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Figure 1.4 The immunological role of epithelial cells in virus-indcued
asthma exacerbations. Adapted from (Message and Johnston, 2001).
21
and IL-5, eosinophil progenitors are developed in bone marrow and
then released into circulation under a phenotypically mature state
(Gould and Sutton, 2008). With a short half-life of 18 hours, eosinophils
do not stay long in peripheral blood before residing in thymus or
gastrointestinal tract under homeostatic conditions. In healthy humans,
the number of mature eosinophils found in the peripheral blood is less
than 400 per mm3 (Rosenberg et al., 2013). However, several
diseases, including asthma, can induce eosinophilia and recruit
eosinophils into inflammatory tissues.
In asthma patients, eosinophils leave the bone marrow,
transport to the site of inflammation and function under the stimulation
of a wide range of pro-inflammation cytokines, chemokines and
adhesion molecules. The most important cytokines are Th2 cytokines
IL-4, IL-5 and IL-13. IL-5 has a central role in all aspects of eosinophil
development, activation and survival via the interaction with IL-5
receptor subunit-α expressed on eosinophil (Takatsu and Nakajima,
2008). IL-4 and IL-13 activate the endothelial and epithelial cells to
produce pro-inflammatory chemokines, which attract and activate
eosinophils. Eotaxin-1/CC-chemokine ligand 11 (CCL11) is a specific
chemokine that have chemoattractive effect on eosinophils. It functions
through CC chemokine receptor (CCR3) on eosinophils, promoting
eosinophilia
cooperatively
with
IL-5
or
via
IL-5-independent
mechanisms (Mould et al., 2000). ICAM-1 and vascular cell adhesion
molecule-1 (VACM-1) are adhesion molecules that involved in
22
transmigration of eosinophils from the vascular bed into the inflamed
tissue. They are expressed by endothelium under the influence of IL-4
and IL-13 (Aceves and Broide, 2008).
Eosinophils are generally considered as terminal effector cells in
allergic asthma. Part of eosinophilsʼ effects on inflammatory response
functions
through
cellular
degranulation.
Eosinophil
piecemeal
degranulation conducts under the effect of several cytokines and
chemokines, including interferon-γ (IFN-γ) and eotaxins (Rosenberg et
al., 2013). The granule contents MBP, EDN, ECP and EPO are
associated with the airway damage and AHR in asthma. Eosinophilderived fibrogenic and growth factors can amplify airway remodeling
and induce mucus secretion, causing asthma exacerbation to some
extent (Kay, 2005). Eosinophils also release a wide range of proinflammatory cytokines
and chemokines
interacting with other
leukocytes, suggesting an additional initiating role in asthma (Figure
1.5).
Eosinophilia was found involved in asthma exacerbations
associated
with
respiratory
virus
infections.
Prolonged
nasal
eosinophilia was detected in allergic patients after common cold with
increase number of eosinophils and other leukocytes (van Benten et
al., 2001). Serum concentration of ECP was elevated in rhinovirusinduced wheezing (Kato et al., 2011). However, the interaction
between viruses and eosinophils needs more exploration. One
23
Figure 1.5 Eosinophils modulate the function of other leukocytes.
Adapted from (Rosenberg et al., 2013).
24
possible function of eosinophils and their secretory mediators is
promoting the antiviral host defense. Researchers also found that
replication and virus loads of RSV were decreased under the effect of
eosinophils (Rosenberg et al., 2009).
EDN and ECP have RNAse
activity and toxicity to single-strand RNA pathogens (Rosenberg and
Domachowske, 2001). EPO catalyzes the production of oxidant
products that are toxic to microorganisms (Gould and Sutton, 2008).
Besides the granule contents, the immune mediators released by
eosinophils, such as IL-6, may also be involved in the antiviral
response to respiratory virus infections (Dyer et al., 2009).
1.2.3.3 Neutrophils
Neutrophils are polymorphonuclear leukocytes involved in innate
immune response to acute inflammation. They are usually the first
leukocytes recruited to inflammatory site and related to immune
defense to microorganisms such as bacteria and viruses. Neutrophils
are generated from myeloid precursors in the bone marrow by the
action of granulocyte colony stimulating factor (G-CSF) and through
several
maturation
stages
(Kolaczkowska
and
Kubes,
2013).
Neutrophils have a short longevity with a circulating half-life of 6-8
hours and a lifespan of 5 days in humans (Summers et al., 2010).
Multiple inflammatory mediators, including IL-8, TNF-α, G-CSF,
leukotriene B4, and also adhesion molecules ICAM-1 and VCAM-1,
regulate and contribute to the recruitment of neutrophils (Summers et
25
al., 2010).
Neutrophils are common leukocytes that eliminate pathogens in
respiratory virus infections. In experimental rhinovirus infection,
peripheral blood neutrophils increased without changes in total white
blood cell counts, corresponding to the increase of G-CSF and IL-8
concentrations (Gern et al., 2000; Kelly and Busse, 2008).
