Attenuation of Airway Hyperreactivity by Gram- Negative Lipopolysaccharide in a
Murine Model of Asthma
By
Daven Jackson-Humbles

A DISSERTATION
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of
Pathobiology - Environmental Toxicology - Doctor of Philosophy
2014

ABSTRACT
ATTENUATION OF AIRWAY HYPERREACTIVITY BY GRAM- NEGATIVE
LIPOPOLYSACCHARIDE IN A MURINE MODEL OF ASTHMA
By
Daven Jackson-Humbles
Asthma exacerbations due to exposures to air pollution and environmental allergens are
common, leading to diminished responses to treatment and increased hospitalizations.
Endotoxin or lipopolysaccharide (LPS) is a component of the cell wall of Gram- negative
bacteria found throughout the environment. Individually, asthma and inhalation of LPS
increases lung inflammation and airway hyperresponsiveness (AHR). Previous studies
have shown that LPS exposure modulates both inflammatory and physiological
responses in the lungs of asthmatic individuals. The primary focus of this work was to
investigate the effects of inhaled LPS exposure on the pathology and physiological
responses in the lung of a murine model of ovalbumin (OVA) - induced allergic asthma.
Additionally, I investigated potential mechanisms that may underlie the dissociation
between airway inflammation and AHR present in allergic mice exposed to LPS (OVALPS). I hypothesized that the attenuation of AHR in OVA-LPS mice was dependent on
the upregulation of the gene responsible for nitric oxide production, Nos2, induced by
recruited neutrophils. Four specific aims were generated.

In aim 1, I studied the

relationships between the cellular character and distribution of lung lesions with
components of AHR in OVA-LPS mice. To do so, naïve and OVA- induced allergic
mice were exposed to LPS by intranasal instillation 48 hours following the final
saline/OVA challenge. AHR was determined and animals were euthanized to collect

tissue samples 24 hours after LPS exposure. There was significant pulmonary
inflammation consisting of eosinophils distributed

within the central airway

compartment composed of perivascular and peribronchiolar regions (OVA group),
neutrophils located in central airway and peripheral lung tissue compartments
composed of alveolar septa and airspaces (LPS group), and OVA-LPS mice had more
severe inflammation combining both cell types distributed in both compartments as well.
AHR was increased in both central airways and the peripheral lung tissue in OVA and
LPS mice.

By comparison, AHR was attenuated in OVA-LPS mice.

In aim 2, I

hypothesized that recruited airway neutrophils contributed to the attenuation of AHR in
OVA-LPS mice. Systemic depletion of neutrophils prior to LPS exposure significantly
lowered BALF neutrophils. Compared to neutrophil sufficient mice, neutrophil depletion
did not alter AHR in OVA-LPS mice. In aim 3, I hypothesized that LPS activation of the
transcription factor, NF-κB, attenuated AHR in OVA-LPS mice. Following treatment with
a

novel NF-κB

inhibitor, OVA-LPS mice

had

significantly decreased BALF

macrophages, eosinophils, and lymphocytes but not neutrophils compared to nontreated mice.

In spite of the decrease in cellularity, AHR significantly increased

suggesting that NF-κB activation contributes to the attenuation of AHR in OVA-LPS
mice. In aim 4, I evaluated the relationship between expression of the genes nitric
oxide synthase-2 (Nos2) and arginase-1 (Arg1) and the attenuation of AHR in OVA-LPS
mice.

Following LPS exposure, Nos2 and Arg1 were increased in OVA-LPS mice.

Treatment with a NF-κB inhibitor significantly blunted the expression of both genes,
suggesting that NF-κB mediated increased expression of Nos2 potentially contributes to
attenuation in AHR, which is reversed with downregulation of NOS2 gene expression.

This dissertation is dedicated to the following people:
the memory of my father, Tommy L. Jackson, my biggest supporter and source
of inspiration;
my mother, Mary, for being my rock and my angel;
my husband, David, for being proud of my work and enduring all of the sacrifices
that have accompanied this process;
my sisters, Stephanie and Cassandra, for being there for me; and
my daughter, Niveah, for showing me the blessings of life.

iv

ACKNOWLEDGMENTS

I would first like to thank God for providing me with the strength and courage to
endure all things in life and the wisdom to seek peace. With highest gratitude, I would
like to recognize everyone who made completion of this dissertation project possible.
First, I would like to acknowledge my mentor, Dr. James Wagner for his
encouragement, guidance, and patience over the years. He has believed in me even
when I lost confidence in myself.

Not only has Jim supported my educational

endeavors, but has been extremely understanding and kind as I experienced major
personal events in my life. I have been blessed to have him as my mentor. Also, I am
grateful to Dr. Jack Harkema for helping me to learn and grow as a scientist as well as a
pathologist. I would also like to thank the rest of my committee members: Drs. Mark
Evans, Ed Robinson, and Kurt Williams for their time, support, insightful comments and
most of all for sharing their knowledge with me.
I’d like to extend special thanks to past and present members of the
Experimental Toxicology laboratory: Lori Bramble, Ryan Lewandowski, Robert Buhs,
Ian Hotchkiss, Luke Hotchkiss, Chanel Redden; and Drs. Katryn Allen, Amanda Audo,
Neil Birmingham, Christina Brandenberger, Echo Hansen and Keisha Williams. Also, I
would like to thank Dr. Ning Li for her support and expertise. Without their assistance I
would not have been able to generate and analyze my data.

Additionally, these

individuals made it a pleasure to work and learn in the laboratory.
I would like to acknowledge Dr. Jetze Tepe and Lauren Azevedo for allowing me
to use their NF-κB inhibitor to further examine my research model.
v

Lauren was

instrumental in helping me administer this treatment in my study and in helping me to
understand its effects.
I am appreciative to the Department of Pathobiology and Diagnostic Investigation
and its leaders: Drs. Jill McCutcheon, Jennifer Thomas, and Willie Reed for giving me
the opportunity to pursue this dissertation work. I am especially grateful for the financial
assistance granted by fellowships awarded by Pfizer Pharmaceuticals, Pfizer Animal
Health, and the Center for Integrative Toxicology at Michigan State University.
Lastly, I would like to thank my family for being as understanding as they have
been supportive. I could not have achieved this or anything else in life without them.
Also, I am extremely thankful to my extended family at Mask Memorial CME Church for
their countless prayers.

vi

TABLE OF CONTENTS
LIST OF TABLES ......................................................................................................... xi
LIST OF FIGURES ....................................................................................................... xii
KEY TO ABBREVIATIONS ........................................................................................ xiv
CHAPTER 1. GENERAL INTRODUCTION AND SPECIFIC AIMS .............................. 1
1.1 ALLERGIC ASTHMA ............................................................................................. 2
1.2 PATHOPHYSIOLOGY OF ASTHMA ..................................................................... 3
1.2.1 Structural Components .............................................................................. 3
1.2.2 Cellular Mediators ...................................................................................... 4
1.2.3 Soluble Mediators ...................................................................................... 5
1.3 NITRIC OXIDE AND ASTHMA .............................................................................. 7
1.4 NF-κB ................................................................................................................... 8
1.5 ANIMAL MODELS ............................................................................................... 10
1.6 DEVELOPMENT OF EXPERIMENTAL ASTHMA ............................................... 12
1.7 AIR POLLUTION ................................................................................................. 13
1.8 ASTHMA AND ENDOTOXIN ............................................................................... 15
1.9 AIRWAY HYPERRESPONSIVENESS ................................................................ 18
1.9.1 Airway Smooth Muscle (ASM) and AHR .................................................. 19
1.9.2 Inflammation and AHR ............................................................................. 20
1.9.3 Measuring AHR ....................................................................................... 20
1.10 RESEARCH GOALS ........................................................................................... 24
1.11 HYPOTHESIS AND AIMS ................................................................................... 25
REFERENCES............................................................................................................ 28
CHAPTER 2. EFFECTS OF INHALED LIPOPOLYSACCHARIDE ON AIRWAY
INFLAMMATION AND AIRWAY HYPERRESPONSIVENESS IN ALLERGIC MICE . 47
2.1 ABSTRACT ......................................................................................................... 48
2.2 INTRODUCTION ................................................................................................. 50
2.3 MATERIALS AND METHODS ............................................................................. 53
2.3.1 Laboratory Animals .................................................................................. 53
2.3.2 Experimental protocol: Development of allergic airway disease and LPS
exposure ............................................................................................................. 53
vii

2.3.3 Necropsy, Lavage Collection, and Tissue Preparation ............................ 55
2.3.4 Bronchoalveolar Lavage Analysis ............................................................ 55
2.3.5 Histopathology ......................................................................................... 56
2.3.6 Morphometry............................................................................................ 56
2.3.7 Real –time PCR of Lung .......................................................................... 57
2.3.8 Airway Hyperresponsiveness (AHR) Measurements ............................... 59
2.3.9 Statistical Analysis ................................................................................... 60
2.4 RESULTS ............................................................................................................ 61
2.4.1 Bronchoalveolar lavage fluid (BALF). ...................................................... 61
2.4.2 BALF Cytokines ....................................................................................... 63
2.4.3 Histopathology ......................................................................................... 65
2.4.4 Morphometric inflammatory cell analysis ................................................. 68
2.4.5 Airway Epithelial Mucus Production ......................................................... 70
2.4.6 Lung tissue mRNA expression ................................................................. 73
2.4.7 Airway Hyperresponsiveness................................................................... 75
2.5 DISCUSSION ...................................................................................................... 77
2.6 SUMMARY .......................................................................................................... 81
REFERENCES............................................................................................................ 83
CHAPTER 3. THE EFFECT OF NEUTROPHIL DEPLETION ON AIRWAY
HYPERRESPONSIVENESS IN ALLERGIC MICE EXPOSED TO INHALED
LIPOPOLYSACCHARIDE ........................................................................................... 90
3.1 ABSTRACT ......................................................................................................... 91
3.2 INTRODUCTION ................................................................................................. 92
3.3 MATERIALS AND METHODS ............................................................................. 95
3.3.1 Laboratory Animals and Treatment protocols .......................................... 95
3.3.2 Depletion of Neutrophils........................................................................... 95
3.3.3 Necropsy, Lavage Collection, and Tissue Preparation ............................ 96
3.3.4 Bronchoalveolar Lavage Analysis ............................................................ 96
3.3.5 Airway Hyperresponsiveness (AHR) Measurements ............................... 97
3.3.6 Statistical Analysis ................................................................................... 98
3.4 RESULTS ............................................................................................................ 99
3.4.1 Bronchoalveolar lavage fluid .................................................................... 99
3.4.2 Airway Hyperresponsiveness................................................................. 101
3.5 Discussion ......................................................................................................... 103
viii

3.6 SUMMARY ........................................................................................................ 106
REFERENCES.......................................................................................................... 107
CHAPTER 4. EFFECTS OF NF-κB INHIBITION ON AIRWAY INFLAMMATION AND
AIRWAY HYPERRESPONSIVENESS IN ALLERGIC MICE EXPOSED TO
LIPOPOLYSACCHARIDE ......................................................................................... 113
4.1 ABSTRACT ....................................................................................................... 114
4.2 INTRODUCTION ............................................................................................... 116
4.3 MATERIALS AND METHODS ........................................................................... 118
4.3.1 Laboratory Animals and Treatment protocols ........................................ 118
4.3.2 NF-κB Inhibition ..................................................................................... 118
4.3.3 Necropsy, Lavage Collection, and Tissue Preparation .......................... 119
4.3.4 Bronchoalveolar Lavage Analysis .......................................................... 120
4.3.5 Histopathology ....................................................................................... 120
4.3.6 Morphometry.......................................................................................... 120
4.3.7 Real –time PCR of Lung ........................................................................ 121
4.3.8 Airway Hyperresponsiveness (AHR) Measurements ............................. 121
4.3.9 Statistical Analysis ................................................................................. 122
4.4 RESULTS .......................................................................................................... 123
4.4.1 Bronchoalveolar lavage fluid .................................................................. 123
4.4.2 Histological examination and morphometry ........................................... 125
4.4.3 Lung gene expression............................................................................ 128
4.4.4 Airway Hyperresponsiveness................................................................. 130
4.5 Discussion ......................................................................................................... 132
4.6 Summary ........................................................................................................... 136
REFERENCES.......................................................................................................... 137
CHAPTER 5. ROLE OF Nos2 AND Arg1 IN THE ATTENUATION OF AIRWAY
HYPERRESPONSIVENESS IN ALLERGIC MICE EXPOSED TO INHALED
LIPOPOLYSACCHARIDE ......................................................................................... 143
5.1 ABSTRACT ....................................................................................................... 144
5.2 INTRODUCTION ............................................................................................... 146
5.3 MATERIALS AND METHODS ........................................................................... 149
5.3.1 Animals and Treatment Protocols. ......................................................... 149
5.3.2 Relative Quantification Real-Time PCR ................................................. 149
5.3.3 Western blot analysis of iNOS and arginase-1 proteins in the lung ....... 150
ix

5.3.4 Immunohistochemistry ........................................................................... 151
5.3.5 Statistical Analysis ................................................................................. 151
5.4 RESULTS .......................................................................................................... 152
5.4.1 Lung tissue Nos2 and Arg1 mRNA expression ...................................... 152
5.4.2 Effects of NF-κB inhibition on pulmonary Nos2 and Arg1 expression .... 154
5.4.3 Protein expression of iNOS and Arg-1 ................................................... 156
5.4.4 Immunohistochemistry for iNOS ............................................................ 156
5.5 DISCUSSION .................................................................................................... 159
5.6 SUMMARY ........................................................................................................ 164
REFERENCES.......................................................................................................... 165
CHAPTER 6. SUMMARY AND CONCLUSIONS ..................................................... 171
REFERENCES.......................................................................................................... 181

x

LIST OF TABLES
Table 1. Qualitative evaluation of Inflammation in the lung ................................... 66
Table 2. Summary of changes in AHR in OVA-LPS mice....................................... 179

xi

LIST OF FIGURES
Figure 1. Classical method of determining lung mechanics using single
compartment model .................................................................................................... 22
Figure 2. Single compartment model with constant phase lung impedance ......... 23
Figure 3. Allergen sensitization, challenge, and LPS exposure protocol ............ 54
Figure 4. BALF of non-allergic and allergic mice exposed to LPS ........................ 62
Figure 5. BALF cytokines in non-allergic and allergic mice exposed to inhaled
LPS ............................................................................................................................... 64
Figure 6. Pulmonary histology of non-allergic and allergic mice exposed to
inhaled LPS .................................................................................................................. 67
Figure 7. Eosinophil and neutrophil distribution in the lung ................................. 69
Figure 8. Airway epithelial mucus............................................................................. 71
Figure 9. Airway mucus gene expression ................................................................ 72
Figure 10. Pulmonary gene expression in non-allergic and allergic mice exposed
to inhaled LPS ............................................................................................................. 74
Figure 11. AHR in non-allergic and allergic mice exposed to LPS ........................ 76
Figure 12. Experimental design for neutrophil depletion study in saline and OVA
mice exposed to LPS .................................................................................................. 96
Figure 13. Comparison of BALF in neutrophil sufficient and neutrophil depleted
mice ............................................................................................................................ 100
Figure 14. Comparison of AHR in neutrophil sufficient and neutrophil depleted
mice ............................................................................................................................ 102
Figure 15. Protocol for development of allergic airway disease and Inhibition of
NF-κB.......................................................................................................................... 119
Figure 16. Comparison of BALF in mice treated and not treated with NF-κB
inhibitor ...................................................................................................................... 124
xii

Figure 17. Pulmonary histology of mice with NF-κB inhibition ........................... 126
Figure 18. Airway mucosubstances and Gob5 gene expression following NF-κB
inhibition .................................................................................................................... 127
Figure 19. Pulmonary gene expression following NF-κB inhibition .................... 129
Figure 20. Effects of NF-κB inhibition on AHR in OVA-LPS mice ........................ 131
Figure 21. Pulmonary gene expression of Nos2 and Arg1 in allergic and nonallergic mice exposed to LPS ................................................................................... 153
Figure 22. Effect of NF-κB inhibition on pulmonary gene expression of Nos2 and
Arg1 ............................................................................................................................ 155
Figure 23. Immunohistochemical staining for iNOS ............................................. 128
Figure 24. Proposed mechanism for attenuation of AHR following endotoxin
exposure in allergic mice ......................................................................................... 180

xiii

KEY TO ABBREVIATIONS

OVA

Ovalbumin

LPS

Lipopolysaccharide

IgE

Immunoglobulin E

Th2

T helper-2

AHR

Airway hyperresponsiveness

IL

Interleukin

IFNγ

Interferon gamma

NO

Nitric oxide

Arg-1

Arginase-1

NOS

Nitric oxide synthase

nNOS

Neuronal Nitric oxide synthase

eNOS

Endothelial Nitric oxide synthase

iNOS

Inducible Nitric oxide synthase

cNOS

Constitutive Nitric oxide synthase

PM

Particulate matter

EPA

Environmental Protection Agency

TLR

Toll-like receptor

MCh

Methacholine

cGMP

Cyclic guanosine monophophate

ONOO
FeNO

-

Peroxynitrite
Fractional exhaled nitric oxide
xiv

HDM

House dust mite

FOT

Forced oscillation technique

MSU

Michigan State University

LPS

Lipopolysaccharide

i.p.

Intraperitoneal

IN

Intranasal

BALF

Bronchoalveolar lavage fluid

H&E

Hematoxylin and eosin

AB/PAS

Alcian Blue /Periodic Acid-Schiff

PAMPs

Pathogen- associated molecular patterns

DAMPs

Damage associated molecular patterns

PRRs

Pattern recognition receptors

NF-κB

Nuclear factor- kappa B

TGF-β

Tissue growth factor β

FEV

Forced expired volume

xv

CHAPTER 1
GENERAL INTRODUCTION AND SPECIFIC AIMS

1

1.1

ALLERGIC ASTHMA
Asthma affects over 25 million people in the United States with annual costs of

$50 billion (Akinbami et al., 2012; AAAAI, 2013). Asthma is a multifaceted, chronic
inflammatory disease of the airways that is associated with reversible airway
obstruction, mucus hyperproduction/hypersecretion in airway epithelium, airway smooth
muscle hyperplasia, and subepithelial fibrosis (Busse and Lemanske, 2001; GINA,
2012). Due to its complexity, there has been a movement by researchers within the
field to classify asthma as several diseases with associated reversible airflow
obstruction, rather than a single disease entity with variation in clinical and pathological
features (Lancet, 2006; Wenzel, 2006).

To this end, a recent consensus report

produced by experts from the European Academy of Allergy and Clinical Immunology
and the American Academy of Allergy, Asthma & Immunology recommended that
asthma be categorized by ‘endotypes’, which focused not only on clinical features as is
done with asthma phenotypes, but also on distinctions of pathology, physiology,
immunology, environmental influences, and response to treatment (Lötvall et al., 2011).
The focus of my research is allergic asthma, an endotype of eosinophilic asthma, which
is one of the most commonly studied asthma phenotypes.
The development of allergic asthma involves first the sensitization to a specific
antigen (allergen) and production of allergen-specific immunoglobulin E (IgE). Exposure
of sensitized subjects to an allergen results in a T helper-2 (Th2) lymphocyte-mediated
inflammatory response, with histamine release, airway infiltration of eosinophils, and
allergen-induced bronchoconstriction (Lötvall et al., 2011).
2

Common allergens

associated with allergic asthma include those antigens derived from house dust mites,
cockroaches, animal dander, and pollens from trees and grasses (Baxi and
Phipatanakul, 2010). Individuals with genetic predispositions to develop atopy have an
increased sensitivity for IgE production directed against these aeroallergens (Mukherjee
and Zhang, 2011).
1.2

PATHOPHYSIOLOGY OF ASTHMA
Clinically, allergic asthma is characterized by variable airway obstruction and

airway hyperresponsiveness (AHR) that is accompanied by airway inflammation and
remodeling. Asthma pathogenesis consists of multiple components, such as structural
changes and inflammation that interact to determine the severity of disease.
1.2.1

Structural Components
Studies have shown that asthmatics have extensive epithelial damage and loss

along with defects in tight junctions (Wan et al., 2000; de Boer et al., 2008; Xiao et al.,
2011). These defects allow allergens, pollutants, toxins, and pathogens to have better
access to the subepithelial airway tissues (Holgate, 2007; Al-Muhsen et al., 2011).
Also, there is goblet cell metaplasia in the central/conducting and peripheral airways
resulting in increased production and secretion of large amounts of mucus (Evans et al.,
2009).

Lungs from individuals with fatal asthma have an increased percentage of

epithelial goblets cells (20-25%) compared to non-asthmatics (5%) and a 20-fold
increase in goblet cells in peripheral airways which are normally not present (Pawankar
et al., 2009).

The increased mucus in the airway lumen contributes to AHR by

decreasing airway lumen size or caliber (Rogers, 2004).
3

A study by Short and

colleagues showed a significant relationship between AHR in asthmatics and airway
caliber or distensibility (Short et al., 2011). A decrease in airway caliber may further
promote airway closure.

