Effects of anti malaria drug artesunate in the treatment of rhinovirus induced asthma exacerbation

4.1 Rhinovirus induces the lung inflammation, mucus production, and inflammatory gene expression in naive mice .... 80 4.6 Artesunate treatment reduces the exacerbation of recruitment of

ANTIMALARIAL DRUG ARTESUNATE IN THE TREATMENT OF RHINOVIRUS-INDUCED ASTHMA

ACKNOWLEDGEMENT

First and foremost, I would like to deeply thank my supervisor Professor Wong Wai-Shiu Fred for his guidance and assistance through my Ph.D studies Without his help and encouragement, I definitely could not overcome so many obstacles in the projects His attitude and discipline will encourage me to continue the research work in the future

I would also like to thank Professor Vincent TK Chow for his invaluable advice and efforts

on my research works

I am grateful to Fera Goh, Khaing Nwe Win, Cheng Chang, Guan Shouping, Lah Lin Chin, Winston Liao, Dong Jinrui, all colleagues in our lab, and friends who helped me in the experiments, shared me with their experience, and supported me

Thanks to National University of Singapore for providing me chances of studying in Singapore

Finally, I would like to extend my sincere gratitude to my parents, my sisters and all my best

friends for their endless love, support, and patience all the time

TABLE OF CONTENTS

ACKNOWLEDGEMENT iii

TABLE OF CONTENTS iv

LIST OF TABLES x

LIST OF FIGURES xi

LIST OF ABBREVIATIONS xiii

1 INTRODUCTION 1

1.1 Asthma 2

1.1.1 Epidemiology and etiology and risk factors of asthma 2

1.1.2 Pathogenesis of asthma 5

1.1.3 Asthma exacerbation 10

1.1.4 Role of NF-κB in allergic airway inflammation 12

1.1.5 Therapeutic strategies in the treatment of asthma exacerbation 14

1.2 Rhinovirus 16

1.2.1 Biology of rhinovirus 16

1.2.1.1 Transmission pathway 16

1.2.1.2 Structure of rhinovirus 17

1.2.1.3 Live temperature of rhinovirus 21

1.2.1.4 Classification of rhinovirus 22

1.2.1.5 A novel family member of rhinovirus family-HRV-C 24

1.2.2 The interaction between rhinovirus and hosts 25

1.2.2.1 The entry and replication of rhinovirus into host cells 25

1.2.2.2 Host immune response to rhinovirus infection: 27

1.2.2.3 The role of NF-κB in rhinovirus infection 30

1.3 Artesunate 34

1.3.1 Effects of artesunate 34

1.3.2 Pharmacokinetics and pharmacodynamics of artemisinins 39

1.3.3 Side effects of artesunate 40

1.4 NF-κB pathway 41

1.4.1 Introduction to NF-κB pathway 41

1.4.2 Role of NF-κB inhibitors in virus infection and allergic airway inflammation 47 2 RATIONALE & AIM 50

3 METHODLOGY 53

3.1 Materials and Reagents 54

3.2 Cell culture 54

3.3 Virus growth 55

3.4 Rhinovirus titration 56

3.5 Virus purification 56

3.6 Viral replication test 57

3.7 Mouse 57

3.8 Rhinovirus-induced lung inflammation mouse model 58

3.9 Rhinovirus-induced asthma exacerbation mouse model 58

3.10 Bronchoalveolar lavage fluid analysis 59

3.11 Total and differential cell count in BALF 59

3.12 Reverse Transcriptase - Polymerase Chain Reaction (RT-PCR) 60

3.12.1 Reverse transcription 61

3.12.2 Polymerase Chain Reaction (PCR) 61

3.12.3 Gel Electrophoresis 61

3.13 Histologic analysis 62

3.14 Airway hyperresponsivenss 63

3.15 ELISA 67

3.15.1 Tissue homogenization and supernatant harvest 67

3.15.2 ELISA 67

3.15.3 Serum total IgE ELISA 67

3.16 NF-κB binding activity measurement 68

3.16.1 Nuclear protein extraction 68

3.16.2 Western Bloting 69

3.16.3 NF-κB transcription factor assay 69

3.17 Statistic analysis 70

4 RESULTS 72

4.1 Rhinovirus induces the lung inflammation, mucus production, and inflammatory gene expression in naive mice 73 4.2 Rhinoviruses exacerbate the lung inflammation in a HDM-induced allergic asthma model 76

4.3 RV infection exacerbate the production of lung cytokines and chemokines 79 4.4 RV infection exacerbate the expression of inflammatory mediators 79 4.5 Effects of RV infection on NF κB translocation and transactivation 80 4.6 Artesunate treatment reduces the exacerbation of recruitment of inflammatory cells in mice with allerfic airway inflammation and inflammatory cell infiltration and mucus production induced by RV infection 87 4.7 Artesunate decreases the production of Th2 related cytokines and chemokines and serum total IgE 90 4.8 Artesunate inhibits the gene expression level of mucus gene Muc5ac 93 4.9 Artesunate partially reversed AHR in RV infected and HDM treated mice 93 4.10 Artesunate reduced the binding activity of NF-κB in RV infected and HDM treated mice 97

