An Investigation into the Mechanisms Driving the …...1 An Investigation into the Mechanisms...

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An Investigation into the Mechanisms Driving the Late Asthmatic Response

Katie Emma Baker

2015

A thesis submitted for the Degree of Doctor of Philosophy

in the Faculty of Medicine, Imperial College London

Respiratory Pharmacology Group

National Heart and Lung Institute

Faculty of Medicine

Imperial College London

Sir Alexander Fleming Building

Exhibition Road

London

SW7 2AZ

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Abstract

Asthma is a disease with increasing global prevalence and patients typically suffer from symptoms

such as wheezing, breathlessness, chest tightness and cough. In allergic asthma, exposure to allergen

can lead to episodes of bronchoconstriction known as the late asthmatic response (LAR).Despite this

being a facet of asthma that patients find debilitating and the fact that this feature of asthma is often

used in clinical research as an end point to study new therapeutic options, the mechanisms driving the

LAR remain unclear. The aim of this thesis was to study the LAR.

Initial studies utilised genetically modified (GM) mice missing key allergic effector cells. The data

showed that CD4+ and CD8+T cells are central to the aetiology of the LAR. Further studies into the role

of the B-cell – mast cell– IgE axis in the murine model indicated that an alternative model was required.

In a rat model, it was found that a mast cell stabiliser Disodium Cromoglycate (DSCG), but not a mast

cell degranulator, compound 48/80, suppressed the LAR. This suggested that DSCG may be inhibiting

LAR independently of its activity on mast cells.

As previous data has shown a role for airway sensory nerves and the TRPA1 ion channel in the LAR,

the impact of DSCG on sensory nerve activity was investigated. The data indicated that DSCG can

inhibit TRPA1 responses in airway sensory nerves and further investigations demonstrated that this

was via its actions on the G-protein coupled receptor, GPR35.

Overall, this body of work provides new insights into the driving mechanisms behind the LAR and may

have uncovered the mystery of the mechanism of action of DSCG. From this, new therapeutic options

for asthmatics may become available in the future.

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Acknowledgements

I would like to thank my supervisors Professor Maria Belvisi and Dr Mark Birrell for providing me with

the opportunity to complete my PhD within such an exciting group. Your help, expertise and

encouragement throughout my PhD has known no bounds and for that I will always be grateful. I

would particularly like to thank Mark for his constant enthusiasm for my project and sense of humour,

especially when things didn’t go to plan. This inspired me to push myself harder and allowed me to

produce pieces of work which I am truly proud of.

I would like to thank supervisor number three, Dr Sarah Maher, for providing emotional support

throughout my time at Imperial College and of course for proof reading this manuscript (any typos can

now be blamed on you – just kidding!). Thank you also to Dr Kristof Raemdonck for introducing me to

the endless delights of the preamplifier and for taking me out with the grownups when things went

wrong, and also when things went right.

A huge thank you goes to the entire Respiratory Pharmacology group who made life enjoyable

throughout my project. In no particular order, thank you to Sara Bonvini (for being my detective

partner), Victoria Jones (for providing an amazing sense of perspective), Liang Yew-Booth (for loving

me unconditionally), Michael Wortley (for allowing me to realise that my own life is actually ok), Matt

Baxter (for making me laugh), James Buckley (for also making me laugh), Eric Dubuis (for being crazy

but brilliant), Ryan Robinson (for being a spaceman), Nicole Dale (for helping me understand Kristof),

Abdel Dekkak (for your technical expertise) and Bilel Dekkak (for being a lovely human being and lab

partner). Special thanks to John Adcock for much time spent in the lab with me on single fibre

recordings and also for teaching me why the 70’s and 80’s were amazing.

I would also like to thank the “Clayponds Crew” and “MRes Crew” who began this journey with me

and have remained close friends. I don’t have room to mention you all individually but you know who

you are. I would particularly like to thank Ian Harrison for introducing me to the oyster card and looking

after me during my first year in London. I would also like to thank Sammy Price for being who she is.

Thank you to my family, Dad, Fiona and Nanan Ruth, for your support, kindness and unconditional

love. Thank you for reminding me what is important in life. I wouldn’t be where I am without you.

Finally, my biggest thanks go to my best friend and partner Dan. Thank you for supporting me

throughout this process. As well as holding my hand and loving me completely you also provided hot

meals and clean pants without which I would not have seen this PhD through to completion. Thank

you for making me smile and for deciding to spend your life with me.

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Statement

This thesis is the product of my own work, except where appropriately referenced. Whole animal

single afferent recording experiments were performed by John Adcock with my assistance (chapter

7). Isolated vagus nerve experiments were performed with the assistance of Dr Sarah Maher (chapter

7). This PhD project was funded by a Medical Research Council (MRC) studentship.

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Copyright Declaration

The copyright of this thesis rests with the author and is made available under a Creative Commons

Attribution Non-Commercial No Derivatives licence. Researchers are free to copy, distribute or

transmit the thesis on the condition that they attribute it, that they do not use it for commercial

purposes and that they do not alter, transform or build upon it. For any reuse or redistribution,

researchers must make clear to others the licence terms of this work.

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Table of Contents

Abstract ................................................................................................................................................... 2

Acknowledgements ................................................................................................................................. 3

Statement ............................................................................................................................................... 4

Copyright Declaration ............................................................................................................................. 5

Table of Contents .................................................................................................................................... 6

List of Figures ........................................................................................................................................ 11

List of Tables ......................................................................................................................................... 13

List of Abbreviations ............................................................................................................................. 14

1 Introduction .................................................................................................................................. 19

1.1 Asthma .................................................................................................................................. 19

1.2 Allergic Sensitisation in Asthma ............................................................................................ 19

1.3 Allergic Asthmatic Responses: The EAR and LAR .................................................................. 21

1.4 Other features of Allergic Asthma ........................................................................................ 22

1.4.1 Airway Remodelling ...................................................................................................... 22

1.4.2 Airway Hyperresponsiveness ........................................................................................ 23

1.5 Cells and Mediators in Allergic Asthma ................................................................................ 24

1.5.1 Dendritic Cells ............................................................................................................... 24

1.5.2 T-Cells ............................................................................................................................ 24

1.5.3 Eosinophils .................................................................................................................... 26

1.5.4 Macrophages................................................................................................................. 28

1.5.5 Neutrophils ................................................................................................................... 28

1.5.6 B-Cells ............................................................................................................................ 29

1.5.7 Mast Cells ...................................................................................................................... 29

1.5.8 Basophils ....................................................................................................................... 30

1.5.9 Immunoglobulins .......................................................................................................... 31

1.5.10 Epithelial Cells ............................................................................................................... 32

1.5.11 Innate Lymphoid Cells ................................................................................................... 33

1.6 Innervation of the Lung ......................................................................................................... 34

1.7 Transient Receptor Potential Receptors and Asthma ........................................................... 37

1.7.1 TRPA1 ............................................................................................................................ 37

1.7.2 TRPV1 ............................................................................................................................ 38

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1.7.3 TRPV4 ............................................................................................................................ 38

1.7.4 TRPM8 ........................................................................................................................... 39

1.8 Aetiology behind the Late Asthmatic Response ................................................................... 39

1.9 Current Asthma Treatments ................................................................................................. 45

1.10 In vivo models of allergic asthma .......................................................................................... 47

1.10.1 Ovalbumin Model ......................................................................................................... 47

1.10.2 Adjuvants ...................................................................................................................... 48

1.10.3 Different Species as Asthma Models ............................................................................ 50

1.11 Thesis Plan ............................................................................................................................. 52

1.11.1 Hypothesis ..................................................................................................................... 52

1.11.2 Aims ............................................................................................................................... 52

2 General Methods .......................................................................................................................... 54

2.1 Introduction .......................................................................................................................... 54

2.2 Animals .................................................................................................................................. 54

2.2.1 Mice............................................................................................................................... 54

2.2.2 Rats ................................................................................................................................ 54

2.2.3 Guinea-Pigs ................................................................................................................... 54

2.3 Validation of Genetically Modified Mice .............................................................................. 54

2.3.1 Cell Preparation............................................................................................................. 55

2.3.2 Staining Protocol ........................................................................................................... 55

2.3.3 Flow Cytometry ............................................................................................................. 57

2.3.4 Histology ....................................................................................................................... 62

2.4 Models of Allergic Asthma .................................................................................................... 63

2.4.1 The OVA Driven Mouse Model of LAR .......................................................................... 63

2.4.2 The OVA Driven Rat Model of LAR ................................................................................ 64

2.5 Assessment of Lung Function ............................................................................................... 64

2.5.1 WBP and Penh ............................................................................................................... 65

2.5.2 Mouse WBP ................................................................................................................... 66

2.5.3 Rat WBP ........................................................................................................................ 66

2.5.4 Data Analyses ................................................................................................................ 66

2.6 Sample Harvesting and Processing ....................................................................................... 67

2.6.1 Blood Sampling ............................................................................................................. 67

2.6.2 Bronchoalveolar Lavage ................................................................................................ 67

2.6.3 Cell Counts .................................................................................................................... 67

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2.6.4 Histology ....................................................................................................................... 67

2.6.5 Measurement of Plasma IgE ......................................................................................... 68

2.7 Isolated Vagus Nerve Preparation ........................................................................................ 70

2.7.1 Nerve Dissection ........................................................................................................... 70

2.7.2 Measurement of Depolarisation ................................................................................... 70

2.7.3 Agonist/Antagonist Experimental Protocols ................................................................. 73

2.7.4 Data Recording and Analysis ......................................................................................... 74

2.8 In Vivo Single Afferent Fibre Recording ................................................................................ 75

2.8.1 Surgery .......................................................................................................................... 75

2.8.2 Recording Equipment .................................................................................................... 75

2.8.3 Nerve Fibre Identification ............................................................................................. 76

2.8.4 Recording Protocol ........................................................................................................ 77

2.8.5 Data Recording and Analysis ......................................................................................... 77

2.9 Statistical Analysis ................................................................................................................. 77

3 Optimisation of animal models of the Late Asthmatic Response ................................................. 79

3.1 Rationale ............................................................................................................................... 79

3.2 Methods ................................................................................................................................ 80

3.2.1 Assessment of Lung Function ....................................................................................... 80

3.2.2 The OVA Driven Rat Model of LAR ................................................................................ 80


3.2.4 Statistical Analysis ......................................................................................................... 81

3.3 Results ................................................................................................................................... 82

3.3.1 Characterisation of the rat OVA-driven LAR model using Budesonide ......................... 82

3.3.2 Characterisation of the mouse OVA-driven LAR model using Budesonide .................. 82

3.4 Discussion .............................................................................................................................. 85

4 Exploring the role of key allergic effector cells in the LAR ............................................................ 89

4.1 Rationale ............................................................................................................................... 89

4.2 Methods ................................................................................................................................ 90

4.2.1 Validation of Genetically Modified Mice by Flow Cytometry ....................................... 90

4.2.2 Validation of Genetically Modified Mice by Histology .................................................. 90


4.2.4 Plasma IgE Analysis ....................................................................................................... 91

4.2.5 Further Exploration of the Enhanced Baseline Response ............................................. 91

4.2.6 Statistical Analysis ......................................................................................................... 91

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4.3 Results ................................................................................................................................... 92

4.3.1 Validation of Genetically Modified Mice ...................................................................... 92

4.3.2 The Role of CD4+ Cells in the OVA Driven Mouse Model of LAR .................................. 96

4.3.3 The Role of CD8+ Cells in the OVA Driven Mouse Model of LAR .................................. 97

4.3.4 The Role of B-Cells in the OVA Driven Mouse Model of LAR ........................................ 98

4.3.5 The Role of Mast Cells in the OVA Driven Mouse Model of LAR .................................. 99

4.3.6 The Role of IgE in the OVA Driven Mouse Model of LAR ............................................ 100

4.3.7 Plasma IgE Levels in the OVA Driven Mouse Model of LAR ........................................ 101

4.3.8 Further Exploration of the Enhanced Baseline Response ........................................... 103

4.4 Discussion ............................................................................................................................ 104

5 Utilising Compound 48/80 and Disodium Cromoglycate as tools to further explore the LAR ... 111

5.1 Rationale ............................................................................................................................. 111

5.2 Methods .............................................................................................................................. 112

5.2.1 Validation of the Compound 48/80 Dosing Protocol .................................................. 112

5.2.2 The OVA Driven Rat Model of LAR .............................................................................. 113

5.2.3 Statistical Analysis ....................................................................................................... 114

5.3 Results ................................................................................................................................. 115

5.3.1 Validation of the Compound 48/80 Dosing Protocol .................................................. 115

5.3.2 The Effect of Compound 48/80 on the LAR ................................................................ 119

5.3.3 The Effect of DSCG on the LAR .................................................................................... 120

5.4 Discussion ............................................................................................................................ 121

6 Further exploration into the role of airway sensory nerves in the LAR ...................................... 126

6.1 Rationale ............................................................................................................................. 126

6.2 Methods .............................................................................................................................. 128


6.2.2 Statistical Analysis ....................................................................................................... 129

6.3 Results ................................................................................................................................. 130

6.3.1 The Effect of Tiotropium and Glycopyrrolate on the LAR ........................................... 130

6.3.2 The Effect of Janssen130 on the LAR .......................................................................... 131

6.3.3 The Effect of XEND0501 on the LAR............................................................................ 132

6.4 Discussion ............................................................................................................................ 133

7 The Role of Disodium Cromoglycate on airway sensory nerves in the LAR ............................... 137

7.1 Rationale ............................................................................................................................. 137

7.2 Methods .............................................................................................................................. 138

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7.2.1 Isolated Vagus Nerve Experiments ............................................................................. 138


7.2.3 Whole Animal Single Afferent Recordings .................................................................. 140

7.2.4 Statistical Analyses ...................................................................................................... 141

7.3 Results ................................................................................................................................. 142

7.3.1 Effect of DSCG on TRPA1 and TRPV1 mediated depolarisation of the isolated vagus

nerve 142

7.3.2 The role of GPR35 Receptor in DSCG mediated inhibition of TRPA1 driven

depolarisation in the isolated vagus nerve ................................................................................. 144

7.3.3 Effect of ML145 on DSCG mediated inhibition of the LAR .......................................... 145

7.3.4 The role of TRPA1 and the GPR35 Receptor in DSCG mediated inhibition of

depolarisation in the Guinea-Pig isolated vagus nerve .............................................................. 146

7.3.5 Effect of DSCG on TRPA1 induced firing of C-fibre afferents ...................................... 148

7.3.6 Effect of ML145 on DSCG mediated inhibition of C-fibre afferent firing .................... 149

7.4 Discussion ............................................................................................................................ 150

8 Summary and Future Studies ...................................................................................................... 154

8.1 Summary ............................................................................................................................. 154

8.2 Thesis Limitations ................................................................................................................ 158

8.3 Future Studies ..................................................................................................................... 160

8.3.1 Further investigation into the Mechanism of Action of DSCG .................................... 160

8.3.2 Further investigate and validate in vivo models ......................................................... 161

8.3.3 Further Exploration of TRPA1 ..................................................................................... 162

8.3.4 Investigate whether the same mechanisms are involved in the LAR on a background of

cigarette smoke exposure under conditions where steroids have limited efficacy ................... 163

References .......................................................................................................................................... 165

9 Appendix ..................................................................................................................................... 208

9.1 Chemicals and Reagents ..................................................................................................... 208

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List of Figures Figure 1.1: Schematic representation of the inflammatory cascade in allergic asthma.. .................... 21

Figure 1.2: The EAR and LAR.. ............................................................................................................... 22

Figure 1.3: Schematic representation of innervation of the airways. ................................................. 35

Figure 1.4: Step-wise approach to asthma treatment. (Adapted from Barnes 2010). ......................... 46

Figure 2.1: Demonstrating the basis of the live/dead cell stain kit.. .................................................... 56

Figure 2.2: Spectral overlap of fluorophores.. ...................................................................................... 58

Figure 2.3: Characterisation of Lymphocyte Populations. .................................................................... 60

Figure 2.4: Characterisation of eosinophil population. ........................................................................ 60

Figure 2.5: Characterisation of neutrophils, alveolar macrophages and inflammatory

monocytes/tissue macrophages. .......................................................................................................... 61

Figure 2.6: Demonstrating histological staining for mast cells in mouse lung tissue sections. ............ 62

Figure 2.7: Timeline demonstrating the protocol used for OVA driven mouse model of LAR ............. 63

Figure 2.8: Timeline demonstrating the protocol used for OVA driven rat model of LAR ................... 64

Figure 2.9: Apparatus setup for isolated vagus nerve depolarisation recordings. ............................... 72

Figure 2.10: Example trace showing protocol for testing inhibitory effect of drugs in the vagus nerve

preparation. .......................................................................................................................................... 73

Figure 2.11: Apparatus setup for in vivo single afferent recording. ..................................................... 76

Figure 3.1: Timeline demonstrating protocol for drug administration in OVA driven rat model of the

LAR ........................................................................................................................................................ 81

Figure 3.2: Effect of Budesonide treatment in the rat OVA-driven LAR. .............................................. 83

Figure 3.3: The effect of Budesonide treatment in the mouse OVA-driven LAR. ................................. 84

Figure 4.1: Validation of Lymphocyte Populations in Genetically Modified Mice by Flow Cytometry. 94

Figure 4.2: Validation of Mast Cell Populations in Genetically Modified Mice by Histology................ 94

Figure 4.3: Validation of Additional Leukocyte Populations in Genetically Modified Mice by Flow

Cytometry. ............................................................................................................................................ 95

Figure 4.4: The Role of CD4+ Cells in the OVA-driven Mouse Model of LAR. ........................................ 96

Figure 4.5: The Role of CD8+ Cells in the OVA-driven Mouse Model of LAR.. ....................................... 97

Figure 4.6: The Role of B-Cells in the OVA-driven Mouse Model of LAR. ............................................. 98

Figure 4.7: The Role of Mast Cells in the OVA-driven Mouse Model of LAR. ....................................... 99

Figure 4.8: The Role of IgE in the OVA-driven Mouse Model of LAR. ................................................. 100

Figure 4.9: Plasma IgE Levels in IgE Deficient Mice. ........................................................................... 101

Figure 4.10: Plasma OVA-Specific IgE Levels in Cell Deficient Mice.................................................... 102

Figure 4.11: Further Exploration of the Enhanced Baseline Response. .............................................. 103

Figure 5.1: Timeline demonstrating protocol for drug administration of Compound 48/80 ............. 112

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Figure 5.2: Timeline demonstrating protocol for final administration of Compound 48/80 in OVA

driven rat model of LAR ...................................................................................................................... 113

Figure 5.3: Timeline demonstrating protocol for DSCG administration in OVA driven rat model of LAR

............................................................................................................................................................ 114

Figure 5.4: Assessment of BAL fluid following treatment with compound 48/80 in naive (C57BL/6)

mice. .................................................................................................................................................... 115

Figure 5.5: Assessment of BAL fluid following treatment with compound 48/80 in naive Brown

Norway Rats. ....................................................................................................................................... 116

Figure 5.6: Validation of Mast Cell depletion by Compound 48/80. .................................................. 118

Figure 5.7: Effect of Compound 48/80 treatment in the rat OVA-driven LAR. ................................... 119

Figure 5.8: Effect of DSCG treatment in the rat OVA-driven LAR. ...................................................... 120

Figure 6.1: The Proposed Mechanisms involved in the LAR. .............................................................. 127

Figure 6.2: Timeline demonstrating protocol for Tiotropium and Glycopyrrolate administration in the

OVA driven rat model of LAR .............................................................................................................. 128

Figure 6.3: Timeline demonstrating protocol for Janssen130 administration in OVA driven rat model

of LAR .................................................................................................................................................. 129

Figure 6.4: Effect of Tiotropium and Glycopyrrolate treatment in the rat OVA-driven LAR. ............. 130

Figure 6.5: Effect of Janssen130 treatment in the rat OVA-driven LAR. ............................................. 131

Figure 6.6: Effect of XEND0501 treatment in the rat OVA-driven LAR. .............................................. 132

Figure 7.1: Timeline demonstrating protocol for ML145 and DSCG administration in the OVA driven

rat model of LAR ................................................................................................................................. 139

Figure 7.2: Recording protocol for in vivo single afferent recording. ................................................. 140

Figure 7.3: Effect of DSCG on TRPA1 and TRPV1 mediated depolarisation of Brown Norway rat vagus

nerve. .................................................................................................................................................. 142

Figure 7.4: Effect of DSCG on TRPA1 and TRPV1 mediated depolarisation of human vagus nerve. .. 143

Figure 7.5: The role of the GPR35 Receptor in DSCG mediated inhibition of TRPA1 driven

depolarisation in the Brown Norway rat isolated vagus nerve. ......................................................... 144

Figure 7.6: Effect of ML145 on DSCG mediated inhibition of the LAR................................................ 145

Figure 7.7: The role of TRPA1 and the GPR35 Receptor in DSCG mediated inhibition of depolarisation

in the Guinea-Pig isolated vagus nerve. ............................................................................................ 147

Figure 7.8: Effect of DSCG on TRPA1 induced firing of C-fibre afferents. ........................................... 148

Figure 7.9: Effect of ML145 on DSCG mediated inhibition of C-fibre afferent firing. ....................... 149

Figure 8.1: Steroid resistance in a murine model of asthma. ............................................................. 163

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List of Tables

Table 2.1: Outlining monoclonal antibody conjugate combinations and dilution factors used to stain

cell populations. .................................................................................................................................... 57