In addition to changes in airway smooth muscle and

epithelium, Holgate and Davies also noted that alterations in neural and vascular
networks along with matrix deposition in airway wall promote the development of AHR
(Holgate and Davies, 2009).
1.2.2

Cellular Mediators
Eosinophils are a common in allergic asthma and recruitment is dependent on

Interleukin (IL)- 5 (Cohn et al., 2004). Eosinophils may play an important role in asthma
pathogenesis by causing damage to airway epithelium and cholinergic nerve receptors.
Also, eosinophils can mediate airway remodeling, mucus hypersecretion, and AHR by
the degranulation and release of toxic enzymes, such as major basic protein, production
of pro-inflammatory cytokines (IL-4, -5, and -13), chemokines (eotaxin), generation of
reactive oxygen species and leukotrienes (Liu et al., 2006; Kudo et al., 2013).
Neutrophils are increased with severe, corticosteroid resistant forms of asthma,
as well as during exacerbation and fatal exacerbation (Jatakanon et al., 1999; Goleva et
al., 2008; Fahy, 2009). The role of neutrophils in the pathogenesis of asthma is not
clear. Nabe and colleagues showed that neutrophils were inducers of the late asthmatic
response, which occurred several hours following allergen challenge, in mice (Nabe et
al., 2011). In a review by Foley and Hamid, they suggest that neutrophils may activate
epithelial cells and eosinophils along, with increasing goblet cell mucus secretion and
vascular permeability in acute asthma (Foley and Hamid, 2007).
4

Lymphocytes are crucial for the development of asthma because naïve CD4+ T
lymphocytes differentiate into (Th2) cells when stimulated during the sensitization
process. These T cells produce Th2 cytokines such as IL-4, -5, and -13 that promote
the expansion of Th2 cells. These T cells activate B lymphocytes to produce allergen
specific immunoglobulins (usually IgE) that bind to and activate mast cells (Cohn et al.,
2004; Shum et al., 2008; Holgate and Sly, 2014).
Mast cells are important in the development of acute asthmatic response. They
can cause mucus secretion, edema, and bronchoconstriction through the release of
mediators, such as histamine and prostaglandins. Also, mast cells promote the Th2
response of IgE production and eosinophilic inflammation by secreting Th2 cytokines,
as well as promoting antigen and Th17 cell neutrophilia (Murphy and O'Byrne, 2010).
Mast cells can induce airway remodeling and has been associated with AHR (Mauad et
al., 2011; Holgate and Sly, 2014).
1.2.3

Soluble Mediators
Th2 cytokines: As mentioned above, IL-4, -5, and -13 are important to maintain

Th2 lymphocyte activation.

IL-4 also promotes Th2 pathways by decreasing the

production of Th1 cells, IFN-γ and IL-12, and induces alternative activation of
macrophages into M2 cells and inhibits classical activation of macrophages into M1
cells (Gordon and Martinez, 2010). As previously stated, IL-5 is a potent stimulus for
eosinophils, leading to their expansion, recruitment and activation during allergy. IL-13
has similar sequence homolog to IL-4, but appears to be a more important mediator of

5

airway hyperresponsiveness, goblet cell metaplasia and mucus hypersecretion, which
all contribute to airway obstruction.
Nitric Oxide (NO) in exhaled breath has been used over the past decade as a
biomarker of airway inflammation in asthmatics. However, NO is a readily soluble,
ubiquitous messenger molecule that mediates various biological functions in several
organs in the body. At low concentrations it functions as a physiological signal, such as
to maintain blood flow as a vasodilator and is a neurotransmitter, while at high
concentrations it is cytotoxic to defend against pathogens (Ricciardolo et al. 2004). In
the lung, notable functions of NO include maintaining airway and vascular tone in
addition to regulating mucus secretion (Reynaert et al. 2005; Zuo et al. 2013). Within
the lung, NO is produced by many cells including nerves, endothelial cells, smooth
muscle cells, respiratory epithelium, type II alveolar cells along with inflammatory cells,
such as macrophages, mast cells, and neutrophils (Gaston et al. 1994; Ricciardolo et al.
2004). In vivo NO has a short half-life of less than 5 seconds due to its reactivity with
biological compounds, such as hemoglobin (Ricciardolo et al. 2004).
NO is produced by the enzyme nitric oxide synthase (NOS), which consists of
three isoforms: constitutively expressed neuronal NOS (nNOS; NOS1) and endothelial
NOS (eNOS; NOS3) isoforms; and inducible NOS (iNOS; NOS2) isoform expressed in
cells. Constitutive NOS enzymes depend on calcium and calmodulin to promote the
release of small amounts of NO (Ricciardolo et al. 2004).

While iNOS is calcium

independent and production of large amounts of NO is induced by pro-inflammatory
cytokines (Aktan 2004; Ricciardolo et al. 2004). Under normal physiologic conditions in
6

the lung, NO causes dilation of blood vessels and dilation of airways by increasing
production of cyclic

guanosine monophosphate (cGMP) causing smooth muscle

relaxation (Dweik et al. 1998). However during disease states, cytotoxic effects are
seen when NO interacts with superoxide to produce peroxynitrite (ONOO -), which has
been associated with the induction of AHR (SadeghiHashjin et al., 1996; Pacher et al.,
2007).
1.3

NITRIC OXIDE AND ASTHMA
The functional role of NO in the pathogenesis of asthma is complex and not

completely understood.

Its effects have been considered beneficial by promoting

bronchodilation and harmful due to edema formation from vasodilation, increased
mucus secretion, and enhancement of Th2 inflammation (Prado et al., 2011). It has
been well documented that increased NO is present in exhaled air (FeNO) and
bronchoalveolar lavage fluid (BALF) from asthmatics (Kharitonov et al. 1994; Barnes
and Liew 1995; Khatri et al. 2003; Anderson et al. 2011).

It has been shown that

elevated FeNO is associated with inflammation, particularly eosinophils, and its
concentration decreases with corticosteroids (Dweik et al. 2010). Also, high FeNO has
been correlated with asthma exacerbation (Guo et al. 2000). Prado and colleagues
showed that NO derived from inflammatory cells can enhance the inflammatory
response (Prado et al. 2006). FeNO is considered an indicator of airway inflammation
(Aytekin and Dweik 2012). Despite its association with inflammation, no definitive link
has been shown between high FeNO and asthma severity (Dweik et al. 2010). Recent
studies suggest that FeNO levels may indicate a subtype of asthma rather than
represent a clinical manifestation of asthma severity (Aytekin and Dweik 2012). For
7

example asthmatics with high FeNO generally were atopic with greater airway
eosinophils, airway reactivity, and obstruction (Dweik et al. 2010).
In animal models of allergic asthma, increase NO levels are present within BALF
in allergic subjects compared to saline control (Yang et al., 2010). Many studies have
been conducted examining the roles of NO production by the different isoforms of NOS.
Decreased bronchodilation due to the deficiency of NO derived from constitutive NOS
(cNOS) has been implicated in development of asthma (de Boer et al. 1999; Meurs et
al. 2003; Maarsingh et al. 2006; Prado et al. 2006).

Conversely increased NO

production by inducible NOS (iNOS) has been correlated with increased inflammation,
injury of airway epithelium, and asthma severity (Prado et al. 2006; Anderson et al.
2011; Zuo et al. 2013).
1.4

NF-κB
Nuclear Factor- kappa B (NF-κB) is a transcription factor involved in the

regulation of a variety of genes involved in cellular processes, immunity, and
inflammation (Hayden and Ghosh, 2008). Inactive NF-κB is bound to inhibitory proteins
of κB family (IκB) and sequestered in the cellular cytoplasm.

Activation of NF-κB

requires the degradation of IκB proteins by IκB kinase (IKK) complex. Activated IKK
phosphorylates IκB proteins which targets them for ubiquitination. The ubiquitinated IκB
proteins are then degraded by the 26S proteasome, and NF-κB enter the cell’s nucleus
to turn on expression of target genes, including its inhibitor IκB (Hayden and Ghosh,
2008; Hayden and Ghosh, 2011; Liu and Chen, 2011).

8

Downstream consequences of NF-κB activation are as diverse as the cells in
which it resides, but are usually associated with activating, or increasing cellular
functions. For example its activation is required for the development and proliferation of
lymphocytes (Kumar et al., 2004; Hayden and Ghosh, 2011). NF-κB regulates the
transcription of proinflammatory cytokines, chemokines, growth factors and cell
adhesion molecules (Kumar et al., 2004; Verma, 2004).

Because of these roles,

investigations into its effects in disease have been widely studied. Persistent NF-κB
activation has been noted with chronic inflammatory conditions such as diabetes,
rheumatoid arthritis, and asthma (Gagliardo et al., 2003; Verma, 2004; Simmonds and
Foxwell, 2008; Clarke et al., 2009; Rial et al., 2012).
NF-κB’s role in inflammation is complex, as it has been shown to modulate both
proinflammatory and anti-inflammatory functions and is tissue specific (Lawrence,
2009). It has been shown to induce proinflammatory genes during the initiation of
inflammation, but is then associated with the expression of anti-inflammatory genes
during the resolution (Lawrence et al., 2001). In intestinal epithelium activation of NFκB is cytoprotective and anti-inflammatory during acute colitis, but is pro-inflammatory
during chronic colitis (Eckmann et al., 2008). In studies by Poynter and colleagues, NFκB activation in lung epithelium promotes inflammation in an acute model of allergic
airway disease in BALB/c mice, and inhibition of NF-κB in the epithelium attenuated
inflammation, cytokines, chemokines, and IgE (Poynter et al., 2002; Poynter et al.,
2004). In addition to its influence on inflammation in asthma, NF-κB has also been
implicated with AHR in experimental asthma depending on the mouse strain and the
presence of inflammation (Pantano et al., 2008; Sheller et al., 2009; Alcorn et al., 2010).
9

Other pulmonary inflammatory diseases are associated with NF-κB activation.
LPS is a common inducer of the canonical pathway of activation, and causes lung injury
through sepsis or by inhalational exposure. Using mice with a transgene for NF-κB
inhibition in airway epithelium, Poynter and colleagues demonstrated that LPS-induced
airway inflammation can be modulated by epithelial NF-κB, and is associated with
increased neutrophilic inflammation and AHR.

However, NF-κB activation not only

occurs in the epithelium, but also in inflammatory cells (Vargaftig, 1997; Poynter et al.,
2003).
1.5

ANIMAL MODELS
Over one hundred years ago researchers at the Rockefeller Institute described

edematous airways and bronchoconstriction in guinea pigs after multiple injections of
horse serum (Auer and Lewis, 1910). By the 1970s, the guinea pig continued to be an
important model for experimental hypersensitivity responses, but other animal models
were developed such as the dog and monkey to understand different aspects of allergic
airways diseases (Patterson

n and Kelly, 1974).

However, over the last 20 years

Mus musculus domesticus (the laboratory mouse) has become the most frequently
used laboratory animal due to the availability to perform detailed analysis of cellular and
molecular responses, in addition to many transgenic and gene knockout strains that can
target specific pathways involved in asthma (Holmes et al., 2011). For example, allergic
airway disease induced by sensitization and challenge to an antigen induces specific
IgE production; Th2 mediated inflammation, airway hyperreactivity and increased
mucus production and secretion, all key features in human asthma (Fuchs and Braun,
2008; Kumar et al., 2008). Furthermore, like humans, these changes can be alleviated
10

by glucocorticoids and β2-adrenergic receptor agonists treatments commonly used to
control asthma symptoms (Takeda and Gelfand, 2009; Stevenson and Birrell, 2010).
Despite the critical role murine models have played in asthma research, they do
have their limitations. Unlike humans, mice do not naturally develop asthma (Takeda
and Gelfand, 2009). As well, there are differences in lung structure, such as the lack of
intrapulmonary airway cartilage, dissimilar branching patterns, and differences in
smooth muscle mass of airways (Hyde et al., 2009). Furthermore, the inflammatory
pattern in mice with acute allergic airway disease is more similar to allergic alveolitis
than the more focal, less intense inflammation present in human asthmatics (Cohn,
2001; Kumar and Foster, 2002; Holmes et al., 2011).

As such, caution has been

advised when interpreting and translating data from mouse studies (Wenzel and
Holgate, 2006).
Ovalbumin (OVA), an antigen that is derived from chicken egg, is a common
allergen used to develop allergic airway disease in animal models. It is inexpensive and
highly purified.

Its purity has allowed immunodominant epitopes that elicit immune

responses to be identified (Lloyd et al., 2014). OVA-sensitized and challenged BALB/c
mice display a robust Th2-type response with IL-4, -5, and -13 induction, IgE
production, eosinophilic airway inflammation, and AHR (Gueders et al., 2009). Recently
the use of environmental antigens with a human relevance has been introduced in
experimental asthma, most notably cockroach- and house dust mite (HDM)-derived
proteins. Whether these antigens will lead to superior animal models of asthma is a
current subject of debate (Phillips et al., 2013).
11

Variations in the pathology produced in OVA-induced allergic airway disease are
due to differences in the strain and gender of mouse (Takeda et al., 2001; Hayashi et
al., 2003; Gueders et al., 2009; Alcorn et al., 2010), the source of OVA, such as
endotoxin-free versus OVA with LPS contamination, (Watanabe et al., 2003; Tsuchiya
et al.,2012) and route and frequency of OVA administrations (Zhang et al., 1997).
1.6

DEVELOPMENT OF EXPERIMENTAL ASTHMA
One of the most commonly utilized protocols for the development of allergic

airway disease in mice is sensitization with ovalbumin (OVA) by intraperitoneal (i.p.)
injection containing an adjuvant, such as aluminum potassium sulfate (Alum). Alum
works by activating the cellular inflammasome (Nalp3) and IL-1 and IL-18 production
(Eisenbarth et al., 2008). Naïve T lymphocytes (CD+ T cells) are primed by dendritic
cell presentation of processed OVA peptides, and differentiate into IL-4 and -13
producing Th2 cells. IL-4 augments the Th2 response by stimulating IgE production
and eosinophilic inflammation.

Additionally, Th2 cell differentiation occurs by the

transcription factor, STAT-6, in the presence of IL-4 (de Lafaille et al., 2010). A second
i.p. injection of OVA amplifies the Th2 response and production of the immunoglobulins
IgG and IgE (Kumar et al., 2008). Once the immune response is generated, the mice
are re-exposed (challenged) with OVA directly into the airways via inhalation (aerosols)
or instillation into the nose or trachea to produce an inflammatory response,
pathological changes to airways, and airway hyperreactivity (AHR) (Bates et al., 2009).

12

1.7

AIR POLLUTION
With the increasing prevalence of asthma world-wide, there has been much

interest in assessing the link between allergens and exposure to additional
environmental agents, such as indoor and outdoor air pollution with the development
and modulation of asthma.

(Peden and Reed, 2010; Mukherjee and Zhang, 2011; Sly

and Holt, 2011). Tobacco smoke has been well recognized to worsen airway symptoms
in asthmatics (Weiss et al., 1999). Outdoor air pollutants such as particulate matter
(PM), ozone, traffic exhaust, and biogenic and agricultural dusts are also important
factors that can worsen asthma symptoms (Zeldin et al., 2006; Jacquemin et al., 2012;
Laumbach and Kipen, 2012).
Particulate matter (PM) is a mixture of solid particles and liquid droplets in the air.
The US Environmental Protection Agency (EPA) has set regulatory standards for PM in
the size ranges of less than 10 m aerodynamic size (PM10; coarse PM), and less than
2.5 m aerodynamic size (PM2.5; fine PM) (USEPA, 2013). PM2.5 is associated with
chronic diseases of the cardiovascular system, as well as compromised lung function,
asthma exacerbation, and obstructive disease. Increases in ambient PM2.5 are related
to allergic airway symptoms and emergency room visits for asthma (Millstein et al.,
2004; Nikasinovic et al., 2006; Li et al., 2011). Cellular and molecular mechanisms of
PM2.5 exacerbation of asthma has been successfully studied in rats (Harkema et al.,
2004; Wagner et al., 2012).
Diesel Exhaust: A substantial component of traffic PM, diesel exhaust emissions
are commonly used in clinical and preclinical studies to act as an adjuvant by promoting
13

allergic responses in humans and mice (Diaz-Sanchez et al., 1997; Diaz-Sanchez et al.,
1999; Li et al., 2009; Acciani et al., 2013). In animal studies diesel exhaust can worsen
responses during allergen challenge (Dong et al., 2005). Epidemiological studies in
children suggest that traffic-related PM2.5 increases asthma and other respiratory
disease (Searing and Rabinovitch, 2011). However, only few studies which have
assessed pollution and sensitization suggest that pollution induces sensitization in
children (Braback and Forsberg, 2009).
Ozone is a gaseous component of photochemical smog that is regulated by EPA
due to its health effects on the cardiorespiratory system (USEPA, 2013).

Airway

inflammation is exacerbated in atopic asthmatics exposed to ozone (Peden et al., 1995;
Michelson et al., 1999; Hernandez et al., 2010), and ozone increases intraepithelial
mucus and Th2 cytokines in nose and lung of allergic rats (Wagner et al., 2002; Wagner
et al., 2007). Studies in human volunteers suggest that ozone may modify asthma
through the same toll-like receptor (TLR4) as endotoxin (Peden, 2011).
Endotoxin or lipopolysaccharide (LPS) is a component of the cell wall of Gram
negative bacteria found throughout the environment. It is found in dust along with solid
and liquid waste produced in various occupational settings ranging from animal
husbandry, cotton industry, agricultural settings, and waste treatment facilities
(Charavaryamath et al., 2005; Heederik et al., 2007; Sigsgaard et al., 2010).
In healthy individuals, inhalation of LPS increases sputum neutrophils and
causes airway obstruction (Kline et al., 1999; Doyen et al., 2012; Möller et al., 2012);
however, this response varies between subjects (Kline et al., 1999). Inhalation of LPS
14

in mice and guinea pigs increases AHR and neutrophilic inflammation in BALF and lung
and decreases lung function (Toward and Broadley, 2000; Card et al., 2006; Hakansson
et al., 2012). In rats, airway endotoxin has been well characterized to induce goblet
cell hypertrophy and increased mucus production (Harkema and Hotchkiss, 1992).
1.8

ASTHMA AND ENDOTOXIN
Despite many years of research, pathways by which endotoxin modifies asthma

have not been definitively elucidated. Evidence for both exacerbation and inhibition of
allergic airway responses by endotoxin has been documented in both laboratory rodents
and humans. The complexities of the endotoxin response in asthmatics are a
consequence of not only the contrasting immunologic activities (i.e., Th1 vs. Th2
pathways), but also the pleiotropic effects endotoxin has on airway epithelium and
inflammatory cells (Doreswamy and Peden, 2011; Peden, 2011). These effects are a
result of the timing and dose of endotoxin exposure in asthmatics.

The protective

effects of endotoxin have generally been seen during asthma development in early life
which has generated the “hygiene hypothesis” which states that childhood infections
prevent the development of allergic disease by enhancing the Th1 immune response
(Von et al., 2000; Wills-Karp et al., 2001; Vercelli, 2006).

In a study by Braun-

Fahrlander and colleagues, they found an inverse relationship between endotoxin
present in mattresses of children and their incidence of having atopic asthma (BraunFahrlander et al., 2002). In mice George et al. (George et al., 2006) found that mice
exposed to corn dust bedding containing endotoxin had decreased pulmonary
inflammation. They also reported that mice exposed to the corn dust bedding in early
life and then removed from the bedding also had attenuated inflammation when
15

exposed to ovalbumin in adult life.

Another study in mice showed a decrease in

eosinophilic inflammation, IgE production, and BALF Th2 cytokines (Delayre-Orthez et
al., 2004). In rats LPS exposure before or early during sensitization prevented OVA
sensitization (Tulic et al., 2000). In summary, most studies suggest that LPS can
interrupt sensitization processes, but the definitive mechanism has yet to be identified.
Studies assessing the effect of endotoxin during allergen challenge have yielded
conflicting results depicting exacerbation or attenuation of the allergic response. These
variable responses have been attributed to differences in the amount of LPS given,
timing of exposure, and other factors, such as gender and genetic variation (Liu, 2002).
Eldrige and Peden reviewed several studies regarding the effect of the timing of LPS
exposure on the modulation of the asthmatic response in people. They found that
asthmatics are more sensitive to LPS based on studies showing that concurrent
allergen and LPS exposure, or LPS exposure following allergen challenge, caused an
enhanced response to endotoxin by airway neutrophilic inflammation, whereas prior
exposure to LPS promoted the allergic response by increasing eosinophilic
inflammation and AHR (Eldridge and Peden, 2000).

Other studies in asthmatic

individuals produced have also had variable results. For example, following allergen
challenge in asthmatics there is an increase in CD14, a cell surface receptor for LPS
binding protein, which is associated with increased neutrophilic inflammation and may
account for increased sensitivity to LPS in asthmatics (Alexis et al., 2001). Inversely, it
has been shown that intranasal challenge with LPS heightened the presence of
eosinophils in the nasal tissue of allergic subjects (Peden et al., 1999). However, a
recent study by Hernandez et al. reported that there was an attenuation in the
16

inflammatory response to inhaled LPS in atopic asthmatics, and was related to the
expression of TLR4 (Hernandez et al., 2012). In addition to disease-dependent receptor
expression, modulation of allergic responses by LPS may be dependent on
polymorphisms to endotoxin receptors CD14 and TLR4 (Simpson and Martinez, 2010).
Studies have shown that the amount of LPS exposure dictates the response in
asthmatics.

Exposure to low doses of LPS is associated with exacerbation of the

allergic inflammation in both asthmatics and allergic mice (Eisenbarth et al., 2002;
Alexis et al., 2004; Alexis et al., 2008; Dong L; Li H, 2009; Bennett et al., 2013). Alexis
and colleagues have shown that inhalation of low doses of LPS in asthmatics increases
genes associated with antigen presentation, innate immunity, and inflammation in
airway cells (Alexis et al., 2008), and they hypothesized that LPS promotes Th2
inflammation in the airways (Alexis et al., 2004).

Also, they showed decrease

phagocytosis by airway phagocytes (Alexis et al., 2003). Bennett et al. performed a
particle deposition study and showed that low dose LPS exposure increased deposition
of inhaled particles in asthmatics (Bennett et al., 2013). In addition, low LPS doses
increased AHR in asthmatics (Boehlecke et al., 2003). In OVA-induced allergic mice,
Dong et al. found that low dose LPS increased recruitment of eosinophils and
neutrophils into the lung, mucus secretion, and Th2 cytokines (Dong L; Li H, 2009).
Conversely, several investigations describe high doses of LPS as protective against
allergy and atopy.

