A mouse model of rhinovirus induced asthma exacerbation 2

Figure 1.4 The immunological role of epithelial cells in virus-indcued asthma exacerbations. Adapted from (Message and Johnston, 2001).   21     and IL-5, eosinophil progenitors are developed in bone marrow and then released into circulation under a phenotypically mature state (Gould and Sutton, 2008). With a short half-life of 18 hours, eosinophils do not stay long in peripheral blood before residing in thymus or gastrointestinal tract under homeostatic conditions. In healthy humans, the number of mature eosinophils found in the peripheral blood is less than 400 per mm3 (Rosenberg et al., 2013). However, several diseases, including asthma, can induce eosinophilia and recruit eosinophils into inflammatory tissues. In asthma patients, eosinophils leave the bone marrow, transport to the site of inflammation and function under the stimulation of a wide range of pro-inflammation cytokines, chemokines and adhesion molecules. The most important cytokines are Th2 cytokines IL-4, IL-5 and IL-13. IL-5 has a central role in all aspects of eosinophil development, activation and survival via the interaction with IL-5 receptor subunit-α expressed on eosinophil (Takatsu and Nakajima, 2008). IL-4 and IL-13 activate the endothelial and epithelial cells to produce pro-inflammatory chemokines, which attract and activate eosinophils. Eotaxin-1/CC-chemokine ligand 11 (CCL11) is a specific chemokine that have chemoattractive effect on eosinophils. It functions through CC chemokine receptor (CCR3) on eosinophils, promoting eosinophilia cooperatively with IL-5 or via IL-5-independent mechanisms (Mould et al., 2000). ICAM-1 and vascular cell adhesion molecule-1 (VACM-1) are adhesion molecules that involved in   22   transmigration of eosinophils from the vascular bed into the inflamed tissue. They are expressed by endothelium under the influence of IL-4 and IL-13 (Aceves and Broide, 2008). Eosinophils are generally considered as terminal effector cells in allergic asthma. Part of eosinophilsʼ effects on inflammatory response functions through cellular degranulation. Eosinophil piecemeal degranulation conducts under the effect of several cytokines and chemokines, including interferon-γ (IFN-γ) and eotaxins (Rosenberg et al., 2013). The granule contents MBP, EDN, ECP and EPO are associated with the airway damage and AHR in asthma. Eosinophilderived fibrogenic and growth factors can amplify airway remodeling and induce mucus secretion, causing asthma exacerbation to some extent (Kay, 2005). Eosinophils also release a wide range of proinflammatory cytokines and chemokines interacting with other leukocytes, suggesting an additional initiating role in asthma (Figure 1.5). Eosinophilia was found involved in asthma exacerbations associated with respiratory virus infections. Prolonged nasal eosinophilia was detected in allergic patients after common cold with increase number of eosinophils and other leukocytes (van Benten et al., 2001). Serum concentration of ECP was elevated in rhinovirusinduced wheezing (Kato et al., 2011). However, the interaction between viruses and eosinophils needs more exploration. One   23   Figure 1.5 Eosinophils modulate the function of other leukocytes. Adapted from (Rosenberg et al., 2013).   24   possible function of eosinophils and their secretory mediators is promoting the antiviral host defense. Researchers also found that replication and virus loads of RSV were decreased under the effect of eosinophils (Rosenberg et al., 2009). EDN and ECP have RNAse activity and toxicity to single-strand RNA pathogens (Rosenberg and Domachowske, 2001). EPO catalyzes the production of oxidant products that are toxic to microorganisms (Gould and Sutton, 2008). Besides the granule contents, the immune mediators released by eosinophils, such as IL-6, may also be involved in the antiviral response to respiratory virus infections (Dyer et al., 2009). 1.2.3.3 Neutrophils Neutrophils are polymorphonuclear leukocytes involved in innate immune response to acute inflammation. They are usually the first leukocytes recruited to inflammatory site and related to immune defense to microorganisms such as bacteria and viruses. Neutrophils are generated from myeloid precursors in the bone marrow by the action of granulocyte colony stimulating factor (G-CSF) and through several maturation stages (Kolaczkowska and Kubes, 2013). Neutrophils have a short longevity with a circulating half-life of 6-8 hours and a lifespan of 5 days in humans (Summers et al., 2010). Multiple inflammatory mediators, including IL-8, TNF-α, G-CSF, leukotriene B4, and also adhesion molecules ICAM-1 and VCAM-1, regulate and contribute to the recruitment of neutrophils (Summers et   25   al., 2010). Neutrophils are common leukocytes that eliminate pathogens in respiratory virus infections. In experimental rhinovirus infection, peripheral blood neutrophils increased without changes in total white blood cell counts, corresponding to the increase of G-CSF and IL-8 concentrations (Gern et al., 2000; Kelly and Busse, 2008). In severe asthma exacerbation, lung neutrophil level was