Table 2.2: Outlining surface marker antigens and relevant conjugated monoclonal antibodies used to

identify various cell populations ........................................................................................................... 59

Table 2.3: Morphology of Cells Counted in BAL Fluid ........................................................................... 68

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List of Abbreviations

5-HT 5-hydroxytryptamine

ACh Acetylcholine

AHR Airway Hyperresponsiveness

APC Antigen Presenting Cell

ASM Airway Smooth Muscle

ATP Adenosine triphosphate

AUC Area Under Curve

BAL Bronchoalveolar lavage (fluid)

BSA Bovine serum albumin

CCR3 CC or β chemokine receptor 3

CD40L CD40 Ligand

CD40R CD40 Receptor

CGRP Calcitonin Gene Related Peptide

CNS Central Nervous System

CTLA4 Cytotoxic T-lymphocyte Antigen 4

DAMPs Damage-associated molecular pattern molecules

DSCG Disodium Cromoglycate

EAR Early Asthmatic Response

ECP Eosinophil Cationic Protein

EDN Eosinophil Derived Neurotoxin

ELISA Enzyme Linked Immunosorbant Assay

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EPX Eosinophil Peroxidase

FBS Fetal Bovine Serum

FceR Fc-epsilon receptor

FcγRs Fc-gamma receptor

FMO Fluorescence Minus One

FWBP Flow Whole Body Plethysmography

GATA3 Trans-acting T-cell-specific transcription factor 3

GI Gastrointestinal

GM Genetically Modified

GPR35 G-protein Coupled Receptor 35

GR Glucocorticoid Receptor

GRE Glucocorticoid Response Element

GWAS Genome Wide Association Study

HAT Histone Acetyltransferase

HDAC Histone Deacetyltransferase

HDM House Dust Mite

i.p. Intraperitoneal

i.t. Intratracheal

ICS Inhaled Corticosteroid

IFN-γ Interferon-gamma

Ig Immunoglobulin

IKK2 Inhibitor of nuclear factor kappa-B kinase subunit beta

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IL Interleukin

ILC Innate Lymphoid Cell

IVC Individually Ventilated Cages

KH Krebs-Henseleit

LABA Long Acting β2-adrenoceptor Agonist

LAMA Long Acting Muscarinic receptor Antagonist

LAR Late Asthmatic Response

LTB4 Leukotriene B4

M (1, 2, 3) Muscarinic receptor (subtypes 1, 2, 3)

MAR-1 Fc epsilon Receptor I alpha subunit targeted antibody

MBP Major Basic Protein

MHC-I Major histocompatibility complex class I

MHC-II Major histocompatibility complex class II

MMPs Matrix Metallopeptidases

MRI Magnetic Resonance Imaging

NF-κB Nuclear factor kappa-light-chain-enhancer of activated B cells

NKA Neurokinin A

NLRP3 NOD-like receptor family, pyrin domain containing 3

OVA Ovalbumin

PAMPs Pathogen Associated Molecular Patterns

PBS Phosphate buffered saline

Penh Enhanced pause

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PgE2 Prostaglandin E2

PMT Photomultiplier tube

PRRs Pattern Recognition Receptors

RARs Rapidly Adapting Stretch Receptors

RORα RAR-related orphan receptor α

ROS Reactive Oxygen Species

RPMI Roswell Park Memorial Institute media

SABA Short Acting β2-adrenoceptor Agonist

SARs Slowly Adapting Stretch Receptors

SP Substance P

ST2 IL-1 family receptor subtype

TCR T -cell receptor

TGF-β Transforming Growth Factor β

Th (cell) T-helper cell

TNF-α Tumour Necrosis Factor - α

TRPA1 Transient receptor potential cation channel, subfamily A, member 1

TRPM8 Transient receptor potential cation channel, subfamily M, member 8

TRPV1 Transient receptor potential cation channel, subfamily V, member 1

TRPV4 Transient receptor potential cation channel, subfamily V, member 4

TSLP Thymic Stromal Lymphopoietin

WBP Whole Body Plethysmography

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Chapter 1

Introduction

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1 Introduction

1.1 Asthma

Asthma is defined as a chronic inflammatory disease of the airways with symptoms such as wheezing,

chest tightness, breathlessness and cough (Lemanske Jr. & Busse, 2003; Levy et al., 2006; Bateman et

al., 2008). It affects approximately 300 million people worldwide and the World Health Organisation

estimates asthma to represent 1% of the total global disease burden. The prevalence of asthma is still

on the rise and escalating healthcare costs are making it a global health concern (Masoli et al., 2004;

Bateman et al., 2008; Akinbami, Moorman & Liu, 2011; Fahy, 2015).

The phenotype of asthma is characterised by airway hyperresponsiveness (AHR), reversible

bronchoconstriction, airway remodelling events and chronic inflammation. A wide range of

environmental and endogenous stimuli have been shown to trigger asthma symptoms including;

allergens, exercise and cold air (Holgate, 2008b, 2008a). Recent attempts at phenotyping asthma have

adopted cluster based approaches whereby patients are grouped according to a multitude of possible

features such as age of onset, atopic status, severity of airways obstruction and medication

requirements. Atopic asthma is associated with allergy (elevated levels of allergen specific IgE) and

atopic sensitisation has been implied as an important determinant in the development of asthma. The

most common etiological allergens include House Dust Mite (HDM), fungal species, animal dander and

plant pollen (Pearce, Pekkanen & Beasley, 1999; Wenzel, 2006). The focus of this thesis will be on

atopic asthma whereby the asthmatic response is thought to occur in allergen sensitised patients upon

which allergen challenge results in a response.

1.2 Allergic Sensitisation in Asthma

The pathogenesis of allergic asthma linking the initial triggers, the inflammatory process and the

resulting asthma phenotype is complex and involves numerous cells and mediators. The immune

response in atopic asthma involves two phases; the sensitisation phase following initial allergen

exposure and the challenge or allergic response phase following re-exposure in sensitised patients.

Allergens in the airways are detected by Antigen Presenting cells (APCs), primarily dendritic cells,

which patrol the airway epithelium and submucosa in order to scan the lumen for invading pathogens.

Upon contact, the APCs phagocytose the allergen. This action, along with the presence of cytokines

such as Thymic Stromal Lymphopoietin (TSLP), triggers the maturation of the APCs during which they

up-regulate their surface expression of major histocompatibility complex class II (MHC-II) molecules

and migrate to the lymph nodes (Ito et al., 2005; Wang et al., 2006; Burgess et al., 2005). Following

the internalisation of allergen, it is processed and loaded into MHC-II molecules at the cell surface.

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The APC then presents the allergen to naive B-cells and T-cells in a process known as antigen

presentation (Hintzen et al., 2006; Neefjes, 1996).

During antigen presentation, when an APC encounters a naive CD4+ T-cell expressing a relevant T-cell

Receptor (TCR) for the allergen, a complex is formed between the MHC-II/antigen complex and the T-

cell. For this to be a success, the T-cell must express the correct TCR for the allergen. However, it must

be noted that it is thought that each TCR can recognise a vast number of antigenic peptides

(Wooldridge et al., 2012). The coupling of the MHC-II/antigen and TCR (along with costimulatory

molecules from the APC) activates the naive CD4+ T-cell to proliferate and differentiate into effector

cells in a process known as clonal selection and expansion. The main effector phenotypes which the

activated CD4+ T-cells differentiate into are predominantly T-helper type 1 (Th1) and T-helper type 2

(Th2), and is dependent on the cytokine environment in which antigen presentation occurs (Buc et al.,

2009; Ito et al., 2005; Wang et al., 2006). Cytokines such as Interleukin-4 (IL-4) promote Th2

polarisation whereas cytokines such as Interferon-γ (IFN-γ) promote Th1 polarisation. In asthma

typically the Th2 endotype is a key hallmark in the pathogenesis of the disease and stimulated Th2

cells are involved in directing a host of cellular immune responses via the release of various cytokines

(Hammad & Lambrecht, 2008).

B-cells also function as APCs. CD40 ligand (CD40L) is expressed on the surface of T-cells and will bind

to CD40 Receptor (CD40R) expressed on the surface of B-cells, though only if the T-cell expresses the

correct TCR for the allergen. This interaction, along with the release of the cytokines IL-4 and IL-13

from the Th2 cell stimulates B-cell activation and triggers the release of allergen specific IgE via a

process known as class switching (McKenzie et al., 1999; Grünig et al., 1998). IgE is known to bind to

high affinity receptor FcR1 which is present on the surfaces of mast cells and basophils (Barnes, 2008).

This action effectively primes the cells to respond to future allergen exposure (see figure 1.1).

21

Figure 1.1: Schematic representation of the inflammatory cascade in allergic asthma. (Adapted from

Holgate 2008).

1.3 Allergic Asthmatic Responses: The EAR and LAR

Further exposure to an allergen following sensitisation results in an allergic response. The allergen

activates sensitised mast cells by inducing the cross linking of the IgE-FcεRI complexes and results in

the release of mediators such as histamine, 5-hydroxytryptamine (5-HT), prostaglandins and cysteinyl

leukotrienes. This results in a reaction characterised by rapid bronchoconstriction, oedema (plasma

extravasation) and mucus secretion which all contribute to airflow obstruction. This immediate

bronchoconstriction is termed the Early Asthmatic Response (EAR) and typically occurs within 1hour

of contact with aeroallergen. This reaction has been well characterised by researchers and occurs via

the IgE-mast cell products axis as highlighted in this section (Barnes, 2008; Holgate, 2008a).

The Late Asthmatic Response (LAR) refers to a more prolonged bronchoconstriction event taking place

approximately 3-8 hours following contact with allergen. The response is characterised by an influx of

inflammatory cells such as eosinophils, mast cells, neutrophils and T cells (Hogan et al., 1998). This

particular feature of asthma is experienced by approximately 50% of asthma sufferers and the

aetiology is less well understood in comparison to that of the EAR (Booij-Noord, Orie & De Vries, 1971;

Booij-Noord et al., 1972). It is also noteworthy that the LAR could be considered to be a more relevant

marker of asthma than the EAR as the bronchoconstriction is more pronounced and long-lived and

the LAR is also often used as an endpoint in clinical studies exploring new therapeutic options with

which to treat asthma (O’Byrne, Dolovich & Hargreave, 1987; Stevens & van Bever, 1989; Muller et

22

al., 1993). Despite this, the specific mechanisms driving the LAR have yet to be elucidated. The focus

of this thesis is to explore the driving mechanisms behind this response.

Figure 1.2: The EAR and LAR. The EAR takes place within 1hr of allergen exposure and is known to

involve IgE cross-linking of allergen on mast cells, degranulation and release of mediators acting

directly on smooth muscle. The LAR takes place 3-8 hrs following exposure. Mechanisms are unclear

but are thought to involve cellular allergic airway inflammation.

1.4 Other features of Allergic Asthma

1.4.1 Airway Remodelling

The asthmatic airway is characterised by several pathological changes in comparison to that of the

non-asthmatic airway, collectively described as airway remodelling. Structural changes within the

epithelium include goblet cell metaplasia and hyperplasia which leads to increases in epithelial mucin

stores. This along with changes in the submucosal gland cells leads to airway obstruction due to

excessive mucus production (Fahy, 2015; Wadsworth, Sin & Dorscheid, 2011). Subepithelial fibrosis is

also a common feature of the asthmatic airway. This term defines the thickening of the basement

membrane due to the enhanced deposition of extracellular matrix factors such as Collagens and

Fibronectins (Karjalainen et al., 2003; Beasley, Roche & Holgate, 1989; Brewster et al., 1990). Another

characteristic of airway remodelling is enhanced airway smooth muscle (ASM) mass caused ASM

23

hypertrophy and hyperplasia (Woodruff et al., 2004; Bara et al., 2010). Increases in the number of

blood vessels serving the asthmatic airway in comparison to non-asthmatics have also been noted

(Siddiqui et al., 2007).

It is widely believed that airway remodelling occurs as a result of the chronic inflammation associated

with asthma. Numerous mediators released during the inflammatory process in the asthmatic airway

have been implicated in this pathological feature. Cytokines such as IL-1β and IL-6 have been shown

to have the capability to enhance ASM proliferation (De et al., 1995). IL-13 and Transforming Growth

Factor β (TGFβ) has also been shown to induce fibrosis (Kenyon et al., 2003; Fichtner-Feigl et al., 2006).

A role for mast cells has been implied as they have the ability to release matrix metallopeptidases

(MMPs) which have been suggested as likely mediators of remodelling and repair (Dahlen, Shute &

Howarth, 1999; Wenzel et al., 2003). Murine models have also highlighted a role for eosinophilia and

IL-5 within remodelling events (Tanaka et al., 2001; Cho et al., 2004; Humbles et al., 2004). However,

it is also of note that some believe inflammation not to be a cause of remodelling, but simply an

observed factor occurring in parallel, as in some cases airway remodelling occurs in the absence of

inflammation (Malmström et al., 2011; Wadsworth, Sin & Dorscheid, 2011).

1.4.2 Airway Hyperresponsiveness

AHR is a cardinal feature of the asthma phenotype. It is defined as an increased sensitivity to inhaled

stimuli resulting in narrowing of the airways which would not usually occur in healthy individuals. This

response manifests as excessive bronchoconstriction and airflow limitation, resulting in shortness of

breath and chest tightness. Stimuli of AHR include pollution, allergens, cold air and spasmogens such

as Methacholine (Spina & Page, 2002).

AHR is largely thought to occur as a result of airway remodelling and inflammation. Indeed,

researchers have described the pathological changes occurring in airway remodelling as predisposing

asthmatics to exaggerated responses to inhaled stimulants by narrowing the baseline calibre and

altering structural elements in such a manner which allows for this (Fahy, 2015). More specifically,

hypertrophy and hyperplasia of ASM have been associated with AHR (Ebina et al., 1993; Lambert et

al., 1993), as has airway wall oedema (Van de Graaf et al., 1991; Kimura et al., 1992; Chung et al.,

1999). Inflammation has been associated with AHR as steroids have been shown to attenuate this

response in asthmatic patients (Djukanović et al., 1992, 1997; Laitinen, Laitinen & Haahtela, 1992;

Chalmers et al., 2002; Clearie et al., 2012). Increases in cytokines (IL-4, IL-5 and IL-13) and eosinophilic

inflammation have also been shown to correlate with asthma severity and AHR (Grünig et al., 1998;

Walter et al., 2001; Taube et al., 2002; Bousquet et al., 1990).

24

1.5 Cells and Mediators in Allergic Asthma

1.5.1 Dendritic Cells

Dendritic cells are APCs and, as previously described, play a central role in allergic sensitisation in

asthma. They are derived from bone marrow pre-cursor cells and originate in the blood. Immature

forms of this cell type are principally found in tissues such as the skin, the lining of the nose, the

airways and the gastrointestinal (GI) tract. In the airway, they patrol the lumen and submucosa

scanning for invading pathogens. Dendritic cells recognise Pathogen Associated Molecular Patterns

(PAMPs) present on invading pathogens via Pattern Recognition Receptors (PRRs) present on their cell

surface. Following this recognition they internalise, process and present the antigen on MHC-II

molecules at the cell surface and migrate to the lymph nodes where antigen presentation takes place

(Hintzen et al., 2006; Wang et al., 2006; Buc et al., 2009; Salazar & Ghaemmaghami, 2013).

During antigen presentation, the dendritic cells promote the polarisation of the naive CD4+ T-cells into

Th1 and Th2 phenotypes, which is determined by the cytokine environment. The mechanism by which

this takes place is not well understood though several have been proposed. The possibility that certain

dendritic cell types are preconditioned to polarise to a specific response (either Th1 or Th2) has been

raised by researchers. Lymphoid derived dendritic cells have been shown to stimulate IL-2 and IFN-γ

production from T-cells which is associated with the Th1 phenotype. Myeloid derived dendritic cells

have been shown to induce the production of IL-4 from T-cells which is allied with a Th2 response

(Pulendran et al., 1999; Hammad et al., 2010). Others have suggested that ingestion of different

pathogens can promote maturation of dendritic cells to differentially express various cytokine profiles.

A role for feedback cytokine regulation from T-cells has also been linked with this theory (Pulendran,

2004; d’Ostiani et al., 2000; Bozza et al., 2002; Edwards et al., 2002). Indeed, various cytokines

originating in Th2 cells such as IL-4, IL-13 and IL-6 have been shown to promote the Th2 response.

Whilst cytokines originating in Th1 cells such as IL-12 have been shown to promote Th1 differentiation

(Eisenbarth, Piggott & Bottomly, 2003). Overall, dendritic cells are known to play a role in controlling

T-cell polarisation. Whether this is due to a predetermined bias to specific endotypes, or via

modulatory effects of microbes and mediators in the pulmonary environment, requires further

elucidation.

1.5.2 T-Cells

1.5.2.1 CD4+ T-Cells

CD4+ T-cells are a subset of lymphocyte termed T-helper cells which mature in the thymus. They are

identified by the expression of a TCR on the cell surface and antigens are presented to them via APCs

25

on MHC-II molecules (Buc et al., 2009; Salazar & Ghaemmaghami, 2013). There are a number of CD4+

T-cell subtypes associated with asthma; Th1, Th2, Th17 and Treg cells which will be discussed in the

following sections.

1.5.2.1.1 Th1/Th2 Cell Paradigm

Th1 cells are responsible for phagocyte dependent protective responses, and produce the cytokines

IL-2 and IFN-γ. In contrast, Th2 cells produce cytokines such as IL-4, IL-5 and IL-13 and have been widely

implicated in the promotion of the chronic inflammation associated with asthma (Romagnani, 1999).

There is an increased presence of Th2 cells and associated cytokines in the airways of asthmatic

patients which has lead to asthma being traditionally described as a Th2 cell driven disease (Robinson

et al., 1992; Bentley et al., 1992).

As previously explained, in the process of antigen presentation, the subtype the CD4+ cells

differentiates into is dependent on the cytokine environment present. IFN-γ and IL-2 drive the

differentiation of Th1 cells, while IL-4 drives the Th2 subtype (Hammad & Lambrecht, 2008; Buc et al.,

2009). The development of the Th1 and Th2 subtypes are described as being mutually inhibitory as

the associated cytokines for each subtype inhibits the differentiation of the other (Varga et al., 2000;

Romagnani, 1999). Th1 development is dependent on the transcription factor T-bet, which regulates

the production of IFN-γ (Salazar & Ghaemmaghami, 2013). Th2 development is dependent on Trans-

acting T-cell-specific transcription factor 3 (GATA3) (Zheng & Flavell, 1997). T-bet has been shown to

inhibit Th2 responses via preventing GATA3 binding to DNA, resulting in the dominance of a Th1

response (Hwang et al., 2005; Usui et al., 2006). Indeed, T-bet expression in asthmatics is reduced

compared with non-asthmatics leading to the speculation that lack of Th1 promoting signals in

asthmatics may allow the Th2 subtype to predominate (Finotto et al., 2002).

1.5.2.1.2 Th2 Cells

Th2 cells have been widely implicated in asthma as playing a major role in promoting innate and

adaptive responses. As previously described, they are important for promoting the production of IgE

from B-cells and produce cytokines such as IL-4, IL-5 and IL-13. This cytokine milieu has been

associated with various features of disease, with enhanced expression found in the airways of

asthmatic patients (Robinson et al., 1992; Jaffar et al., 1999; Cohn, Elias & Chupp, 2004). IL-4 has

mainly been associated with the allergic sensitization process alone and in combination with IL-13

(Corry et al., 1996, 1998; McKenzie et al., 1999). In addition, IL-13 has been shown to be vital to

orchestrating allergic responses such as AHR, inflammation and airway remodelling (Zhu et al., 1999;

Mattes et al., 2001; Walter et al., 2001). IL-5 is a selective eosinophil chemoattractant and activator

and is therefore important in eosinophil recruitment and survival. Indeed, allergen challenge in

26

asthmatic patients results in the influx of activated Th2 cells into the airway, accompanied by

prominent eosinophil recruitment. Eosinophilic inflammation has been associated with many features

of allergic asthma such as AHR and airway remodelling (Finkelman et al., 2010).

1.5.2.1.3 Th17 Cells

Th17 cells express IL-17 and have been widely implicated in asthma. CD4+IL17+ T-cells have been

observed in the lungs of asthmatic patients (Pène et al., 2008). Levels of IL-17 have also been found

to be elevated in the lungs of patients with asthma, with levels correlating with disease severity (Molet

et al., 2001). IL-17 has been suggested to play a role in neutrophil recruitment, possibly via inducing

the production of the neutrophil chemoattractant IL-8 (Bullens et al., 2006). Studies have shown that

the transfer of Th17 cells promotes steroid resistant AHR and inflammation which is associated with

neutrophilia (He et al., 2007; Wilson et al., 2009; McKinley et al., 2008).

1.5.2.1.4 Treg Cells

Unlike other CD4+ cell subsets, regulatory T-cells (Tregs) are important in the suppression of

inflammatory responses. They have been shown to inhibit Th2 cytokine driven asthmatic responses

such as AHR, airway inflammation and remodelling events. They are thought to exert these

suppressive actions through the secretion of the anti-inflammatory cytokine IL-10 and expression of

inhibitory molecules such as cytotoxic T lymphocyte antigen 4 (CTLA4) (Ostroukhova et al., 2004;

Kearley et al., 2005; Kearley, Robinson & Lloyd, 2008). Due to this it has been suggested that a reduced

function of this cell type within the asthmatic lung could be responsible for the enhanced immune

response to allergens observed in asthma (Lloyd & Hessel, 2010).