Simpson and Martinez showed that in asthmatics with certain

genetic traits were protected against the development of atopy by exposure with high
doses of LPS (Simpson and Martinez, 2010). In allergic mice, high doses of inhaled
LPS induced a Th1 response characterized by increased neutrophils and IFN-γ
17

production and decreased mucus secretion (Eisenbarth et al., 2002; Kim et al., 2007;
Dong L; Li H, 2009). Reduced AHR after high LPS treatment was associated with
variants of toll-like receptor 4 (TLR-4), (Senthilselvan et al., 2009). Given the dosedependent effect of LPS on the allergic inflammation, it is not surprising that conflicting
data is present in the epidemiological literature.
1.9

AIRWAY HYPERRESPONSIVENESS
Airway hyperresponsiveness (AHR) is the exaggerated response of airways to a

contractile stimulus (Berend et al., 2008). In 1921 it was first described in asthmatics
following systemic administration of pilocarpine, a cholinergic agonist, (O'Byrne and
Inman, 2003). AHR is one of the main symptoms of asthma, but it can be present in
other airway diseases as well. It is a measure of the relationship between the shortening
of airway smooth muscle tissue and airway remodeling (Walker et al., 2013). AHR is
used clinically to diagnose asthma because airway narrowing is easily induced in
asthmatics; it can be reliably measured, and is often the cause of asthma mortality
(O'Byrne, 2010; Bossé et al., 2013). AHR can predict the development of asthma in
atopic individuals (Berend et al., 2008), asthma severity (Busse, 2010), and the
effectiveness inhaled corticosteroid treatment in asthmatics (Walker et al., 2013).
Similar to asthma the mechanism of AHR is not completely known. AHR can be
categorized as either persistent or variable based its occurrence (early vs. late phase
response), and severity of histopathology (Cockcroft and Davis, 2006; Meurs et al.,
2008; Busse, 2010).

Persistent or chronic AHR is constantly present even in the

absence of a stimulus. It is thought to be associated with chronic inflammation and
18

changes in airway structure, such as subepithelial membrane thickening, smooth
muscle hypertrophy, and fibrosis. As a result airways are less compliant and have
exaggerated response to bronchoconstrictors. Chronic AHR responds poorly to inhaled
corticosteroids, and the association with specific inflammatory cells is inconsistent.
Variable AHR develops and resolves within hours following exposure to allergens or
stimuli.

It has been linked to airway inflammation and is believed to reflect current

asthma severity. Variable AHR can be affected by external exposures to allergens,
respiratory infections, and corticosteroid treatment.
1.9.1

Airway Smooth Muscle (ASM) and AHR
There has been much debate regarding the role of airway smooth muscle (ASM)

in AHR (Gunst and Panettieri, 2012; Paré and Mitzner, 2012).

The basis of this

argument lies in whether the alterations of ASM are due to intrinsic phenotypic changes
or are a response by normal tissue to the modification of the surrounding milieu. There
are many studies that support the theory that the AHR in asthmatics is caused by
inherent abnormalities in ASM that alter its phenotype, such as increases in excitationcontraction

coupling

mechanisms,

hyperplasia,

extracellular

matrix

proteins,

inflammatory chemokine and cytokine production (Tagaya and Tamaoki, 2007; Gunst
and Panettieri, 2012). Wagers et al. found that intrinsic AHR in A/J mice was attributed
to the exaggerated response of ASM (Wagers et al., 2007).

Plant and colleagues

demonstrated that acute OVA mouse models displayed active ASM proliferation that
correlated with AHR in the peripheral airways, while chronic OVA mice developed
hyperplastic and hypertrophic ASM which correlated with increased AHR in central
airways (Plant et al., 2012). Other studies have focused on additional changes within
19

the lung, such as inflammatory mediators as the actual mediators of AHR that amplify
normal ASM contraction in asthmatics (Wagers et al., 2004; Bosse et al., 2010; Paré
and Mitzner, 2012).
1.9.2

Inflammation and AHR
Inflammation is one of the hallmarks of asthma, and its effects on AHR have

been extensively studied.

Inflammatory cells, such as mast cells, eosinophils, T

lymphocytes, neutrophils, alveolar macrophages, and dendritic cells, have been
associated with AHR (Wills-Karp, 1999; Short et al., 2011; Fuchs et al., 2012;
Hakansson et al., 2012; Janssen-Heininger et al., 2012). A study by Elwood et al. found
significant correlations between AHR and the presence of eosinophils during both early
and late responses following OVA challenge and with neutrophils in the late response
only (Elwood et al., 1992). Additional studies have shown that neutrophils can induce
late asthmatic responses in mice (Nabe et al., 2011). Also, neutrophils have been
associated with more severe forms of asthma, such as steroid resistant phenotype
(Jatakanon et al., 1999; Ito et al., 2008; Choi et al., 2012).
1.9.3

Measuring AHR
The detection of AHR can be accomplished through indirect and direct

stimulation. Indirect stimulation, which is not as widely performed, is produced by the
inhalation of agents such as hypertonic saline or mannitol, or performing exercise to
indirectly stimulate AHR via activation of inflammatory cells and the release of
inflammatory mediators (Busse, 2010).

This method indicates the level of current

airway inflammation. Direct stimulation with the use of inhaled agents, such as
20

methacholine (MCh), is the most commonly used method of AHR detection.
Methacholine is a cholinergic agonist that acts on the muscarinic receptors on the
airway smooth muscle causing its contraction. This method best reflects persistent
AHR (Cockcroft and Davis, 2006). In people, the extent of methacholine-induced AHR
is believed to be correlated with asthma severity and has a high association with other
indirect and direct stimuli of AHR, the presence of exhaled nitric oxide, and sputum
eosinophils (Porsbjerg et al., 2008; Tepper et al., 2012).
Our current understanding of lung mechanics in normal and diseased states can
be attributed to studies utilizing animal models. Previously, this data was generated
using unrestrained plethysmography in which pressure changes within a closed
chamber containing an awake, freely roaming animal were measured. Recently, the use
of these pressure changes, calculated as enhanced pause (Penh), as a measurement
of lung function has been shown to be misleading and can only accurately represent
breathing patterns (Bates and Irvin, 2003; Lundblad et al., 2007). Input impedance
utilizing the forced oscillation technique (FOT) is the most accepted technique for
measuring lung function in small laboratory animals (Vanoirbeek et al., 2010). Using a
small animal ventilator, a computer controlled piston determines pressure and flow
within the trachea by measuring volume displacement of the piston within the pump
cylinder (Schuessler and Bates, 1995; Bates and Irvin, 2003).
Measuring lung mechanics in mice is generally accomplished by using two
mathematical models, single compartment and constant-phase models.

The single

compartment model represents the lung as a linear structure composed of a flow21

resistive pipe or airway supplying a single elastic compartment or alveolar tissue (Bates
and Irvin, 2003; Irvin and Bates, 2003) as seen in figure 1. In the presence of an
oscillatory flow, the single compartment model is mathematically described as

RV + EV + Po

Equ.1

where P is pressure at the opening of the pipe, V is flow of gas, V is volume of gas in
the elastic compartment (E), Po is resting applied pressure or positive end-expiratory
pressure.
Figure 1. Diagram of single compartment model. Classical method of determining
lung mechanics using single compartment model consisting of resistance (R) of
conducting airways and elastance (E) of parenchymal tissue (Bates and Irvin, 2003).

Resistance (R) and elastance (E) are determined by fitting recorded values of the other
parameters into the equation using multiple linear regressions (Bates and Suki, 2008).
In this model, lung resistance is a measurement of narrowing of the central airways and
changes within the lung, while lung elastance measures peripheral lung alterations.

22

The constant-phase model established by Hantos et al. (Hantos et al., 1992)
measures R and E over a range of frequencies and provides a better separation of
central and peripheral lung changes (Irvin and Bates, 2003). Broad-band flow signals
are used to determine respiratory system input impedance (Zrs), which is a ratio of
pressure to flow as a function of superimposed oscillation frequency (Kaczka and
Dellaca, 2011).

The Fourier Transform of Eq. 1 produces input impedance; Eq. 2

describes the constant phase model:

Zrs = RN +

I

Equ.2

=

tan

-1

Figure 2. Single compartment model with constant phase lung impedance. RN is
Newtonian resistance;
is the positive square root of -1 or imaginary unit;
is
frequency; G is tissue damping, H is tissue elastance, and I is inertance or gas in the
central airways (Irvin and Bates, 2003; Bates, 2009).

23

Newtonian resistance is the approximation of resistance within the central airways, and
along with inertance composes impedance in airways. However, inertance is negligible
in mice at frequencies fewer than 20 Hertz (Bates and Irvin, 2003; Bates, 2009). Tissue
damping is the dissipative energy in the lung tissue and is related to tissue resistance.
Alterations in tissue damping occur with distortion of peripheral tissue due to airway
constriction, and increases indicate heterogeneity in airflow in the lung.

Tissue

elastance is the stored energy in the lung and is related to tissue stiffness. Increases in
tissue elastance can indicate airway closure or increased tissue stiffness due to
distortion of parenchymal tissue (Bates and Suki, 2008; Bates, 2009).
Applying a sine-wave oscillatory flow signal to the lungs measures dynamic lung
mechanics, and the data is analyzed using the single compartment model, while a
broadband oscillatory flow signal is applied to the lungs to measure respiratory
impedance analyzed by the constant phase model (Bates, 2009). By comparison most
assessments of lung function in asthmatics are evaluated by spirometric tests, such as
forced expired volume in 1 second (FEV1). The magnitude of airway obstruction can be
assessed by measuring FEV1 following the administration of bronchodilator agents and
AHR is assessed following bronchoconstriction (Reddel et al., 2009). However, FEV1 is
not a measurement of direct lung or airway resistance. To directly measure resistance
in people, FOT has recently become more commonly employed (Walker et al., 2013).
1.10

RESEARCH GOALS
LPS is a component of gram-negative bacteria ubiquitously present in particulate

matter of indoor and outdoor air, and has been demonstrated to modulate different
aspects of allergic airway disease. While early life exposure has been linked to
24

decreased atopy in childhood, results from clinical studies suggest asthmatics can
demonstrate either enhanced (Boehlecke et al., 2003) or blunted responses to
endotoxin exposure (Hernandez et al., 2012). Allergic inflammation is driven by Th2
cytokines (e.g., IL-4, -5,-13) and eosinophil granulocytes, whereas LPS activates Th1
pathways (e.g., IFNγ, TNF- ) and produces neutrophilic inflammation. In general, the
actions of these disparate immunity pathways oppose the action of the other, but the
mechanisms for LPS to either exacerbate or attenuate symptoms in asthmatics are
unknown. Furthermore, the fundamental knowledge of the interaction of LPS and
allergen to modulate airway inflammation and hyperreactivity is lacking. In my murine
model of allergic airway disease I demonstrated the attenuation in airway
hyperresponsiveness to methacholine despite an increase in the severity of
inflammation following airway LPS exposure. Therefore, I used this model of allergic
airway disease to identify the cellular and molecular mechanisms whereby airway
exposure to LPS modulates asthma.
1.11

HYPOTHESIS AND AIMS
My central hypothesis is that attenuation of airway hyperresponsiveness (AHR)

by LPS in asthmatic mice is dependent on upregulation of nitric oxide synthase 2 (Nos2)
gene that is associated with the recruitment of neutrophils. The specific aims used to
test this hypothesis are:
Aim 1: to determine the relationships between the cellular character and anatomical
distribution of lung lesions with specific components of AHR in allergic mice exposed to
airway LPS.
25

Aim 2: to determine the role of neutrophils in allergen-induced AHR following LPS
exposure in allergic mice.
Aim 3: to determine the role of nuclear transcription factor kappa beta (NF-κB), a central
modulator of inflammation, in the attenuation of allergen-induced AHR following LPS
exposure in allergic mice.
Aim 4: to determine the role of nitric oxide synthase in allergen-induced AHR in LPS
exposed allergic mice.
In aims 1-3, I used male BALB/c mice as a model of allergic airway disease by
injecting 20 µg of ovalbumin, an allergen, with 1 mg Alum, an adjuvant, into the
peritoneum of each allergic mouse.

Ten days following this injection, mice were

injected again in the peritoneum with 20 µg of ovalbumin, and in addition a 0.5%
ovalbumin solution was instilled through the nares. Control mice received intranasal
instillation of saline. A week later OVA-treated mice were intranasally (IN) challenged
with 0.5% ovalbumin for two consecutive days and control mice received saline. Two
days following last ovalbumin challenge, allergic and non-allergic mice received either 0
or 3 µg of lipopolysaccharide by intranasal instillation.

The next day airway

hyperresponsiveness data was collected and/or animal were euthanized to collect
samples

to

characterize

and

assess

pulmonary

alterations

and

injury

by

histopathological; histochemical (intraepithelial mucosubstances staining by periodic
acid Schiff); immunohistochemical (eosinophils and neutrophils); molecular (gene
expression by real time PCR); and immunological (evaluation of cytokines by flow
cytometry) methods.

26

I found that the attenuation in AHR to methacholine in allergic mice exposed to
LPS was not dependent on the location or severity of inflammation along the airways or
in parenchymal tissue.

For aim 2, neutrophil recruitment by LPS exposure was

prevented by depleting neutrophils using intraperitoneal injection of a neutrophil
depleting antibody for 2 consecutive days prior to LPS exposure.

This study

demonstrated that the presence of neutrophils was not associated with an increase in
AHR in allergic mice. Aim 3 examined the effects of NF-κB activation on the modulation
of allergic airway disease by endotoxin. A novel NF-κB inhibitor was injected i.p. 2 days
following the last OVA challenge and 1 hour prior to LPS or saline exposure. In this
study, I found that NF-κB inactivation does not affect the inflammatory response in late
phase asthma but modified the inflammatory cell mix of the acute inflammatory
response to inhaled LPS in this model. However, its inhibition reversed the attention of
AHR associated with LPS exposure in allergic mice. Specific aim 4 used lung tissues
collected from studies in aims 1 and 3 to analyze the expression of NOS 2 gene
expression.

I observed that NOS2 gene expression was increased in the lungs of

allergic mice exposed to LPS compare to mice with ovalbumin or LPS alone. There
was also a decrease in NOS 2 in the lungs of allergic mice exposed to LPS that were
given the NF-κB inhibitor compared to similar mice which only received the vehicle
control.

The results of these studies demonstrate that LPS can attenuate AHR in

established asthma by increasing gene expression of iNOS.

27

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28

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46

CHAPTER 2
EFFECTS OF INHALED LIPOPOLYSACCHARIDE ON AIRWAY INFLAMMATION
AND AIRWAY HYPERRESPONSIVENESS IN ALLERGIC MICE

47

2.1

ABSTRACT

Lung inflammation is a major characteristic of asthma, and is often used as an indicator
of severity. Nonetheless, inflammation severity has not correlated with asthma control,
and there are conflicting data determining whether increased inflammation is
associated with functional changes in the lung.

In this study, I examined the

relationship of pulmonary inflammation with changes in AHR by using a mouse model
of LPS-induced exacerbation of established OVA- induced allergic airway disease.
Male BALB/c mice were sensitized and challenged with saline or OVA. Two days
following the last challenge, allergic and non-allergic mice received either 0 or 3 µg LPS
by IN. Twenty-four hours later AHR was evaluated and animals were euthanized to
collect tissue samples. A significant inflammatory response consisting of infiltrations of
eosinophils in OVA alone mice and neutrophils in LPS only mice was present in the
BALF and lung. OVA-LPS mice produced a more severe inflammatory cell response
combining both eosinophils and neutrophils.

OVA-associated expression of Il13,

Muc5ac, and Gob5 genes in lung tissue was decreased following LPS exposure in
OVA-LPS mice, while expression of genes for Th1 cytokine Ifng and the regulatory
cytokine Il10 was increased.

AHR to methacholine challenge was increased in the

central airways and the peripheral lung tissue of both OVA and LPS alone mice,
although the magnitude was greater in LPS mice. Despite an increase in inflammation,
AHR was attenuated in OVA-LPS mice compared to both OVA and LPS only.
Morphometric analyses of eosinophils and neutrophils demonstrated that eosinophils in
allergic animals were localized within the central airway compartments (perivascular
48

and peribronchiolar regions). With LPS exposure, neutrophils were present throughout
the central airway and peripheral lung tissue. In OVA-LPS mice, both eosinophils and
neutrophils were distributed in the central and peripheral compartments. OVA and LPS
mice had increase AHR in the central airways and peripheral tissue.

However, in

OVA-LPS mice AHR was attenuated in both of these compartments even with
increased inflammation present in those regions. These findings suggest that AHR is
not linked to inflammation severity.

49

2.2

INTRODUCTION
Asthma is characterized by lung inflammation, reversible airway obstruction,

decreased lung function, and airway hyperresponsiveness (AHR) (Busse and
Lemanske, 2001; Fitzpatrick et al., 2008). Increased inflammation is often used as an
indicator of asthma severity but the relation to functional changes is inconsistent. For
example, elevations of induced sputum biomarkers or exhaled nitric oxide are used to
estimate the severity of airway inflammation but thus far have not been effectively
correlated with asthma control (Louis et al., 2000; Busse, 2010; Walker et al., 2013).
Furthermore, elevations in pulmonary eosinophils and neutrophils that occur in severe
asthmatics are not closely related with AHR (Wenzel et al., 1999; Holgate, 2008;
Busse, 2010). These inconsistencies may be due to the sampling site of inflammation
versus the site of AHR. Decreased airway function in some asthmatics may have
critical contributions from small airways, a region that is difficult and unreliable to
sample (Tulic and Hamid, 2006; Ueda et al., 2006; Burgel, 2011; Sterk and Bel, 2011;
Farah et al., 2012). As such, efforts to specifically target small and/or large airways
with therapeutic interventions would benefit from a deeper understanding of the
relationship between regional inflammatory status and functional changes within
specific airways.
Experimental asthma in laboratory rodents can reproduce many of the acute
features of asthmatic airways, including inflammation, epithelial remodeling, and airway
obstruction and reactivity (Nials and Uddin, 2008; Bates et al., 2009; Hyde et al., 2009).
These models allow the complete examination of the tracheobronchial tree that is
50

difficult to obtain by bronchial biopsy in humans.

Importantly, it is also possible to

conduct invasive respiratory maneuvers that provide a detailed analysis of functional
changes that occur during the development of allergic airways disease. Although the
forced oscillation technique (FOT) is currently considered an emerging technique to
measure pulmonary function in asthmatics (Tepper et al., 2012), it is commonly used to
assess experimental asthma in animals and provides greater specificity in determining
changes the airways (ie. central vs. peripheral airways) (Bates et al., 2009; Vanoirbeek
et al., 2010).
We have previously used allergic Brown Norway rats to show that environmental
agents such as ozone and particulate matter (PM2.5) can exacerbate airway
inflammation and mucus cell metaplasia (Wagner et al., 2007; Wagner et al., 2012).
We also found that ozone-induced epithelial lesions can be enhanced by another
airborne contaminant, lipopolysaccharide (LPS), a component of the cell wall of Gram
negative bacteria (Wagner et al., 2003) that has been shown to be present in
particulate matter in ambient air and occupational settings (Becker et al., 2002; MuellerAnneling et al., 2004).

Despite eliciting a neutrophilic, innate immune, Th1 type

response, inhaled LPS can exacerbate asthma symptoms in humans (Boehlecke et al.,
2003), as well as worsen allergic airway disease in rats (Tulic et al., 2000). However,
the histopathological changes induced by LPS in asthmatic airways have not been
characterized in detail, nor has the influence of LPS-induced neutrophil recruitment on
reactivity of asthmatic airways been described.

51

In the present study we developed a murine model of LPS-induced exacerbation
of asthma to test the hypotheses that 1) LPS induces site-specific neutrophilic lesions
in the allergic lung and that 2) the severity and location of lesions would be correlates
with treatment-related change in airway resistance.

We combined a detailed

morphological assessment of lung tissue with rigorous pulmonary function testing to
explore associations between changes in central airways and peripheral resistance with
anatomical lesions induced by allergen and LPS.

52

2.3

MATERIALS AND METHODS

2.3.1 Laboratory Animals
Male BALB/c mice (Charles River Laboratories, Portage, MI), 6-8 weeks of age
were housed in individual ventilated cages (IVC, Innocage; Innovive, San Diego, CA)
that contained

heat-treated aspen hardwood bedding (Northeastern Product Corp-

NEPCO, Warrensburg, NY). Cages were placed in a rodent ventilated housing system
(Innorack; Innovive, San Diego, CA). Animals were placed 3-4 per cage with ad libitum
food (Harlan Teklad Laboratory Rodents 22/5 diet, Madison, WI) and laboratory grade
acidified water (Aquavive; Innovive, San Diego, CA). Mice were maintained at Michigan
State University (MSU) animal facilities accredited by the Association for Assessment
and Accreditation of Laboratory Animal Care.

Animal rooms were kept at a

temperature of 21-24°C and relative humidity of 45-70%, with a 12 hour light/dark cycle.
All protocols and animal procedures were approved by MSU Institutional Animal Care
and Use Committee.
2.3.2 Experimental protocol: Development of allergic airway disease and LPS
exposure
Mice were randomly assigned to one of four experimental groups consisting of 6
animals. Mice designated to receive the allergen were sensitized to ovalbumin (OVA,
Sigma-Aldrich, St. Louis, MO) by intraperitoneal (i.p.) injection with 0.25 ml saline
containing 20 µg of OVA with 1 mg alum adjuvant (aluminum potassium sulfate, SigmaAldrich) on Day 0. On Day 10, mice were administered a boost with i.p. injection of
53

alum free OVA (20 µg) in 0.25 ml saline along with an intranasal (IN) instillation of 30 µl
0.5% OVA. Delivery of OVA by IN instillation was chosen to produce a robust
inflammatory response in the lung and induce AHR (Swedin et al., 2010) Seven days
later, the mice were challenged IN with 30 µl 0.5% OVA for two consecutive days (Days
17 & 18). All remaining mice received only saline during sensitization and challenge.
Two days following the last OVA or saline challenge (Day 20), mice in allergic and nonallergic groups were divided in half and received IN either 0 µg or 3 µg
lipopolysaccharide (LPS; P.aeruginosa, Sigma-Aldrich) in 30 µl saline.

