75











2. Rationale and objective













76

1. Rational and objective
Globally 300 million people suffer from asthma and the prevalence of asthma still continues
to increase (Pawankar et al., 2012). Asthma is a multifactorial disease, involving a complex

network of molecular and cellular interaction. In addition to genetic predisposition,
environmental factors also mediates the initiation and development of asthma (von Mutius,
2009).

Current therapies for asthma generally rely on SABAs or LABAs and inhaled corticosteroids.
Although these current therapies are relatively effective at controlling symptoms, these
therapies do not change the chronic course of disease. In addition, major concerns about the
systemic effects of inhaled corticosteroids remains. Furthermore, around 15% of asthmatic
patients who suffer from uncontrollable asthmatic symptoms fail to respond well to inhaled
corticosteroids. Currently, there is no established method to prevent asthma. Therefore, the
major unmet needs of this area include better management of the severe forms of the disease
and the developments of curative therapies (Akdis, 2012). Consequently, much research has
been done to better understand the pathophysiology of asthma and to explore novel therapies
for this asthma.

One attractive target for therapeutic intervention would be the NF-κB signaling pathway,
which plays an important role in Th2-mediated inflammation (Edwards et al., 2009). Thus,
the development of specific inhibitors targeting NF-κB signaling pathway is promising for
attenuating allergic airway inflammation. Nonetheless, direct inhibition of NF-κB may not be
a safe approach because NF-κB plays a pivotal role in numerous normal biological functions
and pathological conditions. Therefore, a safer and yet effective anti-inflammatory approach
for attenuation of allergic airway inflammation would be through appropriate and specific
inhibition of signaling molecules which regulate the activation of NF-κB (Uwe, 2008).
Rip-2 has been shown to mediate the formation of functional IKK complex, which is essential
for NF-κB activation; while RPS-3 has been demonstrated to enhance the binding of NF-κB
77

to the κB sites of selected NF-κB targets. Fisetin, on the other hand, is a natural product that
has been reported to interfere with the activity of NF-κB pathway. The objectives of my
project are to examine the potential anti-inflammatory effects of Rip-2 siRNA, RPS-3siRNA,

and fisetin, a bioactive flavonol in mouse asthma model or TNF-α stimulated lung cell lines
and investigate their effects in the regulation of NF-κB pathway. Future aims include
developing efficacious therapies which are easy to comply with and have minimal systemic
side effects.


















78









3. Materials and methods


















79

3.1 Materials and Reagents
Drugs and chemicals used in this Ph.D. project are as follows: Bicinchonic acid (BCA)
protein assay kit, calf bovine serum (CBS); custom control siRNA; fetal bovine serum (FBS);
M-PER Mammalian Protein Extraction Reagent containing phosphatase inhibitor; ON-
TARGETplus Receptor interacting protein (Rip)-2 small interfering RNA (siRNA); ON-
TARGETplus Rip-2 siRNA with in vivo processing; ON-TARGETplus Ribosomal protein
(RP)S3 siRNA, RestoreTM PLUS western blot stripping buffer; and 2 X Sybrgreen master
mix were obtained from Thermo Scientific, Waltham, MA, USA. Acetyl-β-methylcholine

chloride; aluminium hydroxide (Al(OH)
3
; bovine serum albumin (BSA); chicken ovalbumin
(OVA), dimethyl sulfoxide (DMSO); eosin Y, fisetin; G418; harris hematoxylin solution; 10%
neutral buffered formalin; skim milk powder; tissue culture grade 10 X phosphate buffered
saline (PBS); tween-20 were obtained from Sigma-Aldrich, St. Louis, MO, USA. Ammonium
chloride (NH
4
Cl) was obtained from BDH Laboratory Supplies, Poole, England. Agarose;
alkaline phosphatise (AP) conjugated substrate kit; blotting paper, polyvinylidene difluoride
(PVDF) membrane; tetramethylbenzidine (TMB) substrate kit; tetramethylethylenediamine
(TEMED); and 3,3’,5,5’- Triton X-100 were obtained from Bio-Rad laboratories, Hercules,
CA, USA). NF-κB/Secreted alkaline phosphatase (SEAP) gene reporter assay kit was
obtained from Imgenex, San Diego, CA, USA. Anti-β-actin monoclonal antibody, anti-mouse
Rip-2 monoclonal antibody, anti-human RPS-3 monoclonal antibody were obtained from
Abcam, Cambridge, UK. Diethylpyrocarbonate (DEPC)-treated water; Dulbecco’s modified
Eagle medium (DMEM) with 4.5 g/L glucose; Lipofectamine 2000; optimum I reduced serum
medium; Recombinant human tumor necrosis factor (TNF)-α; Roswell Park Memorial
Institute (RPMI) Medium; RNAlater; TRIzol; trypan blue were obtained from Invitrogen,
Carlsbad, CA, USA. Anesthetic mixture (10 l/g ketamine: medetomidine: H
2
O = 3:4:3);
Sterile NaCl 0.9% were obtained from Animal Holding Unit AHU (NUS, Singapore).
Aerosolised isofluorane was obtained from Halocarbon, NJ, USA. Fetal bovine serum (FBS)
was obtained from Hyclone Laboratories, South Logan, Utah, USA). Sodium dodecyl sulfate
80