5 DISCUSSION 98

6 REFERENCE 114

SUMMARY

Rhinovirus causes the majority of virus-induced asthma exacerbations The purpose of this study was to investigate the inflammatory mechanisms underlying this exacerbation phenomenon We combined the mouse model of allergic asthma (using house dust mite [HDM] sensitization and challenge) and rhinovirus (RV) infection (intra-tracheal inoculation with RV-1B, a minor group RV which binds to mouse airway epithelial cells) Mice were sensitized and challenged with HDM or saline and infected with RV or PBS Bronchoalveolar lavage fluid and lung homogenates were tested for Th2 cytokines protein levels and mRNA expression Compared with saline/PBS treated mice, saline/RV treated mice showed significant increase in airway neutrophils, lymphocytes, inflammatory cell infiltration, mucus production and inflammatory chemokines mRNA expression Compared

to HDM/PBS-treated mice, HDM/RV-treated mice showed significant increase in airway neutrophils, eosinophils and lymphocytes The HDM/RV group also showed higher protein level of IL-4, IL-5, IL-13 Besides, the HDM/RV group showed higher mRNA level of eotaxin-1, Muc-5AC Taken together, we have demonstrated a model of RV-1B exacerbation of the Th2 response in HDM-induced allergic asthma

On the basis of the RV-induced asthma exacerbation mouse model, we test the effects of anti-malarial drug Artesunate in the treatment of RV-induced asthma exacerbation Artesunate was administrated after RV infection via i.p route Control mice were injected with DMSO Lung homogenates were examined for inflammatory cells infiltration, and Th2 cytokine protein production and mRNA expressions Airway hyper-responsiveness was measured using direct airway resistance analysis Compared with DMSO treatment group, artesunate significantly inhibited rhinovirus –induced exacerbation in total inflammatory cell count, eosinophil count and lymphocyte count Artesunate also significantly inhibited

rhinovirus-induced exacerbation in IL-4, IL-5, IL-13, IP-10 and Muc5AC mRNA production Meanwhile, artesunate singnificantly inhibited rhinovirus-induced total IgE secretion and airway hyperresponsiveness At last, we also found that artesunate significantly reduced the binding activity of NF-κB Artesunate attenuated the exacerbation

of RV on asthma, and our findings implicated a potential value of artesunate in the treatment

of rhinovirus-induced asthma exacerbation (This work was supported by a BMRC grant 09/1/21/19/595 to WSFW)

LIST OF TABLES

Table Title Page

Table 3-1 Targets and sequences for primer sets of RT-PCR 71

LIST OF FIGURES

Figure Title Page

Figure 1-1 Worldwide spread of asthma 4 Figure 1-2 Pathogenesis of asthma (Adapted from Lemanske RF et al, 2010) 9 Figure 1-3 Structure of the genome of rhinovirus (Adapted from Jacobs et al 2013) 19 Figure 1-4 Structure of the rhinovirus capsid protein (adapted from Friedlander et al 2005) 20 Figure 1-5 Host immune response to rhinovirus infection 29 Figure 1-6 Role of rhinovirus infection in asthma exacerbation 33 Figure 1-7 Cultivar of the Chinese Herb Artemisia annua L (qinhao or sweet wormwood) and its active principle artemisinin (qinhaosu) 37 Figure 1-8 Chemical structure of dihydroartemisinin (DHA) (adapted from Keating 2012) 38 Figure 1-9 Family members of NF-κB proteins, IκB family, IKK family (Adapted from Hayden and Ghosh 2004) 42 Figure 1-10 The three kinds of NF-κB signal transduction pathways (Adapted from Hayden and Ghosh 2004) 44 Figure 1-11 The different levels of action of various inhibitors of the NF-κB signal

transduction pathway (Adapted from Gilmore et al 2006) 49

Figure 3- 1 The Buxco system for measurement of airway hyperresponsiveness 66

Figure 4- 1 RV-1B exposure induces airway inflammation, mucus production, and

Figure 4- 2 Rhinovirus-1b exacerbates the recruitment of inflammatory cells, inflammatory cell infiltration and mucus production in mice with allergic airway inflammation 78 Figure 4- 3 Rhinovirus infection further promoted in the production of cytokines and

chemokines in mouse with allergic airway inflammation 81 Figure 4- 4 Effects of rhinovirus infection on HDM induced gene expression in allergic airway inflammation mouse lung 83 Figure 4- 5 Effects of rhinovirus infection on NF-κB signaling pathway in mouse with allergic airway inflammation 86 Figure 4- 6 Effects of Artesnuate on the exacerbated recruitment of inflammatory cells in mice with allerfic airway inflammation and inflammatory cell infiltration and mucus

production 89 Figure 4- 7 Effects of Artesunate treatment on cytokines and chemokines levels from rhinovirus-induced lung homogenate 92 Figure 4- 8 Effects of artesunate on RV induced MUC5AC mRNA expression in allergic airway inflammation mouse lung 94 Figure 4- 9 Effects of artesunate on rhinovirus-induced AHR exacerbation 95 Figure 4- 10 Effect of artesnuate on rhinovirus-induced NF-κB signaling pathway 97