In severe asthma exacerbation, lung neutrophil level was
increased to a greater degree than eosinophils in some fatal asthma
attacks (Wenzel, 2001). In asthmatic patients suffering from viral
infection, increased sputum neutrophils and cell lysis were detected
and correlated with clinical severity (Wark et al., 2002b). Several
components from neutrophil granules, including metalloproteinases,
elastase, lactoferrin and myeloperoxidase, were reported to be
upregulated in severe asthma or asthma exacerbation, contributing to
chronic inflammation and airway remodeling (Monteseirín, 2009).
Neutrophil elastase has been shown to up-regulated MUC5 proteins
expression, which increase mucus production and lead to airway
obstruction in asthma exacerbation (Zhou et al., 2013). Neutrophil
proteases activated eosinophils in vitro to produce superoxide and proinflammatory cytokines (Hiraguchi et al., 2008). Neutrophils also
contribute the recruitment of DCs and T cells to inflammatory site,
which further aggravate airway inflammation in severe asthma (Ganz,
2003; Yang et al., 2000).
26
1.2.3.4 Macrophages
Macrophages are important cells that are involved in most
aspects of both innate and adaptive immune responses, including
recognition and elimination of pathogens, releasing cytokines and
chemokines, and antigen presentation to other leukocytes like T cells
and DCs (Balhara and Gounni, 2012; Gordon, 2003). They are typically
developed and differentiated from monocytes by series of changes,
triggered by a variety of stimuli (Gordon and Taylor, 2005; Martinez et
al., 2008). According to the cytokines and chemokines they express
and the immune responses they invoke, macrophages can be divided
into two subtypes: the classically activated (M1) macrophage and the
alternatively activated (M2) macrophage. M1 macrophages are induced
by Th1 stimuli such as IFN-γ and LPS, and typically express CXC motif
chemokine 9 and 10 (CXCL9, CXCL10). They participate in Th1
responses to kill intracellular pathogens in a cytokine-dependent
manner. M2 macrophages are stimulated by Th2 cytokines like IL-4
and IL-13, and participate more in phagocytosis of foreign pathogens
and apoptotic cells (Balhara and Gounni, 2012; Biswas and Mantovani,
2010).
Macrophages are the most numerous inflammatory cells in the
airways. With their ability to produce both pro- and anti- inflammatory
mediators, their role in asthma remains to be determined. Alveolar
macrophages from sensitized mice were once transferred and induced
27
pulmonary eosinophilic inflammation in unsensitized mice. However,
whether the inflammation is due to the sensitized macrophages or the
effect of challenge perse remains unclear (Moon et al., 2007). Other
studies have shown that macrophages could induce histamine release
from basophils and mast cells and deteriorate tracheal function through
reactive oxygen radicals, which modulated the contractility of airway
smooth muscle (Yang et al., 2012).
Rhinovirus can attach to airway macrophages despite the
limited replication in these cells (Laza-Stanca et al., 2006). The
interaction between rhinovirus and macrophages induces the release
of multiple pro-inflammatory cytokines and chemokines including IFNγ, TNF-α, IL-8 and macrophage inflammatory protein (MIP-1α) (Kelly
and Busse, 2008). TNF-α promotes T cell activation and chronic airway
remodeling,
and
induces
ICAM-1
expression
to
facilitate
the
recruitment of leukocytes and binding of rhinoviruses (Krunkosky et al.,
2000). IL-8 is a typical chemoattractant for neutrophils, which usually
present in severe asthma. Except the increase of pro-inflammatory
cytokines, rhinovirus exposure also induces impaired antibacterial
immune response in alveolar macrophages
by decrease the
phagocytic ability (Oliver et al., 2008).
The role of macrophages in virus-induced asthma exacerbation
is still unclear. However, studies showed that the pro-inflammatory
cytokines and chemokines released from macrophages under virus
28
stimulation may contribute to the immune response in asthma
exacerbation.
1.2.3.5 Mucus hyper-secretion
The respiratory mucus and the mucociliary clearance are
essential for protection of the airways. Normal mucus consists of 97%
water and 3% solids, with mucins account for nearly 30% of the solids
(Thornton et al., 2008). Mucins, the large glycoproteins with extensive
O-glycan attachment domains, play a crucial role in determining the
physical properties of mucus (Thornton and Sheehan 2004; Fahy and
Dickey 2010). There are 17 genes encoding mucins in human genome.
These mucins can segregate into three families: secreted gel-forming
mucins, membrane-associated mucins and secreted non-gel-forming
mucin (Voynow and Rubin, 2009). Of the secreted gel-forming mucins,
only
MUC5AC
and
MUC5B
are
significantly
produced
in
intrapulmonary airways (Evans et al., 2009; Thornton et al., 2008). In
healthy persons, MUC5AC is mainly produced in goblet cells, while
MUC5B is expressed in the mucous cells of the submucosal gland
(Voynow and Rubin, 2009).