1.5.2.2 CD8+ T-Cells

CD8+ T-cells are known as Cytotoxic T-cells. They also express a TCR on the cell surface and antigens

are presented to them via MHC-I molecules. They have been identified in the lungs of asthmatic

patients, with certain subtypes present in increased proportions (Hirosako et al., 2010). Animal models

have shown that the actions of CD8+ cells can be either protective or injurious within the asthmatic

lung. Studies examining the role of the CD8+γδ subtype showed that with suppression asthma

symptoms worsened, and with adoptive transfer symptoms were improved, highlighting protective

capabilities (Olivenstein et al., 1993b; Allakhverdi et al., 2000; Suzuki et al., 1999). Conversely,

adoptive transfer studies of CD8+αβ cells demonstrated this subtype to exacerbate asthma symptoms

(Isogai et al., 2004).

1.5.3 Eosinophils

Eosinophils are polymorphonuclear granulocytes which develop in the bone marrow from common

myeloid progenitor cells. They are found in the sputum, bronchoalveolar lavage (BAL) fluid and

27

bronchial biopsies of allergic asthmatic patients and as such their presence in the lung is commonly

used as a marker of allergic asthma (Vieira & Prolla, 1979; Gibson, Saltos & Borgas, 2000; Shahana et

al., 2005). They have been shown to release a plethora of toxic proteins such as Major Basic Protein

(MBP), Eosinophil Cationic Protein (ECP), Eosinophil Derived Neurotoxin (EDN) and Eosinophil

Peroxidase (EPX) which have been implicated in the pathogenesis of asthma (Frigas et al., 1981;

Motojima et al., 1989; De Monchy et al., 1985). They have also been shown to release other mediators

such Cysteinyl Leukotrienes and Prostanoids which are potent bronchoconstricting agents, in addition

to Th2 promoting cytokines such as IL-4 and IL-13 (Weller et al., 1983; Nonaka et al., 1995; Woerly et

al., 2002).

The differentiation, survival and migration of eosinophils to the lungs has been shown to be mediated

by a number of chemokines and cytokines. Eotaxin is a chemokine produced by a number of cell types

such as mast cells, macrophages and epithelial cells present within the allergic lung (Liu, Yang & Huang,

2006; Zeibecoglou et al., 1999). It signals via the CC Chemokine receptor-3 (CCR3) receptor which is

expressed on eosinophils and has been shown to mediate the recruitment of this cell type to the lungs

(Sehmi et al., 1997; Griffiths-Johnson et al., 1993). IL-5 is a Th2 cytokine produced by a range of cell

types as previously described and is important for eosinophil differentiation, migration and survival

(Yamaguchi et al., 1988, 1991). Numerous clinical and animal studies have presented evidence linking

Eotaxin, IL-5 and eosinophilia with key hallmarks of asthma. The inhalation of IL-5 by asthmatic

patients was shown to result in enhanced eosinophilia and AHR (Shi et al., 1998). In a murine allergic

asthma model, IL-5 deficient mice exhibited a lack of eosinophilia, inflammation, AHR and airway

remodelling compared with controls. Eosinophil transfer into these knockout mice restored levels of

Th2 cytokines and AHR (Shen et al., 2003). Other studies utilising various genetically modified (GM)

mice in allergic asthma models have also concluded eosinophils, Eotaxin and IL-5 to be key in the

presentation of AHR, airway remodelling events and airway inflammation (Mattes et al., 2002; Justice

et al., 2003; Lee et al., 2004).

The hypothesis that eosinophilic inflammation plays a key role in the presentation of asthma

symptoms is supported by the fact that certain subsets of asthma patients respond well to inhaled

corticosteroids (ICS) resulting in diminished symptoms and airway inflammation (Djukanović et al.,

1992). However, it has also been suggested that eosinophils may not be as important as once thought

within allergic asthma. Certain subsets of asthmatics present little evidence of eosinophilia and some

investigations have shown that AHR does not correlate with inflammation in BAL fluid, sputum and

bronchial biopsies (Crimi et al., 1998; Wenzel et al., 1999; Wenzel, 2005). Animal studies have also

shown that AHR is not dependent on lung and airway eosinophilia (Hessel et al., 1997; Wilder et al.,

28

1999; Humbles et al., 2004). Nevertheless, therapeutics for asthma targeting eosinophilia are

currently in development. Mepolizumab, Benralizumab and Reslizumab are antibody therapeutics

which target IL-5 and have reached at least Phase II in clinical trial and their efficacy assessed. Initial

trials of Mepolizumab concluded that it had no significant effect on asthma exacerbation rates (Flood-

Page et al., 2007). However, these initial trials did not specifically target the subgroup of patients

expected to benefit the most such as those with persistent eosinophilia. More recent studies of

Mepolizumab targeted this subgroup and concluded that the treatment significantly decreased

asthma exacerbations and was corticosteroid sparing (Nair et al., 2009; Haldar et al., 2009; Pavord et

al., 2012; Bel et al., 2014; Ortega et al., 2014). Data from Phase II clinical trials of Benralizumab and

Reslizumab also showed that they were effective in reducing exacerbations and improving asthma

control but only in patients with persistent eosinophilia (Castro et al., 2011, 2014).

Overall, eosinophilia is considered to be a feature of allergic asthma in certain subsets of patients

although their specific role remains controversial.

1.5.4 Macrophages

Macrophages are derived from monocytes generated in bone marrow and develop into macrophages

in tissues such as that of the lung. They are phagocytic in nature, protecting the body from infection

and cleaning up dead and dying cells. Despite being major players in the innate immune system, they

have also been linked with adaptive immunity associated with asthma immunology (Murray & Wynn,

2011).

They have been shown to be one of the predominate cell types in sputum samples taken from

asthmatic patients and release mediators associated with features of asthma such as prostaglandins

and cysteinyl leukotrienes (Woodruff et al., 2001; Holgate et al., 2003). They also release cytokines

such as IL-1β, Tumour Necrosis Factor-α (TNF-α) and IL-6 which may be associated with the promotion

of ongoing inflammation in asthmatic patients (Rincon & Irvin, 2012). Macrophages are capable of

functioning as APCs, ingesting allergens and presenting them to T-cells (Unanue, 1984). They express

Fc receptors on their cell surface and may provide proinflammatory signals in response to allergen

exposure (Melewicz et al., 1982; Borish, Mascali & Rosenwasser, 1991). Alveolar macrophages have

been shown to release leukotrienes in response to IgE or IgE allergen immune complexes which

further highlights this point (Rankin et al., 1982, 1984).

1.5.5 Neutrophils

Neutrophils are polymorphonuclear granulocytes and function as phagocytes within the innate

immune system. They are the immediate defence mechanism against invading pathogens and migrate

29

to the site of infection via multiple chemokine gradients such as that of Leukotriene B4 (LTB4) and IL-

8. They are known to release phagosomes containing high levels of Reactive Oxygen Species (ROS)

which enhance their ability to fight pathogens (Galli, Borregaard & Wynn, 2011; Kolaczkowska &

Kubes, 2013). Their role in asthma is not yet fully understood, although their presence in the asthmatic

lung has been noted (Woodruff et al., 2001). Clinical and Pre-clinical models have also elucidated that

they are recruited to the airways following allergen exposure (Koh et al., 1993; Nocker et al., 1999;

Underwood et al., 2002). They secrete proinflammatory mediators such as IL-6, IL-8 and TNF-α which

have been deemed as important in promoting the chronic inflammation associated with asthma (Cicco

et al., 1990; Dubravec et al., 1990; Strieter et al., 1992). Neutrophils have also been implicated in

steroid resistant asthma exacerbations (Ito et al., 2008).

1.5.6 B-Cells

B-cells undergo development in the bone marrow and then migrate to the lymph nodes. Their major

role is to produce antibodies to fight invading pathogens. They are capable of functioning as APCs and

recognise antigen via B-cell receptors. This recognition results in internalisation, processing and

presentation on MHC-II molecules at the cell surface. T-helper cells (such as Th2 cells in asthma)

expressing the correct TCR for the allergen interact with this complex. CD40L on the T-cell also binds

with CD40R on the B-cell surface. Together these interactions are known as the formation of an

immunological synapse. This, along with cytokines such as IL-4 and IL-13 released from the Th2 cell

results in the promotion of the B-cell to undergo the production of IgE in a process known as class

switching (Grünig et al., 1998; McKenzie et al., 1999). IgE will then bind to high affinity FcεRI receptor

on mast cells and basophils to effectively prime them for future allergen exposure. In addition to IgE

release, the B-cells also undergo clonal selection and expansion to result in the formation of plasma

B-cells and memory B-cells (Mitchison, 2004).

1.5.7 Mast Cells

Mast cells develop from pluripotent hematopoietic stem cells in the bone marrow. They circulate as

CD34+ precursors until they migrate into tissues where they mature into granulocytes (Kirshenbaum

et al., 1992). They are found in various tissue types but are especially numerous at barriers such as the

skin, respiratory tract and GI tract (Sayed et al., 2008). There are two subtypes of mast cell which are

determined based upon their protease content. The first contains only tryptase and is termed MCT.

The second contains tryptase, chymases and carboxypeptidase A and is denoted MCTC. MCT are the

predominate subtype found in the human lung and bronchus. However, similar numbers of both

subtypes are found in the pulmonary vasculature (Irani et al., 1986; Andersson et al., 2009).

30

Mast cells are largely associated with allergic asthma. As previously explained, following the

sensitisation process, allergen binds to allergen specific IgE on high affinity FcεRI receptor at the mast

cell surface. This results in cross linking of the complexes and subsequent mast cell degranulation to

release mediators such as histamine, cysteinyl leukotrienes and prostaglandins. These mediators act

to cause the EAR characterised by rapid bronchoconstriction, oedema and mucus secretion (Barnes,

2008; Holgate, 2008a).

Following this immediate release of mediators, mast cells have also been shown to synthesise and

release a host of cytokines, chemokines and growth factors implicated in the chronic inflammation

associated with asthma. Indeed, activated mast cells have been shown to synthesise and release

cytokines associated with Th2 pathology such as IL-4 and IL-13 (Galli & Tsai, 2012; Bradding, Walls &

Holgate, 2006). Mast cells have also been implicated in airway remodelling events. The number of

mast cells present in ASM bundles is increased in asthmatics compared to healthy controls and they

have been shown to promote the release of TGFβ (important in ASM fibrosis) from ASM (Brightling et

al., 2002; Woodman et al., 2008).

Mast cells are often found in close proximity to nerve terminals and as such are thought to interact

with sensory and parasympathetic neurons (Nilsson et al., 1990; Marshall, 2004). Mast cell mediators

such as histamine and 5-HT have been shown to cause depolarisation of parasympathetic neurons and

antigen mediated bronchoconstriction has been shown to be dependent on mast cell mediator release

(Myers, Undem & Weinreich, 1991; Undem et al., 1995). Overall this implies that mast cell derived

spasmogens may contribute to antigen induced bronchoconstriction via the activation of airway

nerves.

1.5.8 Basophils

Basophils are granulocytes which develop in the bone marrow and then circulate in the peripheral

blood. They are understood to play a similar role to mast cells in allergic disease but their specific

function is less well understood (Voehringer, 2013). They express high affinity FcεRI on their cell

surface and have been shown to respond to IgE stimulation to release mediators such as histamine.

As such they have been implicated in immediate bronchoconstriction events such as the EAR

(Schroeder et al., 1994; Seder et al., 1991; Thompson, Metcalfe & Kinet, 1990; Nabe et al., 2013). They

have also been shown to release Th2 cytokines such as IL-4 and IL-13 amongst other inflammatory

mediators and are thought to orchestrate allergic inflammation (Min et al., 2004; Voehringer, Shinkai

& Locksley, 2004; Kondo et al., 2008). There is also evidence to suggest that basophils function as

APCs. They have been observed to migrate to the lymph nodes upon allergen stimulation and have

31

been found to be important in the differentiation of Th2 cells via IL-4 release (Perrigoue et al., 2009;

Sokol et al., 2009).

1.5.9 Immunoglobulins

Immunoglobulins are antibodies produced by B-cells. There are five main isotypes which have been

identified: IgA, IgD, IgE, IgG and IgM. The main isotype implicated in allergic asthma aetiology is IgE

(Schroeder & Cavacini, 2010). As previously explained, antigen presentation along with Th2 cytokines

induces B-cells to produce IgE in a process known as class switching. Allergen specific IgE then

sensitises mast cells and basophils to future allergen exposure by binding to high affinity FcεRI on the

cell surface. Upon re-exposure, the allergen is recognised by and binds to the antigen binding site on

IgE resulting in activation of the mast cells and basophils (McKenzie et al., 1999; Grünig et al., 1998;

Barnes, 2008). High affinity FcεRI and low affinity FcεRII are both capable of binding IgE and these

receptors are present on, and expression can be induced on, a range of other allergic cells such as

eosinophils, B-cells, T-cells and macrophages. As such, IgE could play a role in the chronic inflammation

associated with asthma (Dullaers et al., 2012). Due to the strong association of IgE and asthma, IgE is

now a target for new therapeutics. Omalizumab is an anti-IgE antibody which targets IgE to prevent it

from binding to high affinity FcεRI. In the clinic this has been shown to inhibit allergen induced

responses such as the EAR and LAR. Reduced frequency of asthma exacerbations and decreased use

ICS were also reported with treatment. A review of the clinical trial data concluded that although

reductions to symptoms were modest, overall treatment improved exacerbations (Normansell et al.,

2014).

IgG is an immunoglobulin with 4 main subtypes (IgG1-4) mainly found in the blood. Its main role has

been identified as protecting the body from pathogen invasion, although roles within allergy and

asthma have been suggested. Increased levels of IgG1 have been reported in the BAL fluid of asthmatic

patients (Out et al., 1991; Kitz, Ahrens & Zielen, 2000). Exposure to allergens such as HDM has been

associated with IgG1 and IgG4 production (Platts-Mills et al., 2001). More specifically, development of

the LAR has been associated with increased serum levels of IgG and IgE, with some studies suggesting

that levels IgG1 rather than IgE is predictive of the LAR following allergen challenge (Pelikan & Pelikan-

Filipek, 1986; Ito et al., 1986). IgG is known to interact with FC gamma receptors (FcγRs) which are

expressed on many effector cell types such as macrophages, eosinophils, neutrophils, basophils, B-

cells and mast cells. Due to this, there is an association of IgG with allergic inflammation (Williams,

Tjota & Sperling, 2012). Studies have demonstrated this by showing that inhalation of anti-allergen-

IgG immune complexes can induce Th2 type airway inflammation and eosinophilia (Hartwig et al.,

2010).

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1.5.10 Epithelial Cells

Epithelial cells line the airways and are capable of releasing a plethora of cytokines in response to cell

damage induced by environmental and endogenous stimuli. These cytokines are thought of as

mediators between innate and adaptive immunity as they have been shown to direct adaptive and

innate cellular responses (Islam & Luster, 2012; Jovanovic, Siebeck & Gropp, 2014). The main

epithelium derived innate cytokines identified in the context of allergic asthma are TSLP, IL-25 and IL-

33 and will be discussed below.

1.5.10.1 TSLP

TSLP is released by epithelial cells and has been associated with the promotion of Th2 responses. This

is understood to occur via promoting dendritic cell maturation. TSLP treatment of dendritic cells has

been shown to promote the proliferation of CD4+ T-cells and production of IL-4, IL-5 and IL-13

(Soumelis et al., 2002). Elevated levels of TSLP have been found in the BAL fluid of asthmatic patients

compared with healthy controls which also correlates with Th2 cytokine production (Ying et al., 2005;

Shikotra et al., 2012). Increased TSLP expression has been shown in animal models of allergic asthma

which also correlates with allergic inflammation, AHR and airway remodelling events (Zhou et al.,

2005; Shi et al., 2008; Chen et al., 2013). TSLP has also been shown to activate mast cells to produce

Th2 cytokines such as IL-13, thus, enhancing mast cell driven responses in the lungs (Jovanovic, Siebeck

& Gropp, 2014). Other observed roles of TSLP in allergic asthma include the suppression of IL-10

production from Tregs and so the reduction of their repressive abilities in terms of allergic inflammation

(Nguyen, Vanichsarn & Nadeau, 2010). TSLP has also been observed to activate sensory nerves which

are increasingly being implicated in allergic asthma (Wilson et al., 2013) (Later discussed in section

1.6).

1.5.10.2 IL-25

IL-25 has also been linked with the promotion of Th2 responses in allergic inflammation. Animal

models have demonstrated that IL-25 induces features of asthma such as AHR, eosinophilia and

remodelling events (Fort et al., 2001; Sharkhuu et al., 2006; Angkasekwinai et al., 2007; Gregory et al.,

2013). IL-25 released by epithelial cells in response to cell damage has been shown to activate

receptors in innate lymphoid type 2 (ILC2) cells (discussed in later section) which respond by secreting

Th2 cytokines. It has also been shown to promote the development of multipotent progenitor cells to

give rise to mast cells, basophils and macrophages (Jovanovic, Siebeck & Gropp, 2014; Saenz et al.,

2010).

33

1.5.10.3 IL-33

IL-33 is also released in response to epithelial cell damage. This cytokine binds to the ST2 receptor (an

IL-1 family receptor) which is found on cell types including Th2 cells, mast cells, basophils, ILC2s and

eosinophils. This induces the release of a plethora of cytokines including IL-4, IL-5 and IL-13 from these

cell types (Imai et al., 2013; Jovanovic, Siebeck & Gropp, 2014). Animal models have shown IL-33 to

exacerbate allergic responses such as inflammation, AHR and airway remodelling (Kurowska-Stolarska

et al., 2008; Oboki et al., 2010; Stolarski et al., 2010; Kouzaki et al., 2011).

1.5.11 Innate Lymphoid Cells

ILCs are a family of effector cells which are increasingly being implicated in inflammatory diseases such

as allergic asthma. They are described as innate as they do not express a TCR and react independently

of specific antigens (Jovanovic, Siebeck & Gropp, 2014). The specific characteristics of these cell types

have not yet been fully elucidated but a classification scheme based on functional criteria such as the

cytokine milieu they produce has been described (Bernink, Mjösberg & Spits, 2013). The subtypes

within this effector cell family are ILC1, ILC2 and ILC3 cells. The subtype most widely implicated in

allergic airways disease are ILC2s which will be discussed in more detail below.

ILC2s were previously known as natural helper cells or nuocytes and require the transcription factors

RAR-related orphan receptor α (RORα) and GATA-3 for effective development (Spits & Cupedo, 2012;

Halim et al., 2012b; Wong et al., 2012; Hoyler et al., 2012). They are responsive to IL-25 and IL-33 and

upon stimulation produce characteristic Th2 cytokines such as IL-5 and IL-13. Due to this they are

associated with a Th2 like immune response and have been linked with hallmarks of allergic asthma

such as enhanced serum IgE levels and eosinophilia (Yu et al., 2014).

In the lung, upon allergen exposure, epithelial cell damage is thought to occur resulting in the release

of IL-33. This cytokine has been shown to activate ILC2s via the ST2 receptor (IL-33 receptor) expressed

on the cell surface, which in turn results in the release of Th2 cytokines and associated asthma

pathology (Yu et al., 2014; Dekruyff et al., 2014). Animal studies have demonstrated that protease

containing allergens damaged epithelial cells causing the release of IL-33 which activated ILC2s,

resulting in enhanced Th2 cytokine levels, eosinophilia and remodelling events. Depletion of the ILC2

population in these investigations resulted in decreased allergic asthma symptoms. Studies have also

shown that ILC2s generally appear to be increased in the lungs of mouse models of allergic airways

disease (Halim et al., 2012a; Wilhelm et al., 2011; Kearley et al., 2009; Barlow et al., 2012).

ILC2s have also been shown to interact with other inflammatory cells in the allergic lung. Indeed, they

are thought to be important in the control of lung eosinophilia due to their production of IL-5

(Nussbaum et al., 2013). Their interaction with mast cells has also been observed. The IL-13 produced

34

by ILC2s has been shown to interact with mast cells, leading to their release of IL-33. Therefore, it

could be suggested that the recognition of allergens by IgE on mast cells could lead to increased IL-33

secretion and activation of ILC2s which then secrete IL-5 to enhance eosinophilia (Hsu, Neilsen &

Bryce, 2010). On the other hand, Th2 cells have also been shown to express ST2 and so this pathway

could also drive their expansion (Dekruyff et al., 2014). It should be noted that these interactions were

observed in mast cells in the skin, but it is thought likely that it could also occur within the allergic lung

(Roediger et al., 2013).

Translational studies in human subjects involving this cell type are in their infancy. However, it has

been reported that a non-B, non-T cell type producing IL-13 and IL-5 is present in the sputum of

asthmatics but not healthy subjects. This cell type has also been shown to express receptors for and

respond to IL-33 by producing IL-5 and Il-13 and has also been shown to increase in number after

allergen inhalation challenge (Allakhverdi et al., 2009). Genome Wide Association Studies (GWAS)

have identified genes for IL-33, ST2 and RORα as susceptibility genes for human asthma (Moffatt et

al., 2010). As products of these genes are associated with ILC2 cell function, these GWAS studies imply

a role for ILC2s in human disease.