LIST OF ABBREVIATIONS

IKK IκB kinase

PCR polymerase chain reaction

expressed and secreted

1.INTRODUCTION

1.1 Asthma

1.1.1 Epidemiology and etiology and risk factors of asthma

Asthma is the most common chronic conditions affecting both children and adults In the whole world, 300 million people suffer from asthma and the prevalence of asthma still continues to increase (FIG.1-1) The prevalence of asthma is higher in western countries compared with the Asian countries (Janson, Anto et al 2001, Asher, Montefort et al 2006) and this trend keeps an increasing (Akinbami, Moorman et al 2012) According to this study, asthma prevalence is higher among children, females and those with family income below the poverty level, and deferrers by race and ethnicity for the period 2008-2010

The causes of asthma is complicated and yet to be clearly understood Several evidence have shown that genetic background is important (March, Sleiman et al 2011) Many genome-wide screenings of asthma and its associated traits have been carried out, and many genetic linkages have been identified in different regions These studies have identified 18 genes associated with allergy and asthma in 11 different populations In particular, there are consistently replicated regions on the long arms of chromosomes 2, 5, 6, 12 and 13 (Subbarao, Mandhane et al 2009) In addition, the interaction between genetic and environmental factors may also be important Observations of migration populations and Germany after reunification have strongly supported the role of local environmental factors, including different types of allergens but likely many lifestyle factors as well, in determining the degree of expression of asthma within genetically similar populations (Cohen, Canino et al 2007, Wang, Wong et al 2008) According to these studies, prevalence rates for asthma among children 13-14 years old were lowest for Chinese children born and grew up in China, intermediate for Chinese children who had migrated during their lifetime to Canada and highest for Chinese children who had been born in

Canada These results strongly suggested gene-by-environment interactions Host factors can also contribute to asthma initiation and pathogenesis For instance, stress, diet, nutrition, socio-economic status and so on Prenatal smoking has been consistently associated with early childhood wheezing (Lewis, Richards et al 1995, Stein, Holberg et al 1999, Tariq, Hakim et al 2000)

Severe or uncontrolled asthma presents high socio-economic burden on a lot of countries The healthcare costs correlate positively with severity of asthma The financial burden of asthma costs up to $1, 300 per patient per year in the developed countries In the United States, 23 million people including seven million children suffer from asthma In developing countries like Vietnam, with gross product per capita of ﹩US1,411, the financial burden of asthma estimates to be US＄184 per patient per year In India, the medication for an asthmatic child can cost up to a third of the family’s income (Pawankar, Canonica et al

2012)

In summary, the rising prevalence, high economic burden and mortality of asthma bog the health-care system worldwide However, the current drugs are only able to control the symptoms None of them are able to eliminate the disease The major unmet in this area include better management of the severe forms of the disease and development of curative therapies Therefore, more efforts are needed to put in this area to elucidate the pathogenesis

of asthma and to explore novel therapies for curing asthma

Figure 1-1 Worldwide spread of asthma (adapted from Asthma: epidemiology, etiology

and risk factors)

1.1.2 Pathogenesis of asthma

Asthma is a heterogeneous disease which is composed of numerous distinct clinical phenotypes It is now accepted that asthma is a chronic inflammation condition, and evidences of inflammation can be observed in mild, moderate and severe disease (Hamid and Tulic 2009)

Many cells are involved in the immune and inflammatory response to allergens in asthma, which include T cells, eosinophils, mast cells and neutrophils (FIG.1-2) The airway epithelial cells are located at the interface between the host and the environment; therefore, they are the first defense against the inhaled antigens Epithelial cells express many pattern recognition receptors (PRRs), which can be activated by the foreign antigens This activation leads to the activation of NF-κB signaling pathway, which results in the production of various of chemokines and cytokines, such as IL-8 and IL-6 (Lambrecht and Hammad 2012)

The bronchial epithelial cells also work as a physical barrier that prevents the access of allergens to lung dendritic cells (Lambrecht and Hammad 2012).The integrity of airway epithelia is maintained by apical tight junction and adherent junctions, which have the ability to keep the cells together and maintain their apico-basal polarity (Xiao, Puddicombe

et al 2011) Based on bronchial biopsy studies, the airways of subjects with asthma have fragile epithelia (Lackie 1997) Exposure to these allergens induces disruption of epithelial junctional proteins and the barrier function of airway epithelium Once the integrity of the epithelial cells has been destroyed, the inhaled allergens will be able to access to the dendritc cells and initiate immune response (Lambrecht and Hammad 2012)

