Modulation of nuclear factor b signaling attenuates allergic airway inflammation 1

IV Table of contents List of publications and conference abstract XIX 1.1 Asthma 1.1.1 Epidemiology of asthma and impetus to develop novel anti- 2 inflammatory agents 1.1.2 Pathophysiol

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

I

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

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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

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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

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Table of contents

List of publications and conference abstract XIX

1.1 Asthma

1.1.1 Epidemiology of asthma and impetus to develop novel anti- 2 inflammatory agents

1.1.2 Pathophysiology / Development of asthma 6

1.1.2.1 Airway Epithelial cells 8

1.1.2.3 B cell IgE production and mast cell activation 25

1.1.2.6 Airway hyperresponsiveness (AHR) 39 1.1.3 Current treatments and future direction for asthma 41

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

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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

3.5 Collection of bronchoalveolar lavage fluid (BALF) from mice 85 3.6 Preparation of BALF for total and differential cell count 85

3.10 Enzyme-linked immunosorbent assay (ELISA)

3.10.1 Cytokines and chemokine levels in BALF or 93 cell culture supernatant

3.11 RNA harvest and mRNA expression quantification

3.11.2 Preparation of lung samples for RNA harvest 95 3.11.3 Preparation of cell culture samples for RNA harvest 95

3.11.5 Reverse transcription (RT)-polymerase chain reaction (PCR) 96

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3.14 NF-κB reporter gene assay

3.14.2 NF-κB/Secreted alkaline phosphatase (SEAP) 100 reporter gene assay

4 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

4.1.3 Rip-2 siRNA suppresses OVA-induced inflammatory cell 109 recruitment and mucus production

4.1.4 Rip-2 siRNA reduces OVA-induced BALF cytokines and 114 serum IgE

4.1.5 Rip-2 siRNA suppresses OVA-induced inflammatory gene 114 expression in lungs

4.1.6 Rip-2 siRNA reduces OVA-induced AHR 114 4.1.7 Rip-2 silencing disrupts NF-κB signaling pathway 116

5.1 Results

5.1.2 Effects of RPS-3 siRNA on TNF-α-induced MUC5AC 128 production

5.1.3 Effects of RPS-3 siRNA on TNF-α-induced inflammatory 132 cytokines and mediators

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5.1.4 Effects of RPS-3 siRNA on NF-κB activity 132

6 Fisetin, a bioactive flavonol, attenuates allergic airway inflammation in mice 145

6.1 Results

6.1.1 Fisetin suppresses OVA-induced inflammatory 146 cell recruitment in BALF

6.1.2 Fisetin reduces OVA-induced airway cell infiltration 146

nd mucus production 6.1.3 Fisetin abates cytokine levels in BALF and serum 150

Ig production

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 158 NHBE cells

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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

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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

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List of tables

1.2 A comprehensive list of known down-stream targets of NF-κB

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List of figures

1.2 Inflammatory and immune cells involved in allergic airway

inflammation

8

1.3 T cells involved in the induction of allergic phenotype 13

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

1.14 Network of the Rip family members in the multiple cellular signaling

pathway

61

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

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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

5.1 Inhibition of RPS-3 mRNA by RPS-3 siRNA in human cell line

NCI-H292 (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

5.10 Effects of RPS-3 siRNA on NF-κB activity in NF-κB/SEAPorter™

HEK293 cell line

140

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

6.10 Effects of fisetin on OVA-induced NF-κB activity in vivo 161

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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 1

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

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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 1 receptor

CysLT2R Cysteinyl leukotriene 2 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

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

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FcεRII Low affinity IgE receptor

FEV1 Forced expiratory volume in 1 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

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

IκB Inhibitor of NF-Κb

LABA Long acting β2 agonist

LPA Lysophophatidic acid

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LPS Lipoploysaccharide

LRR Leucine-rich repeat

LTB4 receptor Leukotriene B4 receptor 1

LTA Lipoteichoic acid

LTRA Leukotriene receptor antagonist

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

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