MODULATION OF NUCLEAR FACTOR-κB SIGNALING ATTENUATES ALLERGIC AIRWAY INFLAMMATION GOH YIQIAN FERA (B.Sc, Hons.) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF PHARMACOLOGY NATIONAL UNIVERSITY OF SINGAPORE 2012 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 Goh Yiqian Fera 18 October 2012 I Acknowledgement I would like to express my gratitude to Professor Fred Wong Wai-Shiu for his kind supervision and guidance in my research work I am grateful for his ideas, thoughts, experience, suggestions and discussion time that have made our work possible I am appreciative of Dr Cheng Chang for sharing her valuable research experience with me, teaching me numerous research techniques, and helping me with my research on bioactive flavonol; Dr Winston Liao Wu Peng for teaching me transfection techniques; Guan Shou Ping for teaching me animal work techniques; Eugene Ho and Peh Hong Yong, for sharing with me their research experience and providing me with useful ideas; Tao Lin, for coming over the weekends to help me with my mouse asthma model; Alan Koh, for helping me with microscopy work; Lah Lin Chin, for helping me and providing useful comments for my project; as well as Genevieve Seow, Jonathan Lim, Khaing, Loh Xinyi, and Zhang Xueyu for their help and constant encouragement I would like specially thank Nadine Upton Erica Gill and Katrina Louisa Tsu Ping Cook, exchange students from King’s College London They stayed back late in the laboratory and came back over the weekends to help me with my projects They have provided invaluable assistance to my projects This project would not have been completed without their contributions My sincere appreciation goes to our collaborators Professor Christopher Thiemermann, A/Professor Alonso Slyvie, Dr Bernard Leung, Dr Gautam Sethi, Dr Li Rui, Dr Muthu Shanmugam, and Dr Nimesh Patel for all the learning opportunities they have given me I wish to thank all professors and seniors from the department of Pharmacology, NUS, from whom I have learnt a lot during my graduate studies II Lastly, I would like to thank my friends and my family for their care and support They are my source of motivation to persevere and strive for excellence in my work III Table of contents Declaration I Acknowledgements II Table of contents IV Summary VIII List of tables X List of figures XI List of abbreviations XIII List of publications and conference abstract XIX Introduction 1.1 Asthma 1.1.1 Epidemiology of asthma and impetus to develop novel anti- inflammatory agents 1.1.2 Pathophysiology / Development of asthma 1.1.2.1 Airway Epithelial cells 1.1.2.2 T cell 11 1.1.2.3 B cell IgE production and mast cell activation 25 1.1.2.4 Eosinophils 27 1.1.2.5 Mucus hypersecretion 30 1.1.2.6 Airway hyperresponsiveness (AHR) 39 1.1.3 Current treatments and future direction for asthma Nuclear Factor (NF)-κB signaling pathway 41 48 1.2.1 Introduction of NF-κB signaling pathway 48 1.2.2 Role of NF-κB pathway in allergic inflammation 55 1.3 Receptor interacting protein (Rip) -2 and NF-κB signaling pathway 57 1.4 Ribosomal protein (RP)S -3 and NF-κB signaling pathway in lung cell lines 62 1.5 Small interfering RNA (siRNA) mechanism 64 1.6 Pharmacology of fisetin 70 IV 1.7 The Animal model of asthma 71 Rationale and objectives 75 Materials and methods 78 3.1 Materials and reagents 79 3.2 siRNA preparation 3.2.1 ON-TARGETplus siRNA without in vivo processing 81 3.2.2 ON-TARGETplus siRNA with in vivo processing 81 3.3 siRNA transfection in vitro 81 3.4 Mouse