Blood and tissue eosinophila are hallmarks of asthma Increased numbers of eosinophils in the bronchial mucosa as well as in the bronchoalveolar lavage (BAL) and sputum are consistent features of asthma, and BAL eosinophilia has been linked to development of the late airway response and asthma severity (Jatakanon, Lim et al 2000, Hamid and Tulic 2009) Eosinophils are able to release prestored granular proteins, including eosinophil cationic protein, eosinophil peroxidase, and major basic protein These potent cytotoxic proteins have been found to be related to the epithelial damage observed in asthmatic patients (Shamri, Xenakis et al 2011) Eosinophils are also able to produce and release oxygen redicals and lipid mediators, such as LTB4, LTC4 and PAF (Ohnishi, Miyahara et al 2008) They are also capable of producing various kinds of cytokines and chemokines, such

as IL-1, 2, 3, 4, 5, 6, 10, 12 and 13, RANTES, MIP-1α and so on In addition, eosinophils also play an important role in airway remodeling via releasing of fibrogenic cytokines, including TGF-β, IL-11, 17 and 25 Together these cytotoxic mediators determine the tissue destructive potency of this inflammatory cell

T cells constitute the majority of lung lymphocytes in normal individuals and can be found

in the airway, alveolar epithelium, and interstitium (Baraldo, Lokar Oliani et al 2007) Once activated, T cells can differentiate and proliferate into mature Th1, Th2, Th17, Th9, Treg, natural killer (NK)T cells, or γδ T cells, depending on the cytokines released by antigen presenting cells (Kaiko, Horvat et al 2008) All the members in this family contribute to the pathogenesis of asthma

Macrophages contribute to inflammatory condition and are a critical cell type in the innate and adaptive immune response Macrophages can produce both pro- and anti-inflammatory mediators, therefore, they act as either negative regulators or positive regulators, depending

on the environments (Bedoret, Wallemacq et al 2009, Byers and Holtzman 2010, Yang,

Kumar et al 2010) The regulatory effects of macrophages in vivo are still unclear

However, more and more evidence indicated that macrophages can positively regulate asthma (Careau, Proulx et al 2006) They found that macrophages are able to protect alveolar macrophage-depleted sensitized rats that received sensitized alveolar macrophages from developing airway hyperresponsiveness compared with that received unsensitized alveolar macrophages

These inflammatory and immune response are regulated by a number of cytokines and chemokines, which are secreted from structural and inflammatory cells, such as the eosinophils, bronchial epithelial cells and airway smooth muscle cells (ASM cells) These inflammatory mediators can be divided into many subgroups For instance, T cell-derived molecules such as the so called-T helper-1 (Th1) cells (IL-2, IFN-γ, and IL-12), Th2 cells (IL-4, 5 9, 13, and 25), Th3 or T regulatory cytokines (IL-10 and TGF-β) and TH17 cells (IL-17A and 17F) Other cytokines include proinflammatory cytokines (IL-1β, 6, and 11, TNF-α, and GM-CSF), growth factors, and chemokines

Mucus is mainly comprised by the mucin glycoproteins, which is composed of up to 2% by mass Under normal conditions, airway mucus protects the host by trapping foreign debris, bacteria and virus and can clear them by ciliary movement However, under chronic inflammation, such as asthma, the mucus could contribute to the pathogenesis of disease (Rogers 2007) Gel forming secreted mucins, including MUC5AC and MUC5B are mainly produced in airway goblet cells The secretion of them is regulated by different mediators, such as IL-13, which is the key regulator, TNF-α, epidermal growth factor receptor (EGFR)

Rogers 2010) Research indicated that the release of mucus are due to the activation of

NF-κB pathway(Evans, Kim et al 2009) The production of mucus contributes to the airway obstruction Several studies have shown that mucus is also able to contribute to airway hyperresponsiveness

Figure 1-2 Pathogenesis of asthma (Adapted from Lemanske RF et al, 2010)

1.1.3 Asthma exacerbation

Severe asthma exacerbations are episodes of increased asthma symptoms, such as dyspnea, cough, wheezing and/or chest discomfort Patients are usually very anxious, hyperventilating, and have a tachycardia (O'Byrne 2007) In 2004, asthma exacerbations resulted in 14.7 million outpatient visits, 1.8 million emergency room visits, 497000 hospitalizations and 4055 deaths in the United States alone While only 20% of asthmatics have had exacerbations requiring treatment in the emergency department or hospitalization, these patients account for more than 80% of total direct costs (Dougherty and Fahy 2009)

Various factors can contribute to the exacerbation of asthma Allergen exposure is important

in host allergic sensitization and as a common precipitant of asthmatic symptom in both children and adults Inhaled allergens, such as house dust mite, cockroach, Alternaria species and cats, play an important role in the pathogenesis of asthma (Bush and Prochnau

2004, Arbes, Gergen et al 2007, Brauer, Hoek et al 2007, Celedon, Milton et al 2007)

Respiratory tract infections caused by pathogens, such as viruses, Chlamydia species, and

Mycoplasma species, have also been implicated in the pathogenesis of asthma (Lemanske,