(SDS) and tris-acetate-EDTA (TAE) were obtained from 1st BASE, Singapore). Anti-mouse
IL-13 monoclonal antibody; biotinylated anti-mouse IL-13 antibody; recombinant murine IL-
13, anti-mouse eotaxin; biotinylated anti-mouse eotaxin; and recombinant mouse eotaxin

(R&D Systems, Minneapolis, MN, USA), Avidin-horseradish peroxidise (HRP); biotinylated
anti-mouse IgE; biotinylated anti-mouse OptEIA™ mouse IFN-γ set, biotinylated anti-mouse
OptEIA™ IgG1; biotinylated antimouse IgG2aOptEIA™ mouse; total IgE set; OptEIA™
mouse IL-4 set; OptEIA™ mouse IL-5 set, OptEIA™ mouse IL-33 set, OptEIA™ human IL-
6 set; and OptEIA™ human IL-8 set were obtained from BD Biosciences Pharmingen, San
Diego, CA, USA. Fluoresave was obtained from Calbiochem, Billerica, MA. Sodium
carbonate (Na
2
CO
3
) was obtained from Kanto Chemical, Tokyo, Japan. Absolute ethanol;
isopropanol; methanol; 1% periodic acid were obtained from Merck, Darmstadt, Germany.
HistoClear, and Histomount were obtained from National Diagnostics, Atlanta, GA, USA);
Avian myeloblastosis virus (AMV) reverse transcriptase; dNTP mix; polymerase chain
reaction (PCR) master mix; and ribonuclease inhibitor were obtained from Promega, Madison,
WI, USA. Anti-IκBα monoclonal antibody; anti-p65 monoclonal antibody were obtained
from Cell Signaling Technology, Beverly, MA, USA. Enhanced chemiluminescent (ECL)
western blotting detection reagents; and hyperfilm were obtained from GE Healthcare,
Piscataway, NJ, USA. HRP-conjugated anti-mouse Ig; HRP-conjugated anti-rabbit Ig; AP-
conjugated anti-mouse Ig; and AP-conjugated anti-rabbit Ig were obtained from Dako,
Glostrup, Denmark. Human MUC5AC ELISA Kit was obtained from USCN, Houston, Texas,
USA. Nuclear extract kit and TransAM™ NF-κB p65 Kit were obtained from ActiveMotif,
Carlsbad, CA, USA




81

3.2 siRNA preparation

3.2.1 ON-TARGETplus siRNA without in vivo processing
ON-TARGETplus siRNA complementary to mouse Rip-2 or RPS-3 mRNA were purchased
from Thermo Scientific (Waltham, MA). Table 3.1 shows the sequences of siRNA used. A
stock solution of siRNA (20 M) was prepared by dissolving every 5 nmole of siRNA in 250
l of 1 x PBS. Briefly, the resuspended siRNA was pipette up and down for three to five
times. Extra care was taken to avoid introduction of bubbles. The siRNA was then vortex for
30 mins at room temperature. Following that, the dissolved siRNA was aliquoted into small
volumes and stored at -30
o
C. The aliquots were limited to freeze-thaw events of no more than
five.
3.2.2 ON-TARGETplus siRNA with in vivo processing
ON-TARGETplus siRNA with in vivo processing complementary to mouse Rip-2 with
sequence S2 (Table 3.1) was dissolved in tissue culture grade 1 X PBS (Sigma-Aldrich, St.
Louis, MO). Every 5 nmole of siRNA was dissolved in 30 l of tissue culture grade 1 X PBS.
Following steps taken to dissolve the siRNA is similar to that listed for ON-TARGETplus
siRNA without in vivo processing.

3.3 siRNA transfection
The mouse macrophage cell line RAW264.7, mouse fibroblast cell line NIH/3T3 (American
Type Culture Collection, Rockville, MD) and NF-κB/ secreted alkaline phosphatase (SEAP)
human embryonic kidney (HEK)293 cell line (IImgenex, San Diego, CA) were maintained in
Dulbecco's modified Eagle medium (DMEM) with 4.5 g/L glucose (Invitrogen, Carlsbad,
CA). In order to maintain the selection of SEAP transfected HEK293, selection agent G418
(Invitrogen, Carlsbad, CA) was added to the maintaining medium. The human bronchial
epithelial cell line BEAS2B and human pulmonary mucoepidermoid carcinoma cell line NCI-


82





siRNA Sequence
Mouse Rip-2 Sequence 1 (S1) 5’-GCUCGACAGUGAAAGAAAU-3’
Mouse Rip-2 Sequence 2 (S2) 5’-ACGAGAAGCCGAAAUA UUA-3’
Mouse Rip-2 Sequence 3 (S3) 5’-CAAAUUUCCCUCAGAAUAA-3’
Human RPS-3 5’-UAGUUAACAGGGUCUCCGCUU-3’
Control 5’-UUCUCCGAACGUGUCACGU-3’

