Human Rhinovirus Infection of Airway Epithelial Cells Modulates Airway Smooth Muscle Migration

University of Calgary PRISM: University of Calgary's Digital Repository

Graduate Studies The Vault: Electronic Theses and Dissertations

2015-12-22 Human Rhinovirus Infection of Airway Epithelial Cells Modulates Airway Smooth Muscle Migration

Shariff, Sami

Shariff, S. (2015). Human Rhinovirus Infection of Airway Epithelial Cells Modulates Airway Smooth Muscle Migration (Unpublished master's thesis). University of Calgary, Calgary, AB. doi:10.11575/PRISM/26398 http://hdl.handle.net/11023/2699 master thesis

University of Calgary graduate students retain copyright ownership and moral rights for their thesis. You may use this material in any way that is permitted by the Copyright Act or through licensing that has been assigned to the document. For uses that are not allowable under copyright legislation or licensing, you are required to seek permission. Downloaded from PRISM: https://prism.ucalgary.ca UNIVERSITY OF CALGARY

Human Rhinovirus Infection of Airway Epithelial Cells Modulates Airway Smooth Muscle

Migration

Sami Shariff

A THESIS

SUBMITTED TO THE FACULTY OF GRADUATE STUDIES

IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE

DEGREE OF MASTER OF SCIENCE

GRADUATE PROGRAM IN CARDIOVASCULAR AND RESPIRATORY PHYSIOLOGY

CALGARY, ALBERTA

DECEMBER, 2015

The traditional paradigm of airway remodeling in asthma has held that remodeling occurs after many years of chronic inflammation. However, studies have confirmed that remodeling changes are observed in children even before the clinical diagnosis of asthma is established.

There is now robust evidence to indicate that children with recurrent human rhinovirus

(HRV)-induced wheezing episodes are at significantly increased risk of developing subsequent asthma. A feature of airway remodeling is increased airway smooth muscle

(ASM) mass with a greater proximity of the ASM to the subepithelial region, and we interrogated the hypothesis that HRV-induced alterations of airway epithelial cell biology might regulate ASM migration. We demonstrated that ASM chemotaxis is GPCR dependent, can be regulated by cAMP and is dependent upon CCL5 release by the epithelium post-HRV infection. These observations substantiate the growing body of evidence that links HRV infections to the subsequent development of asthma.

ii Acknowledgements

Undertaking this graduate project, moving back to Calgary, and all the changes it has brought would not have been possible if Richard had not allowed me to become his student after just one meeting with me. Your trust and belief in me has allowed me to overcome the challenges

I faced throughout my degree and I cannot say thank you enough.

To both Richard and David, you have been mentors and idols for me and have lit the pathway forward for several years. Thank you for all your support, guidance, and criticisms, because without them I would not be who I am today. It truly has been an honor to work with such outstanding scientists and I hope to be as successful in my future endeavors as you are currently in yours. To my committee members, thank you for your time and your input.

To all the members of the Leigh, Proud, Newton, Giembycz and Kelly labs: thank you. Your encouragement, advice, guidance, and support have made these past few years fly past.

Shahina, thank you for being like an older sister and both pushing me and supporting me to achieve greater heights. Suzanne and Cora, thank you for your expertise and knowledge that made overcoming every challenge easier. Sergei, Chris, and Jason, thank you for being the best of friends and colleagues, and for providing consistent boosts to my morale.

Lastly, thank you to my parents, my siblings, and my wife Aleena for understanding the challenges I faced and providing me with overwhelming support. I could not have made it here without your love. I hope to keep making you proud.

iii Table of Contents

Abstract ...... ii

Acknowledgements ...... iii

Table of Contents ...... iv

List of Tables ...... viii

List of Figures and Illustrations ...... ix

1 CHAPTER ONE: INTRODUCTION ...... 1

1.1 Executive Summary ...... 2

1.2 Literature review ...... 3

1.3 Characteristics and definition of asthma ...... 4

1.4 Airway Hyperresponsiveness ...... 5

1.5 Airway Inflammation in Asthma ...... 8

1.6 Airway Remodeling ...... 9

1.7 Origins of Airway Remodeling ...... 11

1.8 Human Rhinovirus ...... 13

1.9 The Airways ...... 17

1.10 Role of the epithelium in asthma ...... 21

1.11 HRV Infection of the epithelium ...... 22

1.12 The Airway Smooth Muscle ...... 30

1.13 Thesis Objectives ...... 40

2 CHAPTER TWO: MATERIALS AND METHODS ...... 41

2.1 Materials ...... 42

2.2 Isolation of HBE and ASM from Human Lung Tissue ...... 44

iv 2.3 HRV-16 ...... 46

2.4 HRV-16 Infection of Bronchial Epithelial Cells ...... 48

2.5.1 GPCR Array ...... 50

2.6 Fluorescence-Activated Cell Sorting (FACS) Analysis ...... 51

2.7 Enzyme Linked Immunosorbent Assay (ELISA) ...... 53

2.7.1 Measurement of CCL5 and CXCL10 protein release ...... 53

2.7.2 Measurement of CXCL8 protein release ...... 54

2.8 Multiplex Chemokine Array Assay (64-Plex) ...... 56

2.9 Filtration of HBE cell supernatants ...... 60

2.10 Migration of ASM ...... 60

2.10.1 Migration in the micro chemotaxis chamber ...... 60

2.10.2 Chemotaxis via the xCELLigence Real Time Cell Analyzer (RTCA) system ...... 62

2.10.3 Determination of chemotaxis versus chemokinesis ...... 68

2.10.4 ASM pertussis toxin treatment ...... 68

2.10.5 β-agonist treatment of ASM ...... 69

2.10.6 ASM treatment with adenylyl cyclase agonist ...... 69

2.10.7 ASM treatment with 8-Br-adenosine 3′, 5′-cyclic monophosphate (cAMP) ...... 70

2.10.8 CCL5 inhibition in conditioned medium ...... 71

2.10.9 Quantification of cell viability ...... 72

2.11 Data management ...... 73

3 CHAPTER THREE: RHINOVIRUS INFECTION OF THE AIRWAY EPITHELIAL CELLS

DRIVES CHEMOTAXIS OF AIRWAY SMOOTH MUSCLE CELLS ...... 74

3.1 Introduction ...... 75

3.2 Hypothesis ...... 76

3.3 Results ...... 76

v 3.3.1 Establishing a positive migratory stimulus ...... 76

3.3.2 Conditioned medium from HRV infected HBE cells drives ASM migration ...... 78

3.3.3 Abolishing the chemotactic gradient attenuates ASM migration ...... 80

3.3.4 Presence of viral capsid is not sufficient for migration ...... 82

3.3.5 Migration is dependent on duration of infection ...... 85

3.3.6 Viral infection of HBE cells is necessary to drive migration ...... 87

3.4 Discussion ...... 89

4 CHAPTER FOUR: HRV-16 INDUCED, HBE CELL MEDIATED, MIGRATION OF ASM

CELLS IS REGULATED BY G-PROTEIN COUPLED RECEPTORS, AND DEPENDENT ON

CCL5 ...... 92

4.1 Introduction ...... 93

4.2 Hypothesis ...... 94

4.3 Results ...... 94

4.3.1 Pertussis toxin treatment prevents ASM chemotaxis to HRV CM ...... 94

4.3.2 Stimulation of the Gαs pathway via formoterol attenuates migration ...... 97

4.3.3 Direct stimulation of adenylyl cyclase attenuates ASM cell migration ...... 99

4.3.4 An analogue of cAMP is able to abolish migration ...... 101

4.3.5 Identification of chemokines that facilitate HRV-induced ASM cell migration ...... 103

4.3.6 GPCR mRNA analysis suggests that CCL5 (RANTES) may be an important chemokine

for HRV-induced ASM chemotaxis ...... 108

4.3.7 Flow cytometry data indicate the presence of several chemokine receptors ...... 110

4.3.8 HRV-induced ASM migration is mediated, at least in part, via the chemokine CCL5 ... 113

4.3.9 Inhibition of soluble CCL5 in HRV infected HBE cell supernatants attenuates ASM cell

chemotaxis ...... 116

vi 4.3.10 HRV-infection of HBE cells obtained from asthmatic subjects results in significantly

greater ASM chemotaxis compared to healthy controls ...... 118

4.4 Discussion ...... 121

5 CHAPTER FIVE: DISCUSSION, CLINICAL RELEVANCE & FUTURE DIRECTIONS ... 125

5.1 General Discussion and Future Work ...... 126

5.2 Limitations ...... 130

5.3 Clinical relevance ...... 133

5.4 Future directions ...... 135

5.5 Conclusion ...... 136

6 CHAPTER SIX: REFERENCES ...... 139

7 CHAPTER SEVEN: APPENDIX ...... 169

vii List of Tables

Table 1.1: Expression of cytokines, growth factors, and chemokines produced by the airway epithelium following HRV infection...... 27

Table 1.2: Summary of agents that modulate ASM migration...... 35

Table 2.1: Antibodies for FACS Analysis ...... 52

Table 2.2: Conditions for FACS Analysis ...... 52

Table 2.3: Multiplex chemokine assay targets ...... 56

Table 2.4: Comparison between MCC and RTCA systems ...... 67

Table 4.1: Results of a 64-chemokine multiplex assay of supernatants of HBE cells post treatment with medium control or HRV-16 infection...... 104

Table 4.3: Characteristics of patients from whom brushings of epithelial cells were obtained...... 119

Table 7.1: All targets of 384-gene GPCR array conducted on 4 different ASM donors...... 170

viii List of Figures and Illustrations

Figure 1.1: Dose-response curves to inhaled direct agonists in non-asthmatic, mild- asthmatic, and severe asthmatic subjects...... 7

Figure 1.2: Asthmatic airways undergo significant structural remodeling...... 10

Figure 1.3: Organization of the HRV genome ...... 15

Figure 1.5: Structural components of epithelial cell-cell and cell-substratum junctions...... 20

Figure 1.6: The pattern recognition receptor (PRR) based response of the epithelium to

HRV infection ...... 25

Figure 1.7: Pathogens associated with virally induced exacerbations of asthma in children

(age 9-11)...... 29

Figure 1.8: Migration of smooth muscle cells...... 37

Figure 1.9: Signaling pathways regulating muscle migration...... 38

Figure 2.1: The Neuro Probe AP48 48-well micro chemotaxis chamber (MCC)...... 62

Figure 2.2: The xCELLigence Real-Time Cell Analyzer (RTCA) system...... 65

Figure 2.3: Migration of ASM cells to PDGF-AB in the RTCA system...... 66

Figure 3.1: Determination of the experimental parameters for ASM migration...... 77

Figure 3.2: Conditioned Medium from HRV infected HBE cells drives ASM migration...... 79

Figure 3.3: ASM migration to conditioned medium from HRV infected HBE cells is chemotactic and directional...... 81

ix Figure 3.4: CXCL8 levels post-filtration of conditioned medium from medium treated (CM) or HRV-16 infected (HRV CM) HBE cells...... 83

Figure 3.5: Removal of virion from conditioned medium from HRV-16 infected HBE cells does not attenuate migration...... 84

Figure 3.6: The ability of conditioned medium from HRV infected epithelial cells to drive

ASM chemotaxis depends upon the duration of infection...... 86

Figure 3.7: Replication deficient HRV-16 is unable to drive a response in HBE cells that stimulates ASM chemotaxis...... 88

Figure 4.1: HRV induced ASM migration is primarily GPCR mediated...... 96

Figure 4.2: β-agonist treatment abolishes the ability of HRV CM to drive ASM chemotaxis. 98

Figure 4.3: Direct activation of adenylyl cyclase inhibits ASM migration to HRV CM...... 100

Figure 4.4: Pre-treatment with a cAMP analogue is able to significantly attenuate ASM cell chemotaxis...... 102

Figure 4.5: Highly up-regulated chemokines post-HRV infection...... 107

Figure 4.6: A 384-panel GPCR array highlights some important chemokine receptors that may be present, based on mRNA-based detection...... 110

Figure 4.7: Flow cytometry analysis of ASM donor cells indicates the presence of several different chemokine receptors on ASM cell membrane surfaces...... 112

Figure 4.8: CCL5 is able to drive ASM migration comparable to HRV CM...... 115

x Figure 4.9: Blockade of soluble CCL5 via an anti-CCL5 antibody results in a significant attenuation of ASM chemotaxis...... 117

Figure 4.10: Epithelial cells obtained from brushings from asthmatic individuals induce significantly greater ASM chemotaxis as compared to healthy controls...... 120

Figure 5.1: Proposed schematic indicating the novel mechanism uncovered in this thesis, by which HRV-16 infection of the airway epithelium results in chemotaxis of ASM cells...... 138

xi List of Symbols, Abbreviations, and Nomenclature

Symbol Definition

8-Br-cAMP 8-bromo-adenosine-3′, 5′-cyclic monophosphate

ABTS 2,2’-azino-bis (3-ethylbenzthiazoline-6-sulphonic acid)

AC Adenylyl Cyclase

AHR Airway Hyperresponsiveness

AJ Adherens junctions

ANOVA Analysis of variance

AP-1 Activator Protein 1

ARP Actin-related protein

ASM Airway smooth muscle

BEBM Bronchial epithelial basal medium

BEGM Bronchial epithelial growth medium cAMP Cyclic adenosine 3′, 5′-monophosphate

CCR CC chemokine-receptor

CD Cluster of differentiation

CDHR3 Cadherin-related family member 3

CIM Cellular invasion and migration

CL Cloverleaf-like domain

CM Conditioned medium (supernatants from medium treated HBE cells)

COAST Childhood Origin of Asthma

CT Cycle threshold

DC Dendritic cells

xii DMEM Dulbecco’s modified eagle medium

DMSO Dimethyl sulfoxide dsRNA Double stranded ribonucleic acid

DTT Dithiothreitol

ECM Extracellular matrix

ELISA Enzyme linked immunosorbent assay

F12 Ham’s F12 medium

FAK Focal adhesion kinase

FBS Fetal bovine serum

FEV1 Forced expiratory volume in 1 second

FSC Forward scatter

FVC Forced vital capacity

GINA Global Initiative for Asthma

GPCR G-protein coupled receptors

GRO Growth related oncogene

HBE Human bronchial epithelial cells

HBSS Hanks’ Balance Salt Solution

HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid

HRV Human Rhinovirus

HRV CM Conditioned medium from HBE cells collected post-human rhinovirus

infection

ICAM-1 Intercellular adhesion molecule 1

ICS Inhaled corticosteroid

xiii IFN Interferon

IL Interleukin

IRES Internal ribosome entry site

IRF Interferon-response factor

JAM Junctional adhesion molecule

LDLR Low-density lipoprotein receptor

MAPK Mitogen-activated protein kinase

MCC Micro chemotaxis chamber

MDA-5 Melanoma differentiation-associated gene 5

MEM Minimum Essential Media

MHC Major histocompatibility complex

MLCK Myosin light chain kinase

MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

MW Molecular weight

NCR Non-coding region

NF-κB Nuclear Factor κB

No-HC Medium without hydrocortisone

ODU Optical density units

PBS Phosphate buffered saline

PDGF Platelet derived growth factor

PDGFR- β Platelet derived growth factor receptor β

PET Polyethylene terephthalate

PI3K Phosphoinositide 3-kinase

xiv PIP2 Phosphatidylinositol 4,5 bisphosphate

PKR Protein kinase R

PRR Pattern recognition receptors

PSF Penicillin-streptomycin-amphotericin B

PTX Pertussis toxin

RANTES Regulated upon activation, normal T-cell expressed and secreted (CCL5)

RIG-I Retinoic acid-inducible gene I

RLR Retinoic acid-inducible gene I like receptors

RMLC Regulatory myosin light chains

RNA Ribonucleic acid

ROCK Rho-activated protein kinase

RSV Respiratory syncytial virus

RT Room temperature

RT-PCR Reverse Transcription Polymerase Chain Reaction

RTCA xCELLigence real-time cell analyzer

RTK Receptor tyrosine kinase

SD Standard deviation

Src Sarcoma tyrosine kinase

SSC Side scatter ssRNA Single-stranded ribonucleic acid

TBS Tris-buffered saline

TCID50 50% tissue culture infectious dose

TGF Transforming growth factor

xv TH (TH) T helper lymphocyte

TIR Toll/Interleukin-1 receptor domain

TJ Tight junctions

TLR Toll-like receptor

TNF Tumor necrosis factor

TRIF Toll/Interleukin-1 receptor domain containing adaptor inducing

interferon-β

UV-HRV Ultraviolet light inactivated human rhinovirus

VEGF Vascular endothelial growth factor

VP Viral protein

WASP Wiskott-Aldrich syndrome proteins

WAVE Wiskott-Aldrich syndrome protein-family verprolin-homologous protein

complex

xvi

1 CHAPTER ONE: INTRODUCTION

1.1 Executive Summary

The origins of the episodic airway dysfunction, termed airway hyperresponsiveness (AHR), that is the hallmark of asthma remains poorly understood. The traditional viewpoint has held that this AHR arises from chronic airway inflammation that, over time, results in airway remodeling. However, recent studies have established that aspects of remodeling are present in young pre-school children, often prior to the clinical diagnosis of asthma, suggesting that something other than longstanding chronic inflammation may be responsible for the pathogenesis of AHR. Long-term birth cohort studies have provided robust evidence that virally induced wheezing episodes – particularly those caused by human rhinovirus (HRV) infections – significantly increase the risk for the subsequent development of asthma. This has led our laboratory to hypothesize that HRV infection plays an important role in the pathogenesis of airway remodeling in asthma. Moreover, as HRV primarily infects the airway epithelium, it stands to reason that the epithelium is most likely a key mediator in this process. Changes in airway smooth muscle morphology and physiology are among the most obvious differentiating factors between asthmatic patients and healthy individuals, and several groups have noticed an increased proximity of the airway smooth muscle mass to the epithelium in asthma. Thus, the program of research undertaken in partial fulfilment of this Masters’ thesis sought to explore the role that

HRV infection of the airway epithelium plays in modulating airway smooth muscle migration.

1.2 Literature review

I conducted a comprehensive literature review of the published research literature in the areas of airway remodeling and smooth muscle migration to ensure that the work contained in this thesis was based on sound scientific principles, would be scientifically and clinically relevant, and that it would provide novel insights into the pathogenesis of airway remodeling in asthma. A thorough understanding of how to perform this procedure was facilitated through a meeting with an experienced research librarian from the University of

Calgary medical sciences library. The PubMed and MEDLINE (OVID) databases were searched using AND/OR phrases and multiple combinations of the following keywords: smooth muscle; involuntary muscle; muscle cell; cell movement; cell motility; cell locomotion; cell migration; migration; chemotaxis; lung; airway; and airway remodeling. In addition, I conducted hand searches and reviewed bibliographies of identified papers. The search was limited to English language papers published between January 2000 and May 2015, dealing specifically with lung biology. The original search identified over 590 papers, commentaries, and reviews of the literature. To address my specific research focus, I excluded all papers that did not describe research primarily examining smooth muscle migration and excluded papers focusing on fibroblast migration. This resulted in the identification of approximately

200 peer-reviewed publications relevant to this thesis project.

1.3 Characteristics and definition of asthma

Asthma is a respiratory disorder primarily composed of episodic airway dysfunction, termed airway hyperresponsiveness (AHR), and is characterized by symptoms such as breathlessness, chest tightness, wheezing, sputum production, and cough. The root cause of these symptoms is bronchoconstriction, secondary to AHR and airway remodeling. Although asthma is a heterogeneous disease that is difficult to define due to a lack of understanding surrounding its pathogenesis, in 2010 the Global Initiative for Asthma (GINA) characterized it as follows1:

“Asthma is a chronic inflammatory disorder of the airways in which many cells and cellular elements play a role. The chronic inflammation is associated with airway hyperresponsiveness that leads to recurrent episodes of wheezing, breathlessness, chest tightness, and coughing, particularly at night or in the early morning. These episodes are usually associated with widespread, but variable, airflow obstruction within the lung that is often reversible either spontaneously or with treatment.”

Asthma is one of the most common diseases worldwide, and it is the most common chronic disease in both children and young adults2,3. Studies have indicated that nearly 10% of

Canadian adolescents and adults, and over 15% of Canadian children have asthma4. Current estimations suggest that there are currently around 300 million people worldwide that have been diagnosed with asthma, with the prevalence projected to increase by an additional 100 million subjects by 20255. The Canadian economic burden associated with the disease in

2010 was estimated to be $2.21 billion, with estimates predicting a doubling in these costs by 20306. With global population numbers projected to rise in the future, it is evident that the economic burden associated with asthma will increase as well5. Moreover, studies from

GINA have found that 1 in 250 deaths worldwide are due to asthma5.

1.4 Airway Hyperresponsiveness

Episodic airway dysfunction has long been recognized to be a characteristic feature of asthma. Henry Hyde-Salter in 1859 described the presence of variable airflow limitation in asthma as, “paroxysmal dyspnea of a peculiar character, generally periodic, with intervals of healthy respiration between attacks”7. A little over 60 years later, Alexander and Paddock demonstrated that a cholinergic agonist pilocarpine8 was able to drive variable airflow obstruction more readily in asthmatic patients as compared to healthy controls. In 1932

Weiss et al. corroborated these data by finding that asthmatic subjects, but not healthy controls, responded to intravenous histamine with bronchoconstriction8,9. However, the concept of AHR was not introduced until the 1940s when Curry10 and Tiffeneau11 provided descriptions of direct bronchial challenge testing using threshold doses of inhaled acetylcholine, histamine, or methacholine to determine the degree of airway responsiveness in asthmatic individuals. In addition, Curry was the first to demonstrate that the severity of disease directly correlated with the magnitude of the bronchoconstrictor response in asthmatic subjects10. Working definitions of asthma subsequently emerged that emphasized that variable airflow limitation is the fundamental abnormality underlying the disease12.

Additionally, AHR was highlighted as a central component of asthma and standardized

5 protocols were developed to measure AHR in response to bronchoconstrictor mediators, which have now become routine in the diagnosis of asthma13-16.

The term AHR is now considered to describe the exaggerated narrowing of the airways in response to normally innocuous stimuli. The presence of AHR gives rise to episodic airflow obstruction, which is assessed using spirometry; spirometry measures the forced expiratory volume in one second (FEV1), and the forced vital capacity (FVC). A reduced FEV1/FVC ratio confirms the presence of airflow obstruction, and the change in FEV1 following the inhalation of a short-acting bronchodilator confirms the presence of post-bronchodilator responsiveness (variable airflow obstruction). Airflow obstruction is present if a patient’s

1,17 pre-bronchodilator FEV1/FVC ratio is less 0.7 . If the improvement in FEV1 in a patient post-bronchodilator treatment is ≥12% and 200ml then the reversibility is considered clinically significant, and is consistent with the diagnosis of asthma18. In clinical practice, challenges arise in those patients in whom a diagnosis of asthma is suspected but who have normal/near-normal lung function; in these individuals the presence of AHR can be assessed by a bronchoprovocation test, using methacholine, histamine or other bronchoconstrictive agents, such as AMP or exercise. Methacholine challenge tests are widely used in Canada, and

AHR is deemed to be present if the concentration of methacholine required to produce a 20%

1,17 fall in FEV1 is ≤8 mg/ml . The change in the patient’s FEV1 to the provocative agent can be plotted as a concentration-response curve (Figure 1.1) allowing us to draw several important conclusions.

Figure 1.1: Dose-response curves to inhaled direct agonists in non-asthmatic, mild- asthmatic, and severe asthmatic subjects. FEV1 is measured in response to increasing logarithmic doses of methacholine/histamine. AHR is expressed as the provocative concentration of methacholine or histamine necessary to cause a 20% decrease in FEV1

19 (PC20, dashed line). Adapted from O’Byrne, et al. .

The leftward shift of the differing classes of asthma patients indicates an increased sensitivity to the provocative agent, and is related to disease severity. In addition, increased

(and subsequent loss of) maximal responses are indicated in groups with higher asthma severity19. These data correlate with clinical findings in asthmatic patients in whom increasing asthma severity is associated with 1) an earlier breakpoint, 2) a steeper slope

(indicating increasing hyperreactivity), and 3) the ultimate loss of the plateau that indicates maximal bronchoconstriction.

1.5 Airway Inflammation in Asthma

Chronic airway inflammation is a central component in the pathogenesis of asthma and involves many different cells and mediators. There is now considerable evidence to support the role of immune-mediated airway inflammation in the pathogenesis of asthma20-

23. The disease onset is most likely multifactorial with various risk factors involved, including genetic determinants and environmental influences, such as allergen exposures and viral infections24. Many cases of asthma begin in childhood and there is a close association with allergic diseases, with estimates suggesting that over 90% of asthmatic children have some form of respiratory allergy25. Most often this is facilitated through a selective expansion of

T-helper lymphocyte (TH) type 2 (TH2) lymphocytes that regulate and secrete a series of inflammatory cytokines including the interleukins (IL) IL-3, IL-4, IL-5, IL-6, IL-9 and IL-13.

This TH2-type cytokine response potentiates the allergic inflammatory cascade that is seen in asthma by promoting B cell isotype switching from IgM to IgE synthesis, and subsequent basophil, eosinophil, and mast cell maturation, recruitment, and survival26. In addition, recent evidence points to other TH subsets including TH9, TH17, and T-regulatory cells as also having important roles in modulating the disease phenotype27.

As a result, most of the current pharmacological treatment strategies for asthma have focused on anti-inflammatory actions, and current asthma guidelines1,17,28 emphasize inhaled corticosteroid (ICS) treatment as the cornerstone of asthma therapy. While ICS treatment is highly effective in attenuating the immune-mediated airway inflammation in asthma, numerous studies confirm that ICS treatments are only marginally effective at

8 attenuating AHR in patients29-35. Thus, it can be concluded that underlying airway inflammation is unlikely to account for all of the components of AHR, and suggests that other mechanisms are likely to account for a major component of AHR.

1.6 Airway Remodeling

Although changes in airway wall structure have been recognized as a feature of asthma for over 80 years36,37, Lynne Reid in 1966 was the first to refer to the term airway remodeling with regard to lung development38. However, the concept of airway remodeling in asthma was only proposed as recently as 199239 and was defined as changes in the composition, content, and organization of the cellular and molecular constituents of the airway wall40-44.

Early pathological investigation involving post-mortem samples led to reports that the airways of asthmatic individuals who died from acute severe asthma demonstrated significant structural changes, as compared to the airways of healthy individuals dying of non-natural causes45-52. These structural changes (see Figure 1.2) include thickening of the airway wall, angiogenesis, hyperplasia and hypertrophy of the airway smooth muscle (ASM), increased proximity of the smooth muscle to the epithelium, and goblet cell hyperplasia45,47-49,51,53,54. Although initial reports indicated that these structural changes were secondary to longstanding inflammation and occurred late in the disease cycle, subsequent findings demonstrated that these structural changes were also present (to varying degrees) in the airways of patients with mild asthma43,53-58.

Figure 1.2: Asthmatic airways undergo significant structural remodeling. Medium- sized airways from a normal and a severe asthmatic patient were sectioned and stained using Movat’s pentachrome stain. The epithelium (Ep) in asthma shows mucous hyperplasia and hyper secretion (blue), and significant basement membrane (Bm) thickening. Smooth muscle (Sm) volume is also increased in asthma. Bv = blood vessel. Scale bar = 100μm.

Adapted from Wadsworth, et al. 59.

The functional effects of airway remodeling can be difficult to quantify clinically and, as a result, much of our understanding comes from theoretical mathematical modeling studies42.

These studies predict that thickening of the airway wall, occurring as a result of ASM hypertrophy and hyperplasia, as well as airway wall inflammation and edema, is able to amplify the effect of ASM shortening and thus play a major role in AHR. Moreno and

10 colleagues60 modeled the effect of inner airway wall thickening upon the degree of luminal narrowing for a given degree of smooth muscle shortening. Their findings reported that increasing inner airway wall thickness produced simulated agonist response curves that closely resembled in vivo airway challenges in asthmatic individuals. Since then this mathematical model has been used in more complex and realistic paradigms in several studies, and the findings have confirmed the predictions made by Moreno’s original study

50,61-63. Additional studies predict that increases in ASM mass result in an increased force of contraction and in an increase in the airways’ ability to generate radial stress and airway narrowing63. These findings suggest that increases in ASM mass, which is consistently found in asthmatic patients43,46,52, is likely to be a major determinant of AHR. Although these modeling studies have only evaluated in isolation certain aspects of remodeling, they and other more recent studies64-67 provide convincing evidence that implicates airway remodeling in contributing to AHR by decreasing airway compliance, elastic recoil, and airway caliber.

1.7 Origins of Airway Remodeling

Although asthma can occur and be diagnosed at any age, symptoms usually occur prior to the age of 6 years25,68. Originally this finding led to the conclusion that asthma is likely to originate in early childhood69, and that airway remodeling only occurs after many years of chronic inflammation. More recently, however, bronchial biopsy studies done in symptomatic pre-school children have confirmed that certain characteristics of remodeling are present prior to the clinical diagnosis of asthma being established70-73. It is also known

11 that airway remodeling is not present in infants (≤ 12 months old) with symptoms of airflow limitation, which suggests that remodeling must occur as a consequence of some initiating factor72. As mentioned above, pediatric studies indicate that while ICS treatment is effective at providing symptom relief, there is no evidence that early prophylactic treatment with ICS or other anti-inflammatory agents impacts on the asthmatic disease progression74,75. It is thus likely that an event independent of the allergic, immune-mediated inflammatory process is responsible for the initiation of airway remodeling in asthma.

