A mouse model of rhinovirus induced asthma exacerbation 4

airflow (Rl= ΔP/V). Cdyn presents the distensibility of the lung and is defined as a change in volume relative to an applied change in pressure (C= ΔV/ΔP). AHR was measured on day 3 after the last challenge. Mice were anesthetized by an injection of 200 μl mouse anaesthetic mixture (ingredients show in 3.1). Tracheotomy was performed and a Y shape cannular was inserted into the trachea. Each mouse was placed in the whole-body plethysmograph chamber and the trachea was connected via the Y shape cannular to pneumotach meter, ventilator and nebulizer. The system was setting at a tidal volume of 200 μl/breath and a breathing rate of 140/min. Methacholine was stored in -20℃ and dissolved in cold PBS and 10 μl of each dose of methacholine was loaded on to the nebulizer. Mice were challenged with aerosolized methacholine for 3 min, and bronchoconstriction was recorded for an additional 5 min for each increasing dose of methacholine. Results are expressed as a percentage of the respective basal values in response to PBS (Cheng et al., 2011). 3.11 Statistical analysis Data are presented as means ± SEM. Statistical analyses were performed with Statistical Product and Service Solutions 13.0 and involved the use of One-way ANOVA followed by Dunnettʼs test to determine significant differences between treatment groups. Significant levels were set at P < 0.05.   52   Chapter 4 Results   53   4.1 Rhinovirus-induced BAL fluid inflammatory cell increases in experimental allergic asthma murine model We performed the experiments on mice with four treatments: PBS challenge (PBS), PBS challenge with RV infection (PBS-RV), OVA challenge (OVA) and OVA challenge with RV infection (OVA-RV). BAL fluid was collected on day 1, 2, 3, 5 and 7 after the last challenge, total and differential cell counts were performed as described in Chapter 3. From the data presented in Figure 4.1, OVA challenge increased total cell, eosinophil and lymphocyte counts in BAL fluid, successfully inducing experimental allergic response in mice. To investigate the effect of RV on allergic mice, we compared data of OVA group and OVA-RV group. RV infection significantly amplified the inflammatory cell infiltration in BAL fluid (day 2 & 3 after the last challenge), especially eosinophils and macrophages. Besides, RV infection also significantly increased the number of neutrophil (day 1 & 2) and lymphocyte (day 5). Thus, RV infection exacerbates the inflammatory cell infiltration in allergic mice around day 2 and day 3 after the last challenge.   54   A Total number of inflammatory cells PBS * * PBS-RV Cell Number ×105 7 6 OVA OVA-RV 5 4 3 2 1 0 1 2 3 5 Day after the last challenge 7     B Macrophages Cell Number ×105 4 * 3 2 1 0 1 2 3 5 Day after the last challenge 7 C Eosinophils Cell Number ×105 4 * * 3 2 1 0 1   2 3 5 Day after the last challenge 7 55   D Cell Number ×105 1.5 * Neutrophils 1 0.5 * 0 1 2 3 5 Day after the last challenge 7 E Lymphocytes Cell Number ×104 2 1.5 * 1 0.5 0 1 2 3 5 Day after the last challenge 7     Figure74.1 Rhinovirus-induced increase in BAL inflammatory cell counts in experimental allergic asthma murine model. Total inflammatory cell counts (A) in BAL fluid obtained at different time points (day 1, 2, 3, 5 and 7 after the last challenge). Differential cell counts were performed on a minimum of 500 cells to identify macrophage (B), eosinophil (C), neutrophil (D), and lymphocyte (E). n = 6 per treatment group. * indicates significant difference between OVA group and OVA-RV group, P < 0.05.   56   4.2 Rhinovirus-induced lung tissue inflammatory cell infiltration in experimental allergic asthma murine model To investigate the effect of RV on inflammatory cell infiltration into the peribronchiolar and perivascular regions of airways, lung tissues were collected on day 1, 3 and 7 after the last challenge. Representative sections of stained samples and respective inflammatory scores are presented in Figure 4.2. The control PBS group rarely showed inflammatory cell infiltration. PBS-RV group presented slight but not significant infiltration compared with PBS group. OVA challenge markedly induced leukocyte infiltration around bronchiolar and vascular regions. Comparing OVA group and OVA-RV group, RV infection substantially increased the infiltration on day 3 after the last challenge. The histological results indicate that RV infection exacerbates the inflammatory cell infiltration in allergic mice, supporting the BAL fluid results.   