Table 3.1: Sequences of siRNA
83

292 (American Type Culture Collection, Rockville, MD) were maintained in RPMI (Sigma-
Aldrich, St. Louis, MO). These cell lines were seeded at 60 to 70 % confluency in antibiotics-
free media one day before transfection. During transfection, the cells were maintained in
GIBCO ™ optimum I reduced serum medium (Invitrogen, Carlsbad, CA). The cells were then
transfected with 100 nM siRNA or negative siRNA control 4 – 6 h at 37°C in OptiMEM
(Invitrogen, Carlsbad, CA) containing Lipofectamine 2000 (Invitrogen, Carlsbad, CA). After
transfection, the cells were allowed to recover in complete DMEM or RPMI for 18, 42, or 66
h before they were analysed for Rip-2, IL-6, IL-8, MUC5AC, and TSLP mRNA and their
protein expression.

NCI-H292 is a human pulmonary mucoepidermoid carcinoma cell line. This cell line
produces MUC2, MUC4 and MUC5AC, which is a major component of mucin that
contributes to the viscosity of mucous. Mucin protein synthesis by H292 can be stimulated by
TNF-α or EGF. Therefore, NCI-H292 is commonly used to study signaling pathway involved
in mucus hypersecretion (Lora et al., 2005; Zhen et al., 2007) and is consequently used in this
study. On the other hand, BEAS2B is a human bronchial epithelial cell line that is developed
by transformation of normal human bronchial epithelial cells. The transformation involved

adenovirus 12-simian virus 40 hybrid virus (Ad12SV40). BEAS-2B form tight junctions but
retain the ability to undergo squamous differentiation in response to serum. BEAS-2B cells
have been used to study pulmonary inflammatory response in a large number of studies
(Verstraelen et al., 2008). Therefore, BEAS-2B was used in this study.

3.4 Mouse asthma model and treatments
Female BALB/c mice, 6-8 wk of age (Interfauna, East Yorkshire, UK) used in this project
were ordered from centre for animal resources and housed in plastic cages (maximum 5
mice/cage) in Animal Holding Unit in the National University of Singapore (NUS) according
to the spirit of Good Laboratory Practice. Animal experiments were performed according to
the Institutional Guidelines for Animal Care and Use Committee of the NUS. Briefly, animal
84

rooms were regulated by automatic timers to provide cycles with 12-14 h of light and 10-12 h
of dark. The temperature in the animal room ranged from 18 °C to 26 °C with an average
temperature of 22 °C. Standard diets generally contained 4-5 % fat and 14% protein. A
minimum of 3 days of acclimatization were given to the mice to adapt to their new
surroundings. Cage bedding was changed thrice a week. Mice were sensitized with 20 µg
OVA and 4 mg Al(OH)
3
in 0.1 ml saline intraperitoneally on day 0 and day 14. For OVA
Challenge, 0.15 g of OVA was dissolved in 15 ml of saline to be aerosolized by a DeVilbiss
Ultra-Neb Large-Volume Ultrasonic Nebulizer (Sunrise Medical Respiratory Products,
Somerset, PA) (Figure 3.2). Mice were then challenged with 1% OVA aerosol for 30 min in a
chamber on days 22, 23 and 24 (Figure 3.2). Average OVA aerosol particle size was less than
5 µm. Saline aerosol was used as a negative control.

Female BALB/c mouse was used because airway inflammation and AHR are easily induced
in this mouse strain. Furthermore, female mice displayed higher serum IgE level and are
more susceptible to airway inflammation than male mice (Melgert et al., 2005). Also, OVA

rather than HDM model was used at the point of study because of the following reasons: (1)
OVA does not occur as an allergen naturally, so it is easy to prevent the mice from OVA
exposure prior to sensitisation; (2) OVA model is more well-characterised than HDM model
in terms of the participation of various leukocytes; and (3) OVA model allows the
sensitisation period and activation of inflammatory response to be completely discerned
(Blanchet et al., 2012; Brewer et al., 1999). Nonetheless, given the model’s growing
popularity, it is likely that the detailed knowledge of the immunopathology induced by HDM
will soon become equivalent to that of the OVA model (Blanchet et al., 2012)

ON-TARGETplus Rip-2 siRNA or control siRNA were prepared as mentioned in section
3.2.2. For drug treatment, Rip-2 siRNA also diluted to 1 nM using 1X PBS (tissue culture
grade). The mice were anesthetized with aerosolised isofluorane (Halocarbon, NJ, USA)
before Rip-2 siRNA (1 and 5 nmol) or control siRNA in 30 µl 1X PBS was administered
85

intratracheally. siRNA was given daily on days 19-21, and 2 h before each OVA aerosol
challenge on days 22-24. Fisetin (0.3, 1, and 3 mg/kg; Sigma-Aldrich, St. Louis, MO) or
vehicle (8 µl DMSO in a total of 20 µl saline) was given by intravenous injection into the tail
vein of the mice 2 h before each OVA aerosol challenge on days 22-24. Saline aerosol was
used as a negative control. Mice were sacrificed 24 h after the last aerosol challenge and
various samples such as lung, BALF (BALF) and serum were collected for subsequent studies.