The most common cause of acute respiratory wheezing illnesses in early childhood are viral infections76,77 and clinical studies have shown that children who develop virally induced wheezing illnesses in the first 2-3 years of life are at significantly increased risk of developing asthma78-81. While this association has now been clearly established and documented, the specific details regarding viral illnesses and their role in asthma pathogenesis are still poorly understood. For instance, although respiratory syncytial virus (RSV) is the most common virus inducing wheezing illnesses in children76,80,81, and while studies indicate that most children develop an RSV infection by age 282, only a minority of these children go on to develop asthma. Indeed, studies have demonstrated that RSV infection is uncommon in children who later develop asthma83-85, suggesting that other viruses may play the role of predisposing children to the subsequent development of asthma. The Childhood Origins of

Asthma (COAST) prospective study86,87 found that human rhinovirus (HRV) infections were the most common cause of infant and childhood respiratory wheezing illnesses (~50% of all viral isolates). The COAST and subsequent studies have confirmed that children who develop

HRV induced wheezing illnesses are at an increased risk of developing subsequent

12 asthma83,84,88,89 with the COAST study reporting that children who had at least 1 moderate- to-severe HRV-associated wheezing illness during infancy had an Odds Ratio of 10 for developing asthma by age 690. This finding has been validated in several subsequent clinical studies84,91 and led us to the hypothesis that HRV infections play a role in the development of airway remodeling and asthma.

1.8 Human Rhinovirus

HRV infections are the predominant cause for the common cold and acute infections in humans92,93. HRV belongs to the picornaviridae family of viruses94 and is a single stranded, positive sense RNA virus. The relatively recent development of sensitive reverse transcription polymerase chain reaction (RT-PCR) assays and other molecular analysis techniques have allowed for a better understanding of HRV pathobiology and has allowed the HRVs to be classified into 3 genetic clades based on their sequence homology. There are thought to be 74 members of the HRV-A clade, 25 members of the HRV-B clade, and at least

11 of the recently discovered HRV-C clade95, though some studies have argued that this could be a gross underestimation96. The mechanism through which clade C viruses may attach to the cell surface was until very recently unknown97. There is evidence that this receptor is distinct from those utilized by clades A and B98, and a recent study has found cadherin- related family member 3 (CDHR3) enables cells to bind and be infected by HRV-C99. HRV clades A and B are further sub-divided on the basis of cell surface receptors utilized for viral entry. Viruses utilizing intercellular adhesion molecule-1 (ICAM-1) belong to the major group of HRV, which consists of more than 90% of clade A and B100-102. Alternatively, the 12

13 known serotype (HRV 1A, 1B, 2, 23, 25, 29, 30, 31, 44, 47, 49, 62) viruses that utilize members of the low-density lipoprotein receptor (LDLR) family belong to the minor group103. Though the specific mechanism for cellular entry by HRV remains to be fully elucidated, studies have suggested that endocytosis may be responsible104.

The HRV genome is 7200 base-pairs in length, and is encapsulated by an icosahedral capsid comprised of 4 viral proteins (VP) designated VP1, VP2, VP3, and VP496,105. The capsid itself has a molecular weight of about 8.5 x 106 Daltons and a diameter of 300 Angstroms106, and is composed of 60 identical copies of each of the four coat VP proteins105. The three larger proteins, VP1, VP2, and VP3 make up the exterior of the capsid, while the smaller VP4 protein lies inside the capsid105. The virion surface has a star shaped plateau possessing a fivefold axis of symmetry surrounded by a canyon. Beneath this canyon floor is a hydrophobic pocket containing a fatty acid known as the fatty acid pocket factor (though this is not present in all

HRV serotypes). In major group HRVs this factor is displaced during viral entry by binding of the capsid to domains 1 and 2 of the ICAM-1 receptor107. Conversely, minor group viruses bind the LDL family receptors near the star shaped plateau108.

The HRV genome is covalently linked at the 5' end to a protein called VPg (virion protein, genome linked)109. Adjacent to the VPg is a 5’-terminal cloverleaf-like motif (CL) that is involved in binding viral and host proteins, thus initiating RNA synthesis. The 5'-noncoding region contains the internal ribosome entry site (IRES), an element that directs translation of the mRNA by internal ribosome binding. Studies have shown that the genome consists of a single open reading frame in the viral RNA that codes a long polyprotein. This polyprotein

14 is cleaved during translation by virus-encoded proteinases to yield final cleavage products, and the full-length product is never observed109.

Figure 1.3: Organization of the HRV genome. The HRV genome (top) consists of a 7200 base pair single-stranded positive sense RNA that encodes all the proteins necessary for replication and virion formation. It is translated into a polyprotein (middle), which is then cleaved by the 2A and 3C proteases into individual proteins (bottom). Flanking the coding regions are the 5’ and 3’ non-coding regions (NCR), alongside a 5’ VPg and 3’ poly-adenosine tail. Adapted from Knipe & Howley109.

The polyprotein is divided into three regions: P1, P2, and P3. The P1 region encodes the previously mentioned viral capsid proteins VP1, VP2, VP3, and VP4. The P2 and P3 regions, however, encode proteins involved in protein processing (2A, 3C) and genome replication

(2B, 2C, 3A, 3B, 3D). More specifically, the 2A protein is a protease that cleaves factors in the

15 cap-dependent translation initiation and is involved in shutting down host cell translation.

The 2B protein is potentially involved in RNA synthesis and is thought to inhibit host cell secretory pathways, whereas 2C is suggested to be involved in vesicle formation. The 3A protein plays a role in inhibition of intracellular transport, and the 3B protein encodes the

VPg. Protein 3C is a protease involved in polyprotein processing and helps generate replication proteins from the viral genome. The 3D protein is a virally encoded RNA- dependent RNA polymerase109.

Only humans and higher non-human primates are known to be susceptible to HRV infection, and primates are generally asymptomatic, meaning that only human infections are clinically relevant110,111. The airway epithelium is the principle site in vivo for HRV infection112,113.

Though classically thought to infect only the upper airways where the temperature is ideal for its replication (33-35°C)114, evidence has emerged the HRV does also infect the lower airways, and can infect both ciliated and non-ciliated epithelium as well as basal cells115,116.

Though ciliated cells have been found to be the chief site of infection in vivo114,116, in vitro model systems have shown basal cells to be more susceptible to HRV infection117. Studies have shown that HRV infection of the airway epithelium is not overtly cytotoxic either in vivo or in vitro117. However, culture models of the airway epithelium do not show uniform infection by HRV, suggesting differential or limited receptor expression of cells in these models, and it is thought these findings are reflective of those found in vivo where infection can be described as patchy and focal118. For instance, while HRV infection of the airways of healthy human volunteers results in a productive infection it does not infect the entire nasal epithelium119. Though the process through which viral shedding occurs is poorly

16 understood, virions can be detected in nasal secretions 8-10 h post-infection, with peak shedding occurring 2 days post-infection119,120. Nevertheless some studies have suggested autophagy121 or apoptosis122 as potential mechanisms for shedding.

1.9 The Airways

The human respiratory system is responsible for gas exchange and can be divided either anatomically into the proximal and distal airways or functionally into the conducting and respiratory airways. The proximal airways are defined as entailing the region containing the trachea and major bronchi, whereas the distal airways entail the regions from the bronchioles to the alveolar ducts and alveolar sacs. The conducting airways extend from the nasal cavity to the larynx, through the large airways consisting of the trachea and the bronchi, and continue into the smaller airways, specifically the terminal bronchioles. These regions do not participate in gas exchange but to conduct and humidify the inhaled ambient air; they also function to remove any particulate matter that may be present113. The conducting airways transition into the respiratory airways at the level of the respiratory bronchioles, which continue as the alveolar ducts, and terminate in the alveoli, which function as the principle sites of gas exchange. Epithelial cells (of different morphologies and functions) line the entire airway surface and act as the primary point of contact for pathogens. Though the classical view of the role of the epithelium was simply as a barrier between host and environment, recent developments in the field have resulted in the realization that it plays a complex role in responding to inhaled environmental agents and pathogens, and can also serve to modulate the airway mucosa123. Some of the known

17 functions of the airway epithelium include: 1) barrier function, 2) mucociliary clearance, 3) secretion of immunomodulatory agents, 4) repair and renewal, 5) pathogen recognition, and

6) regulation of other respiratory cells (immune, smooth muscle, etc.). Additionally, there are 5 major types of differentiated respiratory epithelial cells, whose distribution and prevalence varies throughout the respiratory tree: 1) ciliated cells 2) basal cells 3) non- ciliated secretory/goblet cells 4) alveolar type I cells and 5) alveolar type II cells.

The epithelium in the large airways is expressed as a pseudo-stratified single-layer, columnar epithelium. Much like the upper airways, the predominant cell type to be found are the columnar ciliated cells that function in mucocilliary clearance. We also find basal cells that are connected to the basement membrane and act as progenitor cells, along with goblet cells that work to secrete mucus. As one transitions to the small airways in the bronchioles, the epithelium loses its pseudo-stratified structure and takes on a simple, cuboidal morphology. Furthermore, the cell types move toward an equal distribution of ciliated columnar epithelial cells, and club cells whose primary function is as the progenitor cell in this region. The predominant epithelial cell types found lower in the gas exchange regions are the large, flat, simple squamous type I alveolar epithelial cells, which comprise nearly

90% of the epithelium and have the primary function of gas-exchange. The remaining 10% are composed of the smaller type II alveolar epithelial cells that release surfactant proteins and act as the progenitor cells for repopulation of the alveoli113.

Large Cartilaginous Trachea

Bronchi (primary, secondary, tertiary)

Small Non-cartilaginous Bronchioles (terminal, transitional)

Respiratory Bronchioles

Alveolar ducts

Alveolar sacs

Figure 1.4: Structure of human airways and accompanying epithelium. Adapted from

Wetsel, et al. 124.

The barrier function of the epithelium, briefly described previously, is integral to its ability to regulate host defense and gas exchange. These functions are possible due to the specialized structures that reside in epithelial cell-cell and cell-substratum junctions (see

Figure 1.5).

Figure 1.5: Structural components of epithelial cell-cell and cell-substratum junctions.

Adapted from Proud, et al. 113.

In terms of epithelial cell-cell interactions, the tight junctions (TJ) seen in Figure 1.5 are the most apical components of the junctional complex and their structure varies between different epithelial cell types: the fibrils of the TJs are loosely interconnected in the ciliated cells of the proximal airways but are highly interconnected in the distal airways. High- magnification studies of TJs demonstrate that they are present at zones where conjoined plasma membranes overlap, essentially eliminating the extracellular space in that region.

Initial studies were able to identify both occludin and claudins as important components of

TJs, while later studies also identified a member of the immunoglobulin superfamily termed the junctional adhesion molecule (JAM). However, studies have since identified over 40- different protein components that contribute to the adhesion architecture found in TJs.

Adherens junctions (AJ) are just basal to the TJs and act to hold cells together through tight calcium-dependent links. The proteins responsible for AJs are the cadherins, α-catenin, and

β-catenin. Desmosomes are further basal than the AJs and act as structural spot-welds that hold cells together; in the pseudostratified epithelium they are present primarily on the lateral aspects of columnar cells. They form discrete disc-like plaques which act as adhesion sites for their intermediate filaments (such as desmin, vimentin, and/or cytokeratins), rather than using actin. Hemidesmosomes are important for cell-substratum adhesion; they also utilize a cytoplasmic plaque with intermediate filament linkages to anchor the epithelium.

Like desmosomes, hemidesmosomes are based around intermediate filament interactions

(though hemidesmosomes are missing some of protein components found in desmosomes).

Their action is most commonly seen in the skin where they provide stable adhesion for top layer of the skin against mechanical stress. Gap junctions, while not a component of cell-cell adhesion junctions, are important in intercellular metabolic coupling113.

1.10 Role of the epithelium in asthma

The airway epithelium is widely recognized to be actively involved in modulating both innate and adaptive immune responses in the human airways113. Its proximity to the external environment allows it to act as the first cell type to encounter invasive pathogens.

Recognition of these pathogens is integral to the immune response that follows, which is

21 regulated through the production of epithelium derived inflammatory mediators. It is then interesting to note that several studies have shown that the epithelium present in the airways of asthmatic patients behaves differently to that of non-asthmatic individuals125. The asthmatic epithelium has been shown to have an altered expression of epithelial junction proteins126,127 and to also possess defects in repair mechanisms that result in reduced barrier function. The epithelium is widely considered to be more fragile and easily injured in asthmatic patients127,128. In addition, asthmatic subjects showcase increased epithelial shedding in vivo40, while also displaying deficiencies in their innate host defenses129-132.

These abnormalities are preserved even upon serial passaging in vitro127,133,134 and are considered to play a major role in the increased disease susceptibility and allergy seen in asthma135-137. Given the epithelium’s role as a homeostatic regulator in the respiratory system, it is apparent then as to how this disease state is considered to be a significant contributor to the inflammation and remodeling seen in asthmatic airways138,139.

1.11 HRV Infection of the epithelium

HRV infection in vivo principally targets the airway epithelium with studies indicating that

5-10% of all epithelial cells are infected115,118. It stands to reason than that, since both HRV infections and the epithelium have previously been implicated in playing a role in the airway remodeling seen in asthma, it is the response of the epithelium to HRV infection that is important in the pathogenesis of this remodeling phenomenon. This epithelial immune response to HRV infection can be divided into two categories: 1) responses based upon viral entry, and 2) responses based on viral replication. In the viral binding phase, the HRV capsid

22 is able to attach to ICAM-1 or members of the LDL receptor family, and studies have shown that this interaction can result in early signaling events. Specifically, sarcoma tyrosine kinase

(Src) and phosphoinositide 3-kinase (PI3K) have been seen activated and co-localized following the binding and infection of HRV-39, a major group virus140,141. Studies have also shown activation of the p38 mitogen-activated protein kinase (MAPK) pathway upon ICAM-

1 dependent internalization of HRV-16142,143. Currently there is very little known regarding the signaling events tied to LDL receptor based HRV internalization, though a study in mice demonstrated the HRV-1B internalization resulted in the activation of PI3K144.

Following viral internalization, the cell initially relies on the innate immune system to help provide host defense. Pattern recognition receptors (PRR) have been proposed as sites for recognition of the HRV RNA. These PRRs are able to take advantage of the replication cycle of HRV, wherein there is the production of a double stranded RNA (dsRNA) intermediate, and trigger an immune response. The PRRs that recognize this dsRNA intermediate, and have been shown to be expressed in airway epithelial cells are: Toll-like receptor (TLR)-3, retinoic acid-inducible gene-I (RIG-I) like receptors (RLR): RIG-I and melanoma differentiation- associated gene 5 (MDA-5) (Figure 1.6), and protein kinase R (PKR)145-148. Upon recognition of receptor binding, both TLR3 and the RLR recruit adaptor proteins to activate downstream transcription factors. Thus, TLR3 activation results in the recruitment of the Toll/IL-1 receptor (TIR) domain containing adaptor inducing interferon (IFN)-β (TRIF), while RLR activation results in the recruitment of the IFN-β promoter stimulator 1 (IPS-1). These proteins then activate transcription factors such as nuclear factor kappa-light-chain- enhancer of activated B cells (NF-κB) 149-151 and interferon-response factor (IRF)-1152,153 and

IRF-3154 (Figure 1.6). Additionally, HRV infection has also been shown to induce pro- inflammatory cytokine production through a PKR-dependent pathway155; dsRNA recognition by PKR results in dimerization and autophosphorylation, whereby PKR goes on to affect several effector proteins and eventually various signaling pathways156. Some virally expressed proteins have also been shown to induce replication-dependent signaling such as the HRV 3C protease, which activate activator-protein-1 (AP-1) and results in CXCL8 production157.

Figure 1.6: The pattern recognition receptor (PRR) based response of the epithelium to HRV infection. Following internalization of HRV after binding to ICAM-1, dsRNA is generated via its replication cycle. PRRs within the cell detect the dsRNA, resulting in pathways that converge upon activation of NF-κB and interferon regulatory factors (IRFs).

Adapted from Proud & Leigh 123.

These replication-dependent or replication-independent immune response pathways result in the up-regulation of many different pro-inflammatory cytokines. A subgroup of these cytokines are chemokines, which are characterized as being positively charged proteins between 8-15 kDa that are key in the inflammatory cell recruitment. Signal transduction for many chemokines is mediated through pertussis toxin (PTX) sensitive, seven-

25 transmembrane domain G-protein-coupled receptors (GPCR). There are four broad chemokine families that are classified by arrangement of two N-terminal cysteine residues within their amino acid sequences, and these include the CXC (α), CC (β), C (γ) and CX3C (δ) families. The CXC family of chemokines, which possess a single amino acid between adjacent cysteine residues, are chemotactic for several cell types, including TH1 lymphocytes. The CC chemokines have been shown to be chemotactic for monocytes, TH2 lymphocytes158, and eosinophils159. The C family includes only one member, XCL1, which chemotactic for

160 lymphocytes . Lastly, the CX3C family also consists of only one member, CX3CL1, which is found primarily as a cell membrane protein161. Chemokines are produced by many cell types, including structural cells such as epithelial cells, fibroblasts and smooth muscle cells113, as well as by inflammatory leukocytes. Several of these cytokines, growth factors, and chemokines that are induced by HRV infection of the epithelium in vitro, and are listed in

Table 1.1.

Table 1.1: Expression of cytokines, growth factors, and chemokines produced by the airway epithelium following HRV infection. Adapted from Proud & Leigh and others123,149,162-166.

Cytokines Growth Factors Chemokines

IL-1 Activin A CXCL1,

IL-6 Amphiregulin CXCL5,

IL-10 TGF-α CXCL8,

IL-11 TGF-β CXCL10,

Vascular Endothelial CCL5,

Growth Factor (VEGF) CCL11

Several of the chemokines listed above have been detected in vivo during HRV infection of the airways162,166-168 and are thought to play an important role in the resultant inflammatory cascade that follows. These chemokines have been previously shown to act as chemoattractants for neutrophils and lymphocytes169,170 that, upon reaching the airways, add to the inflammatory milieu by releasing their own pro-inflammatory mediators which have a further damaging effect on the epithelium. Studies have also indicated that several of these same mediators are also efficacious in driving ASM migration and could be contributing to airway remodeling171, discussed in greater detail below.

The adaptive immune response to HRV infection is less well characterized, but the major players are thought to be 1) dendritic cells (DCs), given their role in the antigen presentation to T cells, and 2) airway epithelial cells, which are known to express both class I and class II

27 major histocompatibility complexes (MHC) and thus are able to present antigen and induce cluster of differentiation (CD) 4 positive (CD4+) and CD8+ T cell activation and proliferation172-175. HRV infection has been shown to increase expression of class I MHC and, at least in vivo, results in an infiltration of CD4+ and CD8+ T cells176-178. Approximately 2 weeks post HRV infection there is production of neutralizing antibodies, and titres increase until 4-5 weeks post infection179. Due to the extended period of time it takes for the generation of physiologically relevant levels of HRV antibody titres, it has been proposed that antibody production is not involved in initial clearance of the infection and, instead, provides protection against secondary infections by the same HRV serotype109,180.

Viral infections play an important role in asthma exacerbations. Acute exacerbations in asthma are defined as “a worsening of the patient’s conditions, beyond day-to-day variability associated with the disease, that is sufficient enough to require change in management, or to seek emergency medical intervention”169. While various risk factors such as exposure to allergens or pollutants, and to bacterial infections can be associated with exacerbations, viral infections account for 50-60% of adult and 80-85% of childhood asthmatic exacerbations respectively181,182. Most commonly HRV is the detected pathogen183,184 (Figure 1.7), with studies suggesting that it is responsible for over 60% of all virally induced exacerbations in both children and adults181,182. Severe asthma exacerbations have shown to result in a progressive decline in patient FEV1, suggesting that they may result in a gradual modulation of the airway structure; this relationship, however, is not present in all asthmatics and a causal relationship has not yet been clearly defined185-187. As mentioned previously, HRV infection in vivo does not result in any obvious cytopathic effects188,189 indicating that the

28 exuberant host defense inflammatory response is likely to be implicated in the resultant pathogenesis of these asthmatic exacerbations.

Figure 1.7: Pathogens associated with virally induced exacerbations of asthma in children (age 9-11). Data adapted from Johnston, et al. 181.

A chemokine that may play an important role in this inflammatory cascade, as mentioned above (Table 1.1), is the CC chemokine CCL5. Also known as Regulated on Activation, Normal

T-cell Expressed and Secreted (RANTES), it was first detected in 1988190 as potentially a T- cell specific molecule, and was originally characterized on its ability to regulate T-cell behavior158,191. However, it has subsequently been characterized as molecular regulator of many different cell types, including macrophages, dendritic cells, natural killer T cells, eosinophils, basophils, and smooth muscle cells171,192,193. It acts chiefly through its specific

CC-receptors (CCR) 1, 3, and 5, and its production is predominantly generated through CD8+

T cells, epithelial cells, fibroblasts, and platelets193. Importantly, abnormally increased CCL5 expression has been associated with a wide range of inflammatory disorders and pathologies, including allogeneic transplant rejection, atherosclerosis, arthritis, and atopic dermatitis193. Interestingly, CCL5 has also been implicated in the clearance of common respiratory infections, whereby it is suggested that CCL5 plays a role in attracting virus specific CD4+ and CD8+ T-cells to the site of infection; these cells then activate and proliferate to kill host cells and eliminate the viral threat192. Of note, expression of CCL5 has been demonstrated in several studies to be markedly up-regulated in asthma in both children194 and adults195. As HRV infection is linked to the development of asthma, and the epithelium is the chief site of HRV infection, it thus seems reasonable to suggest that CCL5 production by the epithelium is a key contributor to the pathogenesis of asthma, and potentially to the pathogenesis of airway remodeling. As such, CCL5 has been identified in this project as a potentially important chemokine to study in the context of its role in HRV- induced airway remodeling in asthma.

1.12 The Airway Smooth Muscle

As mentioned above, the airway smooth muscle (ASM) tissue plays arguably one of the most important roles in relation to asthma pathogenesis. Anatomically the ASM circumferentially surrounds the lumen of the airways196 and in healthy individuals has two functions: 1) development and maintenance of the extracellular matrix (ECM) protein environment, and

2) maintaining tissue contraction and elasticity197. In the proximal conducting airways the

30 smooth muscle is found attached to cartilage but in the distal airways the contractile muscle is organized in a helix-antihelix pattern that surrounds the bronchioles198. Studies have found many different individualized cell subpopulations of ASM in the lungs, suggesting both multiple differentiation pathways and different lineages196,197. The ASM cells of the lung are of a phenotype that is distinctly different to that presented by vascular smooth muscle cells197. In contrast to skeletal and cardiac muscle derived from mesodermal precursors, ASM appears to originate from neural crest cells and mesenchymal cells196 and ASM differentiation occurs prior to that of the vasculature197. There is a strikingly different pattern of distribution of smooth muscle myosin heavy chain isoforms in ASM as compared to the pulmonary vasculature. Specifically, the ASM expresses cytosolic smooth muscle markers such as smooth muscle α-actin, myosin, and desmin at mid-gestation in a cranial- to-caudal pattern198. The ECM appears to play an important role in modulating ASM cell responses and cell differentiation199. In cultured ASM, myocytes secrete an array of ECM proteins as well as matrix metalloproteinases, which are key enzymes that regulate the turnover of matrix196.

While healthy ASM is integral to the proper functioning of the airways, its presence becomes detrimental in asthma. One of the most obvious changes in the remodeled airways of asthmatics is the increased smooth muscle mass present and its increased proximity to the epithelium200, with studies conducted almost 90 years ago finding increased smooth muscle mass in the airways of patients who died from asthma37. While many studies have found increased smooth muscle cell populations in asthmatics45,201-203, little information is present on the cellular mechanism(s) involved200. Current theories regarding smooth muscle

31 remodeling in asthma propose that the combination of migration, proliferation, and hypertrophy accounts for the changes observed in severe asthma smooth muscle mass200.

Migration of smooth muscle cells is not unprecedented and is fundamental to the process of developing hollow organs, such as blood vessels and the airways204. However, it has also been implicated in many diseases states such atherosclerosis205, asthma204, and restenosis206 for contributing negatively to disease pathogenesis. Several groups have noted that smooth muscle cells exist in multiple phenotypes, predominantly switching between a “contractile” and a “synthetic” phenotype in situations of tissue repair197,207. While in most cases this switch is natural and helpful, in some cases – as seen in vascular tissue – initially injury results in the recruitment of an inflammatory reaction that alter this response. Specifically, in atherosclerosis, response to tissue injury is multicellular and heavily involves the release of cytokines and growth factors such as platelet derived growth factor (PDGF), VEGF, and angiotensin II to mediate wound repair206. These inflammatory mediators act as chemokines in the damaged vascular lumen that attract a migratory and invasive phenotype of smooth muscle that moves in and grows abnormally205. Groups have noted that the difference between physiological and pathological smooth muscle migration is the failure to cease migration once tissue repair has been completed206. This “synthetic” phenotype has also been seen displayed by ASM in vivo, wherein ASM cells can act to orchestrate and perpetuate airway inflammation by its ability to secrete cytokines (IL-1, IL- 5, IL-6) and chemokines

(CXCL8, CCL5, and eotaxin) and by expressing a variety of cell adhesion molecules (ICAM-1,

VCAM-1, CD44, and integrins)196.

When trying to formulate an understanding of ASM migration it is important to look at other biological systems and cell types for answers. For instance, while eukaryotic cells can use spatial or temporal aspects of chemoattractants to guide movement, prokaryotic cells are constrained due to their small size and thus they must rely upon only temporal information.

As a result, they perform a random movement in all directions that is frequently interrupted by stops. Additionally, if the cell experiences an increase in chemoattractant concentration, stopping periods decrease and one-directional movement is prolonged208,209. In contrast, larger eukaryotic cells are able to respond to differences in chemoattractant concentration

(that are less than 10%) between the front and back of the cell, allowing the cell to process spatial information and move accordingly210. The work of Dr. Peter Devreotes and his colleagues in examining the slime mold Dictyostelium discoideum has provided substantial insight into the mechanism of cell migration because D. discoideum’s ability to sense and navigate gradients of extracellular signals is similar to mammalian neutrophils. D. discoideum is a soil-living amoeba that has a life cycle consisting of four stages: vegetative, aggregation, migration, and culmination. During the aggregation stage of the life cycle, D. discoideum works to release signals to attract individual cells to a central location to establish cell-cell junctions; this section of the life cycle then is perfect for studying migration. The migration process scan be broken down into three components: pseudopod formation, polarization, and directional sensing211. While an unstimulated D. discoideum will extend one or more pseudopodia at random, a chemoattractant signal increases the probability that a pseudopod forms at the leading edge, similar to neutrophils. Studies have indicated that the relative uniformity of pseudopodia, which are unaffected in size, frequency, or lifetime by the chemoattractant, are most likely self-organizing structures. This is important because it

33 indicates that pseudopod formation can occur without having to consider taking in signals from the rest of the cell. Polarization by the chemoattractant results in strong directional movement, and studies with D. discoideum 212 have indicated that this polarization only increases over prolonged migratory periods. This indicates a heightened ability of a cell to reach source of the chemoattractant but polarization would also make it less competent in responding to rapid changes in gradient direction. Directional sensing becomes important as it refers to the bias relating to pseudopod formation to the side of the cell facing the chemoattractant at the highest concentration. Importantly, cells are able to sense and amplify the spatial gradient of a chemoattractant even in the absence of the ability to migrate: cells which had been treated with latrunculin A (which is an inhibitor of actin polymerization) become rounded and stop moving but continue to align their leading edges as detected by PIP3 regulation211,213. Eukaryotic cells are also able to undergo adaptation of chemoattractant mediated responses whereby cells only respond to increments of chemoattractant concentration. While these responses are poorly understood, in D. discoideum receptor phosphorylation negatively controls receptor affinity for chemoattractant ligand214, and the mechanism could be similar in other cell types. Lastly, recent studies with D. discoideum have indicated that PI3K is an important regulator of migration215.

ASM migration is initiated by activation of surface receptors that trigger the cytoskeleton to rearrange in preparation for movement. Some of the receptor types thought to play a role include GPCRs, receptor tyrosine kinases (RTK), and matrix adhesive proteins (such as integrins). Depending on the type of migratory stimulus, ASM cells can be induced into a

34 variety of different types of migration. Specifically, time-lapsed images demonstrate that the lack of a defined chemotactic gradient results in random movement (chemokinesis), the presence of a chemotactic gradient results in directional movement following the gradient

(chemotaxis), or the cells may choose to follow the paths laid out by various ECM proteins

(haptotaxis)204. Presented in Table 1.2 is a summary of some of the pro-migratory and anti- migratory agents currently known for ASM in vitro; these compounds are known to activate signaling cascades that can modulate cytoskeletal remodeling, activation of motor proteins, and/or cellular adhesiveness.

Table 1.2: Summary of agents that modulate ASM migration. Adapted from Gerthoffer204.