57   A. day 1 after the last challenge B. day 3 after the last challenge C. day 7 after the last challenge           58   D Inflammatory scores 3 * PBS PBS-RV OVA 2 OVA-RV 1 0 1 3 Day after the last challenge 7   Figure84.2 Rhinovirus-induced lung tissue inflammatory cell infiltration in experimental allergic asthma murine model. Representative photos (×200 magnification) of histologic examination show the lung tissue eosinophilia on day 1 (A), 3 (B) and 7 (C) after the last challenge. Quantitative analyses of inflammatory cell infiltration (D) in lung sections were performed as described in Chapter 3. Mean scores were obtained from three animals. * indicates significant difference between OVA group and OVA-RV group, P < 0.05..   59     4.3 Rhinovirus-induced airway mucus hyper-secretion in experimental allergic asthma murine model To investigate the effect of RV infection on mucus hypersecretion, lung tissues were collected on day 1, 3 and 7 after the last challenge and PAS staining was conducted. Representative sections and mucus scores are presented in Figure 4.3. As shown in representative photomicrographs, PBS group had no obvious mucus production in airways. OVA challenge induced mucus hyper-secretion on day 3 after last the challenge. Comparing OVA and OVA-RV group, RV infection significantly promoted the mucus hyper-secretion. The hyper-secretion started from day 3 after the last challenge and sustained to day 7, indicating an increasing trend for mucus production in OVA-RV group. Thus, RV infection exacerbates the mucus production in allergic mice.   60   A. day 1 after the last challenge B. day 3 after the last challenge C. day 7 after the last challenge             61      D Mucus scores 3 * PBS PBS-RV 2 * OVA OVA-RV 1 0 1 3 Day after the last challenge 7   Figure94.3 Rhinovirus-induced airway mucus hyper-secretion in experimental allergic asthma murine model. Representative photos (×200 magnification) of histologic examination show the mucus secretion on day 1 (A), 3 (B), 7 (C) after the last challenge. Quantitative analysis of mucus production (D) in lung sections was performed as described in Chapter 3. Mucus scoring was performed in at least three different fields for each lung section. Mean scores were obtained from three animals. * indicates significant difference between OVA group and OVA-RV group, P < 0.05.   62     4.4 Rhinovirus-induced serum IgE production in experimental allergic asthma murine model To evaluate the effect of RV on ongoing Th2 response in vivo, serum levels of total and OVA-specific IgE were determined using ELISA. Serums were collected on day 1, 2, 3, 5 and 7 after the last challenge. The results are presented in Figure 4.4. OVA challenge increased the total IgE levels on day 1 after the last challenge. Comparing OVA group and OVA-RV group, RV infection significantly increased the total IgE levels on day 3 and day 7 after the last challenge (Figure 4.4 A). For the OVA-specific IgE, OVA group showed a significant increase on day 5 and day 7 after the last challenge. Comparing OVA group and OVA-RV group, RV infection significantly increased the OVA-specific IgE on day 3 and day 7 after the last challenge (Figure 4.4 B). Both IgE levels in PBS-RV group do not show significant difference from those of PBS group. Thus, RV infection exacerbates the total IgE and OVA-specific IgE levels in OVA challenged mice.   63   A Total IgE 200 * ng/ml 150 * PBS PBS-RV OVA OVA-RV 100 50 0 1 2 3 5 Day after the last challenge 7 B OVA-specific IgE PBS-RV 0.1 * O.D. PBS OVA OVA-RV * 0.05 0 1 2 3 5 Day after the last challenge 7   Figure104.4 Rhinovirus-induced serum IgE productions in experimental allergic asthma murine model. The serum levels of total IgE (A) and OVA-specific IgE (B) in mice were analyzed by ELISA. n= 3 – 5 per group. * indicates significant difference between OVA group and OVA-RV group, P < 0.05.   