3.5 Collection of BALF from mice
BALF was collected 24 h after the last saline or OVA challenge from all four treatment
groups of mice. Mice were anaesthetized by an intraperitoneal injection of 300 ul of mice
anesthetic mixture (ketamine: medetomidine: H
2
O = 3:4:3). Tracheotomy was carried out and
a cannula (20G) was inserted into the trachea. Ice cold 1 X PBS (0.5 ml X 3) was instilled
into the lungs and a final volume of approximately 1.3 ml of BALF was retrieved from the

lungs.

3.6 Preparation of BALF for total and differential cell count
The BALF was centrifuged at 3000 rpm for 5 min at 4 °C. Supernatant was collected and
stored at -80°C for further analysis. The pellet was resuspended in 200 µl of 8.5 mg/ml
NH
4
Cl (BDH Laboraties supplies, Poole, England) for 5 min at room temperature to remove
red blood cells. The cell suspension was centrifuged at 3000 rpm for 5 min at 4°C and the


86


Figure 3.1 OVA aerosolization system (Taken from Cheng, 2011)

The ultrasonic nebulizer (Sunrise Medical Respiratory Products, Somerset, PA) aerosolizes 1%
OVA into aerosol mist (particle size less than 5 M). The aerosolized OVA was transferred
into the aerosol chamber through a tube. Mice in the chamber were then exposed to the
aerosolized OVA.
87

supernatant was discarded. The cell pellet was then resuspended in 200 µl of RPMI
supplemented with 10 mg/ml bovine serum albumin (BSA) (Sigma-Aldrich,St. Louis, MO) .
A total number of viable cells was enumerated using a haemocytometer (10 µl cell suspension:
10 µl 0.4% trypan blue) under a light microscope (magnification ×100). Following the total
cell count, aliquots (10
5
cells/150 µl) of the cell suspension were cytospined onto a slide in a
Shandon Cytospin 3 (Thermo Electron Corporation, Pittsburgh, PA) at 600 rpm for 10 min at

room temperature. The BALF cells were stained with Liu stain (modified Wright stain).
Briefly, cell smear was stained with 800 µl of Liu A for 30 sec followed by 1600 µl of Liu B
for 90 sec. Differential cell count was then performed on a minimum of 500 leukocytes under
oil immersion lens (magnification ×1000). Four types of inflammatory cells —eosinophils,
macrophages, neutrophils, and lymphocytes — were identified and their respective
percentage in the total inflammatory cells was enumerated, based on standard morphological
criteria and staining (Figure 3.1). The cell count was performed on a single blinded manner
to eliminate bias. The absolute number of four types of inflammatory cells was calculated by
their percentages and total inflammatory cell count.

3.7 Histological examination
Lungs were isolated from the thoracic cavity
24 h after the last OVA or saline challenge. The isolated lungs were fixed in 10 % neutral
buffered formalin solution for at least 48 h, and processed in a tissue processor (Leica
Microsystems, Wetzler, Germany). Briefly, lungs were dehydrated in a serial concentration of
ethanol mixtures (70% 80%  90%  100%) 30 min each and 1 h for 100%, and were
immersed in xylene for 10.5 h. Lungs were then infiltrated with hot paraffin for 3 h and
embedded in paraffin wax. The specimens were then section into 4 µm sections using a
microtome (Leica Microsystems, Wetzler, Germany). The sectioned tissues were then placed
and dried on glass slides before staining.


88






Figure 3.2 Types of inflammatory cells in mouse BALF.

(A) Representative diagram of BALF collected from OVA sensitised and saline challenged
mice (negative control). (B) Representative diagram of BALF collected from OVA sensitised
and OVA challenged mice (positive control).
Abbreviations: Mac, macrophages; Eos, eosinophil; Lym, lymphocyte; Neu, neutrophil.

OVA/Saline
OVA/OVA
Lym
Eos
Neu
Mac
A
B
89

H&E staining was performed to measure the severity of cell infiltration. For H&E staining,
slides were deparaffinized with HistoClear for 10 min and rehydrated in a serial
concentrations of ethanol (100%  90%  70% water) for 2 min each. The sections were
then stained with Harris haematoxylin for 5 min, then washed in the distilled water, and
differentiated in 0.1 % acid alcohol solution for 30 sec. The sections were washed with tap
water for 5 min and counter stained with Eosin for 1 minute. Finally, the sections were
dehydrated in serial concentrations of ethanol solutions (70% 90% 100%) for 30 sec each
before they were immersed in HistoClear for 10 minute. Evaluation of inflammation around
peribronchial and perivascular areas was semi-quantitatively performed in a single-blind
manner as previously described (Myou et al., 2003). A subjective scale (0 - 4) was assigned as
follows: 0: no inflammatory cells; 1: occasional cuffing with few inflammatory cells; 2: most
bronchi or vessels surrounded by a thin layer of inflammatory cells; 3: most bronchi or
vessels surrounded by a thick layer (2 - 4 cells layer deep) of inflammatory cells; 4: most of
bronchi or vessels surrounded by a thicker layer (more than 4 cells layer deep) of
inflammatory cells.