1.6 Innervation of the Lung

The airways are innervated by a number of nerve fibre types. Interactions between nerves and the

inflammation associated with asthma have been postulated by a number of studies and so

understanding airway innervation is advantageous when conducting investigations into the

mechanisms behind allergic asthmatic responses (Zhang et al., 2008; Weigand et al., 2009; Cyphert et

al., 2009).

The vagus nerve supplies all parasympathetic and most sensory nerve fibres to the airways, although

some sensory nerves originate in the dorsal root ganglia (see figure 1.3). The cell bodies of vagal

sensory fibres are found in the jugular and nodose ganglia with peripheral projections to the airways

and central projections to the medulla (Belvisi, 2002). Symptoms of allergic disease such as

bronchoconstriction and mucus hypersecretion have been shown to manifest following inappropriate

activation of these nerve types (Barnes, 1992, 1996).

35

Figure 1.3: Schematic representation of innervation of the airways. The vagus nerve supplies all

parasympathetic and most of the sensory nerve fibres to the airways. Some sensory innervation

originates from the dorsal root ganglia and these sensory fibres run with spinal sympathetic nerves.

(Adapted from Belvisi 2002).

36

Motor control of the airways is predominantly exerted by efferent cholinergic parasympathetic

nerves. These nerves originate in the vagal nuclei of the medulla and travel to the airways via the

vagus nerve where they relay in the parasympathetic ganglia located within the airway wall. The main

neurotransmitter involved in parasympathetic transmission is Acetylcholine (ACh) which binds to

muscarinic receptors (Belvisi, 2002). Subtypes of muscarinic receptor thought to play important roles

in the control of airway tone are M1, M2 and M3 receptors. M1 receptors are located within

parasympathetic ganglia and activation results in the facilitation of cholinergic neurotransmission. M2

receptors are thought to protect against bronchoconstriction by acting as feedback autoreceptors and

inhibiting ACh release. M3 receptors are located on ASM and mucus glands and activation results in

bronchoconstriction and mucus secretion (Barnes, 1992; Patel et al., 1995a; Barnes, 1995, 1996).

Afferent airway sensory nerves are also important in the control of airway tone. The main purpose of

airway sensory nerves is to relay sensory information from the airways to the Central Nervous System

(CNS) in order to initiate appropriate motor outputs elicited via parasympathetic pathways (Coleridge

& Coleridge, 1994). Airway sensory nerve receptors have been classified into categories of nerve fibre

types based on their functional properties namely; slowly adapting stretch receptors (SARs), rapidly

adapting stretch receptors (RARs), and C-fibre receptors. SARs are primarily involved in the control of

breathing pattern. RARs respond to mechanical stimuli such as lung hyper- and hypo-inflation of the

lung. C-fibre receptors respond to chemical stimuli such as mediators released in lung inflammation

and as such are considered to be of most relevance with respect to asthma (McAlexander, Myers &

Undem, 1999; Fox et al., 1993a; Adcock, 2002; Belvisi, 2002).

C-fibres originate in the nodose and jugular ganglia. They are activated by chemical stimuli but not by

mechanical stimuli such as changes in pulmonary volume. As such, activation of these fibres by

chemical stimulation is thought to occur directly rather than indirectly as a result of

bronchoconstriction. Mediators which activate these fibres include inhaled irritants (such as cigarette

smoke and air pollution), aromatic compounds in food and perfume (such as mustard oil, wasabi and

cinnamon) and endogenous inflammatory mediators (such as PGE2) (Coleridge & Coleridge, 1984;

Mohammed, Higenbottam & Adcock, 1993; Riccio & Proud, 1996; Lee & Pisarri, 2001; Undem et al.,

2004). In addition to relaying sensory nerve information to the CNS, the activation of c-fibres has also

been shown to result in local axonal reflexes and the release of neuropeptides such as calcitonin gene

related peptide (CGRP), Substance P (SP) and Neurokinin A (NKA) (Barnes, 1986, 1995). Studies have

shown that the peripheral release of neuropeptides can contribute to inflammatory processes in the

lung resulting in bronchoconstriction, mucus production and oedema (Nasra & Belvisi, 2009). As such,

local effects of stimulation in addition to central reflex events leading to parasympathetic cholinergic

37

effector responses must be considered when studying c-fibres and their role in innervation of the

allergic asthmatic lung.

1.7 Transient Receptor Potential Receptors and Asthma

The transient receptor potential (TRP) superfamily of ion channels are cation-selective

transmembrane proteins consisting of six subfamilies (TRPC, TRPV, TRPM, TRPA, TRPP and TRPML)

based on sequence homology (Clapham, 2003). These receptors are increasingly being identified in a

range of cell types associated with respiratory diseases such as asthma (Banner, Igney & Poll, 2011a;

Prasad et al., 2008). This section will summarise the key roles of four members of the TRP family in

asthma: TRPA1, TRPV1, TRPV4 and TRPM8.

1.7.1 TRPA1

TRPA1 is thought to be mainly expressed in neurons such as airway sensory nerves (Bautista et al.,

2006b; Story et al., 2003; Bandell et al., 2004; Jang et al., 2012; Nassenstein et al., 2008). It has also

been reported to be expressed in non-neuronal tissues such as human and mouse lung; and in asthma

relevant cell types such as CD4+ T-cells, CD8+ T-cells, B-cells and mast cells (Banner, Igney & Poll,

2011a; Prasad et al., 2008). Links between the symptoms of asthma and TRPA1 activation are also

beginning to emerge. For example, studies have shown that environmental irritants usually causing

asthma-like symptoms also activate TRPA1 in the airways (Deering-Rice et al., 2011; Shapiro et al.,

2013).

Roles for TRPA1 in allergic airway inflammation and AHR have been explored by researchers. These

studies have mainly utilised murine models whereby GM strains could be applied. Hox et al (2013)

showed TRPA1 to be involved in non-allergic AHR. In this investigation the authors modelled the

increased incidents of AHR in swimmers in mice via exposure to hyperchlorite. When TRPA1 deficient

mice were applied, the AHR was abolished. Similarly, it has been reported that TRPA1 deficient mice

have reduced inflammation and AHR when applied to ovalbumin (OVA) driven murine models of

allergic asthma (Caceres et al., 2009). Although conversely, a study by Spiess et al (2013) showed that

exposure to acrolein (a known TRPA1 agonist) resulted in a decreased inflammatory response in their

murine allergic asthma model. It should be noted that although acrolein is defined as a TRPA1 agonist,

it is not considered a clean compound. Due to this it could be expected to elicit a number of different

effects in vivo. Animal models utilising more clinically relevant allergens (e.g. HDM) are emerging and

may help to further elucidate the role of TRPA1 in key features of allergic asthma such as inflammation

and AHR.

38

Studies have also indicated a direct role for TRPA1 in the control of airway tone. Andre et al (2008)

reported that activation of TRPA1 channels on isolated guinea-pig bronchus resulted in contraction

which was found to be secondary to the release of neuropeptides via local axons. Within the

Respiratory Pharmacology group, TRPA1 was found to be associated with the characteristic

bronchoconstriction of the LAR (Raemdonck et al., 2012). This will be discussed in more detail in

section 1.8.

1.7.2 TRPV1

Similarly to TRPA1, TRPV1 has been reported to be mainly expressed in airway sensory nerves but is

also expressed in lung tissue (Banner, Igney & Poll, 2011a; Jang et al., 2012; Kunert-Keil et al., 2006).

It is thought to play a role in the control of airway tone as agonists of TRPV1 have been shown to

contract isolated guinea-pig ASM. This constriction has been demonstrated to involve the release of

sensory neuropeptides from finite stores in nerve endings which consequently act upon neurokinin

receptors on ASM (Belvisi et al., 1992). It is currently unclear whether TRPV1 is involved in

bronchospasm in humans and if its action is altered in diseases such as asthma.

TRPV1 is increasingly been shown to be involved in inflammatory processes such as those in allergic

asthma. Animal models have so far been useful in exploring this but results produced are often

conflicting. Rehman et al (2013) reported that TRPV1 modulation inhibited inflammation, AHR and

airway remodelling in an IL-13 driven allergic asthma model. However, other studies have indicated a

protective role for TRPV1 in allergic inflammation (Mori et al., 2011) and some have reported that no

part at all is played by TRPV1 in features of allergic asthma (Raemdonck et al., 2012; Caceres et al.,

2009).

1.7.3 TRPV4

TRPV4 expression has been reported in a number of tissues in the lung including ASM, lung vessel and

alveolar wall (Grant, Amadesi & Bunnett, 2007; Jia et al., 2004; Alvarez et al., 2006; Dietrich et al.,

2006; Yang et al., 2006). It has been shown to have roles in thermosensitisation,

mechanosensitisation, and osmotic stress (Watanabe et al., 2003; Strotmann et al., 2000; Suzuki et

al., 2003; Liedtke & Friedman, 2003a). The bronchial fluid of asthmatics has been reported to be

hypotonic due to airway remodelling and this implies a role for TRPV4 in asthma (Liedtke & Simon,

2004). TRPV4 in isolated human and guinea-pig trachea has also been shown to be activated by

hypotonic solution and results in contraction (Jia et al., 2004).

39

1.7.4 TRPM8

Little TRPM8 expression data has been published but it has been reported to be present in airway

sensory nerves and human bronchial epithelial cells (Peier et al., 2002; Li et al., 2011b). It is a

thermosensor of temperatures approximately 15-28 °C and, consequently, TRPM8 activators include

compounds which elicit a cooling sensation such as menthol and eucalyptol (McKemy, Neuhausser &

Julius, 2002; Zhou et al., 2011).

The role of TRPM8 in airway diseases such as asthma remains fairly unexplored; however, certain links

have been made. Inhalation of cold air has been shown to trigger symptoms of asthma such as

bronchoconstriction, cough, plasma extravasation and mucosal secretion (Carlsen & Carlsen, 2002).

TRPM8, being a thermosensor, has been linked to this (Li et al., 2011b; Fisher, 2011). It has also been

reported to be involved in cold triggered mast cell activation and so further study could implicate it in

inflammatory processes in diseases such as asthma (Cho et al., 2010b).

1.8 Aetiology behind the Late Asthmatic Response

The LAR is experienced by approximately 50% of asthma suffers and is often used as an endpoint in

clinical studies exploring new therapeutic options (Booij-Noord, Orie & De Vries, 1971; Booij-Noord et

al., 1972). A number of hallmarks have been associated with the LAR although a clear picture of the

mechanistic driving factors behind this response has yet to be uncovered. Studies so far exploring this

particular feature of asthma have concluded that it is likely that the LAR is at least in part driven by

inflammation. This is largely based on the fact that the response is accompanied by cellular

inflammatory influx into the lung and that steroid treatment impacts on the response (Bousquet et

al., 2000; Barnes, 2008). Studies have explored the roles of specific inflammatory cell types in this

response. Others have suggested other driving mechanisms behind the LAR such as possible roles for

airway sensory nerves (Raemdonck et al., 2012).

Clinical studies have demonstrated specific roles for T-cells within the LAR. Increased numbers of

activated T-cells have been observed in the bronchial mucosa and BAL fluid of patients with atopic

asthma (Walker et al., 1991; Gerblich, Salik & Schuyler, 1991; Azzawi et al., 1990; Bentley et al., 1993).

Allergen challenge in atopic asthmatics has also been associated with increased numbers of BAL fluid

T-cells at 6 hours post challenge, the LAR time-point (Gratziou et al., 1996). Studies utilising

Cyclosporin A (a T-helper cell inhibitor) to investigate asthma specific endpoints concluded a role for

T-cells within the LAR (Khan et al., 2000). Other studies involving the inhalation of T-cell peptides

derived from feline allergen Fel d 1 demonstrated the induction of an isolated LAR in atopic asthmatics

(Ali et al., 2004b). More specifically, CD4+ T-cells have been identified in increased numbers in

bronchial biopsies from asthmatics and allergen challenge has been shown to be associated with less

40

circulating CD4+ T-cells in isolated early responders compared with dual responders (those displaying

an EAR and LAR) (Muratov et al., 2005).

Animal models have also implicated T-cell subsets in the driving mechanisms behind the LAR. CD4+

and CD8+ T-cells were found to be increased in BAL fluid 4 hours post allergen challenge in the lungs

of dogs with ragweed allergy (Out et al., 2002). Mouse asthma models have demonstrated an increase

in the accumulation of CD4+ T-cells and associated cytokine profile (IL-4, IL-5, IL-13) in the airway

correlated to a LAR (Nabe et al., 2005). The application of anti-CD4 monoclonal antibodies to these

mouse LAR models has also demonstrated a central role for CD4+ cells in this response (Meyts et al.,

2008; Nabe et al., 2011a). Studies have also shown that transferring OVA reactive Th-cell clones into

naïve mice have resulted in an LAR upon OVA challenge (Ohtomo et al., 2009). Rats have also been

traditionally used to model the LAR. Watanabe et al (1995) demonstrated the importance of CD4+ T-

lymphocytes in Brown Norway rats using an adoptive transfer model of the LAR. In this study, naïve

Brown Norway rats received OVA-primed CD4+ T-cells and upon exposure to aerosolised OVA

exhibited a sustained bronchoconstriction (Watanabe et al., 1995).

CD8+ cells have also been explored with respect to the LAR in animal models. Increased numbers of

CD8+ T-cells have been found in the airway submucosa of sensitised Brown Norway Rats exposed to

OVA (Haczku et al., 1995b; Watanabe et al., 1995). It has been shown that depletion of CD8+ cells using

a monoclonal antibody in the Sprague Dawley Rat enhances the LAR (Olivenstein et al., 1993a;

Allakhverdi et al., 2000). Suzuki et al (1999) demonstrated that adoptive transfer of CD8+ T-cells from

sensitised donors into sensitised recipients resulted in a suppression of the LAR upon allergen

exposure(Suzuki et al., 1999). The LAR suppression was associated with a decrease in IL-4 mRNA

positive cells and an increase in IFN-γ positive cells in BAL fluid obtained at the time point of LAR. It

has been shown that administration of exogenous IFN-γ can reduce the LAR (Isogai et al., 2000). It has

also been well documented that IFN-γ can suppress the proliferation of Th2 type CD4+ T-cells

(Gajewski, Goldwasser & Fitch, 1988; Gajewski, Joyce & Fitch, 1989; Young & Hardy, 1995; Nakamura

et al., 1997) and it has been shown to inhibit the infiltration of CD4+ cells into the airways in a mouse

allergic asthma model (Iwamoto et al., 1993). CD8+ T-cells are known producers of IFN-γ (Kemeny et

al., 1994; Li et al., 1995) and so could elicit a protective effect in the allergic airway via this cytokine.

Isogai et al (2003) investigated this further by isolating the highly expressing IFN-γ cells, CD8+γδ T-cells,

from sensitised and unsensitised Brown Norway rats and adoptively transferring them to sensitised

recipients. Upon OVA challenge, the recipient rats displayed a significantly decreased LAR. The levels

of BAL Th2 type cytokines (IL-4, IL-5 and IL-13) were significantly decreased and levels of IFN-γ were

significantly increased in recipients compared to controls, providing further evidence for a suppressive

41

effect of CD8+ cells on CD4+ cells and the LAR via the cytokine IFN-γ (Isogai et al., 2003). In a later

study, the authors again utilised their adoptive transfer model and showed that pre-treatment of

CD8+γδ T-cells with an antisense oligonucleotide to inhibit IFN-γ before transfer into sensitised

recipients resulted in complete recovery of the LAR compared with recipients receiving untreated

CD8+γδ T-cells (Isogai et al., 2007).

Isogai and colleagues combined the studies demonstrating the central role of CD4+ cells in driving the

LAR and those exhibiting the suppressive effects of CD8+ cells in this response. In this investigation

they demonstrated induction of an LAR response by adoptively transferring CD4+ cells into recipient

rats. They then showed that by depleting CD8+ cells using a monoclonal antibody an enhanced LAR

was exhibited (Isogai et al., 2005).

Overall, these studies demonstrate that the balance between CD4+ and CD8+ cells in the allergic airway

could be a likely determinant of the LAR. Levels of these cell types have been explored clinically in the

asthmatic lung. Human subjects experiencing an isolated EAR after undergoing allergen challenge

have been shown to have relative increases in CD8+ cells in the BAL fluid compared to patients

experiencing a dual response who have increased levels of CD4+ cells in their BAL fluid (Gonzalez et

al., 1987). It has also been postulated that T-cell dysfunction is involved in the mechanisms behind

allergic respiratory tract disease (Rola-Pleszczynski & Blanchard, 1981). Studies assessing the effects

of intradermal challenge in humans showed that a higher proportion of CD4+ T-lymphocytes and a

lower proportion of CD8+ T-lymphocytes were accumulated in skin chamber fluids during the late

phase response (Werfel et al., 1995). Although it should also be noted that studies investigating the

patient characteristics indicating an LAR have showed that correlation exists between peripheral T-

lymphocytes and LAR, but not the late phase cutaneous response and LAR(van der Veen et al., 1999).

So far it seems likely that there could be interactions between the CD4+ and CD8+ T-cell populations in

the driving mechanisms of the LAR. CD4+ T-cell – CD8+ T-cell interactions have been demonstrated by

researchers. It has been shown that when a naïve CD8+ T-cell encounters antigen binding to MHC-I

molecules on the surface of an APC which is also bound to a CD4+ T-cell, the expression of

costimulatory molecules can activate the CD8+ T-cell (Azuma et al., 1992). Researchers have also

speculated that cytokines liberated from CD4+ T-cells such as IL-2 and IL-4 may activate the CD8+ T-

cells (Isogai et al., 2005). Direct effects of CD8+ cells on CD4+ cells have been shown through interaction

of Fas ligand-Fas receptor where they have been shown to induce CD4+ T-cell apoptosis (Noble,

Pestano & Cantor, 1998). Further investigations into these interactions and mechanisms further

downstream of them with respect to responses such as the LAR could lead to the discovery of

promising new drug targets.

42

There are a plethora of subsets of CD4+ and CD8+ T-cell which exist. Investigations into more specific

cell types, their associated cytokines and their effects on the LAR have begun. Th2 cells and their

cytokines have been linked to features of asthma extensively. In a study by Molet et al (1999) the

inhibition of IL-4 in CD4+ cells blocked the adoptive transfer of the LAR into naïve animals, highlighting

IL-4 to be essential in this response (Molet et al., 1999).Murine models of asthma have also elucidated

IL-13 to be essential in the LAR (Taube et al., 2002). However, clinical trials targeting Th2 cytokines

individually have yielded disappointing results (Holt & Sly, 2007) implying that targeting individual

cytokines alone are insufficient in inhibiting the LAR (O’Byrne, Inman & Adelroth, 2004). Wenzel and

colleagues have since demonstrated that Pitrakinra, a competitive inhibitor of the IL-4Rα complex,

interferes simultaneously with the actions of IL-4 and IL-13 and significantly attenuates the LAR

(Wenzel et al., 2007).

The inhibitory role of CD8+ T-cells in the LAR has so far been shown to be due to actions of the CD8+γδ

T-cell and the cytokine IFN-γ (Isogai et al., 2000, 2003, 2007). Allergen inhalation challenge in atopic

asthmatic subjects is associated with decreased IFN-γ positive CD8+ T-lymphocytes in peripheral blood

and sputum (Matsumoto et al., 2002; Yoshida et al., 2005) and so a deficiency in this cell type could

account, at least partially, for the LAR in asthmatics. The mechanism by which IFN-γ exerts its

protective effect has been speculated upon by researchers. For example, the phagocytic actions of

alveolar macrophages have been shown to be stimulated by IFN-γ (Nacy et al., 1991; Careau &

Bissonnette, 2004). It has also been postulated that CD8+γδ TCRs are capable of recognising antigen

directly in the absence of antigen processing (Schild & Chien, 1994).

IL-10, a typical anti-inflammatory cytokine, has been shown to be increased in the airway at the time

point of LAR in a murine model and is produced by CD4+ regulatory T-cells (Nabe et al., 2005;

Hawrylowicz & O’Garra, 2005).Afshar et al (2013) demonstrated an increase in T-regulatory cells in

BAL fluid in a murine asthma model following allergen challenge (Afshar et al., 2013). In a clinical study,

Kinoshita et al (2014) showed dual responders to exhibit a diminished percentage of airway T-

regulatory cells after allergen challenge compared with isolated early responders. The authors also

showed fewer T-regulatory cells compared with CD4+ T-cells in the dual responders and the

percentages of T-regulatory cells were correlated to the magnitude of LAR (Kinoshita et al., 2014). It

could therefore be stipulated that an imbalance in the numbers of inhibitory T-regulatory cells is an

important determinant of the LAR in atopic asthmatics.

T-helper 17 cells have been shown to be activated by antigen stimulation in vivo (McKinley et al., 2008;

Wilson et al., 2009). A monoclonal antibody targeting IL-17 in a murine model was found to suppress

the LAR. The authors of this paper also demonstrated an increase in the numbers of CD4+IL17+ cells

43

(T-helper 17 cells) correlated with the LAR and concluded this cell type to be the origin of the LAR

driving cytokine IL-17 (Mizutani et al., 2012). In clinic, it has recently been shown that the Th17/Treg

ratio is significantly enhanced in dual responders compared to isolated early responders post allergen

challenge. It has, therefore, been proposed that this imbalance post allergen challenge is an important

contributor to the development of the LAR (Singh et al., 2014).