increased to a greater degree than eosinophils in some fatal asthma attacks (Wenzel, 2001). In asthmatic patients suffering from viral infection, increased sputum neutrophils and cell lysis were detected and correlated with clinical severity (Wark et al., 2002b). Several components from neutrophil granules, including metalloproteinases, elastase, lactoferrin and myeloperoxidase, were reported to be upregulated in severe asthma or asthma exacerbation, contributing to chronic inflammation and airway remodeling (Monteseirín, 2009). Neutrophil elastase has been shown to up-regulated MUC5 proteins expression, which increase mucus production and lead to airway obstruction in asthma exacerbation (Zhou et al., 2013). Neutrophil proteases activated eosinophils in vitro to produce superoxide and proinflammatory cytokines (Hiraguchi et al., 2008). Neutrophils also contribute the recruitment of DCs and T cells to inflammatory site, which further aggravate airway inflammation in severe asthma (Ganz, 2003; Yang et al., 2000).   26   1.2.3.4 Macrophages Macrophages are important cells that are involved in most aspects of both innate and adaptive immune responses, including recognition and elimination of pathogens, releasing cytokines and chemokines, and antigen presentation to other leukocytes like T cells and DCs (Balhara and Gounni, 2012; Gordon, 2003). They are typically developed and differentiated from monocytes by series of changes, triggered by a variety of stimuli (Gordon and Taylor, 2005; Martinez et al., 2008). According to the cytokines and chemokines they express and the immune responses they invoke, macrophages can be divided into two subtypes: the classically activated (M1) macrophage and the alternatively activated (M2) macrophage. M1 macrophages are induced by Th1 stimuli such as IFN-γ and LPS, and typically express CXC motif chemokine 9 and 10 (CXCL9, CXCL10). They participate in Th1 responses to kill intracellular pathogens in a cytokine-dependent manner. M2 macrophages are stimulated by Th2 cytokines like IL-4 and IL-13, and participate more in phagocytosis of foreign pathogens and apoptotic cells (Balhara and Gounni, 2012; Biswas and Mantovani, 2010). Macrophages are the most numerous inflammatory cells in the airways. With their ability to produce both pro- and anti- inflammatory mediators, their role in asthma remains to be determined. Alveolar macrophages from sensitized mice were once transferred and induced   27   pulmonary eosinophilic inflammation in unsensitized mice. However, whether the inflammation is due to the sensitized macrophages or the effect of challenge perse remains unclear (Moon et al., 2007). Other studies have shown that macrophages could induce histamine release from basophils and mast cells and deteriorate tracheal function through reactive oxygen radicals, which modulated the contractility of airway smooth muscle (Yang et al., 2012). Rhinovirus can attach to airway macrophages despite the limited replication in these cells (Laza-Stanca et al., 2006). The interaction between rhinovirus and macrophages induces the release of multiple pro-inflammatory cytokines and chemokines including IFNγ, TNF-α, IL-8 and macrophage inflammatory protein (MIP-1α) (Kelly and Busse, 2008). TNF-α promotes T cell activation and chronic airway remodeling, and induces ICAM-1 expression to facilitate the recruitment of leukocytes and binding of rhinoviruses (Krunkosky et al., 2000). IL-8 is a typical chemoattractant for neutrophils, which usually present in severe asthma. Except the increase of pro-inflammatory cytokines, rhinovirus exposure also induces impaired antibacterial immune response in alveolar macrophages by decrease the phagocytic ability (Oliver et al., 2008). The role of macrophages in virus-induced asthma exacerbation is still unclear. However, studies showed that the pro-inflammatory cytokines and chemokines released from macrophages under virus   28   stimulation may contribute to the immune response in asthma exacerbation. 1.2.3.5 Mucus hyper-secretion The respiratory mucus and the mucociliary clearance are essential for protection of the airways. Normal mucus consists of 97% water and 3% solids, with mucins account for nearly 30% of the solids (Thornton et al., 2008). Mucins, the large glycoproteins with extensive O-glycan attachment domains, play a crucial role in determining the physical properties of mucus (Thornton and Sheehan 2004; Fahy and Dickey 2010). There are 17 genes encoding mucins in human genome. These mucins can segregate into three families: secreted gel-forming mucins, membrane-associated mucins and secreted non-gel-forming mucin (Voynow and Rubin, 2009). Of the secreted gel-forming mucins, only MUC5AC and MUC5B are significantly produced in intrapulmonary airways (Evans et al., 2009; Thornton et al., 2008). In healthy persons, MUC5AC is mainly produced in goblet cells, while MUC5B is expressed in the mucous cells of the submucosal gland (Voynow and Rubin, 2009). Mucus hyper-secretion is common in asthmatic patients and is one of the major symptoms of asthma exacerbation. Airway obstruction by a plugging of mucus and cells contributes to