Twenty-four

hours following LPS exposure (Day 21) pulmonary function testing and/or necropsy of
mice was performed (Fig 3).

Figure 3. Allergen sensitization, challenge, and LPS exposure protocol

54

2.3.3 Necropsy, Lavage Collection, and Tissue Preparation
Mice were anesthetized with an i.p. injection of with sodium pentobarbital (10
mg; Fatal Plus, Vortech Pharmaceuticals, Dearborn, MI). Blood was collected from the
caudal vena cava for separation of plasma, and animals were euthanized by transection
of the abdominal aorta. The lungs and heart were harvested en bloc. The lungs were
instilled twice with 800 µL saline via the cannulated trachea to collect BALF. After
lavage the right lung lobes were ligated, separated and stored in RNAlater (Qiagen) for
RNA isolation or snap frozen in liquid nitrogen for storage. The left lobe was inflated
with 10% neutral buffered formalin to a pressure of 30 cm H2O for 1 hour and then
stored in a large volume of the same fixative.
2.3.4 Bronchoalveolar Lavage Analysis
Total BALF leukocytes were counted with a hemocytometer and cytological
slides were prepared using a Cytospin centrifuge (Shandon). Slides were stained with
Diff-Quick (Dade Behring, Newark, DE) and cell differential (macrophages, eosinophils,
neutrophils, and lymphocytes) were counted. The remaining BAL fluid was centrifuged,
and the supernatants were analyzed for inflammatory cytokine concentrations of
interferon-gamma (IFN-γ), interleukin (IL)-17, IL-6, and tumor necrosis factor-alpha
(TNF- ).

All cytokine kits were purchased as either Flex Set reagents or as

preconfigured cytometric bead array kits (BD Biosciences, San Jose, CA). Cytokine
analysis was performed using a FACSCalibur flow cytometer (BD Biosciences). 50 μl of
BALF was added to the antibody-coated bead complexes and incubation buffer.
Phycoerythrin-conjugated secondary antibodies were added to form sandwich
55

complexes. After acquisition of sample data using the flow cytometer, cytokine
concentrations were calculated based on standard curve data using FCAP Array
software (BD Biosciences).
2.3.5 Histopathology
Following fixation, two traverse sections of the left lung were taken along the
axial airway at the levels of the 5th and 11th generation (G5 and G11) to examine the
proximal and distal airways (Harkema and Hotchkiss, 1992).

The tissues were

embedded in paraffin and stained with hematoxylin and eosin (H&E) for routine
histological examination, Alcian blue periodic-acid-Schiff stain (AB/PAS) to identify
intraepithelial mucosubstances, and immunohistochemical stains to detect eosinophils
(major basic protein, MBP; Mayo Clinic, AZ) and neutrophils (NIMP-R14; Serotec,
Raleigh, NC).
2.3.6 Morphometry
Estimation of the amount of the intraepithelial mucosubstances in epithelium
lining axial airways (G5 and G11) was performed as specified in previous studies
(Wagner et al., 2003). Using a computerized image analysis system, the volume density
(Vs) of AB/PAS-stained mucosubstances was quantified by calculating the area of
positive stained mucosubstances from the automatically circumscribed perimeter of
stained material using a Dell XPS 400 computer and Scion Image (Scion Corporation).
The length of the basal lamina beneath the surface epithelium was calculated from the
contour length on the digitized image. The volume of stored mucosubstances per unit of
56

surface area of epithelial basal lamina was estimated using the method described in
detail by (Harkema et al., 1987). The Vs of intraepithelial mucosubstances is expressed
as nanoliters of intraepithelial mucosubstances per mm square of basal lamina.
Inflammatory cells were quantified by using digitalized histological slides
captured by a slide scanner (VS110, Olympus).

At 20x magnification, lung tissue

images were captured by systematic random sampling. Digital images were uploaded to
the web based Stepanizer program, a computer based software tool for stereological
evaluation of digital images (Tschanz et al., 2011). A point grid was superimposed over
random images of sampled lung to count the numbers of points positively stained for
eosinophils or neutrophils within the regions of interest. The data was expressed as
percent of positive staining points within a tissue region / total points counted in the
tissue region. The lung (left lobe) was classified into regions consisting of bronchioles
with adjacent blood vessels (bronchovascular), terminal bronchioles, alveolar walls or
interstitium, and the airspaces.
2.3.7 Real –time PCR of Lung
Total RNA was isolated from the right caudal lung lobe using Rneasy Mini Kit
(Qiagen, Valencia, CA) according to manufacturer’s instructions.

Tissues were

homogenized using Qiagen’s TissueLyser II Bead Beater and three 2.8mm Zirconium
Oxide beads in 600µl buffer RLT containing β -Mercaptoethanol. Homogenate was then
centrifuged at 12,000g for 3 minutes and RNA was purified from the supernatant using
the RNeasy capture column. Eluted RNA was diluted 1:5 with Rnase free water and
quantified using a Nanodrop 1000 spectrophotometer (Thermo Fisher Scientific,
57

Waltman, MA). Reverse transcription was accomplished by using High Capacity cDNA
Reverse Transcription Kit reagents (Applied Biosystems). Each RT reaction was run in
a 50 l reaction volume containing 5 g of total RNA with cDNA Master Mix prepared
according to the manufacturer’s protocol. The reaction mixture was incubated in a in
GeneAmp PCR System 9700 Thermocycler PE (Applied Biosystems, Foster City, CA)
at 25ºC for 10 Minutes, 37ºC for 2 hours, then held at 4º.
Relative quantitative mRNA expression analysis of targeted genes for molecular
analysis included Gob5, Muc5ac, TNF, IFN, IL-4,-5,-6, -10 -13s was conducted on an
ABI

RISM 7900 HT Sequence Detection System at Michigan State University’s

Research Technology Support Facility using Taqman Gene Expression Assay reagents
(Applied Biosystems). 2µl cDNA and 8µl reagents were dispensed (in duplicate) into a
384-well reaction plate. The cycling parameters were 48 C for 2 minutes, 95 C for10
minutes, and 50 cycles of 95 C for 15 seconds followed by 60 C for 1 minute
Gene expression levels were reported as fold-change (FC) of mRNA in
experimental samples compared to a control sample. Real-time PCR amplifications
were relatively quantified using the Ct method. Following the PCR, amplification plots
(change in dye fluorescence versus cycle number) were examined and a dye
fluorescence threshold within the exponential phase of the reaction was set for the
target gene and the endogenous references. The cycle number at which each amplified
product crosses the set threshold represents the CT value. The amount of target gene
normalized to its endogenous reference was calculated by subtracting the geometric
mean of the CTs from endogenous controls (Arbp, Gusb, and Gapdh) from the target
58

gene CT (delta Ct (Ct)) This normalization strategy has been utilized for accurate RTPCR expression profiling in biological samples with small expression differences
(Vandesompele et al., 2002). The Ct value for the experimental sample is subtracted
from the Ct value of the corresponding control sample (Ct). The FC in experimental
samples relative to control samples is then calculated as: FC= 2-Ct.
2.3.8 Airway Hyperresponsiveness (AHR) Measurements
On day 21, some of the mice were anesthetized by i.p. sodium pentobarbital (100
mg/kg). A tracheostomy was performed to insert an 18 gauge cannula which was
attached to a mechanical ventilator with a computer controlled piston pump (flexiVent;
Scireq, Montreal, Canada). Mice were ventilated at a respiratory rate of 150 breaths /
minute, tidal volume of 10 ml/kg, and positive end expiratory pressure (PEEP) of 2-3 cm
H2O.

Incremental concentrations (0, 1.25, 2.5, 5, 10, and 20 mg/ml) of a

bronchoconstrictor, acetyl-β-methacholine (MCh, Sigma-Aldrich) were delivered into the
trachea via an attached nebulizer (Aeroneb; Aerogen, Galaway, Ireland). Prior to each
MCh response curve, two deep inspirations were given. Following MCh administration,
12 perturbation maneuvers consisting of alternating measurements of sinusoidal, single
frequency, oscillations (SnapShot) and broadband, multi-frequency, oscillations (Quickprime) were performed. From the collected data, the flexiVent software calculated total
respiratory system resistance using the single compartment model, as well as airway
resistance, tissue damping, and tissue elastance using the constant phase model
(Hantos et al., 1992).

The mean of the responses for each concentration of

methacholine was determined. A dose- response curve was generated. The lowest
59

dose of MCh with the greatest variation in response between groups was at 10 mg/ml.
Therefore, the data were expressed as the percent change at 10 mg/ml compared to
baseline for each group.
2.3.9 Statistical Analysis
For BALF and AHR analysis, data were expressed as group means + the
standard error of the mean (mean + SEM). Grubb’s outlier test was used to determine
and remove outliers.

A two-way analysis of variance (ANOVA) was performed to

determine statistical differences. Significant results were further analyzed with StudentNewman-Keuls post hoc test to make direct comparison between groups. Significant
differences between group means were based on p values set at p < 0.05. For RTPCR, statistical differences of Ct values between groups were determined with twoway ANOVA with Student-Newman-Keuls post hoc test to make direct comparison
between groups; p  0.05. SigmaPlot statistical software was used for data analysis
(Systat Software Inc, San Jose, CA).

60

2.4

RESULTS

2.4.1 Bronchoalveolar lavage fluid (BALF).
Seventy-two hours following the final allergen challenge, OVA elicited a
significant accumulation of inflammatory cells in BALF (Fig.4), consisting of
predominately of eosinophils and macrophages. Twenty-four hours following exposure,
intranasal LPS caused an increase in BALF cellularity due primarily to neutrophils in
non-allergic (LPS) mice. In allergic mice given ovalbumin and then followed with LPS
(OVA-LPS; black bars), total BALF inflammatory cells were two-fold greater than that
elicited by either ovalbumin or endotoxin alone, with the increase due to neutrophils.

61

Figure 4. BALF of non-allergic and allergic mice exposed to LPS. Total cells,
macrophages, eosinophils, neutrophils, and lymphocytes were determined in BALF
collected from non-allergic BALB/c mice which received saline during sensitization and
challenge (open bars); and allergic mice sensitized and challenged to OVA (solid bars)
grouped by exposure to L S (0 or 3 μg) as described in Materials and Methods. Values
are expressed as mean + SE (n=5-6). Horizontal lines indicate significant difference; p
< 0.05.

62

2.4.2 BALF Cytokines

At 72 hours after the last allergen challenge, there were no significant changes in
any cytokine evaluated in OVA mice compared to saline control mice. LPS treatment
induced a significant accumulation of TNF and IL-6 in BAL fluid that was similar in nonallergic (LPS alone) (Fig. 5 A-B). By comparison, in allergic mice given LPS (OVALPS), concentrations in IFN-γ, and IL-17 were increased compared to either OVA or
LPS mice.

63

Figure 5. BALF cytokines in non-allergic and allergic mice exposed to inhaled
LPS. Concentrations in BAL fluid of IFN, IL-17, TNF and IL-6 were determined by
cytometric bead assays as described in Materials and Methods. Values are expressed
as mean+ SE (n=5-6). Horizontal lines indicate significant difference; p < 0.05.

64

2.4.3 Histopathology

The treatment-related responses I observed in BALF cellularity was confirmed
with microscopic examination of the lung. Qualitative evaluation of inflammation in
peribronchial, perivascular, and lung interstitial regions was determined by scoring
histological slides based on the following criteria: 0 – no / minimal inflammation; 1 –
occasional airways and adjacent blood vessels with aggregate of cells one layer thick
and occasional inflammatory cells present in alveolar spaces and interstitium; 2 –
airways / blood vessels with cellular cuffing of 2-5 layers thick and inflammation in 1030% of the interstitium and airspace; and 3 - airways / blood vessels with cellular cuffing
of >5 cell layers thick and inflammation in > 30% of the interstitium and airspace (Table
1). Representative lesions are depicted histologically in Figure 6. Non-sensitized, saline
mice did not show any histopathology in the lung (Fig 6A). OVA caused a moderate to
severe

inflammation

consisting

of

predominately

lymphocytes,

eosinophils,

macrophages, and lesser numbers of neutrophils in peribronchiolar and perivascular
regions. Inflammation occasionally extended into adjacent alveolar attachments (Fig
6B). LPS elicited a mild neutrophilic influx and edema surrounding bronchioles and
blood vessels which extended into adjacent alveolar attachments and airspaces (Fig
6C). OVA-LPS also induced peribronchiolar and perivascular inflammation similar to
OVA alone. However, inflammation was more severe and characterized by increased
neutrophils along with lymphocytes, eosinophils, and macrophages in these mice.
Inflammatory cells were present throughout the lung interstitial tissue and within alveolar
spaces (Fig 6D).
65

Table 1. Qualitative evaluation of Inflammation in the lung. Numerical scores are
defined as follows: 0 – no / minimal inflammation; 1 – airways / adjacent blood vessels
with aggregate of cells one layer thick and occasional inflammatory cells present in
alveolar spaces and interstitium; 2 – airways / blood vessels with cellular cuffing of 2-5
layers thick and inflammation in 10-30% of the interstitium and airspace; and 3 - airways
/ blood vessels with cellular cuffing of >5 cell layers thick and inflammation in > 30% of
the interstitium and airspace.

Inflammation Score
Group

Peribronchiolar

Perivascular

Alveoli / Alveolar
Space

Saline

0

0

0

OVA

2

3

1

LPS

1

1

1

OVA-LPS

3

3

3

66

Figure 6. Pulmonary histology of non-allergic and allergic mice exposed to
inhaled LPS. Light photomicrographs (H&E) of lungs showing no inflammation (saline,
A) and increased inflammation surrounding airways and blood vessels in mice
sensitized and challenged with OVA along with mucous cells metaplasia within airway
epithelium (OVA, B). LPS alone produced a milder inflammatory response (LPS, C).
OVA-LPS mice had a inflammatory pattern similar to both OVA and LPS with
peribronchiolar and perivascular inflammation and thickening of airway epithelium due
to mucus metaplasia and extension of inflammation into the alveolar tissue and spaces
(OVA-LPS, D). Scale bar 100 μm

67

2.4.4 Morphometric inflammatory cell analysis

To better characterize the location and distribution of eosinophils and neutrophils
associated with different treatments morphometric analysis was performed on the
proximal sections of lungs (Fig. 7). Percentages of positive-staining cells for eosinophils
and neutrophils was determined by point counting in multiple reference sites which
included bronchioles and adjacent blood vessels (peribronchovascular), terminal
bronchioles, lung interstitial tissue, and the alveolar spaces. OVA mice had a significant
influx of eosinophils which were predominately located in peribronchovascular regions
and around terminal bronchioles (Figs 7A, B), with scant accumulation in alveolar region
(Fig 7C, D). Accumulation of neutrophils was not significant in these mice compared to
saline mice. In comparison, LPS mice had neutrophils accumulations located in all
studied compartments.

In OVA-LPS mice, there were larger percentages of

inflammatory cells within the peribronchovascular region and terminal bronchioles. The
peribronchovascular region contained a nearly equal distribution of eosinophils and
neutrophils, while the terminal bronchioles had larger numbers of neutrophils. There
was a significant decrease in the percentage of neutrophils in the alveolar wall of OVALPS mice compared to LPS mice.

68

Figure 7. Eosinophil and neutrophil distribution in the lung. Lung sections were
immunohistochemically stained for neutrophils or eosinophils as described in Methods.
Data are expressed as the percent of positive-staining eosinophils or neutrophils per the
total counted points in the reference tissue: (A) peribronchiolar and perivascular region,
(B) terminal bronchioles; (c) alveolar wall; and (D) airspace. Significantly different from
(*) saline group; (+) LPS group; (#) OVA group p<0.05; n=5-6.

69

2.4.5 Airway Epithelial Mucus Production

There was no AB/PAS positive staining in saline mice (Fig 8 A). OVA caused
remodeling of the airway epithelium characterized by marked mucous cell metaplasia
present in the axial airways, small bronchioles, and pre-terminal bronchioles as
indicated by an increased staining of intraepithelial mucosubstances with AB/PAS (Fig 8
B). Minimal intraepithelial mucosubstances was present in LPS alone mice (Fig 8 C).
Mucus was decreased in OVA-LPS compared to OVA mice (Fig. 8 D). Morphometric
analysis determined that intraepithelial mucosubstances were significantly decreased in
OVA-LPS mice when compared to OVA alone mice (Fig 8 E).

70

Figure 8.
Airway epithelial mucus. Increased intraepithelial mucus in airways, a
feature of asthma, was analyzed by staining with AB/PAS (A-D). Staining was
increased in allergic mice (B, D), however, LPS blunted this increase in OVA-LPS mice
as shown in (D). Also, quantification of mucus showed that LPS decreased OVAinduced intraepithelial airway mucosubstances (E). Horizontal lines indicate significant
difference; p < 0.05. B, bronchiole; BV, blood vessel; arrowheads, intraepithelial mucus.
Scale bar 100 μm.

71

Figure 9. Airway mucus gene expression. (A) mRNA of mucus producing genes,
Gob-5 and MUC5AC. Data are reported as the fold increase of the group mean + SEM.
(B) Volume density of intraepithelial mucosubstances in the proximal airway (G5).
Significantly different from a- saline group; b- OVA group; c- LPS group; horizontal line
indicates significant difference between specified groups, p<0.05; n=6/group.

72

2.4.6 Lung tissue mRNA expression

Expression of genes involved in mucus overproduction, Gob-5, and goblet cell
metaplasia, Muc5ac, were both induced in OVA mice, but in OVA-LPS mice both genes
had decreased expression when compared to OVA alone (Fig. 9). These changes are in
alignment with the decreased presence of intraepithelial mucosubstances in OVA-LPS
mice. OVA up-regulated Il13, but in comparison Il13 was decreased in OVA-LPS mice
(Fig 10, A). Il10 was increased in both OVA and OVA-LPS mice (Fig 10, B). In both
LPS and OVA-LPS mice there was an increase expression of Ifng (Fig 10, C). Both
OVA and LPS have an increase in expression of Il17a (Fig 10, D). Both LPS and OVALPS had significant increases in Il23a mRNA expression compared to saline and ova
alone animals, respectively (Fig 10, E). In addition, inducible nitric oxide synthase,
NOS2, was elevated in only OVA-LPS mice (Fig 10, F).

73

Relative Gene Expression
Fold Change

Relative Gene Expression
Fold Change

Relative Gene Expression
Fold Change

Figure 10. Pulmonary gene expression in non-allergic and allergic mice exposed
to inhaled LPS. Relative expressions of mRNA encoding for cytokines IL-13, IL-10,
IFN-γ, IL-17a and IL-23a and the enzyme, NOS2, were determined in right caudal lobes
by RT-PCR and described in Materials and Methods. Values are expressed as fold
increase of mRNA expression compared to control (Saline) animals (n=5-6). Horizontal
lines indicate significant differences between specified groups, p<0.05.

74

2.4.7 Airway Hyperresponsiveness

Alterations in total lung resistance due to hyperresponsiveness elicited by
aerosolized β-methacholine (10 mg/ml) were significantly increased by OVA alone and
LPS alone compared to saline. However, the hyperreactivity present with OVA or LPS
was significantly decreased in OVA mice exposed to LPS (Fig 11, A). Also, similar
increases in OVA and LPS mice with significant decreases in OVA-LPS mice were
present in airway resistance, tissue damping, and tissue elastance (Fig 11, B-D). OVA
mice displayed prominent changes in tissue damping and tissue elastance with a
greater than 4-fold increase over saline animals (Fig 11, C-D). While in LPS mice, the
predominant change occurred in tissue elastance which displayed an almost 7-fold
increase over saline mice (Fig 11, D).

75

Figure 11. Measurement of AHR in non-allergic and allergic mice exposed to LPS.
Methacholine-induced changes in total lung resistance (A), airway resistance (B), tissue
damping (C) and tissue elastance (D) were measured in intubated and ventilated mice
using the Flexivent system as described in Materials and Methods. Values are
expressed as the percent increase from baseline value after administration of 10 mg/ml
of methacholine (n=12-14). Horizontal line indicates significant difference between
specified groups, p<0.05.