64     4.5 Rhinovirus-induced expression of other inflammatory markers in experimental allergic asthma murine model To investigate the effect of rhinovirus on other inflammatory markers expression in allergic asthma mice, lung tissues were collected on day 1, 2, 3, 5 and 7 after the last challenge and mRNA levels were examined by quantitative real-time PCR. The gene expressions in PBS group were regarded as the negative control and the gene expressions in other groups were presented as fold change over baseline (Figure 4.5). MUC5AC is an important protein in mucus. Its expressions at different time points were shown in Figure 4.5 (A). OVA challenge markedly increased MUC5AC expression from day 2 after the last challenge. Comparing OVA group and OVA-RV group, RV infection significantly enlarged the expression on day 5 and 7 after the last challenge. The effect of RV infection on the expression of Th2 cytokines IL4 and IL-13 is shown in Figure 4.5 (B, C). OVA challenge alone increased the expression of IL-4 and IL-13. Comparing OVA group and OVA-RV group, RV infection significantly increased the expression of IL-4 (day 3) and IL-13 (day 5 and 7). RV infection also influenced the expression of several chemokines. OVA challenge increased the expression of eotaxin-1, MCP-1 and CXCL-10. Comparing OVA group and OVA-RV group, RV   65   infection significantly amplified the expression of eotaxin-1, MCP-1 and CXCL-10 (Figure 4.5 D, E, F). In conclusion, RV infection exacerbated the expression of several inflammatory genes in allergen-challenged mice. A Fold increase over baseline MUC5AC 70 PBS 60 PBS-RV 50 OVA OVA-RV 40 * 30 * 20 10 0 1 2 3 5 Day after the last challenge 7 B Fold increase over baseline IL-4 3 * PBS-RV OVA 2 OVA-RV 1 0 1   PBS 2 3 5 Day after the last challenge 7 66   C Fold increase over baseline IL-13 10 9 8 7 6 5 4 3 2 1 0 PBS PBS-RV OVA OVA-RV * * 1 2 3 5 Day after the last challenge 7   D Fold increase over baseline Eotaxin-1 * 18 16 14 12 10 8 6 4 2 0 PBS-RV OVA OVA-RV * 1   PBS 2 3 5 Day after the last challenge * 7 67   E Fold increase over baseline MCP-1 7 * 6 * PBS PBS-RV OVA 5 OVA-RV 4 3 2 1 0 1 2 3 5 Day after the last challenge 7 F Fold increase over baseline CXCL-10 8 * 7 PBS PBS-RV OVA 6 * 5 OVA-RV 4 3 2 1 0 1 2 3 5 Day after the last challenge 7   Figure114.5 Rhinovirus-induced inflammatory marker expression changes in experimental asthmatic lungs. The gene expression changes of MUC5AC (A), IL-4 (B), IL-13 (C), eotaxin-1 (D), MCP-1 (E) and CXCL-10 (F) were measured by real-time PCR. Lung sections were collected on day 1, 2, 3, 5 and 7 after the last challenge. Reactions were run in triplicate and three independent experiments. The relative quantity of target gene expression was automatically normalized by β-actin as an internal control and values shown were expressed as fold change over the PBS group. n=3-5 per group * indicates significant difference between OVA group and OVA-RV group, P < 0.05.   68     4.6 Rhinovirus-induced AHR in experimental allergic asthma murine model To investigate the effect of rhinovirus on AHR in response to different concentrations of methacholine, both airway resistance (Rl, A) and dynamic compliance (Cdyn, B) were measured in mechanically ventilated mice. Rl is defined as the pressure driving respiration divided by flow. Cdyn refers to the distensibility of the lung and is defined as the change in volume of the lung produced by a change in pressure across the lung. The PBS group served as the negative control. Considering the inflammation cells infiltration and the mucus production degrees, we conducted the experiment on day 3 after the last challenge. Data are presented in Figure 4.6. OVA challenge slightly changed the RI (P=0.07, 8 mg/ml) and Cdyn in response to methacholine. Comparing OVA group and OVARV group, RV infection slightly increased the RI (P=0.13, 8 mg/ml) and decreased Cdyn, with an increased trend in the difference between these two groups. RV infection may have an effect on AHR in allergic mice.   69   A RI (% baseline) Resistance 1600 1400 1200 1000 800 600 400 200 0 PBS PBS-RV OVA OVA-RV 0 0.5 1 2 Methacholine (mg/ml) 4 8   B Compliance Cdyn (% baseline) 120 100 80 60 PBS 40 PBS-RV OVA 20 OVA-RV 0 0 0.5 1 2 Methacholine (mg/ml) 4 8   Figure124.6 Rhinovirus-induced AHR in experimental allergic mice. Airway responsiveness of mechanically ventilated mice in response to aerosolized methacholine was measured on day 3 after the last challenge. AHR is expressed as percentage change from the baseline level of lung resistance (Rl, A) and dynamic compliance (Cdyn, B). Rl is defined as the pressure driving respiration divided by flow. Cdyn refers to the distensibility of the lung and is defined as the change in volume of the lung produced by a change in pressure across the lung. n= 5 per treatment group.   