the morbidity and mortality of asthma (Kuyper et al., 2003). Pathologic mucus has high viscosity and elasticity, leading to the poor mucus clearance in the   29   airways. In asthma, airway remodeling is characterized by increased surface epithelial mucin production and bronchial microvessels (Fahy and Dickey 2010). Many common mediators in asthma, including neutrophil elastase, ECP, mast cell chymase and leukotrienes, can trigger the hyper-secretion of mucus (Thornton and Sheehan, 2004). Th2 cytokine IL-13 is also involved in mucus plugging by regulate mucus cell metaplasia (Hays and Fahy, 2003). Mucus overproduction also presents in common cold, suggesting a role of rhinovirus in this process. Experimental infection with rhinovirus-16 in undifferentiated bronchial epithelial cells induced secretion of MUC5AC, the major mucin components of mucus (He et al. 2004). Clinical study revealed that rhinovirus infection in volunteers also induced the mucin production, following the increase of IL-8 in nasal lavages (Yuta et al., 1998). The expressions of MUC5AC and MUC5B depend on the activation of gene transcription factors under the regulation of several signaling pathways (Voynow and Rubin, 2009). Signal transducer and activator of transcription (STAT) 6 signaling is crucial for IL-13regulated MUC5AC induction and AHR in asthma (Evans et al., 2009). Nuclear factor-κB (NF-κB) and epidermal growth factor receptor (EGFR) are involved in rhinovirus-induced MUC5AC in human experimental model (Hewson et al., 2010). However, in animal model, NF-κB contributes less in MUC5AC transcription (Pantano et al.,   30   2008). 1.2.3.6 Airway Hyper-responsiveness (AHR) As a clinical symptom in asthma, the degree of AHR is usually in proportion to the severity of asthma. Provokers such as viral infection and allergen exposure can enhance the underlying AHR in asthmatic patients (Cockcroft and Davis, 2006). It is suggested that AHR has two components: the variable component associated with airway inflammation and the persistent one related to structural changes of airways. The former one is reflected mainly by changes that response to indirect stimuli, whereas the latter is reflected by the airway responsiveness under direct stimuli (Brannan and Lougheed, 2012; Cockcroft and Davis, 2006). Airway remodeling is a feature related to several respiratory inflammation diseases, usually defined as the changes in structural components during growth or in response to injury and inflammation. The structural changes including epithelial thickening, goblet cell hyperplasia, and airway smooth muscle hypertrophy and hyperplasia, contribute to the overall thickening of the airway wall in asthma (Berend et al., 2008). Airway inflammation also plays a role in AHR in asthma. The correlation between peripheral blood eosinophilia and AHR was demonstrated, along with the effect of IL-5 and IL-13 (Chen et al.,   31   2009; Kuperman et al., 2002; Schwartz et al., 2012). The presence of mast cells in airway smooth muscle is also associated with AHR in asthma (Brightling et al., 2002; Schultz et al., 2010). However, the relationship between inflammatory cell infiltration and AHR were also brought to question by some other studies (Berend et al., 2008). IL-5 blocking antibody reduced sputum eosinophils but failed to suppress AHR in asthmatics (Leckie et al., 2000). Dissociation between inflammatory cells and AHR also presented in patients with mild asthma (Crimi et al., 1998). Rhinovirus infection also induces AHR. Experimental rhinovirus16 infection in volunteers with mild asthma induced AHR to histamine. The aggravation of symptoms was accompanied with an increased level of IL-8, which indicates rhinovirus may function through inflammatory mediators (Grünberg et al., 1997). With the regulation of IL-13, neonatal rhinovirus infection induced AHR in mice, further supporting the role of inflammatory mediators in virus-associated AHR (Schneider et al., 2012). 1.3 Animal models for asthma exacerbation Asthma exacerbations, especially virus-induced ones, have brought heavy burden to the society. Inhaled corticosteroid (ICS) therapy has long been used in asthma control and was proved to be the most efficient anti-inflammatory treatment (Chung et al., 2009). However, corticosteroid insensitivity happens in severe asthma   32   patients, who count for approximately 5-10% of the asthmatic population (Robinson et al., 2003). The effect of ICS treatment on asthma exacerbation control showed no clinically significant benefit in children (Doull et al., 1997). Meanwhile, corticosteroid therapy can usually induce side effects including oral candidiasis, hoarseness and dysphonia (Chung et al., 2009). Considering the unclear mechanism of virus-associated asthma exacerbation, it is necessary to establish experimental models for further investigation. In vitro model of rhinovirus infection was established in BEAS2B cells, a transformed continuous cell line, and increased expression of IL-8, RANTES and eotaxins were detected in this cellular model (Papadopoulos et al., 2001). Other epithelial cell lines, such as A549, 16HBE and HEp-2 were also