76

2.5

DISCUSSION

In this study I sought to determine the relationship of pulmonary inflammation
with changes in AHR by using a mouse model of LPS-induced exacerbation of
established allergic airway disease. My approach was to match airway lesions with
airway compartment-specific AHR, specifically by morphometric evaluation of the site,
severity and types of inflammatory cells in lesions, and by partitioning airway resistance
as arising from either lung tissue or conducting airways using forced oscillation
techniques. My results demonstrate that LPS worsened the inflammation in allergic
mice by eliciting a robust recruitment of neutrophils, but that AHR was attenuated in
these mice. As such, rather than exacerbate AHR as I hypothesized, my data suggests
that LPS-induced signaling for the recruitment and/or activation of neutrophils may
contribute to a decrease in allergic airway reactivity.
Previous studies that have characterized the airway inflammatory responses to
OVA or LPS in mice describe infiltration of eosinophils and neutrophils, respectively
(Kung et al., 1994; Hakansson et al., 2012). In the current study I show that eosinophils
and lymphocytes were primarily concentrated around bronchioles and blood vessels in
OVA mice (i.e., conducting airways). By comparison in LPS mice, neutrophils were
localized in these same perivascular and peribronchial regions, but lesions also
extended to a mild to moderate alveolitis and extravasation of neutrophils into airspaces
(i.e., both conducting airways and tissues). Others have described that airway challenge
with OVA or LPS alone can cause AHR that is associated temporally with airway
inflammation in mice (Gueders et al., 2009; Hakansson et al., 2012). My current results
77

are similar to these reports, as I measured AHR in mice 72 hours after OVA challenge
or 24 hours after LPS exposure. Furthermore, LPS elicited a neutrophilic recruitment
and AHR in both airway and tissue compartments, suggesting a link between
inflammation and functional changes. However severity of airway inflammation and
AHR were not associated in the same compartments in OVA mice, where eosinophilic
involvement was concentrated around airways, while most of the measured resistance
came from tissues.
While plausible hypotheses have been proposed for the contribution of
granulocytes and their products to the development of AHR (Wardlaw et al., 2002),
supporting evidence in experimental asthma models has been inconsistently reported.
Some findings demonstrate critical roles for inflammatory cell influxes (Webb et al.,
2001; Walsh et al., 2008), while others indicate that eosinophils and/or neutrophils are
unnecessary to manifest AHR (Birrell et al., 2003; Shen et al., 2003; Cui et al., 2005;
Siegle et al., 2006). In humans with fatal asthma, airway lesions are characterized by
widespread but unequally distributed eosinophils throughout the lung, while neutrophils
are localized along peribronchiolar-alveolar attachments (Simoes et al., 2005). Similar
to my own findings, others researchers using allergic BALB/c mice describe eosinophil
infiltration that is predominately in peribronchiolar regions, and is correlated with
increases in total lung resistance and dynamic compliance (Takeda et al., 2001). In the
present study, treatment of allergic mice with LPS did not change eosinophil localization
in this region; however it did result in decreased AHR that putatively arises from
obstruction in these conducting airways. As such, if eosinophils contribute to local AHR
in my model, then LPS may act directly on these cells to modulate their production of
78

inflammatory or bronchodilatory mediators. Alternatively, LPS-induced co-localization of
neutrophils along airways may be involved in the modulation of AHR in this region.
The primary pathological feature associated with reversal of AHR in LPS-treated
allergic mice is widespread infiltration of neutrophils in all the compartments of the lung
(i.e., both tissues and airways). Potential neutrophil-derived mediators associated with
AHR include elastase (Koga et al., 2013) and IL-17 (Kudo et al., 2012), which had
increased gene expression and presence in BAL fluid of OVA-LPS mice. Along with IL23, IL-17 is associated in asthmatics with a more severe, neutrophil-associated
phenotype (Cosmi et al., 2011), and at least one report suggests that IL-17 can activate
asthmatic smooth muscle cells (Dragon et al., 2014). However I did not observe
increased AHR with PMN infiltration in the current study. Alternatively, elaboration of the
bronchodilator mediators from LPS-stimulated neutrophils such as nitric oxide (NO)
(Prado et al., 2006) and prostaglandin E2 (Tanaka et al., 2005) may underlie the
inhibition of AHR I observed in OVA-LPS mice. Localization of neutrophils in both the
airways and tissues is consistent with their potential influence to reduce airway reactivity
that I measured in both of these lung compartments.
Rodriquez and colleges have shown that LPS exposure during OVA challenge
decreased BALF concentrations of IL-5 and -13 (Rodriguez et al., 2003). In my studies
these Th2 cytokines were below the limit of detection in BALF in both OVA and OVALPS mice. However, the OVA-associated expression of the IL-13 gene in lung tissue
was decreased following LPS exposure in OVA-LPS mice, suggesting that LPS may be
downregulating allergic, Th2-type pathways.

This change in immune pathways is

supported by the pulmonary expression of the Th1 cytokine IFN- and the regulatory
79

cytokine IL-10 that coincide with the decrease in Il-13 expression in OVA-LPS mice. My
results are similar to others, who report that inhaled LPS can induce a Th1 response
characterized by increased neutrophils, IFN-γ production and decreased mucus
secretion in allergic mice (Eisenbarth et al., 2002; Kim et al., 2007; Dong L; Li H, 2009).
Simpson and Martinez showed that asthmatics with certain genetic traits were protected
against the development of atopy by exposure with high doses of LPS (Simpson and
Martinez, 2010). The effect of LPS on allergic airway responses may be dosedependent in mice, as Dong et al. found that low dose LPS increased recruitment of
eosinophils and neutrophils into the lung, mucus secretion, and Th2 cytokines (Dong L;
Li H, 2009). Future studies that employ lower doses of LPS than those used in my
protocol are needed to determine dose-sensitive pathways of LPS exposure in preexisting asthma.
In addition to airway inflammation, goblet cell metaplasia and mucus
hypersecretion are hallmarks of human asthma that are reproduced in the OVA BALB/c
mouse model (Long et al., 2006; Pawankar et al., 2009). Inhibition of mucus secretion
can protect from allergic AHR in mice (Agrawal et al., 2007), which suggests that
luminal accumulation of mucus might contribute to airway obstruction. In the current
study, I describe OVA-induced mucus cell metaplasia and hyperplasia, along with
induction of genes associated with mucus production and secretion, Gob-5 and
MUC5ac. In allergic mice treated with LPS these mucous responses were significantly
reduced. Therefore, it is possible that the inhibition of AHR by LPS is due to changes in
mucin secretion.

80

2.6

SUMMARY
Sensitization and challenge with OVA caused mucous cell metaplasia, airway

eosinophil accumulation, and AHR in central airways and peripheral tissues in lungs of
BALB/c mice. Introduction of LPS into airways with pre-existing allergic inflammation
caused an accumulation of neutrophils into the lung, no change in eosinophils, but led
to a reduction in AHR. Therefore my hypothesis that the contributions from the central
airways and peripheral tissues to AHR in allergic mice exposed to endotoxin are
dependent on the location and severity of neutrophilic lesions in the lung was not
supported by the data. Indeed, the increased inflammation in LPS-treated allergic mice,
specifically of neutrophils, was related to a decrease in AHR. As such my data
suggests that neutrophils or a neutrophil product may contribute to the decrease in
OVA-induced AHR. Approaches to test this possibility include blocking neutrophil
migration, activation, or the activity of its potential mediators. Two potential neutrophilderived mediators that have bronchodilatory effects are nitric oxide and the
prostaglandin PGE2. Another possibility is that LPS had direct effects on cells
responsible for AHR, such as eosinophils, airway smooth muscle cells, epithelial cells,
or airway macrophages. However cell receptors such as CD14 or TLR4 are required for
activation by LPS, and such activation usually results in NF-B-mediated production of
inflammatory cytokines.

In general this response may oppose the Th2 responses

induced by OVA, and my data of decreased IL-13 in OVA-LPS support this possibility.
Taken together there are several reasonable explanations for the effects of LPS to
reverse allergic AHR. In the next few chapters I will focus on the role of neutrophils,

81

which were the most obvious histological change that accompanied the reduction of
AHR in allergic lungs.

82

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89

CHAPTER 3

THE EFFECT OF NEUTROPHIL DEPLETION ON AIRWAY
HYPERRESPONSIVENESS IN ALLERGIC MICE EXPOSED TO INHALED
LIPOPOLYSACCHARIDE

90

3.1

ABSTRACT

The innate immune response initiates pathways that lead to activation of adaptive
immunity.

Allergic asthma is an abnormal immune response involving adaptive

immunity, and research has focused primarily on this response. Recent knowledge
suggests that innate immunity may play a critical role in asthma.

Therefore,

examination into the role of the innate immune system in the development and
exacerbation of allergic asthma is a topic of ongoing studies to identify novel targets for
the treatment and prevention of asthma. Previously, I determined that endotoxin, a
commonly used agent to study innate immunity, exacerbated the inflammatory response
in the lungs of allergic mice by significantly increasing neutrophils (Chapter 2). In this
study, I set forth to determine the role of neutrophils in allergen-induced AHR following
LPS exposure in allergic mice. Mice were sensitized and challenged to OVA as
previously described. Prior to LPS exposure, neutrophils were systemically depleted
from the mice using an anti-Ly6g antibody. AHR was assessed 24 hours after LPS.
Total BALF cells were significantly decreased in OVA mice, due to reductions in
neutrophils along with eosinophils and lymphocytes.

Also, BALF cellularity was

significantly decreased in OVA-LPS mice as a result of large decreases in neutrophils
along with lymphocytes.

Neutrophil depletion did not affect AHR when comparing

neutrophil sufficient and neutrophil deficient mice in OVA and OVA-LPS groups. These
results suggest that neutrophils are not linked to the attenuation of AHR in OVA-LPS
mice.

91

3.2

INTRODUCTION
Cells of the innate immune system provide immediate and general protection

from pathogens by receptor-mediated identification of molecular structures present on
microorganisms, known as pathogen-associated molecular patterns (PAMPs) or
produced by damage cells, damage associated molecular patterns (DAMPs). These
pattern recognition receptors (PRRs), such as toll-like receptors (TLRs) are present on
a variety of cells including respiratory epithelium, dendritic cells, macrophages, mast
cells, and neutrophils (Condon et al., 2011; Minnicozzi et al., 2011; Parker and Prince,
2011; Liu et al., 2014). The innate immune response initiates pathways to lead to
activation of the adaptive immune system which provides long lasting protection to a
specific antigen presented by dendritic cells or other antigen presenting cells.
Allergic asthma is an aberrant immune response predominately associated with
lymphocytes involved in the adaptive immunity. However, examination into the role of
the innate immune system in the development and exacerbation of allergic asthma is a
topic of ongoing studies. LPS, a prototypical PAMP recognized by TLR4, is often used
to study innate immunity (Rajaiah et al., 2013). It has been shown that the dose and
timing of LPS exposure can play a major role in asthma development and exacerbation
(Delayre-Orthez, et al., 2004; Delayre-Orthez, et al., 2005; Kim et al., 2007). Studies
have shown that during allergic sensitization exposure to high doses prevents asthma
(Von et al., 2000). This finding has become the basis of the “hygiene hypothesis” which
states that the decreased prevalence of childhood bacterial, viral, and parasitic
infections in westernized countries contributes to the increase in asthma development in
92

those societies (Braun-Fahrlander et al., 2002). Additionally, it was demonstrated that
exposure to low doses of LPS promote asthma development (Eisenbarth et al., 2002;
Piggott et al., 2005). However, the stimulation of innate immunity in the response to
endotoxin in asthmatics is controversial. Studies in humans and in experimental models
have shown that asthmatic response can be either exacerbated or inhibited by LPS
(Michel et al., 1989; Michel et al., 1991; Braun-Fahrlander et al., 2002; Liu, 2002).
Although asthma is affiliated with Th2 mediated adaptive immune response
characterized predominately by eosinophilic inflammation, the function of neutrophils is
critical to the pathogenesis in some forms of the disease.

Neutrophils have been

associated with severe, steroid resistant forms of asthma and asthma exacerbations
(Jatakanon et al., 1999; Wenzel et al., 1999), in addition to being prominent in asthma in
the elderly (Nyenhuis et al., 2010) and the obese (Scott et al., 2011).
In a rodent model of LPS modulation of asthma, we recently described the
attenuation AHR that was associated with widespread infiltration of neutrophils into all
compartments of the lung (Chapter 2).

The contribution of neutrophils to AHR in

asthmatic airways is unclear. Compared to eosinophilic asthma, a less severe AHR has
been indentified in asthmatics with neutrophilic subtype (Baines et al., 2014). At least
one report in a model of RSV-exacerbation of asthma suggests that neutrophils are
associated with AHR inhibition (Aeffner and Davis, 2012). A similar role for neutrophilmediated reversal of AHR may explain the attenuation by LPS in our endotoxin-asthma
model. In the current study, I systemically depleted neutrophils prior to IN instillation of

93

LPS in allergic BALB/c mice to test the hypothesis that attenuation of AHR in LPSexposed allergic mice is dependent on the recruitment of airway neutrophils..

94

3.3

MATERIALS AND METHODS

3.3.1 Laboratory Animals and Treatment protocols
Male BALB/c mice (Charles River Laboratories, Portage, MI), 6-8 weeks of age
were

housed

and maintained

as described in Chapter 2 MSU animal facilities

accredited by the Association for Assessment and Accreditation of Laboratory Animal
Care. Mice were randomly assigned to one of six experimental groups consisting of 6
animals as follows: 1) animals were assigned to saline, OVA, or OVA-LPS groups and
2) half of each group were depleted of neutrophils prior to IN LPS (Fig 12).
3.3.2 Depletion of Neutrophils
Non-allergic and allergic groups were divided in half and on Days 19 and 20 mice
received 0 or 250 µg i.p. of a neutrophil depleting antibody (anti-Ly6g [RB6-8C5],
Abcam; Cambridge, MA). One hour following the last injection of antibody, the control
and neutrophil-depleted mice groups were divided in half and received either IN saline
or LPS (Fig 12).

Neutrophil depletion was confirmed from peripheral blood smears

prepared from blood collected at necropsy.

95

Figure 12. Experimental design for neutrophil depletion study in saline and
OVA mice exposed to LPS.

3.3.3 Necropsy, Lavage Collection, and Tissue Preparation
Mice were anesthetized with an i.p. injection of with sodium pentobarbital
(10 mg; Fatal Plus, Vortech Pharmaceuticals, Dearborn, MI). Blood was collected from
the caudal vena cava for separation of plasma, and animals were euthanized by
transection of the abdominal aorta. The lungs and heart were harvested en bloc. The
lungs were instilled twice with 800 µL saline via the cannulated trachea to collect BALF.
After lavage the right lung lobes were ligated, separated and stored in RNAlater
(Qiagen) for RNA isolation or snap frozen in liquid nitrogen for storage. The left lobe
was inflated with 10% neutral buffered formalin to a pressure of 30 cm H2O for 1 hour
and then stored in a large volume of the same fixative.
3.3.4 Bronchoalveolar Lavage Analysis

96

Total BALF leukocytes were counted with a hemocytometer and cytological
slides were prepared using a Cytospin centrifuge (Shandon). Slides were stained with
Diff-Quick (Dade Behring, Newark, DE) and cell differential (macrophages, eosinophils,
neutrophils, and lymphocytes) were counted. The remaining BAL fluid was centrifuged,
and stored at -20°C.
3.3.5 Airway Hyperresponsiveness (AHR) Measurements
On day 21, some of the mice were anesthetized by i.p. sodium
pentobarbital (100 mg/kg).

A tracheostomy was performed to insert an 18 gauge

cannula which was attached to a mechanical ventilator with a computer controlled
piston pump (flexiVent; Scireq, Montreal, Canada). Mice were ventilated at a respiratory
rate of 150 breaths / minute, tidal volume of 10 ml/kg, and positive end expiratory
pressure (PEEP) of 2-3 cm H2O. Incremental concentrations (0, 1.25, 2.5, 5, 10, and 20
mg/ml) of a bronchoconstrictor, acetyl-β-methacholine (MCh, Sigma-Aldrich) were
delivered into the trachea via an attached nebulizer (Aeroneb; Aerogen, Galaway,
Ireland).

Prior to each MCh response curve, two deep inspirations were given.

Following MCh administration, 12 perturbation maneuvers consisting of alternating
measurements of sinusoidal, single frequency, oscillations (SnapShot) and broadband,
multi-frequency, oscillations (Quick-prime) were performed.

From the collected data,

the flexiVent software calculated total respiratory system resistance using the single
compartment model, as well as airway resistance, tissue damping, and tissue elastance
using the constant phase model (Hantos et al., 1992). The mean of the responses for
each concentration of methacholine was determined.
97

A dose- response curve was

generated. The lowest dose of MCh with the greatest variation in response between
groups was at 10 mg/ml. Therefore, the data were expressed as the percent change at
10 mg/ml compared to baseline for each group.
3.3.6 Statistical Analysis
Data were expressed as group means + the standard error of the mean (mean +
SEM).

Grubb’s outlier test was used to determine and remove statistical outliers.

SigmaPlot statistical software (Systat Software Inc, San Jose, CA) was used to perform
t-tests to make direct comparison between neutrophil- sufficient and depleted mice.
Significant differences between group means were based on p values set at < 0.05.

98

3.4

RESULTS

3.4.1 Bronchoalveolar lavage fluid
Seventy-two hours following the final allergen challenge, ovalbumin elicited a
significant accumulation of cells in BALF (Fig. 13), consisting predominately of
eosinophils and macrophages, with lesser numbers of lymphocytes. Twenty-four hours
following allergen exposure, instillation of intranasal LPS caused a two-fold increase in
total cells that were due to a robust increase in neutrophils. Treatment with the
neutrophil-depleting antibody decreased neutrophil numbers to control levels in OVALPS mice, but also caused reductions in eosinophils and lymphocytes in OVA mice, and
in lymphocytes of OVA-LPS mice.

99

Figure 13. Comparison of BALF in neutrophil sufficient and neutrophil depleted
mice. Total cells, macrophages, eosinophils, neutrophils, and lymphocytes were
determined in BALF collected from BALB/c mice in the following groups: Saline
(control), OVA, and OVA-LPS. Mice from each group were also treated i.p. with 250 l
saline vehicle (clear bars) or neutrophil-depleting antibody (PMN antibody; anti-Ly6g,
solid bars) as described in Materials and Methods. Values are expressed as mean +
SE (n=5-6). Horizontal bars represent significant difference between indicated groups,
p < 0.05.

100

3.4.2 Airway Hyperresponsiveness
OVA sensitization and challenge caused airway hyperreactivity to methacholine
as indicated by an increase in total lung resistance (Fig 14A), central airway resistance
(Fig14B), tissue damping (Fig 14C), and elastance (Fig 14D). Instillation with LPS in
allergic mice attenuated central airway resistance and elastance, and caused mild
decreases in total resistance and tissue damping. Using the single compartment model
to determine total lung resistance, there were no significant differences in AHR in mice
that were depleted of neutrophils compared to neutrophil sufficient mice in saline, OVA,
and OVA-LPS groups (Fig 14 A).

Further analysis using the constant phase model to

partition the airways showed no significant changes in airway resistance with neutrophil
depletion in saline, OVA, and OVA-LPS mice (Fig 14B). Analyses of peripheral tissue
changes show a significant increase in tissue damping in saline mice depleted of
neutrophils compared saline neutrophil sufficient mice.

However, there were no

significant changes in OVA and OVA-LPS groups regardless of treatment (Fig 14C).
Neutrophil depletion caused a significant decrease in tissue elastance in OVA mice, but
no significant differences were notable with neutrophil depletion in OVA-LPS mice (Fig
14D).
Figure 14. Comparison of AHR in neutrophil sufficient and neutrophil depleted
mice. Methacholine-induced changes in total lung resistance (A), airway resistance (B),
tissue damping (C) and tissue elastance (D) were measured in intubated and ventilated
mice using the FlexiVent system as described in Materials and Methods. Values are
expressed as the percent increase from baseline value after administration of 10 mg/ml
of methacholine (n=4-6). Horizontal bars represent significant difference between
indicated groups, p < 0.05.

101

Figure 14. Comparison of AHR in neutrophil sufficient and neutrophil depleted mice. Methacholine-induced
changes in total lung resistance (A), airway resistance (B), tissue damping (C) and tissue elastance (D) were measured in
intubated and ventilated mice using the FlexiVent system as described in Materials and Methods. Values are expressed
as the percent increase from baseline value after administration of 10 mg/ml of methacholine (n=4-6). Horizontal bars
represent significant difference between indicated groups, p < 0.05.

102

3.5

Discussion
From my initial study (Chapter 2), I found that airway LPS induced a robust and

widespread recruitment of neutrophils into lung tissue, and at the same time decreased
allergic AHR in OVA-LPS mice. Activated neutrophils have the potential to make several
bronchoactive mediators that may alter the hyperreactivity of the lung to methacholineinduced contraction of airway smooth muscle.

In the current study I eliminated

neutrophils prior to LPS treatment to test the hypothesis that neutrophils or a neutrophil
product may contribute to the decrease in OVA-induced AHR. However depletion of
neutrophils had no effect on AHR. Both OVA-induced increases, and LPS-induced
decreases in airway resistance were similar in neutrophil-sufficient and -deficient mice.
These results suggest that, despite their large presence in lung tissue, neutrophils do
not affect AHR in OVA-LPS mice.
Other mouse models of AHR show strong associations between neutrophilic
inflammation and increased airway resistance. Increased AHR has been associated
with neutrophils and IL-17 production from Th17 lymphocytes in response to different
immunologic stimuli (Wilson et al., 2009; Mizutani et al., 2012a). Although we did not
observe a large neutrophil influx in our OVA model, neutrophils and neutrophil-derived
products are actually required in some acute allergic AHR protocols using OVAsensitized mice (Riesenfeld et al., 2010; Mizutani et al., 2012b; Koga et al., 2013). In the
current study however, treatment with neutrophil-depleting antibody blocked the minor
increase in BALF neutrophils in OVA mice, but it also reduced BALF eosinophils by
more than 50% in these mice. AHR was unaffected in allergic mice, which suggests that
103

neither neutrophils nor eosinophils make a major contribution to allergic AHR in our
OVA sensitization and challenge protocol. If AHR had been altered by treatment with
the neutrophil antibody, further studies would be needed to determine if it were
eosinophils or neutrophils were critical, since both were reduced by treatment.
Airway neutrophil infiltration has been linked to endotoxin-induced AHR in a
number of animal models (Horie et al., 1988; Schwartz et al., 2001; Starkhammar et al.,
2012). Like allergen-induced AHR, studies in laboratory animals suggest that endotoxin
may elicit a biphasic response, with early and late AHR phases that are probably driven
by different mechanisms (Cochran et al., 2002). Increased breathing frequency and
bronchoconstriction 24 hours after LPS challenge in rats is dependent on neutrophils,
whereas changes airway function at 2 hours was not (Spond et al., 2004). In allergic
guinea pigs exposed to inhaled LPS, AHR occurred by 1 hour, but airways were
hyporeactive 48 hours later when neutrophils in BALF were significantly elevated
(Toward et al., 2005; Sharma et al., 2009). We observed no relation of neutrophils in
LPS effects on AHR in the current study, but an important difference is that we exposed
allergic airways that had ongoing inflammation and epithelial remodeling. In model of
repeated pulmonary LPS exposure mice became tolerant to AHR with downregulation
of muscarinic receptors in lung tissue, but maintained airway neutrophilia (Natarajan et
al., 2010). A similar tolerance may occur in our allergic mice given a further
inflammatory stimulus of endotoxin when, despite additional inflammatory cell
recruitment, smooth muscle cells respond with downregulation in contractile pathways.