Pro-Migratory Agents Anti-Migratory Agents

Growth Factors and ECM Proteins Β-adrenergic Other anti-

Cytokines agonists migratory agents

IL-1β Collagen (I, III, V) Dibutyryl cAMP Fluticasone

IL-8 (CXCL8) Fibronectin Formoterol Pertussis toxin

Platelet-Derived Laminin Forskolin Prostaglandin E2

Growth Factor Cilomilast

(PDGF)

RANTES (CCL5)

After receptor activation by one of these listed pro-migratory agents, Ca2+, phosphatidylinositol 4,5 bisphosphate (PIP2), and small G proteins activate a series of signaling cascades; the pathway associated with PDGF is amongst the best studied (though

35 many other ligands share similar cascades)204. The binding of PDGF to the PDGF receptor-β

(PDGFR-β) results in the recruitment of PI3K and phospholipase C. This then leads to changes in myoplasmic calcium concentrations, the hydrolysis of PIP2, and the downstream activation of several MAPKs216. These intermediates then act in concert with G-proteins that trigger nucleation of F-actin filaments at the minus end by recruitment of the actin-related protein (ARP) complexes 2/3, while also promoting uncapping at the plus ends. The formins mDia1 and mDia2 act together with profiling to then promote the extension of new actin filaments. Filament branching is also activated by the small G-proteins through the recruitment of the Wiskott-Aldrich syndrome proteins (WASP) and the WASP-family verprolin-homologous protein (WAVE) complex. These proteins then provide positive feedback into the actin nucleation and branching process by activating components of the previously mentioned ARP2/3 complex. Conversely, in order for the cell to migrate properly, there also needs to be actin depolymerization in the trailing edge of the cell. Specifically, increases in intracellular Ca2+ result in the downstream activation of the protein gelsolin, which acts as a severing protein that cuts existing actin filaments. Another protein, cofilin, works in with gelsolin to enhance the turnover of actin filaments. Altogether, these processes allow for the cell to appropriately remodel its intracellular matrix for and during migration217.

Figure 1.8: Migration of smooth muscle cells. This is a schematic diagram representing the migration of a muscle cell. The leading edge is designated by a crosshatched pattern on the right side of the diagram. Inset A showcases the leading edge as an area of high actin polymerization and depolymerization. Inset B showcases the nascent focal contacts (red bars) that form to allow the cell to attach to a matrix and migrate. Adapted from

Gerthoffer218.

At the leading edge, in order to gain footholds for forward movement, migratory cells develop points of focal adhesion between cellular membrane and the ECM. These adhesion points form during the process of actin polymerization and provide adhesion for the cellular lamellipodia for cell propulsion. On the other hand, in the trailing edge of the cell these points of focal adhesion are disassembled to allow a release from the anchoring to the ECM. This is

37 a fluid, dynamic process that occurs throughout migration and is said to involve many different proteins such as paxillin219,220, vinculin221, talin220, focal adhesion kinase (FAK)219-

221, and Src 219-222. However, currently there are not many studies that have examined focal contact characteristics in ASM cells204.

Figure 1.9: Signaling pathways regulating muscle migration. Activation of GPCRs or receptor tyrosine kinases (RTK) results in signaling cascades that result in actin filament remodeling and regulation of myosin II motors (which generate traction force). Adapted from Gerthoffer218.

The actual force(s) necessary to propel the migratory cell forward can be generated by two difference sources simultaneously. Firstly, as mentioned above, forward momentum is generated by actin polymerization that leads to the protrusion of the cellular membrane at the leading edge. Secondly, as discussed below, propulsion is generated by the enzymatic activity of myosin motors that provide a force that is transmitted through the focal adhesions to the ECM. Smooth muscle myosin II in particular plays a large role and is regulated by the

Ca2+-calmodulin based activation of myosin light chain kinase (MLCK), which acts to phosphorylate the regulatory myosin light chains (RMLC). Though it has not yet been shown to do so in smooth muscle cells, RhoA activation of Rho-activated protein kinase (ROCK) can potentially result in Ca2+ independent activation of myosin II through the direct phosphorylation of RMLCs by ROCK223. At present, however, there is relatively small amount present in the literature regarding how myosin motors can be regulated204.

Numerous studies have examined the ability of ASM cells to migrate to inflammatory mediators and cytokines224-228. Groups have shown that several chemokines implicated in asthma such as CXCL8227, CCL5229, and CCL11230 are able to drive smooth muscle migration.

A study by Takeda et al. (2009) showed that treatment of a epithelial basal cell line BEAS-2B with tumor necrosis factor-alpha (TNF-alpha) resulted in a supernatant that could drive smooth muscle migration226. Aside from soluble molecules, it is now understood that the

ECM environment likely plays a significant role in directing ASM migration in asthma pathogenesis, and it is likely interplay between soluble and matrix-bound signals that is responsible for this behavior204. Our laboratory has demonstrated the release of numerous remodeling mediators from the bronchial epithelium following HRV infection112, several of

39 which have already been implicated in driving smooth muscle cell migration (Tables 1.1 and

1.2). Given that airway remodeling is often present before the diagnosis of asthma is established and likely requires an early life trigger, and given that HRV-induced wheezing episodes are linked heavily with the subsequent development of asthma, it is reasonable to hypothesize that the inflammatory response following epithelial infection with HRV could result in smooth muscle migration, which is considered to be an important component of asthmatic airway remodeling. Thus, the hypothesis of this thesis was that HRV-16 infection of HBE cells in vitro up-regulates the production of certain chemotactic inflammatory mediators, which then facilitate ASM chemotaxis.

1.13 Thesis Objectives

Objectives

The overall objective of this Masters thesis was to determine whether HRV infection of human bronchial epithelial (HBE) cells resulted in production of chemotactic inflammatory mediators that were able to drive ASM migration, and to delineate the cellular mechanism(s) by which this ASM migration is regulated.

Aim 1: To characterize the magnitude and kinetics of ASM migration in response to HRV infected HBE supernatants

Aim 2: Identify basic intracellular mechanisms by which ASM migration to HRV infected HBE supernatants occurs.

2 CHAPTER TWO: MATERIALS AND METHODS

2.1 Materials

The following materials and reagents were purchased from indicated suppliers:

• ACEA Biosciences (San Francisco, CA, USA): Cellular Invasion and Migration (CIM)

plates.

• Ambion (Austin, TX, USA): DNA-free DNase I kit

• American Type Tissue Collection (Manassas, VA, USA): HRV-16 and WI-38 human

fetal lung fibroblasts.

• BD Biosciences (Franklin Lakes, NJ, USA): 5 and 50 cc syringes, mouse anti-CD181

PE-cy5 antibody, mouse anti-CD193 BV421 antibody, and mouse IgG2b AF647

isotype control antibody.

• Corning Life Sciences (Lowell, MA, USA): cell culture plates, dishes and flasks.

• Life Technologies (Carlsbad, CA, USA): 96-well optical reaction plates, TaqMan

universal PCR mastermix, RNase inhibitor, TaqMan GPCR array micro fluidic card,

Hanks’ balanced salt solution (HBSS), Ham’s F12 medium, fetal bovine serum (FBS),

penicillin-streptomycin-amphotericin B (PSF), 4-(2-hydroxyethyl)-1-

piperazineethanesulfonic acid (HEPES), gentamicin, Minimum Essential Media

(MEM), Dulbecco’s modified eagle medium (DMEM), L-glutamine, non-essential

amino acids, sodium pyruvate, TRIzol Reagent, GIBCO RNase/DNase-free double

distilled water, Oligo (dt) primer, 5X first strand buffer, SuperScript III reverse

transcriptase, random primers/hexamers, Cryogenic vials, 96-well Immulon 4 ultra-

high binding polystyrene microtiter plates, Collagen I (rat tail), and Typsin-EDTA.

• Lonza (Walkersville, MD, USA): Bronchial epithelial cell basal growth medium

(BEBM) and additives (bovine pituitary extract, epidermal growth factor,

epinephrine, gentamicin/amphotericin B, hydrocortisone, insulin, retinoic acid,

transferrin and triiodothyronine).

• Neuro Probe (Gaithersburg, MD, USA): 48-well micro chemotaxis chamber (Boyden

chamber), and 8-micron polycarbonate filters.

• New England Biolabs (Ipswich, MA, USA): 20X bovine serum albumin (BSA).

• Roche (Mississauga, ON, Canada): pronase, anti-protease tablets, and 2,2’-azino-bis

(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS).

• R & D Systems (Minneapolis, MN, USA): recombinant CCL5, CXCL8, and CXCL10

protein; CCL5, CXCL8, and CXCL10 capture and biotinylated detection antibodies for

ELISA, mouse anti-CCR1 AF488 antibody, unlabeled mouse IgG1 isotype control

antibody, mouse anti-CCR PE antibody, and mouse IgG2A PE isotype control antibody.

• Thermo Scientific (Rochester, NY, USA): Cryogenic vials, 96-well Immulon 4 ultra-

high binding polystyrene microtiter plates, Collagen I (rat tail), and Typsin-EDTA.

• VWR (Mississauga, ON, Canada): eppendorf tips and tubes, stripettes, Phase Lock Gel-

Heavy tubes, RNAse/DNase free tubes, and isopropanol.

All other materials and reagents were purchased from Sigma-Aldrich (Oakville, ON,

Canada).

2.2 Isolation of HBE and ASM from Human Lung Tissue

Normal, non-transplanted human lungs were obtained through a tissue retrieval service

(International Institute for the Advancement of Medicine, Edison, NJ, USA). Ethical approval was obtained from both the Conjoint Health Research Ethics Board of the University of

Calgary and the Internal Ethics Board of the International Institute for the Advancement of

Medicine (Edison, NJ, USA). The lungs were received 24-36 hours post-surgical removal.

For HBE extraction, the trachea and bronchi were dissected out and incubated in an enzymatic solution of sterile filtered Ham’s F12 medium (F12), which contained 1 mg/mL pronase and 1 μg/mL gentamicin for 30-40 h at 4°C. Post-treatment the tissue were cut longitudinally, and then placed into F12 containing 20% fetal bovine serum (FBS); the cells were subsequently removed through forceful jetting of the luminal surface via a 5 mL syringe. The cell suspension was then centrifuged in the F12/FBS medium (153 x g, 8 min, room temperature (RT)) and re-suspended in fresh 20 mL F12/FBS medium. Cells were stained with erythrosin B to identify viable, non-ciliated epithelial cells and cell counts were performed using a haemocytometer. Cells were then suspended in a 1:1 solution of freezing media (2X penicillin/streptomycin in L15 media and 2X DMSO). Cells were stored at 5.0 x

105 cells/vial and kept in liquid nitrogen until later use. HBE cells were characterized through cytokeratin staining as described previously231.

For ASM extraction, tissue was sectioned from the main bronchus and placed into antibiotic

(1% penicillin-streptomycin-amphotericin B/Fungizone; PSF) containing Dulbecco’s

Modified Eagle Medium (DMEM; supplemented with 4.5 g/L D-glucose, 2mM L-glutamine,

110 mg/mL sodium pyruvate) for 1 hour. The tissue was then longitudinally cut and dissected under a stereoscopic microscope. The epithelium was gently removed and the individual smooth muscle bundles were dissected from the surrounding tissue and subsequently isolated. The bundles were suspended in an enzymatic solution in Dulbecco’s

Modified Eagle Medium (DMEM; supplemented with 4.5 g/L D-glucose, 2mM L-glutamine,

110 mg/mL sodium pyruvate) containing 1 mg/mL collagenase at room temperature. The solution was then placed onto an orbital shaker at 60 rpm for 2 hours. The resulting enzymatically dispersed cell solution was centrifuged (153 x g, 8 min, RT) and re-suspended in DMEM supplemented with 10% FBS and antibiotics (PSF), before being plated into a 25

2 cm tissue culture flask. ASM cells grown in submersion culture at 37°C in 5% CO2 and confluent cells were passaged onto 175 cm2 tissue culture flasks. Cell lines were expanded until passage 3-4 at which point they were trypsinized and re-suspended in freezing media

(1:1 solution of penicillin streptomycin-amphotericin-B and DMSO). Cells were stored at 2 x

106 cells/vial and kept in liquid nitrogen until later use.

Prior to experiments being performed, HBE cells were thawed and immediately re- suspended into F12 medium containing 20% FBS. Cells were then centrifuged (153 x g, 8 min, RT), re-suspended in Bronchial Epithelial Growth Medium (BEGM) supplemented with

5% FBS, and plated onto 6-well plates. Four days later the medium was replaced and the HBE cells were grown in submersion culture in BEGM at 37°C in 5% CO2 until they reached desired levels of confluence. BEGM consists of Bronchial Epithelial Basal Medium (BEBM) supplemented with growth factors (bovine pituitary extract, insulin, retinoic acid,

45 hydrocortisone, epinephrine, triiodothyronine, transferrin and human epithelial growth factor) and antibiotics (Gentamicin-1000 and 0.5% penicillin-streptomycin-amphotericin

B/Fungizone). It has been previously confirmed that this method of isolation and culture produces cells of an epithelial cell nature as was confirmed through cytokeratin staining231.

Concurrently, ASM cells were thawed and immediately re-suspended into DMEM containing

10% FBS. Cells were then centrifuged (153 x g, 8 min, RT), re-suspended DMEM supplemented with 10% FBS and 1% PSF, and plated on to 175-cm2 tissue culture flasks

(T175). The ASM cells were in submersion culture in DMEM (supplemented with 10% FBS and 1% PSF) at 37°C in 5% CO2 until they reached desired levels of confluence. ASM cells were grown and used for ensuing experiments up to passage 8. This method of smooth muscle isolation has been previously confirmed to result in a >95% population of cells that were positive for ASM markers232.

2.3 HRV-16

Stocks of HRV-16 were generated through propagation of the virus through infection of WI-

38 fetal lung fibroblast cells. A single vial of WI-38 fibroblasts (ATCC) was first cultured in submersion culture in a 75-cm2 tissue culture flask (T75) in WI-38 medium (Minimum

Essential Medium (MEM), 10% FBS, 2 mM L-glutamine, 1% non-essential amino acids, 1% sodium pyruvate, and 1% PSF). Upon reaching confluence, the cells were lifted and split first into 4 175-cm2 tissue culture flasks, then secondly into 16 175-cm2 tissue culture flasks.

Upon reaching confluence, WI-38 cells were then infected with supernatants containing

HRV-16 from previous WI-38 preparations for approximately 28 hours at 34°C and 5% CO2, until observable signs of cytotoxicity were detected (indicated by marked rounding of cells).

Supernatants were then collected, centrifuged (425 x g, 15 min, 4°C) and stored at -80°C for subsequent reinfection. The collected supernatant was replaced with fresh WI-38 medium, and the flasks were scraped using a cell scraper. The cell lysate that was obtained was then sonicated on ice (Fisher Scientific Sonic Dismembrator Model 500; 15 seconds, 50% amplitude) and centrifuged (425 x g, 15 minutes, 4°C). Supernatants were collected and stored at -80°C for subsequent viral purification.

Stocks of HRV-16 were purified through sucrose density centrifugation to remove cellular contaminants originating from WI-38 fibroblasts. Prior to centrifugation, RNase A (80

μL/100 mL of crude lysate) was added to the crude cell lysates derived from the WI-38 fibroblasts to remove any single stranded RNA (ssRNA; unpackaged virus), and incubated for 30 min at 34°C. Subsequently, N-laurosarcosine (10 mg/100 mL crude lysate) and β- mercaptoethanol (100 μL/100 mL crude lysate) were added to break up cellular membranes and reduce disulphide bonds respectively. The crude stock was then underlayed with a 30% sucrose solution (30% sucrose, 20 mM Tris-acetate, 0.1 M NaCL) and centrifuged (28,000 rpm, 5 h, 16°C) using a Beckman Coulter SW28 rotor in a Beckman Coulter Ultracentrifuge

Optima XL-100K. After centrifugation, supernatant layers, containing both WI-38 medium and products were removed via aspiration leaving only the sucrose layer. The derived HRV-

16 containing sucrose layer was combined with a solution of F12/50mM HEPES in a 1:1 ratio, sterile filtered via a 0.45 μm filter, and stored at -80°C (termed: purified HRV-16). Separately,

47 the pelleted virus was re-suspended in F12/50 mM HEPES, filtered under sterile conditions using a 0.45 μm filter, and stored at -80°C (termed: HRV pellet).

Stocks of replication-deficient HRV-16 were generated by first adding 1 mL of HRV-16 pellet into wells of a 6-well tissue culture plate in sterile conditions inside a biosafety cabinet. The plate lid was then removed and a Spectroline model XX-15F high-intensity, short-wavelength

(254 nm) UV lamp was positioned above the plate. The viral stock was then exposed to UV light for 5 minutes and HRV-16 replication deficiency was subsequently confirmed by the inability of the virus to replicate and/or cause cytopathic effects in the WI-38 fibroblasts.

Titres of the HRV-16 stock were assessed by infection of confluent WI-38 fibroblasts. The cells were grown to confluence on 96-well plates and were infected with serial half-log dilutions (purified HRV 1/300 – 1/106; HRV pellet 1/3000 – 1/109). Following a 5-day infection period (34°C; 5% CO2), the cells were fixed in methanol (1 min) and stained with

0.1% crystal violet (20 min). Plates were then washed with distilled water and allowed to dry (48 h). Plates were then analyzed using a microplate reader (Bio-rad Benchmark) at 570 nm. The data from the spectrophotometric analysis were used to calculated the 50% tissue

233 culture infectious dose (TCID50) using the Reed-Muench method .

2.4 HRV-16 Infection of Bronchial Epithelial Cells

HBE cells were grown to 80-90% confluence (37°C; 5% CO2) in full BEGM medium prior to removal of hydrocortisone for 16 hours (BEGM – No HC), which was done to reduce any

48 modulatory effects of the hydrocortisone on the HBE cells. Thereafter, the medium was aspirated off and the cells were washed with Hank’s Balance Salt Solution (HBSS) prior to

5.5 fresh BEBM being added for 4 hours. HBE cells were then infected with 10 TCID50 U/ml

(MOI ~1.0) for 24 hours at 34°C with 5% CO2. The supernatants from 6 individual lung donors were then pooled for each experimental condition to generate stocks of conditioned medium (CM) and HRV-conditioned medium (HRV CM) for migration experiments.

2.5 Real-time polymerase chain reaction

RNA was isolated from sample cells using TRIzol reagent, as per the manufacturer’s instructions. Briefly, following experimental interventions, cells were washed with HBSS, and then 500 μL/well of TRIzol reagent was added. Pipetting TRIzol repeatedly onto the cells induced cellular lysis and the resulting homogenate was collected and placed into Phase Lock

Gel tubes. Chloroform was then added (0.2 mL/1 mL TRIzol homogenate), mixed vigorously

(15 seconds), and centrifuged (12,000 x g, 10 minutes). The aqueous layer, containing the

RNA, was transferred to an RNase/DNase free tube and precipitated via the addition isopropanol (500 μL/mL TRIzol) followed by centrifugation (12,000 x g, 10 minutes). The remaining RNA pellet was washed with 1 mL of 75% ethanol and centrifuged (7500 x g, 5 minutes). The ethanol was carefully removed and the pelleted RNA was air dried for 10 minutes prior to being re-suspended in 20 μL of RNase/DNase-free H2O. Spectrophotometric analysis at 260 nM was performed to determine RNA concentrations, and sample purity was assessed with 260 nm/280 nm ratio readings. Isolated RNA was then stored at -80°C until later use.

DNase treatment was performed according to the manufacturer’s protocol, using the DNA- free DNase I kit. Briefly, 10 μg of RNA was incubated with DNase I (2 units) and 10X DNase buffer for 25 minutes at 37°C. DNase inactivation reagent (5 μL) was then added and the samples were incubated at room temperature for 2 minutes prior to centrifugation (10,000 x g, 1 minutes). The treated RNA was then transferred to a new RNase/DNase free tube.

Spectrophotometric analysis at 260 nM was performed to determine RNA concentrations, and sample purity was assessed with 260 nm/280 nm ratio readings. Isolated RNA was stored at -80°C until later use. 4 μg of input RNA from ASM cells was reverse transcribed into cDNA in a 20 μL reaction mixture containing 1 μL Oligo(dT)20, 250 ng of random primers, and 1 μL of 10 mM dNTP mix. Samples were heated to 65°C for 5 min and then incubated on ice for 1 minute before addition of 4 μL 5X first strand buffer, 1 μL 0.1 M dithiothreitol (DTT) and 1 μL Superscript 3 reverse transcriptase. The samples were then placed into the Techne

Flexigene thermocycler and subjected to the following conditions: 50°C for 60 min and 70°C for 15 min. The cDNA samples were then collected and stored at -80°C.

2.5.1 GPCR Array

The cDNA samples from ASM cells were used to evaluate the presence or absence of GPCR mRNA, using the TaqMan® Array Micro Fluidic Card as per the manufacturer’s instructions.

Briefly, the reaction component was generated by combining 3.6 μg of cDNA dissolved in 450

μL nuclease-free water and 450 μL of TaqMan® Universal PCR Master Mix. This was then loaded into 8 reservoirs (100 μL each) on the microfluidic card before being centrifuged (331 x g, 2 min). The card was then sealed using the TaqMan® Array Micro Fluidic Card Sealer and

50 the reservoirs of the card were cut off. The card was then analyzed using the Applied

Biosystems Model 7900HT Fast Real-time PCR system and the SDS Software Suite (v2.4) wherein the manufacture provided array card protocol was loaded as follows: 50°C for 2 minutes, 94.5°C for 10 minutes, and 40 cycles at 97°C for 30 seconds and 59.7°C for 1 minute.

The resulting data were imported into RQ Manager (v1.2.1) for analysis, with the baseline set to automatic baseline and manual cycle threshold (CT) was set to 0.2 (as per manufacturer instructions). These data were analyzed using relative quantification methods where the resulting CT values for each individual gene measured were compared across donors for indication of relative expression. A full list of genes that were examined can be found in

Appendix Table 7.1.

2.6 Fluorescence-Activated Cell Sorting (FACS) Analysis

ASM cells were suspended through use of a non-enzymatic cell dissociation buffer (Life

Technologies). Cells were then centrifuged (331 x g, 8 min) prior to being re-suspended in

1500 μL of FACS buffer (0.5% BSA, 2 mM EDTA, 1X PBS, pH 7.2). 100 μL of the cell suspension was added to fresh round bottom test tubes. The manufacturer’s specified concentration of fluorescently labeled antibody(s) was then added and the cells were allowed to incubate in the dark at 4°C for 2 hours (for antibodies used see Table 2.1; for experimental conditions studied, see Table 2.2). The cells were then washed by the addition of 1 mL of FACS buffer followed by centrifugation (600 x g, 10 min). The resultant supernatant was decanted off and the cells were fixed by the addition of 200 μL of 2% paraformaldehyde and 500 μL of FACS buffer, and allowed to incubate in dark at 4°C for 30 min. The ASM cells were then washed

51 by the addition of 1 mL of FACS buffer followed by centrifugation (600 x g, 10 min). Pelleted cells were then re-suspended in 200 μL of FACS buffer and stored at 4°C until they were read for FACS analysis. FACS analysis was performed in the Flow Cytometry core facility, which is a part of the Center for Advanced Technologies at the University of Calgary and further information about this protocol is available on their website.

Table 2.1: Antibodies for FACS Analysis

Antibody Target Type Fluorophore

IgG1 Isotype IgG1 APC

IgG2B Isotype IgG2B Alexa Fluor 647

CCR1 IgG2B PerCP

CCR3 IgG2B BV421

CCR5 IgG2B PE

CXCR1 IgG2B PE-Cy 5

CXCR3 IgG1A APC

Table 2.2: Conditions for FACS Analysis

Tube Number Antibodies Present

1 Cells only control

2 IgG1 Isotype Control

3 IgG2B Isotype Control

4 CCR1

5 CCR3

6 CCR5

7 CXCR1

8 CXCR3

9 CCR3, CCR5, CXCR1, CXCR3

10 CCR1, CCR5, CXCR1, CXCR3

11 CCR1, CCR3, CXCR1, CXCR3

12 CCR1, CCR3, CCR5, CXCR3

13 CCR1, CCR3, CCR5, CXCR1

14 CCR1, CCR3, CCR5, CXCR1, CXCR3

2.7 Enzyme Linked Immunosorbent Assay (ELISA)

2.7.1 Measurement of CCL5 and CXCL10 protein release

Supernatants from treated HBE cells were collected and assayed on 96-well Immulon 4 plates, using matched antibody pairs and recombinant protein for human CCL5 and CXCL10.

All the steps in this procedure were performed at room temperature, using minimum incubation times. The plates were first coated overnight at room temperature with monoclonal anti-human CCL5 (2 μg/mL) or CXCL10 (3 μg/mL) antibody, diluted in 1X phosphate buffered saline (PBS). The following day the plates were washed four times with

ELISA wash buffer (0.05% Tween-20, diluted in 1X PBS; pH 7.4), before nonspecific binding sites were blocked with blocking buffer (1% BSA, 5% sucrose, diluted in 1X PBS) at 300

μL/well for 1 hour. After the blocking step the wells were washed again with ELISA wash

53 buffer prior to the addition of 100 μL/well of recombinant CCL5 (2000 pg/mL – 31.25 pg/mL) or CXCL10 (3000 pg/mL – 23.44 pg/mL), which were each dissolved in ELISA diluent (0.1% BSA, 0.05% Tween-20, dissolved in 1X Tris-buffered saline (TBS)), to duplicate wells. Samples were prepared with three serial dilutions (e.g. ½, ¼, ⅛) in ELISA diluent to ensure sample readings were within a linear range of the standard curve. Samples were then added to duplicate wells also at 100 μL/well, and allowed to incubate for 2 hours. Post- incubation, all the wells were washed with ELISA wash buffer and 100 μL/well biotinylated anti-human CCL5 (10 ng/mL) or CXCL10 (300 ng/mL) antibody was added and allowed to incubate for 2 hours. Post-incubation the plates were washed again with ELISA wash buffer and allowed to incubate with 100 μL/well of streptavidin peroxidase (1 μg/mL) for 30 minutes. Finally, the wells were washed again and developed with the addition of 100

μL/well 1:1000 H2O2/ABTS in citrate phosphate buffer (29.4 mM citric acid, 41.7 mM sodium phosphate, pH 4.3) for up to 30 minutes in the dark. The development reaction was terminated by the addition of 100μL/well of 2 mM sodium azide and absorbances were read at 405 nm using the Bio-Rad Benchmark microplate reader. Protein concentrations were determined through interpolation of the linear region of the standard curve (Bio-Rad

Microplate Manager II ver. 2.248). The sensitivities of both the CCL5 and the CXCL10 assays are stated by the manufacturer as being 30 pg/mL, respectively.

2.7.2 Measurement of CXCL8 protein release

Supernatants from treated HBE cells were collected and assayed on 96-well Immulon 4 plates, using matched antibody pairs and recombinant protein for human CXCL8 (in-house

54 assay). All the steps in this procedure were performed at room temperature, using minimum incubation times. The plates were first coated overnight at room temperature with polyclonal CXCL8 antibody (1:1200) diluted in 0.1 M carbonate buffer (pH 9.6). The following day, plates were washed four times with ELISA wash buffer before nonspecific binding sites were blocked with a blocking buffer (1:100 rabbit serum diluted in 1% BSA,

0.05% Tween-80, 1X PBS) at 100 μL/well for 30 minutes. After the blocking step, the wells were washed again with ELISA wash buffer and 100 μL/well of recombinant CXCL8 (7500 pg/mL – 59 pg/mL), which was dissolved in ELISA diluent (1% BSA, 0.05% Tween-80, dissolved in 1X PBS), was added to duplicate wells. Samples were prepared with three serial dilutions (e.g. ½, ¼, ⅛) in ELISA diluent to ensure sample readings were within linear range of the standard curve. Samples were then added to duplicate wells also at 100 μL/well, and allowed to incubate for 90 minutes. Post-incubation, all the wells were washed with ELISA wash buffer, and 100 μL/well biotinylated anti-human CXCL8 (1:1200 dissolved in ELISA diluent) antibody was added and allowed to incubate for 90 min. Post-incubation the plates were washed again with ELISA wash buffer and allowed to incubate with 100 μL/well of streptavidin peroxidase (1 μg/mL) for 30 minutes. Lastly, the wells were washed again and developed with the addition of 100 μL/well 1:1000 H2O2/ABTS in citrate phosphate buffer

(29.4 mM citric acid, 41.7 mM sodium phosphate, pH 4.3) for up to 15 minutes in the dark at

37°C. The development reaction was halted by the addition of 100μL/well of 2 mM sodium azide, and absorbances were read at 405 nm using the Bio-Rad Benchmark microplate reader. Protein concentrations were determined through interpolation of the linear region of the standard curve (Bio-Rad Microplate Manager II ver. 2.248). The sensitivity of the assay is stated by the manufacturer as being 60 pg/mL.

2.8 Multiplex Chemokine Array Assay (64-Plex)

The 64-chemokine multiplex protein assay was performed by Eve Technologies (Calgary, AB,

Canada) based on the MILLIPEX® MAP assay kit developed by Millipore (Billerica, MA, USA).

Briefly, supernatants from HBE cells treated with medium control or infected by HRV-16 were collected and centrifuged (200 x g, 8 minutes); the pelleted cellular debris was discarded and supernatants were placed into fresh tubes and sent for multiplex analysis. The samples were run against a panel of 64 different chemokines (see Table 2.3). Detection of the target analyte is dependent on binding to a fluorescent bead-capture antibody conjugate.

During an incubation period, the sample-bead conjugate interacts with a detection antibody cocktail that contains biotinylated antibodies specifically targeting individual target analytes. The react mixture is then incubated with Streptavidin-Phycoerythrin conjugates to complete the reaction at/on the surface of each individual microsphere bead. There can be up to 100 uniquely colored fluorescent bead sets; the samples were analyzed using the Bio-

Plex 200 apparatus (Bio-rad, Hercules, CA, USA). Individual analyte values and other assay details are available on Eve Technologies' website or in the Milliplex protocol.