Periodic acid-fluorescence Schiff staining (PAFS) was performed to determine the extent of
mucus production. PAFS allows visualization of mucus through covalent bonding of sulfited
acriflavine to mucin glycoconjugates. Mucin granules emit red fluorescence when excited at
380-580 nm and observed at 600-650 nm using a confocal microscope (Leica TCS SP5, Leica
Microsystems, Deerfield, IL, USA). Noncovalent linkage of acriflavine to nucleic acid, nuclei
and cytoplasm results in green fluorescence when excited at 380-500 nm and observed at 450-
475 nm (Evans et al., 2009). Briefly, the slides were deparaffinized with HistoClear for 10
min and rehydrated in a serial concentrations of ethanol (100%  90%  70%  deionised
water) and each for 2 min. The sections were immersed in 1 % periodic acid (Merck,
Darmstadt, Germany) for 10 min then thrice in distilled water for 2 min. The subsequent steps
were carried out in the dark. The sections were treated with Schiff reagent for 20 min, and
then washed in running tap water for 5 min before being dipped in 1 % acid alcohol for 5 min
90

twice, which is the differentiation step. After differentiation, the sections were washed in
running tap water again for 5 min. Finally, the sections were dehydrated in serial
concentrations of ethanol solutions (70% 90% 100%) for 30 sec each before they were
immersed in HistoClear for 10 min. The slides were left to dry in the dark before they were
mounted with Fluoresave (Calbiochem, Billerica, MA). Mucus production by goblet cells in
the airway epithelium was assessed blinded and scored based on a 5 point grading system
(Tanaka et al., 2001). According to the percentage of area covered by mucus within the
bronchi rings, score (0-4) were assigned: 0, no goblet cells; 1 <25%; 2, 25-50%; 3, 50-75%;
and 4, >75%. This same scoring system was adopted from the H&E staining.
In both H&E and PAFS staining, bronchioles with the maximum internal diameter twice the
minimum internal diameter were excluded from the analysis. The scoring for inflammatory
cell infiltration and mucus secretion was performed in 2 – 4 preparations of each mouse. The
mean scores were calculated from 4 – 5 mice per treatment group.

3.8 AHR measurement

In this project, AHR was measured in mouse asthma model. The mouse asthma model of
allergen-induced AHR is widely used because the immunology of the mice is well described
and many immunological tools and genetically altered strains are available. The methods to
assess AHR in mice vary. There are two commonly used approaches —non-invasively, by
barometric plethysmography of unrestrained mice; or invasively, by measuring lung
resistance in anesthetized, trachesotomized mice.

The latter method was used in this project because it imposes no stress on the anesthetized
mouse and the inhalation exposure is focused to the lungs. Furthermore, it establishes gold
standard parameters. Also the invasive method provides a direct measurement of the changes
in pulmonary resistance (Rl) and dynamic compliance (Cydn) in response to increasing
concentration of inhaled methacholine. Rl measures the resistance pressure in airway divided
91

by the flow; Cydn measures the volume changes to the concomitant elastic recoil pressure
changes between end inspiration and expiration (Busse, 2010).

To measure of AHR, Buxco’s modular system was used in our study. Mouse AHR was
assessed by measuring the changes of two parameters — airway resistance (Rl) and dynamic
compliance (Cdyn) — in response to increasing concentration of nebulized methacholine
(0.5-8.0 mg/ml; Sigma-Aldrich, St. Louis, MO) recorded using a whole-body plethysmograph
chamber (Buxco, Sharon, CT). RI (P/V) is defined as the pressure driving respiration
divided by air flow; while Cdyn (V/P) is defined as the distensibility of the lung during a
change in volume relative to an applied change in pressure. Figure 1.10 shows the setup of the
apparatus in the experiment.

AHR was measured 24 h after the last saline or OVA challenge. Mice were anaesthetized by
an intraperitoneal injection of 200 µl of an anesthetic mixture (ketamine: medetomidine: H
2
O

= 3: 4: 33, Parnell, Alexandria NSW, Australia & Pfizer, Auckland, New Zealand). After the
mice have been anesthetized, tracheotomy was performed where a small transverse incision
was made on the exposed trachea. The mouse was placed on a warming bed in the whole
body plethysmograph chamber and the trachea was connected to a Y shape cannular and
attached to a pneumotach, ventilator, and nebuliser. Mice were challenged with aerosolised
methacholine for 3 min. Methacholine was prepared by dissolving acetyl-β-methylcholine
(Sigma-Aldrich, St. Louis, MO) in 1X PBS. Bronchoconstriction was recorded for an
additional 5 min for every increasing dose of methacholine. Results were expressed as a
percentage of the respective basal values in response to 1 X PBS.