The LAR has also been linked to the B-cell – Mast cell – IgE products axis. Some clinical studies have

indicated a prominent role for these cell types and mediators in the LAR. Increased numbers of mast

cells and increased levels of IgE in BAL fluid has been associated with the magnitude of the LAR

(Peebles et al., 2001; Crimi et al., 1991). Patients being treated with corticosteroids (which inhibit the

LAR) have been shown to have decreased numbers of mast cells in the smooth muscle and epithelium

(James et al., 2012). Disodium Cromoglycate, a known mast cell stabiliser, has been shown to inhibit

the LAR in asthmatic patients (Cockcroft & Murdock, 1987; Pepys et al., 1968, 1974; Booij-Noord, Orie

& De Vries, 1971; Cieslewicz et al., 1999b). Anti-IgE monoclonal antibodies have also been shown to

suppress allergen induced late phase bronchoconstriction (Busse et al., 2001; Holgate et al., 2004,

2005; Fahy et al., 1997).

In contrast, other clinical studies have alluded to mechanisms independent of B-cells, Mast cells and

IgE to be important in driving the LAR. In asthmatic patients, Durham et al(1984) failed to see an

association between total or allergen specific IgE and the LAR. Total IgE has been shown to be a poor

indicator of the LAR (Tschopp et al., 1998). In the African population, serum levels of IgE have even

been reported to be higher in non-asthmatics compared to asthmatics (Scrivener & Britton, 2000).

Studies involving the inhalation of T-cell peptides derived from the feline allergen Fel d 1

demonstrated the induction of an isolated LAR without an accompanying EAR, implying the LAR to be

T-cell dependent and IgE independent (Ali et al., 2004a). Khan et al (2000) demonstrated that while

Cyclosporin A decreased the LAR in patients, no effects were seen on the EAR indicating that the LAR

occurs independently of the B-cell-Mast cell- IgE products axis.

Animal models of the LAR have also resulted in conflicting conclusions being drawn on the role of B-

cells, Mast cells and IgE in the LAR. Mizutani et al (2012) demonstrated that mice sensitised with OVA-

specific IgE displayed an LAR upon OVA challenge. Inhibitors of mast cell products such as Cysteinyl

Leukotrienes have been shown to inhibit the LAR at least partially (Nabe et al., 2002; Roquet et al.,

1997). Nabe et al (2005) demonstrated a correlation between the presence of mast cells and the ability

to display an LAR in a mouse asthma model. However, the authors in this investigation also exhibited

that the LAR was not associated with IgE as Dexamethasone was shown to suppress the LAR but not

plasma levels of IgE(Nabe et al., 2005).The same authors also more recently showed that applying an

44

antibody targeting mast cells did not attenuate the LAR (Nabe et al., 2013). As previously described,

authors adoptively transferring CD4+ and CD8+ cells into rat models of the LAR noted that there were

no changes in the EAR and serum IgE levels where there were effects upon the LAR (Watanabe et al.,

1995; Isogai et al., 2003). Interestingly, McMenamin et al. (1995) showed that CD8+γδ T-cells suppress

plasma IgE levels, although the authors did not demonstrate a functional link to the LAR.

Eosinophilia and the LAR have been traditionally associated. This is mainly due to the fact that steroid

treatment has been shown to suppress both eosinophilia and the LAR (Kidney et al., 1997; Cieslewicz

et al., 1999b; Inman et al., 2001). Conversely, targeting eosinophilia via the application of monoclonal

antibodies has been shown to have minimal impact on the LAR in pre-clinical models and in clinic

(Flood-Page et al., 2007). Although it must be noted that, as previously described in section 1.5.3, the

interpretation of clinical studies utilising antibodies targeting eosinophils is currently under review.

The aetiology of the LAR has also been associated with sensory nerve interactions. Raemdonck et al

(2012) showed that the LAR but not the EAR was lost under general anaesthesia in pre-clinical asthma

models. The application of ruthenium red (a non-selective cation channel blocker) also resulted in

inhibition of the LAR, confirming a role for sensory nerves. Tiotropium, a long acting muscarinic

receptor antagonist (LAMA) also attenuated the LAR, suggesting a role for cholinergic constrictor

responses in the driving mechanisms behind the response. This overall led to the conclusion that

airway sensory nerves, a central reflex component and cholinergic constrictor responses are involved

in the driving mechanisms behind the LAR. These studies also concluded that the specific TRP channel

TRPA1 was also associated with the LAR via the application of specific TRP channel blockers. It was

suggested as a possibility that TRPA1 channels present upon airway sensory nerves could be triggered

directly by allergen challenge, leading to the bronchoconstriction that is the LAR (Raemdonck et al.,

2012). Indeed, the functional relevance of TRPA1 on airway sensory nerves has been well

characterised, although the role of TRPA1 present on inflammatory cell types remains to be elucidated

and so could also plausibly play a role in the LAR (Grace et al., 2012a).

45

1.9 Current Asthma Treatments

The current gold standard in asthma therapy involves the application of inhaled glucocorticoids in

combination with short acting and long acting β2-adrenoceptor agonists. Inhaled glucocorticoids treat

the chronic inflammation associated with asthma, whereas the β2-adrenoceptor agonists provide

symptomatic relief against reversible bronchoconstriction (Mullane, 2011). This combination has been

shown to provide effective asthma control in asthmatics in clinic and has been shown to be beneficial

compared with glucocorticoid treatment alone (Aubier et al., 1999; Zetterström et al., 2001; Lalloo et

al., 2003).

β2-adrenoceptor agonists induce the relaxation of ASM in order to promote airway conductance. They

bind to β2-adrenoceptors on ASM which induces intracellular signalling cascades. This ultimately

results in the relaxation of ASM. A number of secondary mechanisms of action have also been

described for β2-adrenoceptor agonists including anti-inflammatory and anti-proliferative effects

(Barnes, 1999; Yan et al., 2011; Shore & Moore, 2003; Kotlikoff & Kamm, 1996).

Glucocorticoids are anti-inflammatory and are thought to exert their effects via a number of

mechanisms. They are known to bind to Glucocorticoid Receptors (GR) in the cytoplasm of various cell

types. This results in the GR dimerizing and migrating to the nucleus of the cell where it binds to a

Glucocorticoid Response Element (GRE) (a location in the promoter region of steroid responsive

genes). This results in the up regulation of anti-inflammatory gene transcription or the down

regulation of pro-inflammatory gene transcription (Barnes, 1998; Barnes, Pedersen & Busse, 1998).

During inflammatory states, the activation of proinflammatory transcription factors results in the

increased expression of inflammatory cytokines, chemokines and adhesion molecules. Transcription

factors are thought to act via two main mechanisms. They bind to promoter regions of DNA and recruit

co-activator molecules to the DNA transcription factor complex whilst aiding in the binding of RNA

polymerase to catalyse gene transcription. They may also act via modulating the chromatin structure.

DNA is usually wound around core histones (a structure known as chromatin). In basal condition this

is a closed structure and results in minimal gene expression. The binding of transcription factors

induces Histone acetylation by Histone Acetyltransferase (HAT) and results in the opening of the

structure. This enables increased binding of the DNA transcription factor complex to result in

increased transcription. Glucocorticoids are thought to inhibit inflammation through recruitment of

Histone Deacetyltransferases (HDACs). HDACs counteract the activity of HATs and reduce DNA

transcription by increasing DNA winding and hence reducing the binding of transcription factors

(Barnes, 1998; Barnes, Pedersen & Busse, 1998). Steroids have also been shown to interact directly

with proinflammatory transcription factors such as NF-κB. In basal states, NF-κB is sequestered in the

46

cytoplasm. HDACs have been shown to induce GR deacetylation causing it to bind to the NF-κB

complex to prevent it from translocating to the nucleus where it would usually promote the

transcription of pro-inflammatory genes (Ito, Barnes & Adcock, 2000; Ito et al., 2006; Kagoshima et

al., 2001).

In the clinic the treatment of asthma is approached in a stepwise manner. Patients who present with

mild intermittent symptoms are treated with inhaled short acting β2-adrenoceptor agonists (SABA)

such as salbutamol. More severe cases are treated with a combination of low dose ICS (e.g.

Budesonide or Beclamethasone) and Long Acting β2-adrenoceptor agonists (LABA) such as Salmeterol

or Formoterol. Even more severe cases gain a higher dose of ICS or oral corticosteroid such as

Prednisolone. If symptoms are still poorly controlled, add on therapies are incorporated into

treatment regimes such as leukotriene antagonists, Phosphodiesterase inhibitors or Anticholinergics.

(Barnes, 2010; Bateman et al., 2008) (See Figure 1.4).

Despite the available effective therapies, approximately 5-10% of asthma patients have uncontrolled

symptoms. These patients account for a huge amount of healthcare spending, highlighting an urgent

need for new therapeutics.

Figure 1.4: Step-wise approach to asthma treatment. (Adapted from Barnes 2010).

47

1.10 In vivo models of allergic asthma

Pre-clinical models closely mimicking the cardinal features of asthma are useful in understanding

the mechanisms driving the disease and in identifying novel therapeutic targets. They are also

invaluable in further investigating existing therapeutics in terms of their efficacy and side effect

profile where in clinic this would limit development. Asthma is a complex disease comprised of

many symptoms and influencing factors and so not surprisingly animal models of asthma do not

exhibit all features of the disease. Experimentally induced animal models of asthma tend to mimic

certain aspects of the disease with a focus on specific asthma endpoints. The purpose of this thesis

is to investigate the mechanisms driving the LAR and as such this will be the endpoint of interest

focused upon in this body of work.

1.10.1 Ovalbumin Model

Laboratory animal do not naturally develop allergic asthma. Classical models of allergic asthma utilise

OVA to induce asthma like symptoms. The protocols used to induce allergic reactions involve two

distinct phases: sensitisation and challenge. In the first phase, animals are sensitised to OVA by a

systemic intraperitoneal injection of OVA plus an adjuvant such as AlumTM (later described in section

1.10.2). The challenge phase is typically a topical dose (i.e. intranasal, aerosol or intratracheal) of OVA

only. Different research groups employing these models may vary in their approach to these protocols

in terms of doses, sensitisation times and challenge times but the general principle of a sensitisation

phase followed by a challenge phase remains consistent.

Systemic sensitisation in OVA-driven models of asthma results in a robust increase in total and OVA-

specific IgE (Beck & Spiegelberg, 1989). Following this phase, OVA challenge has been shown to induce

multiple asthma-like symptoms such as EAR, LAR, non-specific AHR, allergic airway inflammation and

airway remodelling events (Stevenson & Belvisi, 2008). As such, OVA-driven models of asthma are

considered extremely robust and have been used extensively as a platform to profile novel

therapeutics and delineate disease mechanisms.

There are also disadvantages associated with this model. OVA models require systemic sensitisation

and atopy in asthma is associated with the lungs. Due to this, systemic sensitisation is believed to be

less clinically relevant than other topical routes (Gregory et al., 2009). However, some groups have

shown that while topical sensitisation may initially produce increases in IgE, this response has been

reported to decrease with time and is not considered to be robust (Holt, Batty & Turner, 1981).

Tolerance to OVA as an inducer of allergic responses has also been widely reported as a practical

disadvantage to the model (Van Hove et al., 2007; Swirski et al., 2002). Acute challenge protocols (e.g.

48

2 weeks) have been shown to result in allergic asthmatic responses, however, these responses have

been shown to dissipate following chronic OVA challenge (e.g. 6-8 weeks) (Yiamouyiannis et al., 1999).

Few people tend to develop an allergy to OVA and so the use of this as an allergen has been

questioned. Models utilising more disease relevant allergens such as HDM are currently under

development. So far success has been achieved in modelling features of asthma such as AHR, airway

inflammation and remodelling events using this allergen (Verheijden et al., 2015; Sivapalan et al.,

2014; Mariñas-Pardo et al., 2014). However, none of these recently developed HDM-driven models

display an EAR and LAR. As such, the use of a HDM driven model in this thesis was unfortunately not

a possibility.

The need for an adjuvant in the OVA-driven model has also been noted as a disadvantage in terms of

clinical relevance. Although it should be noted that adjuvant free models have also been described

(Renz et al., 1992; Hessel et al., 1995; Blyth et al., 1996; De Bie et al., 1996; Besnard et al., 2011).

Adjuvants will be discussed in more detail in the next section.

1.10.2 Adjuvants

Adjuvants are widely used in vaccinations to aid in the development of immunity and have been

adapted for use in preclinical models of allergic disease to aid the sensitisation process. One of the

most widely used adjuvants is AlumTM which is comprised of aluminium hydroxide and magnesium

hydroxide. Despite being widely used in animal models of allergic disease and in human vaccinations,

the mechanism of action of AlumTM and other adjuvants remains poorly understood. Nevertheless,

research into these mechanisms has resulted in the suggestion of some theories which will be

discussed below.

The first theory proposed on the mechanism of action of adjuvants is the depot theory. It has been

suggested that adjuvants form a depot to allow the slow uptake and presentation of allergens by APCs.

Indeed, dendritic cells and allergen specific T-cells have been found around AlumTM precipitates. It has

also been shown that the effects of adjuvants can be transferred between animals via the

transplantation of these precipitates (Kool, Fierens & Lambrecht, 2012). However, others have also

suggested that depot formation and the slow uptake of allergens may not be important in the

mechanisms surrounding the effects of adjuvants. Researchers have shown that antigen presentation

by APCs predominantly occurs 6-24 hours following allergen-adjuvant injection and the removal of the

adjuvant depot 2 hours following injection did not affect B-cell and T-cell responses (Hutchison et al.,

2012). Other investigators also showed that mice deficient in fibrin (a key component of the adjuvant

49

depot) had comparable immune responses to wildtype mice following allergen-adjuvant

administration (Munks et al., 2010).

Adjuvants have also been shown to act on immune cell populations. It has been shown that adjuvants

are capable of promoting monocyte differentiation into dendritic cell phenotypes with enhanced

expression of co-stimulatory molecules such as MHC-II molecules. Enhanced IL-4 expression has also

been reported as an effect of adjuvants. Overall, this has led to the proposal of the theory that Alum

may act on T-cells to produce IL-4 which in turn promotes the increased expression of MHC-II

molecules on dendritic cells derived from monocytes to enhance their antigen presentation

capabilities (Seubert et al., 2008; Ulanova et al., 2001).

Another theory surrounding the mechanisms behind adjuvant activity is that they can induce innate

inflammatory responses which result in the promotion of allergen uptake by APCs. This is based on

the fact that organic molecules such as LPS have a similar effect on the immunological response to

that of adjuvants. Their effects are likely to be mediated via PAMPs and DAMPs to result in the release

of inflammasome mediated cytokines (Eisenbarth et al., 2002). Indeed, AlumTM has been shown to

induce the release of IL-1β and IL-18 from inflammatory cell populations via activation of the

inflammasome which has been shown to be NLRP3 dependent (Li et al., 2008; Hornung et al., 2008;

Kool et al., 2008a). AlumTM has also been shown to produce endogenous agents which activate the

inflammasome. Uric acid acts as a danger signal and is released from dying cells. Necrotic cells which

release Uric acid have been found around AlumTM injection sites (Goto & Akama, 1982, 1984; Goto et

al., 1997; Shi, Evans & Rock, 2003). Uric acid has been shown to be capable of activating the NLRP3

inflammasome resulting in the release of IL-1β and IL-18 (Martinon et al., 2006). In vivo experiments

have also shown that AlumTM injection induced Uric acid release in the peritoneum which resulted in

the activation and migration of APCs and T-cell proliferation. These effects were abolished with

Uricase treatment (Kool et al., 2008b). Other studies have contradicted this mechanism of action. It

has been proposed that the inflammasome is important in AlumTM-induced IL-1β production but this

does not contribute to adjuvant effects (McKee et al., 2009). One study showed that Alum plus OVA

application was able to induce the production of extracellular ATP (another DAMP) but this was shown

not to contribute to AlumTM-induced immunity (Idzko et al., 2007; Kool et al., 2011).

Other proposed mechanisms of action of adjuvants such as AlumTM include that it may stick to

dendritic cells and interact with cell membrane lipids to promote high affinity binding of dendritic cells

to CD4+ T-cells via adhesion molecules (Flach et al., 2011). A role for PGE2 acting independently of the

inflammasome has also been proposed in the mechanisms behind AlumTM driven immunogenicity

(Kuroda et al., 2011).

50

Overall, theories behind the mechanisms driving the adjuvant activity of AlumTM remain inconclusive

and further investigation into this aspect of animal model development is warranted.

1.10.3 Different Species as Asthma Models

Animal models of allergic asthma have been established in various species such as the C57BL/6 and

BALB/c mouse strains, the Brown Norway rat and the Dunkin-Hartley guinea-pig. Various different

aspects of allergic asthma have been successfully modelled in these species such as EAR, LAR, allergic

airway inflammation, AHR and airway remodelling events. The guinea-pig is less commonly used as an

allergic asthma model in comparison with mice and rats due to an enhanced basal level of eosinophils

in the lung and as such this section will focus mainly on rat and mouse models of asthma (Rothenberg

et al., 1995). Although many features of allergic asthma have been successfully modelled in rats and

mice, the following sections will highlight models of bronchoconstriction in response to allergen as

the focus of this thesis is the LAR.

1.10.3.1 Rat Models

The Brown Norway rat is considered to be the most suitable rat strain for use as an allergic asthma

model. This particular strain is a high IgE producer, it produces robust responses to allergens (distinct

EAR and LAR) and the infiltration of allergic airway inflammation is considered to be similar to that

seen in asthmatic patients (Murphey et al., 1974; Eidelman, Bellofiore & Martin, 1988; Elwood et al.,

1992; Haczku et al., 1995a). This particular model is considered advantageous due to the robustness

of response to allergen challenge in comparison to other species used as allergic asthma models.

1.10.3.2 Mouse Models

The mouse is considered to be an advantageous model of allergic asthma due to the possibility of the

application of GM strains and the fact that it comprises a highly characterised immune system.

Classically, BALB/c mice were the preferred strain for developing allergic asthma models but more

recently the use of the C57BL/6 strain has increased due to more GM strains being available on this

genetic background.

The EAR and LAR have been largely overlooked in murine asthma models in comparison to other

features of asthma such as AHR and allergic inflammation. This may reflect the difficulty in achieving

these endpoints in mice. Despite this, some research groups have reported the EAR and LAR in mouse

models. Cieslewicz et al (1999) reported that in C57BL/6 mice systemic sensitisation and aerosolised

challenge with OVA resulted in an EAR 5-30 minutes after challenge, followed by an LAR which peaked

at 6 hours after challenge. The LAR displayed was considered to be clinically relevant due to the time

point and a strong association with allergic airway inflammation. The EAR, however, was questionable

in terms of relevance due to the fact that it was not dependent on IgE and mast cells (Cieslewicz et al.,

51

1999a). Nabe et al (2005) demonstrated in BALB/c mice that systemic sensitisation plus 4 intratracheal

doses of OVA resulted in an EAR and LAR. The EAR was associated with IgE and mast cells and the LAR

with allergic airway inflammation (Nabe et al., 2005). In contrast, de Bie et al (2000) observed an EAR

but no LAR in their adjuvant free OVA model (de Bie et al., 2000).

So far, success in modelling the EAR and LAR in mouse models of allergic asthma has been limited. As

such, little data is available on the characterisation of these models. Further evaluation of these

models will hopefully highlight their strengths and weaknesses with respect to human disease in the

future.

52

1.11 Thesis Plan

The LAR is experienced by approximately 50% of asthma sufferers and is routinely used as a clinical

endpoint when exploring new therapeutic options (Booij-Noord et al., 1972; O’Byrne, Dolovich &

Hargreave, 1987; Stevens & van Bever, 1989; Muller et al., 1993). Despite this, the specific

mechanisms driving the LAR remain unclear. Inhaled glucocorticoids are known to significantly

attenuate the LAR implying a role for airway inflammation within the response (Bousquet et al., 2000;

Barnes, 2008). Within the Respiratory Pharmacology group, animal models of the LAR have also

suggested possible roles for airway sensory nerves, central reflexes and parasympathetic cholinergic

constrictor responses (Raemdonck et al., 2012). The overall aim of this thesis was to build upon this

body of work in order to further explore the driving mechanisms behind the LAR.

1.11.1 Hypothesis

The hypothesis that formed the basis of this project was;

“Mast cells and airway sensory nerves are central to the driving mechanisms behind the LAR”.

1.11.2 Aims

The aims of this thesis are to:

Validate the clinical relevance of mouse and rat models of the LAR using the clinically relevant

glucocorticosteroid Budesonide (Chapter 3).

Explore the roles of key allergic effector cells in the LAR via the application of GM mice to

preclinical asthma models (Chapter 4).

Further explore the role of mast cells in the LAR via the application of Disodium Cromoglycate

and Compound 48/80 to preclinical asthma models (Chapter 5).

Further characterise the role of airway sensory nerves in the LAR via the application of

Pharmacological tools to animal models of the LAR (Chapter 6).

Investigate the possibility that Disodium Cromoglycate attenuates the LAR via a mechanism

of action on airway sensory nerves using in vivo models and isolated vagus nerve experiments

(Chapter 7).

53

Chapter 2

General Methods

54

2 General Methods

2.1 Introduction

This chapter outlines a general methodology for experimental protocols used throughout this thesis.

Specific details of individual experimental protocols and statistical analyses are detailed in the

methods section of individual chapters.