Table 2.3: Multiplex chemokine assay targets

Sensitivity

Number Analyte (pg/mL)

1 6CKine 18.9

2 BCA-1 0.5

3 CTACK 0.8

4 EGF 2.7

5 ENA-78 7.0

6 Eotaxin 1.2

7 Eotaxin-2 3.7

8 Eotaxin-3 8.7

9 FGF-2 1.8

10 Flt-3 ligand 2.6

11 Fractalkine 6.0

12 G-CSF 0.5

13 GM-CSF 9.5

14 GRO 10.1

15 I-309 0.5

16 IFN- α2 2.9

17 IFN-γ 0.1

18 IL-1ra 2.9

19 IL-1α 3.5

20 IL-1β 0.4

21 IL-2 0.3

22 IL-3 2.1

23 IL-33 4.6

24 IL-4 0.6

25 IL-5 0.1

26 IL-6 0.3

27 IL-7 1.8

28 IL-8 0.2

29 IL-9 0.7

30 IL-10 0.3

31 IL-12 (p40) 7.4

32 IL-12 (p70) 0.4

33 IL-13 0.4

34 IL-15 0.4

35 IL-16 6.9

36 IL-17A 0.2

37 IL-20 25.6

38 IL-21 3.3

39 IL-23 28.6

40 IL-28A. 7.2

41 IP-10 0.3

42 LIF 4.2

43 MCP-1 0.9

44 MCP-2 3.0

45 MCP-3 2.0

46 MCP-4 6.7

47 MDC (CCL22) 3.7

48 MIP-1d 3.5

49 MIP-1α 4.5

50 MIP-1β 7.2

51 PDGF-AA 0.1

52 PDGF-AB/BB 7.3

53 RANTES 1.0

54 sCD40L 4.9

55 SCF 3.4

56 SDF-1a+B 55.8

57 TARC 0.4

58 TGFα 0.4

59 TNF- α 0.1

60 TNF-β 1.9

61 TPO 18.5

62 TRAIL 2.0

63 TSLP 2.2

64 VEGF 5.8

2.9 Filtration of HBE cell supernatants

Conditioned medium (both CM and HRV CM) from HBE cells were filtered through a PALL

Life Sciences MicrosepTM centrifugal filtration system, with a specified 30,000 MW cut off.

This was chosen as the molecular weight of HRV is estimated to be 8,500,000 MW234, allowing us to be confident that the filter will remove the virus. The filter membranes were first blocked through the use of a 0.5% BSA solution (dissolved in DMEM) that was allowed to incubate at room temperature with the filter before it was centrifuged through the device

(1000 x g, 10 min). Sterile HBSS was used to wash the filter membranes (1000 x g, 10 min) prior to the loading of supernatant samples. Samples were centrifuged (1000 x g, 10 min) and both the retentate and filtrate were collected and stored at -80°C until later use.

2.10 Migration of ASM

2.10.1 Migration in the micro chemotaxis chamber

A modified version of the Boyden Chamber235 known as the Neuro Probe AP48 48-well micro chemotaxis chamber (MCC) shown below (Figure 2.1), was initially selected as the gold- standard assay to measure migration; this decision was based on the longstanding publication track record validating the Boyden Chamber as a valid tool to assess cellular migration. The apparatus consists of four components: the bottom chamber where chemoattractants are added, the polycarbonate filter of a desired pore size that the cells must migrate through, a perforated cushion that holds the filter in place, and the top chamber where the cells are directly added in suspension. The cells in the assay are brought into

60 contact with the filter as they settle and if the chemoattractants are able to illicit a migratory response then the cells will change shape and migrate through the filter onto its lower side.

All conditions were performed in six-replicates for the samples in all experiments. The bottom wells of the apparatus were filled with 25 μL of the chemotactic stimulus of interest.

These wells were then covered by an 8-micron polycarbonate filter membrane that had been coated with a 0.1% Collagen I (rat tail) solution for 30 minutes. The filter membrane was then held in place by a perforated cushion and then the apparatus is assembled and allowed to incubate at room temperature for 15 minutes. The ASM cells were suspended via trypsinization and placed into the top wells of the chamber at 40,000 cells per well (50 μL of

800,000 cell/mL solution in DMEM). The chamber was then allowed to incubate for 4 hours at 37°C and 5% CO2. The experiment was concluded by the disassembly of the apparatus where the non-migratory cells were scraped off the top of the filter membrane and migratory cells were fixed and stained using the Diff-quick stain solution. Three randomized visual fields were counted at 200x magnification across the six-replicates per condition before the counts were averaged.

The benefit of this approach is that it allows for the counting of individual cells post- migration, and to clearly visualize differences (differences in cells migrating through) between experimental conditions. However, the technique is limited in that it does not provide information about migration in real-time, and running concurrent experiments on a single plate for different durations is not possible. In addition, this assay proved to be sensitive to static forces, due to the dry ambient atmosphere in the laboratory, whereby the

61 filter membrane would sometimes fold upon itself during the staining protocol and this would necessitate that the experiment be discarded.

a Top of Boyden Chamber

50 μL wells b Perforated cushion

Filter

25 μL wells

Figure 2.1: The Neuro Probe AP48 48-well micro chemotaxis chamber (MCC). (a) A top down schematic view of the MCC. Cells in suspension are added, and conditions are replicated six times to allow for consistency. (b) A side schematic view of the MCC highlighting how the chamber comes together. Cells are added to the 50 μL wells, chemoattractants are added to the 25 μL wells.

2.10.2 Chemotaxis via the xCELLigence Real Time Cell Analyzer (RTCA) system

To address some of the limitations of the MCC, the xCelligence Real-Time Cell Analyzer

(RTCA) system (Figure 2.2) was selected as a complimentary assay to measure ASM cell migration. The main migration chamber resembles the 48-well modified chemotactic

62 chamber, as described above but it uses a filter membrane lined with gold-plated microelectrodes to detect migration in real-time. As with the modified Boyden Chamber, migrating cells move through the porous membrane and adhere to the bottom of the filter.

However, in the RTCA system, this adhesion of migratory cells is recognized as increased impedance for the alternating current that runs through the plate. As a result, the apparatus is able to detect small changes in cellular adhesion, and can thus quantify, in real time, the migration of cells via its docking station and software.

The system utilizes Cellular Invasion and Migration (CIM) plates that mimic the design of a modified Boyden Chamber. The top and bottom wells are separated by a polyethylene terephthalate (PET) 8-micron porous membrane that is coated with a 0.1% Collagen I (rat tail) solution for 30 minutes. The membrane is then rinsed with HBSS and the chemotactic stimuli of interest are pipetted in duplicate into the wells in the bottom chamber of the CIM plate (165 μL/well), after which the apparatus is assembled. To moisten the filter and to facilitate migration, the top wells are partially filled with the negative control (DMEM; 50

μL/well), and the plate is allowed to incubate for 1 hour at 37°C at 5% CO2. Post-incubation, a background scan of the plate is performed by the RTCA Software (2.0), whereby each well is zeroed to ensure subsequent standardization and consistency in measurements. For the specific experiments described in this thesis, ASM cells were then suspended via trypsinization and added to the wells in the top chamber of the CIM plate at 80,000 cells/well

(100 μL of 800,000 cells/mL solution in DMEM). The plate was then incubated at room temperature for 15 minutes prior to being placed into the RTCA apparatus for migration

63 measurement for 4 hours at 37°C and 5% CO2. Migration impedance data were normalized at the earliest time point to control for differences in well loading and cell settling.

a b

Figure 2.2: The xCELLigence Real-Time Cell Analyzer (RTCA) system. The core components of the RTCA system are the laptop with RTCA software (a), the cellular-invasion and migration (CIM) plates (b), and the 3-plate reader (c). A cross section of the CIM plates

(d) is also present. Adapted from Bird236.

One of the potential advantages of the RTCA system is that it allows for a more comprehensive and integrated migration assay protocol. Figure 2.3 illustrates the migration of ASM cells in response to 300 ng/mL of PDGF-AB (serving as a positive control) over a 12- hour time period. Additionally, some of the advantages and disadvantages of the RTCA system as compared to the MCC are listed below in Table 2.4.

Figure 2.3: Migration of ASM cells to PDGF-AB in the RTCA system. A migration plot illustrating cell index (indirect measure of cell migration in the RTCA) over 12h of a singular representative ASM cell sample was provided to demonstrate migration plateauing around

4h.

Table 2.4: Comparison between MCC and RTCA systems

System Advantages Disadvantages

Boyden Proven technology Only end-point assay

Direct cell counts Cell counting can be subject

to error

Six replicates of each No check of cell viability

condition

Relatively inexpensive Difficulty handling apparatus

(such as filter paper)

RTCA Real-time data Expensive

Measures cell viability Indirect cell counts

Counts all cells migrating Difficult to visualize under

microscope

High throughput

Can normalize to account

for cell loading errors

2.10.3 Determination of chemotaxis versus chemokinesis

To examine whether the migratory response of ASM cells was chemotactic (directional) or chemokinetic (random, non-specific movement), experiments were designed to eliminate the chemotactic gradient in the xCELLigence RTCA system. Various chemotactic stimuli were added to the wells of the bottom chamber of CIM plates (165 μL) in duplicate. The PET 8- micron filter was coated with a 0.1% collagen I (rat tail) solution for 30 minutes, prior to being washed with HBSS. The chamber was assembled and either the negative control condition (DMEM) or experimental chemotactic stimulus condition were added to the top wells. The chemotactic gradient was considered preserved in conditions where the negative control was added to the top wells, but was considered abolished if the same chemotactic stimulus was present in both the top and bottom wells. The chamber was allowed to incubate at 37°C at 5% CO2 for 1 hour, prior to being zeroed by the RTCA software (v2.0). The ASM cells were trypsinized and added to the wells in the top chamber of the CIM plate at 80,000 cells/well, and allowed to incubate for 15 minutes prior to being placed into the RTCA system for 4 hours at 37°C and 5% CO2. Migration impedance data were normalized at the earliest time point to control for differences in well loading and cell settling.

2.10.4 ASM pertussis toxin treatment

To examine what whether migration of ASM cells to HBE supernatants was dependent on

GPCRs, pertussis toxin (PTX) experiments were utilized, on the basis that PTX acts to prevent the subunits of the Gi pathway from inhibiting adenylyl cyclase activity. Thus, ASM cells were

68 grown to confluence in 75 cm2 tissue culture flasks prior to being treated with 100 ng/mL of

PTX or medium control for 12 hours at 37°C and 5% CO2. Immediately following treatment the ASM cells washed with HBSS and were trypsinized and used for migration in the xCELLigence RTCA system.

2.10.5 β-agonist treatment of ASM

To examine the role of the Gs pathway of GPCR signally, the β-agonist formoterol was used on the basis of its ability to strongly activate a Gs pathway as well as its well established reputation as an effective asthma therapy. ASM cells were grown to confluence in 175 cm2 tissue culture flasks prior to being washed with HBSS, and were trypsinized and suspended in 2 mL DMEM at 800,000 cells/mL. The solution was then divided into 1 mL aliquots where one aliquot was treated with formoterol (100 nM) treatment and the other aliquot was treated with the DMSO (0.01%) vehicle control. The ASM cells were then added to top chamber of the xCELLigence RTCA CIM plate, at 80,000 cells/well and allowed to incubate for 15 minutes prior to being placed into the RTCA system for 4 hours at 37°C and 5% CO2.

Migration impedance data were again normalized at the earliest time point to control for differences in well loading and cell settling.

2.10.6 ASM treatment with adenylyl cyclase agonist

To examine the role of adenylyl cyclase activity in modulating the migratory capacity of ASM cells NKH-477 was used, which is a water soluble analogue of forskolin and thus allowed us

69 to use water as the vehicle control as opposed to DMSO. ASM cells were grown to confluence in 175 cm2 tissue culture flasks prior to being washed with HBSS; they were then trypsinized and suspended in 2 mL DMEM at 800,000 cells/mL. The solution was then divided into 1 mL aliquots where one aliquot was received NKH-477 (10 μM) treatment and the other aliquot received medium control. ASM cells were then added to top chamber of the xCELLigence

RTCA CIM plate at 80,000 cells/well and allowed to incubate for 15 minutes, prior to being placed into the RTCA system for 4 hours at 37°C and 5% CO2. Migration impedance data were normalized at the earliest time point to control for differences in well loading and cell settling.

2.10.7 ASM treatment with 8-Br-adenosine 3′, 5′-cyclic monophosphate (cAMP)

To further expand upon the role of Gs signaling and intracellular cAMP in modulating the ability of ASM cells to migrate to supernatants from HRV infected HBE cells, a cAMP analogue was used. ASM cells were grown to confluence in 175 cm2 tissue culture flasks prior to being washed with HBSS, and were trypsinized and suspended in 2 mL DMEM at 800,000 cells/mL.

As before, the solution was then divided into 1 mL aliquots, where one aliquot was received

8-Bromoadenosine 3′, 5′-cyclic monophosphate (8-Br-cAMP; 50 μM) treatment and the other aliquot received medium control. The compound 8-Br-cAMP was chosen because it is both cell permeate and resistant to degradation by cAMP phosphodiesterase. The cells were added to top chamber of the xCELLigence RTCA CIM plate at 80,000 cells/well and allowed to incubate for 15 minutes prior to being placed into the RTCA system for 4 hours at 37°C

70 and 5% CO2. Migration impedance data were normalized at the earliest time point to control for differences in well loading and cell settling.

2.10.8 CCL5 inhibition in conditioned medium

To investigate the ability of the chemokine CCL5 to modulate the migration of ASM cells to condition medium from HRV infected HBE cells, an anti-CCL5 antibody (R&D Systems) was utilized to block the chemokine in the medium (thus preventing it from interacting with its receptor). Conditioned medium (CM and HRV CM) from experimentally treated HBE cells was subjected to monoclonal anti-human CCL5 antibody or IgG1 isotype control (R&D

Systems) at 140 ng/mL for 1 hour at room temperature. These conditioned media were then used as chemoattractants, and loaded in duplicate at 165 μL/well into the bottom chamber of the xCELLigence RTCA system CIM plate. Additionally, ASM cells were grown to confluence in 175 cm2 tissue culture flasks prior to being washed wish HBSS, and were treated with mouse serum (10%, dissolved in DMEM), to block the possible interaction between the ASM cells and the mouse antibodies, for 15 minutes. ASM cells were then trypsinized and added to the wells in the top chamber of the CIM plate at 80,000 cells/well, after which the plate was allowed to incubate for 15 minutes prior to being placed into the RTCA system for 4 hours at 37°C and 5% CO2.

2.10.9 Quantification of cell viability

The viability of ASM cells, following various experimental interventions, was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The assay assesses the mitochondrial dehydrogenase enzyme (succinate dehydrogenase) of viable cells in its ability to cleave the tetrazolium rings of MTT. The MTT compound is originally yellow in color but, following cleavage by the succinate dehydrogenase, it forms dark purple formazan crystals. The number of surviving cells have been previously shown to be directly proportional to the amount of formazan formed237.

After various experimental interventions, the cellular (either HBE or ASM) supernatants were aspirated off and the MTT reagent (1 mg/mL, dissolved in HBSS) was added (500

μL/well in 6-well plates) and incubated at 37°C for 90 min. The soluble reagent was then aspirated and the formazan crystals were then solubilized by the addition of DMSO (1 mL/well in 6-well plates). A small sample (100 μL) was then added to a 96-well plate and the color was quantified through spectroscopic analysis using the Bio-Rad Benchmark microplate reader. The optical density units (ODU) were measured at 570 nm for all the samples and the percent viability was calculated as per the following equation:

�� = ×100% �� (��/��ℎ�� )

2.11 Data management

The data obtained from ASM chemotaxis experiments conducted in the RTCA system were normalized in the RTCA Software (2.0) at the first measured time point prior to averaging the cell index values from duplicate wells. These data were then transferred into GraphPad

Prism 6 software (GraphPad Software, CA, USA) for data storage and statistical analysis.

Cell counts from the MCC were conducted as described (section 2.10.1) and also compiled and recorded into GraphPad Prism 6. All data files were backed-up regularly (>1 week) through the utilization of an enterprise back-up solution Time Machine (Apple, CA, USA) that utilized a personal external hard drive as well as a complimentary back-up through the cloud data management system OneDrive (Microsoft, CA, USA). A one-way ANOVA was performed with the Newman-Keuls post-hoc comparison to establish significance for all the data graphs presented in this thesis (*p<0.05, **p<0.01, ***p<0.001).

3 CHAPTER THREE: RHINOVIRUS INFECTION OF THE AIRWAY EPITHELIAL CELLS

DRIVES CHEMOTAXIS OF AIRWAY SMOOTH MUSCLE CELLS

3.1 Introduction

The human airway epithelium is the main interface between the lung parenchyma and the external environment. Therefore, it has important roles not only as both a physical barrier and a modulator of host defense, but also as a key homeostatic regulator. Although the pathogenesis of asthma remains to be fully elucidated, several studies have pointed to the epithelium as having an important influence in this process125,137,238. Airway remodeling in a key facet of asthma as discussed above and HRV infections have been highlighted as a pathogen that may play an important role. As HRV principally infects the airway epithelium119,138 it seems reasonable to assume that the epithelium’s response upon infection may be inducing remodeling.

ASM in healthy individuals circumferentially surrounds the lumen of the airways196, and serves to provide the dual functions of developing/maintaining the ECM protein environment, as well as maintaining tissue contraction and elasticity197. In asthmatic airways, one of the most distinct changes is the increased smooth muscle mass present and its increased proximity to the epithelium37,200. ASM migration is not unprecedented, being fundamental to organ development204 and it has been implicated in many diseases states such atherosclerosis205, asthma204, and restenosis206 for impacting negatively on disease pathogenesis. Given that i) remodeling occurs early in age, ii) HRV infections are associated with an increased risk for developing asthma, iii) previous data indicated that HRV infection of HBE cells facilitates the release of airway remodeling mediators, and iv) studies have demonstrated an increase in ASM mass in the airways of asthmatic individuals, we now

75 sought to investigate whether HRV infection of the airway epithelial cells resulted in the production of chemoattractant mediators that would induce ASM migration.

3.2 Hypothesis

HRV infection of the human airway epithelium results in the release of chemotactic mediators that drive ASM migration.

3.3 Results

3.3.1 Establishing a positive migratory stimulus

It was essential to first establish the migratory behavior and dynamics of the ASM cells to be used for this project. Thus, we used platelet-derived growth factor (PDGF-AB) as a positive control mediator when assaying ASM migration; it has been previously established as a potent chemotactic stimulus for ASM cells and other mesenchymal cell types239,240. As the

MCC is an endpoint assay a 4 hour migratory period was chosen, which has been shown to be the optimal time of migration for ASM cells based on studies from several other groups226,241. Migration in the RTCA was assessed for a total of 12 hours. Both systems, the

MCC (Figure 3.1a) and the RTCA (Figure 3.1b) systems demonstrated clearly that PDGF-AB is able to significantly drive migration of ASM cells. In addition, the RTCA system confirmed a plateau in migration capacity of the ASM cells at the 4 hour time point (Figure 2.3), matching closely with previous studies as mentioned above. A concentration of 300 ng/mL

76 of PDGF-AB proved to induce significantly more migration in both the MCC (p<0.001) and the RTCA system (p<0.05).

a b

Figure 3.1: Determination of the experimental parameters for ASM migration. (a) 4h migration of ASM cells to various concentrations of PDGF-AB in the MCC, represented as fold increase of migratory behavior as compared to medium (negative) control (expressed as 1).

PDGF-AB at 300 ng/mL was most effectively able to drive ASM migration in the MCC (n=4; p<0.001). Migration in the RTCA system to PDGF-AB was also evaluated at 4h for comparison to the MCC (a) and 300 ng/mL was again most effectively able to drive ASM migration (n=5; p<0.05).

3.3.2 Conditioned medium from HRV infected HBE cells drives ASM migration

Stocks of combined supernatants from HBE cells treated with medium control (CM) or HRV-

16 for 24 hours (as described in Chapter 2) were obtained and used as chemoattractants in both the MCC and the RTCA system. The CM was able to drive a migratory response that was higher than the medium alone (serving as a negative control); however the HRV CM condition was able to drive migration at a significantly higher rate over the 4 hour migratory period in both the MCC (p<0.05; Figure 3.2a) and the RTCA system (p<0.001; Figure 3.2b).

This indicates that while HBE cells constitutively release modulators that moderately drive

ASM migration, HRV-16 infection of HBE cells results in an increased or differential production of chemoattractant mediators that significantly enhance ASM migration. In addition, the HRV-16 virus alone, innoculated into the DMEM medium control, was not able to significantly drive migration of the ASM cells. These data indicate that ASM migration to the supernatants from HBE cells is dependent upon viral infection of the HBE cells themselves and not simply upon the interaction of the ASM cells with free virus in suspension. In addition, the similarity in data found in both the MCC and the RTCA system led to the validation of the RTCA system as an assay to measure ASM migration and as such it was used for all future migration experiments.

a 2.0 b 4

*** 1.8 *** * *

3 1.6

1.4

Over Medium Control 2 Over Medium Control Fold Increase In Migration Fold Increase In Migration 1.2

1.0 1 CM CM Medium Medium HRV CM HRV CM PDGF-AB HRV virion Figure 3.2: Conditioned Medium from HRV infected HBE cells drives ASM migration.

Migration of ASM cells was over 4h was assessed in the MCC (a) to conditioned medium from either medium treated (CM) or HRV-16 infected (HRV CM) epithelial cells (n=3, *p<0.05). In the RTCA system (b), ASM cell migration was additionally assessed to PDGF-AB (positive

5.5 control, 300 ng/mL) and the HRV virion (10 TCID50 U/ml) (n=3). Although CM was able to drive migration greater than medium control, HRV CM was significantly higher in both (a) the MCC and (b) the RTCA assays (***p<0.001, *p<0.05).

3.3.3 Abolishing the chemotactic gradient attenuates ASM migration

To examine whether the migratory response of the ASM cells, shown above, was chemotactic

(directional) or chemokinetic (random, non-specific movement), an experiment was designed to eliminate the chemotactic gradient in the xCELLigence RTCA system. Various chemotactic stimuli were added into the wells of the bottom chamber of CIM plate (165

μL/well) in duplicate (as per protocol in Chapter 2). However, when the experiment was set up, either the negative control condition (DMEM) or an experimental chemotactic stimulus condition was added to the top wells in addition to the ASM cells. The chemotactic gradient was considered preserved in conditions where the negative control was added to the top wells but was considered abolished if the same chemotactic stimulus was present in both the top and bottom wells. As seen in Figure 3.3, while both CM and HRV were able to drive ASM migration as compared to the medium control, both conditions were significantly (p<0.001) attenuated when there was no gradient present. In addition, while there was a significant difference between CM and HRV CM when the chemotactic gradient was present, this difference was abolished when the gradient was eliminated. These data indicate that the

ASM cells are migrating directionally, i.e. chemotactically, and in response to the gradient created by either the CM or HRV CM conditions.

3.0 *** *** *** ***

2.5

2.0 Over Medium Control

Fold Increase In Migration 1.5

1.0 Medium + - - - - CM - + + - - HRV CM - - - + + No Gradient - - + - +

Figure 3.3: ASM migration to conditioned medium from HRV infected HBE cells is chemotactic and directional. Migration of ASM cells over 4h was assessed in the RTCA system to conditioned medium from either medium treated (CM) or HRV-16 infected (HRV

CM) epithelial cells, with either the gradient present (grey bars) or the gradient absent (black bars) (n=3, ***p<0.001). Although CM and HRV CM are both able to drive migration, this migration is significantly attenuated when the gradient is abolished.

3.3.4 Presence of viral capsid is not sufficient for migration

While the migration seen in the previous experiments was most likely due to the release of chemotactic mediators from HBE cells following HRV infection, it is still possible that the

HRV-16 virus in suspension (present in the HRV CM) could be acting as a modulator of chemotactic behavior. While it was not able to drive migration on its own (Figure 3.3), the question remained whether removing it from the conditioned medium altogether would alter the migratory response of the ASM cells. As per the protocol in Chapter 2, centrifugal filtration devices were used to remove >30,000 MW particles from the supernatants collected from both the CM and HRV CM conditions. There was initial concern that the filtration process could result in a loss of smaller molecules as well but assessment of CXCL8 protein (MW 11,098) both pre and post filtration (Figures 3.4a and 3.4b) indicated that most of the smaller chemotactic molecules had been retained.

a b 6000 100

82% 79% 80

4000

2000 filtration (%) post Percent CXCL8 remaining remaining CXCL8 Percent CXCL8 Protein (pg/mL) 20

0 0 CM + + - - CM Filtered HRV CM Filtered HRV CM - - + + Filtered - + - +

Figure 3.4: CXCL8 levels post-filtration of conditioned medium from medium treated

(CM) or HRV-16 infected (HRV CM) HBE cells. Both CM and HRV CM were passed through a 30,000 MW filter and (a) CXCL8 levels were assessed via ELISA and (b) displayed as percentage of levels prior to filtration(see Chapter 2). Though CXCL8 levels were reduced post filtration, approximately >79% was retained, suggesting that a majority of smaller chemotactic molecules would also be retained post-filtration.

Both filtered and unfiltered CM and HRV CM were used to examine their effects on ASM chemotaxis (Figure 3.5). Notably, filteration did not alter the ability of HRV CM to drive ASM migration but significantly altered the ability of CM alone to drive ASM migration (p<0.001).

This indicates that the presence of some larger, constitutively secreted molecules could be driving the ASM cell’s migratory response in the CM condition and also demonstrates the

HRV-16 virion itself plays no role in the HRV CM’s ability to drive chemotaxis. In contrast, it

83 seems likely that the HRV CM response is driven in large part by small molecular mediators, such as chemokines, and further suggests the mechanism of action may be through GPCRs, though this remains to be properly elucidated.

4 NS

*** 3 Over Medium Control Fold Increase In Migration 2

1 Medium + - - - - - PDGF-AB - + - - - - HRV CM - - + + - - CM - - - - + + Filtered - - - + - +

Figure 3.5: Removal of virion from conditioned medium from HRV-16 infected HBE cells does not attenuate migration. Migration of ASM cells over 4h was assessed in the

RTCA system to PDGF-AB (300 ng/mL), conditioned medium from either medium treated

(CM), HRV-16 infected (HRV CM) epithelial cells, and to filtered CM and HRV CM (white bars)

(n=3, ***p<0.001). There was no significant difference between HRV CM pre-and-post filtration.

3.3.5 Migration is dependent on duration of infection

Having observed that HRV CM was consistently able to drive migration at significantly higher levels than CM (Figure 3.2, 3.3), we then asked whether the duration of HRV-16 infection of

HBE cells influenced this outcome. Thus, HBE cells from multiple donors were infected for periods of 3, 9, 15 and 24 hours prior to collecting the supernatants and utilizing them as chemoattractant media in the RTCA system (Figure 3.6). While the ASM chemotaxis was greater in response to the CM condition as compared to the medium control, the HRV CM greater migration at 3 and 9 hours, and significantly greater migration at 15 and 24 hours

(p<0.001; Figure 3.6). As mentioned previously (see Chapter 1), the responses elicited from

HBE cells following HRV infection can be classified as a result of viral binding or viral replication. However, these data indicate that the ASM chemotaxis induced by HRV infection of HBE cells is most likely due to viral replication, and that viral binding alone is not sufficient to promote ASM migration.

*** ***

2 Over Medium Control Fold Increase In Migration

1 Medium + ------PDGF-AB - + - - - - - CM (24h) - - + - - - - HRV CM (3h) - - - + - - - HRV CM (9h) - - - - + - - HRV CM (15h) - - - - - + - HRV CM (24h) ------+

Figure 3.6: The ability of conditioned medium from HRV infected epithelial cells to drive ASM chemotaxis depends upon the duration of infection. Migration of ASM cells over 4h was assessed in the RTCA system in response to PDGF-AB (300 ng/mL), conditioned medium (CM; 24h) or HRV-16 infected HBE cell conditioned medium (HRV CM) at multiple time points (3, 9, 15, 24h; n=4, ***p<0.001). Although the CM was able to drive modest ASM migration, HRV CM was able to do so at significantly greater levels at 15h and 24h, indicating a likely dependence on viral replication within the HBE cells.

3.3.6 Viral infection of HBE cells is necessary to drive migration

While Figure 3.6 was able to show that the duration of infection is important for the ability of supernatants from HRV infected HBE cells to drive ASM chemotaxis, it was not definitive in demonstrating the need for viral replication. To evaluate whether HRV-16 replication within the HBE cells was necessary to drive ASM chemotaxis, HRV-16 was rendered replication deficient by treatment with high-intensity UV-light (see Chapter 2). This replication deficient HRV (UV-HRV) was then used to infect HBE cells prior to their supernatants being harvested and used as chemoattractant media. UV-inactivation of HRV-

16 prior to infection resulted in significantly decreased ASM migration as compared to the normal HRV CM condition (Figure 3.7; p<0.05). These data indicate that viral binding and internalization alone by the HBE cells is not sufficient for ASM chemotaxis.

** 6 *

3 Over Medium Control Fold Increase In Migration 2

1 Medium + - - - PDGF-AB - + - - HRV CM - - + - UV-HRV CM - - - +

Figure 3.7: Replication deficient HRV-16 is unable to drive a response in HBE cells that stimulates ASM chemotaxis. Migration of ASM cells over 4h was assessed in the RTCA system in response to PDGF-AB (300 ng/mL) or conditioned medium from either HRV-16

(HRV CM) or UV-inactivated HRV-16 (UV-HRV CM) infected epithelial cells (n=3, **p<0.01,

*p<0.05). Although UV-HRV CM was able to drive migration to modest levels compared to medium control, it was significantly less than the HRV CM indicating the HRV-16 replication within the HBE cells is necessary to maintain the conditioned medium’s ability to drive ASM chemotaxis.

