severity, exacerbation, age on set, etc.) (Bel, 2004; Wenzel, 2006; Zedan et al., 2013). Among them, allergic asthma might be counted for the largest phenotype, especially in children (Wenzel, 2006). Environmental factors play a crucial role in asthma development. Typical environmental triggers for asthma include allergens in air and diet, air pollutants, respiratory viruses and tobacco smoke (Mukherjee and Zhang, 2011). One possible theory that explains the relationship between environmental risks and asthma development is “ hygiene hypothesis”. The hypothesis suggests that a lacking of childhood exposure to infection might increase the susceptibility of certain inflammatory disorders (Strachan, 2000). This hypothesis is also supported by recent studies focusing on the correlation between farm living and a low risk of atopy (Ege et al., 2011; Stevens et al., 2011). In contrast, early viral infections seem less relevant to the hygiene hypothesis, with an increased risk of persistent wheezing after viral infection (Fishbein and Fuleihan, 2012). The genetic background also contributes to asthma development. More than 100 major or minor susceptibility genes are involved in asthma development (Hammad and Lambrecht, 2008). The products of those genes are involved in many aspects of asthma,   4   including innate immunity and immune-regulation, T helper 2 (Th2)-cell differentiation and effector functions, epithelial biology and mucosal immunity, lung functions and airway remodeling (Vercelli, 2008). However, the linkage between the genotypes and phenotypes of asthma is still far from conclusive. Gene-environment interaction might change the effect of a gene on different phenotypes. Other factors, like social and psychological factors, have also been found to contribute to the complexity of asthma phenotypes (Drake et al., 2008). 1.1.3 Pathophysiology of asthma Though there are several phenotypes of asthma, inflammatory cell infiltration, mucus hyper-secretion and airway hyper- responsiveness (AHR) are three characteristic features that can be found in most asthma cases. The airway accumulation of Th2 cells, which predominately secrete interleukin 4 (IL-4), IL-5, IL-13, has a central role in the pathogenesis of asthma, particular the allergen-related (Kim et al., 2010). When allergens enter the body, antigen present cells (APCs) such as dendritic cells (DCs) are activated and take up the allergens to draining lymph nodes. The activated DCs present the antigens to naïve CD4+ T cells and drive them to Th2 deviation (Hammad and Lambrecht, 2008). Allergen-specific Th2 cells secrete several cytokines including IL-4, IL-5 and IL-13. IL-4 amplifies the Th2 response by promoting Th2 lymphocyte differentiation, and activates B cells switch   5   towards immunoglobulin E (IgE) synthesis with the help of IL-13 (Mamessier and Magnan, 2006). IL-5 is associated with the differentiation and activation of eosinophils, which is a pivotal effector cell in asthma pathophysiology. IL-13 can induces the pathophysiological features of asthma without the effect of IgE and eosinophils (Wills-Karp et al., 1998). Under the presence of IL-4, IL-13, and other molecules, B cells undergo isotype switching and synthesize IgE, which can bind to high affinity IgE receptors (FcεRI) on the surface of mast cells (Stone et al., 2010). The crosslinking of allergens, IgE and FcεRI on the surface activates mast cells, leading to the release of granule content including histamine, leukotriene (LT) C4, LTD4, LTE4 and prostaglandin D2. These mediators can change airway smooth muscle (ASM) activity and induce mucus hyper-secretion (Gould and Sutton, 2008). Eosinophils are granulocytes that participate in variety of inflammatory processes, especially asthma. They are often present in the airways of allergic asthmatics and correlate with disease severity (Wardlaw et al., 2000). In response to inflammatory stimuli of IL-5, IL13 or other inflammatory chemokines such as eotaxins, eosinophils are recruited into the inflammatory site, and release pro-inflammatory mediators including granule-stored cationic proteins, newly synthesized eicosanoids and cytokines (Gould and Sutton, 2008). The major cationic proteins are major basic protein (MBP), eosinophil cationic   6   protein (ECP), eosinophil derived neurotoxin (EDN), and eosinophil peroxidase (EPO). These proteins can induce airway damage and contribute to airway hyper-responsiveness (AHR) (Holgate, 2008; Wardlaw et al., 2000). Airway mucus hyper-secretion is a feature of asthma that contributes to morbidity and mortality, with the obvious features including goblet cell hyperplasia, mucus plugging and submucosal gland hypertrophy (Rogers, 2004). In healthy individuals, mucus is a film of slippery secretion that covers and protects the airway epithelium (Evans et al., 2009; Rogers, 2004). Mucins are the high molecular weight glycosylated proteins that consist in mucus (Thornton and Sheehan, 2004). In the airways of asthmatics, airway mucins may be greatly secreted, leading to airway obstruction and hyper- responsiveness. The regulation of IL-13 were found associating with mucus overproducing in allergic asthma, with