Jackson et al 2005, Kusel, de Klerk et al 2007, Jackson, Gangnon et al 2008, Kelly and Busse 2008, Newcomb and Peebles 2009) The outcomes of these infections are demonstrated to be dependent on the interaction between the pathogen and the host—these outcomes can be inception, exacerbation or prevention During infancy, certain kinds of viruses, including human rhinovirus and RSV, have been shown to be potent in initiating asthmatic phenotype (Kusel, de Klerk et al 2007, Jackson, Gangnon et al 2008) However, these opinions should be interpreted carefully, because most of the infants have been infected by these viruses and the initiation of asthmatic phenomenon may be the outcome of

the combination of genetic and environmental factors Secondly, in patients with established asthma, viral upper respiratory tract infection plays a key role in inducing asthma exacerbation, which results in hospitalization and morbidity Rhinovirus is the most common inducer of virus-induced asthma exacerbation This can be the results of the interaction between host immune response, allergen exposure, and virus infection There are evidences that asthmatic patients have abnormal response to rhinovirus infection (Contoli, Message et al 2006) Rhinovirus infection of the primary epithelial cells from asthmatic patients also results in higher viral yield, compared with the infection of primary bronchial epithelial cells from normal control subjects (Wark, Johnston et al 2005) Further investigation revealed a deficiency in interferon-β in these asthmatic epithelial cells Thirdly, and paradoxically, there are studies indicating viral infection can indeed prevent the progression of asthma exacerbation Clinical reports showed that about 5-10% of adult asthmatic patients have acute worsening of asthmatic symptoms to non-steroidal anti-inflammatory drugs (NSAIDs) Patients that are sensitive to NSAIDs may react differently

to different drugs, depending on the potency of this drug to inhibit the activity of the COX-1 enzyme (Hamad, Sutcliffe et al 2004) Patients that are sensitive to aspirin are sensitive to all other NSAIDs The principle of the sensitivity to aspirin is not so clear However, research in this filed indicated that it might involve the modulation of eicosanoid production (Fischer, Rosenberg et al 1994) It has been suggested that NSAIDs increased the production of asthma-provoking eicosanoids, such as cysteinyl leukotrienes and hydroxyeicosatetraenoic acids Meanwhile, it reduced the formation of prostaglandins, which works in maintaining normal airway function There is evidence indicating that mast cell activation also occurs during this procedure

Other risk factors that trigger asthma exacerbation include psychosocial factors, exercises and so on Research has found that stress is a risk factor for asthma expression in some children (Liu, Coe et al 2002) The mechanism by which this happens is still not clear Several studies indicated that stress might promote the disease by increasing the expression

of cytokines (Chen, Hanson et al 2006)

1.1.4 Role of NF-κB in allergic airway inflammation

Cumulative evidences have suggested the NF-κB signaling pathway plays an important role

in the initiation and development of asthma in both animal models and human asthma patients (Charokopos, Apostolopoulos et al 2009, Imanifooladi, Yazdani et al 2010) Persistent activation of NF-κB was detected in both bronchial biopsies and induced sputum from asthmatic patients (Gagliardo, Chanez et al 2003) Compared with those from normal subjects, clinical samples from severe asthmatic subjects have higher levels of IκBβ and p65

in the peripheral blood mononuclear cells, p65 was shown to have greater activation status (Gagliardo, Chanez et al 2003) Broide David et al showed that IKKβ was essential in the immune response in mouse strain in which the bronchial epithelial IKKβ was selectively knocked down, compared with that in wild type mice In this study, they found that IKKβ knockdown inhibited mucus production and eosinophils infiltration, which are the hallmarks

of allergic airway inflammation (Broide, Lawrence et al 2005)

NF-κB also takes an important part in the production of a lot of inflammatory cytokines and chemokines that are closely related to allergic asthma inflammation pathogenesis (Lin, Lin

et al 2000) Lin, Lin et al showed that, compared with the non-treated normal rats, OVA sensitized and challenged rats showed greater production of Th2 related cytokines in the lung Allergic inflammation in asthma has been shown to be Th2 dominant These results

indicated that NF-κB activation is essential in driving Th2 inflammation In addition, NF-κB activation has also been shown to promote the production of growth factors, such as VEGF, which is important in bronchial smooth muscle thickning

Both classical and non-classical pathways have been shown to be important in asthma pathogenesis P50, the central player of the non-classical NF-κB pathway, has been shown

to be essential in the eosinophilia caused by OVA administration (Yang, Cohn et al 1998)

In this study, they also demonstrated that p50 is important in secretion of 1α and 1β, which acts as chemoattractant for the recruitment of lymphocytes to the sites of inflammation

MIP-On the basis of the function of NF-κB in the pathogenesis of asthma, manipulation of

NF-κB pathway can be a promising method to fight against asthma Coticosteriods, which has long been used to control asthma, have also been shown to be a potent NF-κB inhibitor (Auwardt, Mudge et al 1998)