3.4 Discussion

HRV infections in early age are very common242 and have been implicated in the development of asthma3,242 (see Chapter 1). In addition, the epithelium is an important regulator of the immune response in the airways243, and its dysfunction has been postulated to play a role in the development of asthma125. Importantly, pathological changes in the smooth muscle tissue in the airways are amongst the most commonly seen differences between asthmatics and healthy individuals63,203,244.

In this chapter, we demonstrated for the first time that HRV-16 infection of primary HBE cells leads to the release of factors that drive ASM cell migration. In addition, we are the first group to employ the xCELLigence RTCA system to monitor ASM cell migration in real-time.

This novel utilization of the system allowed us to demonstrate that maximal ASM migration does plateau at approximately the 4 hour time period, though it does continue to occur up to

12 hours. Moreover, we demonstrated that while the CM and HRV CM were able to drive migration independently, abolishing the chemotactic gradient resulted in significant decreases to their ability to do so. These data support the argument that the ASM migration was chemotactic and directional in nature, and not simply chemokinetic.

Once it was established that HRV CM was able to drive ASM migration we sought to determine whether the presence of HRV in the medium alone was able to drive or modulate

ASM migration. The virus itself was not able to drive ASM chemotaxis in the RTCA system, and additional experiments aimed at removing the virus from the medium via centrifugal

89 filtration indicated that the virus does not play a direct role on ASM, in terms of modulating the observed chemotaxis. Thus, while it was evident that HRV-16 contact/infection was not the vehicle for driving the migration of ASM cells, the question remained whether the observed chemotaxis was being driven through viral binding to the HBE cells alone, or through internalization and replication within the HBE cells (see Chapter 1). A short- duration (3 h) infection of HBE cells with HRV-16 was not able to significantly drive ASM migration compared to the CM (24 h) condition, while longer-duration infections (15 h, 24 h) were able to drive a robust and significantly higher migratory response. Moreover, the inability of UV-treated replication deficient HRV-16 to drive a robust chemotactic response through UV-HRV CM (as compared to HRV CM) clearly demonstrated that viral binding alone was not sufficient to drive ASM migration, and that viral internalization and replication was necessary to drive ASM migration.

Given the validity and repeatability of the xCELLigence RTCA systems results compared to the MCC, which has previously been used by many other studies225,226,245, we elected to continue this program of research using the the xCELLigence RTCA system to further assess

ASM cell migration.

In summary, the data presented in this chapter describe the novel observation that HRV infection of airway epithelial cells results in chemotaxis of ASM cells in vitro. UV-HRV conditioned medium was unable to drive ASM chemotaxis, indicating that this phenomenon requires viral internalization and replication inside the HBE cell to occur. The focus of the

90 following chapters is to now further elucidate the cellular mechanisms by which HRV infection of HBE cells is able drive this observed ASM chemotaxis.

4 CHAPTER FOUR: HRV-16 INDUCED, HBE CELL MEDIATED, MIGRATION OF ASM

CELLS IS REGULATED BY G-PROTEIN COUPLED RECEPTORS, AND DEPENDENT ON

CCL5

4.1 Introduction

In the previous chapter, we demonstrated that HRV infection of HBE cells resulted in a supernatant that drove chemotactic ASM cell migration. There are many different chemotactic mediators that have previously been detected following HRV infection of HBE cells (see Chapter 1), and more than one may be playing a key role in inducing ASM cell chemotaxis. Based on our experimental data in Chapter 3, in which HRV in the medium alone was unable to drive ASM chemotaxis, and the fact that filtration of the medium from the HRV- infected HBE cells did not impact on this cell migration, we now questioned whether HRV infection of HBE cells could result in the production of small molecule chemokines (<30,000

MW). Moreover, we questioned whether these chemokines might likely act via G-protein coupled receptors (GPCRs) present on ASM cells. Thus, we designed a series of experiments to i) identify the chemokines necessary for HRV-induced, HBE cell mediated, ASM cell migration, and ii) to determine the potential role of GPCRs in the ability of HRV in the ability of HRV infection of HBE cells to drive ASM migration.

Many chemotactic factors are known to regulate cell migration through GPCRs246. The discovery that intervention with Bordella pertussis toxin (PTX) on cells blocks the ability of

CXCL8 to drive neutrophil chemotaxis led to hypothesis that its receptor is coupled to

247 heterotrimeric Gi proteins . This paradigm that has since been broadly confirmed in studies of other receptors to other established chemokines as well. The activated Gαi subunits associated with these Gi heterotrimeric proteins inhibit adenylyl cyclase, while also

248 initiating cell migration . However, although initial studies indicated that Gαi subunits

93 were the mechanistic drivers behind cellular migration, it has been since demonstrated that

249 receptors can induce chemotaxis by utilizing the βγ subunits instead . Nevertheless, Gs and

Gq, both of which do not inhibit adenylyl cyclase, are unable to drive chemotaxis even when expressed on cells that are capable of migration 248. This indicates that the βγ subunits may be necessary, but are not be sufficient for chemotaxis, and that cAMP regulation may be playing a larger role in this process.

4.2 Hypothesis

The overarching hypothesis for the work detailed in this chapter is that HRV-16 induced migration of ASM cells is regulated, in part, through the release of one or more chemokines from HBE cells post HRV infection, and that the mechanisms by which this chemotaxis occurs is likely GPCR dependent and can be regulated by intracellular cAMP levels.

4.3 Results

4.3.1 Pertussis toxin treatment prevents ASM chemotaxis to HRV CM

Before undertaking experiments to determine the exact chemotactic mediator(s) that might be driving the migratory response in the ASM cells (as seen in Chapter 3), it was necessary to establish what intracellular signaling pathways might be involved in this process. To do so, we utilized PTX to evaluate its effect in blocking the HRV-16 driven, HBE cell mediated,

ASM chemotaxis. As described previously, ASM cells were grown to confluence T75 flasks prior to being treated with 100 ng/mL of PTX or medium control for 12 hours at 37°C and

5% CO2, prior to being used for migration in the xCELLigence RTCA system (see Chapter 2).

ASM cell viability, following PTX treatment, was assessed using trypan blue and erythrocin

B; no differences were found between the medium treated and PTX treated conditions thereby indicating that the given concentration of PTX was non-cytotoxic.

Results from these initial experiments indicated that the HRV-infected HBE supernatant- driven ASM chemotaxis is mediated, at least in part, by a GPCR-based mechanism.

Specifically, PTX treatment was able to significantly attenuate the chemotactic response of the ASM cells to HRV CM when compared to the untreated condition (Figure 4.1).

Interestingly, migration to PDGF-AB was not effected by the PTX treatment, which is most likely because PDGF-AB acts primarily through a RTK pathway independent of GPCRs. As

PTX targets the Gαi pathway, it substantiates our premise that the mediators present in the

HRV CM are small molecule chemokines that act through Gi based GPCRs in particular.

3.0

*** 2.5

2.0 Over Medium Control

Fold Increase In Migration 1.5

1.0 Medium + + - - - - PDGF-AB - - + + - - HRV CM - - - - + + PTX - + - + - +

Figure 4.1: HRV induced ASM migration is primarily GPCR mediated. Migration of ASM cells pre-treated (12h) with either medium control or PTX (100 ng/mL) was assessed in the

RTCA system to PDGF-AB (300 ng/mL) and to conditioned medium from HRV-16 infected

HBE cells (HRV CM) over 4h (n=3, ***p<0.001). Migration of the ASM cells to the HRV CM was significantly attenuated following PTX treatment, indicating that the HRV CM likely utilizes mediators that act through a GPCR based mechanism.

4.3.2 Stimulation of the Gαs pathway via formoterol attenuates migration

In Figure 4.1 we demonstrated PTX was able to significantly attenuate ASM cell migration, likely by inhibition of a Gi pathway. We therefore asked the question as to whether stimulation of a Gs pathway, through a β2-agonist, is able to attenuate migration; this has clinical relevance as β2-agonists are widely used in the treatment of asthma and may have an impact on the potential treatment of airway remodeling. Thus, ASM cells were pre-treated with to formoterol (100 nM) or the DMSO (0.01%) vehicle control for 15 minutes prior to migration assays being run in the RTCA system. ASM cell viability was assessed via the MTT assay at the end of the 4 hour experiment (Figure 4.2b; see Chapter 2).

The results indicate that the chemotactic response of ASM cells are significantly attenuated by formoterol pre-treatment in both the CM and HRV CM experimental conditions (Figure

4.2a). These data support the premise that this ASM chemotaxis is driven through a pathway that is likely sensitive to cAMP. In particular, activation of Gαs by formoterol was able to abolish migration induced by both CM and HRV CM (p<0.001). Surprisingly, although PTX was unable to affect the ability of PDGF-AB to driven ASM chemotaxis (Figure 4.1), pre- treatment of ASM cells with formoterol was able to significantly attenuate this chemotactic response (Figure 4.2a). This suggests that both RTK- and GPCR mediated migration are likely to be regulated by shared pathways. Neither the formoterol nor the DMSO treatments had any effect on ASM cell viability (Figure 4.2b).

a b 6 150 ***

100 4 ***

Over Medium Control 50 Fold Increase In Migration

2 hours) (4 Viability Percent

1 Medium + + ------0 PDGF-AB - - + + - - - - DMEM DMSO Formoterol CM - - - - + + - - HRV CM ------+ + Formoterol - + - + - + - +

Figure 4.2: β2-agonist treatment abolishes the ability of HRV CM to drive ASM chemotaxis. (a) Migration of ASM cells pre-treated with either DMSO or formoterol (100 nM) over 4h was assessed in the RTCA system to the following chemoattractants: PDGF-AB

(300 ng/mL); conditioned medium from medium treated (CM) or HRV-16 infected HBE cells

(HRV CM) (n=3, ***p<0.001). Migration of ASM cells to the HRV CM was significantly attenuated following formoterol treatment, indicating that the HRV CM induced migration is dependent on a pathway that is regulated by downstream signalling by the Gs receptor. (b)

ASM cell viability 4 hour post treatment with DMEM, DMSO, or formoterol (100 nM) was assessed through the MTT assay.

4.3.3 Direct stimulation of adenylyl cyclase attenuates ASM cell migration

Having confirmed that ASM chemotaxis to HRV-infected HBE cell supernatants is regulated by both Gi (Figure 4.1) and Gs (Figure 4.2) in opposing ways, we next sought to determine which downstream signaling event could be tying the two pathways together. It is important to note that blocking the Gi pathway’s ability to inhibit adenylyl cyclase with PTX attenuated migration to approximately the same extent as using formoterol as a Gs agonist to stimulate adenylyl cyclase activity. Thus, we reasoned that the logical meeting point for the pathways is adenylyl cyclase, and therefore elected to use an agonist for adenylyl cyclase, namely NKH-

477 (which is a water-soluble forskolin analogue) to investigate this hypothesis. Thus, ASM cells that were grown to confluence were pre-treated with either NKH-477 (10 μM) or the medium control for 15 minutes prior to being utilized for migration assay experiments in the

RTCA system. ASM cell viability was assessed via the MTT assay the end of the 4 hour experiment.

These data confirm the findings from the previous experiments (Figure 4.2) that indicated that ASM cell migration to supernatants from HRV-infected HBE cells is at least partially regulated by cAMP. Specifically, it was demonstrated that short-term (15 min) activation of adenylyl cyclase (15 min pre-treatment with NKH-477) prior to migration was sufficient to abrogate the migratory capacity of the ASM cells (Figure 4.3a). The main role of adenylyl cyclase is to generate cAMP from ATP, which then modulates multiple downstream signaling events. These data indicate that cAMP is likely to be closely involved in regulating ASM

99 migration to HRV infected HBE cell supernatants. Cellular viability was not affected by NKH-

477 treatments over the 4 hour duration of the experiment (Figure 4.3b). a b 4 150

***

3 100

***

2 50 Over Medium Control Fold Increase In Migration Percent Viability (4 hours) (4 Viability Percent

1 0 Medium + + ------DMEM NKH-477 PDGF-AB - - + + - - - - CM - - - - + + - - HRV CM ------+ + NKH-477 - + - + - + - +

Figure 4.3: Direct activation of adenylyl cyclase inhibits ASM migration to HRV CM. (a)

Migration of ASM cells treated with either DMEM or NKH-477 (10 μM) over 4h was assessed in the RTCA system to the following chemoattractants: PDGF-AB (300 ng/mL); conditioned medium from medium treated (CM) or HRV-16 infected HBE cells (HRV CM) (n=6,

***p<0.001). Migration of the ASM cells to the HRV CM was significantly attenuated following

NKH-477 treatment indicating that the HRV CM induced migration utilizes a pathway that is regulated by downstream signalling events modulated by adenylyl cyclase. (b) ASM cell viability was assessed via the MTT assay at the end of the 4h experiment.

100

4.3.4 An analogue of cAMP is able to abolish migration

Having confirmed that ASM chemotaxis to supernatants from HRV infected HBE cells is regulated through a GPCR mediated pathway that is sensitive to adenylyl cyclase activity, we next hypothesized that the specific downstream mediator that is likely to be involved in this process is cAMP. We therefore sought to determine whether using a cell permeating cAMP analogue would modulate ASM cell chemotaxis in our experimental model system. Thus,

ASM cells were pre-treated with either the cAMP analogue 8-bromoadenosine 3′, 5′-cyclic monophosphate (8-Br-cAMP; 50 μM) or the medium control for 15 minutes prior to migration assays being performed in the RTCA system. ASM cell viability was assessed via the MTT assay at the end of the 4 hour experiment.

Results from these experiments demonstrated that the ASM cell chemotaxis to HRV CM was significantly attenuated by pre-treatment with 8-Br-cAMP, providing some evidence that this process is regulated through elevated cAMP levels (Figure 4.4a). Specifically, the ASM cell migration to both PDGF-AB and HRV CM were significantly attenuated (p<0.001) after a short-term (15 min) pre-treatment with 8-Br-cAMP. Interestingly, while ASM migration to

HRV CM was completely abrogated following pre-treatment with 8-Br-cAMP, the positive control PDGF-AB was able to induce a modest, but significantly less ASM chemotaxis despite pre-treatment with 8-Br-cAMP. This is consistent with the data seen in Figures 4.2a and

Figure 4.3a, and further supports our earlier premise that PDGF-AB signaling shares some redundancies with the signaling pathways activated by HRV infected HBE cell supernatants.

101

Cellular viability was not affected by 8-Br-cAMP pre-treatments for the 4 hour duration of the experiment (Figure 4.4b).

a b 6 150 ***

100 4 ***

3 50 Over Medium Control Percent Viability (4 hours) (4 Viability Percent Fold Increase In Migration 2

1 0 Medium + - - - - - DMEM 8-Br-cAMP PDGF-AB - - + + - - HRV CM - - - - + + 8-Br-cAMP - + - + - +

Figure 4.4: Pre-treatment with a cAMP analogue is able to significantly attenuate ASM cell chemotaxis. (a) Migration of ASM cells pre-treated with either DMEM or 8-Br-cAMP (50

μM) was assessed in the RTCA system in response to PDGF-AB (300 ng/mL) and to conditioned medium from HRV-16 infected HBE cells (HRV CM) (n=3, ***p<0.001). 8-Br- cAMP pre-treatment was able to significantly attenuate migration of ASM cells in response to both PDGF-AB and HRV CM, thereby indicating that cAMP analogues can effectively modulate ASM chemotaxis. (b) ASM cell viability was assessed through the MTT assay at the end of the 4 hour experiment.

102

4.3.5 Identification of chemokines that facilitate HRV-induced ASM cell migration

Having performed experiments to identify one of the pathways of regulating ASM chemotaxis to supernatants from HRV infected HBE cells, we next sought to identify which component(s) in the HRV infected HBE cell supernatants are driving this chemotactic response. We reasoned that these are likely to be chemotactic molecules of less than 30,000

MW (see Chapter 3), and in the previous sections of this chapter it was apparent that any such relevant chemokine is likely to bind to Gi receptors as a part of its mechanism of action.

We recognize that there are potentially many chemokines that are up-regulated following

HRV infection of HBE cells, and, in order to initially screen for the most likely candidates, a

64-chemokine multiplex protein assay was performed on our samples by Eve Technologies

(Calgary, AB, Canada), using the MILLIPEX® MAP assay kit developed by Millipore (Billerica,

MA, USA). Briefly, supernatants from HBE cells treated with medium control or infected by

HRV-16 were collected and centrifuged (200 x g, 8 minutes). The pelleted cellular debris was discarded and the supernatants were placed into fresh tubes and sent for multiplex analysis

(see Chapter 2). The table below (4.1) represents pooled data from experiments using HBE cells obtained from six independent lung donors. The multiplex assay approach was chosen over standard sandwich ELISA techniques because it allows for a higher throughput and examination of more chemotactic molecules (increased potential outcomes) from smaller sample volumes, thus conserving the sample. Table 4.1 lists the results from the multiplex assay experiments, and confirms that CXCL10, CXCL8, CCL5, IL-6, GRO, and TRAIL had the highest fold increase in expression by the HBE cells post HRV infection. Figure 4.5 illustrates

103 these 6 up-regulated chemokines, expressed as a fold expression (Log10) over baseline expression in medium treatment condition.

Table 4.1: Results of a 64-chemokine multiplex assay of supernatants of HBE cells post treatment with medium control or HRV-16 infection.

(OOR – out of range of the assay, below threshold of detection).

Medium HRV

Treatment Infection

Chemokine (pg/mL) (pg/mL)

CXCL10 6.41 13776.12

CXCL8 61.46 5990.58

GRO 118.27 3787.93

CCL5 2.04 675.90

VEGF 61.16 476.36

IL-1RA 25.28 320.14

Fractalkine 42.78 174.51

PDGF-AA 42.56 158.45

MCP-1 4.07 105.82

MDC 23.75 91.74

FGF-2 21.88 74.73

GM-CSF 2.71 35.06

IL-1a 3.93 32.81

104

Eotaxin 7.04 31.16

IL-6 0.42 27.66

ENA-78 OOR 25.29

IFNa2 4.18 24.96

G-CSF 2.15 19.60

PDGF-BB 2.49 13.86

MCP-3 4.34 12.80

IL-12p40 1.89 10.99

MCP-4 OOR 10.34

TGF-A 1.98 9.66

TNFa OOR 9.41

Flt-3L 4.20 5.84

MIP-1b 1.16 5.84

TRAIL 0.15 5.60

IL-7 OOR 5.42

MCP-2 OOR 4.83

IFNg 0.41 4.49

6CKine OOR 4.26

MIP-1a OOR 4.19

IL-20 5.33 4.12

IL-1b 0.22 3.10

EGF 1.41 2.74

105

sCD40L 3.20 2.66

IL-23 OOR 2.54

IL-28A OOR 2.19

Eotaxin-2 0.13 2.12

SCF OOR 1.97

IL-4 OOR 1.96

IL-15 0.22 1.80

TNFb 0.90 1.68

IL-12p70 1.11 1.59

IL-13 OOR 1.48

IL-21 OOR 0.87

IL-10 0.49 0.79

BCA-1 0.16 0.67

IL-3 0.10 0.64

I-309 0.05 0.52

Eotaxin-3 OOR 0.48

IL-17A 0.09 0.40

IL-9 0.11 0.31

IL-5 0.14 0.16

TARC OOR 0.05

IL-16 OOR OOR

LIF OOR OOR

106

TSLP OOR OOR

MIP-1d OOR OOR

IL-2 OOR OOR

TPO OOR OOR

IL-33 OOR OOR

CTACK OOR OOR

SDF-1a+B OOR OOR

1 Over Medium Control (log10) Fold Increase In Protein Expression

0 CXCL10 CCL5 CXCL8 IL-6 TRAIL GRO

Figure 4.5: Highly up-regulated chemokines post-HRV infection. A multiplex assay was performed on the combined supernatants of six HBE donors, post-HRV infection or medium control. Out of the 64-chemokines measured in the multiplex assay of supernatants from

HBE cells, the 6 chemokines with the greatest fold increase in secretion post HRV-16 infection compared to medium treatment alone are shown above.

107

4.3.6 GPCR mRNA analysis suggests that CCL5 (RANTES) may be an important

chemokine for HRV-induced ASM chemotaxis

Having demonstrated that the supernatants of HRV infected HBE cells are able to drive migration through GPCRs, and having identified several chemokines in these HBE cell supernatants that are most likely capable of modulating this response, we next sought to investigate whether the ASM cells expressed GPCR receptors capable of binding the up- regulated chemokines, described above. We therefore performed a 384-GPCR-panel mRNA array and focused on those receptors that best matched with the most highly up-regulated chemokines identified in Table 4.1 and Figure 4.5. The mRNA from ASM cells extracted from four different lung donors was extracted, expanded into cDNA and utilized to evaluate the presence or absence of mRNA based on the threshold detectability of the assay, as described in the Methods chapter (see Chapter 2).

The data from the complete mRNA array indicate that mRNA from 381 known receptors was detected in any of the 4 donors (Appendix Table 7.1). Interestingly, only 153 receptors were expressed consistently in all four donors, indicating that inter-donor variances are a major factor in what is, admittedly, a small clinical sample size for these experiments. We next analyzed these receptors to identify those receptors that could potentially bind to the six chemokines that were up-regulated following HRV infection of HBE cells (Table 4.1 and

Figure 4.5). Interestingly, we only detected mRNA for CCR1 and CCR3 consistently in all four donors (Figure 4.6). While this is consistent with another group’s finding that CCR3 is found on ASM cells230, these data conflict with a previous report by that ASM cells also express

108

CXCR1, CXCR2 and CXCR3250, as while these specific receptors were detected on ASM cells isolated from individuals within the study cohort of 4 donors, we did not consistently detect mRNA for these receptors on ASM cells of all 4 donors (Figure 4.6). Thus, while sample size for these experiments is small, and while the validity of these data are likely to be limited by both the small sample size and the inter-subject variability that exists between clinical material from 4 different donors, our data suggest that – since CCR1 and CCR3 are receptors for the chemokine CCL5 (RANTES) – CCL5 may have a major role in modulating ASM chemotaxis, particularly as we have also shown it to be up-regulated in the supernatants of

HBE cells following HRV infection (Table 4.1 and Figure 4.5).

109

20 CT

0 CCR1 CCR3 CCR5 CXCR1 CXCR2 CXCR3

Figure 4.6: A 384-panel GPCR array highlights some important chemokine receptors that may be present, based on mRNA-based detection. The mRNA from ASM cells isolated from 4 lung donors was collected, converted to cDNA, and utilized for this array. Each individual data point represents one of four different ASM donors. A cycle threshold (CT) of

40 represents undetectable levels of the particular receptor based on manufacturer instructions.

4.3.7 Flow cytometry data indicate the presence of several chemokine receptors

Based on our data obtained from GPCR mRNA analysis, we next wanted to confirm our findings using another distinct but complimentary assay. Thus, we conducted a flow

110 cytometry study to identify potentially relevant cell surface receptors, utilizing ASM cells obtained from 3 of the 4 ASM donors used in the GPCR mRNA experiments. Briefly, ASM cells from 3 different lung donors were suspended through the use of a non-enzymatic cell dissociation solution, prior to being resuspended in FACS buffer and allowed to incubated with specific antibodies for 2 hours at 4°C before being washed, fixed, and analyzed in the the Flow Cytometry core facility at the University of Calgary. ASM cells were placed into suspension, utilizing a non-enzymatic solution to prevent the chance of receptor loss during the process, prior to being incubated with anti-CCR1, anti-CCR3, anti-CCR5, anti-CXCR1 and anti-CXCR3 antibodies (as described in Chapter 2). ASM cells were then passed through a flow-cytometer for a multi-color analysis and receptor detection.

A representative figure for 1 of the 3 donors examined is shown in Figure 4.7a, and confirms that a small percentage of the ASM cells present express CXCR1, CXCR3, CCR1, CCR3 and

CCR5. These data seemed to contradict those of the GPCR mRNA array from the previous section. In particular, they demonstrate that CCR5, CXCR1, and CXCR3 are clearly present on the plasma membrane of at least a minority of the total ASM population (Figure 4.7b and

4.7c). While only a small percentage of the ASM cells express these receptors, their presence is at least consistent with our thesis that CXCL10, CCL5, and CXCL8, either alone or in combination, are likely to be relevant chemokines responsible for ASM migration following

HRV infection of HBE cells. It is possible that the use of the non-enzymatic cellular dissociation solution to try to lift a cell resulted in receptor internalization, which could explain the expression of the receptors in only a small percentage of the total ASM population.

111 a

b c Antibody

Section Target Type Percent Expression

P4 CCR3 IgG2B 1.8%

P5 CXCR3 IgG1A 5.7%

P6 CCR1 IgG2B 1.7%

P7 CXCR1 IgG2B 3.9%

P8 CCR5 IgG2B 1.2%

Figure 4.7: Flow cytometry analysis of ASM donor cells indicates the presence of several different chemokine receptors on ASM cell membrane surfaces. (a) Chemokine receptor expression as detected by flow cytometry. ASM cell population was evaluated through forward scatter (FSC) and side scatter (SSC), prior to being evaluated for the presence or absence of the relevant fluorophore. (b) The populations from (a) that correspond to a particular cell surface receptor are designated alongside percent of population expressing the receptor (n=3). (c) Percent of population expressing a particular receptor.

112

4.3.8 HRV-induced ASM migration is mediated, at least in part, via the chemokine

CCL5

In the previous sections we were able to identify chemokines and their relevant receptors that could potentially be involved in the pathogenesis of ASM cell chemotaxis, as described earlier in this thesis. It is also possible that more than one chemokine in combination could be driving this migratory response, as several of them have been previously reported to be capable of driving ASM chemotaxis in various different model systems171,225. To determine whether a combination of the three highest expressed chemokines (based on fold expression day, Figure 4.5) could drive ASM migration to any greater degree than any of the 3 chemokines alone, we performed the following series of experiments. The chemokines CCL5,

CXCL8 and CXCL10 were combined in concentrations that approximated the concentrations at which these chemokines were presented in the 64-chemokine multiplex assays for the

HRV infected HBE supernatants (HRV CM) experiments. Thus, to mimic the HRV CM experiments, we generated a chemotactic solution comprising of CCL5, CXCL8, and CXCL10 in concentrations of 500 pg/mL, 5000 pg/mL and 10000 pg/mL (comparable to 675.90,

5990.58, 13776.12 pg/mL; Table 4.1) to achieve a ratio of 1:10:20 to mimic concentrations of these chemokines in HRV CM.

The data obtained demonstrate that the capacity of each chemokine to drive ASM cell chemotaxis varied greatly. Surprisingly, CXCL10 alone was barely able to drive any chemotactic response at all when utilized at a concentration similar to that present in the

HRV CM. CXCL8 alone was able to drive a modest chemotactic response, but this was not

113 significantly different that that seen with CM control. Only CCL5 alone was able to drive migration that was significantly greater than CM control, and was comparable to the magnitude of ASM migration seen with HRV CM (Figure 4.8). Importantly, the combination condition of all 3 chemokines was able to drive migration to a significantly greater degree than the CM control, and to levels that were higher than CCL5 alone, and comparable to the

HRV CM condition (Figure 4.8). Based on these data, in which the magnitude of the ASM cell migratory response was not significantly different between CCL5 alone and the combination of CCL5, CXCL8, and CXCL10, we concluded that CCL5 is the chemokine predominantly responsible for the ability of HRV CM to drive ASM chemotaxis in our model system.

114

2.5

*** *** *** 2.0

1.5 Over Medium Control Fold Increase In Migration

1.0 Medium + ------CXCL10 - + - - + - - CXCL8 - - + - + - - CCL5 - - - + + - - CM - - - - - + - HRV CM ------+

Figure 4.8: CCL5 is able to drive ASM migration comparable to HRV CM. Migration of

ASM cells to CXCL10 (10 ng/mL), CXCL8 (5 ng/mL), CCL5 (0.5 ng/mL) and to conditioned medium (CM) or HRV-16 infected HBE cell medium (HRV CM) was assessed (n=3,

***p<0.001). While CXCL10 is unable to drive ASM migration, CXCL8, CCL5, and the combination of all three chemokines are able to drive migration to varying degrees. Only

CCL5 alone and the combination of all 3 chemokines are able to drive migration that is comparable to the HRV CM condition, suggesting that CCL5 may be the major chemotactic mediator.

115

4.3.9 Inhibition of soluble CCL5 in HRV infected HBE cell supernatants attenuates

ASM cell chemotaxis

In the previous sections we demonstrated the CCL5, alone and in combination with CXCL8 and CXCL10, is able to act as a robust chemoattractant for the ASM cells at concentrations that match those present in the supernatants of HBE cells following infection with HRV.

However, it was still not explicitly clear whether CCL5 is the major chemokine responsible for driving this chemotactic response, as receptors for both CXCL8 and CXCL10 are also present on ASM cells, suggesting that they could potentially be playing a modulating role. We thus designed the following series of experiments to evaluate whether blocking CCL5 in HRV

CM could significantly attenuate ASM migration. Supernatants from HRV infected epithelial cells were collected and allowed to incubate with either an anti-CCL5 antibody (mouse anti- human IgG1; 140 ng/mL; R&D Systems) or an isotype control (mouse IgG1 isotype control;

140 ng/mL; R&D Systems) for 1 hour at room temperature prior to being utilized as chemoattractants in the RTCA system. Importantly, ASM cells were treated with mouse serum (10% solution in DMEM; Sigma-Aldrich; 15 min) prior to the migration experiments so as to prevent any interaction of the ASM cells with the isotype or target antibodies (see chapter 2).