increased expression of two important mucins, MUC5AC and MUC5B (Zhen et al., 2007). AHR is a key clinical feature of asthma and an indicator of disease severity. Referring to the mechanism of mediated bronchoconstriction, the stimuli of AHR segregate into direct stimuli such as methacholine and indirect stimuli such as allergens. The direct stimuli function directly on receptors on the airway smooth muscle while the indirect ones cause bronchoconstriction via the release of mediators from inflammatory cells (Brannan and Lougheed, 2012;   7   Cockcroft and Davis, 2006). Another hypothesis suggests that the pathogenesis of AHR is characterized by epithelial, microbial and inflammatory triggers on one hand and abnormalities of effector airway structures such as smooth muscle cells on the other hand. Cytokines and chemokines act as mediators that link and aggravate triggers and effectors (Lommatzsch, 2012). 1.2 Asthma exacerbation 1.2.1 Epidemiology of asthma exacerbation The natural history of asthma consists of relatively stable periods and outbreak periods with significant exacerbation of symptoms. While there is no clear consensus definition, the term “exacerbation” is usually associated with severe asthma, as an episode of acute deterioration. According to a task force conducted by American Thoracic Society/ European Respiratory Society, asthma exacerbation were graded into severe, moderate and mild, and the recommended definition of a severe asthma exacerbation for clinical trials includes one of following: 1. Use of systemic corticosteroids, or an increase from a stable maintenance dose, for at least 3 days. 2. A hospitalization or emergency department visit because of asthma, requiring systemic corticosteroids (Reddel et al., 2009). Though the definition of asthma exacerbation is still contentious, exacerbation symptoms produce significant cost for healthcare systems and bring a heavy burden to the patients and the society. In   8   2007, asthma exacerbations resulted in 1.75 million emergency department visits and 456,000 asthma hospitalizations in the United States alone (Akinbami et al., 2011). Hospitalization constitutes nearly one third of the total $14.7 billion in US annual asthma-related health care expenditures (American Lung Association, 2012). The risks of asthma exacerbations differ within age and also between the sexes. According to data from Canada and New Zealand from 1995 to 1999, the rates of hospital admission for asthma decline throughout childhood; while in adults aged 18-70 years, the risk of asthma exacerbation increased slightly for every year of age (Johnston and Sears, 2006). Boys have higher risk for asthma exacerbation than girls, however, after 20s, women have roughly three times the risk of a severe exacerbation than men (Skobeloff et al., 1992). This difference may indicate the contributory role of hormonal influences on asthma exacerbation, although no clear mechanism was found. Except age and sex, race and ethnicity contribute to the risk of asthma exacerbation, with a greater risk of emergency department visits in African American with asthma (Erickson et al., 2007). An important character of asthma exacerbation is its seasonal cycles of eruption. According to the data from 2001 to 2005 in Ontario, the hospitalization and emergency department visit associated with asthma exacerbation were obviously increased in autumn and weeks around New Year (Figure 1.1). Among school-age children, the   9   hospitalization was rapidly increased in mid-August and reached the peak around half a month after school return. The autumn peak of older aged groups was some one week later than that of school children, which suggests that school children may be the primary vectors of agents causing asthma exacerbation. Another outbreak of asthma exacerbation in December to January was obvious in adults groups, which is related less to school children (Johnston and Sears, 2006; Sears, 2008). The seasonal pattern of asthma exacerbations requiring hospital admission has been found in many northern hemisphere countries, also in some southern countries at corresponding seasons (Lister et al., 2001). 1.2.2 Factors inducing asthma exacerbation Many factors can induce asthma exacerbation, including allergens, pollution, bacteria infection and respiratory viral infection. Among them, respiratory viral infection is the most frequent trigger. Since the early 1970s, respiratory viral infections have been confirmed to be associated with asthma exacerbation in adults and children (Lambert and Stern, 1972). Viruses were found in approximately 80% of wheezing episodes in school-aged children, and among the respiratory tract viruses detected in those circumstances, rhinoviruses were the most frequently identified (Johnston et al., 1995). In addition to rhinovirus, other respiratory tract viruses including   10      Figure 1.1 The annual cycle of asthma exacerbation in children 2 to 15 years, adults 16 to 49 years, and adults older. Adapted from (Sears 2008)   11     influenza virus, respiratory syncytial virus (RSV), parainfluenza virus, adenovirus and bocavirus have also been detected in asthma exacerbation