asthma model and treatments 83 3.5 Collection of bronchoalveolar lavage fluid (BALF) from mice 85 3.6 Preparation of BALF for total and differential cell count 85 3.7 Histological examination 87 3.8 AHR measurement 90 3.9 Lymphocyte antigen recall experiment 93 3.10 Enzyme-linked immunosorbent assay (ELISA) 3.10.1 Cytokines and chemokine levels in BALF or cell culture supernatant 93 3.10.2 Immunoglobulin (Ig) levels in serum 94 3.10.3 MUC5AC level in cell lysate 94 3.11 RNA harvest and mRNA expression quantification 3.11.1 Storage of lung samples 95 3.11.2 Preparation of lung samples for RNA harvest 95 3.11.3 Preparation of cell culture samples for RNA harvest 95 3.11.4 RNA harvest 96 3.11.5 Reverse transcription (RT)-polymerase chain reaction (PCR) 96 3.11.6 Realtime (RT)-PCR 96 3.11.7 Semi-quantitative PCR 96 3.12 Immunoblotting 97 V 3.12.1 Lung protein extraction 97 3.12.2 Tissue protein nuclear extraction 97 3.12.3 Cell line protein extraction 98 3.12.4 Immunoblot 98 3.13 NF-κB transactivation assay (TransAM) 98 3.14 NF-κB reporter gene assay 3.14.1 NF-κB luciferase gene reporter assay 99 3.14.2 NF-κB/Secreted alkaline phosphatase (SEAP) reporter gene assay 100 3.15 Band intensity analysis 100 3.16 Statistical analysis 101 Rip-2 gene silencing attenuates allergic airway inflammation in mice 104 4.1 Results 4.1.1 In vitro characterization of Rip-2 siRNA 105 4.1.2 Rip-2 silencing in vivo 105 4.1.3 Rip-2 siRNA suppresses OVA-induced inflammatory cell recruitment and mucus production 109 4.1.4 Rip-2 siRNA reduces OVA-induced BALF cytokines and serum IgE 114 4.1.5 Rip-2 siRNA suppresses OVA-induced inflammatory gene expression in lungs 114 4.1.6 Rip-2 siRNA reduces OVA-induced AHR 114 4.1.7 Rip-2 silencing disrupts NF-κB signaling pathway 116 4.2 Discussion 116 Anti-inflammatory effects of RPS3 siRNA in vitro 127 5.1 Results 5.1.1 RPS-3 gene silencing in vitro 128 5.1.2 Effects of RPS-3 siRNA on TNF-α-induced MUC5AC production 128 5.1.3 Effects of RPS-3 siRNA on TNF-α-induced inflammatory cytokines and mediators 132 VI 5.1.4 Effects of RPS-3 siRNA on NF-κB activity 5.2 Discussion 132 141 Fisetin, a bioactive flavonol, attenuates allergic airway inflammation in mice 145 6.1 Results 6.1.1 Fisetin suppresses OVA-induced inflammatory cell recruitment in BALF 146 6.1.2 Fisetin reduces OVA-induced airway cell infiltration nd mucus production 146 6.1.3 Fisetin abates cytokine levels in BALF and serum Ig production 150 6.1.4 Fisetin prevents OVA-specific lymphocyte responses in viv o 154 6.1.5 Fisetin blocks OVA-induced inflammatory gene expression 154 6.1.6 Fisetin attenuates OVA-induced AHR in mice 158 6.1.7 Fisetin disrupts NF-κB DNA activity in vivo 158 6.1.8 Effects of fisetin on TNF-α-induced NF-κB activation in NHBE cells 158 6.2 Discussion 159 Conclusion 167 References 170 Appendix 233 VII Summary Asthma is an inflammatory lung disorder that accounts for significant morbidity and mortality worldwide Corticosteroid remains the first-line of treatment of this disease However, such treatment is associated with side-effects and does not change the chronic cause of the disease Therefore, there is an unmet medical need to develop novel anti-inflammatory therapy Studies have associated pathogenesis of allergic airway inflammation with persistent nuclear factor (NF)-κB signaling pathway activation This association has made NF-κB, a master pro-inflammatory transcription factor, an attractive therapeutic target for asthma However, NF-κB is essential for both normal biological