2.2 Animals

All experimental procedures were conducted in accordance with UK Home Office Guidelines for

animal welfare based on the Animals (Scientific Procedures) Act of 1986. All animals were housed in

individually ventilated cages (IVC) and a 12 hour light-dark cycle maintained. Prior to and during

experimental periods, food and water was supplied ad libitum.

2.2.1 Mice

C57BL/6 wildtype (Harlan, UK), mast-cell knockout [KitW-sh/HNihrJaeBsmJ] (Jackson Laboratories Ltd,

USA), B-cell knockout [B6J-IgHtm1 Mom](SchwImMR collaboration group, Switzerland), CD4 knockout

[B6-CD4tm1 Mak](SchwImMR collaboration group, Switzerland), CD8 knockout[B6-CD8atm1 Mak]

(SchwImMR collaboration group, Switzerland) and IgE knockout [BALB/c-IgEtm1Led](SchwImMR

collaboration group, Switzerland)male mice aged 8-12 weeks were grown in house. Official strain

names are given in square brackets. Original breeding pairs were purchased from suppliers as

indicated.

2.2.2 Rats

Male Brown Norway Rats (200 g -225 g) were purchased from Charles River Laboratories (UK). Animals

were housed for at least 7 days prior to experimental procedures being carried out.

2.2.3 Guinea-Pigs

Male Dunkin-Hartley Guinea-Pigs (300 g -350 g) were purchased from Charles River Laboratories (UK).

Animals were housed for at least 7 days prior to experimental procedures being carried out.

2.3 Validation of Genetically Modified Mice

Commercial assays for genotyping were not available for all knockout mice. Therefore, it was decided

to validate the GM mice in terms of their lung cell populations via the use of flow cytometry and

histology. This was advantageous as it was possible to determine that the mice were in fact deficient

of their specific knockout cell type as well as observing any changes in baseline levels of other

leukocyte populations occurring as a result of genetic modification.

55

2.3.1 Cell Preparation

Naïve animals were culled via overdose with Sodium Pentobarbitone (200 mg/kg i.p.). The systemic

circulation was then perfused. This was done by opening the thoracic cavity and making an incision in

the left ventricle and the left atria of the heart. The left ventricle was then cannulated and the systemic

circulation perfused with sterile saline (0.9%) at approximately 100 mmHg pressure. The purpose of

this was to remove contaminating systemic cell populations.

The lungs were removed, weighed and finely chopped using a Mcllwain tissue chopper (Campden

Instruments Ltd, UK) and transferred to 1 ml Roswell Park Memorial Institute media (RPMI)/Penicillin-

Streptomycin. This was then added to 4mls RPMI supplemented with 10% Foetal Bovine Serum (FBS),

collagenase (1 mg/ml) and DNase (25 g/ml) and incubated in a water bath with gentle agitation for

1 hour at 37 °C. Following this, samples were filtered using a 70 µm mesh sieve. The samples then

underwent two rounds of centrifugation at 807 g, discarding the supernatant and resuspending the

pellet in 10 mls RPMI (supplemented as previous) each time. For each sample, a total leukocyte cell

count was performed using a Sysmex XP-300 cell counter (Sysmex Ltd., UK).

2.3.2 Staining Protocol

Cells were stained for surface markers as detailed in Snelgrove et al (2008). Briefly, cell suspensions

were resuspended at 1x106 cells/ml and 200 µl transferred to the relevant wells of a 96-well v-

bottomed staining plate (Sigma, UK). The plates were centrifuged (1398 g, 2 minutes, room

temperature) before the supernatant was discarded. The cells were stained using 50 µl per well

(diluted 1/1000 in PBS) of a fixable near-IR dead cell stain kit for 633/635 nm excitation (Invitrogen,

UK) and incubated for 15 minutes at room temperature. The basis of the live/dead cell stain kit is

detailed in figure 2.1. A further 150 µl PBS per well was added before the plates were centrifuged

(1398 g, 2 minutes, room temperature) and the supernatant discarded.

Cell suspensions were then stained for surface markers using the relevant monoclonal antibody

conjugate stains as detailed in Table 2.1. The stains were prepared in PBA (PBS containing 0.1%

(wt/vol) sodium azide and 1% (wt/vol) BSA) before 30 µl of the relevant stain was added to each well

and incubated for 30 minutes on ice. A further 150 µl per well PBA was added, the plate centrifuged

(1398 g, 2 minutes, room temperature) and the supernatant discarded. The cells were washed a

further two times by adding 200 µl PBA, centrifuging (1398 g, 2 minutes, room temperature) and

discarding the supernatant. The cells were then fixed by adding 50 µl per well of 4% formaldehyde

(diluted in PBS) and incubating at room temperature for 15 minutes. Following this a final wash in 100

µl PBA was performed by resuspending and centrifuging (1398 g, 2 minutes, room temperature)

56

before a final resuspension in 200 µl PBA was performed. The plates were sealed and stored at 4 °C

until Flow Cytometry was performed (Snelgrove et al., 2008).

Figure 2.1: Demonstrating the basis of the live/dead cell stain kit. Assays are based upon the ability

of the fluorescent reactive dye to react with cellular amines. The reactive dye can permeate the

compromised membranes of necrotic cells and react with amines on the interior of the cell as well

as on the cell surface. Viable cells are stained only on the cell surface. The difference in staining

intensity between live and dead populations is typically 50-fold more intense in the dead

population. (Adapted from Invitrogen Molecular Probes Experimental Protocols).

57

Stain Monoclonal Antibody Conjugates Dilution in PBA

Stain 1 CD19 (FITC)

NKp46 (PE)

CD4 (PercP)

CD8 (APC)

100x

200x

200x

200x

Stain 2 Gr-1 (FITC)

F4/80 (PE)

CD11b (PercP)

CD11c (APC)

100x

100x

400x

200x

Stain 3 Gr-1 (FITC)

SiglecF (PE)

CD11b (PercP)

CD11c (APC)

100x

200x

400x

200x

Table 2.1: Outlining monoclonal antibody conjugate combinations and dilution factors used to stain cell populations.

2.3.3 Flow Cytometry

Data was acquired on a BD FACS Canto II (BD Biosystems, UK) and 30000 events per sample were

analyzed using FlowJo analysis programme (Treestar Ashland, USA). Forward scatter and side scatter

gates were used to exclude debris, and dead cells were excluded using a fixable near-IR dead cell stain

kit for 633 or 635-nm excitation (See figure 2.1). Cell types were characterised by their forward and

side scatter profiles and by their specific phenotypes (See Table 2.2 and figures 2.3, 2.4 and 2.5).

Unstained controls were used to account for the auto-fluorescence of the samples and the

Photomultiplier tube (PMT) gains set accordingly. Single stained controls were used to account for

spectral overlap and the flow cytometer compensations set accordingly (See figure 2.2). Gates to

assess positive and negative staining were set using standard fluorescence minus one (FMO) controls

for each monoclonal antibody conjugate. This particular type of control contains every fluorophore

conjugated antibody in the stain apart from the one you are controlling for. As a result of performing

this control, you can discard negative staining by gating it out and thus determine a positive

population.

58

Figure 2.2: Spectral overlap of fluorophores. Spectra from fluorophores FITC and PE with two band-

pass filters superimposed. Some of the light from FITC will pass through the filter used to collect

light from PE (indicated by arrow). By using single stained controls, compensations can be set on

the flow cytometer to account for this (Adapted from Ormerod 2008).

59

Cell Type Surface marker Phenotype Monoclonal Antibody

Conjugate

B-cells CD19+ Rat anti-mouse CD19 (FITC)

CD4+ T-cells CD4+ CD4 (PerCP)

CD8+ T-cells CD8+ CD8 (APC)

Neutrophils Ly-6Ghigh

CD11bhigh

CD11clow

F4/80low

Ly6G (FITC)

CD11b (PerCP)

CD11c (APC)

F4/80 (PE)

Eosinophils CD11bhigh

CD11clow

Ly6Glow

SiglecFhigh

CD11b (PerCP)

CD11c (APC)

Ly6G (FITC)

SiglecF (PE)

Alveolar Macrophages CD11blow-intermediate

CD11chigh

F4/80high

Ly-6Glow

CD11b (PerCP)

CD11c (APC)

F4/80 (PE)

Ly6G (FITC)

Inflammatory

monocytes/tissue

macrophages

CD11bhigh

CD11clow

F4/80high

CD11b (PerCP)

CD11c (APC)

F4/80 (PE)

Table 2.2: Outlining surface marker antigens and relevant conjugated monoclonal antibodies used to identify various cell populations

60

Figure 2.3: Characterisation of Lymphocyte Populations. (A) Lymphocytes were defined as small,

non-granular cells (based upon their forward and side-scatter profiles) and gated upon this

characteristic. Within the lymphocyte gate, cells were classified according to their expression of

surface antigens; (B) CD19+ cells defined as B-cells (red box); (C) CD4+ cells defined as CD4+

lymphocytes (green box) and CD8+ cells defined as CD8+ lymphocytes (purple box). All gates were

set using the relevant Fluorescence Minus One (FMO) controls.

Figure 2.4: Characterisation of eosinophil population. Cells were characterised by their forward

and side scatter profiles; and then according to their CD11b and CD11c expression.

CD11clow/CD11bhigh cells were gated for (purple box). These cells were then characterised in terms

of their SiglecF and Ly-6G expression. SiglecFhigh/Ly-6Glow cells were classified as eosinophils (red

box).

61

Figure 2.5: Characterisation of neutrophils, alveolar macrophages and inflammatory

monocytes/tissue macrophages. Cells were characterised by their forward and side scatter profiles;

and then according to their CD11b and CD11c expression. CD11chigh/CD11blow cells were gated for

(green box) and then characterised in terms of F4/80 and Ly-6G expression. F4/80 high/Ly6G low

cells (orange box) were classified as alveolar macrophages. CD11clow/CD11bhigh cells (purple box)

were also classified in terms of F4/80 and Ly-6G expression. F4/80low/Ly-6Ghigh cells were classified

as neutrophils (red box). F4/80high cells (blue box) were classified as inflammatory monocytes/tissue

macrophages [N.B. A forward/side scatter profile of this gate was performed and any characteristic

eosinophils were gated out. Eosinophil populations were determined using a different stain (see

figure 2.4) due to the number of fluorophores which could be detected by the FACS machine being

a limiting factor].

62

2.3.4 Histology

Mast cells were identified using a standard Toluidine blue histological stain. Mice were culled via

overdose with Sodium Pentobarbitone and the systemic circulation perfused as detailed in section

2.3.1. The trachea was then cannulated and the lungs superfused with formalin before being placed

in formalin for 24 hours. Following this, they were transferred into 70% ethanol until paraffin wax

embedding and slicing could take place.

The paraffinized lung samples were cut into 4 µm sections. The sections were stained using a standard

toluidine blue and eosin counterstain protocol as in Yagil et al (2012). Briefly, the lung sections were

dewaxed using Histochoice clearing agent ® (Sigma, UK) and rehydrated in a series of ethanol dilutions

(100%, 90%, 70%). The slices were then washed in deionised water and stained in 0.1% Toluidine Blue

(Sigma, UK) for 5mins. Sections were then washed in distilled water before counterstaining with Eosin-

Phloxine solution (Sigma, UK) for 1 min. A final wash in distilled water then took place before the slices

were dehydrated using a series of ethanol dilutions (70%, 90%, 100%). The slices were left to dry at

room temperature and mounted onto glass slides(Yagil et al., 2012).

The stained sections were analysed under light microscopy at x40 magnification, the observer blinded

to the specimen identities. The numbers of mast cells per slide (3 slides per lung sample) were counted

(figure 2.6).

Figure 2.6: Demonstrating histological staining for mast cells in mouse lung tissue sections. Mast

cells in Toluidine Blue and eosin counterstained tissue sections were identified by characteristic dark

purple staining (see arrows). Mast cells were counted on a per slide basis under light microscopy

(X40 magnification) by a blinded observer (3 slides per lung sample).

63

2.4 Models of Allergic Asthma

The OVA driven rat and mouse models of LAR have previously been developed ‘in house’ (Raemdonck

et al 2011). Studies should ideally contain 4 control groups as follows: Saline-Sensitised/Saline

Challenged, Saline-Sensitised/OVA-challenged, OVA-Sensitised/Saline-Challenged and OVA-

Sensitised/OVA-Challenged. This is important as it allows the determination of whether responses are

allergic (in sensitised mice) or innate occurring independently of sensitisation. Following a short time-

course study, it was deemed practical for the studies included in this thesis, to include just 2 groups;

Saline-Sensitised/OVA-Challenged and OVA-Sensitised/OVA-Challenged. Throughout the studies,

AlumTM diluted 1:1 with saline was used as an adjuvant during the sensitisation of animals. AlumTM

refers to 20 mg.ml-1 aluminium hydroxide and 20 mg.ml-1 magnesium.

2.4.1 The OVA Driven Mouse Model of LAR

Male mice (20 g-25 g) were sensitised with OVA in AlumTM (50 µg per mouse, 500 µl i.p.) or vehicle

(AlumTM diluted 1:1 with saline, 500 µl i.p) on days 0 and 14 (see figure 2.7). On day 28, mice were

given a single intra-tracheal (i.t.) dose of OVA (2% OVA, 25 µl). For the i.t. dosing, mice were placed in

a Perspex chamber connected to an anaesthetic machine (Bowring Medical Engineering Ltd, Witney,

UK) and exposed to 4% isoflurane in 0.5% oxygen until sufficiently anaesthetised. Mice were dosed

into the trachea using a dosing gavage and then monitored until fully recovered.

Figure 2.7: Timeline demonstrating the protocol used for OVA driven mouse model of LAR

64

2.4.2 The OVA Driven Rat Model of LAR

Male Brown Norway rats (200 g-225 g) were sensitised with OVA in AlumTM (100 µg per rat, 1 ml i.p.)or

vehicle (AlumTM diluted 1:1 with saline, 1 ml i.p) on days 0, 14 and 21 (see figure 2.8). On day 28, rats

were placed in a Perspex chamber and challenged with1% (w/v) aerosolised OVA for 30 minutes.

Figure 2.8: Timeline demonstrating the protocol used for OVA driven rat model of LAR

2.5 Assessment of Lung Function

The LAR is characterised by bronchoconstriction occurring approximately 3-8 hours following allergen

exposure. It is therefore essential to assess lung function in order to successfully model this feature of

asthma.

Resistance and Compliance is a specific way of assessing lung function in animal models of asthma.

However, the technique is highly invasive and requires anaesthesia, tracheal intubation and artificial

ventilation; meaning that the technique must be performed under non-physiological conditions. This

method is also terminal which results in the prevention of further endpoints (e.g. inflammation) from

being assessed in the same animal. Raemdonck et al (2012) showed that anaesthesia abolishes the

LAR, meaning that for the purpose of this body of work, Resistance and Compliance was not deemed

a viable technique for the measurement of lung function.

Whole Body Plethysmography (WBP) is a non-invasive method of assessing enhanced pause (Penh) in

animal models of asthma. As part of this technique, the animals are conscious, unrestrained and

therefore have more natural breathing patterns. Penh was used as an indicator of lung function

throughout this thesis and is discussed below.

65

2.5.1 WBP and Penh

Flow Whole Body Plethysmography (FWBP) encompasses a pneumotachograph in its wall (measuring

pressure changes between the main chamber and reference chamber) and was used for all studies

measuring lung function within this thesis.

Within WBP, respiration is considered as the exchange of air between the body and the chamber. As

this process occurs, pressure changes within the WBP are recorded as the respiratory waveform.

During inspiration air is drawn into the animal from the chamber (chamber pressure decreases), which

results in thoracic displacement (chamber pressure increases). The increase in pressure due to

thoracic displacement is greater than the previously decreased pressure. This is due to conditioning

(heating and humidification) of the air as it moves from chamber conditions to body conditions and

results in a net increase in pressure during inspiration. It should also be noted that nasal flow always

lags behind thoracic flow. This means that as the thorax expands there is a delay before air is drawn

into the lungs due to airway resistance. This difference is greater in a state of constriction. The

difference between the two waveforms makes up an FWBP waveform and Penh is calculated from this

waveform. Penh is “a non-dimensional parameter based on a characteristic change in the expiratory

wave shape of the unrestrained plethysmography box signal” (Lomask, 2006).

There is some controversy over the relevance of using Penh in investigations of lung function. Penh

has been widely used in AHR studies in mice (Pennock et al., 1979; Hamelmann et al., 1997; Dohi et

al., 1999; Cieslewicz et al., 1999a)and it has been shown to correlate with airway resistance

(Hamelmann et al., 1997). Penh has also been shown to correlate with allergen induced bronchial

changes (Dohi et al., 1999). However, studies also exist which dispute the use of Penh as an indicator

of lung function. The main arguments are that the contribution of the nasal component means that

the respiratory signal obtained cannot strictly be used to represent changes within the airways and

that the contribution of air conditioning may also interfere with pressure change recordings (Adler,

Cieslewicz & Irvin, 2004; Bates et al., 2004; Sly et al., 2005; Lundblad et al., 2007).

Although some controversy still exists surrounding the use of Penh, the technique was deemed

necessary for this body of work. Raemdonck et al (2012) showed that the neurological output of the

CNS must be preserved in order to observe the LAR and so a conscious modelling system was required

(Raemdonck et al., 2012).There are currently no alternative ways of attaining these measurements

without using restraint, and Home Office restrictions would not allow the use of restraint for the

prolonged periods of time necessary to achieve the LAR response. Therefore, Penh was used as an

arbitrary measure of airway constriction in order to assess lung function within this thesis.

66

2.5.2 Mouse WBP

Immediately following recovery from i.t. dosing, mice were placed in WBP chambers (Buxco

Electronics, USA) and pressure changes continuously recorded by Buxco Finepointe Software (Buxco

Electronics, USA). The average Penh was recorded at 10 minute intervals for a total duration of 12

hours and was used for analysis.

2.5.3 Rat WBP

Following OVA aerosol, rats were placed in rat WBP chambers (Buxco Electronics, USA). Pressure

changes were recorded from 30 minutes following the end of OVA challenge using Buxco Finepointe

Software (Buxco Electronics, USA). The average Penh was recorded at 10 minute intervals for a total

duration of 5 hours and was used for analysis. Drugs were dosed around the OVA challenge as detailed

in the relevant chapters.

2.5.4 Data Analyses

The Penh average was plotted against time following challenge for rat and mouse studies. The Area

Under Curve and Penh average at peak response for the various experimental groups was calculated

using Graphpad Prism Software (Graphpad Software Inc., USA) and used for statistical analyses as

detailed in the relevant chapters.

67

2.6 Sample Harvesting and Processing

2.6.1 Blood Sampling

Animals were euthanized via overdose with Sodium Pentobarbitone (200 mg/kg i.p.). Blood was

attained via cardiac puncture with a heparinised syringe. Samples were then centrifuged (1398 g, 10

minutes, 4 °C) and the plasma supernatant transferred to eppendorfs and stored at -20 °C until

subsequent analysis.

2.6.2 Bronchoalveolar Lavage

The trachea was exposed via blunt dissection and cannulated. A syringe was used to introduce RPMI

media (Life Technologies) into the lungs for 30 seconds, before being withdrawn. For mice 0.3 mls was

introduced 3 times. For rats 3 mls was introduced 2 times. The samples were then pooled to give 1

sample per animal, before being prepared for total and differential cell counts.

2.6.3 Cell Counts

The total leukocyte cell counts in BAL fluid were attained using a Sysmex XP-300 automated cell

counter (Sysmex Ltd., UK).

Slides for differential cell counts were prepared using 100 µl BAL fluid in a cytospin (807 g, 5 minutes,

room temperature, low acceleration) (Shandon, Runcorn, UK). Slides were stained using a Hema-tek

2000 automated slide stainer (Ames Co., Elkhart, USA) using ACCUSTAIN ® modified Wright-Giemsa

stain (Sigma). Slides were analysed under light microscopy at x40 magnification by an observer blinded

to the specimen identities. Differential counts were completed on 200 cells per slide using standard

morphological criteria (See Table 2.3). The percentage of macrophages/monocytes, neutrophils,

eosinophils and lymphocytes were calculated. Macrophages and monocytes were counted as one

group. Other cell types such as epithelial cells and red blood cells were ignored.

2.6.4 Histology

The trachea was exposed via blunt dissection and cannulated. The lungs were superfused with

formalin, before being placed in formalin for 24 hours. Following this, lungs were transferred into 70%

ethanol until paraffin wax embedding and slicing could take place. A standard toluidine blue staining

protocol and eosin counter stain was then performed upon the formalin fixed lung tissue slices. The

numbers of mast cells were analysed as previously described (section 2.3.4).

68

Neutrophil

Multi-lobed, darkly stained

nucleus. Granular, but

lightly stained, cytoplasm

Macrophage/Monocyte

Large cell with a free shape.

Darkly stained nucleus and a

very large cytoplasm

Lymphocyte

Small cell with little to no

cytoplasm. A single very

darkly stained nucleus

Eosinophil

Multi-lobed nucleus which

often assumes a donut or

figure 8 appearance.

Granular cytoplasm which

stains pink

Table 2.3: Morphology of Cells Counted in BAL Fluid. (Adapted from Blumenreich, 1990)

2.6.5 Measurement of Plasma IgE

The level of plasma IgE was used as a marker of allergic sensitisation and measured using Enzyme

Linked Immunosorbant Assay (ELISA). This technique was also used in order to ascertain that the IgE

knockout mice were deficient of IgE.