patients (Jackson et al., 2011). Influenza is a common infection during the winter months. Studies of H1N1 influenza A found that hospitalization and mortality in infected patients were associated with the diagnosis of asthma (Plessa et al., 2010). Respiratory syncytial virus (RSV) is the major pathogen causing bronchiolitis in infants, which usually occur between December and February. RSV infection is less frequent in older children and young adults, but related to 7% asthma hospitalization in those above 65 years old (Falsey, 2005). Study about RSV infection and family history of asthma suggested that RSV infections might contribute more on asthma predisposition than asthma exacerbation (Sigurs et al., 2000). The annual cycle of asthma outbreak and respiratory viral infections also indicates the association between them. Rhinovirus infections occur throughout the year, and they are more common in autumn and late spring. Both the rate of rhinoviral illness and rhinovirus yield markedly peak in autumn, especially September (Monto, 2002) (Figure 1.2). As mentioned above, the seasonal peaks of asthma exacerbation in children usually occur in autumn, around the weeks of school return, and followed by a peak in older adolescents and young adults a week later. The September epidemic of asthma exacerbation coincides with the autumn outbreak of rhinovirus, suggesting the central role of rhinovirus in asthma exacerbation (Johnston and Sears,   12      Figure 1.2 Variation in the frequency of rhinovirus isolation in sampled illnesses and rates of rhinoviral respiratory illness. (Adapted from(Monto, 2002).   13     2006). Family and community studies about viral respiratory infection found that the most likely introducers of viral infection to family were children in various age categories, who are at particular risk of viral respiratory infection (Monto, 2003). So the one-week late of adultsʼ asthma exacerbation could be explained by the family transmission of rhinovirus infection. The September epidemic was investigated in Canada with limiting recruitment of asthma exacerbation children, about 62% of cases were infected with respiratory viruses and rhinovirus accounted for two thirds of them (Johnston et al., 2005). Another peak of asthma hospitalization in adults occurs between December and January, the common season of RSV and influenza virus infection, supporting the linkage between respiratory viruses and asthma exacerbation (Johnston and Sears, 2006; Monto, 2003). Bacterial infection has long been shown to be involved in asthma exacerbation (Berkovich et al., 1970; Maffey et al., 2010). Mycoplasma pneumonia and Chlamydophila pneumonia are two common bacteria that are associated with asthma exacerbations (Brar et al., 2012). In a study using PCR to classify the infectious cause of asthma exacerbation, infection rates of 4.5% and 2.2% for C pneumonia and M pneumonia were reported in children hospitalized with acute asthma (Maffey et al., 2010). Viral infections were also present in many bacteria-related asthma exacerbation, which indicates the cofactor role of bacteria (Brar et al., 2012; Wark et al., 2002a).   14   Environmental allergens are important triggers for acute attack in allergic asthma. Allergens evoke an acute allergic response in sensitized individuals, causing the eosinophilic infiltration of airways via Th2-driven IgE mechanism. The inflammatory activation increases mucus production and cause airway obstruction, leading to asthma exacerbation. Many studies have shown allergen responsiveness is enhanced by exposure to other exacerbation trigger like smoking, pollution, and respiratory viral infection. The risk of hospital admission with acute asthma in adults was markedly increased with combination of sensitizing allergens and viral infection (Green et al., 2002b). In children, combination of viral infection and allergen exposure also increase the risk of asthma hospitalization (Murray et al., 2006). Though the effects are less than those of virus and aeroallergen, evidence has shown that exposure to air pollutions contributes to asthma exacerbation. Nitrogen dioxide (NO2) is both an indoor and outdoor pollutant that increases respiratory symptoms in children with asthma, and elevated personal levels of NO2 are associated with increased severity of virus-induced exacerbations (Chauhan et al., 2003; McConnell et al., 2003). Another pollutant, cigarette smoking, can induce a non-eosinophilic phenotype in asthma and increase the associated hospital admission (Thomson et al., 2004). Investigation in Scotland indicated a reduction of 18.2% per year for asthma-related hospitalization in children, after the implementation of a public smoking ban (Mackay et al., 2010).   