Results obtained from these experiments confirm that inhibition of soluble CCL5 in HRV infected HBE supernatants significantly attenuated ASM cell migration to the HRV CM

(p<0.001) when compared to the isotype control antibody (Figure 4.9). These data provide

116 further robust validation that CCL5 is the major chemokine present in HRV infected HBE supernatants that is involved in the pathogenesis of downstream ASM chemotaxis.

*** *** 3

2 Over Medium Control Fold Increase In Migration

1 Medium + + - - - - - PDGF-AB ------+ HRV CM - - - + + + - Isotype - + - - + - - Anti-CCL5 - - + - - + -

Figure 4.9: Blockade of soluble CCL5 via an anti-CCL5 antibody results in a significant attenuation of ASM chemotaxis. Migration of ASM cells to following chemoattractants was assessed in the RTCA system: PDGF-AB (300 ng/mL); conditioned medium from HRV-16 infected HBE cells (HRV CM) that was untreated, treated with isotype control, or treated with anti-CCL5 for 1 hour (n=3, ***p<0.001).

117

4.3.10 HRV-infection of HBE cells obtained from asthmatic subjects results in

significantly greater ASM chemotaxis compared to healthy controls

Having elucidated that CCL5 is the major chemokine involved in the pathogenesis of downstream ASM chemotaxis following HRV infection of HBE cells, we questioned whether this process might be different in cells from asthmatic individuals. Data presented thus far have been exclusively generated using both HBE and ASM cells obtained from previously healthy lung donors. Recognizing that the airway epithelium from asthmatic individuals is altered125, we designed the following series of experiments to assess whether HBE cells obtained from bronchial brushings from both healthy control subjects and from asthmatic subjects would identify any differences in the degree of ASM cell chemotaxis observed in our model system. HBE cells were obtained via bronchial brushings from well-characterized asthmatic subjects (n=3), as well as from bronchial brushings of healthy control individuals

(n=3). Cells were then grown to confluence infection with HRV-16 or treatment with medium control. The resultant supernatants were then used as chemoattractants in the xCELLigence

RTCA system to assess ASM cell chemotaxis, as described before (see Chapter 2).

Results from these experiments indicate that the supernatants from HBE cells obtained via bronchial brushing of asthmatic individuals were able to robustly induce chemotaxis of ASM cells in our system. Indeed, HBE cells from asthmatic brushings drove ASM cell migration that was nearly 3 folds higher than that seen in experiments using HBE cells from healthy control brushings (p<0.001). These data are consistent with the findings from other groups, who have reported that the asthmatic airway epithelium has a differential capacity to

118 produce inflammatory cytokines and chemokines125, and may explain in part why such a large increase in smooth muscle mass is seen in the airways of asthmatic patients.

Table 4.3: Characteristics of patients from whom brushings of epithelial cells were obtained.

Normal Asthmatic

Characteristics (SD) (SD)

Age, yr 36 (10.5) 47.7 (9.3)

Male/Female 3/0 3/0

FEV1/FVC 0.79 (0.04) 0.62 (0.06)

FEV1 % Predict 102.4 (6.1) 77 (9.6)

119

11 *** 10

Over Medium Control 4 Fold Increase In Migration 3

1 Medium + - - - PDGF-AB - + - - HRV CM - - + + Asthmatic - - - +

Figure 4.10: Epithelial cells obtained from brushings from asthmatic individuals induce significantly greater ASM chemotaxis as compared to healthy controls.

Migration of ASM cells was assessed in the RTCA system to the following chemoattractants:

PDGF-AB (300 ng/mL); conditioned medium from HRV-16 infected HBE cells (HRV CM) from healthy or asthmatic individuals (n=3). Supernatants from HBE cells from asthmatic individuals were able to drive migration that was significantly higher than non-asthmatic brushings (***p<0.001).

120

4.4 Discussion

In this chapter, we have confirmed the novel observation that chemotaxis of ASM in response to supernatants from HRV infected HBE cells is mediated through a Gi based, cAMP sensitive pathway. Moreover, we have demonstrated that CCL5 is likely to be an important chemokine involved in this process. A number of studies have previously identified that ASM cell migration is sensitive to PTX204 intervention, but some studies have indicated it is also

249 possible to have migration not tied to Gαi . Our PTX data clearly confirmed that the migratory mechanism was tied to Gi as a 12-hour pre-treatment resulted in a significant attenuation of the chemotactic potential of HRV CM. This finding is in agreement with the data presented in this current chapter, in which we demonstrate that ASM chemotaxis occurs through a CCL5 chemokine-mediated pathway.

Moreover, treatment with the long-acting β-agonist, formoterol, provides additional verification that the mechanism of action for ASM chemotaxis to HRV infected HBE cell supernatants is through Gi. Specifically, we identified that the activation of Gαs by formoterol has the same effect as the inhibition of Gαi by PTX. This finding resulted in us redirecting our focus towards adenylyl cyclase, and its potential role in modulating ASM chemotaxis in response to HRV infected HBE cell supernatants. Treatment of ASM cells with NKH-477, which is a direct agonist for adenylyl cyclase, abrogated ASM chemotaxis indicating that cAMP formation serves as a negative regulator for ASM chemotaxis in our model. When the

ASM cells were treated with 8-Br-cAMP, a cAMP analogue, it was also able to abrogate ASM

121 chemotaxis to HRV infected HBE cell supernatants, thereby confirming that elevated levels of intracellular cAMP are able to attenuate migration.

The filtration studies from the previous chapter led us to conclude that the factors likely responsible for driving chemotaxis in our system were molecules of less than 30,000 MW, indicating that chemokines are likely the mediators. Data from the 64-chemokine multiplex assay identified 6 chemokines present in HRV infected HBE cell supernatants. Of these,

CXCL8226 and CCL5229 both have been previously been shown to induce ASM migration in different disease states, and were therefore considered as strong chemotactic candidates in our experimental model. When we examined what receptors might be present on the ASM cell surface to identify which target molecule could be involved, we encountered conflicting results. Although the data from the 384-plex GPCR mRNA array was able to effectively detect mRNA for both CCR1 and CCR3 consistently in all 4 donors, the data from the flow cytometry analysis demonstrated that a population of ASM cells expressed CCR1, CCR3, CCR5, CXCR1 and CXCR3. This could indicate that both CCR1 and CCR3 are consistently regulated in expression in ASM cells through receptor cycling and need the constant generation of new receptor mRNA, while it is also possible other receptors are not regulated in this way thus explaining the lack of detectable mRNA levels.

When CCL5, CXCL8, and CXCL10 were used alone or in combination as chemoattractants for

ASM cells in the RTCA system, it became evident that CCL5 alone, and in combination with

CXCL8 and CXCL10, was able to drive ASM chemotaxis at a magnitude that approximated that seen with HRV infected HBE cell supernatants. While this clearly pointed to CCL5 as

122 being the major chemotactic mediator present in HRV infected HBE cell supernatants, we were able to further confirm this observation through a series of anti-CCL5 blocking studies.

Specifically, by utilizing a monoclonal antibody to block CCL5 directly in the HRV infected

HBE cell supernatants and then utilizing the blocked solution as a chemoattractant, we were able to confirm the importance of HBE secreted CCL5 in driving HRV induced ASM migration.

Lastly, up until this point, the HBE cells from which the HRV-induced supernatants were obtained were from previously healthy individuals. However, we know that the airway epithelium in asthmatic individuals is significantly different from that of healthy control individuals125,128,131,134, and thus, by infecting HBE cells obtained from brushings from mild asthmatics and normal subjects with HRV, we were able to verify, for the first time, that ASM chemotaxis is exaggerated in HRV infected asthmatic HBE cell supernatants. This novel finding, that the asthmatic HBE cells, post-HRV infection, are able to drive ASM cell migration at a nearly a three fold greater rate than HBE cells obtained from healthy individuals, could account for the increased smooth muscle mass present in airway remodeling in asthma.

In conclusion, the data presented in this chapter describe the novel findings that HRV infection of the bronchial epithelium drives ASM chemotaxis, and that this process can be suppressed through activation of the Gs signaling pathway or inhibition of the Gi signaling pathway. Moreover, adenylyl cyclase is involved in the regulation of this system, whereby the generation of cAMP is able to inhibit chemotaxis to HRV infected HBE cell supernatants; this finding was confirmed through our experiments utilizing the cAMP analogue 8-Br-cAMP.

Our data utilizing a 64-chemokine multiplex assay, a 384-plex GPCR mRNA array, and

123 performing flow cytometry analysis resulted in the selection of CXCL10, CXCL8, and CCL5 as potential chemokines responsible for driving ASM chemotaxis from within the HRV CM.

Importantly, we were able to identify the chemokine CCL5 as a key component of HRV CM in its ability to drive migration, alone and in combination with CXCL8 and CXCL10. Finally, the utilization of an anti-CCL5 antibody to block the soluble CCL5 in HRV infected HBE cell supernatants, and the attenuation of the ASM chemotaxis that resulted, identified a key role for CCL5 in driving this response. Taken together, these observations provide insights into the mechanisms by which HRV infection of the bronchial epithelium is able to drive ASM cell chemotaxis. Lastly, the results reported in this chapter also identify several additional and intriguing areas for investigation, which are beyond the scope of this Masters thesis but which will be discussed in more detail in Chapter 5 under future directions.

124

5 CHAPTER FIVE: DISCUSSION, CLINICAL RELEVANCE & FUTURE DIRECTIONS

125

5.1 General Discussion and Future Work

While the traditional paradigm with regard to the pathogenesis of airway remodeling in asthma has held that remodeling occurs after many years of chronic inflammation, studies have now confirmed that remodeling changes can be observed in pre-school children prior to the diagnosis of asthma being made70. Moreover, there is now robust evidence to indicate that children with recurrent HRV-induced wheezing episodes are at a significantly increased risk of developing subsequent asthma90. These findings have resulted in the general hypothesis that HRV infection may be involved in the pathogenesis of asthma and airway remodeling, although the mechanisms by which this might occur have remained unclear. The airway epithelium is the major site of HRV infection and it is proposed that the inflammatory responses within the epithelium to infection could contribute to the remodeling process in susceptible individuals; this inflammatory response could cascade to recruit and effect multiple other inflammatory and structural cell types. As a continuation of the research program that has been investigating the mechanisms by which HRV infections may contribute to, or initiate airway remodeling, the overall objective of this Master of Science thesis was to explore the role of HRV infection of the airway epithelium in modulating airway smooth muscle migration.

The results reported in Chapter 3 constitute the novel observation that HRV infection of HBE cells results in the generation of cell supernatants that were capable of inducing ASM chemotaxis. We are the first group to utilize the RTCA system, having validated it alongside the MCC, to demonstrate ASM migration in real-time in response to chemoattractant

126 supernatants from HRV infected HBE cells. In addition, we were able to show that ASM migration peaked at 4 hours, and was dependent on a chemotactic gradient. As discussed in

Chapter 1, eukaryotic cells such as ASM cells and D. discoideum are able utilize both temporal and spatial information to guide in migration211, and by abolishing the chemotactic gradient and confirming a significant decrease in migratory capacity, we can surmise that ASM cell chemotaxis is likely dependent on spatial information in order to proceed. In addition, given that the HRV in suspension itself was unable to drive migration, it is evident that the inflammatory response of the HBE cells to HRV-16 infection is capable of driving ASM cell migration. Finally, by utilizing UV-light to render HRV replication deficient, we demonstrated that viral replication within HBE cells is necessary to initiate the inflammatory responses that can induce robust ASM chemotaxis.

In Chapter 4 we investigated the mechanisms through which HRV infection of HBE cells is able to modulate ASM migration. Specifically, we confirmed for the first time that chemotaxis of ASM to supernatants from HRV infected HBE cells is driven through a Gi based, cAMP sensitive pathway. Moreover, we demonstrated that the chemokine CCL5, which is up- regulated following HRV infection of HBE cells, is likely a key mediator in this process. Using

PTX to block GPCRs, and thereby abrogate ASM migration to HRV infected HBE cell supernatants, we were able to confirm the role of Gi in modulating ASM migration. We also established an inhibitory role for cAMP, in that the β-agonist formoterol and the adenylyl cyclase agonist NKH-477, both of which increase intracellular cAMP, completely abolished

ASM migration to HRV infected HBE cell supernatants. Finally, when ASM cells were pre- treated with the cAMP analogue, 8-Br-cAMP, chemotaxis to the HRV infected HBE cell

127 supernatants was again abolished, further validating our premise that intracellular cAMP is capable of modulating ASM cell migration.

The results from both Chapters 3 and 4 allowed us to focus down on to which factors in the supernatants collected from HRV infected HBE cells might be responsible for driving ASM chemotaxis. Processing the HRV infected HBE supernatants through a 30,000 MW filtration device did not result in a significant attenuation of the previously observed ASM chemotaxis, indicating that any potential mediators were likely <30,000 MW – and, based on this size assumption, likely to be a chemokine. The involvement of GPCRs, namely Gi, provided further support of this hypothesis and we therefore performed a 64-chemokine multiplex protein assay on the HRV infected HBE cell supernatants to detect which chemokines are most highly up-regulated in HBE cells post-HRV infection. In combination with a GPCR mRNA array and

FACS analysis, we were able to identify CCL5 as a chemokine likely to be responsible for driving this ASM chemotactic response. Utilizing anti-CCL5 antibodies to block this chemokine in the HRV infected HBE cell supernatants, we observed a significant decrease in

ASM cell chemotactic capacity, and thus we were able to confirm that CCL5 is, alone and possibly in combination with other chemokines, a key modulator of ASM migration that is present in HRV infected HBE cell supernatants. Finally, we presented the novel findings that supernatants from HRV infected HBE cells obtained from asthmatic patients were able to drive migration at significantly greater magnitudes than supernatants from HRV infected

HBE cells obtained from healthy individuals. These data thus add further credence to the premise that the airway epithelium in asthmatic individuals is dysfunctional125.

128

The model that we propose for this system is as follows: firstly, HRV infection of HBE cells results in internalization and replication of the virus. This viral replication results in the production of ssRNA and dsRNA which are detected within the epithelium by PRRs. This

PRR-directed, innate host-defense response results in the production of a chemokine/cytokine milieu. The chief regulatory chemokine within this milieu is CCL5, which acts through its receptors CCR1, CCR3 and CCR5, present on the ASM cell surface, to cause cytoskeletal rearrangement and migration of the ASM. This system can be blocked through PTX or β2-agonist treatment due to an inherent relationship between cAMP and migration. Specifically, an intracellular increase in cAMP results in hyper-polarization of the

ASM sarcoplasm, which prevents the myosin motors within the cell from acting. As the cell lacks the capacity to generate force despite still being able to form focal adhesions, it is unable to migrate. This is also likely why 8-br-cAMP is able to prevent migration.

The findings presented in this thesis are novel because they provide a direct link, in vitro, between HRV infection and a characteristic pathological change seen in asthma – namely airway remodeling. Importantly these observations demonstrate that HRV infection of the airway epithelium provokes a host immune response that directly promotes ASM chemotaxis. This could be one of the initial steps by which HRV infections might facilitate airway remodeling and result in the characteristic pathological changes seen in airway remodeling, such as ASM hypertrophy and hyperplasia, and a greater proximity of the smooth muscle to the basement membrane region, which is clearly evident in histological sections of patients with severe asthma.

129

5.2 Limitations

The experimental models utilized to evaluate ASM cell migration throughout this thesis were all in vitro models, and these do not necessarily reflect what might be occurring in vivo in asthmatic individuals undergoing naturally occurring HRV infections. Moreover, the HBE cells culture models utilized mainly basal cells that were grown in a submerged monolayer

(Chapter 2) and may not completely reflect the pathobiology of the pseudostratified columnar epithelium found in the airways, in vivo. Thus it can be difficult to infer whether

HRV infection of the airway epithelium in vivo could result in the same inflammatory response seen in our culture cells, although our group has previously published reports confirming that the observed in vitro observations also occur during in vivo natural cold clinical studies, thereby providing some validation for our current model systems 251.

In addition, because of the relative scarcity of smooth muscle donors present in the tissue bank of our laboratory, the sample size for individual experiments were often limited. Each n value represents an individual line of smooth muscle cells extracted from a previously healthy lung donor. While the HBE cell extraction method described in Chapter 2 has been established in our laboratory for over 10 years, the ASM extraction technique was completely new to the laboratory environment, and needed substantial refining before it yielded optimal numbers of ASM cells for our migration experiments. As a result, we initially were unable to generate a cell bank with as large a number of smooth muscle cells – though this is no longer the case. Throughout the course of my Master’s project I was able to build upon techniques acquired through collaboration with other smooth muscle laboratories to

130 develop a technique that results in tissue extraction with a very high success rate of isolating high quality ASM cells.

The filtration studies presented in Chapter 3 demonstrated a decrease in migration in only the CM and not in the HRV CM. A limitation of this study is that we are unaware of any HBE secreted compounds greater than the molecular cut off of the centrifugal filtration system and thus the difference between the CM pre-and-post filtration can not be explained. If allowed to speculate, it is possible that some perpetually secreted regulatory chemokines present in the CM adhered to the filtration membrane and thus this difference in migratory capacity can be explained by a decreased presence of these chemokines.

The data presented in Chapter 4, involving the epithelial cells obtained from bronchial brushings of both asthmatic patients and healthy individuals also possess a low sample size.

This is because brushings are only obtained as part of specific research studies, require subsequent isolation and culture, that results in relatively small numbers of cells that can be utilized for experimentation; often the brushings will not grow or can become infected early on in this process and are rendered useless. Thus, it was challenging to obtain epithelial cells from 3 well characterized asthmatics and 3 healthy controls for this comparison study. In addition, the asthmatics that were utilized were not assessed as severe asthmatics, as per the guidelines described in Chapter 1, and we were unable to provide data about the potential differences between HBE cells obtained from severe asthmatics, and healthy controls, in their inflammatory response to HRV infection and the resultant modulation of the ASM. Lastly, we were unable to use ASM cells obtained from asthmatic subjects because

131 of the logistical challenges associated with their extraction. Nevertheless, it is possible that these cells differ from the ASM cells from healthy individuals that we have utilized in this project, and i) could express a great number of chemotactic receptors, ii) have dysfunction in their intracellular pathways (Gi or cAMP), and iii) could potentially be more sensitive to migratory stimuli and thus migrate at greater magnitudes to supernatants from HRV infected

HBE cells.

The model systems utilized to evaluate migration of ASM cells, namely the MCC and the RTCA systems, both have benefits and drawbacks (partially described in Chapter 2). While the move from the MCC to the RTCA system allowed us to measure ASM cell migration in real- time, the system’s high operating costs did not allow for replicates greater than two to be utilized during experiments. In addition, neither system allowed the operator to directly visualize the cells in real-time thus making it difficult to understand what percentage of the

ASM population was migrating in response to the specific chemoattractants utilized. When suspending the cells prior to migration, our use of trypsin may potentially have affected the chemotactic results by the enzymes serine protease mechanism of action damaging or removing surface receptors on the ASM cells. Furthermore, the RTCA system relies on cellular adhesion to the gold micro-electrodes (through which it passes an alternating current) to detect impedance and thus quantify migration; this could result in the apparatus mistakenly observing cell hypertrophy or hyperplasia as migration. Nevertheless, several other groups have utilized the xCELLigence RTCA system to successfully differentiate between cell migration, proliferation, and cellular viability236,252,253, and we do not believe that these latter issues are likely to have impacted on the results presented in this thesis.

132

Nevertheless, there are competing theories on what role migration, hypertrophy and hyperplasia might be playing in causing ASM remodeling in asthma200. While there are numerous studies that indicate that there is an increase in smooth muscle mass in asthma201,254,255, there are several potential pitfalls with this work. Firstly, muscle contraction can exaggerate the thickness of the ASM layer. Furthermore, the ASM layer is not necessarily uniform and can contain significant elements of connective tissue 256. A study done by Benayoun and colleagues257 found there to be an absence of Ki67 nuclear staining

(a marker for cell cycle traversal) thus indicating that hypertrophy contributes more to airway remodeling than does hyperplasia. Conversely, it is possible that hyperplasia and migration work side-by-side to induce changes in smooth muscle morphology in asthma.

Particularly, it is possible that migration of ASM cells towards the lumen of the airway underlies the appearance of myofibroblasts in the subepithelium of asthmatic airways258. In support of this, segmental challenge of asthmatic airways increases the number of myofibroblasts in the airway subepithelium259. It is most likely then that hypertrophy, hyperplasia, and migration are playing a concurrent role in ASM remodeling in asthma.

5.3 Clinical relevance

Smooth muscle mass is increased dramatically in asthmatic patients as compared to healthy controls (see Chapter 1) and migration of smooth muscle cells is not unprecedented, being fundamental to the process of developing hollow organs, such as blood vessels and the airways204. However, in many diseases states such as atherosclerosis205 and restenosis206 muscle migration is regarded as a negative contributor to disease pathogenesis. It is

133 therefore very likely that ASM migration could be contributing to the airway remodeling seen in asthma, possibly facilitating disease progression. The studies detailed in this thesis were able to demonstrate that HRV infection of the airway epithelium, in vitro, resulted in an inflammatory response that initiates and drives ASM migration, thus highlighting a key potential therapeutic targets for further development. As shown in this thesis, cAMP elevating agents, including the commonly used asthma β-agonist therapy formoterol, and the forskolin analogue NKH-477, were able to effectively abolish ASM migration, thereby preventing the ability of HRV infection of the epithelium to drive an aspect of airway remodeling. Consequently, it is feasible that β-agonist treatment during an HRV infection of a susceptible individual during childhood could halt or attenuate the remodeling seen in early asthma. The data presented in this thesis provide a possible mechanism to target for preventing or attenuating airway remodeling following HRV-infection in susceptible individuals.

134

5.4 Future directions

The contents of this thesis have provided a new avenue for future research into HRV infections and their relationship with the pathogenesis of asthma. We have identified CCL5 as a key modulator of the chemotactic response seen in ASM cells to HRV infection of the airway epithelium but, as seen in Chapter 3, there are several other mediators secreted by

HBE cells post HRV-infection that are also highly up-regulated. Future studies should focus on delineating what other chemokines and growth factors could be responsible in modulating the ability of ASM to migrate to supernatants from infected airway epithelial cells. Specifically, it was seen that CXCL8 (IL-8) was able to drive migration on its own in ASM cells, thereby suggesting a potential additional target for future research. Other groups have already confirmed that ASM cells migrate to CXCL8226, and have also identified the GRO family of chemokines as a potential important factor for ASM migration225.

While we have been able to demonstrate that β2-agonist treatment and other methods for modulating intracellular ASM cAMP levels are effective in regulating the chemotactic response of ASM cells to supernatants from HRV infected HBE cells, it is likely there are also other pathways capable of regulating migration. As mentioned in Chapter 1, several studies have cited the involvement of MAPKs in non-specific migratory pathways present in the cell216,218; it would thus be valuable to utilize different intracellular kinase antagonists to elucidate exactly which pathways could potentially be responsible for driving the ASM cell migratory response.

135

Our studies utilizing airway brushings from asthmatic patients and healthy individuals were able to indicate that marked differences exist between the two groups in their ability to drive

ASM chemotaxis post-HRV-infection, but we have not yet been able to explore the cellular mechanisms that might underlie these variations. In future studies it would be ideal to utilize a comparison between the epithelium present in mild, moderate, and severe asthmatic patients as well as healthy individuals, and conducting a gene array that examines up- regulated genes post HRV-infection. Furthermore, future studies should look to investigate whether there are any differences present between the migratory capacities of ASM tissue present in asthmatic patients as compared to healthy controls.

5.5 Conclusion

In conclusion, the results presented in this thesis represent the novel findings on the ability of HRV infection of the airway epithelium to drive ASM chemotaxis in vitro. HRV infection of the epithelium results in the up-regulated production and release of various different chemokines and remodeling mediators, with the key modulator identified in this thesis being

CCL5. Supernatants generated from HRV treated epithelial cells were able to consistently drive chemotactic, directional, migration of the ASM cells that was sensitive to the chemokine gradient, and thus apparently dependent on spatial awareness of the cell to the gradient. The effects of CCL5 in facilitating ASM cell chemotaxis was confirmed to be acting through a

GPCR-based mechanism, and the subsequent downstream signaling was confirmed to be regulated, at least in part, by levels of intracellular cAMP. The role of CCL5 as the key modulator was further validated through antibody-based blocking studies that prevented

136

ASM cell chemotaxis. These proposed mechanisms described in this thesis, through which

HRV infection of the airway epithelium drives ASM remodeling and migration, are summarized in Figure 6.1.

This body of work is the first to show that HRV infection of the airway epithelium results in the production of factors that drive ASM cell chemotaxis, and that this migration can be regulated through changes in intracellular cAMP. These observations provide insights into the pathogenesis of airway remodeling in asthma, and substantiate the growing body of evidence that links HRV infections to the development of airway remodeling and thus to the increased risk of developing asthma.

137

HRV

Infection

Airway Epithelium

HRV UV-Light Replication

Production

Of CCL5

(24 h)

Smooth Muscle Pertussis Toxin GPCR Dependent Formoterol

Internal NKH-477 Pathway 8-Br-cAMP

CHEMOTAXIS

Figure 5.1: Proposed schematic indicating the novel mechanism uncovered in this thesis, by which HRV-16 infection of the airway epithelium results in chemotaxis of

ASM cells. The contribution and elucidation of other chemokines involved in this process is beyond the scope of this thesis.

138

6 CHAPTER SIX: REFERENCES

139

1. GINA Report. Global Strategy for Asthma Management and Prevention. 1–119

(2014).

2. To, T. et al. Global asthma prevalence in adults: findings from the cross-sectional

world health survey. BMC Public Health 12, 204 (2012).

3. Gavala, M. L., Gern, J. E. & Bertics, P. J. Rhinoviruses, allergic inflammation, and

asthma. Immunological Reviews 242, 69–90 (2011).

4. Chapman, K. R. Asthma in Canada: missing the treatment targets. Canadian Medical

Association Journal 178, 1027–1028 (2008).

5. Masoli, M., Fabian, D., Holt, S., Beasley, R.Global Initiative for Asthma (GINA)

Program. The global burden of asthma: executive summary of the GINA

Dissemination Committee Report. Allergy 59, 469–478 (2004).

6. Hermus, G., Stonebridge, C., Goldfarb, D., Theriault, L. & Bounajm, F. Cost Risk

Analysis for Chronic Lung Disease in Canada. Conference Board of Canada (2012). at

7. Salter, H. On the Ætiology of Asthma. British Medical Journal 1, 538–540 (1859).

8. Pascoe, C. D., Wang, L., Syyong, H. T. & Paré, P. D. A Brief History of Airway Smooth

Muscle's Role in Airway Hyperresponsiveness. J Allergy (Cairo) 2012, 768982–8

(2012).

9. Weiss, S., Robb, G. P. & Ellis, L. B. The systemic effects of histamine in man: with

special reference to the responses of the cardiovascular system. Archives of Internal

… 49, 360 (1932).

10. Curry, J. J. The action of histamine on the respiratory tract in normal and asthmatic

subjects. J. Clin. Invest. 25, 785–791 (1946).

140

11. Tiffeneau, R. & Beauvallet, M. Epreuve de bronchoconstriction et de

bronchodilation par aerosols. Bull Acad Med 129, 165–8 (1945).

12. Ciba Foundation Guest Symposium. Terminology, Definitions, and Classification of

Chronic Pulmonary Emphysema and Related Conditions: A Report of the

Conclusions of a Ciba Guest Symposium*. Thorax 14, 286–299 (1959).

13. Cockcroft, D. W., Killian, D. N., Mellon, J. J. & Hargreave, F. E. Bronchial reactivity to

inhaled histamine: a method and clinical survey. Clin. Allergy 7, 235–243 (1977).

14. Chai, H. et al. Standardization of bronchial inhalation challenge procedures. J.

Allergy Clin. Immunol. 56, 323–327 (1975).

15. Shapiro, G. G. & Sinon, R. A. Bronchoprovocation committee report. Journal of

Allergy and Clinical Immunology (1992). doi:10.1016/0091-6749(92)90387-H

16. Inman, M. D., Hamilton, A. L., Kerstjens, H. A., Watson, R. M. & O'Byrne, P. M. The

utility of methacholine airway responsiveness measurements in evaluating anti-

asthma drugs. J. Allergy Clin. Immunol. 101, 342–348 (1998).

17. Lougheed, M. D. et al. Canadian Thoracic Society 2012 guideline update: diagnosis

and management of asthma in preschoolers, children and adults. Can Respir J 19,

127–164 (2012).

18. Bousquet, J. et al. GINA guidelines on asthma and beyond*. Allergy 62, 102–112

(2007).

19. O'Byrne, P. M., Gauvreau, G. M. & Brannan, J. D. Provoked models of asthma: what

have we learnt? Clinical & Experimental Allergy 39, 181–192 (2009).

20. Wardlaw, A. J., Dunnette, S., Gleich, G. J., Collins, J. V. & Kay, A. B. Eosinophils and

mast cells in bronchoalveolar lavage in subjects with mild asthma. Relationship to

141

bronchial hyperreactivity. Am Rev Respir Dis 137, 62–69 (1988).

21. Brusasco, V., Crimi, E., Gianiorio, P., Lantero, S. & Rossi, G. A. Allergen-induced

increase in airway responsiveness and inflammation in mild asthma. J. Appl. Physiol.

69, 2209–2214 (1990).

22. Robinson, D. S., Bentley, A. M., Hartnell, A., Kay, A. B. & Durham, S. R. Activated

memory T helper cells in bronchoalveolar lavage fluid from patients with atopic

asthma: relation to asthma symptoms, lung function, and bronchial responsiveness.

Thorax 48, 26–32 (1993).