70     Chapter 5 Discussion   71   5.1 Development of rhinovirus-induced asthma exacerbation mouse model Rhinovirus infection is widely regarded as the major factor causing asthma exacerbation and leading to the emergency department visits for asthmatic patients. Efforts were made in this project to investigate the biological consequences of rhinovirus-induced asthma exacerbation in mice. Animal models are instrumental undertaking of the pathogenesis of asthma exacerbation. Airway inflammatory cells infiltration, mucus hyper-secretion and AHR are three hallmark features of asthma (Busse and Rosenwasser, 2003). During the exacerbation episode, triggers like respiratory virus infections, allergen exposure, and other factors aggravate these symptoms. In our mouse model, allergen challenge followed by rhinovirus infection induced pulmonary infiltration of inflammatory cells such as eosinophils and neutrophils, into the airways, supported also by the significant increase of total cell counts and differential cell counts in BAL fluid (Figure 4.1). We observed an increase in the infiltration of inflammatory cells into peribronchiolar and perivascular tissues shown by histological examinations (Figure 4.2). Study on asthma management strategy showed that treatment directed towards normalization of sputum eosinophil count reduced asthma exacerbations in patients, indicating a crucial role of eosinophil in   72   asthma symptoms aggravation (Green et al., 2002a). Rhinovirus infection alone rarely induces eosinophilia in airways, but experimental rhinovirus 16 infection can promote ECP increase, correlating with hyper-responsiveness in asthmatic subjects (Grünberg et al., 2000). Besides eosinophilia, neutrophilia was also detected in exacerbation patients, with a higher degree in virus-induced exacerbation (Nair et al., 2012). Th2 cytokines and chemokines participate in the complex process of inflammatory cells (mostly eosinophils) recruitment. IL-4 and IL-13 can induce the expression of adhesion molecules in endothelial cells and facilitate the trafficking of inflammatory cells. IL-13 upregulates expression of chemokines such as eotaxin-1 and eotaxin-2 in airways (Matsukura et al., 2001). The C-C chemokine eotaxin-1 (CCL11) is a specific chemoattractant causing selective infiltration of eosinophils in lung tissue (Bhardwaj and Ghaffari, 2012). Increased expression of eotaxin contributes to the airway eosinophilia and asthma severity (Zietkowski et al., 2010). Our gene expression data reveal that allergen challenge followed by rhinovirus infection induced an increase in IL-4, IL-13 expression (Figure 4.5 B, C). The expression of eotaxin-1 (Figure 4.5 D) was also significantly amplified by rhinovirus infection on day 2 after post challenge, which was correlated with the increase of eosinophils in BAL fluid and histological sections. Considering the eosinophilic chemoattractive effects of these cytokines and chemokines, it is possible for rhinovirus to amplify the eosinophilia   73   through enhancement of the Th2 cytokines and chemokines expression. Airway mucus plugging is long regarded as a principle cause of death in asthma, with mucus hyper-secretion and goblet cell hyperplasia detected in specimens from patients died of asthma attacks (Aikawa et al., 1992; Molfino, 2010). From the histological examination of mucus production in lung tissue sections, we found that rhinovirus infection amplified the mucus plugging and airway obstruction in airways (Figure 4.3). After rhinovirus infection, mucus hyper-secretion and airway obstruction persisted up to day 7 post challenge, and even had a higher mucus score than the earlier days. Rhinovirus infection alone typically can induce mucus overproduction in airways (Schneider et al., 2012; Yuta et al., 1998). Mucin protein expression was also detected in rhinovirus-infected bronchial