functions and pathological conditions Therefore, total inhibition of NF-κB may not be a safe approach Appropriate and specific negative-regulation of the aberrantly elevated NF-κB activity pathway may be a novel anti-inflammatory strategy for allergic airway inflammation The objectives of my project were to investigate the potential anti-inflammatory action of molecules that can interfere with NF-κB signaling pathway: (1) Rip-2 siRNA; (2) RPS-3 siRNA; and (3) bioactive flavonol fisetin The potential anti-inflammatory effect of Rip-2 siRNA was examined in an ovalbumin (OVA)induced mouse asthma model A potent and selective Rip-2 siRNA given intratracheally knocked down Rip-2 expression in OVA-challenged lungs, and reduced OVA-induced increases in total and eosinophil counts, and Th2 cytokines and eotaxin levels in bronchoalveolar lavage fluid Rip-2 silencing blocked OVA-induced inflammatory cell infiltration and mucus hypersecretion as observed in lung sections, and mRNA expression of adhesion molecules and inflammatory mediators in lung tissues Also, elevation of serum OVA-specific IgE level in mouse asthma model was markedly suppressed by Rip-2 siRNA Furthermore, Rip-2 siRNA-treated mice produced significantly less airway hyperresponsiveness induced by methacholine Mechanistically, Rip-2 siRNA was found to enhance cytosolic level of IκB, and block p65 nuclear translocation and DNA binding activity in lung tissues from OVA-challenged mice The potential anti-inflammatory effect of RPS-3 siRNA was examined using TNF-α-stimulated lung cell lines Transfection of RPS-3 siRNA in lung cell lines significantly knock-down RPS-3 expression VIII The down-regulation of RPS-3 expression was accompanied by suppressed NF-κB activation For the first time, we observed that down-regulation of RPS-3 suppressed TNF-α-induced MUC5AC expression and IL-8 production None of these effects were reproducible by control siRNA Finally, the potential anti-inflammatory effect of fisetin was investigated in OVA-induced mouse asthma model Intravenous administration of fisetin dose-dependently inhibited OVA-induced increases in total cell count, eosinophil count, and Th2 cytokine levels recovered in bronchoalveolar lavage fluid It attenuated OVA-induced lung tissue eosinophilia and airway mucus production, mRNA expression of adhesion molecules, chitinase, and inflammatory mediators in lung tissues, and airway hyperresponsiveness to methacholine Fisetin blocked NF-κB subunit p65 nuclear translocation and DNA-binding activity in the nuclear extracts from lung tissues of OVA-challenged mice In normal human bronchial epithelial cells, fisetin repressed TNF-α-induced NF-κB-dependent reporter gene expression In this Ph.D project, I have demonstrated that intervention of NF-κB signaling pathway is linked to attenuation of inflammation These results suggest that Rip-2 siRNA, RPS-3 siRNA, and fisetin may have therapeutic potential for treatment of allergic airway inflammation IX List of tables Table Title Page 1.1 The economic burden of asthma 1.2 A comprehensive list of known down-stream targets of NF-κB signaling pathway relevant to asthma 47 1.3 siRNA therapeutics in clinical trial 51 1.3 siRNA in animal studies 67 3.1 Sequences of siRNA 82 3.2 Primer sets for RT-PCR 103 X List of figures Figure Title Page 1.1 World map of the prevalence of clinical asthma 1.2 Inflammatory and immune cells involved in allergic airway inflammation 1.3 T cells involved in the induction of allergic phenotype 13 