15   1.2.2.1 Rhinovirus Rhinovirus was first discovered in 1956 and determined to be the major cause of common cold. Common cold is a viral infectious disease of upper respiratory tract, with the symptoms including nasal stuffiness and sneezing, sometimes sore throat and cough (Heikkinen and Järvinen, 2003). By using RT-PCR and culture, rhinovirus was detected in approximately 30% to 80% of the common cold cases, and was the most frequent isolated virus in several community studies of respiratory viral infections (Kesson, 2007). Rhinovirus-associated respiratory infections occur in all populations and all ages throughout the year. The infections exhibit in a seasonal pattern and peak in autumn and late spring (Bartlett and Johnston, 2008). The onset of infection symptoms occurs after a 1-2 days incubation period and peak symptoms appear at 2-4 days, usually last for 5-7 days in total (Heikkinen and Järvinen, 2003). According to several experimental studies of natural transmission of rhinovirus infection, direct contact and aerosol inhalation are two possible routes for infection spread via virus-contaminated respiratory secretions (Bartlett and Johnston, 2008). Early epidemiologic studies based on family and community showed that infection risk decrease with age, with the highest rates 12 times per year in children while 2 to 5 times in adults (Badger et al., 1953; Monto et al., 1987). Belonging to the family Picornaviridiea, rhinoviruses are small   16   (approximately 30 nm) non-enveloped single-stranded positive sense RNA viruses (Bartlett and Johnston, 2008). The particles are coated with a protein capsid consists of 60 copies of protomers. Each protomer comprises of four viral capsid proteins (VP1 to VP4) and arranges around a fivefold axis to form a pentamer (Kennedy et al., 2012) (Figure 1.3). Twelve pentamers form the icosahedral capsid shell that coat the viral genomic RNA. The rhinovirus genome contains about 7400 nucleotides, forming a single open reading frame with untranslated regions (UTRs) at both termini. The 5ʼ terminus UTR is linked to a virus–encoded protein VPg which initiates (Rollinger and Schmidtke, 2011). The difference of the amino acid in one or more capsid proteins leads to different antigenic properties; based on antibody neutralization, the human rhinovirus genus were classified into different serotypes. Currently, there are more than 100 known serotypes around the world (Rollinger and Schmidtke, 2011). According to their receptor tropism, rhinovirus can also be classified into two groups: about 90% of rhinoviruses (major group) bind to the intercellular adhesion molecule 1 (ICAM-1) while the remains (minor group) utilize the low-density lipoprotein (LDL) receptor family (Greve et al., 1989; Hofer et al., 1994). The inability to bind nonhuman ICAM-1 for major group rhinoviruses leads to rhinovirusʼs high degree of species specificity, which is a great obstacle for animal model establishment (Bartlett et al., 2008).   17   Figure 1.3 The structure of rhinovirus. (a) The icosahedral formatted protein shell of rhinovirus. (b) The location of VP 1-4 in a protomer unit. The canyon is the likely point of ICAM-1 contact. Adapted from(Kennedy et al., 2012).   18   1.2.3 Pathophysiology of asthma exacerbation Respiratory viruses, particular rhinovirus, are important triggers for asthma exacerbations. However, the details of the pathophysiology mechanism are still unknown. Some viruses such as influenza A virus infection can cause cytotoxic activation of T lymphocytes and lead to apoptosis in bronchiolar epithelial cells (Lowy, 2003). These responses contribute to airway structural change and may aggravate asthma symptoms. However, as the key trigger in asthma exacerbation, rhinovirus infections seldom induce cytopathic effect in respiratory epithelial cells (Papadopoulos et al., 2000). Thus, rhinovirus infection induced immune responses and their roles in asthma exacerbation become the focus of attention. 1.2.3.1 Epithelial cells Epithelium is the barrier between host and the outside environment. As the first cell type that contacts the inhaled environmental pathogens, epithelial cells play a crucial role in the immune responses in the airway inflammation. Through secreting and producing several families of molecules, including enzymes, permeabilizing peptides, protease inhibitors and others, epithelial cells prevent and neutralize microorganisms from entering the host (Schleimer et al., 2007). However, in asthmatics, the barrier function is abnormal due to the increased permeability and fragility of epithelia (Lambrecht and Hammad, 2012). Under the stimulation of allergens,   19   epithelial cells secrete multiple cytokines and chemokines, which directly and indirectly affect other cells such as DCs, B cells and T cells, leading to Th2 polarization and production of various inflammatory molecules (Schleimer et al., 2007). Respiratory virus infections in epithelial cells usually cause the necrosis of airway epithelium. However, little cellular damage was shown in bronchial epithelial cells exposed to rhinoviruses, the major cause in asthma exacerbation (van Kempen et al., 1999). Rhinovirus replicates in airway epithelial cells via attaching to ICAM-1 or LDL receptor. The infection itself can up-regulate the expression of ICAM-1 to amplify the binding and infection (Grünberg et al., 2000). Infection in bronchial epithelial cells also induces the production of a wide range of cytokines and chemokines, including IL-6, IL-8, RANTES, eotaxins and CXCL10 (Kelly and Busse, 2008). Those pro-inflammatory mediators are able to induce infiltration of leukocytes and secretion of other cytokines and chemokines, which contribute to aggravate asthma symptoms (Figure 1.4). 