Mucus hyper-secretion is common in asthmatic patients and is
one of the major symptoms of asthma exacerbation. Airway obstruction
by a plugging of mucus and cells contributes to the morbidity and
mortality of asthma (Kuyper et al., 2003). Pathologic mucus has high
viscosity and elasticity, leading to the poor mucus clearance in the
29
airways. In asthma, airway remodeling is characterized by increased
surface epithelial mucin production and bronchial microvessels (Fahy
and Dickey 2010). Many common mediators in asthma, including
neutrophil elastase, ECP, mast cell chymase and leukotrienes, can
trigger the hyper-secretion of mucus (Thornton and Sheehan, 2004).
Th2 cytokine IL-13 is also involved in mucus plugging by regulate
mucus cell metaplasia (Hays and Fahy, 2003).
Mucus
overproduction
also
presents
in
common
cold,
suggesting a role of rhinovirus in this process. Experimental infection
with rhinovirus-16 in undifferentiated bronchial epithelial cells induced
secretion of MUC5AC, the major mucin components of mucus (He et
al. 2004). Clinical study revealed that rhinovirus infection in volunteers
also induced the mucin production, following the increase of IL-8 in
nasal lavages (Yuta et al., 1998).
The expressions of MUC5AC and MUC5B depend on the
activation of gene transcription factors under the regulation of several
signaling pathways (Voynow and Rubin, 2009). Signal transducer and
activator of transcription (STAT) 6 signaling is crucial for IL-13regulated MUC5AC induction and AHR in asthma (Evans et al., 2009).
Nuclear factor-κB (NF-κB) and epidermal growth factor receptor
(EGFR) are involved in rhinovirus-induced MUC5AC in human
experimental model (Hewson et al., 2010). However, in animal model,
NF-κB contributes less in MUC5AC transcription (Pantano et al.,
30
2008).
1.2.3.6 Airway Hyper-responsiveness (AHR)
As a clinical symptom in asthma, the degree of AHR is usually in
proportion to the severity of asthma. Provokers such as viral infection
and allergen exposure can enhance the underlying AHR in asthmatic
patients (Cockcroft and Davis, 2006). It is suggested that AHR has two
components:
the
variable
component
associated
with
airway
inflammation and the persistent one related to structural changes of
airways. The former one is reflected mainly by changes that response
to indirect stimuli, whereas the latter is reflected by the airway
responsiveness under direct stimuli (Brannan and Lougheed, 2012;
Cockcroft and Davis, 2006).
Airway remodeling is a feature related to several respiratory
inflammation diseases, usually defined as the changes in structural
components during growth or in response to injury and inflammation.
The structural changes including epithelial thickening, goblet cell
hyperplasia, and airway smooth muscle hypertrophy and hyperplasia,
contribute to the overall thickening of the airway wall in asthma (Berend
et al., 2008).
Airway inflammation also plays a role in AHR in asthma. The
correlation between peripheral blood eosinophilia and AHR was
demonstrated, along with the effect of IL-5 and IL-13 (Chen et al.,
31
2009; Kuperman et al., 2002; Schwartz et al., 2012). The presence of
mast cells in airway smooth muscle is also associated with AHR in
asthma (Brightling et al., 2002; Schultz et al., 2010). However, the
relationship between inflammatory cell infiltration and AHR were also
brought to question by some other studies (Berend et al., 2008). IL-5
blocking antibody reduced sputum eosinophils but failed to suppress
AHR in asthmatics (Leckie et al., 2000). Dissociation between
inflammatory cells and AHR also presented in patients with mild
asthma (Crimi et al., 1998).
Rhinovirus infection also induces AHR. Experimental rhinovirus16 infection in volunteers with mild asthma induced AHR to histamine.
The aggravation of symptoms was accompanied with an increased
level of IL-8, which indicates rhinovirus may function through
inflammatory mediators (Grünberg et al., 1997). With the regulation of
IL-13, neonatal rhinovirus infection induced AHR in mice, further
supporting the role of inflammatory mediators in virus-associated AHR
(Schneider et al., 2012).
1.3
Animal models for asthma exacerbation
Asthma exacerbations, especially virus-induced ones, have
brought heavy burden to the society. Inhaled corticosteroid (ICS)
therapy has long been used in asthma control and was proved to be
the most efficient anti-inflammatory treatment (Chung et al., 2009).
However, corticosteroid insensitivity happens in severe asthma
32
patients, who count for approximately 5-10% of the asthmatic
population (Robinson et al., 2003). The effect of ICS treatment on
asthma exacerbation control showed no clinically significant benefit in
children (Doull et al., 1997). Meanwhile, corticosteroid therapy can
usually induce side effects including oral candidiasis, hoarseness and
dysphonia (Chung et al., 2009). Considering the unclear mechanism of
virus-associated asthma exacerbation, it is necessary to establish
experimental models for further investigation.
In vitro model of rhinovirus infection was established in BEAS2B cells, a transformed continuous cell line, and increased expression
of IL-8, RANTES and eotaxins were detected in this cellular model
(Papadopoulos et al., 2001). Other epithelial cell lines, such as A549,
16HBE and HEp-2 were also used (Guan et al., 2008). However, the
systemic immune responses in asthma and rhinovirus infection are
complex and involve the correlation of multiple inflammatory cells.
Thus, it is necessary to establish in vivo models for further
investigation.