epithelial cells from asthmatics (Hewson et al., 2010). IL-13 and IL-4 contribute to the increase of airway mucus content and goblet cell hyperplasia in the airways of murine animals (Cho et al., 2010). MUC5AC is the principle gel-forming mucin and a dominant protein expressed in goblet cells (Evans et al. 2009; Fahy 2002). The examination of MUC5AC mRNA expression revealed that rhinovirus infection further enhanced the expression of allergeninduced MUC5AC (Figure 4.5 A). Although the expressions on day 5 and 7 post challenge were decreased, there were still significant   74   differences between allergen-challenged mice with or without infection. However, the expression of MUC5AC seems not high enough to explain the rhinovirus amplified mucus hyper-secretion, especially the continued increase on day 7 post challenge. Considering the possible role of other mucin protein including MUC5B in mucus hyper-secretion, and the effect of other cytokines and chemokines, more investigations on the mechanism of rhinovirus-amplified mucus hyper-secretion are warranted. Airway hyper-responsiveness was a characteristic feature of asthma with a complex mechanism. It is regarded that different components of AHR are related with different mechanisms. The transient AHR is commonly associated with acute airway inflammation involving eosinophils, while the persistent AHR is related to the structural airway changes result from airway inflammation (Cockcroft and Davis, 2006). Th2 cytokines IL-4 and IL-13 were found to be related to allergen-induced AHR (Kinyanjui et al., 2013; Mattes et al., 2001). Increase in IL-13 is sufficient to induce AHR in mice independent of IgE and eosinophilia (Kibe et al., 2003). However, there is also evidence showing that AHR can occur without the increase in IL-4 and IL-13 (Proust et al., 2003). Airway remodeling such as changes in smooth muscle, collagen deposition and mucus glands, is considered as another possible mechanism of AHR, especially the persistent ones (Cockcroft and Davis, 2006; Homer and Elias, 2000). Based on the cell infiltration and mucus hyper-secretion data, we   75   chose the day 3 post challenge as the time point to measure the effect of rhinovirus on AHR. In our model, the allergen-challenged rhinovirusinfected mice showed an increased trend of AHR to increasing concentration of methacholine, which is stronger than that of respective non-infected ones, though not statistically significant (Figure 4.6). Despite the critical role of inflammatory cells in asthma AHR, study has shown that AHR in asthma exacerbation may be independent of eosinophilic inflammation (Siegle et al., 2006). Considering the high mucus score on day 5 and 7 post challenge and the importance of mucus overproduction and airway obstruction, there is a possibility for rhinovirus to induce even more stronger bronchial responsiveness in allergic subjects in later time points. Taken together, in our mouse asthma exacerbation model, rhinovirus infection significantly amplified the airway inflammatory cell infiltration and mucus hyper-secretion in allergic mice, and also contributed to the AHR. The protocol in establishing our mouse model is based on that of Johnstonʼs team with a lower dose of OVA in allergen challenge. Our model showed rapid acute airway inflammation with eosinophils that is occurred more promptly than Johnstonʼs. This model of asthma exacerbation may help us to explore the role of rhinovirus in the pathogenesis of asthma exacerbation.   76   5.2 Increased chemokine expression in rhinovirus-induced allergic airway disease Aside from Th2 cytokines, rhinovirus infection also induced the expression of several chemokines in allergen-challenged mice, which may also contribute to the mechanism of rhinovirus-associated asthma exacerbation. CXCL10 (also known as interferon-γ inducible protein-10) is a CXC chemokine that can bind to a primary receptor CXCR3, which is usually expressed at high level on surface of activated T cells, memory T cells and NK cells (Müller et al., 2010). The characteristics that CXCL10 is capable of recruiting dendritic cells and CD8+ T cells, makes it a more Th1-related chemokine in virus infection, with a possible role in viral clearance (Lindell et al., 2008; Spurrell et al., 2005). The