used (Guan et al., 2008). However, the systemic immune responses in asthma and rhinovirus infection are complex and involve the correlation of multiple inflammatory cells. Thus, it is necessary to establish in vivo models for further investigation. Allergen sensitization and respiratory virus infection are two major risk factors for asthma exacerbation and subsequent asthma development. Researchers now pay attention to the relationship between these two factors and their combination effect on acute asthma attack and persistent asthma. Several birth cohort studies have shown that atopic children have higher risks to develop prolonged   33   asthma in school age with respiratory viral infections in infancy or during early years of life (Kusel et al., 2007; Jackson et al., 2008). Compared with non-sensitized children, children sensitized to aeroallergens developed greater risk of severe viral wheezing, especially the rhinovirus-induced ones (Jackson et al., 2012). These results indicate the interaction of allergen sensitization and respiratory virus infections in asthma development and exacerbation, which support the use of allergen sensitization combined with virus infection to establish asthma exacerbation model. Traditionally, in vivo asthma models were established in animals sensitized and challenged by allergens. Many species of animals, such as mice, rats, guinea pigs, monkeys, were used to establish asthma models (Zosky and Sly, 2007). Among them, mouse models of asthma were the most commonly used due to their resemblance features of human asthma. With the sensitization and challenge by allergens, mouse asthma models are easy to develop asthma symptoms including eosinophilic infiltration, mucus hyper-secretion and AHR. Female Balb/c mice were commonly used as the higher prerulane of asthma exacerbation in women and a good susceptibility to inflammation in female mice (Melgert et al., 2005). The commonly used allergen for asthma mouse model is ovalbumin (OVA), which induces a classic Th2 phenotype immune response and is not normally exposed to mice (Shore and Shapiro, 2009; Zosky and Sly, 2007). Aluminium hydroxide is frequently used with OVA as an adjuvant to promote the   34   Th2 responses of the immune system. Progress has been made in animal model of asthma exacerbation by the combination of allergen sensitization and virus infections. RSV infections prior or after allergen sensitization were conducted in murine models, and increased AHR and airway inflammation were demonstrated when infection were performed challenge exposure, after allergen sensitization (Peebles et al., 2001a; Kondo et al., 2004; You et al., 2006; Aeffner and Davis, 2012). Sendai and RSV infection were also established in guinea pig models and induced clinically relevant symptoms (Riedel et al., 1996; Dakhama et al., 1999). Efforts were also made to establish asthma exacerbation models via rhinovirus infection, the major cause of human asthma exacerbation. However, more than 90% of rhinovirus serotypes use human ICAM-1 to enter cells and cannot bind to mouse ICAM-1. Due to the host cell tropism of rhinovirus, it is difficult to establish a rhinovirus-induced asthma exacerbation model in mouse. Non-human animals susceptible to rhinovirus are chimpanzees and gibbons. Despite the fact that those models were used in some experiments, It is not a common choice due to their high cost and impracticability (Huguenel et al., 1997). Attempts were made to transfect mouse cells with viral RNA. By alternate passage of major group serotype rhinovirus 16 between   35   human HeLa cells and ICAM-1 expressing mouse L cells, a variant 16/L virus was isolated and able to produce high levels of infection in mouse cells (Harris and Racaniello, 2003). Another study suggests that changes in viral polypeptides permit the viral growth in mouse cells (Harris and Racaniello, 2005). Investigations were also made to establish a transgenic mouse expressing a mouse-human ICAM-1 chimera. Domains 1 and 2 of mouse ICAM-1 were replaced with the equivalent human domains, and rhinovirus 16 successfully infected and replicated in mouse cells expressing the chimeric ICAM-1 (Tuthill et al., 2003). Depending on this cellular model, great achievement in major-group rhinovirus infection of transgenic mice was obtained. Bartlett et al. (2008) have successfully infected the transgenic mice expressing a mouse-human ICAM-1 receptor with rhinovirus-16, resulting in the outcomes similar to those in minor group rhinovirus infection model. The similarity of immune responses to the major and minor group of rhinovirus infections suggests the possibility to investigate the rhinovirus-induced asthma exacerbation with minor group serotypes. The minor group serotype rhinovirus 1B was used in several studies to investigate the rhinovirus-related respiratory diseases (Newcomb et al., 2008; Nagarkar et al., 2009). However, few models of rhinovirus-induced asthma exacerbation were reported, except the work of Bartlettʼs group. In their acute inflammation model, mice were   36   sensitized with OVA and rhinovirus infection was preformed during allergen challenge. Increased eosinophilic infiltration and airway hyperresponsiveness were observed in mice challenged by allergen and rhinovirus. Similar pathologic symptoms were observed in experimental infections in human, with increased Th2 immune response in asthmatics comparing with normal subjects (Fraenkel et al., 1995; Message et al., 2008). Due to the limitations of animal models, such as anatomic and physiologic differences by species, and the lack of chronicity in inflammation, it is hard to completely mimic the symptoms and immune responses in human asthma exacerbations. Despite the shortcoming, the investigation on animal models has contributed much to the exploration of asthma exacerbation, enhancing our understanding of this disease.   