Allergen sensitization and respiratory virus infection are two
major risk factors for asthma exacerbation and subsequent asthma
development. Researchers now pay attention to the relationship
between these two factors and their combination effect on acute
asthma attack and persistent asthma. Several birth cohort studies have
shown that atopic children have higher risks to develop prolonged
33
asthma in school age with respiratory viral infections in infancy or
during early years of life (Kusel et al., 2007; Jackson et al., 2008).
Compared
with
non-sensitized
children,
children
sensitized
to
aeroallergens developed greater risk of severe viral wheezing,
especially the rhinovirus-induced ones (Jackson et al., 2012). These
results indicate the interaction of allergen sensitization and respiratory
virus infections in asthma development and exacerbation, which
support the use of allergen sensitization combined with virus infection
to establish asthma exacerbation model.
Traditionally, in vivo asthma models were established in animals
sensitized and challenged by allergens. Many species of animals, such
as mice, rats, guinea pigs, monkeys, were used to establish asthma
models (Zosky and Sly, 2007). Among them, mouse models of asthma
were the most commonly used due to their resemblance features of
human asthma. With the sensitization and challenge by allergens,
mouse asthma models are easy to develop asthma symptoms
including eosinophilic infiltration, mucus hyper-secretion and AHR.
Female Balb/c mice were commonly used as the higher prerulane of
asthma exacerbation in women and a good susceptibility to
inflammation in female mice (Melgert et al., 2005). The commonly used
allergen for asthma mouse model is ovalbumin (OVA), which induces a
classic Th2 phenotype immune response and is not normally exposed
to mice (Shore and Shapiro, 2009; Zosky and Sly, 2007). Aluminium
hydroxide is frequently used with OVA as an adjuvant to promote the
34
Th2 responses of the immune system.
Progress has been made in animal model of asthma
exacerbation by the combination of allergen sensitization and virus
infections. RSV infections prior or after allergen sensitization were
conducted in murine models, and increased AHR and airway
inflammation were demonstrated when infection were performed
challenge exposure, after allergen sensitization (Peebles et al., 2001a;
Kondo et al., 2004; You et al., 2006; Aeffner and Davis, 2012). Sendai
and RSV infection were also established in guinea pig models and
induced clinically relevant symptoms (Riedel et al., 1996; Dakhama et
al., 1999).
Efforts were also made to establish asthma exacerbation
models via rhinovirus infection, the major cause of human asthma
exacerbation. However, more than 90% of rhinovirus serotypes use
human ICAM-1 to enter cells and cannot bind to mouse ICAM-1. Due
to the host cell tropism of rhinovirus, it is difficult to establish a
rhinovirus-induced asthma exacerbation model in mouse. Non-human
animals susceptible to rhinovirus are chimpanzees and gibbons.
Despite the fact that those models were used in some experiments, It
is not a common choice due to their high cost and impracticability
(Huguenel et al., 1997).
Attempts were made to transfect mouse cells with viral RNA. By
alternate passage of major group serotype rhinovirus 16 between
35
human HeLa cells and ICAM-1 expressing mouse L cells, a variant
16/L virus was isolated and able to produce high levels of infection in
mouse cells (Harris and Racaniello, 2003). Another study suggests that
changes in viral polypeptides permit the viral growth in mouse cells
(Harris and Racaniello, 2005).
Investigations were also made to establish a transgenic mouse
expressing a mouse-human ICAM-1 chimera. Domains 1 and 2 of
mouse ICAM-1 were replaced with the equivalent human domains, and
rhinovirus 16 successfully infected and replicated in mouse cells
expressing the chimeric ICAM-1 (Tuthill et al., 2003). Depending on
this cellular model, great achievement in major-group rhinovirus
infection of transgenic mice was obtained. Bartlett et al. (2008) have
successfully infected the transgenic mice expressing a mouse-human
ICAM-1 receptor with rhinovirus-16, resulting in the outcomes similar to
those in minor group rhinovirus infection model. The similarity of
immune responses to the major and minor group of rhinovirus
infections suggests the possibility to investigate the rhinovirus-induced
asthma exacerbation with minor group serotypes.
The minor group serotype rhinovirus 1B was used in several
studies to investigate the rhinovirus-related respiratory diseases
(Newcomb et al., 2008; Nagarkar et al., 2009). However, few models of
rhinovirus-induced asthma exacerbation were reported, except the
work of Bartlettʼs group. In their acute inflammation model, mice were
36
sensitized with OVA and rhinovirus infection was preformed during
allergen challenge. Increased eosinophilic infiltration and airway hyperresponsiveness were observed in mice challenged by allergen and
rhinovirus. Similar pathologic symptoms were observed in experimental
infections in human, with increased Th2 immune response in
asthmatics comparing with normal subjects (Fraenkel et al., 1995;
Message et al., 2008).
Due to the limitations of animal models, such as anatomic and
physiologic differences by species, and the lack of chronicity in
inflammation, it is hard to completely mimic the symptoms and immune
responses in human asthma exacerbations. Despite the shortcoming,
the investigation on animal models has contributed much to the
exploration of asthma exacerbation, enhancing our understanding of
this disease.