1.4 Components of airway allergic inflammation 17 1.5 Inflammatory mediators derived from eosinophil 29 1.6 Impact of mucus hypersecretion on airway obstruction in asthma 32 1.7 Molecular control of goblet cell metaplasia in asthma 34 1.8 Signaling pathway involved in TNF-α-mediated mucin synthesis 37 1.9 Molecular mechanism involved in IL-1β -mediated mucin expression 38 1.10 Members of NF-κB family 52 1.11 Canonical and non-canonical pathway of NF-κB 54 1.12 The nuclear regulation of NF-κB 58 1.13 Domain organization of the Rip family 59 1.14 Network of the Rip family members in the multiple cellular signaling pathway 61 1.15 NF-κB mechanism of action 63 1.16 Mechanism of siRNA silencing 66 1.17 The structure of fisetin 71 3.1 OVA aerosolization system 86 3.2 Types of inflammatory cells in mouse BALF 88 3.3 Invasive system 92 4.1 Sequence dependent inhibition of Rip-2 mRNA and protein expression by S2 in mouse cell lines 106 4.2 Inhibition of Rip-2 protein expression and mRNA by S2 in mouse lungs 107 4.3 Effects of S2 on OVA-induced inflammatory cell recruitment and mucus hypersecretion 110 XI 4.4 Effects of S2 on OVA-induced BALF cytokine and chemokine levels and serum Ig production 113 4.5 Effects of S2 on inflammatory gene expression in allergic airway inflammation 115 4.6 Effects of S2 on OVA-induced AHR 117 4.7 Effects of S2 on NF-κB activity 118 5.1 Inhibition of RPS-3 mRNA by RPS-3 siRNA in human cell line NCIH292 (lung mucoepidermoid carcinoma) 129 5.2 Inhibition of RPS-3 mRNA by RPS-3 siRNA in human cell line BEAS-2B (lung epithelial cell line) 130 5.3 Effects of RPS-3 siRNA on MUC5AC expression in NCI-H292 cells 131 5.4 Effects of RPS-3 siRNA on IL-8 expression in NCI-H292 cells 133 5.5 Effects of RPS-3 siRNA on IL-8 expression in BEAS-2B cells 134 5.6 Effects of RPS-3 siRNA on IL-6 expression in NCI-H292 cells 135 5.7 Effects of RPS-3 siRNA on IL-6 expression in BEAS-2B cells 136 5.8 Effects of RPS-3 siRNA on TSLP expression 137 5.9 Effects of RPS-3 siRNA on p65 DNA-binding activity 138 5.10 Effects of RPS-3 siRNA on NF-κB activity in NF-κB/SEAPorter™ HEK293 cell line 140 6.1 Differential cell counts in BALF 147 6.2 Effects of fisetin on BALF cell infiltration 148 6.3 Effect of fisetin on PBMC 149 6.4 Histological examination of lung tissue 151 6.5 Effects of fisetin on OVA-induced BALF cytokine and chemokine levels 152 6.6 Effects of fisetin on OVA-induced serum Ig production 153 6.7 Effects of fisetin on OVA-specific responses in vivo 155 6.8 Effects of fisetin on pulmonary mRNA expression of inflammatory genes 157 6.9 Effects of fisetin on OVA-induced AHR 160 6.10 Effects of fisetin on OVA-induced NF-κB activity in vivo 161 XII List of abbreviations Ad12SV40 Adenovirus 12-simian virus 40 hybrid virus AHR Airway hyperresponsiveness Al(OH)3 Aluminium hydroxide AMCase Acidic mammalian chitinase AMD Age related macular degeneration AMV Avian mammalian chitinase ANK Ankyrin repeats AP Alkaline phosphatase AP-1 Activating protein-1 APC Antigen presenting cell ASM Airway smooth muscle ASO Antisense oligonucleotide ATP Adenosine Triphosphate BALF Bronchoalveolar lavage fluid BCA Bicinchonic acid BLT1 Leukotriene B4 receptor BSA Bovine serum albumin Cdyn Dynamic compliance c/EBP CCAAT/enhancer binding protein cAMP Cyclic adenosine monophosphate CARD Caspase activation and recruitment domain CBP cAMP response element-binding protein (CREB) binding protein CCL Chemokine ligand CCR Chemokine receptor CD Cluster of differentiation XIII CD40L Cluster of