92


Figure 3.3 Invasive system (Taken from Cheng, 2011)
(A) Whole body plethysmograph; (B) Ventilator for supplying air to the tracheostomized
mouse and for acquiring data; and (C) Nebulizer for aerosolizing methacholine to the mouse
in the plethysmograph

93

3.9 Lymphocyte antigen recall experiments
Thoracic lymph nodes (3-4 nodes per mouse) were extracted from the mice (n = 4 per group).
The isolated lymph nodes were passed through cell strainers to prepare a single-cell
suspension. The single cell suspension was then cultured at 2 x 10
6
cells/ml in RPMI 1640
medium supplemented with 2 mM L-glutamine, 100 IU/ml antibiotics, 25 mM HEPES buffer,
and 10 % fetal calf serum (FCS). The cultured cells were stimulated with or without 200
g/ml OVA; Con-A (10 g/ml) was set as a positive control. Concanavalin A is known for its
ability to stimulate mouse T-cell and cause T-cells to release an array of cytokines and

chemokines. Concanavalin A serves as a positive control to check that the T-cells are viable
and capable of secreting cytokines. Supernatant from parallel triplicate cultures were
harvested 72 h after OVA stimulation for cytokine analysis.

3.10 Enzyme-linked immunosorbent assay (ELISA)
3.10.1. Cytokines and chemokine levels in BALF or cell culture supernatant
Levels of mouse IL-4, IL-5, IL-13, IL-33, IFN-γ, and eotaxin in the BALF supernatant and
levels of human IL-6 and IL-8 from cell culture supernatant were measured by ELISA (BD
Biosciences Pharmingen, San Diego, CA, USA for mouse IL-4, mouse IL-5, mouse IL-33,
human IL-6, and human IL-8; R&D Systems, Minneapolis, MN, USA for mouse IL-13,
mouse eotaxin, and mouse IFN-γ) according to the manufacturer’s instructions. Briefly, 50 µl
of diluted capture antibody (diluted to the appropriated concentration in relevant coating
buffer) was coated onto every well of the ELISA plate (NUNC, Denmark). The plate was
sealed with parafilm and incubated overnight at 4°C. After the antibody has been coated onto
the wells of the plate, the coating buffer was aspirated and the plate was washed with wash
buffer (PBS with 0.05% Tween-20) to remove unbound antibody. The plate was then blocked
with 200 µl assay diluent buffer (1X PBS with 10% heat inactivated FBS) for 2 h. After 2 h,
the plate was washed and 50 µl of standards or BALF samples were added and incubated for
2 h at room temperature. After incubation, the plate was washed and incubated with
biotinylated detection antibodies and streptavidin-HRP conjugates for another one to 2 h.
94

Subsequently, HRP substrate solution was added to every well of the washed plate and
incubated for another 30 min in the dark. The reaction between HRP and HRP substrate was
stopped by adding 50µl of 1 M H
2
SO
4
. Finally, the plate was read at 450 nm (reference filter
570 nm) using an automatic microplate reader (Sunrise Tecan, Austria). The detection limits

were 4 pg / ml for 1L-4 and IL-5; 15.6 pg/ml for IL-12, IL-13, and IFN-γ; and 2 pg/ml of
eotaxin.
3.10.2. Immunoglobulin (Ig) levels in serum
Cardiac puncture was performed to collect blood from the mouse 24 h after the last OVA or
saline challenge. Blood collected was left at clot for 3h at room temperature. In order to
harvest the serum, the blood was subjected to centrifugation at 3000 rpm for 5 min at 4
o
C.
Serum was harvested from the top layer of the supernatant and stored at -80
o
C for further
analysis.
Serum level of total IgE, OVA-specific IgE, OVA-specific IgG1 and OVA-specific IgG2a
were assayed using ELISA kits (BD Biosciences Pharmingen, San Diego, CA, USA total IgE;
R&D Systems, Minneapolis, MN, USA OVA-specific IgE, IgG, and IgG2a). ELISA plates
were incubated with IgE capture antibody or 20g/ml OVA coating buffer (OVA-specific
ELISA). The steps that follow were similar as that for cytokine ELISA. The detection limit
was 2 ng/ml for total IgE.
3.10.3. MUC5AC level in cell lysate
RPS-3 siRNA transfected NCI-H292 and BEAS-2B were stimulated with 50 ng/ml of TNF-α
for 24 h, as performed by Lora et al., 2005, and harvested using lysis buffer M-PER
Mammalian Protein Extraction Reagent. The protein harvested was quantified using
bicinchoninic acid (BCA) protein assay kit. Levels of human MUC5AC in the cells were
measured by ELISA (USCN, Houston, Texas, USA) according to the manufacturer’s
instructions. Antibody specific to MUC5AC was already coated on the microtiter plate
provided in the kit. 100 l of standards or 50 g of proteins per cell lysate were added to the
appropriate microtiter plate wells and topped up to 100 l with assay diluent then incubated
for 2 h. Following that, biotin-conjugated antibody preparation specific for MUC5AC and
95


Avidin conjugated to Horseradish Peroxidase (HRP) were added to each microplate well and
incubated. After incubation, TMB substrate solution was added to each well. Only wells that
contain MUC5AC turn blue. The amount of MUC5AC present was proportional to the
intensity of the blue. The enzyme-substrate reaction was terminated by the addition of stop
solution. The color change was measured spectrophotometrically at a wavelength of 450. The
concentration of MUC5AC in the samples was then determined by comparing the O.D. of the
samples to the standard curve.