37
Chapter 2 Rational
38
Asthma affects nearly 300 million people of all ages and all
ethnic background (Masoli et al., 2004). The exacerbations of its
symptoms usually cause the mortality and bring heavy social and
economic burdens. Respiratory virus infections, particular rhinovirus
infections, were associated with asthma exacerbation as a crucial
trigger. However, the mechanism of rhinovirus associated asthma
exacerbation is still unclear and needs more investigation. Thus,
animal models including mouse models would contribute an important
role to the investigation.
In this thesis we established a mouse model of rhinovirusinduced asthma exacerbation. Due to the host cell tropism of
rhinovirus, it is difficult to establish a mouse model with the major group
of rhinovirus. Infection of rhinovirus 1B in transgenic mice induced
similar immune responses as that to major group rhinovirus (Bartlett et
al., 2008). Thus, with a modification on allergen challenge, we treat
mice with combination of allergen challenge and infection of minor
group
Rhinovirus
1B
to
imitate
the
virus-mediated
asthma
exacerbation. The time courses of inflammation, mucus production,
and inflammatory genes expression, along with AHR were examined to
further investigating the effect of viruses on allergic asthma
exacerbation.
39
Chapter 3 Materials and methods
40
3.1
Materials
Eagleʼs minimum essential medium (EMEM) (ATCC, USA), fetal
bovine serum (FBS) (Hyclone Laboratories, South Logan, Utah),
trypsin, polyethylene glycol (PEG6000) (Sigma-Aldrich, USA), cell
culture tested sodium chloride (NaCl) (Sigma); chicken ovalbumin
(OVA) (Sigma), phosphate buffered saline (PBS), aluminium hydroxide
hydrate (Al(OH)3) (Sigma), saline; mouse anesthetic mixture (ketamine:
medetomidine: H2O=3:4:33) obtained from Animal Holding Unit (AHU)
(NUS, Singapore), bovine serum albumin (BSA) (Sigma, Singapore),
Roswell Park Memorial Institute (RPMI) -1640 medium (Hyclone
Laboratories, South Logan, Utah), ammonium chloride (NH4Cl) (BDH
Laboratory Supplies, Poole, England), trypan blue (Invitrogen,
Carlsbad, CA, USA); Liu A and Liu B staining dye, histological
mounting medium (Histomount) (National Diagnostics, CA, USA);
Xylene, paraffin wax, eosin Y, Harris hematoxylin solution, Mayerʼs
hematoxylin solution, periodic acid solution, Schiffʼs reagent, 10%
neutral buffered formalin, absolute ethanol, histoclear (National
Diagnostics, CA, USA); RNAlater (Ambion, Austin, Texas), Trizol
(Ambion, Life Technologies, USA), chloroform, isopropanol (Merck,
Darmstadt, Germany); avian myeloblastosis virus (AMV) reverse
transcriptase, dNTP mix, oligo(dT)15 primer and ribonuclease inhibitor
(RNasin) (Promega, Madison, USA), diethylpyrocarbonate (DEPC)treated water, sodium acetate (CH3COONa) (1st BASE, Singapore),
41
Maxima SYBR Green/ROX qPCR Master Mix (2X) (Thermo Scientific,
USA); Mouse IgE ELISA Set (BD OptEIA), Tween-20, 10×PBS,
Substrate solution A and B (Bio-Rad), 2 N H2SO4.; methacholine
(Sigma-Aldrich).
3.2
Virus culture and purification
HeLa Ohio cells (ATCC, USA) were cultured in EMEM with 10%
FBS in 5% CO2 incubator at 37℃ to reach the 70-80% confluence.
Rhinovirus 1B was seeded in HeLa Ohio cells with 2% FBS EMEM.
The infected cells were cultured in 5% CO2 incubator at 33℃ for 16 –
24 h until cytopathic effect was observed. The infected cells with
supernatant were harvested by using cell scrapers. The mixture was
centrifuged at 1200 rpm 4℃ for 10 min to collect the cells and remove
the supernatant. The cell pellet was washed with 1×PBS once and resuspended in PBS after centrifugation. The cell suspension was frozen
and thawed twice to break down the cell membrane and release the
virus. Afterwards, cell debris was removed by 4500 rpm centrifugation,
and the virus in the supernatant was precipitated with 0.5 M NaCl with
7% PEG 6000 on ice for 1 h. After precipitation, virus was centrifugated
at 4500 rpm for 60 min and then dissolved in 1×PBS. Virus
concentration was conducted by using Amicon Uitra-15 centrifugal
filtration devices (Millipore, USA). Concentrated virus solution was
aliquoted and stored in -80℃ (Bartlett et al., 2008).
42
3.3
Virus titration
HeLa Ohio cells were seeded in a flat bottom 96 well cell culture
microplate (Corning, USA) overnight to reach 80% above confluence.
The medium was removed and 1×PBS was added to wash the cells by
a multi-channel micropipette. Virus stock was 10 serial diluted with 2%
FBS EMEM medium and each dilution was aliquoted to infect 8 wells of
cells. The plate was incubated in 5% CO2 incubator at 33℃ for 5 days
until the cytopathic effect (CPE) (cell rounding and detachment from
the monolayer, Figure 3.1) was observed under microscope. The
infection situation of each well was recorded to calculate the virus titer.