differentiation 40 ligand CFTR Cystic fibrosis transmembrane conductance regulator CRTH2 Chemoattractant homologous receptor expressed on Th2 cells CTLA Cytotoxic T-lymphocyte antigen CXCR Chemokine (C-X-C motif) receptor CysLT Cysteinyl leukotrienes CysLT1R Cysteinyl leukotriene receptor CysLT2R Cysteinyl leukotriene receptor DAMP Danger-associated molecular pattern DMSO Dimethyl sulfoxide DNA Deoxyribonucleotide DOTAP 1,2-Dioleoyl-3-trimethylammonium-propane ECM Extracellular matrix ECP Eosinophil cationic Protein EDN Eosinophil-derived neurotoxin EGF Epidermal growth factor EGFR Epidermal growth factor receptor EMSA Electrophoretic mobility shift essay Eos Eosinophil EP2 Prostaglandin E2 receptor EPO Eosinophil peroxidase ER Endoplasmic reticulum ERK Extracellular signal regulated kinase FBS Fetal bovine serum FCS Fetal calf serum FcεRI High affinity IgE receptor XIV FcεRII Low affinity IgE receptor FEV1 Forced expiratory volume in second FOXP3 Forkhead box P3 H&E Hematoxylin and Eosin GATA trans-acting T cell-specific transcription factor GM-CSF Granulocyte-macrophage colony-stimulating factor hCLCA1 human calcium-activated chloride channel hnRNP K Heterogeneous nuclear protein K ICAM-1 Intercellular adhesion molecule-1 ICOS Inducible costimulatory molecules IFN Interferon Ig Immunoglobulin IKK Inhibitor of NF-κB kinase IL Interleukin IL-4R IL-4 receptor IN Intranasal iNOS Inducible nitric oxide synthase IP Intraperitoneal IRE Inositol-requiring enzyme IT Intratracheal IV Intravenous IκB Inhibitor of NF-Κb JAK Janus kinase KD Kinase domain LABA Long acting β2 agonist LPA Lysophophatidic acid XV LPS Lipoploysaccharide LRR Leucine-rich repeat LTB4 receptor Leukotriene B4 receptor LTA Lipoteichoic acid LTRA Leukotriene receptor antagonist Lym Lymphocyte Mac Macrophage MAPK Mitogen-activated protein kinases MARCKS Myristoylated alanine-rich C-kinase substrate MBP Major basic protein MCP Monoctye chemotatic protein MEKK Mitogen-activated protein/extracellular signal-regulated kinase kinase MHC Major histocompatitbility complex MSK Mitogen and stress activating kinases Myd Myeloid differentiation primary response gene NEMO NF-κB essential modulator Neu Neutrophil NFAT Nuclear factor activated T cell NF-κB Nuclear factor-κB NHBE Normal human bronchial epithelial NH4Cl Ammonium chloride NKT Natural killer cell NOD Nucleotide oligomerisation domain NLRP NOD-like receptor protein NPRA Natriuretic peptide receptor A OVA Ovalbumin XVI PAF Platelet activating factor PAMP Pathogen associated molecular patterns PBS Phosphate buffered saline PBMC Peripheral blood mononuclear cell PCR Polymerase chain reaction PDE Phosphodiesterase PG Prostaglandin PKA Protein Kinase A PKAc Protein Kinase A catalytic subunit PLA2 Phospholipase A2 PRR Pattern recognition receptors RANKL Receptor activator NF-κB ligand RANTES Regulated and normal T cell expressed and secreted Rel v-rel reticuloendotheliosis viral oncogene homolog RHD Rel homology domain RHIM Rip homolytic interaction domain Rip Receptor interacting protein RISC RNA-induced silencing complex Rl Lung resistance RNA Ribonucleic acid RNAi RNA interference Roc/COR Ros of complex proteins/C-terminal of Roc ROR Orphan retinoic acid nuclear receptor ROS Reactive Oxygen Species SCF Stem cell factor SDS Sodium dodecyl sulfate XVII SiRNA Small interfering RNA SIT Specific immunotherapy SLPI Secretory Leukocyte Protease Inhibitor SNALPs Stable nucleic acid-lipid particules SPDEF SAM pointed domain-containing Ets transcription factor SphK