2.6.5.1 Total IgE

Total IgE levels were measured using BD OptEIATM set for mouse immunoglobulin E (BD Biosciences,

Oxford, UK) in accordance with the manufacturer’s instructions. Briefly,96 well plates (Thermo

Scientific, Rockford, USA) were coated with 100 µl of specific capture antibody diluted to working

concentration in coating buffer (0.1 M Sodium Carbonate, pH 9.5) and incubated at 4 °C overnight.

Wells were washed three times with wash buffer (0.05% Tween-20 in PBS) in order to remove any

unbound antibody. Wells were then incubated for 1 hour at room temperature with assay diluent

(10% FBS in PBS) in order to block non-specific binding. Following this, the wells were washed three

times with wash buffer; and 100 µl of the standards (serial dilutions in assay diluent) and plasma

69

samples (diluted 1:10 with assay diluent) were added and incubated for 2 hours at room temperature.

Assay diluent was used as a negative control. The plate was then washed five times with wash buffer

before 100 µl of substrate solution was added (1:1 mixture of H2O2 and tetramethylbenzidine; R&D

Systems). The plate was incubated in the dark at room temperature for 15-30 minutes. If the cytokine

of interest was present then the reaction with the substrate solution produced a blue colour. The

reaction was stopped with the addition of 50 µl of 2 N H2SO4 when the colour change was seen in the

standards. The plate was read at wavelengths of 450 nm and 570 nm using a spectrophotometer

(BiotekPowerWave XS Plate Reader). The absorbance was calculated by taking the absorbance at 570

nm away from the absorbance at 450 nm. The standard curve was used to measure the amount of IgE

in each sample minus the negative control values. The upper limit of detection was 100 ng/ml and

lower limit of detection was 1.6 ng/ml.

2.6.5.2 Allergen Specific IgE

OVA specific IgE was also measured using ELISA. This was an adapted protocol from BD Biosciences.

Briefly, OVA (20 µg/ml in PBS) was added to 96 well plates (Thermo Scientific, Rockford, USA) at 50

µl/well and incubated overnight at 4 °C. Plates were washed three times with wash buffer (0.05%

Tween-20 in PBS) and 50 µl of the plasma samples were added neat. Plasma from IgE deficient mice

was used as a negative control. The samples were incubated overnight at room temperature and then

washed three times as previous. Biotinylatedanti-IgE (2 µg/ml, 50 µl/well, BD Biosciences) was added

and incubated for 1 hour shaking at room temperature. The plates were washed three times with

wash buffer before 100 µl of substrate solution was added (1:1 mixture of H2O2 and

tetramethylbenzidine; R&D Systems). This was incubated for 15-30 minutes as done for the total IgE

assay before the reaction was stopped using 2 N H2SO4. The absorbance was read at 450 nm using a

spectrophotometer (BiotekPowerWave XS Plate Reader). Unlike for the Total IgE assays, there was no

commercially available quantitative OVA-specific IgE assay available. Due to this, the absorbances

were simply used for qualitative assessment of the samples.

70

2.7 Isolated Vagus Nerve Preparation

2.7.1 Nerve Dissection

Male Brown Norway rats (200 g-250 g; Charles River Laboratories UK) were euthanized via overdose

with Sodium Pentobarbitone (200 mg/kg i.p.). Skin was removed via midline incision and the trachea

and thorax exposed. The ribcage, connective tissue and smooth muscle was removed to observe the

vagal nerves running from the base of the skull, parallel to the trachea, to the heart. The vagal nerves,

caudal to the nodose ganglia, were removed and placed in Krebs-Henseleit (KH) solution (mM: NaCl

118; KCl 5.9; MgSO4 1.2; CaCl2 2.5; NaH2PO4 1.2; NaHCO3 25.5 and glucose 5.6; pH 7.4) and bubbled

with 95% O2/5% CO2.

Human lung samples were received from various locations in the US via the International Institute for

the Advancement of Medicine (IIAM: NJ, US). The tissue received was donor tissue unsuitable for

transplant, and was approved by local Human Tissue Ethics Committee. Whole lungs were received in

isotonic saline on ice, and within 48 hrs of cross-clamp. On receipt, the whole lungs were placed in KH

solution and bubbled with 95%O2/5% CO2. Vagal nerves running parallel to the trachea and bronchi

were dissected free of the lungs and placed in oxygenated KH solution.

Following procurement of the tissue, the vagal nerve was desheathed and cut into 10-15 mm sections

before being placed back into oxygenated KH solution where it remained for the duration of the

experiments. Throughout, care was taken not to stretch or damage the nerve in anyway.

2.7.2 Measurement of Depolarisation

The desheathed nerve was mounted in a “grease gap” recording chamber by drawing it longitudinally

through a narrow channel (measuring 2 mm diameter by 10 mm length) in a custom made Perspex

block (See figure 2.9). Petroleum jelly was injected through a side arm into the centre of the channel,

covering the centre of the nerve. This was done to create an area of high chemical and electrical

resistance such that the passage of ions and solutions was prevented between the two ends of the

nerve trunk. One end of the nerve trunk was superfused with KH solution at a rate of 2 ml/min in order

to allow the delivery of test compounds. The other end was perfused with just KH solution. The KH

solution applied was constantly bubbled with 95%O2/5% CO2 and maintained at 37 °C via the use of a

water bath and thermal jacket. Hollow glass rods (1.5 mm diameter) were inserted into Ag/AgCl pellet

half-cell electrodes (Mere 2 Flexible reference electrodes, World Precision Instruments) and the air

spaces filled with KH solution. The electrodes were positioned at each end of the nerve trunk such

that the glass rods made contact with the nerve. Potential difference was recorded via a DAM50

differential amplifier unit (World Precision Instruments) which amplified the DC potential signal (X10

71

gain, high filter 1 KHz, sample rate 5 Hz) which was then recorded on a chart recorder (LectroMed 2,

Lectromed, UK [now Digitimer, UK]).

This system indirectly measures compound depolarisation of the membranes of all nerve fibres

contained within the vagus nerve. It does not measure action potentials or firing of the nerve. This is

done by recording the difference in ionic potential between the two ends of the nerve. In the end of

the nerve that is perfused with KH solution containing test compounds, once a sufficient concentration

of activating ligand to achieve depolarisation is achieved, there is an influx of Na+ ions from the KH

solution into the axon cytosol (from high Na+ to low Na+). This has the net effect of depolarisation (see

figure 2.9). The now decreased concentration of positive ions in the KH solution near to the electrode

leaves the Cl- ions in a ‘freer’ state to react with the electrode pellet. The difference between the

increased potential on the electrode at the perfused end and the unaltered potential at the reference

end of the nerve trunk is amplified and recorded via the amplifier unit and chart recorder.

For this technique it should be noted that the depolarisation measured may not be of sufficient

magnitude to generate an action potential and so the depolarisation measured is not necessarily equal

the firing of a nerve fibre. Another point to make is that the vagus nerve supplies innervating fibres,

not just to the airways, but also to other organs such as the heart and gut. It should also be considered

that the cell bodies and nerve termini are not present in the isolated vagus trunk. This means that

there is the possibility of differences in terms of receptors and associated signalling proteins between

the nerve axon and the nerve terminals. However, it should also be noted that there is a good

correlation between compounds eliciting an effect using this technique and in in vivo studies (Birrell

et al., 2009a; Grace et al., 2012b; Freund-Michel et al., 2010). This system is also amenable to

pharmacology, meaning that many compounds can be tested on tissue from the same animal, thus,

reducing the overall numbers of animals used.

72

Figure 2.9: Apparatus setup for isolated vagus nerve depolarisation recordings. Vagus nerve was

drawn into a channel in a Perspex chamber and the two ends of the nerve trunk isolated chemically

and electrically with petroleum jelly. KH solution was constantly perfused onto the left side of the

nerve where test compounds could be applied at a rate of 2 ml/min. The right side of the nerve was

bathed in just KH solution. Ag/AgCl half-cell electrodes were placed in contact with both sides of

the vagus nerve. The signal from the electrodes was passed through an amplifier and the signal

output documented on a chart recorder.

73

2.7.3 Agonist/Antagonist Experimental Protocols

The following protocol was used to measure depolarisation (stimulation) and inhibition of stimulation

of the vagus nerve. There were several variations in the incubation timings and concentrations of

drugs, details of which can be found in the relevant results chapters.

Initially, 2-3 control stimulations were performed in order to attain two depolarisations of consistent

magnitude. This involved incubating with stimulation drug for 2 minutes, before washing with KH

solution in order to return to baseline depolarisation, and repeating. Following this, the nerve was

incubated with drug for 10 minutes, before reapplying the stimulating agent in the presence of drug

for 2 minutes. The nerve was then washed with KH solution for 10 minutes until baseline

depolarisation was achieved. A final application of stimulating agent was applied to the nerve for 2

minutes in order to ascertain that the nerve was still viable. A single concentration of drug was tested

on any single piece of nerve from 1 individual animal or patient (See figure 2.10).

A B C D

Figure 2.10: Example trace showing protocol for testing inhibitory effect of drugs in the vagus nerve preparation.

(A) Two control stimulations were evoked to achieve 2 stimulations of consistent magnitude. (B) The nerve was

incubated with drug for 10 minutes before (C) applying the stimulating agent in the presence of the drug. (D)

After washing to re-establish baseline, a final application of stimulating agent was applied in order to confirm

nerve viability.

74

2.7.4 Data Recording and Analysis

The chart recorder was calibrated such that a scale of 1 cm: 0.1 mV was set. The magnitude of

depolarisation was calculated by taking a measurement from the baseline (prior to application of

stimulating agent) to the peak of the response evoked by the stimulating agent. In order to calculate

inhibition of a stimulus by a drug, the average magnitude of 2 consistent control stimulations was

taken. The magnitude of depolarisation in the presence of the inhibitory drug was then compared to

the control stimulations in order to give a % inhibition as per the following equation:

% 𝐼𝑛ℎ𝑖𝑏𝑖𝑡𝑖𝑜𝑛 = 100 − (𝑅𝑒𝑠𝑝𝑜𝑛𝑠𝑒 𝑖𝑛 𝑝𝑟𝑒𝑠𝑒𝑛𝑐𝑒 𝑜𝑓 𝑣𝑒ℎ𝑖𝑐𝑙𝑒 𝑜𝑟 𝑑𝑟𝑢𝑔

𝑀𝑒𝑎𝑛 𝑜𝑓 𝑖𝑛𝑖𝑡𝑖𝑎𝑙 𝑐𝑜𝑛𝑡𝑟𝑜𝑙 𝑟𝑒𝑠𝑝𝑜𝑛𝑠𝑒𝑠 × 100)

75

2.8 In Vivo Single Afferent Fibre Recording

2.8.1 Surgery

Male Dunkin Hartley guinea-pigs (400 g -650 g; Harlan UK) were anaesthetised with Urethane (1.5 g/kg

i.p.) and supplemented with additional Urethane as required. Body temperature was monitored via

the use of a rectal probe and maintained (37 °C) using a heated blanket and control unit (Harvard

Apparatus, UK). A midline incision was performed to expose the trachea. The trachea was cannulated

and the animal artificially ventilated (tidal volume 10 ml/kg; 50-60 breaths per minute) using a small

animal ventilator (Ugo Basile, Italy). The right carotid artery was cannulated so that systemic arterial

blood pressure and heart rate could be continuously measured. This was done using a pressure

transducer (either Gould P2X3L or MEMSCAP SP 844), linked to the Spike 2 software data acquisition

system via a CED Micro1401 interface. Tracheal pressure was also similarly monitored using an air

pressure transducer (either SenSymb647 or MEMSCAP SP 844) attached to a side arm of the tracheal

cannula. The right jugular vein was cannulated in order to allow the delivery of Vecuronium Bromide

which was delivered initially at 0.1 mg/kg and then supplemented every 20 minutes at 0.05 mg/kg.

This was used in order to paralyse the animal such that spontaneous muscle spasm didn’t interfere

with the single afferent fibre recordings. Due to this application, the depth of anaesthesia was

assessed throughout the experiment by monitoring heart rate and blood pressure. The cervical vagus

nerves were dissected free and cut at the central end. The skin and muscle around the initial incision

were sutured to a metal ring to form a well which was filled with light mineral oil. The left vagus nerve

was desheathed and the nerve fibre placed on the recording electrode, the sheath placed on the

reference electrode. The nerve was teased under a binocular microscope until recordings from a single

nerve fibre type could be recorded.

2.8.2 Recording Equipment

Bipolar Teflon-coated platinum electrodes were used for recording purposes. The electrodes were

connected to a pre-amplifier NL100K head stage (Digitimer, UK) to record action potentials. The signal

was amplified using a NL104 amplifier (Digitimer, UK) and then filtered using a NL125 filter module

(Digitimer, UK). The signal was then passed through a Humbug noise reducer (Automate Scientific, US)

and then input into the Micro1401 interface and the Spike 2 software package. The software allows

pulse train counting over selected time periods. Signals were also monitored on a Tektronix DP02012

digital storage Oscilloscope (BidSync, US) and fed through an audio amplifier to a loud speaker (See

figure 2.11).

76

Figure 2.11: Apparatus setup for in vivo single afferent recording. Cervical vagus nerves were

dissected free and cut at the central end. The left vagus nerve was desheathed and teased until

recordings from a single fibre type could be observed. Action potentials were recorded via platinum

recording electrodes, a digital oscilloscope and output into the Spike 2 software package. Tracheal

pressure was also recorded in this manner.

2.8.3 Nerve Fibre Identification

Single nerve fibres can be identified as belonging to one of three major groups of airway sensory nerve

endings: Slowly Adapting Stretch Receptors (SARs), irritant receptors (rapidly adapting stretch

receptors, RARs, Aδ-fibres) and pulmonary/bronchial C-fibre receptors. C-fibres were identified using

several criteria outlined in Adcock et al (2003). They were defined as having no obvious pattern of

spontaneous discharge, non-responding to hyperinflation/hyperdeflation and responding to capsaicin

aerosol. At the end of the experiment, the fibre type was verified by assessing the conduction velocity

in order to make sure a slow conducting non-myelinated C-fibre had been attained as opposed to a

fast conducting myelinated Aδ-fibre. This was done by stimulating the nerve using bipolar silver

electrodes and stimulator (Grass Products-Natus Neurology Inc., US) set to a suprathreshold voltage

of 0.5 ms, 1 Hz. The time from stimulus to response and the distance between the stimulating and

recording electrode was measured. A C-fibre was defined as having action potential conduction

velocity of under 1m.s-1as opposed to the faster conducting Aδ-fibres, ranging from 2.0 m.s-1to 23 m.s-

1 (Adcock et al., 2003; Fox et al., 1993b; Riccio, Myers & Undem, 1996; Mazzone, 2004).

Figure 2.5. Diagram of apparatus for single fibre nerve recordings

Top: Anaesthetised guinea pigs were carefully dissected to reveal the

vagus nerves, which were both cut posterior to the vagal ganglia. The left

vagus nerve was teased, and a single nerve was placed onto a platinum

recording electrode, connected to a digital oscilloscope, outputted to

spike2 software on a computer to record nerve action potentials. Animals

were ventilated and tracheal pressure was monitored via an intra-tracheal

cannula. Nebulised drugs were delivered via a side-arm of the tracheal

cannula. Bottom: representative traces showing effect of capsaicin on

tracheal pressure and action potentials

nebulised delivery

ventilation

platinum electrode

tracheal pressure (PT)

urethane NMBs – vecuronium bromide

vagus nerve fibre

Capsaicin

5 s

amplifier)

1 min

Caps (100 µM) 15 s

Tra

ch

eal

Pre

ssu

re

(cm

H2O

)

Ac

tio

n

Po

ten

tia

ls

(mV

)

77

2.8.4 Recording Protocol

Following identification of a suitable C-fibre, a 2 minute baseline recording took place. Following this,

capsaicin (100 µM) was administered by aerosol for 15 seconds using an Aerogen Nebuliser (Buxco,

US) which was connected by a side arm in line with the ventilator and tracheal cannula for delivery

into the airways. Changes in nerve fibre firing, intratracheal pressure and blood pressure were

recorded until baseline levels were re-established. Two reproducible responses to acrolein aerosol (10

mM, 1.0 min) were established and after a suitable interval, either Disodium Cromoglycate (DSCG; 10

mg/kg) or Vehicle was administered i.t. and incubated for 30 minutes. Following this, a response to

acrolein was attained before a final capsaicin response took place. Guinea pigs were euthanized by an

overdose of pentobarbitone (i.p.) at the end of the experiment.

2.8.5 Data Recording and Analysis

Action potentials and tracheal pressure were recorded using the Spike 2 software package. The data

from this software was output into Microsoft Excel (Microsoft, US) and GraphPad Prism 5 (Graphpad

Software Inc., USA). Data was recorded and expressed as number of peak impulses per second

(peak.impulses.S-1).

2.9 Statistical Analysis

All data was analysed using GraphPad Prism 5. Data was expressed as mean ± s.e.m. Multiple

measurements were analysed by one-way ANOVA, non-parametric Kruskal-Wallis test and post-

hoc comparisons were performed by Dunn’s multiple comparison test, comparing selected

columns to a control. Additionally, a non-parametric Mann Whitney test, with two-tailed p values,

was carried out to determine statistical significance between two groups. Differences were

considered statistically significant if p<0.05. The figures have been graphically presented on a

range-specific axis.

78

Chapter 3

Optimisation of Animal Models of the

Late Asthmatic Response

79

3 Optimisation of animal models of the Late Asthmatic Response

3.1 Rationale

The LAR is a key symptom of allergic asthma with susceptible patients typically experiencing this form

of bronchoconstriction 3-8 hours following allergen exposure. Glucocorticoids are effective clinically

in significantly inhibiting this feature of asthma, although the driving mechanisms behind the LAR

remain unclear (Murai et al., 2005; Kidney et al., 1997; Miyagawa et al., 2009; Inman et al., 2001; Leigh

et al., 2002).

Pre-clinical models closely mimicking the cardinal features of asthma are useful in understanding the

mechanisms driving the disease. Within the Respiratory Pharmacology group, the LAR has been

modelled successfully in Brown Norway rats and C57BL/6 mice (Raemdonck et al 2012). The overall

aim of this thesis is to explore the mechanisms driving the LAR, therefore, it was necessary to utilise

animal models of LAR for this purpose. The Brown Norway rat model of LAR has been used traditionally

to explore this feature of asthma(Stevenson & Belvisi, 2008). It was also considered advantageous to

also use the mouse model of LAR as this would open up the possibility of applying GM mice to the

model. This would enhance the toolbox for exploration of the LAR as various knockout mice could be

applied to this model in order to ascertain the importance of specific cells and mediators in an

observable functional response.

Preceding these investigations it was considered important to assess the clinical relevance of these

models. Therefore, the aim of this chapter was to characterise the OVA driven rat and mouse models

using the clinically relevant glucocorticoid steroid Budesonide. The results of this investigation would

determine which models were taken forward for further understanding the driving mechanisms

behind the LAR.

80

3.2 Methods

The following methods were carried out as detailed in chapter 2. Details of individual experiments are

described below.

3.2.1 Assessment of Lung Function

Due to the uncertainty of the role of inflammation within the LAR (see section 3.4), it was decided that

when investigating the driving mechanisms behind the LAR within this thesis, the functional response

of the LAR was measured as opposed to an inflammatory response. The LAR was measured via the use

of whole body plethysmography (WBP; Penh) as described in chapter 2. It was not possible to use

invasive resistance measurements as anaesthesia has been shown to inhibit the response(Raemdonck

et al., 2012). Due to this, a conscious modelling system was required. It was considered unethical and

Home Office restrictions would not allow for the use of restraint for the prolonged periods of time

required to observe the LAR. There are currently no alternative methods for measuring lung function

in a conscious, unrestrained model. Therefore, Penh was used as an arbitrary measure of airway

constriction in order to assess lung function in this chapter and throughout this thesis.


Male Brown Norway rats (200-225g) were sensitised and challenged as detailed in general methods

section 2.4.2. On the day of allergen challenge, rats were dosed orally with Budesonide (0.03, 0.3, 3

and 30 mg/kg at 2 ml/kg) or vehicle (0.5% methylcellulose, 0.2% Tween80 in water) 60 minutes before

allergen challenge and 30 minutes following the end of allergen challenge (See figure 3.1). Two

Budesonide doses were administered pre and post antigen stimulant as per standard practice in order

to ensure adequate coverage for the duration of the LAR measurement period. Penh was recorded

from 30 minutes after the final drug administration for a period of 5 hrs as detailed in general methods

section 2.5.3.

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Figure 3.1: Timeline demonstrating protocol for drug administration in OVA driven rat model of the

LAR


Male C57BL/6 mice (20-25g) were sensitised and challenged as detailed in general methods 2.4.1. On

the day of allergen challenge, mice were dosed orally with Budesonide (3 mg/kg at 1 ml/kg) or vehicle

(0.5% methylcellulose, 0.2% Tween80 in water) 60 minutes before allergen challenge. Immediately

following recovery from allergen challenge, mice were placed in WBP chambers and Penh recorded

for 12 hours as detailed in general methods section 2.5.2.

3.2.4 Statistical Analysis

Data were analysed using Graphpad Prism 5. Data was expressed as mean ±s.e.m. Non-parametric

Mann Whitney-U tests with two-tailed p-values were carried out in order to assess statistical

significance between 2 groups. Differences were considered statistically significant if p<0.05.