In addition, the upstream activator, phosphoinositide 3-kinase (PI-3K) has been shown to play a part in the pathogenesis of allergic disease, such as mast cell differentiation and activation(Ali, Bilancio et al 2004), eosinophils infiltration(Thomas, Edwards et al 2009), Th2 cytokines production(Park, Lee et al 2010) and allergen induced airway hyperresponsiveness (AHR) (Park, Lee et al 2010) PI-3K deficient mice have been shown

to have reduced levels of allergen-induced eosinophilic inflammation and airway remodeling (Ali, Bilancio et al 2004, Nashed, Zhang et al 2007, Lim, Cho et al 2009, Takeda, Ito et al 2009) PI-3K specific inhibitors have been shown to have important roles

vivo (Duan, Aguinaldo Datiles et al 2005, Lee, Lee et al 2006, Doukas, Eide et al 2009)

In vitro studies also indicated that PI-3K inhibitors were able to suppress eosinophil

migration, mast cells migration and degranulation (Ali, Bilancio et al 2004, Ali, Camps et

al 2008) Thus PI-3K inhibitors seemed to be a potential therapeutic drug for allergic airway disease

Collectively, these findings suggest that the constitutive activation of NF-κB pathway is one

of the primary causes in the pathogenesis of asthma and pharmacological agents targeting the NF-κB cascade may be promising in the treatment of asthma

1.1.5 Therapeutic strategies in the treatment of asthma exacerbation

There are currently some drugs for the treatment of rhinovirus-induced asthma exacerbation Inhaled corticosteroids (ICSs) are effective in the treatment of persistent asthma and childhood wheezing episodes (Pauwels, Lofdahl et al 1997) ICSs reduce asthma exacerbations among patient with persistent asthma but could not eliminate these events completely In addition, the ineffectiveness of this drug has been identified as a risk factor for asthma exacerbations related to rhinovirus infection (Venarske, Busse et al 2006)

Another kind of drug candidate, leukotriene receptor antagonists, called montelukast, when taken prophylatically or therapeutically, can mildly ameliorate but not eliminate asthma exacerbations, including those induced by viral infections According to one study, the use

of montelukast over the course of a year reduced the number of exacerbations by – 30%, however, these results should be interpreted with caution because the use of oral corticosteroids presumably prescribed for sever exacerbations were not reduced (Bisgaard, Zielen et al 2005)

The optimal choice for prevention of rhinovirus-induced exacerbations would be the development of anti-rhinovirus drug However, the vaccine is still not yet available because

of the antigenic diversity of more than 100 serotypes

IFNs have also been studied as therapeutic agents for rhinovirus infection According to one study, intranasal IFN-α has been shown to be effective in reducing transmission of rhinovirus-related colds in family members However, the adverse side effects, such as nasal obstruction limited its use (Hayden, Albrecht et al 1986) Other studies showed that IFN-β has similar anti-rhinovirus activities in vitro, but not effective when used in vivo (Sperber and Hayden 1989, Sperber, Levine et al 1989)

Pleconaril, which targets the viral capsid, has been shown to have modest benefit in reducing the severity and duration of common cold when taken within 24 hours from the onset of the cold symptoms (Hayden, Herrington et al 2003, Pevear, Hayden et al 2005) However, it was not approved in US because of its potential of induce viral resistance

Rhinovirus 3C protease is required for virus replication and the gene is quite conservative in the viral genome, which makes it a sensitive point for drug targeting (Rotbart 2000, Maugeri, Alisi et al 2008) Rupintrivir, which blocks the function of rhinovirus 3C protease, has been

shown to reduce the production of IL-6 and IL-8 in vitro (Zalman, Brothers et al 2000)

Clinical trials also showed that intranasal inoculation of rupintrivir reduced both the viral titer and the cold symptom score (Hayden, Turner et al 2003)

Other drug candidates, which target the virus, such as anti-ICAM-1, which blocks the binding of the virus to the cellular receptor (Hayden, Gwaltney et al 1988), could also be other kinds of promising drugs More effects need to be put in the development of these compounds to reduce the side effects to minimum

1.2 Rhinovirus

Respiratory viral infections, especially rhinovirus infection, are the major causes of asthma exacerbation With the help of new techniques such as RT-PCR, respiratory viral infections are identified in majority of asthma exacerbations in both children (80–85%) and adults (75–80%), with rhinovirus accounting for approximately 60% of all these infections (Johnston 2007) Rhinovirus, one cause of common flu, is a single-stranded small RNA

virus which belongs to the Picornaviridae family (Friedlander and Busse 2005) Since first

identified in 1953, rhinovirus has been shown to be a big family that includes more than 100 serotypes