1.2.3.2 Eosinophils Eosinophils are granulocytes first described in 1879 with the capacity to be stained by acid aniline dyes. Eosinophil progenitors develop from pluripotent hematopoietic stem cells that express CD34, IL-5 receptor and CC-chemokine receptor 3 (CCR3). Under the stimulation of macrophage-colony stimulation factors (GM-CSF), IL-3   20   [...]... age and sex, race and ethnicity contribute to the risk of asthma exacerbation, with a greater risk of emergency department visits in African American with asthma (Erickson et al., 2007) An important character of asthma exacerbation is its seasonal cycles of eruption According to the data from 2001 to 2005 in Ontario, the hospitalization and emergency department visit associated with asthma exacerbation. .. with asthma exacerbations (Brar et al., 2012) In a study using PCR to classify the infectious cause of asthma exacerbation, infection rates of 4.5% and 2.2% for C pneumonia and M pneumonia were reported in children hospitalized with acute asthma (Maffey et al., 2010) Viral infections were also present in many bacteria-related asthma exacerbation, which indicates the cofactor role of bacteria (Brar et al.,... nearly one third of the total $14.7 billion in US annual asthma- related health care expenditures (American Lung Association, 2012) The risks of asthma exacerbations differ within age and also between the sexes According to data from Canada and New Zealand from 1995 to 1999, the rates of hospital admission for asthma decline throughout childhood; while in adults aged 18-70 years, the risk of asthma exacerbation. .. 1.2 Asthma exacerbation 1.2.1 Epidemiology of asthma exacerbation The natural history of asthma consists of relatively stable periods and outbreak periods with significant exacerbation of symptoms While there is no clear consensus definition, the term exacerbation is usually associated with severe asthma, as an episode of acute deterioration According to a task force conducted by American Thoracic Society/... (Figure 1.2) As mentioned above, the seasonal peaks of asthma exacerbation in children usually occur in autumn, around the weeks of school return, and followed by a peak in older adolescents and young adults a week later The September epidemic of asthma exacerbation coincides with the autumn outbreak of rhinovirus, suggesting the central role of rhinovirus in asthma exacerbation (Johnston and Sears,  ... Wark et al., 200 2a)   14   Environmental allergens are important triggers for acute attack in allergic asthma Allergens evoke an acute allergic response in sensitized individuals, causing the eosinophilic infiltration of airways via Th2-driven IgE mechanism The inflammatory activation increases mucus production and cause airway obstruction, leading to asthma exacerbation Many studies have shown allergen... one-week late of adultsʼ asthma exacerbation could be explained by the family transmission of rhinovirus infection The September epidemic was investigated in Canada with limiting recruitment of asthma exacerbation children, about 62% of cases were infected with respiratory viruses and rhinovirus accounted for two thirds of them (Johnston et al., 2005) Another peak of asthma hospitalization in adults occurs... December and January, the common season of RSV and influenza virus infection, supporting the linkage between respiratory viruses and asthma exacerbation (Johnston and Sears, 2006; Monto, 2003) Bacterial infection has long been shown to be involved in asthma exacerbation (Berkovich et al., 1970; Maffey et al., 2010) Mycoplasma pneumonia and Chlamydophila pneumonia are two common bacteria that are associated... for asthma exacerbations However, the details of the pathophysiology mechanism are still unknown Some viruses such as influenza A virus infection can cause cytotoxic activation of T lymphocytes and lead to apoptosis in bronchiolar epithelial cells (Lowy, 2003) These responses contribute to airway structural change and may aggravate asthma symptoms However, as the key trigger in asthma exacerbation, rhinovirus. .. Eosinophils are granulocytes that participate in variety of inflammatory processes, especially asthma They are often present in the airways of allergic asthmatics and correlate with disease severity (Wardlaw et al., 2000) In response to inflammatory stimuli of IL-5, IL13 or other inflammatory chemokines such as eotaxins, eosinophils are recruited into the inflammatory site, and release pro-inflammatory mediators ... of asthma   1.1.3   Pathophysiology of asthma   1.2   Asthma exacerbation   1.2.1   Epidemiology of asthma exacerbation   1.2.2   Factors inducing asthma exacerbation. .. and aggravate triggers and effectors (Lommatzsch, 2012) 1.2 Asthma exacerbation 1.2.1 Epidemiology of asthma exacerbation The natural history of asthma consists of relatively stable periods and... with a greater risk of emergency department visits in African American with asthma (Erickson et al., 2007) An important character of asthma exacerbation is its seasonal cycles of eruption According