104

Reports

of

LPS-induced

hyporesponsiveness

are

not

unprecedented.

Intratracheally instilled LPS in mice causes hyporesponsiveness to 5-HT-induced
contraction that is dependent on TNF and IL-6 (Brandolini et al., 2001). Airway
hyporesponsiveness after LPS exposure has been linked to NO production that can be
modulated by epithelial caveolin (Hsia et al., 2012) and by surfactant protein A (Pastva
et al., 2011). Epithelial cell-dependent mechanisms that downregulate AHR are not
limited to NO, but include potential mediators such as prostaglandins, acetylcholine, and
inflammatory cytokines (Vanhoutte, 2013). An emerging hypothesis proposes that
intermediate, subepithelial cells (e.g., fibroblasts) transduce signals from activated
epithelium to smooth muscle via soluble mediators to promote airway dilation
(Vanhoutte, 2013).
Lastly,

intrinsic

changes

to

the

smooth

muscle

cells

may

mediate

hyporesponsiveness in OVA-LPS mice. As previously mentioned, decreases in receptor
numbers or receptor desensitization are possible explanations. Induction of nitric oxide
synthase (iNOS) in smooth muscle by LPS would provide an immediate source of nitric
oxide to promote relaxation. Both upregulation of iNOS and kinin receptors in smooth
muscle are mediated by NF-κB (Zhang et al., 2013).

Signal transduction from L S

binding to TLR4 or CD14 can lead to NF-κB activation in airway epithelium and smooth
muscle to bypass neutrophils or other inflammatory cells (Safholm et al., 2011; Tully et
al., 2013). As such, down modulation of smooth muscle cell responsiveness and/or
activation of epithelial cells suggest plausible neutrophil-independent pathways by
which LPS can inhibit AHR.

105

3.6

SUMMARY
Despite a robust infiltration of neutrophils into all lung compartments, their

depletion had no effect on AHR in allergic mice given LPS. Therefore my hypothesis
that attenuation of AHR in endotoxin-exposed allergic mice is dependent on the
recruitment of airway neutrophils was not supported by the data. There is significant
evidence that neutrophils produce both bronchoconstrictive and bronchodilatory
mediators that are required for AHR in some models of allergen or LPS models of
airway disease. However my results indicate that neutrophils are not involved in this
particular model of LPS exposure to airways with ongoing allergic inflammation. The
depleting antibody also reduced eosinophils and lymphocytes by 50%, but this decrease
did not alter AHR when compared to mice that did not receive the antibody. Potential
cellular mediators in my studies include lymphocytes, which have large infiltrations in
allergic mice, airway macrophages, epithelial cells, and smooth muscle cells, among
others. Epithelium and smooth muscle cells are intimately involved to control airway
opening, and lymphocytes and macrophages can produce inflammatory mediators to
modulate AHR. All these cell types should be responsive to LPS via the TLR4 or CD14
receptor. In the next chapter I will focus on the primary downstream transcription factor
of LPS activation, NF-κB.

106

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107

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Chu H W. 1999. Evidence That Severe Asthma Can Be Divided Pathologically into Two
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Wilson R H, Whitehead G S, Nakano H, Free M E, Kolls J K and Cook D N. 2009.
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Zhang Y, Cardell L-O, Edvinsson L and Xu C-B. 2013. Mapk/Nf-Kb-Dependent
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CHAPTER 4

EFFECTS OF NF-κB INHIBITION ON AIRWAY INFLAMMATION AND AIRWAY
HYPERRESPONSIVENESS IN ALLERGIC MICE EXPOSED TO
LIPOPOLYSACCHARIDE

113

4.1

ABSTRACT

Inhalation of particulate matter can exacerbate symptoms in asthmatics by increasing
antigen induced inflammation, as well as, stimulate innate inflammatory responses.
Recent knowledge suggests that induction of toll-like receptor-4 (TLR4), nuclear factor
kappa-B (NF-κB), tumor growth factor-beta (TGF), and genes associated with airway
remodeling are critical for PM-mediated responses in allergic airways.

Current

therapeutic strategies for asthma target the asthma- related adaptive immune
response. Therefore, investigations into a broader set of inflammatory immune targets
are needed to address exaggerated responses to air particulates in asthmatics.
Endotoxin or LPS is ubiquitously present in the environment and is a component in PM
of occupational, agricultural and urban air. LPS binds to TLR4 and activates both NFκB and TGFpathways. NF-κB is an important mediator of the immune response and
has recently been identified as a therapeutic target for multiple chronic inflammatory
diseases.

In chapter 2, I described the effects of airway LPS to enhance airway

inflammation but attenuate AHR in mice with pre-existing allergic airways disease. I
hypothesized that LPS activation of the transcription factor, NF-κB, attenuated AHR in
OVA-LPS mice. In this study I sought to determine the role of NF-κB in the attenuation
of AHR in OVA-LPS mice. To do this, I used OVA to establish allergic airway disease
as described in Chapter 2. Prior to LPS exposure, mice were treated with a novel
inhibitor of NF-κB. Comparisons between animals not treated and treated with the NFκB inhibitor were made between IN instilled saline, OVA, and OVA-LPS groups. .
There were no changes in inflammation or AHR in saline and OVA groups. In OVALPS mice, NF-κB inhibition did not affect the recruitment of neutrophils by LPS.
114

However, in the BALF there were significant decreases in macrophages, eosinophils,
and lymphocytes.

Furthermore, NF-B inhibition reversed the attenuation in AHR.

These findings suggest that attenuation of AHR in OVA-LPS mice is mediated by NFκB -dependent pathways.

115

4.2

INTRODUCTION
Inhalation of particulate matter (PM) from both indoor and outdoor air pollution

can exacerbate airway inflammatory symptoms in asthmatics (Breysse et al., 2010;
Kelly and Fussell, 2011). Pharmaceutical interventions that target allergic inflammationspecific pathways have been developed against IgE, histamines, and leukotrienes.
More often prescribed are corticosteroids that target multiple, non-specific pathways in
immune, inflammatory and epithelial cells, but that often cause undesirable side-effects.
Results from both human and animal studies suggest that PM exposure can
exacerbate antigen-induced, ‘allergic pathways’, as well as can induce additional
‘innate’ inflammatory responses to worsen airway pathology and function. Findings
from genomics studies suggest that induction of toll-like receptor-4 (TLR4), nuclear
factor kappa-B (NF-κB), tumor growth factor-beta (TGF), and genes associated with
airway remodeling are critical for PM-mediated responses in allergic airways (Wang et
al., 2008; Heidenfelder et al., 2009; Vawda et al., 2014). As such therapeutic strategies
to prevent exaggerated responses to air particulates in asthmatics needs to encompass
a broader set of inflammatory immune targets.
Endotoxin is a lipopolysaccharide (LPS) component of the cell wall of Gram
negative bacteria and is found in PM of occupational, agricultural and urban air. LPS
binds to TLR4 and activates both NF-κB and TGFpathways, and in both human and
animals can exacerbate allergic airways responses (Kawai and Akira, 2007; Chen et
al., 2008; Doreswamy and Peden, 2011). We have recently described novel effects of
airway LPS to enhance airway inflammation but at the same time attenuate airway
116

hyperresponsiveness (AHR) in mice with pre-existing allergic airways disease (Chapter
2). While the underlying mechanism is unknown, the primary pathological feature
associated with reversal of allergic AHR was widespread infiltration of neutrophils into
all compartments of the lung and decreased storage of intraepithelial mucous. NF-κB
activation in neutrophils can lead to downstream production of bronchoactive products
including nitric oxide and prostaglandins that might modulate airway reactivity (Tanaka
et al., 2005; Prado et al., 2006), and secretagogues such as elastase that may promote
production and hypersecretion of mucus that contributes to airway obstruction (Koga et
al., 2013).
Interrupting the NF-κB pathway may be an effective approach to mitigating the
adverse effects of PM-induced responses in asthmatic airways. Activation of NF-κB has
been shown in lungs of asthmatics (Hart et al., 2000), and allergic mice (Poynter et al.,
2002; Sheller et al., 2009), and may be linked to AHR in experimental asthma
(Donovan et al., 1999). Furthermore NF-κB also plays a role in promoting endotoxininduced lung inflammation (Poynter et al., 2003). The potential role that NF-κB plays in
endotoxin-induced modulation of allergic airway disease has not been examined.
Inactive NF-κB is sequestered in the cell cytoplasm by binding to the protein IκB(nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha),
and is released after inflammatory signal transduction pathways lead to the IκB kinase
(IKK)-β - mediated phosphorylation and subsequent proteasomal degradation of IκB- .
As a result, specific NF-κB proteins can translocate to the nucleus where inflammatory
gene transcription is activated. There are several types of inhibitors that target different
117

aspects of the NF-κB activation pathway (Edwards et al., 2009). A common approach
to disrupt NF-κB function is to interfere with phosphorylation or proteasome degradation
pathways. Members of our research team have used a novel imidazoline compound to
selectively inhibit the 20S catalytic core of the proteasome and prevent IKKdegradation, which in previous studies has blocked NF-κB-induced cytokine production
in vitro (Kahlon et al., 2009; Lansdell et al., 2013). In the current study I used this novel
proteasome inhibitor to test the hypothesis that LPS- mediated alterations of allergic
AHR are dependent on NF-κB activation. By intervening with the inhibitor immediately
prior to LPS administration, I hoped to limit the effects of NF-κB inhibition on allergic
processes, and rather target the NF-κB-mediated responses to LPS exposure in preexisting allergic airways.
4.3

MATERIALS AND METHODS

4.3.1 Laboratory Animals and Treatment protocols
Male BALB/c mice (Charles River Laboratories, Portage, MI), 6-8 weeks of age
were

housed

and maintained

as described in Chapter 2 MSU animal facilities

accredited by the Association for Assessment and Accreditation of Laboratory Animal
Care. Mice were randomly assigned to one of six experimental groups consisting of 6
animals as follows: 1) animals were assigned to saline, OVA, or OVA-LPS groups and
2) each group was divided in half and were administered the vehicle or NF-κB inhibitor
treatment prior to IN LPS (Fig 15).
4.3.2 NF-κB Inhibition

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On Day 20, non-allergic and allergic animals were randomly divided into 2 groups
to receive 0 or 50 mg/kg i.p. of the NF-κB proteasome inhibitor, TCH-013 in a vehicle
comprised of 3:7 propylene glycol: 5% dextrose (gift from J. Tepe, MSU) 1 hour prior to
IN LPS exposure (Fig 15).

Figure 15 Protocol for development of allergic airway disease and inhibition of
NF-κB

4.3.3 Necropsy, Lavage Collection, and Tissue Preparation
Mice were anesthetized with an i.p. injection of with sodium pentobarbital (10 mg;
Fatal Plus, Vortech Pharmaceuticals, Dearborn, MI).

Blood was collected from the

caudal vena cava for separation of plasma, and animals were euthanized by transection
of the abdominal aorta. The lungs and heart were harvested en bloc. The lungs were
instilled twice with 800 µL saline via the cannulated trachea to collect BALF. After
lavage the right lung lobes were ligated, separated and stored in RNAlater (Qiagen) for
RNA isolation or snap frozen in liquid nitrogen for storage. The left lobe was inflated
119

with 10% neutral buffered formalin to a pressure of 30 cm H2O for 1 hour and then
stored in a large volume of the same fixative.
4.3.4 Bronchoalveolar Lavage Analysis
Total BALF leukocytes were counted with a hemocytometer and cytological
slides were prepared using a Cytospin centrifuge (Shandon). Slides were stained with
Diff-Quick (Dade Behring, Newark, DE) and cell differential (macrophages, eosinophils,
neutrophils, and lymphocytes) were counted. The remaining BAL fluid was centrifuged,
and the supernatants were stored at -20°C.
4.3.5 Histopathology
Following fixation, two traverse sections of the left lung were taken along the
axial airway at the levels of the 5th and 11th generation (G5 and G11) to examine the
proximal and distal airways (Harkema and Hotchkiss, 1992).

The tissues were

embedded in paraffin and stained with H&E for routine histological examination and
AB/PAS to identify intraepithelial mucosubstances.
4.3.6 Morphometry
From the left lung lobe, estimation of the amount of the intraepithelial
mucosubstances in epithelium lining the G5 axial airways was performed on histological
slides stained with AB/PAS. Slides were scanned and digitalized with a slide scanner
(VS110, Olympus).

Using a specialized mucus quantification APP (Visopharm,

Denmark), the G5 airway was manually selected as the region of interest in the scanned
120

images.

The APP automatically highlights the basal lamina beneath the surface

epithelium and the positive staining of mucusubstance to determine the area of mucus
relative to the basal lamina expressed and μm2/μm.

4.3.7 Real –time PCR of Lung
Total RNA was isolated from the right caudal lung lobe using Rneasy Mini Kit
(Qiagen, Valencia, CA) according to manufacturer’s instructions. Briefly, reverse
transcription was accomplished by using High Capacity cDNA Reverse Transcription Kit
reagents (Applied Biosystems). Quantitative mRNA expression analysis was conducted
on an ABI PRISM 7900 HT Sequence Detection System at Michigan State University’s
Research Technology Support Facility using Taqman Gene Expression Assay reagents
(Applied Biosystems).
Targeted genes for molecular analysis included Resistin like alpha (Retnla;
Fizz1), Chloride channel calcium activated 3 (Clca3; Gob5), Interferon gamma (Ifng),
Interleukin 10 (Il10), Il-12b, -13, and -17a and chemokine ligand 2 (Ccl2; MCP1).
Relative gene expression was normalized to endogenous controls (Arbp, Gusb, and
Gapdh). Data was expressed as fold-increase in RNA expression compared to control
animals, which were set at a value of 1.
4.3.8 Airway Hyperresponsiveness (AHR) Measurements
On day 21, mice designated for lung function measurement were anesthetized
and evaluated for AHR with incremental doses of methacholine as outlined in Chapter
2. The mean of the responses for each concentration of methacholine was determined.
121

A dose- response curve was generated. The lowest dose of MCh with the greatest
variation in response between groups was at 10 mg/ml.

Therefore, the data were

expressed as the percent change at 10 mg/ml compared to baseline for each group.
4.3.9 Statistical Analysis
BALF data were expressed as group means + the standard error of the mean
(SEM). Grubb’s outlier test was used to determine and remove outliers.

Statistical

differences were determined with two-way ANOVA with Student-Newman-Keuls post
hoc test to make direct comparison between groups; p  0.05. For RT-PCR, statistical
differences of Ct values between groups were determined with t-tests to make direct
comparison between groups (SigmaPlot, Systat Software Inc, San Jose, CA).

122

4.4

RESULTS

4.4.1 Bronchoalveolar lavage fluid
Consistent my previous results, sensitization and challenge to OVA induced a
significant accumulation of total cells in BALF (Fig 16A). The increase consisted mostly
of macrophages and eosinophils (Fig16 A, B, C). Allergic mice given IN LPS (OVALPS) had a significant increase in total cells compared to OVA mice, which was due to a
robust infiltration of neutrophils and a minor increase in lymphocytes (Fig16 A, D).
Treatment with the NF-κB inhibitor TCH-013 prior to LPS instillation reduced
macrophage, eosinophil and lymphocytes in only OVA-LPS mice. Interestingly, there
was no effect of NF-κB inhibition on neutrophil accumulation elicited by LPS. Lastly,
treatment with TCH-013 resulted in small increases in macrophages and lymphocytes in
saline sensitized and challenged mice (Fig 16 B, E).

123

Figure 16 Comparison of BALF in mice treated and not treated with NF-κB
inhibitor. Total cells, macrophages, eosinophils, neutrophils, and lymphocytes were
determined in BALF collected from Saline, OVA and OVA-LPS mice that received
Vehicle (clear bars) or an Inhibitor (cross hatched bars) of NF-B (50 μg of TCH-013,
i.p.) as described in Methods. Values are expressed as mean + SE (n=4-6). Horizontal
bars represent significant difference between indicated groups, p < 0.05

124

4.4.2 Histological examination and morphometry
Following treatment with the NF-κB inhibitor, non-sensitized, mice did not show
any histological lesions in the lung (Fig 17A).
by treatment with TCH-013.

OVA- induced lesions were not altered

Lungs from OVA mice had focally extensive

peribronchiolar and perivascular inflammation, which predominated around the axial
airway and large diameter bronchioles with occasional extension into distal terminal
bronchioles. Airway inflammation was predominately comprised of mononuclear cells
(lymphocytes and macrophages) with lesser numbers of eosinophils (Fig 17B).

OVA

caused airway epithelial remodeling characterized by mucous cell metaplasia,
identifiable by AB/PAS staining, affecting the axial airway and large diameter
bronchioles (Fig 17D). Treatment with TCH-013 did not significantly alter the lesions in
OVA-LPS mice. Exposure to LPS in OVA mice produced a mixed inflammatory influx in
peribronchiolar and perivascular regions. Prominent inflammation surrounded the axial
airway, large diameter bronchioles, and extended around many terminal bronchioles.
Airway inflammation was characterized by mainly mononuclear cells (lymphocytes,
macrophages and smaller numbers of multinucleate cells) and moderate numbers of
granulocytes (eosinophils and neutrophils).

Additionally, this mixed inflammation

extended into the lung parenchyma and airspaces (Fig 17C). Morphometrically, the
area of AB/PAS stained mucosubstances per basal lamina length in treated animals
was not different from untreated animal (Fig 18A). Increases in stored mucous were
accompanied by increased expression of Gob5 in both OVA and OVA-LPS mice
compared to saline mice (Fig 18B). Inhibition of NF-κB had no effect on Gob-5
expression.
125

Figure 17.
Pulmonary histology of mice with NF-κB inhibition.
Light
photomicrographs (H&E) of lungs from mice treated with the NF-κB inhibitor, TCH-013
showing no inflammation (saline, A), peribronchiolar and perivascular inflammation and
thickening of airway epithelium due to mucous cell metaplasia (OVA/OVA-LPS, B/C),
extension of inflammation into the alveolar tissue and spaces (OVA-LPS, C). AB/PAS
staining of intraepithelial mucus in axial airway (OVA, D). A, bronchiole; BV, blood
vessel, and AA, axial airway. Scale bar 100 μm

126

Figure 18. Airway mucosubstances and Gob5 gene expression following NF-κB
inhibition. (A) Area of mucosubstance per length of basal lamina of the proximal
airway (G5). (B) mRNA Gob-5,involved in mucus secretion. Data were reported as the
fold increase of the group mean + SEM. Horizontal line indicates significant difference
between specified groups, p<0.05; n=6/group.

Gob5

127

4.4.3 Lung gene expression
Sensitization and challenge with OVA caused increased expression of Ifng and
Il10 (Fig 19, A-B). Also, OVA increased Fizz1 and Il13. However, in OVA-LPS mice,
induction of Fizz1 and Il13 were decreased compared to OVA mice (Fig 19, C-D),
suggesting Th2 pathways were down-regulated with LPS treatment. Furthermore,
induction of IL12b and Mcp1 occurred only in OVA-LPS mice (Fig 19, E-F). The only
effect of treatment with TCH-013 was the reduction of Il13 expression in OVA mice.

128

Figure 19. Pulmonary gene expression following NF-κB inhibition. Relative
expressions of mRNA encoding for Ifng, Il10, Fizz1, Il13, Il12b, and Mcp1 were
determined in right caudal lobes by RT-PCR as described in Materials and Methods.
Values are expressed as fold increase of mRNA expression compared to control
(Saline) animals (n=5-6). Horizontal bars represent significant difference between
indicated groups, p < 0.05.

129

4.4.4 Airway Hyperresponsiveness
Treatment with the NF-κB inhibitor, TCH-013, did not significantly alter airway or
tissue resistance caused by methacholine challenge in either Saline or OVA mice (Fig
20, A-D). However, AHR was significantly increased in OVA-LPS mice treated with the
inhibitor. Specifically, increases were measured in total lung resistance (200%, Fig20
A), central airways resistance (180%, Fig 20 B), tissue resistance (200%, Fig 20 C) and
tissue elastance (400%, Fig 20 D).

130

Figure 20. Effects of NF-κB inhibition on AHR in OVA-LPS mice. Methacholine-induced changes in total lung
resistance (A), airway resistance (B), tissue damping (C) and tissue elastance (D) were measured in intubated and
ventilated mice using the FlexiVent system as described in Materials and Methods. Values are expressed as the percent
increase from baseline value after administration of 10 mg/ml of methacholine (n=12-14). Horizontal bars represent
significant difference between indicated groups, p < 0.05.