1.2.1 Biology of rhinovirus

1.2.1.1 Transmission pathway

At the very beginning, the rhinovirus transmission from person to person was thought to be via direct contact or fomites In 1976, a study by D'Alessio, J et al showed that transmission rarely occurs unless the following conditions are fulfilled: (1) enough virus was recovered from the donor’s nasal washing, (2) the donor had virus on his hands and anterior nares, (3)

he was at least moderately symptomatic and (4) he spent many hours with his spouse (D'Alessio, Peterson et al 1976) According to this study, the person-to-person transmission was so dependent on time spent together and the shedding of large amount of virus It seems possible that rhinovirus was mainly transmitted via the direct contact or fomites routes This opinion was later strengthened by other studies Hendley Owen et al showed that transmission of rhinoviruses from infected person under natural conditions might proceed

by transfer of virus from hands of the infected person to an intermediary surface or directly

to the fingers of the susceptible recipient Infection then results from self-inoculation of eyes

or nose with virus on their fingers (Hendley, Wenzel et al 1973, Gwaltney, Moskalski et al 1978) However, later on in 1987, rhinoviruses transmission was shown to be chiefly via

aerosol route (Dick, Jennings et al 1987) Researchers showed that the infection rate of restrained recipients, who could not touch their faces and could only have been infected by aerosol, and that of unrestrained recipients, who could have been infected by aerosol, by direct contact, or by indirect fomite contact, was not significantly different This result indicated that aerosol route might be the only effective way of transmission

1.2.1.2 Structure of rhinovirus

Picornaviruses is one of the largest families of virus and can cause various of diseases, from

a common cold to foot-and-mouth disease Picornavirions has been divided into ten genera:

ebterovirus, cardiovirus, aphthovirus, erbovirus, hepatovirus, kobuvirus, parechovirus, teschovirus and rhinovirus Human rhinoviruses, like other picornaviruses, are icosahedral

assemblies of 60 protomers that envelop a single positive strand RNA (FIG.1-3) Each protomer includes four peptides, VP1, VP2, VP3 and VP4 VP1-VP3 forms the capsid while VP4 is inhered to the genome RNA (FIG.1-4) The external viral radius is around 150Å and the total molecular weight is about 8.5×106 KD (Olson, Kolatkar et al 1993) There is a canyon on the surface of the viron, which is around 12 Å deep and 12-15 Å wide This canyon has been shown to be the binding site of the cellular receptors and the residues inside this pocket has been shown to be more conserved compared with the residues in other site of the surface These conserved sites have been shown to be the binding sites of the receptors and thus block the binding of the neutralizing binding sites of antibodies (Kim, Smith et al 1989) Blockage of the binding site with several compounds could also stabilize the viron and actually many drugs act at the entry level by binding to these sites and thus inhibit the binding of the virus to the host cells(Cox, Buontempo et al 1996, Rotbart 2000) The RNA, which is crosslinked to VP4, is translated into a single polyprotein and then processed stepwise into smaller proteins(Pallansch, Kew et al 1984) Cleavage of the

are mediated by the proteins that are released from the precursor (Hanecak, Semler et al 1982)

Figure 1-3 Structure of the genome of rhinovirus (Adapted from Jacobs et al 2013)

Figure 1-4 Structure of the rhinovirus capsid protein (adapted from Friedlander et al 2005)

This figure shows the transverse section through the center of a pentamer depicting entry of its cellular receptor, ICAM-1, and the location of the drug-binding pocket just beneath the canyon floor Definition of abbreviations: ICAM-1 = intercellular adhesion molecule-1

1.2.1.3 Living temperature of rhinovirus

Human respiratory system has different temperature distribution from the nose to the pulmonary peripheral edge Researchers in one group showed that under quiet condition under room temperature, the average temperature ranged from 32.0 ± 0.05 ℃ in the upper tracheal to 35.5 ± 0.3℃ in the subsegmental bronchi The temperature at the lung periphery

is comparatively higher than that in the upper tracheal

Rhinovirus is considered as the most common cause for upper respiratory tract infection causes However, there are accumulative evidences indicating that rhinovirus can also cause infection in the lower respiratory tract Clinical studies showed that human rhinovirus was frequently found in the lower airways in infants with recurrent respiratory symptoms (Malmstrom, Pitkaranta et al 2006) Bronchoscopy was performed and the presence of HRV was examined using in situ hybridization HRV was found in 45% of the infants with recurrent respiratory symptoms and it was shown to be related to lung dysfunction These results indicated that rhinovirus could cause infection in the lower airway and impair lung function subsequently

In addition, rhinovirus has been shown to replicate effectively in the primary bronchial epithelial cells (Papadopoulos, Sanderson et al 1999) Nikolaos, Papadopoulos et al cultured different serotypes of rhinovirus at both 33 ℃ and 37 ℃ for several passages They found that the majority of the serotypes and wild-type viruses replicated slightly better at 33 ℃ than 37 ℃ However, titers achieved after one or more replication cycles at 37 ℃ were still high enough to initiate infection And some of the serotypes even replicated better at 37 ℃ These results indicated that rhinoviruses are capable to grow at higher temperature and they may have sufficient ability to infect the lower airway

Whether the epithelial cells in the lower airway have higher susceptibility to rhinovirus infection is another problem Study by Anne, Mosser et al demonstrated that, compared with the adenoidal epithelial cells, the primary bronchial epithelial cells have the same frequency of being infected (Mosser, Brockman-Schneider et al 2002)