37   Chapter 2 Rational   38   Asthma affects nearly 300 million people of all ages and all ethnic background (Masoli et al., 2004). The exacerbations of its symptoms usually cause the mortality and bring heavy social and economic burdens. Respiratory virus infections, particular rhinovirus infections, were associated with asthma exacerbation as a crucial trigger. However, the mechanism of rhinovirus associated asthma exacerbation is still unclear and needs more investigation. Thus, animal models including mouse models would contribute an important role to the investigation. In this thesis we established a mouse model of rhinovirusinduced asthma exacerbation. Due to the host cell tropism of rhinovirus, it is difficult to establish a mouse model with the major group of rhinovirus. Infection of rhinovirus 1B in transgenic mice induced similar immune responses as that to major group rhinovirus (Bartlett et al., 2008). Thus, with a modification on allergen challenge, we treat mice with combination of allergen challenge and infection of minor group Rhinovirus 1B to imitate the virus-mediated asthma exacerbation. The time courses of inflammation, mucus production, and inflammatory genes expression, along with AHR were examined to further investigating the effect of viruses on allergic asthma exacerbation.   39   Chapter 3 Materials and methods   40   3.1 Materials Eagleʼs minimum essential medium (EMEM) (ATCC, USA), fetal bovine serum (FBS) (Hyclone Laboratories, South Logan, Utah), trypsin, polyethylene glycol (PEG6000) (Sigma-Aldrich, USA), cell culture tested sodium chloride (NaCl) (Sigma); chicken ovalbumin (OVA) (Sigma), phosphate buffered saline (PBS), aluminium hydroxide hydrate (Al(OH)3) (Sigma), saline; mouse anesthetic mixture (ketamine: medetomidine: H2O=3:4:33) obtained from Animal Holding Unit (AHU) (NUS, Singapore), bovine serum albumin (BSA) (Sigma, Singapore), Roswell Park Memorial Institute (RPMI) -1640 medium (Hyclone Laboratories, South Logan, Utah), ammonium chloride (NH4Cl) (BDH Laboratory Supplies, Poole, England), trypan blue (Invitrogen, Carlsbad, CA, USA); Liu A and Liu B staining dye, histological mounting medium (Histomount) (National Diagnostics, CA, USA); Xylene, paraffin wax, eosin Y, Harris hematoxylin solution, Mayerʼs hematoxylin solution, periodic acid solution, Schiffʼs reagent, 10% neutral buffered formalin, absolute ethanol, histoclear (National Diagnostics, CA, USA); RNAlater (Ambion, Austin, Texas), Trizol (Ambion, Life Technologies, USA), chloroform, isopropanol (Merck, Darmstadt, Germany); avian myeloblastosis virus (AMV) reverse transcriptase, dNTP mix, oligo(dT)15 primer and ribonuclease inhibitor (RNasin) (Promega, Madison, USA), diethylpyrocarbonate (DEPC)treated water, sodium acetate (CH3COONa) (1st BASE, Singapore),   41   Maxima SYBR Green/ROX qPCR Master Mix (2X) (Thermo Scientific, USA); Mouse IgE ELISA Set (BD OptEIA), Tween-20, 10×PBS, Substrate solution A and B (Bio-Rad), 2 N H2SO4.; methacholine (Sigma-Aldrich). 3.2 Virus culture and purification HeLa Ohio cells (ATCC, USA) were cultured in EMEM with 10% FBS in 5% CO2 incubator at 37℃ to reach the 70-80% confluence. Rhinovirus 1B was seeded in HeLa Ohio cells with 2% FBS EMEM. The infected cells were cultured in 5% CO2 incubator at 33℃ for 16 – 24 h until cytopathic effect was observed. The infected cells with supernatant were harvested by using cell scrapers. The mixture was centrifuged at 1200 rpm 4℃ for 10 min to collect the cells and remove the supernatant. The cell pellet was washed with 1×PBS once and resuspended in PBS after centrifugation. The cell suspension was frozen and thawed twice to break down the cell membrane and release the virus. Afterwards, cell debris was removed by 4500 rpm centrifugation, and the virus in the supernatant was precipitated with 0.5 M NaCl with 7% PEG 6000 on ice for 1 h. After precipitation, virus was centrifugated at 4500 rpm for 60 min and then dissolved in 1×PBS. Virus concentration was conducted by using Amicon Uitra-15 centrifugal filtration devices (Millipore, USA). Concentrated virus solution was aliquoted and stored in -80℃ (Bartlett et al., 2008).   