23. Cockcroft, D. W. & Davis, B. E. Mechanisms of airway hyperresponsiveness. J. Allergy

Clin. Immunol. 118, 551–9– quiz 560–1 (2006).

24. Lemanske, R. F. & Busse, W. W. 6. Asthma: Factors underlying inception,

exacerbation, and disease progression. J. Allergy Clin. Immunol. 117, S456–61

(2006).

25. Gern, J. E., Lemanske, R. F., Jr. & Busse, W. W. Early life origins of asthma. J. Clin.

Invest. 104, 837–843 (1999).

26. Holgate, S. T. Innate and adaptive immune responses in asthma. Nature Medicine

18, 673–683 (2012).

27. Locksley, R. M. Asthma and Allergic Inflammation. Cell 140, 777–783 (2010).

28. NHLBI. Expert Panel Report 3: Guidelines for the Diagnosis and Management of

Asthma--Summary Report 2007. 1–74 (2007).

29. Woolcock, A. J., Yan, K. & Salome, C. M. Effect of therapy on bronchial

hyperresponsiveness in the long-term management of asthma. Clin. Allergy 18,

165–176 (1988).

142

30. Djukanović, R. et al. Effect of an inhaled corticosteroid on airway inflammation and

symptoms in asthma. Am Rev Respir Dis 145, 669–674 (1992).

31. Jeffery, P. K. et al. Effects of treatment on airway inflammation and thickening of

basement membrane reticular collagen in asthma. A quantitative light and electron

microscopic study. Am Rev Respir Dis 145, 890–899 (1992).

32. van Grunsven, P. M., van Schayck, C. P., Molema, J., Akkermans, R. P. & van Weel, C.

Effect of inhaled corticosteroids on bronchial responsiveness in patients with

‘corticosteroid naive’ mild asthma: a meta-analysis. Thorax 54, 316–322 (1999).

33. Boulet, L. P. et al. Airway hyperresponsiveness, inflammation, and subepithelial

collagen deposition in recently diagnosed versus long-standing mild asthma.

Influence of inhaled corticosteroids. American Journal of Respiratory and Critical

Care Medicine 162, 1308–1313 (2000).

34. Sont, J. K. et al. Clinical control and histopathologic outcome of asthma when using

airway hyperresponsiveness as an additional guide to long-term treatment. The

AMPUL Study Group. American Journal of Respiratory and Critical Care Medicine

159, 1043–1051 (1999).

35. Leigh, R. et al. Effects of montelukast and budesonide on airway responses and

airway inflammation in asthma. American Journal of Respiratory and Critical Care

Medicine 166, 1212–1217 (2002).

36. Ellis, A. G. THE PATHOLOGICAL ANATOMY OF BRONCHIAL ASTHMA. The American

Journal of the Medical Sciences 136, 407 (1908).

37. Huber, H. L. & Koessler, K. K. The pathology of bronchial asthma. Arch Intern Med

30, 689 (1922).

143

38. Reid, L. The Embryology of the Lung. Ciba Foundation Symposium-Development of

the Lung 109–130 (John Wiley & Sons, Ltd., 1967).

doi:10.1002/9780470719473.ch7

39. Bousquet, J. et al. Asthma: a disease remodeling the airways. Allergy 47, 3–11

(1992).

40. Bousquet, J., Jeffery, P. K., Busse, W. W., Johnson, M. & Vignola, A. M. Asthma. From

bronchoconstriction to airways inflammation and remodeling. American Journal of

Respiratory and Critical Care Medicine 161, 1720–1745 (2000).

41. Boulet, L. P. et al. Airway inflammation and structural changes in airway hyper-

responsiveness and asthma: an overview. Can. Respir. J. 5, 16–21 (1998).

42. McParland, B. E., Macklem, P. T. & Pare, P. D. Airway wall remodeling: friend or foe?

J. Appl. Physiol. 95, 426–434 (2003).

43. Jeffery, P. K. Remodeling in asthma and chronic obstructive lung disease. American

Journal of Respiratory and Critical Care Medicine 164, S28–S38 (2001).

44. Hirota, N. & Martin, J. G. Mechanisms of airway remodeling. CHEST 144, 1026–1032

(2013).

45. Dunnill, M. S., Massarella, G. R. & Anderson, J. A. A comparison of the quantitative

anatomy of the bronchi in normal subjects, in status asthmaticus, in chronic

bronchitis, and in emphysema. Thorax 24, 176–179 (1969).

46. Dunnill, M. S. The pathology of asthma, with special reference to changes in the

bronchial mucosa. J. Clin. Pathol. 13, 27–33 (1960).

47. Takizawa, T. & Thurlbeck, W. M. Muscle and mucous gland size in the major bronchi

of patients with chronic bronchitis, asthma, and asthmatic bronchitis. Am Rev

144

Respir Dis 104, 331–336 (1971).

48. Hossain, S. Quantitative measurement of bronchial muscle in men with asthma. Am

Rev Respir Dis 107, 99–109 (1973).

49. Sobonya, R. E. Quantitative structural alterations in long-standing allergic asthma.

Am Rev Respir Dis 130, 289–292 (1984).

50. James, A. L., Pare, P. D. & Hogg, J. C. The Mechanics of Airway Narrowing in Asthma.

Am Rev Respir Dis 139, 242–246 (1989).

51. Saetta, M., Di Stefano, A., Rosina, C., Thiene, G. & Fabbri, L. M. Quantitative structural

analysis of peripheral airways and arteries in sudden fatal asthma. Am Rev Respir

Dis 143, 138–143 (1991).

52. Carroll, N., Elliot, J., Morton, A. & James, A. The structure of large and small airways

in nonfatal and fatal asthma. Am Rev Respir Dis 147, 405–410 (1993).

53. Laitinen, L. A., Heino, M., Laitinen, A., Kava, T. & Haahtela, T. Damage of the airway

epithelium and bronchial reactivity in patients with asthma. Am Rev Respir Dis 131,

599–606 (1985).

54. Jeffery, P. K., Wardlaw, A. J., Nelson, F. C., Collins, J. V. & Kay, A. B. Bronchial biopsies

in asthma. An ultrastructural, quantitative study and correlation with

hyperreactivity. Am Rev Respir Dis 140, 1745–1753 (1989).

55. Haahtela, T., Laitinen, A. & Laitinen, L. A. Using biopsies in the monitoring of

inflammation in asthmatic patients. Allergy 48, 65–9– discussion 84–6 (1993).

56. Laitinen, L. A. et al. Bronchial biopsy findings in intermittent or ‘early’ asthma. J.

Allergy Clin. Immunol. 98, S3–6– discussion S33–40 (1996).

57. Chetta, A. et al. Airways remodeling is a distinctive feature of asthma and is related

145

to severity of disease. CHEST 111, 852–857 (1997).

58. Jeffery, P. K., Laitinen, A. & Venge, P. Biopsy markers of airway inflammation and

remodelling. Respiratory Medicine 94 Suppl F, S9–15 (2000).

59. Wadsworth, S., Sin, D. & Dorscheid, D. Clinical update on the use of biomarkers of

airway inflammation in the management of asthma. J Asthma Allergy 4, 77–86

(2011).

60. Moreno, R. H., Hogg, J. C. & Paré, P. D. Mechanics of airway narrowing. Am Rev

Respir Dis 133, 1171–1180 (1986).

61. Paré, P. D., Macklem, P. T. & Seow, C. Y. Mechanics of airway narrowing. Physiologic

Basis of … (2005).

62. Wiggs, B. R., Moreno, R. & Hogg, J. C. A model of the mechanics of airway narrowing.

Journal of Applied … (1990).

63. Lambert, R. K., Wiggs, B. R., Kuwano, K., Hogg, J. C. & Paré, P. D. Functional

significance of increased airway smooth muscle in asthma and COPD. J. Appl.

Physiol. 74, 2771–2781 (1993).

64. Bai, T. R. & Knight, D. A. Structural changes in the airways in asthma: observations

and consequences. Clin. Sci. 108, 463–477 (2005).

65. Ward, C. et al. Reduced airway distensibility, fixed airflow limitation, and airway

wall remodeling in asthma. American Journal of Respiratory and Critical Care

Medicine 164, 1718–1721 (2001).

66. Brackel, H. J. et al. Central airways behave more stiffly during forced expiration in

patients with asthma. American Journal of Respiratory and Critical Care Medicine

162, 896–904 (2000).

146

67. Gelb, A. F., Licuanan, J., Shinar, C. M. & Zamel, N. Unsuspected loss of lung elastic

recoil in chronic persistent asthma. CHEST 121, 715–721 (2002).

68. Sears, M. R. et al. A longitudinal, population-based, cohort study of childhood

asthma followed to adulthood. The new england journal of medicine 349, 1414–

1422 (2003).

69. Saglani, S. & Bush, A. The early-life origins of asthma. Current Opinion in Allergy and

Clinical Immunology 7, 83–56 (2007).

70. Payne, D. N. R. et al. Early thickening of the reticular basement membrane in

children with difficult asthma. American Journal of Respiratory and Critical Care

Medicine 167, 78–82 (2003).

71. Pohunek, P., Warner, J. O., Turzikova, J., Kudrmann, J. & Roche, W. R. Markers of

eosinophilic inflammation and tissue re-modelling in children before clinically

diagnosed bronchial asthma. Pediatr Allergy Immunol 16, 43–51 (2005).

72. Saglani, S. et al. Airway remodeling and inflammation in symptomatic infants with

reversible airflow obstruction. American Journal of Respiratory and Critical Care

Medicine 171, 722–727 (2005).

73. Saglani, S. et al. Early detection of airway wall remodeling and eosinophilic

inflammation in preschool wheezers. American Journal of Respiratory and Critical

Care Medicine 176, 858–864 (2007).

74. Guilbert, T. W. et al. Long-term inhaled corticosteroids in preschool children at high

risk for asthma. The new england journal of medicine 354, 1985–1997 (2006).

75. Bisgaard, H., Hermansen, M. N., Loland, L., Halkjaer, L. B. & Buchvald, F. Intermittent

inhaled corticosteroids in infants with episodic wheezing. The new england journal

147

of medicine 354, 1998–2005 (2006).

76. Wright, A. L., Taussig, L. M., Ray, C. G., Harrison, H. R. & Holberg, C. J. The Tucson

Children's Respiratory Study. II. Lower respiratory tract illness in the first year of

life. Am. J. Epidemiol. 129, 1232–1246 (1989).

77. Gern, J. E. et al. Relationships among specific viral pathogens, virus-induced

interleukin-8, and respiratory symptoms in infancy. Pediatr Allergy Immunol 13,

386–393 (2002).

78. Samet, J. M., Tager, I. B. & Speizer, F. E. The relationship between respiratory illness

in childhood and chronic air-flow obstruction in adulthood. Am Rev Respir Dis 127,

508–523 (1983).

79. Martinez, F. D. et al. Asthma and wheezing in the first six years of life. The Group

Health Medical Associates. N. Engl. J. Med. 332, 133–138 (1995).

80. Stein, R. T. et al. Respiratory syncytial virus in early life and risk of wheeze and

allergy by age 13 years. The Lancet 354, 541–545 (1999).

81. Sigurs, N., Bjarnason, R., Sigurbergsson, F. & Kjellman, B. Respiratory syncytial virus

bronchiolitis in infancy is an important risk factor for asthma and allergy at age 7.

American Journal of Respiratory and Critical Care Medicine 161, 1501–1507 (2000).

82. Openshaw, P. J., Dean, G. S. & Culley, F. J. Links between respiratory syncytial virus

bronchiolitis and childhood asthma: clinical and research approaches. Pediatr.

Infect. Dis. J. 22, S58–64– discussion S64–5 (2003).

83. Reijonen, T. M., Kotaniemi-Syrjänen, A., Korhonen, K. & Korppi, M. Predictors of

asthma three years after hospital admission for wheezing in infancy. Pediatrics

106, 1406–1412 (2000).

148

84. Kotaniemi-Syrjänen, A. et al. Rhinovirus-induced wheezing in infancy--the first sign

of childhood asthma? J. Allergy Clin. Immunol. 111, 66–71 (2003).

85. Thomsen, S. F. et al. Exploring the association between severe respiratory syncytial

virus infection and asthma: a registry-based twin study. American Journal of

Respiratory and Critical Care Medicine 179, 1091–1097 (2009).

86. Lemanske, R. F. The childhood origins of asthma (COAST) study. Pediatr Allergy

Immunol 13 Suppl 15, 38–43 (2002).

87. Lemanske, R. F. et al. Rhinovirus illnesses during infancy predict subsequent

childhood wheezing. J. Allergy Clin. Immunol. 116, 571–577 (2005).

88. Duff, A. L. et al. Risk factors for acute wheezing in infants and children: viruses,

passive smoke, and IgE antibodies to inhalant allergens. Pediatrics 92, 535–540

(1993).

89. Rakes, G. P. et al. Rhinovirus and respiratory syncytial virus in wheezing children

requiring emergency care. IgE and eosinophil analyses. American Journal of

Respiratory and Critical Care Medicine 159, 785–790 (1999).

90. Jackson, D. J. et al. Wheezing Rhinovirus Illnesses in Early Life Predict Asthma

Development in High-Risk Children. American Journal of Respiratory and Critical

Care Medicine 178, 667–672 (2008).

91. Kusel, M. M. H. et al. Early-life respiratory viral infections, atopic sensitization, and

risk of subsequent development of persistent asthma. J. Allergy Clin. Immunol. 119,

1105–1110 (2007).

92. Proud, D. et al. Gene Expression Profiles during In VivoHuman Rhinovirus Infection.

American Journal of Respiratory and Critical Care Medicine 178, 962–968 (2008).

149

93. Mäkelä, M. J. et al. Viruses and bacteria in the etiology of the common cold. Journal

of Clinical Microbiology 36, 539–542 (1998).

94. Tacon, C. E. et al. Human Rhinovirus Infection Up-Regulates MMP-9 Production in

Airway Epithelial Cells via NF- B. American Journal of Respiratory Cell and Molecular

Biology 43, 201–209 (2010).

95. Lau, S. K. P. et al. Clinical Features and Complete Genome Characterization of a

Distinct Human Rhinovirus (HRV) Genetic Cluster, Probably Representing a

Previously Undetected HRV Species, HRV-C, Associated with Acute Respiratory

Illness in Children. Journal of Clinical Microbiology 45, 3655–3664 (2007).

96. Palmenberg, A. C. et al. Sequencing and Analyses of All Known Human Rhinovirus

Genomes Reveal Structure and Evolution. Science 324, 55–59 (2009).

97. McErlean, P. et al. Distinguishing molecular features and clinical characteristics of a

putative new rhinovirus species, human rhinovirus C (HRV C). PLoS ONE 3, e1847

(2008).

98. Bochkov, Y. A. et al. Molecular modeling, organ culture and reverse genetics for a

newly identified human rhinovirus C. Nature Medicine 17, 627–632 (2011).

99. Bochkov, Y. A. et al. Cadherin-related family member 3, a childhood asthma

susceptibility gene product, mediates rhinovirus C binding and replication.

Proceedings of the National Academy of Sciences 112, 5485–5490 (2015).

100. Greve, J. M. et al. The major human rhinovirus receptor is ICAM-1. Cell 56, 839–847

(1989).

101. Staunton, D. E. et al. A cell adhesion molecule, ICAM-1, is the major surface receptor

for rhinoviruses. Cell 56, 849–853 (1989).

150

102. Tomassini, J. E. et al. cDNA cloning reveals that the major group rhinovirus receptor

on HeLa cells is intercellular adhesion molecule 1. Proceedings of the National

Academy of Sciences 86, 4907–4911 (1989).

103. Hofer, F. et al. Members of the low density lipoprotein receptor family mediate cell

entry of a minor-group common cold virus. Proceedings of the National Academy of

Sciences 91, 1839–1842 (1994).

104. Fuchs, R. & Blaas, D. Uncoating of human rhinoviruses. Reviews in Medical Virology

20, 281–297 (2010).

105. Laine, P. Phylogenetic analysis of human rhinovirus capsid protein VP1 and 2A

protease coding sequences confirms shared genus-like relationships with human

enteroviruses. Journal of General Virology 86, 697–706 (2005).

106. Rossmann, M. G. Viral cell recognition and entry. Protein Sci. 3, 1712–1725 (1994).

107. Staunton, D. E., Dustin, M. L., Erickson, H. P. & Springer, T. A. The arrangement of

the immunoglobulin-like domains of ICAM-1 and the binding sites for LFA-1 and

rhinovirus. Cell 61, 243–254 (1990).

108. Hewat, E. A. et al. The cellular receptor to human rhinovirus 2 binds around the 5-

fold axis and not in the canyon: a structural view. EMBO J. 19, 6317–6325 (2000).

109. Knipe, D. M. & Howley, P. Fields Virology. (Lippincott Williams & Wilkins, 2001).

110. Dick, E. C. Experimental infections of chimpanzees with human rhinovirus types 14

and 43. Proc. Soc. Exp. Biol. Med. 127, 1079–1081 (1968).

111. Pinto, C. A. & Haff, R. F. Experimental infection of gibbons with rhinovirus. Nature

224, 1310–1311 (1969).

112. Leigh, R. et al. Human rhinovirus infection enhances airway epithelial cell

151

production of growth factors involved in airway remodeling. Journal of Allergy and

Clinical Immunology 121, 1238–1245.e4 (2008).

113. Proud, D. The Pulmonary Epithelium in Health and Disease. (John Wiley & Sons,

2008). doi:10.1002/9780470727010

114. Arruda, E. et al. Localization of human rhinovirus replication in the upper

respiratory tract by in situ hybridization. Journal of Infectious Diseases 171, 1329–

1333 (1995).

115. Mosser, A. G. et al. Similar Frequency of Rhinovirus-Infectible Cells in Upper and

Lower Airway Epithelium. Journal of Infectious Diseases 185, 734–743 (2002).

116. Papadopoulos, N. G. et al. Rhinoviruses infect the lower airways. J. Infect. Dis. 181,

1875–1884 (2000).

117. Jakiela, B., Brockman-Schneider, R., Amineva, S., Lee, W.-M. & Gern, J. E. Basal Cells

of Differentiated Bronchial Epithelium Are More Susceptible to Rhinovirus

Infection. American Journal of Respiratory Cell and Molecular Biology 38, 517–523

(2008).

118. Mosser, A. G. et al. Quantitative and Qualitative Analysis of Rhinovirus Infection in

Bronchial Tissues. American Journal of Respiratory and Critical Care Medicine 171,

645–651 (2005).

119. Winther, B., Gwaltney, J. M., Mygind, N., Turner, R. B. & Hendley, J. O. Sites of

rhinovirus recovery after point inoculation of the upper airway. JAMA 256, 1763–

1767 (1986).

120. Harris, J. M. & Gwaltney, J. M. Incubation periods of experimental rhinovirus

infection and illness. Clin. Infect. Dis. 23, 1287–1290 (1996).

152

121. Jackson, W. T. et al. Subversion of cellular autophagosomal machinery by RNA

viruses. PLoS Biol. 3, e156 (2005).

122. Deszcz, L., Gaudernak, E., Kuechler, E. & Seipelt, J. Apoptotic events induced by

human rhinovirus infection. J. Gen. Virol. 86, 1379–1389 (2005).

123. Proud, D. & Leigh, R. Epithelial cells and airway diseases. Immunological Reviews

242, 186–204 (2011).

124. Wetsel, R. A., Wang, D. & Calame, D. G. Therapeutic potential of lung epithelial

progenitor cells derived from embryonic and induced pluripotent stem cells. Annu.

Rev. Med. 62, 95–105 (2011).

125. Holgate, S. T. Epithelium dysfunction in asthma. Journal of Allergy and Clinical

Immunology 120, 1233–1244 (2007).

126. de Boer, W. I. et al. Altered expression of epithelial junctional proteins in atopic

asthma: possible role in inflammation. Can. J. Physiol. Pharmacol. 86, 105–112

(2008).

127. Bayram, H., Rusznak, C., Khair, O. A., Sapsford, R. J. & Abdelaziz, M. M. Effect of

ozone and nitrogen dioxide on the permeability of bronchial epithelial cell cultures

of non-asthmatic and asthmatic subjects. Clin Exp Allergy 32, 1285–1292 (2002).

128. Bucchieri, F. et al. Asthmatic bronchial epithelium is more susceptible to oxidant-

induced apoptosis. American Journal of Respiratory Cell and Molecular Biology 27,

179–185 (2002).

129. Gern, J. E. et al. Bidirectional interactions between viral respiratory illnesses and

cytokine responses in the first year of life. J. Allergy Clin. Immunol. 117, 72–78

(2006).

153

130. Papadopoulos, N. G., Stanciu, L. A., Papi, A., Holgate, S. T. & Johnston, S. L. A defective

type 1 response to rhinovirus in atopic asthma. Thorax 57, 328–332 (2002).

131. Wark, P. A. B. et al. Asthmatic bronchial epithelial cells have a deficient innate

immune response to infection with rhinovirus. Journal of Experimental Medicine

201, 937–947 (2005).

132. Contoli, M. et al. Role of deficient type III interferon-λ production in asthma

exacerbations. Nature Medicine 12, 1023–1026 (2006).

133. Hackett, T.-L. et al. Induction of epithelial-mesenchymal transition in primary

airway epithelial cells from patients with asthma by transforming growth factor-

beta1. American Journal of Respiratory and Critical Care Medicine 180, 122–133

(2009).

134. Kennedy, J. L. et al. Comparison of viral load in individuals with and without asthma

during infections with rhinovirus. American Journal of Respiratory and Critical Care

Medicine 189, 532–539 (2014).

135. Talbot, T. R. et al. Asthma as a risk factor for invasive pneumococcal disease. The

new england journal of medicine 352, 2082–2090 (2005).

136. Biscione, G. L., Corne, J., Chauhan, A. J. & Johnston, S. L. Increased frequency of

detection of Chlamydophila pneumoniae in asthma. European Respiratory Journal

24, 745–749 (2004).

137. Holgate, S. T., Roberts, G., Arshad, H. S., Howarth, P. H. & Davies, D. E. The role of the

airway epithelium and its interaction with environmental factors in asthma

pathogenesis. Proceedings of the American Thoracic Society 6, 655–659 (2009).

138. Polito, A. J. & Proud, D. Epithelia cells as regulators of airway inflammation. J.

154

Allergy Clin. Immunol. 102, 714–718 (1998).

139. Holgate, S. T. et al. Epithelial-mesenchymal interactions in the pathogenesis of

asthma. J. Allergy Clin. Immunol. 105, 193–204 (2000).

140. Newcomb, D. C. et al. Phosphatidylinositol 3-kinase is required for rhinovirus-

induced airway epithelial cell interleukin-8 expression. Journal of Biological

Chemistry 280, 36952–36961 (2005).

141. Bentley, J. K. et al. Rhinovirus activates interleukin-8 expression via a Src/p110beta

phosphatidylinositol 3-kinase/Akt pathway in human airway epithelial cells.

Journal of Virology 81, 1186–1194 (2007).

142. Wang, X. et al. Syk is downstream of intercellular adhesion molecule-1 and

mediates human rhinovirus activation of p38 MAPK in airway epithelial cells. The

Journal of Immunology 177, 6859–6870 (2006).

143. Lau, C. et al. Syk associates with clathrin and mediates phosphatidylinositol 3-

kinase activation during human rhinovirus internalization. The Journal of

Immunology 180, 870–880 (2008).

144. Newcomb, D. C. et al. Human rhinovirus 1B exposure induces phosphatidylinositol

3-kinase-dependent airway inflammation in mice. American Journal of Respiratory

and Critical Care Medicine 177, 1111–1121 (2008).

145. Guillot, L. et al. Involvement of toll-like receptor 3 in the immune response of lung

epithelial cells to double-stranded RNA and influenza A virus. Journal of Biological

Chemistry 280, 5571–5580 (2005).

146. Imaizumi, T. et al. Involvement of retinoic acid-inducible gene-I in the IFN-

{gamma}/STAT1 signalling pathway in BEAS-2B cells. European Respiratory Journal

155

25, 1077–1083 (2005).

147. Matsukura, S. et al. Role of RIG-I, MDA-5, and PKR on the expression of

inflammatory chemokines induced by synthetic dsRNA in airway epithelial cells.

Int. Arch. Allergy Immunol. 143 Suppl 1, 80–83 (2007).

148. Gern, J. E. et al. Double-stranded RNA induces the synthesis of specific chemokines

by bronchial epithelial cells. American Journal of Respiratory Cell and Molecular

Biology 28, 731–737 (2003).

149. Sanders, S. P., Proud, D., Siekierski, E. S., Porter, J. D. & Richards, S. M. Nitric oxide

inhibits rhinovirus-induced cytokine production and viral replication in a human

respiratory epithelial cell line. Journal of Virology 72, 934–942 (1998).

150. Zhu, Z. et al. Rhinovirus stimulation of interleukin-6 in vivo and in vitro. Evidence

for nuclear factor kappa B-dependent transcriptional activation. J. Clin. Invest. 97,

421–430 (1996).

151. Kim, J., Proud, D., Sanders, S. P., Siekierski, E. S. & Casolaro, V. Role of NF-kappa B in

cytokine production induced from human airway epithelial cells by rhinovirus

infection. The Journal of Immunology 165, 3384–3392 (2000).

152. Zaheer, R. S., Koetzler, R., Holden, N. S., Wiehler, S. & Proud, D. Selective

transcriptional down-regulation of human rhinovirus-induced production of

CXCL10 from airway epithelial cells via the MEK1 pathway. J. Immunol. 182, 4854–

4864 (2009).

153. Koetzler, R. et al. Nitric oxide inhibits human rhinovirus-induced transcriptional

activation of CXCL10 in airway epithelial cells. J. Allergy Clin. Immunol. 123, 201–

208.e9 (2009).

156

154. Wang, Q. et al. Role of double-stranded RNA pattern recognition receptors in

rhinovirus-induced airway epithelial cell responses. J. Immunol. 183, 6989–6997

(2009).

155. Edwards, M. R. et al. Protein kinase R, IkappaB kinase-beta and NF-kappaB are

required for human rhinovirus induced pro-inflammatory cytokine production in

bronchial epithelial cells. Mol. Immunol. 44, 1587–1597 (2007).

156. Pindel, A. & Sadler, A. The role of protein kinase R in the interferon response. J.

Interferon Cytokine Res. 31, 59–70 (2011).

157. Funkhouser, A. W. et al. Rhinovirus 16 3C protease induces interleukin-8 and

granulocyte-macrophage colony-stimulating factor expression in human bronchial

epithelial cells. Pediatr. Res. 55, 13–18 (2004).

158. Schall, T. J., Bacon, K., Toy, K. J. & Goeddel, D. V. Selective attraction of monocytes

and T lymphocytes of the memory phenotype by cytokine RANTES. Nature 347,

669–671 (1990).

159. Ponath, P. D., Qin, S. & Ringler, D. J. Cloning of the Human eosinophil

chemoattractant, eotaxin. Journal of Clinical … 97, 604–612 (1996).

160. Houck, J. C. & Chang, C. M. The purification and characterization of a lymphokine

chemotactic for lymphocytes--lymphotactin. Inflammation 2, 105–113 (1977).

161. Bazan, J. F. et al. A new class of membrane-bound chemokine with a CX3C motif.

Nature 385, 640–644 (1997).

162. Donninger, H. et al. Rhinovirus induction of the CXC chemokine epithelial-

neutrophil activating peptide-78 in bronchial epithelium. J. Infect. Dis. 187, 1809–

1817 (2003).

157

163. Schroth, M. K. et al. Rhinovirus Replication Causes RANTES Production in Primary

Bronchial Epithelial Cells. American Journal of Respiratory Cell and Molecular

Biology 20, 1220–1228 (2012).

164. Subauste, M. C., Jacoby, D. B., Richards, S. M. & Proud, D. Infection of a human

respiratory epithelial cell line with rhinovirus. Induction of cytokine release and

modulation of susceptibility to infection by cytokine exposure. J. Clin. Invest. 96,

549–557 (1995).

165. Terajima, M. et al. Rhinovirus infection of primary cultures of human tracheal

epithelium: role of ICAM-1 and IL-1beta. Am. J. Physiol. 273, L749–59 (1997).

166. Einarsson, O., Geba, G. P., Zhu, Z., Landry, M. & Elias, J. A. Interleukin-11: stimulation

in vivo and in vitro by respiratory viruses and induction of airways

hyperresponsiveness. J. Clin. Invest. 97, 915–924 (1996).

167. Proud, D. et al. Increased levels of interleukin-1 are detected in nasal secretions of

volunteers during experimental rhinovirus colds. J. Infect. Dis. 169, 1007–1013

(1994).

168. Terán, L. M. et al. RANTES, macrophage-inhibitory protein 1alpha, and the

eosinophil product major basic protein are released into upper respiratory

secretions during virus-induced asthma exacerbations in children. J. Infect. Dis.

179, 677–681 (1999).

169. Traves, S. L. & Proud, D. Viral-associated exacerbations of asthma and COPD.

Current Opinion in Pharmacology 7, 252–258 (2007).

170. Kelly, J. T. & Busse, W. W. Host immune responses to rhinovirus: mechanisms in

asthma. J. Allergy Clin. Immunol. 122, 671–82– quiz 683–4 (2008).

158

171. Chang, Y. et al. TH17 cytokines induce human airway smooth muscle cell migration.

Journal of Allergy and Clinical Immunology 127, 1046–1053.e2 (2011).

172. Kalb, T. H., Chuang, M. T., Marom, Z. & Mayer, L. Evidence for accessory cell function

by class II MHC antigen-expressing airway epithelial cells. American Journal of

Respiratory Cell and Molecular Biology 4, 320–329 (1991).