3.11 RNA harvest and mRNA expression quantification
3.11.1. Storage of lung samples
Lung samples from mice were extracted 24 h after the last OVA or saline aerosol challenge.
The lung samples were kept in RNAlater (Invitrogen, Carlsbad, CA), which is an aqueous
tissue storage solution that rapidly permeates the tissues and preserves the RNA integrity of
the tissues. The lung samples were kept at 4
o
C overnight to allow the RNAlater to permeate
into the lung tissues. After that the samples were kept at -80
o
C for long term storage.
3.11.2 Preparation of lung samples for RNA harvest
In order to isolate RNA from frozen lung tissues were thawed and removed from the
RNAlater with a pair of clean forceps. The lung tissues were homogenized in Trizol reagent
(Invitrogen, Carlsbad, CA). The lung tissues were disrupted with a homogenizer
(SilentCrusher M, Heidolph Elektro GmbH & Co, Kelheim, German). To reduce degradation
of RNA during homogenization, lung tissues were kept in ice bath throughout the process.
The homogenate was centrifuged at high speed 12,000g for 10 min at 4
o
C to spin down the
unwanted membranes and cell components. The supernatant was transferred to a fresh tube
for subsequent RNA harvest.

3.11.3. Preparation of cell culture samples for RNA harvest
Cells were cultured on 6-wells plate were washed with ice cold PBS twice. 1 ml of TRIzol
(Invitrogen, Carlsbad, CA) was added to flush the adherent cells, which would be dislodged
96

from the wells by TRIzol. Dislodged cells were transferred to a fresh tube for further analysis.
The samples were stored in -80
o
C freezer for long term storage.
3.11.4. RNA harvest
Samples in TRIzol reagent were placed at room temperature for 5 min. This incubation
allowed for complete dissociation of proteins. Chloroform was then added and the sample
was shook vigorously. The samples were then incubated at room temperature and subjected to
centrifugation. Following centrifugation, the cleared supernatant from every sample was
transferred to another 1.5 ml tube. Isopropanol was added to the supernatant, resulting in
precipitation of RNA. The precipitated RNA was washed using 75 % absolute ethanol. The
ethanol was removed and the RNA pellet was dissolved in nuclease-free water. RNA
concentration was measured using a nanodrop ND-1000 spectrophotometer (Thermo Fisher
Scientific Inc, Waltham, MA, USA). In order to ensure that the RNA extracted were of
sufficient purity, the A260/A280 (DNA/protein) and A260/A230 (DNA/Organic
contaminants) ratio were recorded. Ratios between 1.9 and 2.0 suggest an acceptable level of
purity of the RNA extracted.
3.11.5 Reverse transcription (RT) - polymerase chain reaction (PCR)
Using oligodT (Promega, Fitchburg, WI) as the primer and AMV reverse transcriptase
(Promega, Fitchburg, WI), 1 g of RNA was transcribed into cDNA. The transcription was
performed using a multiwell thermal cycle (GeneAmp PCR system 2700, Applied Biosystems,
Foster City, CA).
3.11.6 Realtime(RT)-PCR
Real time PCR was performed using ABI 7500 cycler in a 20 l reaction volume system
containing the following: 1l cDNA (1g), 10 l 2 X Sybrgreen master mix (Thermo Fisher

Scientific Inc, Waltham, MA), 1 l forward primer (10 M), 1 l reverse primer (10 M),
and 7 l nuclease-free water. Table 3.2 shows the sequences of the primers.
3.11.7 Semi-quantitative-PCR
Synthesized cDNA was used as a template for PCR amplification. PCR amplification were
performed using 1 l cDNA template in a 25 l reaction volume containing the following: 1)
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1 l of cDNA (1 g/l); 12.5 l 2X PCR master mix (50 units/ml TaqDNA polymerase, 400
M dATP, 400M dCTP, 400 M dTTP, and 3 mM MgCl
2
) (Promega, Fitchburg, WI), 9.5 l
nuclease-free water (Promega, Fitchburg, WI), 1l forward primer (10 M) (1
st
Base,
Singapore) and 1 l reverse primer (10 M) (1
st
Base, Singapore). 9 l of PCR-amplified
product was added to 1 l of 10 X DNA tracking dye. Samples were loaded onto 2 % agarose
gel with GelGreen staining and were subjected electrophoresis performed at 100 V for
approximately 30 min. Bands were visualised using the ultraviolet transillumination. β-actin,
which is a housekeeping gene, was used as an internal control to normalise for variations in
loading and sample concentrations. Table 3.2 shows the sequences of the primers.