42   3.3 Virus titration HeLa Ohio cells were seeded in a flat bottom 96 well cell culture microplate (Corning, USA) overnight to reach 80% above confluence. The medium was removed and 1×PBS was added to wash the cells by a multi-channel micropipette. Virus stock was 10 serial diluted with 2% FBS EMEM medium and each dilution was aliquoted to infect 8 wells of cells. The plate was incubated in 5% CO2 incubator at 33℃ for 5 days until the cytopathic effect (CPE) (cell rounding and detachment from the monolayer, Figure 3.1) was observed under microscope. The infection situation of each well was recorded to calculate the virus titer. TCID50 (50% tissue culture infectivity dose) method was used for viral titer assessment (Reed and Muench, 1938). The calculation was conducted through the “Reed & Muench Calculator” (Lindenbach, 2004).     Figure63.1 Cytopathic effect in Rhinovirus 14 infected HeLa cells. (A) Normal HeLa cells. (B) CPE in HeLa cells infected with Rhinovirus 14. Adapt from (Gaudernak et al., 2002)   43   3.4 Mouse model of asthma exacerbation The six-week-old female BALB/c mice for this project were ordered from Animal Holding Unite (AHU) and housed in plastic cages (maximum 5 mice per cage) in the Animal Vivarium of Centre for Life Science in National University of Singapore. The breeding room in Animal Vivarium is regulated by automatic timers to provide 12 h of light and 12 h of dark cycles. The temperature in the animal room ranges from 18 to 26℃ with an average of 22℃. Maintenance diets contain 4-5% fat and 14% protein. The cage bedding was changed every two days. Mice were sensitized with 50 μg OVA and 2 mg Al(OH)3 in 0.2 ml saline intra-peritoneally on day 0, and challenged on day 9, 10, 11 with either intranasal 25 μg OVA in 30 μl PBS or 30 μl PBS only. During the third challenge on day 11, mice were infected with 5×106 TCID50 Rhnivirus-1B in 25 μl PBS or same volume PBS only (Bartlett et al., 2008). Animal experiments were performed according to the Institutional Guidelines for Animal Care and Use Committee of National University of Singapore. 3.5 Bronchoalveolar lavage (BAL) fluid collection BAL fluid was collected at different time points (day 1, 2, 3, 5, 7) after the last challenge. Mice were anaesthetized with 0.35 ml mouse anaesthetic mixture (ingredients show in 3.1) by intra-peritoneally injection. Tracheotomy was carried out and a blunt needle (20G) was   44   inserted into mouse trachea. The Lungs was instilled and washed with 3 syringes of 0.5 ml ice-cold PBS (Cheng et al., 2011). 3.6 Total and differential BAL fluid cell counts BAL fluid was centrifuged at 3000 rpm for 5 min at 4 ℃ . Supernatant was collected and stored in -80℃ for further experiment. The remaining pellet was suspended and washed with 200 μl 0.875% NH4Cl at room temperature for 5 min. The cell suspension was centrifuged at 3000 rpm for 5 min at 4℃ and the supernatant was discarded. The cell pellet was re-suspended with 200 μl of RPMI media with 1% BSA. The total number of viable cells was counted by using a hemocytometer under microscope (10 μl cell suspension with 10 μl trypan blue, magnification ×100). After that, the cell suspension was diluted to 106 cells/ml and a 200 μl aliquot was cytospined to a slide in a Shandon cytospin 3 (Thermo Electro Corporation, USA), with a centrifugation at 600 rpm for 10 min at room temperature. The BAL fluid cells were stained with a modified Wright Staining. The slide was first stained with 0.8 ml Liu A for 30 sec and then 1.6 ml Liu B for 90 sec. Differential cell count was conducted with a minimum of 500 leukocytes under a microscope (magnification ×1000). Four different types of leukocytes (macrophage, eosinophil, lymphocyte and neutrophil) were identified and their percentages were counted (Cheng et al., 2011).   45   3.7 Histological examination Lungs were collected from mice at different time points (day 1, 3, 7 after last challenge), fixed in 10% neutral buffered formalin solution for at least two days and processed in a tissue processor (Leica Microsysterms, Wetzler, Germany). Dehydration of the lung samples was conducted in a serial concentration of ethanol (70% - 80% - 90% 100%, 30 min each and 2 h for 100%). The samples were then placed in xylene 3.5 h for 3 times. Samples were infiltrated in hot molten paraffin wax for another 3 h. Tissues were then embedded in paraffin wax, fixed and sectioned into 5 μm pieces using a microtome (Leica Microsysterms, Wetzler, Germany). The sectioned tissues were then placed and dried on glass slides. Harris Hematoxylin and Eosin (H&E) staining was performed to measure the severity of inflammatory cell infiltration. Slides were placed in histoclear for 10 min and rehydrated in a serious concentration of ethanol (100% - 100% - 90% - 70% - distill water) and each for 2 min. The sections were then stained with Harris hematoxylin for 5 min, washed in the distilled water, and differentiated in 0.1% acid alcohol solution for 30 sec. After washed with tap water for 5 min, the sections were stained with Eosin for 30 sec and washed with distilled water. Afterwards, the sections were dehydrated in a series of ethanol solutions (70%-90%-100%-100%) each for 30 sec and immersed in histoclear for 10 min. Evaluation of inflammation around peribronchial   46   and perivascular areas was semi-quantitatively performed in a blinded manner. Inflammatory scale (0 - 