173. Salik, E. et al. Antigen trafficking and accessory cell function in respiratory

epithelial cells. American Journal of Respiratory Cell and Molecular Biology 21, 365–

379 (1999).

174. Kalb, T. H., Yio, X. Y. & Mayer, L. Human airway epithelial cells stimulate T-

lymphocyte lck and fyn tyrosine kinase. American Journal of Respiratory Cell and

Molecular Biology 17, 561–570 (1997).

175. Oei, E. et al. Accessory cell function of airway epithelial cells. American Journal of

Physiology - Lung Cellular and Molecular Physiology 287, L318–31 (2004).

176. Heinecke, L., Proud, D., Sanders, S., Schleimer, R. P. & Kim, J. Induction of B7-H1 and

B7-DC expression on airway epithelial cells by the Toll-like receptor 3 agonist

double-stranded RNA and human rhinovirus infection: In vivo and in vitro studies.

J. Allergy Clin. Immunol. 121, 1155–1160 (2008).

177. Papi, A. et al. Rhinovirus infection induces major histocompatibility complex class I

and costimulatory molecule upregulation on respiratory epithelial cells. J. Infect.

Dis. 181, 1780–1784 (2000).

178. Fraenkel, D. J. et al. Lower airways inflammation during rhinovirus colds in normal

and in asthmatic subjects. American Journal of Respiratory and Critical Care

Medicine 151, 879–886 (1995).

159

179. Cate, T. R., Rossen, R. D., Douglas, R. G., Butler, W. T. & Couch, R. B. The role of nasal

secretion and serum antibody in the rhinovirus common cold. Am. J. Epidemiol. 84,

352–363 (1966).

180. Gern, J. E., Dick, E. C., Kelly, E. A., Vrtis, R. & Klein, B. Rhinovirus-specific T cells

recognize both shared and serotype-restricted viral epitopes. J. Infect. Dis. 175,

1108–1114 (1997).

181. Johnston, S. L. et al. Community study of role of viral infections in exacerbations of

asthma in 9-11 year old children. BMJ 310, 1225–1229 (1995).

182. Nicholson, K. G., Kent, J. & Ireland, D. C. Respiratory viruses and exacerbations of

asthma in adults. BMJ 307, 982–986 (1993).

183. Friedlander, S. L. & Busse, W. W. The role of rhinovirus in asthma exacerbations. J.

Allergy Clin. Immunol. 116, 267–273 (2005).

184. Khetsuriani, N. et al. Prevalence of viral respiratory tract infections in children with

asthma. J. Allergy Clin. Immunol. 119, 314–321 (2007).

185. O'Byrne, P. M. et al. Severe exacerbations and decline in lung function in asthma.

American Journal of Respiratory and Critical Care Medicine 179, 19–24 (2009).

186. Bai, T. R., Vonk, J. M., Postma, D. S. & Boezen, H. M. Severe exacerbations predict

excess lung function decline in asthma. European Respiratory Journal 30, 452–456

(2007).

187. Covar, R. A., Spahn, J. D., Murphy, J. R., Szefler, S. J.Childhood Asthma Management

Program Research Group. Progression of asthma measured by lung function in the

childhood asthma management program. American Journal of Respiratory and

Critical Care Medicine 170, 234–241 (2004).

160

188. Turner, R. B., Hendley, J. O. & Gwaltney, J. M. Shedding of infected ciliated epithelial

cells in rhinovirus colds. J. Infect. Dis. 145, 849–853 (1982).

189. Winther, B., Brofeldt, S., Christensen, B. & Mygind, N. Light and scanning electron

microscopy of nasal biopsy material from patients with naturally acquired common

colds. Acta Otolaryngol. 97, 309–318 (1984).

190. Schall, T. J. et al. A human T cell-specific molecule is a member of a new gene family.

The Journal of Immunology 141, 1018–1025 (1988).

191. Schall, T. J. Biology of the rantes/sis cytokine family. Cytokine 3, 165–183 (1991).

192. Grayson, M. H. & Holtzman, M. J. Chemokine Complexity. American Journal of

Respiratory Cell and Molecular Biology 35, 143–146 (2012).

193. Appay, V. & Rowland-Jones, S. L. RANTES: a versatile and controversial chemokine.

Trends Immunol. 22, 83–87 (2001).

194. Rojas-Ramos, E. et al. Role of the chemokines RANTES, monocyte chemotactic

proteins-3 and -4, and eotaxins-1 and -2 in childhood asthma. European Respiratory

Journal 22, 310–316 (2003).

195. Alam, R. et al. Increased MCP-1, RANTES, and MIP-1alpha in bronchoalveolar lavage

fluid of allergic asthmatic patients. American Journal of Respiratory and Critical Care

Medicine 153, 1398–1404 (2012).

196. Amrani, Y. & Panettieri, R. A. Airway smooth muscle: contraction and beyond. The

International Journal of Biochemistry & Cell Biology 35, 272–276 (2003).

197. Low, R. B. & White, S. L. Lung smooth muscle differentiation. The International

Journal of Biochemistry & Cell Biology 30, 869–883 (1998).

198. Gunst, S. J. & Tang, D. D. The contractile apparatus and mechanical properties of

161

airway smooth muscle. European Respiratory Journal 15, 600–616 (2000).

199. Yang, Y., Beqaj, S., Kemp, P., Ariel, I. & Schuger, L. Stretch-induced alternative

splicing of serum response factor promotes bronchial myogenesis and is defective

in lung hypoplasia. J. Clin. Invest. 106, 1321–1330 (2000).

200. Bentley, J. K. & Hershenson, M. B. Airway Smooth Muscle Growth in Asthma:

Proliferation, Hypertrophy, and Migration. Proceedings of the American Thoracic

Society 5, 89–96 (2008).

201. Ebina, M., Takahashi, T., Chiba, T. & Motomiya, M. Cellular Hypertrophy and

Hyperplasia of Airway Smooth Muscles Underlying Bronchial Asthma: A 3-D

Morphometric Study. Am Rev Respir Dis 148, 720–726 (1993).

202. Redington, A. E. & Howarth, P. H. Airway wall remodelling in asthma. Thorax 52,

310–312 (1997).

203. Woodruff, P. G. et al. Hyperplasia of smooth muscle in mild to moderate asthma

without changes in cell size or gene expression. American Journal of Respiratory and

Critical Care Medicine 169, 1001–1006 (2004).

204. Gerthoffer, W. T. Migration of airway smooth muscle cells. Proceedings of the

American Thoracic Society 5, 97–105 (2008).

205. Rudijanto, A. The role of vascular smooth muscle cells on the pathogenesis of

atherosclerosis. Acta Med Indones 39, 86–93 (2007).

206. Louis, S. F. & Zahradka, P. Vascular smooth muscle cell motility: From migration to

invasion. Exp Clin Cardiol 15, e75–85 (2010).

207. Hirota, J. A., Nguyen, T. T. B., Schaafsma, D., Sharma, P. & Tran, T. Airway smooth

muscle in asthma: Phenotype plasticity and function. Pulmonary Pharmacology &

162

Therapeutics 22, 370–378 (2009).

208. Berg, H. C. A physicist looks at bacterial chemotaxis. Cold Spring Harb. Symp. Quant.

Biol. 53 Pt 1, 1–9 (1988).

209. Bourret, R. B. & Stock, A. M. Molecular information processing: lessons from

bacterial chemotaxis. Journal of Biological Chemistry 277, 9625–9628 (2002).

210. Zigmond, S. H. Ability of polymorphonuclear leukocytes to orient in gradients of

chemotactic factors. The Journal of Cell Biology 75, 606–616 (1977).

211. Van Haastert, P. J. M. & Devreotes, P. N. Chemotaxis: signalling the way forward.

Nat. Rev. Mol. Cell Biol. 5, 626–634 (2004).

212. Devreotes, P. & Janetopoulos, C. Eukaryotic chemotaxis: distinctions between

directional sensing and polarization. Journal of Biological Chemistry 278, 20445–

20448 (2003).

213. Parent, C. A., Blacklock, B. J., Froehlich, W. M., Murphy, D. B. & Devreotes, P. N. G

protein signaling events are activated at the leading edge of chemotactic cells. Cell

95, 81–91 (1998).

214. Caterina, M. J., Devreotes, P. N., Borleis, J. & Hereld, D. Agonist-induced loss of ligand

binding is correlated with phosphorylation of cAR1, a G protein-coupled

chemoattractant receptor from Dictyostelium. Journal of Biological Chemistry 270,

8667–8672 (1995).

215. Sasaki, A. T. et al. G protein-independent Ras/PI3K/F-actin circuit regulates basic

cell motility. The Journal of Cell Biology 178, 185–191 (2007).

216. Bornfeldt, K. E. et al. Platelet-derived growth factor. Distinct signal transduction

pathways associated with migration versus proliferation. Ann. N. Y. Acad. Sci. 766,

163

416–430 (1995).

217. Prass, M., Jacobson, K., Mogilner, A. & Radmacher, M. Direct measurement of the

lamellipodial protrusive force in a migrating cell. The Journal of Cell Biology 174,

767–772 (2006).

218. Gerthoffer, W. T. Mechanisms of Vascular Smooth Muscle Cell Migration. Circulation

Research 100, 607–621 (2007).

219. Tang, D., Mehta, D. & Gunst, S. J. Mechanosensitive tyrosine phosphorylation of

paxillin and focal adhesion kinase in tracheal smooth muscle. Am. J. Physiol. 276,

C250–8 (1999).

220. Smith, P. G., Garcia, R. & Kogerman, L. Mechanical strain increases protein tyrosine

phosphorylation in airway smooth muscle cells. Exp. Cell Res. 239, 353–360 (1998).

221. Opazo Saez, A. et al. Tension development during contractile stimulation of smooth

muscle requires recruitment of paxillin and vinculin to the membrane. Am. J.

Physiol., Cell Physiol. 286, C433–47 (2004).

222. Krymskaya, V. P. et al. Src is necessary and sufficient for human airway smooth

muscle cell proliferation and migration. The FASEB Journal 19, 428–430 (2005).

223. Totsukawa, G. et al. Distinct roles of ROCK (Rho-kinase) and MLCK in spatial

regulation of MLC phosphorylation for assembly of stress fibers and focal

adhesions in 3T3 fibroblasts. The Journal of Cell Biology 150, 797–806 (2000).

224. Hedges, J. C. et al. A role for p38(MAPK)/HSP27 pathway in smooth muscle cell

migration. Journal of Biological Chemistry 274, 24211–24219 (1999).

225. Al-Alwan, L. A. et al. Autocrine-regulated airway smooth muscle cell migration is

dependent on IL-17-induced growth-related oncogenes. Journal of Allergy and

164

Clinical Immunology 130, 1–15 (2012).

226. Takeda, N. et al. Epithelium-derived chemokines induce airway smooth muscle cell

migration. Clinical & Experimental Allergy 39, 1018–1026 (2009).

227. Govindaraju, V. et al. Interleukin-8: novel roles in human airway smooth muscle cell

contraction and migration. AJP: Cell Physiology 291, C957–C965 (2006).

228. Parameswaran, K. Extracellular matrix regulates human airway smooth muscle cell

migration. European Respiratory Journal 24, 545–551 (2004).

229. Kuo, P. L., Hsu, Y. L., Huang, M. S., Chiang, S. L. & Ko, Y. C. Bronchial Epithelium-

Derived IL-8 and RANTES Increased Bronchial Smooth Muscle Cell Migration and

Proliferation by Kruppel-like Factor 5 in Areca Nut-Mediated Airway Remodeling.

Toxicological Sciences 121, 177–190 (2011).

230. Joubert, P. et al. CCR3 expression and function in asthmatic airway smooth muscle

cells. The Journal of Immunology 175, 2702–2708 (2005).

231. Churchill, L. et al. Cyclooxygenase metabolism of endogenous arachidonic acid by

cultured human tracheal epithelial cells. Am Rev Respir Dis 140, 449–459 (1989).

232. Kaur, M. et al. Effect of 2-adrenoceptor agonists and other cAMP-elevating agents

on inflammatory gene expression in human ASM cells: a role for protein kinase A.

AJP: Lung Cellular and Molecular Physiology 295, L505–L514 (2008).

233. Reed, L. J. & Muench, H. A SIMPLE METHOD OF ESTIMATING FIFTY PER CENT

ENDPOINTS. Am. J. Epidemiol. 27, 493–497 (1938).

234. Olson, N. H. et al. Structure of a human rhinovirus complexed with its receptor

molecule. Proceedings of the National Academy of Sciences 90, 507–511 (1993).

235. Boyden, S. The chemotactic effect of mixtures of antibody and antigen on

165

polymorphonuclear leucocytes. Journal of Experimental Medicine 115, 453–466

(1962).

236. Bird, C. & Kirstein, S. Real-time, label-free monitoring of cellular invasion and

migration with the xCELLigence system. Nature methods 6, (2009).

237. Mosmann, T. Rapid colorimetric assay for cellular growth and survival: application

to proliferation and cytotoxicity assays. J. Immunol. Methods 65, 55–63 (1983).

238. Davies, D. E. The role of the epithelium in airway remodeling in asthma. Proceedings

of the American Thoracic Society 6, 678–682 (2009).

239. Carlin, S. M., Roth, M. & Black, J. L. Urokinase potentiates PDGF-induced chemotaxis

of human airway smooth muscle cells. American Journal of Physiology - Lung

Cellular and Molecular Physiology 284, L1020–L1026 (2003).

240. Ito, I. et al. Platelet-derived growth factor and transforming growth factor-beta

modulate the expression of matrix metalloproteinases and migratory function of

human airway smooth muscle cells. Clinical & Experimental Allergy 39, 1370–1380

(2009).

241. Koziol-White, C. J., Goncharova, E. A., Cao, G., Krymskaya, V. P. & Panettieri, R. A., Jr.

DHEA-S inhibits human neutrophil and human airway smooth muscle migration.

BBA - Molecular Basis of Disease 1822, 1–5 (2012).

242. Jackson, D. J. & Lemanske, R. F. The role of respiratory virus infections in childhood

asthma inception. Immunol Allergy Clin North Am 30, 513–22– vi (2010).

243. Knight, D. A. & Holgate, S. T. The airway epithelium: structural and functional

properties in health and disease. Respirology 8, 432–446 (2003).

244. Black, J. L. & Roth, M. Intrinsic asthma: is it intrinsic to the smooth muscle? Clinical

166

& Experimental Allergy 39, 962–965 (2009).

245. Goncharova, E. A., Billington, C. K. & Irani, C. Cyclic AMP-Mobilizing Agents and

Glucocorticoids Modulate Human Smooth Muscle Cell Migration. American Journal

of Respiratory Cell and Molecular Biology 29, 19–27 (2003).

246. Yang, L. V., Radu, C. G., Wang, L., Riedinger, M. & Witte, O. N. Gi-independent

macrophage chemotaxis to lysophosphatidylcholine via the immunoregulatory

GPCR G2A. Blood 105, 1127–1134 (2005).

247. Peveri, P., Kernen, P., Tscharner, von, V., Walz, A. & Baggiolini, M. Mechanism of

neutrophil activation by NAF, a novel monocyte-derived peptide agonist. FASEB J. 2,

2702–2706 (1988).

248. Thelen, M. Dancing to the tune of chemokines. Nat Immunol 2, 129–134 (2001).

249. Neptune, E. R. & Bourne, H. R. Receptors induce chemotaxis by releasing the

betagamma subunit of Gi, not by activating Gq or Gs. Proceedings of the National

Academy of Sciences 94, 14489–14494 (1997).

250. Saunders, R. et al. Airway smooth muscle chemokine receptor expression and

function in asthma. Clinical & Experimental Allergy 39, 1684–1692 (2009).

251. Proud, D., Sanders, S. P. & Wiehler, S. Human rhinovirus infection induces airway

epithelial cell production of human beta-defensin 2 both in vitro and in vivo. The

Journal of Immunology 172, 4637–4645 (2004).

252. Roshan Moniri, M. et al. Dynamic assessment of cell viability, proliferation and

migration using real time cell analyzer system (RTCA). Cytotechnology 67, 379–386

(2014).

253. Moniri, M. R. et al. TRAIL-engineered pancreas-derived mesenchymal stem cells:

167

characterization and cytotoxic effects on pancreatic cancer cells. Cancer Gene Ther

19, 652–658 (2012).

254. Regamey, N. et al. Increased airway smooth muscle mass in children with asthma,

cystic fibrosis, and non-cystic fibrosis bronchiectasis. American Journal of

Respiratory and Critical Care Medicine 177, 837–843 (2008).

255. Plant, P. J., North, M. L., Ward, A., Ward, M. & al, E. Hypertrophic airway smooth

muscle mass correlates with increased airway responsiveness in a murine model of

asthma. American journal of … 46, 532–540 (2012).

256. Thomson, R. J., Bramley, A. M. & Schellenberg, R. R. Airway muscle stereology:

implications for increased shortening in asthma. American Journal of Respiratory

and Critical Care Medicine 154, 749–757 (2012).

257. Benayoun, L., Druilhe, A., Dombret, M.-C., Aubier, M. & Pretolani, M. Airway

Structural Alterations Selectively Associated with Severe Asthma. American Journal

of Respiratory and Critical Care Medicine 167, 1360–1368 (2003).

258. Brewster, C. E. P. et al. Myofibroblasts and Subepithelial Fibrosis in Bronchial

Asthma. American Journal of Respiratory Cell and Molecular Biology 3, 507–511

(2012).

259. Gizycki, M. J., Adelroth, E., Rogers, A. V., O'Byrne, P. M. & Jeffery, P. K. Myofibroblast

involvement in the allergen-induced late response in mild atopic asthma. American

Journal of Respiratory Cell and Molecular Biology 16, 664–673 (1997).

168

7 CHAPTER SEVEN: APPENDIX

169

Table 7.1: All targets of 384-gene GPCR array conducted on 4 different ASM donors.

Target Average CT (n=4)

Eukaryotic 18S rRNA 12.8385

actin, beta 23.04275

adenylate cyclase activating

polypeptide 1 (pituitary) receptor

type I 40

adrenomedullin receptor 40

adenosine A1 receptor 30.02025

adenosine A2a receptor 32.54075

adenosine A2b receptor 29.297

adenosine A3 receptor 40

adrenergic, alpha-1A-, receptor 36.7335

adrenergic, alpha-1B-, receptor 31.918

adrenergic, alpha-1D-, receptor 33.03675

adrenergic, alpha-2A-, receptor 40

adrenergic, alpha-2B-, receptor 40

adrenergic, beta-1-, receptor 40

adrenergic, beta-2-, receptor, surface 29.10275

adrenergic, beta-3-, receptor 40

angiotensin II receptor, type 1 40

angiotensin II receptor, type 2 40

angiotensin II receptor-like 1 40

arginine vasopressin receptor 1A 40

170

arginine vasopressin receptor 1B 40

arginine vasopressin receptor 2

(nephrogenic diabetes insipidus) 40

beta-2-microglobulin 18.862

brain-specific angiogenesis inhibitor

1 36.01625

brain-specific angiogenesis inhibitor

2 29.54275

brain-specific angiogenesis inhibitor

3 38.515

bradykinin receptor B1 25.2155

bradykinin receptor B2 23.21725

Burkitt lymphoma receptor 1, GTP

binding protein (chemokine (C-X-C

motif) receptor 5) 40

bombesin-like receptor 3 40

chromosome 11 hypothetical protein

ORF4 26.30125

complement component 3a receptor

1 32.467

complement component 5 receptor 1

(C5a ligand) 30.4225

calcitonin receptor 40

calcitonin receptor-like 30.81225

171

calcium-sensing receptor

(hypocalciuric hypercalcemia 1,

severe neonatal

hyperparathyroidism) 40

chemokine binding protein 2 36.00525

cholecystokinin A receptor 29.3215

cholecystokinin B receptor 40

chemokine (C-C motif) receptor 1 31.6485

chemokine (C-C motif) receptor 10 37.09

chemokine (C-C motif) receptor 2 40

chemokine (C-C motif) receptor 3 34.813

chemokine (C-C motif) receptor 4 38.656

chemokine (C-C motif) receptor 5 40

chemokine (C-C motif) receptor 6 40

chemokine (C-C motif) receptor 7 40

chemokine (C-C motif) receptor 8 40

chemokine (C-C motif) receptor 9 40

chemokine (C-C motif) receptor-like

1 24.00725

chemokine (C-C motif) receptor-like

2 36.27375

CD97 antigen 25.8905

172

cadherin, EGF LAG seven-pass G-type

receptor 1 (flamingo homolog,

Drosophila) 36.13525

cadherin, EGF LAG seven-pass G-type

receptor 2 (flamingo homolog,

Drosophila) 33.39325

cadherin, EGF LAG seven-pass G-type

receptor 3 (flamingo homolog,

Drosophila) 33.53675

cholinergic receptor, muscarinic 1 38.40025

cholinergic receptor, muscarinic 2 25.497

cholinergic receptor, muscarinic 3 40

cholinergic receptor, muscarinic 4 34.82025

cholinergic receptor, muscarinic 5 34.107

chemokine-like receptor 1 37.705

cannabinoid receptor 1 (brain) 38.97075

cannabinoid receptor 2

(macrophage) 40

corticotropin releasing hormone

receptor 1 40

corticotropin releasing hormone

receptor 2 40

chemokine (C-X3-C motif) receptor 1 40

chemokine (C-X-C motif) receptor 3 40

173

chemokine (C-X-C motif) receptor 4 40

chemokine (C-X-C motif) receptor 6 39.232

cysteinyl leukotriene receptor 1 35.359

cysteinyl leukotriene receptor 2 40

dopamine receptor D1 32.54425

dopamine receptor D2 36.69325

dopamine receptor D3 38.75

dopamine receptor D4 38.9905

dopamine receptor D5 38.97975

Epstein-Barr virus induced gene 2

(lymphocyte-specific G protein-

coupled receptor) 29.57275

endothelial differentiation,

sphingolipid G-protein-coupled

receptor, 1 26.379

endothelial differentiation,

lysophosphatidic acid G-protein-

coupled receptor, 2 22.09925

endothelial differentiation,

sphingolipid G-protein-coupled

receptor, 3 25.50025

endothelial differentiation,

lysophosphatidic acid G-protein-

coupled receptor, 4 30.0635

174

endothelial differentiation, G-

protein-coupled receptor 6 40

endothelial differentiation,

lysophosphatidic acid G-protein-

coupled receptor, 7 28.97075

endothelial differentiation,

sphingolipid G-protein-coupled

receptor, 8 37.9345

endothelin receptor type A 28.34425

endothelin receptor type B 26.1525

EGF, latrophilin and seven

transmembrane domain containing 1 28.87525

egf-like module containing, mucin-

like, hormone receptor-like 1 40

egf-like module containing, mucin-

like, hormone receptor-like 2 40

egf-like module containing, mucin-

like, hormone receptor-like 3 40

coagulation factor II (thrombin)

receptor 24.62925

coagulation factor II (thrombin)

receptor-like 1 31.286

coagulation factor II (thrombin)

receptor-like 2 25.59425

175

coagulation factor II (thrombin)

receptor-like 3 40

FKSG83 40

formyl peptide receptor 1 40

formyl peptide receptor-like 1 40

formyl peptide receptor-like 2 36.7365

follicle stimulating hormone receptor 40

Duffy blood group 40

frizzled homolog 1 (Drosophila) 28.985

frizzled homolog 10 (Drosophila) 39.03925

frizzled homolog 2 (Drosophila) 26.29725

frizzled homolog 3 (Drosophila) 33.28

frizzled homolog 4 (Drosophila) 25.7735

frizzled homolog 5 (Drosophila) 32.55925

frizzled homolog 6 (Drosophila) 25.24825

frizzled homolog 7 (Drosophila) 22.70725

frizzled homolog 8 (Drosophila) 33.1115

frizzled homolog 9 (Drosophila) 34.943

gamma-aminobutyric acid (GABA) B

receptor, 1 27.30875

galanin receptor 1 40

galanin receptor 2 35.569

galanin receptor 3 40

176

glyceraldehyde-3-phosphate

dehydrogenase 18.6065

glucagon receptor 38.9845

glucosaminyl (N-acetyl) transferase

2, I-branching enzyme 33.84125

growth hormone releasing hormone

receptor 40

growth hormone secretagogue

receptor 37.70775

gastric inhibitory polypeptide

receptor 35.78325

glucagon-like peptide 1 receptor 40

glucagon-like peptide 2 receptor 40

gonadotropin-releasing hormone

receptor 40

gonadotropin-releasing hormone

(type 2) receptor 2 40

G protein-coupled bile acid receptor

1 38.98525

putative G protein coupled receptor 25.93125

G protein-coupled receptor 1 28.091

G protein-coupled receptor 10 40

G protein-coupled receptor 101 40

G protein-coupled receptor 103 40

177

G protein-coupled receptor 110 40

G protein-coupled receptor 111 39.21925

G protein-coupled receptor 112 40

G protein-coupled receptor 113 36.4565

G protein-coupled receptor 114 40

G protein-coupled receptor 115 38.99025

G protein-coupled receptor 116 40

G protein-coupled receptor 119 40

G protein-coupled receptor 12 40

G protein-coupled receptor 120 40

G protein-coupled receptor 123 40

G protein-coupled receptor 124 25.4785

G protein-coupled receptor 125 26.792

G protein-coupled receptor 126 28.63275

G protein-coupled receptor 128 40

G protein-coupled receptor 133 32.248

G protein-coupled receptor 135 30.764

G protein-coupled receptor 141 40

G protein-coupled receptor 142 40

G protein-coupled receptor 143 39.00275

G protein-coupled receptor 145 40

G protein-coupled receptor 146 33.47875

G protein-coupled receptor 147 40

G protein-coupled receptor 148 40

178

G protein-coupled receptor 15 38.98325

G protein-coupled receptor 150 40

G protein-coupled receptor 152 40

G protein-coupled receptor 153 27.745

G protein-coupled receptor 160 33.53

G protein-coupled receptor 161 27.24975

G protein-coupled receptor 17 40

G protein-coupled receptor 171 34.5045

G-protein coupled receptor 173 27.9025

G protein-coupled receptor 174 40

G protein-coupled receptor 18 40

G protein-coupled receptor 19 33.764

G protein-coupled receptor 20 35.70675

G protein-coupled receptor 21 29.818

G protein-coupled receptor 22 29.3865

G protein-coupled receptor 23 30.8125

G protein-coupled receptor 24 36.739

G protein-coupled receptor 25 40

G protein-coupled receptor 26 38.267

G protein-coupled receptor 27 40

G protein-coupled receptor 3 31.4075

G protein-coupled receptor 30 35.4215

G protein-coupled receptor 31 40

G protein-coupled receptor 32 40

179

G protein-coupled receptor 34 30.85525

G protein-coupled receptor 35 36.406

G protein-coupled receptor 37

(endothelin receptor type B-like) 28.427

G-protein coupled receptor 37 like 1 40

G protein-coupled receptor 39 31.924

G protein-coupled receptor 4 34.9785

G protein-coupled receptor 40 40

G protein-coupled receptor 43 40

G protein-coupled receptor 44 34.48075

G protein-coupled receptor 45 34.237

G protein-coupled receptor 50 40

G protein-coupled receptor 51 29.78125

G protein-coupled receptor 52 29.17575

G protein-coupled receptor 54 38.9895

G protein-coupled receptor 55 36.8555

G protein-coupled receptor 56 29.989

G protein-coupled receptor 6 40

G protein-coupled receptor 61 37.97925

G protein-coupled receptor 62 34.795

G protein-coupled receptor 63 30.56575

G protein-coupled receptor 64 37.2325

G protein-coupled receptor 65 36.335

G protein-coupled receptor 68 28.5415

180

G protein-coupled receptor 7 40

G protein-coupled receptor 73 38.876

G protein-coupled receptor 73-like 1 40

G protein-coupled receptor 74 39.23775

G protein-coupled receptor 75 40

G protein-coupled receptor 77 40

G protein-coupled receptor 78 38.2055

G protein-coupled receptor 8 40

G protein-coupled receptor 81 37.726

G protein-coupled receptor 82 31.16825

G protein-coupled receptor 83 35.75775

G protein-coupled receptor 84 38.98375

G protein-coupled receptor 85 29.1945

G protein-coupled receptor 87 35.14575

G-protein coupled receptor 88 40

G protein-coupled receptor 92 39.38025

G protein-coupled receptor 97 40

G protein-coupled receptor, family C,

group 5, member A 26.98675

G protein-coupled receptor, family C,

group 5, member B 25.81825

G protein-coupled receptor, family C,

group 5, member C 40

181

G protein-coupled receptor, family C,

group 5, member D 39.2425

G protein-coupled receptor, family C,

group 6, member A 40

glutamate receptor, metabotropic 1 37.9265

glutamate receptor, metabotropic 2 37.22725

glutamate receptor, metabotropic 3 40

glutamate receptor, metabotropic 4 37.96325

glutamate receptor, metabotropic 5 39.2375

glutamate receptor, metabotropic 6 40

glutamate receptor, metabotropic 7 40

glutamate receptor, metabotropic 8 40

gastrin-releasing peptide receptor 33.91975

glucuronidase, beta 24.604

hypocretin (orexin) receptor 1 40

hypocretin (orexin) receptor 2 38.017

histone deacetylase 3 25.82175

hydroxymethylbilane synthase 26.84775

hypoxanthine

phosphoribosyltransferase 1 (Lesch-

Nyhan syndrome) 24.54625

histamine receptor H1 32.0095

histamine receptor H2 37.93025

histamine receptor H3 40

182

histamine receptor H4 38.257