expression of CXCL10 has been associated with RSVinduced AHR and mucus production (Lindell et al., 2008). Other than viral infections, CXCL10 is also found to be involved in various autoimmune diseases, many of which deploy Th1 response (Bochner et al., 2003; Lee et al., 2009). Despite its Th1 character, CXCL10 has recently been detected in some Th2 allergic conditions. Slight but significant increase of CXCL10 expression was found in patients with allergic asthma, and in allergic animal models, with a relation to increased eosinophilia, AHR and IL-4 levels (Bochner et al., 2003; Medoff et al., 2002; Miotto et al.,   77   2001). Recently, studies focused more on the role of CXCL10, as a biomarker for clinical disease severity in rhinovirus-induced asthma exacerbation. Serum CXCL10 levels in asthmatic patients with viral infections were significant higher than those non-infected. The increase of serum CXCL10 is associated with more severe airflow obstruction, indicating CXCL10 as a potential biomarker for rhinovirus-induced asthma exacerbation (Wark et al., 2007). Through the analysis of CXCL10 mRNA expression in our mouse model, we found that rhinovirus infection prolonged CXCL10 expression in allergen-challenged mice from day 1 post challenge (Figure 4.5 F). Though predominately expressed on memory and activated T cells, CXCR3, the receptor for CXCL10 was highly upregulated in mouse eosinophils when infected with parasite Schistosoma japonicum (He et al., 2004a). By treatment with antiCXCR3 mAb, CXCL10 induced eosinophil chemotaxis was blocked in human peripheral eosinophils in vitro (Tan et al., 2000). Moreover, the stimulation with CXCL10 on eosinophils isolated from healthy subjects increased their adhesion to ICAM-1, along with enhanced EDN, EPO and several cytokines and chemokines productions (Takaku et al., 2011). Considering the effects of CXCL10 on eosinophil recruitment and functions, it is possible that rhinovirus amplified and prolonged CXCL10 expression in allergen-challenged mice and contributed a role in the increased eosinophilia in asthma exacerbation model. Besides eosinophils, CXCR3 was also abundantly expressed by ex vivo human   78   lung mast cells (Brightling et al., 2005a). Along with the expression of CXCL10 by airway smooth muscle in asthmatics, the correlation of CXCL10 and CXCR3 facilitated the mast cell migration to airway smooth muscle, contributing to the AHR and airway remodeling in asthma (Brightling et al., 2005b). MCP-1 is another chemokine that its experimental level was significantly increased in our mouse asthma exacerbation model. MCP1 is regarded as a chemoattractant for monocytes, memory T cells and dendritic cells during inflammation (Yadav et al., 2010). MCP-1 and its cognate receptor CCR2 also contribute a role in respiratory infections. Infection with rhinovirus or RSV increased the expression of MCP1 in macrophages or lower airway epithelial cells (Gotera et al., 2012; Hall et al., 2005). The increased expression of MCP-1, along with other inflammatory chemokines such as RANTES and MIP-α, were detected in bronchial tissue and lavage fluid from asthmatic patients (Conti and DiGioacchino, 2001; Dhaouadi et al., 2013; Saad-El-Din Bessa et al., 2012). In agreement with the clinical findings, animal OVA asthma model also expressed high level of MCP-1 (Tsuchiya et al., 2012). Several studies indicated the possibility of MCP-1 involving in asthma exacerbations and acute asthma attack. Significant increase of MCP-1 was detected in sputum from patients with an aggravation of symptoms after acute asthma attack, compared with that from recovered patients   79   (Kurashima et al., 1996). Asthmatic children had an increased nasal aspirate MCP-1 level during virus-positive weeks, correlating with respiratory tract symptoms (Lewis et al., 2012). In addition, asthmatic children in Taiwan showed increased serum MCP-1 level in acute attack, comparing with asymptomatic state, while healthy children showed the lowest level of MCP-1 (Chan et al., 2009). In our mouse model, the rhinovirus-induced asthma exacerbation mice showed early rise and prolonged expression of MCP-1 (Figure 4.5 E). Considering the chemoattractive effect of MCP1 on monocytes and macrophages, the enhancement of macrophages in