ANTIMALARIAL DRUG ARTESUNATE IN THE TREATMENT OF RHINOVIRUS-INDUCED ASTHMA EXACERBATION TAO LIN (B.Sc) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF PHARMACOLOGY NATIONAL UNIVERSITY OF SINGAPORE 2014 DECLARATION I hereby declare that this thesis is my original work and it has been written by me in its entirety I have duly acknowledged all the sources of information which have been used in the thesis This thesis has also not been submitted for any degree in any university previously Tao Lin 22 Jan 2014 ii 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 iii TABLE OF CONTENTS ACKNOWLEDGEMENT iii TABLE OF CONTENTS iv LIST OF TABLES x LIST OF FIGURES xi LIST OF ABBREVIATIONS xiii INTRODUCTION 1.1 Asthma 1.1.1 Epidemiology and etiology and risk factors of asthma 1.1.2 Pathogenesis of asthma 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 iv 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 RATIONALE & AIM 50 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 v 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 RESULTS 72 vi 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 DISCUSSION 98 REFERENCE 114 vii 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 viii 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) ix LIST OF TABLES Table Title Page Table 3-1 Targets and sequences for primer sets of RT-PCR 71 x Muniz-Junqueira, M I., S M Barbosa-Marques and L F Junqueira, Jr (2013) "Morphological changes in eosinophils are reliable markers of the severity of an acute asthma exacerbation in children." 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Am J Physiol 273(4 Pt 1): L814-824 156 ... 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. .. 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... 2005) 14 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