Modulation of nuclear factor b signaling attenuates allergic airway inflammation 1

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.

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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
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

1. Introduction

1

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

1.1.2.2 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
1. 2 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

2. Rationale and objectives

75

3. 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

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

4.1.2 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

5. 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

6. 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

7. Conclusion

167

8. References

170

9. 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.
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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.

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

5

103

X


List of figures
Figure

Title

Page

1.1

World map of the prevalence of clinical asthma

4

1.2

Inflammatory and immune cells involved in allergic airway
inflammation

8

1.3

T cells involved in the induction of allergic phenotype

13

1.4

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

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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

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 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

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 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

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 1
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

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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 9

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