OVA-challenged infected mice may attributable to rhinovirus-induced MCP-1 expression. Recently, a similar result was found in a rhinovirusinduced mouse model of allergic airway disease. The mice with combined OVA and rhinovirus treatment showed an increase in MCP-1 expression, with the enhanced hyper-responsiveness and inflammation due to the MCP-1 increase (Schneider et al., 2013). Depletion of macrophage in allergen-challenged mice decreased the rhinovirusinduced eosinophilia and hyper-responsiveness, suggesting an indirect effect of MCP-1 on eosinophil recruitment (Nagarkar et al., 2010). Eotaxin-1 is long considered as a key attractant for eosinophils through specifically binding to the CCR3 receptor (Bisset and SchmidGrendelmeier, 2005). Enhanced expression of eotaxin-1 and CCR3 were detected in both human atopic asthmatics and animal asthma   80   models, with increased AHR and eosinophilia (Asosingh et al., 2010; Miotto et al., 2001; Zietkowski et al., 2010). The increased eotaxin-1 expression was also associated with rhinovirus infection, detected in both bronchial epithelial cells in vitro and allergic mouse model in vivo (Nagarkar et al., 2010; Papadopoulos et al., 2001). The amplified eotaxin-1 expression was detected in our mouse model, with the significant increase on day 2, 5, 7 post challenge (Figure 4.5 D). Not only in eosinophils, CCR3 was also expressed on basophils, mast cells and Th2 lymphocytes, suggesting other possible roles of eotaxin-1 in asthma pathophysiology in addition to eosinophils attraction (Amerio et al., 2003; Collington et al., 2010). 5.3 Immune response in virus-induced allergic asthma exacerbation Previous studies indicated that there is an imbalance between Th1 and Th2 immune response in asthmatics, with the Th2 as the main response. However, virus infections, usually considered as Th1 response stimuli in host defense, augment the Th2 response in asthma exacerbation. Two hypotheses about the role of virus infection in asthma exacerbation come out: 1. Virus infections do not trigger the Th1 response but enhance the Th2 response, leading to the imbalance in immune response with a deficient Th1 response and a strong Th2 response; 2. Virus infections stimulate the Th1 response, and the productions cooperate with Th2 components, finally leading to an   81   enhanced Th2 response and worsen symptoms. Several investigations suggested that a deficient Th1 response in asthmatic patients might contribute to the inflammatory symptoms during virus-exacerbated wheezing. Interferons are important cytokines in anti-viral immune response and belonging to Th1 response. Deficient production of IFN-α, IFN-γ and IFN-λ to virus infection were reported in asthmatic primary bronchial epithelial cells, comparing with the normal subjects (Wark et al., 2005; Contoli et al., 2006; Gehlhar et al., 2006). Moreover, virus-induced IFN-γ was inversely associated with viral shedding in blood mononuclear cells (Papadopoulos et al., 2002). With the fact that asthmatic bronchial epithelial cells were more effective to be infected by rhinovirus, it is suggested that deficient IFN responses in asthmatic patients contribute to incomplete viral clearness, leading to a more severe viral inflammation (Kloepfer and Gern, 2010). The Toll-like receptors (TLRs) recognize pathogen-associated molecular patterns and activate innate immunity during virus infection, and are considered to induce production of type I interferons. TLR7 deficiency were reported to interact with Pneumovirus in development of asthma pathology in mice (Kaiko et al., 2013). On the other hand, recent in vitro infection experiment conducted in human bronchial epithelial cells from asthmatic and healthy subjects indicated that rhinovirus-induced interferon production is not deficient in well controlled asthma (Sykes et al., 2013a). In   82   [...]