3.12 Immunoblotting
3.12.1 Lung protein extraction
In order to extract proteins from lung samples, the lung lobes were cut into small pieces,
placed in lysis buffer M-PER Mammalian Protein Extraction Reagent containing phophatase
inhibitor (Thermo Scientific, Waltham, MA) , then homogenised using a homogeniser
(SilentCrusher M, Heidolph Elektro GmbH & Co, Kelheim, German). Lung tissue lysates
were then incubated on ice for at least 30 min. Total protein concentrations were determined

using bicinchoninic acid (BCA) protein assay kit (Thermo Fisher Scientific Inc, Waltham,
MA, USA).
3.12.2 Tissue protein nuclear extraction
Nuclear proteins from lung tissues were extracted using Active Motif nuclear protein
extraction kit (Active Motif, Carlsbad, CA). Extraction process was carried out according to
protocol stated in the extraction kit. Briefly, the lung tissues were homogenised in 1X
Hypotonic Buffer supplemented with DTT and Detergent. The homogenised tissues were
incubated on ice for 15 min. After incubation, the tissues were centrifuged for 10 min. The
supernatants, which contain the cytoplasmic proteins, were collected in a fresh eppendoff tube.
However, at that point, the pellet still contained cytoplasmic protein. Therefore, the pellet was
subjected to another round of resuspension and centrifugation. The supernatant, which still
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contained cytoplasmic protein, was pooled with the cytoplasmic protein collected earlier. The
remaining pellet was resuspend in lysis buffer to lyse the nucleus. The lysed nucleus was
centrifuged to remove any debris. Finally, the supernatant that contained the nuclear protein
was harvested and stored at -80
O
C for further analysis. The protein concentration in the
nuclear extract was determined using BCA protein assay kit.
3.12.3 Cell line protein extraction
In order to extract proteins from cell lines, the cells were incubated for at least 30 min at 4
o
C
in the same lysis buffer used for lung samples. Incubation allowed for cells to be lysed and
proteins to be released. After lung tissue were homogenised or the cells were lysed, they were
subjected to centrifugation (12,000 rpm, 15 min). Supernatant containing the protein was
harvested and total protein concentrations were determined using bicinchoninic acid (BCA)
protein assay kit (Thermo Fisher Scientific Inc, Waltham, MA).
3.12.4 Immunoblot

30 g of protein extracted were subjected to 10 % SDS-PAGE in a Trans-Blot tank (Bio-Rad
Laboratories, Hercules, CA) and transferred to PVDF membrane tank (Bio-Rad Laboratories,
Hercules, CA). The membrane with the transferred protein was subsequently blocked in 5 %
skim milk (Sigma-Aldrich, St. Louis, MO) in Tween-Tris-buffer saline (TTBS) for at least 2 h
at room temperature. The blocked membrane was then probed with various primary
antibodies in 1 % skim milk (Sigma-Aldrich, St. Louis, MO) in TTBS overnight at 4
o
C.
Following that, the membrane was washed with TTBS to remove unbound primary antibodies
before secondary antibody was added. Secondary antibody probes for the presence of primary
antibody. The membrane was developed on hyperfilms using ECL reagents (HRP) (Thermo
Fisher Scientific Inc, Waltham, MA, USA) or Alkaline Phosphatase (AP) reagents (Bio-Rad
Laboratories, Hercules, CA, USA). β-actin or Tata-Binding Protein (TBP) was used as a
loading control.

3.13 NF-κB transactivation assay (TransAM)
99

p65 DNA-binding activity was assessed by TransAM™ NF-κB p65 transcriptional factor
assay kit (Active Motif, Carlsbad, CA), which comes with a 96-well plate coated with
immobilized oligonucleotide containing the NF-κB consensus DNA binding sequence (5’-
GGGACTTTCC-3’). Briefly, 10 g of nuclear protein or total protein from every sample was
added to separate wells of the plate. The protein was incubated in the well for 1 h. Primary
antibody specific for p65 was then added and incubated. Subsequently, HRP-conjugated
secondary antibody specific to p65 antibody was added to each well. Following that,
developing solution was added to allow for colorimetric reaction to occur. To inhibit the
colorimetric reaction, stop solution was added. The plate was read at 450 nm with reference
wavelength of 655nm. The reading was done using a microplate reader (Sunrise Tecan,
Austria).


3.14 NF-κ
κκ
κB reporter gene assay
3.14.1 NF-κB luciferase gene reporter assay
Normal human bronchial cells (NHBE) (Lonza, Walkersville, MD, USA) were cultured in
optimised bronchial epithelial bullet kit medium supplemented with the following: bovine
pituitary extract ( 2 ml), insulin (0.5 ml), hydrocortisone (0.5 ml), GA-1000 (0.5 ml), retinoic
acid (0.5 ml), transferring (0.5 ml), triiodothytonine (0.5 ml), epinephrine (0.5 ml), and
human EGF (0.5 ml). Cell of passage number ranging between three and six were seeded onto
6-wells plate at 1.0 X 10
5
cells/well. Using Lipofectamine, the cells were transfected with NF-
κB responsive elements linked to luciferase reporter gene. The transfected cells were pre-
treated with fisetin (10 M, 25 M, or 50 M), 0.01% DMSO or plasmid expressing
dominant negative IκB for 6 h before stimulation with 1 nM TNF-α for 24 h. Cells were lysed
in luciferase lysis buffer. The luciferase activity was quantified using a luminometer and
normalized to β-galactosidase activity as previously described (Goh et al., 2012). All
luciferase experiments were repeated thrice.
TNF-α was used as a stimulant in the in vitro studies to study NF-κB signaling pathway
because this stimulant has been implicated in the pathophysiologic mechanism of several