TCID50 (50% tissue culture infectivity dose) method was used for viral
titer assessment (Reed and Muench, 1938). The calculation was
conducted through the “Reed & Muench Calculator” (Lindenbach,
2004).
Figure63.1 Cytopathic effect in Rhinovirus 14 infected HeLa cells. (A)
Normal HeLa cells. (B) CPE in HeLa cells infected with Rhinovirus 14.
Adapt from (Gaudernak et al., 2002)
43
3.4
Mouse model of asthma exacerbation
The six-week-old female BALB/c mice for this project were
ordered from Animal Holding Unite (AHU) and housed in plastic cages
(maximum 5 mice per cage) in the Animal Vivarium of Centre for Life
Science in National University of Singapore. The breeding room in
Animal Vivarium is regulated by automatic timers to provide 12 h of
light and 12 h of dark cycles. The temperature in the animal room
ranges from 18 to 26℃ with an average of 22℃. Maintenance diets
contain 4-5% fat and 14% protein. The cage bedding was changed
every two days.
Mice were sensitized with 50 μg OVA and 2 mg Al(OH)3 in 0.2
ml saline intra-peritoneally on day 0, and challenged on day 9, 10, 11
with either intranasal 25 μg OVA in 30 μl PBS or 30 μl PBS only.
During the third challenge on day 11, mice were infected with 5×106
TCID50 Rhnivirus-1B in 25 μl PBS or same volume PBS only (Bartlett et
al., 2008). Animal experiments were performed according to the
Institutional Guidelines for Animal Care and Use Committee of National
University of Singapore.
3.5
Bronchoalveolar lavage (BAL) fluid collection
BAL fluid was collected at different time points (day 1, 2, 3, 5, 7)
after the last challenge. Mice were anaesthetized with 0.35 ml mouse
anaesthetic mixture (ingredients show in 3.1) by intra-peritoneally
injection. Tracheotomy was carried out and a blunt needle (20G) was
44
inserted into mouse trachea. The Lungs was instilled and washed with
3 syringes of 0.5 ml ice-cold PBS (Cheng et al., 2011).
3.6
Total and differential BAL fluid cell counts
BAL fluid was centrifuged at 3000 rpm for 5 min at 4 ℃ .
Supernatant was collected and stored in -80℃ for further experiment.
The remaining pellet was suspended and washed with 200 μl 0.875%
NH4Cl at room temperature for 5 min. The cell suspension was
centrifuged at 3000 rpm for 5 min at 4℃ and the supernatant was
discarded. The cell pellet was re-suspended with 200 μl of RPMI media
with 1% BSA. The total number of viable cells was counted by using a
hemocytometer under microscope (10 μl cell suspension with 10 μl
trypan blue, magnification ×100). After that, the cell suspension was
diluted to 106 cells/ml and a 200 μl aliquot was cytospined to a slide in
a Shandon cytospin 3 (Thermo Electro Corporation, USA), with a
centrifugation at 600 rpm for 10 min at room temperature. The BAL
fluid cells were stained with a modified Wright Staining. The slide was
first stained with 0.8 ml Liu A for 30 sec and then 1.6 ml Liu B for 90
sec. Differential cell count was conducted with a minimum of 500
leukocytes under a microscope (magnification ×1000). Four different
types of leukocytes (macrophage, eosinophil, lymphocyte and
neutrophil) were identified and their percentages were counted (Cheng
et al., 2011).
45
3.7
Histological examination
Lungs were collected from mice at different time points (day 1,
3, 7 after last challenge), fixed in 10% neutral buffered formalin solution
for at least two days and processed in a tissue processor (Leica
Microsysterms, Wetzler, Germany). Dehydration of the lung samples
was conducted in a serial concentration of ethanol (70% - 80% - 90% 100%, 30 min each and 2 h for 100%). The samples were then placed
in xylene 3.5 h for 3 times. Samples were infiltrated in hot molten
paraffin wax for another 3 h. Tissues were then embedded in paraffin
wax, fixed and sectioned into 5 μm pieces using a microtome (Leica
Microsysterms, Wetzler, Germany). The sectioned tissues were then
placed and dried on glass slides.
Harris Hematoxylin and Eosin (H&E) staining was performed to
measure the severity of inflammatory cell infiltration. Slides were
placed in histoclear for 10 min and rehydrated in a serious
concentration of ethanol (100% - 100% - 90% - 70% - distill water) and
each for 2 min. The sections were then stained with Harris hematoxylin
for 5 min, washed in the distilled water, and differentiated in 0.1% acid
alcohol solution for 30 sec. After washed with tap water for 5 min, the
sections were stained with Eosin for 30 sec and washed with distilled
water. Afterwards, the sections were dehydrated in a series of ethanol
solutions (70%-90%-100%-100%) each for 30 sec and immersed in
histoclear for 10 min. Evaluation of inflammation around peribronchial
46
and perivascular areas was semi-quantitatively performed in a blinded
manner. Inflammatory 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: a thick layer (2 - 4 cells layer deep) of inflammatory cells; 4: a
thicker layer (more than 4 cells layer deep) of inflammatory cells (Myou
et al., 2003).