131

4.5

Discussion
We have previously described a LPS exacerbation model of asthma the co-

existing effects of enhanced inflammatory cell recruitment and reduced airway AHR.
The primary pathological feature associated with changes in AHR in LPS-treated
allergic mice was widespread infiltration of neutrophils into all compartments of the lung.
In the current study, inhibition of NF-B had no effect on LPS-stimulated neutrophil
recruitment, but rather led to a dramatic increase in AHR. Furthermore, these changes
in airway physiology were associated with decreases in airway macrophages,
eosinophils and lymphocytes. As such our results suggest that inflammation is inversely
related to AHR in this model, and that this novel relationship is mediated by NF-Bdependent pathways.
Activation of NFB plays a central role in the adverse airway inflammatory and
hyperreactivity responses to both allergen and LPS-stimulation in laboratory rodents. In
BALB/c OVA models of asthma the inhibition of NF-B is a common mechanism for
therapeutic compounds to reduce eosinophil recruitment, Th2 cytokine production,
mucous cell metaplasia and AHR (Desmet et al., 2004; Lee et al., 2005; Bao et al.,
2007; Kim et al., 2007; Shimizu et al., 2012). Likewise, the neutrophil recruitment and
airway injury caused by LPS can be blocked by NF-B inhibition (Chuang et al., 2013;
De Stefano et al., 2013). We designed the protocol to minimize the effects of NF-B
inhibition on allergic processes, by dosing with TCH-013 approximately 48h after the
last OVA challenge. However we did see a small decrease in BALF lymphocytes in
OVA mice, and a modest depression in resistance in their central airways and
132

peripheral tissues. These results suggest that ongoing allergic airway responses can be
interrupted with an NF-κB inhibitor, and does not require interventions at the time of
OVA challenge as was done in other mouse studies mentioned above.
Our protocol was designed to target the NF-κB-mediated responses to LPS
exposure in pre-existing allergic airways. We expected that treatment with TCH-013
would reverse the marked recruitment of BALF neutrophils that were associated with
LPS. Instead, eosinophils, macrophages, and lymphocytes were the inflammatory cells
that were blocked by NF-κB inhibition in OVA-LPS mice. One explanation is that the
dose of TCH-013 was insufficient to affect LPS-induced airway neutrophilia, but was
capable of modulating chemokines specific for eosinophil and macrophages. Cytokines
that promote eosinophil accumulation include eotaxin, IL-5 and GM-CSF, all of which
activate NF-B in eosinophils (Ip et al., 2005). Airway macrophages in asthmatic
airways with LPS exposure may be a mixed population of M1 and M2 subtypes
(classically or alternatively activated, respectively), where M1 cells may be more
sensitive to NF-B inhibition than M2 cells (Porta et al., 2009; Liao et al., 2011).
Compared to OVA mice, both eosinophils and macrophages were trending toward
reductions in allergic mice given LPS, and blocking NF-B pathways may have further
augmented these reductions.

We did not determine if chemokine induction or

production was changed by TCH-013 treatment, which might explain the reduced
accumulation of inflammatory cells in BALF.
Binding and activation of TLR4 in airway macrophages and epithelial cells by
endotoxin should stimulate neutrophil chemokine production (e.g., MIP-2, KC) (Schmal
et al., 1996; Krakauer, 2002; Skerrett et al., 2004) by pathways that involve NF-B
133

activation (Mizgerd et al., 2004). Therefore we were surprised that TCH-013 –induced
reductions in BALF PMN accumulation were not significant. We used a dose of the
inhibitor that was effective in reducing circulating TNF in LPS-treated BALB/c mice
(Lansdell et al., 2012), and that in dose ranging studies inhibited airway LPS-induced
neutrophil recruitment by 60% (data not shown).

It is possible that LPS-induced

neutrophil accumulation in inflamed allergic airways involves activation of additional cell
populations (epithelial cells, macrophages, eosinophils, Th17 cells) that do not
participate in healthy, naïve airways. Higher doses of TCH-013 may be required to
neutralize NF-B in this enhanced inflammatory environment.

Future studies are

required to test this hypothesis. We did notice however, that treatment with TCH-013
resulted in a moderate decrease in the severity of peribronchial/perivascular
inflammation, mostly by reducing the density of neutrophils in these regions. This finding
supports the possibility that we have some effects on neutrophil recruitment and that
higher doses of the inhibitor may further decrease neutrophil accumulations.
Despite the lack of significant effects on airway neutrophil accumulation,
inhibition of NF-kB led to a dramatic increase in airway resistance in OVA-LPS mice.
Inhaled methacholine binds to muscarinic receptors (Gq) on airway smooth muscle,
which activates phospholipase C to release of inositol trisphosphate, and is followed by
an increase in intracellular calcium that interacts with contractile proteins. Studies in
allergic mice and guinea pigs demonstrate that enhanced airway contractility is due to
an increase in Rho protein, which is critical to Rho-kinase mediated smooth muscle
contraction (Schaafsma et al., 2004; Witzenrath et al., 2008). Results in vascular
smooth muscle show that LPS-induced NF-kB activation leading to an increase in iNOS
134

expression and downregulation of Rho and Rho-associated kinase activity (Wei et al.,
2006).
It is well established that upregulation of iNOS in smooth muscle by LPS is
mediated by NF-kB activation (Hattori et al., 2003; Pingle et al., 2003; Gomez et al.,
2005). Therefore, the large increase in AHR with NF-kB inhibition we observed in LPSOVA mice may be explained by changes to smooth muscle cells, and not related to
inflammatory lesions we described in lung tissues.
A limitation of this study is that we did not confirm that NF-kB was affected by our
treatment with TCH-013. Although we used doses that were effective in other animal
models of inflammation (Lansdell et al., 2012) and TCH-013 selectively inhibit the 20S
catalytic core of the proteasome and

prevents IKK-

degradation (Lansdell et al.,

2013), we did not confirm the effects in pulmonary tissue from treated animals. TCH013 clearly had effects on AHR and inflammatory cell infiltrations, but the effects on the
proteasome need to be assessed.

135

4.6

Summary
Intervention with an NF-kB inhibitor in an OVA-LPS mouse model of asthma

exacerbation produced some interesting and unexpected results. The attenuation of
allergic AHR was reversed, with airway resistance in OVA-LPS mice being even greater
than in OVA mice. However NF-kB had no effect on LPS induced neutrophil influx, the
major histopathological difference from OVA mice. While results in Chapter 3 suggested
no role for neutrophils in modulating AHR, the lack of effect by TCH-013 on their
infiltration is puzzling, especially when eosinophil granulocytes were decreased in these
same mice. Although the effects on inflammatory cell populations were unexpected, the
results support the hypothesis that LPS-mediated alterations of allergic AHR were
dependent on NF-κB activation.
Airway LPS activates many cell types in the lung that can influence AHR. Smooth
muscle contraction induced by methacholine can be opposed by NO produced internally
or by neighboring cells in the airway wall, including epithelial cells and fibroblasts.
Among many inflammatory genes, LPS is well documented to upregulate iNOS by NFkB pathways in both epithelial and smooth muscle cells. In the next chapter the role
iNOS in LPS modulation of allergic AHR will be assessed.

136

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142

CHAPTER 5

ROLE OF Nos2 AND Arg1 IN THE ATTENUATION OF AIRWAY
HYPERRESPONSIVENESS IN ALLERGIC MICE EXPOSED TO INHALED
LIPOPOLYSACCHARIDE

143

5.1

ABSTRACT

Nitric Oxide (NO) is thought to be both beneficial and deleterious to the asthmatic lung.
This dual function appears to be both dose and cell specific. NO is a broncho- and
vaso-dilator with anti-inflammatory properties. High NO concentration, commonly
associated with iNOS induction, has been implicated in increased inflammation,
promoting pathology, and AHR in asthma.

In this study, I examined pulmonary

expression of Nos2 as well as Arg1 mRNA, to determine if changes in these genes
were associated with the attenuation of AHR to methacholine displayed in mice
sensitized and challenged with OVA followed by inhalation exposure to LPS.

In

addition, I evaluated tissues from my studies of mice with established allergic airways
disease treated with the vehicle or NF-κB inhibitor prior to LPS exposure to determine if
the reversal of the attenuation in AHR present in OVA-LPS mice was associated with
changes in Nos2. Lung tissues taken from BALB/c mice with OVA-induced allergic
airways disease with and without LPS exposure and with or without treatment with a
novel NF-κB inhibitor were analyzed by RT-PCR. Also, tissues were stained with an
anti-iNOS antibody to determine protein expression within the lung.

Compared to

saline mice neither Nos2 nor Arg1 expression were upregulated in LPS mice and OVA
mice had no change in Nos2, but had a significant increase in Arg1 expression. OVALPS mice had significant increase in Nos2 and Arg1 expression compared to all other
groups. Inhibition of NF-κB did not affect Nos2 and Arg1 expression in saline and OVA
mice; however, there was a significant attenuation in expression of both genes in OVALPS mice. Furthermore, staining for the iNOS protein was not present in saline mice
with or without NF-κB inhibition. In OVA mice, positive staining of iNOS was
144

predominately present in the epithelium of small pre-terminal bronchioles and terminal
bronchioles, ciliated epithelial cells in the central conducting airway, and small numbers
of positive staining inflammatory cells (macrophages) were in peribronchiolar and
perivascular tissue. NF-κB inhibition did not affect positive iNOS staining in OVA mice.
In OVA-LPS mice positive iNOS staining in the lung displayed a similar pattern as OVA
mice, however, there was increased positive staining in inflammatory cells, consistent
with macrophages and neutrophils, present in parenchymal tissue and alveolar spaces
in addition to peribronchiolar and perivascular regions.

Following NF-κB inhibition,

positive staining was less intense and was present in fewer inflammatory cells
compared to vehicle treated OVA-LPS mice. These results support a role for iNOSmediated NO production in the attenuation of AHR to methacholine in my model of
allergic mice exposed to LPS.

145

5.2

INTRODUCTION
Nitric oxide (NO) is a biological mediator of diverse physiological functions

produced in a variety of cell types in many tissues (Ricciardolo et al., 2004). In the lung
NO has been shown to be produced by neutrophils, macrophages, airway epithelium,
fibroblasts, smooth muscle cells, and nonadrenergeric noncholinergic nerves (Gaston
et al., 1994). Under normal physiological conditions, NO is a dilator of airways and
blood vessels (Prado et al., 2011). The role(s) of NO in lung diseases, such as asthma,
has not been definitively established. In spite of its known contribution to
bronchodilation, NO measured in exhaled breath (FeNO) has been shown to be
increased in asthmatics with severe asthma phenotypes, and has therefore been used
to estimate airway inflammation (Dweik et al., 2010; Aytekin and Dweik, 2012;
Yamamoto et al., 2012). High levels of NO may promote pro-inflammatory responses,
such as an increase in neutrophils, eosinophils, and monocytes (Prado et al., 2006). I
hypothesized that the upregulation of lung expression of Nos2 was associated with
attenuation of AHR in OVA-LPS mice.
NO is produced by the metabolism of L-arginine by the enzyme nitric oxide
synthase (NOS), which exists as three isoforms: neuronal NOS (nNOS or NOS1),
inducible NOS (iNOS or NOS2), and endothelial NOS (eNOS or NOS3). Compared to
constitutive isoforms (also referred to as cNOS), iNOS produces large amounts of NO,
nM to μM respectively (Meurs et al., 2003;

rado et al., 2006). It has been proposed

that increased NO production during oxidative stress in asthmatic lungs results in the
-

formation of peroxynitrite (ONOO ) from the reaction of NO with superoxide (O2-).
146

Further evidence demonstrates the importance of spatial and temporal production of
NO to airway dilatation in asthma. For example impaired release of NO is associated
with downregulation of eNOS in bronchial epithelium after allergen-induced AHR in
asthmatics (Ricciardolo et al., 2001). Thus, the role of NO in asthma pathogenesis is
isoform-and tissue-specific.
Alterations in NO production during asthma may be dictated by the competition
for L-arginine, the common substrate of both NOS and arginase (Arg) enzymatic
pathways (Maarsingh et al., 2009; Vonk et al., 2010). Increased arginase-1 (Arg-1)
activity is associated with airway remodeling, decreased NO production by cNOS; and
upregulation of iNOS and its increased production of peroxynitrite (Benson et al., 2011).
Under normal physiological conditions, Arg-1 and NOS enzymes are counterregulatory, with each producing intermediate metabolites that are inhibitory to the other
enzyme (Meurs et al., 2003; Maarsingh et al., 2008).
Paradoxically, iNOS upregulation could potentially alleviate airway constriction by
producing concentrations of NO that counteract the effects of Arg-1 (Hjoberg et al.,
2004). In the study reported in chapter 2, I showed that while allergic (OVA) mice
exposed to LPS had increased pulmonary inflammation, they also had attenuated
allergen-associated AHR when induced by inhaled methacholine. Importantly, these
mice demonstrated an upregulation in the NOS2 gene (i.e, iNOS).

In the study

reported in chapter 4, NF-κB inhibition reversed the inhibitory effect of L S on allergenassociated AHR. The goal of the present investigation was to distinguish alterations in
gene and protein expression of iNOS and Arg-1 in airway tissues in order to test the
147

hypothesis that attenuation of AHR by LPS in asthmatic mice is dependent on iNOS
expression in the lungs. Previously, I have shown that LPS exposure increases iNOS
gene expression in allergic mice (Chapter 2). In the current investigation I expect that
OVA-LPS mice treated with a NF-κB inhibitor will have a decrease in iNOS gene
expression that is associated with the increase in AHR.

148

5.3

MATERIALS AND METHODS

5.3.1 Animals and Treatment Protocols.
Evaluated tissues were taken from BALB/c mice sensitized, challenged, and
exposed to LPS as previously described in Chapter 2. Some animals were treated with
the TCH-013, an inhibitor of NF-κB, as described in detail in Chapter 4.
5.3.2 Relative Quantification Real-Time PCR
Right caudal lung lobes were removed and placed in RNAlater (Qiagen,
Valencia, CA) where it was kept at 4°C for 24 hours and then stored at -20°C until
processed and analyzed. Total RNA was isolated from the right caudal lung lobe using
Rneasy Mini Kit (Qiagen, Valencia, CA) according to manufacturer’s instructions.
Tissues were homogenized using Qiagen’s TissueLyser II Bead Beater and three
2.8mm Zirconium Oxide beads in 600µl buffer RLT containing β -Mercaptoethanol.
Homogenates were then centrifuged at 12,000g for 3 minutes and RNA was purified
from the supernatant using the RNeasy capture column. Eluted RNA was diluted 1:5
with Rnase free water and quantified using the Nanodrop 1000 spectrophotometer
(Thermo Fisher Scientific, Waltman, MA). Reverse transcription was accomplished by
using High Capacity cDNA Reverse Transcription Kit reagents (Applied Biosystems).
Each RT reaction was run in a 50 l reaction volume containing 5 g of total RNA with
cDNA Master Mix prepared according to the manufacturer’s protocol. The reaction
mixture was incubated in a in GeneAmp PCR System 9700 Thermocycler PE (Applied
Biosystems, Foster City, CA) at 25ºC for 10 Minutes, 37ºC for 2 hours, then held at 4º.
149

Relative quantitative mRNA expression analysis of Nos2 and Arg1 genes was
conducted on an ABI PRISM 7900 HT Sequence Detection System at Michigan State
University’s Research Technology Support Facility using Taqman Gene Expression
Assay reagents (Applied Biosystems). 2µl cDNA and 8µl reagents were dispensed (in
duplicate) into a 384-well reaction plate. The cycling parameters were 48 C for 2
minutes, 95 C for10 minutes, and 50 cycles of 95 C for 15 seconds followed by 60 C
for 1 minute
Gene expression levels were reported as fold-change (FC) of mRNA in
experimental samples compared to a control sample. Real-time PCR amplifications
were relatively quantified using the Ct method. Following the PCR, amplification plots
(change in dye fluorescence versus cycle number) were examined and a dye
fluorescence threshold within the exponential phase of the reaction was set for the
target gene and the endogenous references. The cycle number at which each amplified
product crosses the set threshold represents the CT value. The amount of target gene
normalized to its endogenous reference was calculated by subtracting the geometric
mean of the CTs from endogenous controls (Arbp, Gusb, and Gapdh) from the target
gene CT (delta Ct (Ct)) This normalization strategy has been utilized for accurate RTPCR expression profiling in biological samples with small expression differences
(Vandesompele et al., 2002). The Ct value for the experimental sample is subtracted
from the Ct value of the corresponding control sample (Ct). The FC in experimental
samples relative to control samples is then calculated as: FC= 2-Ct.
5.3.3 Western blot analysis of iNOS and arginase-1 proteins in the lung
150

The cranial lung lobes were homogenized as described previously (Kang et al.,
2010). Briefly, frozen lungs were ground and sonicated in 7 M urea containing protease
inhibitors for 10 seconds (3 times) and centrifuged at 13,000g for 15 minutes at 4°C.
Following protein quantification, supernatants were pooled from the same group. 15 µg
of total lysate protein from pooled samples were subject to electrophoresis on 10-20%
SDS-polyacrylamide gels before transferring to PVDF membranes. HRP-conjugated
secondary antibody (GE Life Sciences) followed by enhanced chemiluminescent ECL
substrate was used to develop the blots according to the manufacturer’s instructions.
Primary antibodies were used to detect the expression of iNOS (AbCam, Cambridge,
MA) and Arg-1 (Santa Cruz, CA) proteins.
5.3.4 Immunohistochemistry
Paraffin-embedded sections of the proximal lung (G5) were prepared from
formalin-fixed tissues and prepare by routine immunohistochemical techniques to detect
iNOS (sc-7271, Santa Cruz Biotechnology).
5.3.5 Statistical Analysis
Data were expressed as group means + the standard error of the mean (mean +
SEM).

Grubb’s outlier test was used to determine and remove statistical outliers.

Statistical differences between Ct values between groups were statistically determined
with two-way ANOVA with Student-Newman-Keuls post hoc test to make direct
comparison between groups; p  0.05 (SigmaPlot, Systat Software Inc, San Jose, CA).

151

5.4

RESULTS

5.4.1 Lung tissue Nos2 and Arg1 mRNA expression
To assess differences in the expression of genes involved in L-arginine
metabolism, the pulmonary expression of the Nos2 gene that produces the protein
iNOS, and the Arg1 gene that induces arginase-1, were evaluated by RT-PCR. Twentyfour hours following LPS exposure, Nos2 expression was not significantly increased in
LPS or OVA alone mice (Fig 21 A, saline, black bar; OVA, white bar). OVA-LPS mice
had significant, 3-fold increase in Nos2 expression compared to OVA alone (Fig 21 A;
OVA, black bar).

Arg1 was upregulated by 3-fold in LPS mice (Fig 21 B; saline, black

bar) in reference to control mice. There was a 9-fold increase in the expression of Arg1
in OVA mice (Fig 21 B; OVA, white bar). Additionally, OVA-LPS mice had significant, 3fold increases in Arg1 expression compared to OVA alone (Fig 21 B; OVA, black bar).

152

Figure 21. Pulmonary gene expression of Nos2 and Arg1 in allergic and nonallergic mice exposed to LPS. Relative expressions of mRNA for Nos2 (A) and Arg1
(B) were determined as described in Materials and Methods. Values are expressed as
fold increase of mRNA expression compared to control (Saline) animals (n=5-6).
Horizontal lines indicate significant difference; p < 0.05.

Nos 2

153

5.4.2 Effects of NF-κB inhibition on pulmonary Nos2 and Arg1 expression
Nos2 expression in OVA mice was not significantly different from control mice,
regardless of treatment with NF-κB inhibitor (Fig 22 A; diagonal striped bar). In OVALPS mice, Nos2 expression was increased almost 8-fold, however treatment with the
NF-κB inhibitor TCH-013 blocked expression by 50%, such that relative increases were
less than 4-fold compared to control mice treated with vehicle (Fig 22 A, black bars).
OVA-LPS mice had significant increased expression of Nos2 compared to OVA alone
mice regardless of treatment. Similarly, OVA mice had an almost 30-fold increase in
expression compared to saline mice, which was not significantly affected with the NF-κB
inhibitor (Fig 22 B; diagonal striped bar). There was a 60-fold and 2-fold increase in
Arg1 expression in OVA-LPS animals compared to saline mice and OVA alone mice
respectively. This increase was significantly inhibit by 50% with the NF-κB inhibitor
treatment when compared to untreated OVA-LPS mice resulting in expression levels
similar to treated OVA mice (fig 5-2 B; black bars).

154

Figure 22. Effect of NF-κB inhibition on pulmonary gene expression of Nos2 and
Arg1. In lung lobes from control and NF-κB inhibitor treated (TCH-013) mice, relative
expressions of mRNA for NOS2 and Arg1 were determined. Values are expressed as
fold increase of mRNA expression compared to control (Saline) animals (n=5-6).
Horizontal lines indicate significant difference; p < 0.05.

155

5.4.3 Protein expression of iNOS and Arg-1
Using frozen right cranial lung lobes, protein was extracted and analyzed by
western blot. After two separate runs iNOS protein could not be visualized in the tissue
samples. Arg-1 protein was detected in the lung tissues of OVA and OVA-LPS mice,
but not in LPS alone mice (data not shown).

However, quantification of protein

expression could not be obtained from the study samples. These results showed that
western blot analysis of iNOS and Arg-1 proteins did not work in the desired manner.
Several factors could have contributed to the inability of this approach to generate
reliable data. The use of suboptimal antibodies for the tissues used in this study could
be a major factor in ineffective data generation. Therefore, more stringent optimization
of the antibodies used in this study to determine best dilutions and incubation times may
have be needed. Also, the quantity and quality of the samples used may not have been
adequate to generate data. For example, the protein of interest may not be abundantly
present in the entire tissue. Therefore, maximizing the signal of the protein by only
analyzing specific tissue, such as airway epithelium, may generate more protein than
utilizing the entire lung tissue. This issue may be particularly true for iNOS protein
expression.
5.4.4 Immunohistochemistry for iNOS
Proximal (G5) sections of lung tissue were stained with an anti-iNOS antibody to
detect iNOS positive cells.

Saline mice had occasional positive staining of airway

epithelium in central and peripheral airways; however, there were no positive cells in
mice treated with NF-κB (Fig 5-3 A-B).

In OVA mice, the axial airways contained

positive intracytoplasmic staining only in ciliated epithelial cells. More diffuse positive
156

staining was present in the epithelium of smaller peripheral airways, pre-terminal
bronchioles, and distal terminal bronchioles.