4) was assigned as follows: 0: no inflammatory cells; 1: occasional cuffing with few inflammatory cells; 2: most bronchi or vessels surrounded by a thin layer of inflammatory cells; 3: a thick layer (2 - 4 cells layer deep) of inflammatory cells; 4: a thicker layer (more than 4 cells layer deep) of inflammatory cells (Myou et al., 2003). Periodic Acid - Schiff reagent staining was performed to determine the extent of mucus secretion. Slides were deparaffinized with histoclear for 10 min and rehydrated in a serious concentration of ethanol (100%-100%-90%-70%-distilled water, each for 2 min). The sections were immersed in the periodic acid for 5 min, washed with distilled water, and placed in Schiffʼs reagent for 15 min. Slides were then washed with tap water for 5 min and stained in Mayerʼs hematoxylin for 90 sec. the sections were dehydrated in a series of ethanol solutions (70% -90%-100%-100%, each for 2 min) and cleaned with histoclear for 10 min. Goblet cell hyperplasia in the airway epithelium was assessed blinded and scored based on a 5-point grading system. Mucus scores (0-4) were assigned according to the percentage of PAS-positive mucin-producing cells within the bronchi rings: 0, 0; 1, 75%. The same calculation system was adopted from the H&E staining (Grünig et al., 1998).   47   3.8 Immunoglobulin E levels in serum Blood was collected on the sacrificing day by cardiac puncture and allowed to clot for at least 2 h before centrifugation at 3000 rpm for 10 min at 4°C. Serum was extracted from the top layer of the supernatant and stored at -80°C. Serum level of total IgE and OVA-specific IgE were measured by ELISA kit, according to the manufacturerʼs instructions. Platecoating was conducted by adding 100 μl of coating liquid (diluted capture antibodies for total IgE and OVA solution for specific IgE) to each well of ELISA plates (NUNC, Demark). The ELISA plates were sealed and incubated overnight at 4 °C. After coating, the ELISA plates were washed three times with washing buffer (1xPBS with 0.05% Tween-20) and blocked with 200 μl assay diluent buffer (1xPBS with 10% heat-inactive FBS) for 1 h at room temperature. After washing off blocking buffer, 100 μl of standards and samples were loaded into plates. After 2 h incubation at room temperature, the plates were washed 5 times and incubated with biotinylated anti-mouse IgE monoclonal antibody and streptavidin-horseradish peroxidase conjugate (SAv-HRP) for 1 h. After washing 7 times, 100 μl of substrate solution for HRP was added and incubated for another 30 min in dark. To stop the reaction, each well was added 50 μl of stopping solution (2 N H2SO4). The plates were read at 450nm (reference filter 570 nm) using an automatic microplate reader   48   (Sunrise, TCAN, Austria). 3.9 Reverse transcription-polymerase chain reaction (RT-PCR) Lungs were isolated from the thoracic cavity at different time points (day 1, 2, 3, 5, 7 after the last challenge) and stored in RNAlater at -80℃. Before RNA isolation, lung tissues were removed from the RNAlater and immersed in 1 ml Trizol, and homogenized on ice using a homogenizer (Heidolph). The homogenate was centrifuged at 12000 g 4℃ for 10 min. The supernatant was collected and incubated for 5 min at room temperature. Then, the homogenized sample was mixed with 0.2 ml of chloroform, followed by vigorous shake for 15 sec and room temperature incubation for 3 min. A centrifugation was conducted at 12000 g for 15 min at 4℃, and the aqueous phase of the sample was collected. The aqueous phase was mixed with 0.5 ml isopropanol and incubated at room temperature for 10 min. After centrifuged 12000 g for 10 min at 4℃, the pellet was washed with 1 ml of 75% ethanol by vortex and centrifugation at 7500 g for 5 min at 4℃. The RNA pellet was air-dried and re-suspended in RNase-free water. Incubation in a water bath was conducted at 55-60℃ for 10 min to make the RNA completely dissolved. Ethanol precipitation was conducted to purify the RNA. 3 M CH3COONa was added into RNA to make the final concentration 0.3 M. The mixture was vortexed with 3 times volume of ethanol for 10 sec and stored at -20℃ overnight or -80℃ for 2 hours. Afterwards, the   49   mixture was centrifugated at 13000 g for 20 min at 4℃ to discard the supernatant. The pellet was vigorously washed with 200 μl of 95% ethanol and dried under vacuum with a lyophilizer and re-suspended with RNase-free water. The amount and purity of RNA was measured by the spectrophotometer (NanoDrop ND-1000, Thermo Fisher Scientific Inc, Waltham, MA, USA). Both A260/A280 (DNA/protein) and A260/A230 (DNA/organic contaminants) ratios were recorded as an indicator of purity of RNA. An acceptable level of purity for RNA extracts should be around 1.9 to 2.1 for both A260/A280 and A260/A230 ratio. cDNA was synthesized from 1 μg total RNA together with an oligo dT primer and AMV reverse transcriptase by a multiwell thermal cycler (GeneAmp PCR system 2700, Applied Biosystems, USA). Real time PCR was performed on ABI 7500 cycler in a 20 μl reaction system containing 1 μl cDNA (1μg), 10 μl 2 x SYBR master mix, 1 μl forward primer (10 μM), 1 μl reverse primer (10 μM) and 7 μl nuclease-free water. The primers were obtained from 1st base and the respective sequences are shown in Table 3.1. 