Periodic Acid - Schiff reagent staining was performed to
determine the extent of mucus secretion. Slides were deparaffinized
with histoclear for 10 min and rehydrated in a serious concentration of
ethanol (100%-100%-90%-70%-distilled water, each for 2 min). The
sections were immersed in the periodic acid for 5 min, washed with
distilled water, and placed in Schiffʼs reagent for 15 min. Slides were
then washed with tap water for 5 min and stained in Mayerʼs
hematoxylin for 90 sec. the sections were dehydrated in a series of
ethanol solutions (70% -90%-100%-100%, each for 2 min) and cleaned
with histoclear for 10 min. Goblet cell hyperplasia in the airway
epithelium was assessed blinded and scored based on a 5-point
grading system. Mucus scores (0-4) were assigned according to the
percentage of PAS-positive mucin-producing cells within the bronchi
rings: 0, 0; 1, 75%. The same
calculation system was adopted from the H&E staining (Grünig et al.,
1998).
47
3.8
Immunoglobulin E levels in serum
Blood was collected on the sacrificing day by cardiac puncture
and allowed to clot for at least 2 h before centrifugation at 3000 rpm for
10 min at 4°C. Serum was extracted from the top layer of the
supernatant and stored at -80°C.
Serum level of total IgE and OVA-specific IgE were measured
by ELISA kit, according to the manufacturerʼs instructions. Platecoating was conducted by adding 100 μl of coating liquid (diluted
capture antibodies for total IgE and OVA solution for specific IgE) to
each well of ELISA plates (NUNC, Demark). The ELISA plates were
sealed and incubated overnight at 4 °C. After coating, the ELISA plates
were washed three times with washing buffer (1xPBS with 0.05%
Tween-20) and blocked with 200 μl assay diluent buffer (1xPBS with
10% heat-inactive FBS) for 1 h at room temperature. After washing off
blocking buffer, 100 μl of standards and samples were loaded into
plates. After 2 h incubation at room temperature, the plates were
washed 5 times and incubated with biotinylated anti-mouse IgE
monoclonal
antibody
and
streptavidin-horseradish
peroxidase
conjugate (SAv-HRP) for 1 h. After washing 7 times, 100 μl of
substrate solution for HRP was added and incubated for another 30
min in dark. To stop the reaction, each well was added 50 μl of
stopping solution (2 N H2SO4). The plates were read at 450nm
(reference filter 570 nm) using an automatic microplate reader
48
(Sunrise, TCAN, Austria).
3.9
Reverse transcription-polymerase chain reaction (RT-PCR)
Lungs were isolated from the thoracic cavity at different time
points (day 1, 2, 3, 5, 7 after the last challenge) and stored in RNAlater
at -80℃. Before RNA isolation, lung tissues were removed from the
RNAlater and immersed in 1 ml Trizol, and homogenized on ice using a
homogenizer (Heidolph). The homogenate was centrifuged at 12000 g
4℃ for 10 min. The supernatant was collected and incubated for 5 min
at room temperature. Then, the homogenized sample was mixed with
0.2 ml of chloroform, followed by vigorous shake for 15 sec and room
temperature incubation for 3 min. A centrifugation was conducted at
12000 g for 15 min at 4℃, and the aqueous phase of the sample was
collected. The aqueous phase was mixed with 0.5 ml isopropanol and
incubated at room temperature for 10 min. After centrifuged 12000 g
for 10 min at 4℃, the pellet was washed with 1 ml of 75% ethanol by
vortex and centrifugation at 7500 g for 5 min at 4℃. The RNA pellet
was air-dried and re-suspended in RNase-free water. Incubation in a
water bath was conducted at 55-60℃ for 10 min to make the RNA
completely dissolved.
Ethanol precipitation was conducted to purify the RNA. 3 M
CH3COONa was added into RNA to make the final concentration 0.3
M. The mixture was vortexed with 3 times volume of ethanol for 10 sec
and stored at -20℃ overnight or -80℃ for 2 hours. Afterwards, the
49
mixture was centrifugated at 13000 g for 20 min at 4℃ to discard the
supernatant. The pellet was vigorously washed with 200 μl of 95%
ethanol and dried under vacuum with a lyophilizer and re-suspended
with RNase-free water.
The amount and purity of RNA was measured by the
spectrophotometer (NanoDrop ND-1000, Thermo Fisher Scientific Inc,
Waltham, MA, USA). Both A260/A280 (DNA/protein) and A260/A230
(DNA/organic contaminants) ratios were recorded as an indicator of
purity of RNA. An acceptable level of purity for RNA extracts should be
around 1.9 to 2.1 for both A260/A280 and A260/A230 ratio.
cDNA was synthesized from 1 μg total RNA together with an
oligo dT primer and AMV reverse transcriptase by a multiwell thermal
cycler (GeneAmp PCR system 2700, Applied Biosystems, USA). Real
time PCR was performed on ABI 7500 cycler in a 20 μl reaction system
containing 1 μl cDNA (1μg), 10 μl 2 x SYBR master mix, 1 μl forward
primer (10 μM), 1 μl reverse primer (10 μM) and 7 μl nuclease-free
water. The primers were obtained from 1st base and the respective
sequences are shown in Table 3.1.