The nature of remaining stable may also contribute to the transmission ability of rhinovirus (Reagan, McGeady et al 1981) Kevin, Reagan et al showed that rhinovirus was able to keep the capability of infection for 24 hours at both 6 ℃ and 23 ℃ in the liquid state After evaporation at 37 ℃, rhinovirus could still keep sufficient infectivity on the dry surface Their findings can explain the transmission route of rhinovirus, which is a non-airborne virus

The reason why rhinovirus is so sensitive to temperature is not clear There is evidence showing that temperature affects the binding, uncoating and assembly of rhinovirus particles (Oliveira, Ishimaru et al 1999)

1.2.1.4 Classification of rhinovirus

There are more than 100 serotypes of rhinovirus, which are classified into two main groups receptors (Bartlett, Walton et al 2008) based on their cellular receptors The major serotypes bind to intercellular adhesion molecular-1 (ICAM-1), whereas the minor serotypes bind to the low-density lipoprotein receptor The major kind of receptor was identified first Jeffery

M G et al found that major serotype bound to a 95 KD cell surface glycoprotein They sequenced this protein and found that the sequence is identical to the human ICAM-1 (Greve, Davis et al 1989) Later on, another study conducted by Gordon A et al showed that rhinovirus binds to different cellular receptors, which indicated that there could be another kind of cellular receptor (Abraham and Colonno 1984) In the year of 1993, Franz

H et al purified a protein that binds to minor group RV from Hela cell supernatants The

amino acid sequence was identical to human low density lipoprotein receptor (LDLR) (Hofer, Gruenberger et al 1994) There could be other cellular receptors that are used by the

RV Markete V et al found that HRV-89 (major group) was able to grow in the Hela cell line which was deficient in production of ICAM-1 In this experimental system, HRV-89 was shown to use the heparin sulfate proteoglycan as the receptor However, this HRV was shown to be less stable at low pH and high temperature (Greve, Davis et al 1989, Vlasak, Goesler et al 2005) The receptors for the newly identified HRV-C haven’t been found out

so far

Later on, another classification criterion was established based on the drug screening system Some antiviral compounds were found to be particularly active against one subset of rhinovirus while being inactive or less active than the average against another subset of rhinovirus Andries and Dewint at, al found that one of the antiviral compounds, WIN51771, had an antiviral spectrum clearly distinctive from a consensus spectrum or other capsid binding compounds, although all of them had been shown to bind to the same binding site Multivariate analysis of all the capsid binding compounds resulted that human rhinoviruses were classified into two groups-human rhinovirus-A (susceptible) and human rhinovirus-B (not susceptible)(Andries, Dewindt et al 1990) HRV-B group contains twice (67) more than HRV-A group (33) The minor serotype rhinoviruses, without exception, all fall into the HRV-B group Further investigation showed that the antiviral spectrum was not only related to the amino acid sequence in the binding site, but also related to the whole VP1 protein amino acid sequence And recently studies showed that it was consistent with the phylogenetic analysis of VP4/VP2 sequences (Andries, Dewindt et al 1990, Lau, Yip et al 2007)

The classification on the basis of cellular receptor specify and antiviral compounds

divergence between polioviruses and group A rhinoviruses, both sharing a similar antiviral compound-binding site Next, a further divergence from rhinovirus group A happened, resulting in group B rhinovirus with the same receptor specificity but a different antiviral compound binding site At last, a group of minor receptor-binding virus branched off (Andries, Dewindt et al 1990)

1.2.1.5 A novel family member of rhinovirus family-HRV-C

With the development of new techniques such as real-time PCR, more and more new viral species were discovered In 2007, a new rhinovirus group, HRV-C was firstly detected in America Lee Wai-Ming et al conducted a study to type the HRVs in nasal secretions of infants with frequent respiratory illness by comparing of a 260-bp variable sequence in the 5’ noncoding region with the homologous sequences of the 101 known serotypes (Lee, Kiesner

et al 2007) Of the 103 types of rhinoviruses identified in this study, surprisingly, 54 (52.4%) HRVs did not match any of the known serotypes and had 12–35% nucleotide divergence from the nearest reference HRVs Of these novel viruses, 9 strains (17 HRVs) segregated from HRVA, HRVB and human enterovirus into a distinct genetic group (‘‘C’’) None of these new strains could be cultured in traditional cell lines, until recently HRV-C15 was successfully grown in human sinuses cells (HSEC)(Ashraf, Brockman-Schneider et al 2013) Since then, HRV-C was repeatedly detected worldwide (Lau, Yip et al 2009, Arden and Mackay 2010, Piralla, Baldanti et al 2011)

The cellular receptor of HRV-C has jet to be identified Research has shown that it is neither ICAM-1 nor LDLR (Bochkov, Palmenberg et al 2011) The failure of growth in existing cell culture system may also indicate the lack of proper cellular receptors in these cell cultures, because when the full length viral RNA-transcripts synthesized in vitro were