Sphingosine kinase STAT Signal transducer and activator of transcription TAE Tris-acetate-Ethylenediaminetetraacetic acid (EDTA) TAK TGF-β activated kinase TAS2R Agonists of bitter taste receptors TCR T cell receptor TEMED Tetramethylethylenediamine TGF-β Transforming growth factor β TH T-helper TLR Toll-like receptor TMB Tetramethylbenzidine TNF Tumor necrosis factor Treg T-regulatory cells TSLP Thymic stromal lymphopoietin protein UPR Unfolded protein response UV Ultraviolet VCAM-1 Vascular cell adhesion molecule-1 VEGF Vascular endothelial growth factor VLA Very late antigen XBP X box-binding protein XVIII List of Publications and conference abstracts Publications Liao W, Goh FY, Betts RJ, Kemeny DM, Tam J, Bay BH, Wong WS 2011 A novel anti-apoptotic role for apolipoprotein L2 in IFN-γ-induced cytotoxicity in human bronchial epithelial cells J Cell Physiol Feb; 226(2):397-406 Cheng C, Ho WE, Goh FY, Guan SP, Kong LR, Lai WQ, Leung BP, Wong WS 2011 Anti-malarial drug artesunate attenuates experimental allergic asthma via inhibition of the phosphoinositide 3kinase/Akt pathway PloS One 6(6):e20932 Epub 2011 Jun Patel NS, Nandra KK, Brines M, Collino M, Wong WF, Kapoor A, Benetti E, Goh FY, Fantozzi R, Cerami A, Thiemermann C 2011 A nonerythropoietic peptide that mimics the 3D structure of erythropoietin reduces organ injury/dysfunction and inflammation in experimental hemorrhagic shock Mol Med Sep-Oct;17(9-10):883-92 Goh FY, Upton N, Guan S, Cheng C, Shanmugam MK, Sethi G, Leung BP, Wong WS 2012 Fisetin, a bioactive flavonol, attenuates allergic airway inflammation through negative regulation of NF-κB Eur J Pharmacol Mar 15;679(1-3):109-16 Epub 2012 Jan 20 Nandra KK, Takahashi K, Collino M, Benetti E, Wong WS, Goh FY, Suzuki K, Patel NS, Thiemermann C.2012 Acute treatment with bone marrow-derived mononuclear cells attenuates the organ injury/dysfunction induced by hemorrhagic shock in the rat Shock Jun;37(6):592-8 Li R, Cheng C, Chong SZ, Lim AR, Goh YF, Locht C, Kemeny DM, Angeli V, Wong WS, Alonso S 2012 Attenuated Bordetella pertussis BPZE1 protects against allergic airway inflammation and contact dermatitis in mouse models Allergy Oct;67(10):1250-8 Goh FY, Cook K, Upton N, Tao L, Lah LC, Wong WS Rip-2 Gene silencing attenuates allergic airway inflammation J Immuno Revision, 2012 Conference abstracts Cold Spring Harbor Asia Conferences: Frontiers of Immunology in Health and Diseases Suzhou, China (September 3rd - 7th 2012) Rip-2 silencing attenuates allergic airway inflammation in mice Goh FY, Cook KTP, Upton N, Tao L, Lah LC and Wong WSF Presented by Professor Fred Wong Wai-Shiu 7th Annual RNAi Conference 2012: Gene Regulation by Small RNAs Oxford, United Kingdom (March 27th - 29th 2012) Rip-2 silencing attenuates allergic airway inflammation in mice Goh FY, Cook KTP, Upton N, Tao L, Lah LC and Wong WSF Poster presented by Goh Yiqian Fera XIX ... Development of asthma 1. 1.2 .1 Airway Epithelial cells 1. 1.2.2 T cell 11 1. 1.2.3 B cell IgE production and mast cell activation 25 1. 1.2.4 Eosinophils 27 1. 1.2.5 Mucus hypersecretion 30 1. 1.2.6 Airway. .. in IL -1? ? -mediated mucin expression 38 1. 10 Members of NF-? ?B family 52 1. 11 Canonical and non-canonical pathway of NF-? ?B 54 1. 12 The nuclear regulation of NF-? ?B 58 1. 13 Domain organization of the... 59 1. 14 Network of the Rip family members in the multiple cellular signaling pathway 61 1 .15 NF-? ?B mechanism of action 63 1. 16 Mechanism of siRNA silencing 66 1. 17 The structure of fisetin 71