3.10 Measurement of airway hyper-responsiveness (AHR) Lung resistance (RI) and dynamic compliance (Cdyn) in response to increasing concentrations of methacholine (0.5 - 8.0 mg/ml) were measured using a whole-body plethysmograph chamber (Buxco, Sharon, CT). Rl is the ratio of driving pressure to the rate of   50   [...]... Inc, Waltham, MA, USA) Both A2 60 /A2 80 (DNA/protein) and A2 60 /A2 30 (DNA/organic contaminants) ratios were recorded as an indicator of purity of RNA An acceptable level of purity for RNA extracts should be around 1.9 to 2. 1 for both A2 60 /A2 80 and A2 60 /A2 30 ratio cDNA was synthesized from 1 μg total RNA together with an oligo dT primer and AMV reverse transcriptase by a multiwell thermal cycler (GeneAmp... and Davis, 20 12) Sendai and RSV infection were also established in guinea pig models and induced clinically relevant symptoms (Riedel et al., 1996; Dakhama et al., 1999) Efforts were also made to establish asthma exacerbation models via rhinovirus infection, the major cause of human asthma exacerbation However, more than 90% of rhinovirus serotypes use human ICAM-1 to enter cells and cannot bind to mouse. .. ethnic background (Masoli et al., 20 04) The exacerbations of its symptoms usually cause the mortality and bring heavy social and economic burdens Respiratory virus infections, particular rhinovirus infections, were associated with asthma exacerbation as a crucial trigger However, the mechanism of rhinovirus associated asthma exacerbation is still unclear and needs more investigation Thus, animal models... species, and the lack of chronicity in inflammation, it is hard to completely mimic the symptoms and immune responses in human asthma exacerbations Despite the shortcoming, the investigation on animal models has contributed much to the exploration of asthma exacerbation, enhancing our understanding of this disease   37   Chapter 2 Rational   38   Asthma affects nearly 300 million people of all ages and all... Sly, 20 07) Among them, mouse models of asthma were the most commonly used due to their resemblance features of human asthma With the sensitization and challenge by allergens, mouse asthma models are easy to develop asthma symptoms including eosinophilic infiltration, mucus hyper-secretion and AHR Female Balb/c mice were commonly used as the higher prerulane of asthma exacerbation in women and a good.. .20 08) 1 .2. 3.6 Airway Hyper-responsiveness (AHR) As a clinical symptom in asthma, the degree of AHR is usually in proportion to the severity of asthma Provokers such as viral infection and allergen exposure can enhance the underlying AHR in asthmatic patients (Cockcroft and Davis, 20 06) It is suggested that AHR has two components: the variable component associated with airway inflammation and the... respiratory virus infections in asthma development and exacerbation, which support the use of allergen sensitization combined with virus infection to establish asthma exacerbation model Traditionally, in vivo asthma models were established in animals sensitized and challenged by allergens Many species of animals, such as mice, rats, guinea pigs, monkeys, were used to establish asthma models (Zosky and... room in Animal Vivarium is regulated by automatic timers to provide 12 h of light and 12 h of dark cycles The temperature in the animal room ranges from 18 to 26 ℃ with an average of 22 ℃ Maintenance diets contain 4-5% fat and 14% protein The cage bedding was changed every two days Mice were sensitized with 50 μg OVA and 2 mg Al(OH)3 in 0 .2 ml saline intra-peritoneally on day 0, and challenged on day 9,... injury and inflammation The structural changes including epithelial thickening, goblet cell hyperplasia, and airway smooth muscle hypertrophy and hyperplasia, contribute to the overall thickening of the airway wall in asthma (Berend et al., 20 08) Airway inflammation also plays a role in AHR in asthma The correlation between peripheral blood eosinophilia and AHR was demonstrated, along with the effect of. .. rhinovirus may function through inflammatory mediators (Grünberg et al., 1997) With the regulation of IL-13, neonatal rhinovirus infection induced AHR in mice, further supporting the role of inflammatory mediators in virus-associated AHR (Schneider et al., 20 12) 1.3 Animal models for asthma exacerbation Asthma exacerbations, especially virus -induced ones, have brought heavy burden to the society Inhaled corticosteroid ... Inc, Waltham, MA, USA) Both A2 60 /A2 80 (DNA/protein) and A2 60 /A2 30 (DNA/organic contaminants) ratios were recorded as an indicator of purity of RNA An acceptable level of purity for RNA extracts... were also made to establish asthma exacerbation models via rhinovirus infection, the major cause of human asthma exacerbation However, more than 90% of rhinovirus serotypes use human ICAM-1 to... with asthma exacerbation as a crucial trigger However, the mechanism of rhinovirus associated asthma exacerbation is still unclear and needs more investigation Thus, animal models including mouse