Additionally, there were occasional

positive staining mononuclear cells morphologically consistent with macrophages
present in peribronchiolar and perivascular tissue and alveolar spaces. NF-κB inhibition
did not alter staining in OVA mice (Fig 23 C-D). Compared to OVA alone, lungs from
OVA-LPS animals had similar positive staining in airway epithelium consisting of
staining in the ciliated epithelium of the axial airways and more diffuse staining in preterminal and terminal bronchioles, however, there were increased numbers of positive
staining inflammatory cells morphologically consistent with macrophages and
neutrophils in peribronchiolar and perivascular tissue, alveolar septum, and alveolar
spaces (Fig 23 E-F). Positive iNOS staining was less intense in the airway epithelium
and inflammatory cells following NF-κB inhibition.

157

Figure 23. Immunohistochemical staining for iNOS. Lung sections without or with
inhibition of NF-κB, respectively, were taken from saline (A and B), OVA (C and D), and
OVA-LPS (E and F) mice and were probed with an anti-iNOS antibody. Positive
staining is indicated by cytoplasmic brown staining. Arrows indicate positive staining in
airway epithelium. AA, axial airway; BV, blood vessel; TB, terminal bronchioles; AD,
alveolar duct. Original magnification, x20.

158

5.5

DISCUSSION
In this study my goal was to determine if changes in pulmonary iNOS following

LPS exposure was related to the attenuation of AHR in asthmatic mice. I evaluated
alterations in the expression of iNOS (NOS2) and arginase-1 (Arg1) genes in lungs from
mice with or without OVA induced allergic airways disease exposed to intranasal saline
or endotoxin, as well as in OVA and OVA-LPS mice treated with a novel NF-κB inhibitor.
Results demonstrated that in OVA-LPS mice with the attenuated AHR phenotype, iNOS
is upregulated compared to OVA mice, and NF-κB inhibition decreased iNOS
expression in OVA-LPS mice, which is associated with a reversal of the attenuated AHR
response. Additionally, Arg1 expression was enhanced in OVA-LPS animals compared
to OVA alone. This enhancement was also decreased with NF-κB inhibition. These
results support my hypothesis that attenuation of airway hyperresponsiveness by LPS in
asthmatic mice is dependent on Nos2 expression in the lungs. Moreover, the data
show increased Arg1 expression is not necessary for the induction of AHR in asthma
exacerbation. These findings could provide a better understanding of mechanisms by
which environmental factors, such as endotoxin, can affect the asthmatic response
which will lead to improved models of therapy for future studies.
Dysregulation of arginine metabolism has been associated with many diseases,
such as vascular hypertension, sickle cell anemia, diabetes, and asthma (Morris et al.,
2005; Benson et al., 2011; El-Bassossy et al., 2013). Current knowledge indicates that
metabolism is controlled by opposing enzymes, particularly arginase and nitric oxide
synthase. Arg-1 utilizes arginine to produce urea and ornithine, a precursor of proline,
while, iNOS metabolizes arginine to produce citrulline and NO (Morris, 2008). Both
159

enzymes produce intermediate mediators that can effectively inhibit the opposing
enzyme providing homeostasis in arginine metabolism.
NO deficiency due to decreased arginine substrate that is diverted to increase
arginase activity is a proposed mechanism of asthma pathogenesis (Meurs et al., 2002;
Larsson et al., 2005). Th2 cytokines IL-4 and -13 that are produced during asthma
promote arginase activity (Hesse et al., 2001). It has been shown that asthmatics have
increased arginase activity in sputum and decreased plasma arginine (Morris et al.,
2004). Similarly, in asthma animal models there is increased expression and activity of
arginase-1 (Kenyon et al., 2008; North et al., 2009; Maarsingh et al., 2011). Increased
arginase has been associated with airway remodeling and AHR.

NO is a

bronchodilator, however, there have been many reports of its detrimental effects in
asthma. These effects appear to be dose and cell specific.

High NO concentrations

cause vasodilation with edema, mucus hypersecretion, and enhanced Th2 inflammation
contributing to asthma pathology (Prado et al., 2011).

Also, it has been shown that

large concentrations of NO interact with reactive oxygen species to produce
peroxynitrite, which causes tissue damage and is may contribute to AHR
(SadeghiHashjin et al., 1996; Saleh et al., 1998).

Similar to asthmatics and other

models of asthma, OVA induced a significant increase in arginase-1 gene expression
compared to saline control mice in my model of allergic airway disease. However, there
was no increase in iNOS expression, and even a slight decrease in gene expression
compared to control animals.

160

LPS is one of the major inducers of the iNOS in responsive cells such as
macrophages, epithelial and smooth muscle (Beck et al., 1999). However, it has been
shown that LPS coinduces Arg-1 and iNOS (Sonoki et al., 1997). Inhaled LPS causes
increased inflammation, primarily neutrophils, and increases AHR to methacholine
(Hakansson et al., 2012).
entirely known.

The mechanism of AHR following LPS inhalation is not

It is thought to be mediated through toll like receptor 4 (TLR-4)

(Hollingsworth et al., 2004). In my studies, LPS treatment induced increased pulmonary
neutrophils and AHR (Chapter 2). Twenty-four hours following LPS exposure, these
mice had a small increase in iNOS mRNA but it was not significantly different from
control animals. However, there was a significant increase in Arg-1 mRNA. These
findings may be due to the specific time point of analysis. Sonoki and colleagues
identified that iNOS mRNA and protein began to decrease 12 hours after i.p. LPS
administration in mice, while Arg-1 mRNA and protein began to increase at 12 hours
following LPS (Sonoki et al., 1997).
Inhaled LPS produces variable responses indicating that some people are less
sensitive to its effects than others (Kline et al., 1999).

It has been shown that

asthmatics can be more sensitive to LPS and require smaller doses to produce AHR
(Michel et al., 1989). However, several studies of experimental asthma have shown an
attenuation of AHR with LPS exposure in OVA allergic animals (Tulic et al., 2002;
Rodriguez et al., 2003).

In my studies, OVA-LPS mice had attenuated AHR to

methacholine compared to OVA and LPS mice alone.

This attenuation was

accompanied by an increase in iNOS and Arg-1. In the study by Rodriguez et al, NOS2
deficient animals did not have an attenuation in AHR when exposed to LPS (Rodriguez
161

et al., 2003). These data support a role for iNOS modulation of AHR in my study. Also,
the data suggest a more novel role for arginase in the induction of AHR. In this study,
AHR in OVA-LPS mice was not associated with increased Arg1 expression, but rather
decrease expression in the lung.
The many roles of NO in asthma pathogenesis are complex.

NO has been

associated with enhanced inflammation and promotion of the pathological features of
asthma, such as mucus hypersecretion, edema, and increased Th2 mediated
inflammation (Prado et al., 2011).

However, NO is a biological mediator of

bronchodilation and produces anti-inflammatory effects ((Maarsingh et al., 2011). It has
been suggested that NO can inhibit the transcription factor NF-κB. In my investigations,
NF-κB inhibition reversed the attenuation in AHR in OVA-LPS mice (Chapter 4) and
attenuated the increase in iNOS and Arg-1 in these mice.

The increase in iNOS

suggests that LPS enhances the production of NO in allergic mice. These results are in
accordance with previous studies that have shown NO to decrease methacholine
induced AHR in asthmatics (Kacmarek et al., 1996) and reversed methacholine
bronchoconstriction in dogs

(Brown et al., 1994).

These findings support my

hypothesis that NO may contribute to the attenuation of AHR in OVA-LPS mice.
Since iNOS is regulated predominately at the transcriptional level by NF-κB, de
novo synthesis is required for gene activation and protein production (Korhonen et al.,
2005). Increased iNOS expression has been demonstrated in smooth muscle, airway
epithelium and inflammatory cells such as macrophages and neutrophils. Evaluation of
iNOS expression in lung tissue by immunohistochemistry revealed that OVA increase
162

expression of iNOS predominately in epithelial cells of smaller peripheral airways and
terminal bronchioles, while only ciliated epithelial cells were positive in the axial airway.
OVA-LPS mice displayed a similar pattern of iNOS expression as OVA in alone mice
with an additional increase in iNOS positive staining inflammatory cells consistent with
macrophages and neutrophils in alveolar parenchyma and airspaces. Previous studies
have demonstrated similar staining patterns for iNOS with OVA sensitization and
challenge (Trifilieff et al., 2000; Zimmermann et al., 2003). NF-κB inhibition did not alter
positive iNOS staining in OVA or OVA-LPS mice.

163

5.6

SUMMARY
At 72 hours after OVA challenge, there was not an upregulation of Nos2

expression in the lung, however positive staining with anti-iNOS antibody was present in
peripheral airway and terminal bronchial epithelium. Exposure to LPS augmented this
iNOS expression in allergic mice with positive staining of cells in areas of inflammation
that were predominately macrophages and polymorphonuclear cells. NF-κB inhibition
attenuated the increase in Nos2 expression in OVA-LPS mice and lessened staining
intensity of iNOS.

Evaluation of Arg1 expression showed that OVA increased

expression which was augmented with LPS exposure in OVA-LPS mice.

NF-κB

inhibition also attenuated this increase in OVA-LPS mice, but did not affect OVA mice
alone.

These

results

support

my

hypothesis

that

attenuation

of

airway

hyperresponsiveness by LPS in asthmatic mice is dependent on Nos2 expression. This
increased Nos2 may contribute to increase nitric oxide production in the lungs resulting
in bronchodilation.

This study contributes to body of research that supports the

potential use of NO as pharmacological modulator of asthma. However, it is important
to confirm these findings by quantifying iNOS protein and determining NO production in
lung, which was not be determined in this study.

Additional studies should be

conducted to address the limitations in this study involving iNOS and Arg-1 protein
quantification and confirming NO production in OVA-LPS mice.

Although iNOS

expression was reduced by NF-κB inhibition, further analysis of direct iNOS inhibition
should be conducted to exclude the contribution of modification of cytokines or proteins,
such as Arg-1, to the effects seen in my model.

164

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.

170

CHAPTER 6

SUMMARY AND CONCLUSIONS

171

Effective treatment of asthma includes diminishing symptoms, improving lung
function and preventing the number and severity of exacerbations (Johnston and Sears,
2006; Reddel et al., 2009). Asthma exacerbation increases health risks to asthmatics,
places a significant burden on the healthcare system, and contributes to large financial
costs; therefore, it is considered to be an important clinical outcome (Reddel et al.,
2009). Recent research suggests that induction of TLR-4, NF-κB, TGF-β, and genes
associated with airway remodeling are critical for PM-mediated responses in allergic
airways (Wang et al., 2008; Heidenfelder et al., 2009; Vawda et al., 2014). As an
activator of TLR-4 and NF-κB LPS is a potential model of exacerbation of an asthmatic
lung by environmental triggers, such as PM and ozone.

Epidemiological and

experimental studies have shown that inhaled LPS can modulate the asthmatic
response (Eldridge and Peden, 2000; Bennett et al., 2013). In this work, I explored the
effects of inhaled LPS exposure on established allergic airways disease to identify the
cellular and molecular mechanisms whereby airway exposure to LPS modulates
asthma; and to test my hypothesis that attenuation of AHR by LPS in asthmatic mice is
dependent on the induction of Nos2 that is associated with the recruitment of
neutrophils.
Following airway LPS exposure in non-allergic mice, I documented a significant
increase in neutrophils in the BALF and histological examination of the lungs showed
increased neutrophils infiltrating into peribronchiolar and perivascular regions with
extension into alveolar septa and airspaces. Also, LPS significantly increased AHR to
methacholine.

By comparison, sensitization and challenge to OVA caused airway

epithelial remodeling characterized by mucous cell metaplasia and hypertrophy and
172

produced peribronchiolar and perivascular inflammation comprised of predominately
lymphocytes with large numbers of eosinophils and macrophages, and fewer
neutrophils. Additionally, OVA increased AHR. In allergic mice, LPS exacerbated the
inflammatory response by causing a significant increase in neutrophils along with
elevations in eosinophils and lymphocytes.

Histologically, OVA-LPS mice had

increased peribronchiolar and perivascular inflammation along with increased
infiltrations of inflammatory cells, predominately neutrophils, within the alveolar septa
and in airspaces. Surprisingly, OVA-LPS mice had an attenuation of AHR compared to
both LPS and OVA groups.

In addition, OVA-LPS mice had increased mRNA

expression of Nos2, a gene that induces iNOS protein expression to produce NO, a
bronchodilator.
Reports have shown that airway challenge with OVA or LPS causes AHR that is
associated with inflammation (Gueders et al., 2009; Hakansson et al., 2012). My results
are similar in that AHR was detected at time points near maximal inflammatory cell
infiltration, 72 hours after OVA challenge and 24 hours following LPS exposure (Faffe
et al., 2000; Lommatzsch et al., 2006). Conversely, there are studies that indicate
dissociation between inflammation and AHR (Crimi et al., 1998; Tournoy et al., 2000).
Also, my findings are in accordance with these studies because OVA-LPS mice had
greater lung inflammation but their airways exhibited a decrease in reactivity to
methacholine compared to OVA and LPS mice.

Inhaled LPS can induce a Th1

response characterized by increased neutrophils, IFN-γ production and decreased
mucus secretion in allergic mice (Eisenbarth et al., 2002; Kim et al., 2007; Dong et al.,
2009). Similar findings were present in my study in which there was increased
173

pulmonary expression of IFN-γ and IL-10 that coincided with decrease expression of IL13 in OVA-LPS mice.

This result suggests that LPS may down-regulate the Th2

inflammatory pathway.
Neutrophil derived mediators, such as elastase and IL-17, have been associated
with AHR (Kudo et al., 2012; Koga et al., 2013). However, a recent study suggests that
neutrophils contribute to the attenuation of AHR in allergic airways (Aeffner and Davis,
2012). The influx of neutrophils was a prominent change in the lungs of OVA-LPS mice
in my study.

Therefore, I proposed that that the influx of neutrophils potentially

contributed to the attenuation of AHR. Depletion of neutrophils significantly reduced the
numbers of BALF neutrophils, along with reducing eosinophils and lymphocytes.
However, depleting neutrophils did not affect AHR in mice. This result suggests that
neither neutrophils nor eosinophils are needed for the development of AHR. This lack
of association between inflammation and AHR has been noted in prior works (Tournoy
et al., 2000; Janssen-Heininger et al., 2012).
NF-κB is an important regulator of the immune response. Its activation has been
associated with adverse airway inflammatory and hyperreactivity responses to both
allergen and LPS-stimulation (Pantano et al., 2008; Sheller et al., 2009). Inhibition of
NF-κB has been shown to reduce eosinophil recruitment, Th2 cytokine production,
mucous cell metaplasia, AHR, and neutrophil recruitment in murine models (Desmet et
al., 2004; Kim et al., 2007; Bao et al., 2009). Based on the implications that NF-κB
activation occurs during both asthma and acute LPS exposure and my findings of
increased Nos2 expression, I hypothesized that NF-κB was involved in attenuation of
AHR in OVA-LPS mice.
174

In my study, inhibition of NF- κB had no effect on LPS-stimulated neutrophil
recruitment, and led to an increase in AHR. These changes in airway physiology were
associated with decreases in airway macrophages, eosinophils and lymphocytes. The
reduction in airway inflammation, particularly eosinophils, is consistent with other OVA
models in which NF-κB has been inhibited (Poynter et al., 2004). In that study, NF- κB
repression did not alter AHR in OVA mice. This finding is consistent with my results, as
inhibition of NF-κB did not affect AHR in OVA mice. In my OVA-LPS model, there are
two pathways of activation of NF-κB, which has been shown to play an important role in
pathogenesis of asthma and development of AHR (Stacey et al., 1997). However, the
data show a disparity between inflammation and AHR associated with asthma resulting
in increased inflammation while decreasing AHR.

These results demonstrate the

complex nature of the immune system.
NO deficiency in the lung is a proposed mechanism of asthma pathogenesis
(Meurs et al., 2002; Larsson et al., 2005). Biologically, NO is a bronchodilator, however,
there have been many reports of its detrimental effects in asthma.
appear to be dose and cell specific.

These effects

High NO concentrations cause vasodilation with

edema, mucus hypersecretion, and enhanced Th2 inflammation contributing to asthma
pathology. Additionally, high concentrations of NO can interact with reactive oxygen
species to produce peroxynitrite causing tissue damage and potentially contributing to
AHR (SadeghiHashjin et al., 1996; Saleh et al., 1998).

Since OVA-LPS mice had

increase expression of Nos2, I tested the hypothesis that increased Nos2 expression
contributed to the attenuation of AHR.

175

Analysis of mRNA expression for Nos2 showed an upregulation of this gene in
OVA-LPS mice but not OVA or LPS alone.

However when NF-κB was inhibited,

expression of Nos2 was significantly decreased in OVA-LPS mice. This decreased
expression was accompanied by the reversal of attenuation of AHR. These findings
suggest that Nos2 expression contributed to the attenuation of AHR. Additionally, I
analyzed the expression of Arg1 which induces arginase-1 enzyme that regulates the
functional activity of iNOS enzyme. The Arg-1 competes with iNOS for substrate Larginine, metabolism. Arg1 expression was increased in OVA-LPS mice. It has been
previously demonstrated that LPS can induce expression of both Nos2 and Arg1
(Sonoki et al., 1997). The mechanism and function of LPS-induced arginase induction is
not completely known.

In vitro studies have indicated that MAPK phosphatase 1

switches L-arginine metabolism from NO to ornithine, a metabolite of Arg-1, following
LPS stimulation and may contribute to the down regulation of inflammation
al., 2007).

(Nelin et

Increased expression of iNOS suggests an increase in NO production.

Additional studies which focus on direct function of iNOS using NOS inhibition should be
conducted to confirm that increased iNOS activity is needed for the attenuation of AHR.
Also, quantification of NO should be measured to verify that increased NO production is
the mediator of attenuation of AHR in OVA-LPS mice.
Although induction of iNOS appears to be a very promising mechanism of LPSassociated attenuation of AHR, there are other plausible pathways that can contribute to
this phenomenon. The physical attributes of the allergic airways resulting in reduced
caliber may contribute to this finding.

Undoubtedly, LPS exposure can change the

inflammatory milieu switching it from a Th2 to Th1 mediated response which could
176

attenuate AHR. Furthermore, LPS decreased IL-13 cytokine in BALF and expression in
the lung, as well as, downregulating the expression of Gob5 and Muc5ac which are all
associated with mucin secretion. These findings are consistent with the decreased
volume density of intraepithelial mucosubstances in OVA-LPS mice. Inhibition of mucus
secretion was found to be protective against AHR allergic in mice (Agrawal et al., 2007).
Another possibility is inflammation and airways of allergic mice may respond differently
to LPS. TNF- induced by L S has associated with AHR development

(Choi et al.,

2005). It has been demonstrated that monocytes from asthmatics produce less TNFwhen stimulated by fungi than monocytes from healthy individuals (Johannessen et al.,
2008). My results showed that OVA-LPS mice had less concentrations of TNF-

in

BALF than LPS mice, and therefore, may contribute to diminished AHR.
These results clearly show that the attenuation of AHR in allergic mice is
associated with the upregulation of both Nos2 and Arg1 following inhaled LPS exposure
(24 hours), and that this expression is associated with activated NF-κB (Table 2).
Increased expression of Nos2 and the presence of iNOS protein suggest that NO
production is increased in OVA-LPS mice; however, NO production was not determined
in these studies nor was iNOS protein quantified. Therefore, increased NO production
was not confirmed as the mediator of attenuation of AHR in OVA-LPS mice. Additional
factors that could be addressed include a time course evaluation of LPS post exposure
in allergic mice to determine if attenuation of AHR occurs prior to or extends past 24
hour post exposure to LPS.

Also, lymphocytes are particularly important in the

pathogenesis allergic asthma, for this reason it is important to determine if lymphocyte
derived mediators could contribute to this attenuation in AHR. Other limiting factors
177

include the lack of specificity of the antibody used to deplete neutrophils. The loss of
these non-neutrophil cells could affect AHR. Lastly,

complete NF-κB inhibition could

not be confirmed. Treatment with the inhibitor compound undoubtedly affected airway
inflammation and AHR, however, its effects on proteasomal inhibition needs further
assessment.
Results from this study demonstrate that severity of airway inflammation does not
affect the severity of AHR.

Additionally, neutrophils were not associated with the

inhibition of AHR in OVA-LPS mice. NO can play multiple roles in pulmonary disease.
My results suggest that NO can be beneficial to physiological function in allergic airway
disease as depicted in Figure 24. To briefly summarize the mechanism of attenuation of
AHR in OVA-LPS mice, I propose that 1) the effects of LPS could be enhanced by
increased TLR4 signaling promoted by OVA (Rodriguez et al., 2003).

2) NF-κB

activation is increased by both OVA- induced allergic airway disease and inhaled LPS
which 3) promotes iNOS activity and 4) increases NO production resulting in the
inhibition of AHR induced by allergic airway disease.

178

Table 2. Summary of changes in airway hyperresponsiveness in OVA-LPS mice.
The attenuation of AHR in OVA-LPS mice was influence by several factors. LPS
exposure produces an attenuation of AHR in allergic mice. Severe lung inflammation
was present in central airways and peripheral tissue; however, this was associated with
a less responsive phenotype to methacholine. There was no change in AHR when
neutrophils were depleted. AHR significantly increased following NF-κB inhibition.
Reversal of AHR attenuation was associated with decreased expression of Nos2 and
Arg1.

179

Figure 24. Proposed mechanism for attenuation of AHR following LPS exposure
in allergic mice. In allergic mice, OVA induce the expression of Arg-1 resulting in
airway remodeling and AHR. Exposure of allergic airways to LPS leads to NF-κB
activation, and a preferential increase of iNOS, and less expression of Arg-1. The role
of Arg-1 is unknown, but may regulate iNOS activity through inhibition. NOS
metabolism of arginine produces NO and citrulline. Arginine can be produced from
recycling citrulline, increasing its bioavailability for NOS metabolism. Increased
bronchoprotective NO is produced which inhibits AHR.

180

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