3.10
Measurement of airway hyper-responsiveness (AHR)
Lung resistance (RI) and dynamic compliance (Cdyn) in
response to increasing concentrations of methacholine (0.5 - 8.0
mg/ml) were measured using a whole-body plethysmograph chamber
(Buxco, Sharon, CT). Rl is the ratio of driving pressure to the rate of
50
[...]... Inc, Waltham, MA, USA) Both A2 60 /A2 80 (DNA/protein) and A2 60 /A2 30 (DNA/organic contaminants) ratios were recorded as an indicator of purity of RNA An acceptable level of purity for RNA extracts should be around 1.9 to 2. 1 for both A2 60 /A2 80 and A2 60 /A2 30 ratio cDNA was synthesized from 1 μg total RNA together with an oligo dT primer and AMV reverse transcriptase by a multiwell thermal cycler (GeneAmp... and Davis, 20 12) Sendai and RSV infection were also established in guinea pig models and induced clinically relevant symptoms (Riedel et al., 1996; Dakhama et al., 1999) Efforts were also made to establish asthma exacerbation models via rhinovirus infection, the major cause of human asthma exacerbation However, more than 90% of rhinovirus serotypes use human ICAM-1 to enter cells and cannot bind to mouse. .. ethnic background (Masoli et al., 20 04) The exacerbations of its symptoms usually cause the mortality and bring heavy social and economic burdens Respiratory virus infections, particular rhinovirus infections, were associated with asthma exacerbation as a crucial trigger However, the mechanism of rhinovirus associated asthma exacerbation is still unclear and needs more investigation Thus, animal models... species, and the lack of chronicity in inflammation, it is hard to completely mimic the symptoms and immune responses in human asthma exacerbations Despite the shortcoming, the investigation on animal models has contributed much to the exploration of asthma exacerbation, enhancing our understanding of this disease 37 Chapter 2 Rational 38 Asthma affects nearly 300 million people of all ages and all... Sly, 20 07) Among them, mouse models of asthma were the most commonly used due to their resemblance features of human asthma With the sensitization and challenge by allergens, mouse asthma models are easy to develop asthma symptoms including eosinophilic infiltration, mucus hyper-secretion and AHR Female Balb/c mice were commonly used as the higher prerulane of asthma exacerbation in women and a good.. .20 08) 1 .2. 3.6 Airway Hyper-responsiveness (AHR) As a clinical symptom in asthma, the degree of AHR is usually in proportion to the severity of asthma Provokers such as viral infection and allergen exposure can enhance the underlying AHR in asthmatic patients (Cockcroft and Davis, 20 06) It is suggested that AHR has two components: the variable component associated with airway inflammation and the... respiratory virus infections in asthma development and exacerbation, which support the use of allergen sensitization combined with virus infection to establish asthma exacerbation model Traditionally, in vivo asthma models were established in animals sensitized and challenged by allergens Many species of animals, such as mice, rats, guinea pigs, monkeys, were used to establish asthma models (Zosky and... room in Animal Vivarium is regulated by automatic timers to provide 12 h of light and 12 h of dark cycles The temperature in the animal room ranges from 18 to 26 ℃ with an average of 22 ℃ Maintenance diets contain 4-5% fat and 14% protein The cage bedding was changed every two days Mice were sensitized with 50 μg OVA and 2 mg Al(OH)3 in 0 .2 ml saline intra-peritoneally on day 0, and challenged on day 9,... injury and inflammation The structural changes including epithelial thickening, goblet cell hyperplasia, and airway smooth muscle hypertrophy and hyperplasia, contribute to the overall thickening of the airway wall in asthma (Berend et al., 20 08) Airway inflammation also plays a role in AHR in asthma The correlation between peripheral blood eosinophilia and AHR was demonstrated, along with the effect of. .. rhinovirus may function through inflammatory mediators (Grünberg et al., 1997) With the regulation of IL-13, neonatal rhinovirus infection induced AHR in mice, further supporting the role of inflammatory mediators in virus-associated AHR (Schneider et al., 20 12) 1.3 Animal models for asthma exacerbation Asthma exacerbations, especially virus -induced ones, have brought heavy burden to the society Inhaled corticosteroid ... Inc, Waltham, MA, USA) Both A2 60 /A2 80 (DNA/protein) and A2 60 /A2 30 (DNA/organic contaminants) ratios were recorded as an indicator of purity of RNA An acceptable level of purity for RNA extracts... were also made to establish asthma exacerbation models via rhinovirus infection, the major cause of human asthma exacerbation However, more than 90% of rhinovirus serotypes use human ICAM-1 to... with asthma exacerbation as a crucial trigger However, the mechanism of rhinovirus associated asthma exacerbation is still unclear and needs more investigation Thus, animal models including mouse