... (Tsuchiya et al., 2012) Several studies indicated the possibility of MCP-1 involving in asthma exacerbations and acute asthma attack Significant increase of MCP-1 was detected in sputum from patients with an aggravation of symptoms after acute asthma attack, compared with that from recovered patients   79   (Kurashima et al., 1996) Asthmatic children had an increased nasal aspirate MCP-1 level during virus-positive... Chapter 5 Discussion   71   5.1 Development of rhinovirus- induced asthma exacerbation mouse model Rhinovirus infection is widely regarded as the major factor causing asthma exacerbation and leading to the emergency department visits for asthmatic patients Efforts were made in this project to investigate the biological consequences of rhinovirus- induced asthma exacerbation in mice Animal models are... our mouse model is based on that of Johnstonʼs team with a lower dose of OVA in allergen challenge Our model showed rapid acute airway inflammation with eosinophils that is occurred more promptly than Johnstonʼs This model of asthma exacerbation may help us to explore the role of rhinovirus in the pathogenesis of asthma exacerbation   76   5.2 Increased chemokine expression in rhinovirus- induced allergic... instrumental undertaking of the pathogenesis of asthma exacerbation Airway inflammatory cells infiltration, mucus hyper-secretion and AHR are three hallmark features of asthma (Busse and Rosenwasser, 2003) During the exacerbation episode, triggers like respiratory virus infections, allergen exposure, and other factors aggravate these symptoms In our mouse model, allergen challenge followed by rhinovirus. .. on asthma management strategy showed that treatment directed towards normalization of sputum eosinophil count reduced asthma exacerbations in patients, indicating a crucial role of eosinophil in   72   asthma symptoms aggravation (Green et al., 200 2a) Rhinovirus infection alone rarely induces eosinophilia in airways, but experimental rhinovirus 16 infection can promote ECP increase, correlating with... 2012; Hall et al., 2005) The increased expression of MCP-1, along with other inflammatory chemokines such as RANTES and MIP-α, were detected in bronchial tissue and lavage fluid from asthmatic patients (Conti and DiGioacchino, 2001; Dhaouadi et al., 2013; Saad-El-Din Bessa et al., 2012) In agreement with the clinical findings, animal OVA asthma model also expressed high level of MCP-1 (Tsuchiya et al.,... and facilitate the trafficking of inflammatory cells IL-13 upregulates expression of chemokines such as eotaxin-1 and eotaxin-2 in airways (Matsukura et al., 2001) The C-C chemokine eotaxin-1 (CCL11) is a specific chemoattractant causing selective infiltration of eosinophils in lung tissue (Bhardwaj and Ghaffari, 2012) Increased expression of eotaxin contributes to the airway eosinophilia and asthma severity... feature of asthma with a complex mechanism It is regarded that different components of AHR are related with different mechanisms The transient AHR is commonly associated with acute airway inflammation involving eosinophils, while the persistent AHR is related to the structural airway changes result from airway inflammation (Cockcroft and Davis, 2006) Th2 cytokines IL -4 and IL-13 were found to be related... concentration of methacholine, which is stronger than that of respective non-infected ones, though not statistically significant (Figure 4. 6) Despite the critical role of inflammatory cells in asthma AHR, study has shown that AHR in asthma exacerbation may be independent of eosinophilic inflammation (Siegle et al., 2006) Considering the high mucus score on day 5 and 7 post challenge and the importance of. .. significantly increased in our mouse asthma exacerbation model MCP1 is regarded as a chemoattractant for monocytes, memory T cells and dendritic cells during inflammation (Yadav et al., 2010) MCP-1 and its cognate receptor CCR2 also contribute a role in respiratory infections Infection with rhinovirus or RSV increased the expression of MCP1 in macrophages or lower airway epithelial cells (Gotera et al., ... instrumental undertaking of the pathogenesis of asthma exacerbation Airway inflammatory cells infiltration, mucus hyper-secretion and AHR are three hallmark features of asthma (Busse and Rosenwasser,... Chapter Discussion   71   5.1 Development of rhinovirus- induced asthma exacerbation mouse model Rhinovirus infection is widely regarded as the major factor causing asthma exacerbation and leading... increase of MCP-1 was detected in sputum from patients with an aggravation of symptoms after acute asthma attack, compared with that from recovered patients   79   (Kurashima et al., 1996) Asthmatic