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3.3 Results

3.3.1 Characterisation of the rat OVA-driven LAR model using Budesonide

OVA challenge resulted in a marked LAR in OVA sensitised rats. This response peaked at 2-3 hours and

lasted for up to 6 hours after challenge. The saline sensitised rats did not display a response following

OVA challenge (figure 3.2).

Budesonide treatment in the OVA sensitised/OVA challenged rats resulted in a significant attenuation

of the LAR at doses of 0.3 mg/kg, 3 mg/kg and 30 mg/kg in terms of both Penh AUC and Penh Peak

responses when compared with vehicle treated rats. Budesonide treatment in the control saline

sensitised/OVA challenged rats did not have any impact on baseline Penh recordings when compared

with vehicle treated animals (figure 3.2).

3.3.2 Characterisation of the mouse OVA-driven LAR model using Budesonide

OVA challenge resulted in a marked LAR in OVA sensitised mice. This response peaked at 3 hours and

lasted for up to 8 hours after challenge. The saline sensitised mice did not display a response following

OVA challenge (figure 3.3).

A dose of 3 mg/kg of Budesonide was chosen for use in the mouse model which was based on an

effective dose in the rat model. Budesonide treatment resulted in a significantly decreased LAR in the

OVA sensitised/OVA challenged mice in terms of both Penh AUC and Penh Peak response when

compared with vehicle treated rats (figure 3.3). As no effects on the Penh baseline recordings were

found with respect to Budesonide administration in the rat model, it was not considered necessary to

include the Saline sensitised/OVA-challenged Budesonide treated group in this subsequent mouse

study. This is compliant with the 3R’s.

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Figure 3.2: Effect of Budesonide treatment in the rat OVA-driven LAR. 1 hour following allergen

challenge rats were placed in WBP chambers and Penh recorded for a further 5 hours. Data is

expressed as A) Penh average over the recording time period; B) Penh Area Under Curve; and C)

Penh average at peak response. Data expressed as mean ± s.e.m. n=12. Mann-Whitney U-test.

*p<0.05 Vehicle/Saline compared to Vehicle/Ovalbumin. #p<0.05 Budesonide (0.03 mg.kg-1, 0.3

mg.kg-1, 3 mg.kg-1 and 30 mg.kg-1)/Ovalbumin compared to Vehicle/Ovalbumin.

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Figure 3.3: The effect of Budesonide treatment in the mouse OVA-driven LAR. Immediately

following recovery from allergen challenge, mice were placed in WBP chambers and Penh recorded

for 12 hours. Data is expressed as A) Penh average over the recording time period; B) Penh Area

Under Curve; and C) Penh average at peak response. Data expressed as mean ± s.e.m. n=5-8. Mann-

Whitney U-test. *p<0.05 Vehicle/Saline compared to Vehicle/Ovalbumin. #p<0.05 Budesonide (3

mg.kg-1)/Ovalbumin compared to Vehicle/Ovalbumin.

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3.4 Discussion

The aim of this chapter was to characterise the rat and mouse OVA driven models of LAR in terms of

their time points and clinical relevance with respect to human LAR. The results of this chapter would

determine which animal models would be taken forward to further investigate the driving

mechanisms behind the LAR within this thesis.

In human asthmatic patients, the LAR typically occurs 3-8 hours following allergen exposure

(Raemdonck et al., 2012). Both the rat and mouse models exhibited in this chapter demonstrated

response time points similar to these. The rat model exhibited a response peaking at 2-3 hours, lasting

for up to 6 hours after challenge. The mouse model response peaked at 3 hours and returned to

baseline at 8 hours post challenge.

It could, however, be noted that the responses appear to begin earlier than those shown in clinic, with

both models beginning to respond at 1hour post allergen challenge. It was, therefore, desirable to

characterise these models to show that they were displaying a true LAR and not an EAR. The EAR takes

place up to 1 hour following allergen exposure and the mechanisms driving the response have been

well established by researchers. Allergen challenge induces the cross-linking of IgE-FcR1 complexes

on mast cells which in turn causes mast cell degranulation to release preformed mediators such as

histamine, leukotrienes and prostaglandins which act locally to result in bronchoconstriction

(EAR)(Barnes, 2008; Holgate, 2008a; Buc et al., 2009). The mechanisms driving the EAR are thought to

be different to that of the LAR as in clinic steroids have been shown to inhibit LAR but not the EAR in

asthmatics(Booij-Noord, Orie & De Vries, 1971; Cockcroft & Murdock, 1987; Kidney et al., 1997; Leigh

et al., 2002). Therefore, in order to ascertain if the bronchoconstriction seen in the animal models in

this chapter was reminiscent of an EAR or an LAR, the clinically relevant glucocorticoid steroid

Budesonide was employed in the models. Budesonide was successful in significantly attenuating the

responses in both models, thus demonstrating that both models were displaying bronchoconstriction

consistent with the LAR, not the EAR, and were clinically relevant. Glucocorticoid steroids are the

mainstay of treatment in asthma and treat the chronic inflammation associated with the disease

(Barnes, 1998). Their anti-inflammatory mechanism is thought to occur by modulating the

transcription of genes within inflammatory cells. Steroids bind to GRs in the cytoplasm in various cell

types which results in the dimerization and migration of the GR to the nucleus. Here, it binds to a GRE

– a location on the promoter region of steroid responsive genes- and causes either up regulation of

anti-inflammatory gene transcription or down regulation of pro-inflammatory gene transcription

(Barnes, Pedersen & Busse, 1998). Steroids have also been reported to interact directly with

transcription factors such as NF-κB(Ray & Prefontaine, 1994), and also recruit Histone Deacetylases

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(Kagoshima et al., 2001; Ito, Barnes & Adcock, 2000; Ito et al., 2006) to ultimately result in the

attenuation of cellular inflammation in the airways (see Introduction section 1.9).

The fact that steroids are anti-inflammatory and attenuate the LAR could mean that cellular

inflammation is the main driving factor behind this response. However, in the rat OVA driven model

of LAR it has previously been shown within the Respiratory Pharmacology group, that while a single

OVA challenge results in a robust LAR, a temporal link to cellular inflammation is not apparent within

this particular model. Studies have also indicated that depleting cellular inflammation does not always

attenuate the LAR. Indeed, targeting eosinophilia via the application of anti-IL-5 antibodies in animal

models and in the clinic has been shown to have minimal effect on the development of the LAR (Nag

et al., 2003; Molet et al., 1999; Leckie et al., 2000; Flood-Page et al., 2007; Kips et al., 2003).

Inflammatory responses also result in oedema which has been reported as important in the LAR.

However, Magnetic Resonance Imaging (MRI) studies have shown that no temporal link between

oedema following allergen challenge and the LAR exists. In these investigations in animal models,

allergen challenge resulted in an LAR at 3 hours post challenge, but oedema changes were not seen

until 6 hours after challenge and did not subside until 96 hours after challenge (Beckmann et al., 2001;

Tigani et al., 2007). These data suggest that oedema is not involved in the driving mechanisms behind

the LAR.

Overall, it seems that the LAR is not temporally linked to cellular inflammation, although low levels of

inflammatory cells such as eosinophils are present at the time of the response. Eosinophil depletion

does not attenuate the LAR. Due to this, it could be hypothesised that only small levels of cellular

inflammation are required to induce the LAR with other mechanisms driving the response taking over

later on. It has previously been shown by the Respiratory Pharmacology group that the LAR maybe

mediated by TRPA1 channels on airway sensory nerves (Raemdonck et al., 2012). Therefore, allergen

challenge could lead to the production of an endogenous TRPA1 ligand which stimulates receptors on

airway sensory nerves, leading to an LAR (see Introduction section 1.8). Glucocorticoid steroids such

as Budesonide are successful in attenuating the LAR. It has also been reported that an IKK-2 inhibitor

successfully blocked NF-κB translocation and inhibited the development of the LAR in vivo (Birrell et

al., 2005). This implies that the endogenous mediator(s) responsible for TRPA1 activation in the

development of the LAR are under transcriptional control. In the present models employed, the LAR

begins 1-2 hours after challenge, so synthesis of an endogenous TRPA1 ligand in the mechanisms

behind the LAR would need to occur quickly. In vitro, transcription factor activation can be detected

as soon as 1hour after stimulation (Birrell et al., 2005, 2008) and in vivo can be detected at 2 hours

following stimulation (Eltom et al., 2011; Rastrick et al., 2013) meaning that it is possible for mediators

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to be produced to stimulate airway sensory nerves within the LAR timescale. Factors surrounding this

hypothesis will be further investigated in the subsequent chapters of this thesis.

In summary, this chapter has introduced the Brown Norway rat and C57BL/6 mouse models of LAR

and demonstrated that they are suitably clinically relevant with respect to response time points and

inhibition by glucocorticoid steroid. As a result of this, it was decided to take both these models

forward for further investigating the driving mechanisms behind the LAR. The mouse model was

considered a particularly advantageous tool as GM mice could now be applied to the model.

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Chapter 4

Exploring the Role of Key Allergic

Effector Cells in the LAR

89

4 Exploring the role of key allergic effector cells in the LAR

4.1 Rationale

The aetiology of the LAR is widely reported to involve the infiltration of numerous inflammatory cells

and mediators (Aalbers et al., 1993a, 1993b). This thesis aims to investigate the driving mechanisms

behind the LAR. Therefore, it was considered desirable to explore the roles of key allergic effector cells

within this response.

The LAR has been linked to the B-cell-IgE-Mast cell products axis (Barnes, 2008; Holgate, 2008a).

Patients being treated with corticosteroids have been shown to have decreased numbers of mast cells

in the smooth muscle and epithelium (James et al., 2012). DSCG, a known mast cell stabiliser, has been

shown to inhibit the LAR in asthmatic patients and in animal models of the LAR (Cockcroft & Murdock,

1987; Pepys et al., 1968, 1974; Booij-Noord, Orie & De Vries, 1971). The IgE antibody, Omalizumab,

has also been shown to suppress allergen induced late phase bronchoconstriction (Busse et al., 2001;

Holgate et al., 2004, 2005).

Evidence for IgE independent mechanisms driving the LAR also exist. Clinical studies have shown that

sensitisation to allergens and the accompanying allergen specific IgE is not always predictive of an LAR

(Hatzivlassiou et al., 2010).Studies involving the inhalation of T-cell peptides derived from the feline

allergen Fel d 1demonstrated the induction of an isolated LAR without an accompanying EAR, implying

the LAR to be T-cell dependent and IgE independent (Ali et al., 2004a). Other studies investigating the

effects of Cyclosporin A (a T-helper cell inhibitor) on asthma specific endpoints has pointed to a role

for T-cells within the LAR (Khan et al., 2000).

In Chapter 3, the mouse and rat OVA-driven models of the LAR were demonstrated to be clinically

relevant with respect to the response time points and inhibition of the response with the

corticosteroid Budesonide. Investigations utilising monoclonal antibodies targeting key effector cells

in animal models of the LAR have yielded conflicting results. Issues concerning the specificity of certain

antibodies have also been raised. For example, the antibody MAR-1, targeting the high affinity

receptor FcεRI has not only been shown to inhibit mast cell driven processes, but has been shown to

activate mast cells(Kojima et al., 2007; Ohnmacht et al., 2010; Voehringer, 2013). This uncertainty is

less than desirable when trying to delineate the mechanisms behind a response. Therefore, it was

decided to employ GM mice to investigate the key allergic effector cells driving the LAR which has not

yet been reported on within the associated literature.

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The first aim of this chapter was to validate and confirm the phenotype of CD4-/-, CD8-/-, B-cell-/- and

mast-cell-/- mice. The second aim was to apply these mice to the OVA-driven mouse model of the LAR

to determine the role of these cell types within the response.

4.2 Methods


described below.

4.2.1 Validation of Genetically Modified Mice by Flow Cytometry

Wildtype (C57BL/6) and GM (B-cell-/- , CD4-/- , CD8-/- and Mast-cell-/-) naive male mice (20-25g) were

culled, their systemic circulations perfused and their lung tissue isolated which underwent standard

enzymatic digest in order to obtain a leukocyte population. Leukocytes were counted, stained and

underwent flow Cytometry as described in general methods section 2.3.3. Lymphocytes were gated

for based on their forward/side scatter profile and then assessed in terms of their characteristic

expression of surface markers to identify B-cells, CD4+ cells and CD8+ cells. This was to ascertain that

the relevant knockouts were deficient of their corresponding cell type and to determine that there

was no compensation in terms of these cell populations for knocking out a particular cell type.

The lung tissue leukocyte populations were also assessed for eosinophils, neutrophils,

monocytes/tissue macrophages and alveolar macrophages. This was also done using their

characteristic forward/side-scatter profiles and surface expression profiles as detailed in Snelgrove et

al. 2008 and in general methods section 2.3.3. This was done to validate that each genetic modification

had no downstream effects such as the up regulation or down regulation of other inflammatory cell

populations.

4.2.2 Validation of Genetically Modified Mice by Histology

Wildtype (C57BL/6) and GM (B-cell-/- , CD4-/- , CD8-/- and Mast-cell-/-) naive male mice (20-25g) were

assessed for the presence of mast cells within the lung tissue in order to confirm that the mast cell-/-

mice were deficient of mast cells, and to verify that there were no effects in terms of the mast cell

population in the other GM strains. Briefly, mice were culled, their systemic circulations perfused and

their lung tissue isolated. Lung tissue was fixed and stained using a standard Toluidine Blue stain. The

tissue slices were mounted on slides and assessed using light microscopy by a blinded observer as

detailed in general methods section 2.3.4.


Following phenotypic validation it was decided that the Wildtype and GM (B-cell-/- , CD4-/- , CD8-/-,

Mast-cell-/- and IgE-/-) could be applied to the OVA-driven mouse model of LAR. Male mice (20-25g)

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were sensitised, challenged and Penh recorded for 12 hours as described in general methods sections

2.4.1 and 2.5.2.

4.2.4 Plasma IgE Analysis

Following Penh recordings, mice were culled via overdose with Sodium Pentobarbitone (200 mg/kg

i.p.) and a heparinised blood sample obtained via cardiac puncture. The samples were processed as

detailed in general methods section 2.6.1 and the plasma assessed for Total and OVA-specific IgE via

ELISA (see general methods section 2.6.5).

4.2.5 Further Exploration of the Enhanced Baseline Response

After observing such a prominent increase in Penh in the saline sensitised mast cell-/- and IgE-/- strains,

it was considered necessary to further investigate these GM strains with respect to various aspects of

the experimental protocol.

The first aspect considered was the effect of the anaesthetic (isoflurane) on these mice. Naïve male

wildtype (C57BL/6), mast cell-/- and IgE-/-mice (20-25g) were either placed directly into, or anesthetised

using isoflurane and allowed to recover as previously described in general methods section 2.4.1

before being placed into WBP chambers and Penh recorded for 12 hours.

The second part of the protocol investigated with respect to these mice was the intratracheal

challenge method to assess if the responses observed were an innate response to the presence of

OVA or a result of the intratracheal dosing itself. Naïve male wildtype (C57BL/6), mast cell-/-and IgE-/-

mice (20-25g) were anaesthetised, dosed i.t. with either saline or OVA in saline and allowed to recover

as described previously (general methods section 2.4.1). They were then placed in WBP chambers and

Penh recorded for 12 hours.


Data were analysed using Graphpad Prism 5. Data was expressed as mean ±s.e.m. Multiple

measurements were analysed by one-way ANOVA, non-parametric Kruskal-Wallis test and post-hoc

comparisons were performed by Dunn’s multiple comparison test, comparing selected columns to a

control. Additionally, a non-parametric Mann Whitney test, with two-tailed p values, was carried out

to determine statistical significance between two groups. Differences were considered statistically

significant if p<0.05.

With respect to the observations of Penh within the mouse model of LAR, non-parametric Mann

Whitney test was used to assess observations between specific groups. The group comparison

wildtype (Saline) vs. wildtype (OVA) was of interest as this confirmed a positive response to validate

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that the sensitisation and challenge protocols were effective within the experiments. Comparisons

were made between the Wildtype (Saline) and KO (Saline) groups in order to confirm that the genetic

modification conferred no effect on the baseline response (negative control). The group comparison

wildtype (OVA) vs. KO (OVA) was the comparison of interest in order to assess the question of whether

the cells of interest were key within the LAR response. The groups KO (Saline) and KO (OVA) were not

compared as this was of little interest in addressing the main research question. The main area of

interest was the effect of the KO in the OVA response when compared with wildtype. The other group

comparisons served as positive and negative controls. If it had been decided to compare the groups

KO (Saline) vs. KO (OVA) then multiple measurements would have been analysed using one-way

ANOVA, non-parametric Kruskal-Wallis test and post-hoc comparisons performed using Dunn’s

multiple comparison test.

4.3 Results

4.3.1 Validation of Genetically Modified Mice

The CD4-/-, CD8-/- and B-cell-/- GM mice were all confirmed to be deficient of their specific knockout

cell via flow cytometry (See figure 4.1). Negligible levels of CD4+ cells were found in the CD4 knockout

mice compared with wildtype mice (CD4 KO 0.47 ± 0.38 cells/mg tissue vs. wildtype 427.3 ± 61.62

cells/mg tissue). Levels of CD8+ T-cells were also found to be negligible in CD8 knockout mice

compared with wildtype mice (CD8 KO 0.4 ± 0.3 cells/mg tissue vs. wildtype 266.5 ± 39.22 cells/mg

tissue), as were levels of CD19+ cells in B-cell knockout mice compared with wildtype mice (B-cell KO

8.11 ± 0.93 cells/mg tissue vs. wildtype 905.2 ± 145.9 cells/mg tissue). Lung tissue populations of CD4+,

CD8+ and CD19+ cells in the knockout mice were found to be comparable to those seen in wildtype

mice where the cell type was not the deficient population (see figure 4.1).

The mast cell knockout mice were confirmed to be deficient of lung mast cells using the standard

histological stain Toluidine Blue on lung tissue slices. No significant differences with respect to the

mast cell population in the other GM strains were found compared with the wildtype group (see figure

4.2).

Baseline levels of eosinophils, neutrophils, alveolar macrophages and monocytes/tissue macrophages

were assessed for by flow cytometry in wildtype mice compared with the various transgenic mice. No

significant differences in these cell types were found across the GM strains compared with the

wildtype group (see figure 4.3).

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Following confirmation that the GM mice were deficient of the relevant effector cells and their

inflammatory baseline leukocyte levels were comparable to those seen in the wildtype group, they

were applied to the OVA driven mouse model of the LAR.

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Figure 4.1: Validation of Lymphocyte Populations in Genetically Modified Mice by Flow Cytometry.

Leukocytes from the lung tissue of Wildtype (C57BL/6), B-cell-/- , CD4-/- , CD8-/- and Mast-cell-/-

naïve male mice were attained via enzymatic digest. Flow Cytometry was used to assess the

numbers of: A) CD4+ cells; B) CD8+ cells and C) CD19+ cells. Data is expressed as mean cell number

per mg of lung tissue ± s.e.m. n=6-8. One-way ANOVA, Kruskal-Wallis test with post-hoc

comparisons using Dunn’s multiple comparison test. *p<0.05 compared to Wildtype control group.

Figure 4.2: Validation of Mast Cell Populations in Genetically Modified Mice by Histology. Lung

tissue slices were obtained from Wildtype C57BL/6, B-cell-/- , CD4-/- , CD8-/- and Mast-cell-/- naïve male

mice. They were stained utilising a standard Toluidine Blue histological stain and numbers of mast

cells per slide were counted (x40 magnification) by a blinded observer. Data is expressed as mean

cell number per slide ±s.e.m. n=6-8.One-way ANOVA, Kruskall-Wallis test with post-hoc

comparisons using Dunn’s multiple comparison test. #p<0.05 compared to Wildtype control group.

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Figure 4.3: Validation of Additional Leukocyte Populations in Genetically Modified Mice by Flow

Cytometry. Leukocytes from the lung tissue of Wildtype (C57BL/6), B-cell-/- , CD4-/- , CD8-/- and Mast-

cell-/- naïve male mice were attained via enzymatic digest. Flow Cytometry was used to assess the

numbers of: A) Eosinophils; B) Neutrophils; C) Alveolar Macrophages and Monocytes/Tissue

Macrophages. Data is expressed as mean cell number per mg of lung tissue ± s.e.m. n=6-8. One-way

ANOVA, Kruskall-Wallis test with post-hoc comparisons using Dunn’s multiple comparison test.

*p<0.05 compared to Wildtype control group.

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4.3.2 The Role of CD4+ Cells in the OVA Driven Mouse Model of LAR

The results of this model showed that OVA challenge resulted in a marked LAR in wildtype OVA

sensitised mice which peaked at 3 hours and lasted for up to 8 hours after challenge, compared to

saline sensitised wildtype mice. The saline sensitised CD4-/- mice displayed a baseline response

comparable to the saline sensitised wildtype group. A significant attenuation of response was

observed in the OVA sensitised CD4-/- mice compared to OVA sensitised wildtype group in terms of

both Penh AUC and Penh Peak response (figure 4.4).

Figure 4.4: The Role of CD4+ Cells in the OVA-driven Mouse Model of LAR. Immediately following

recovery from allergen challenge, mice were placed in WBP chambers and Penh recorded for 12

hours. Data is expressed as A) Penh average over the recording time period; B) Penh Area Under

Curve; and C) Penh average at peak response. Data expressed as mean ± s.e.m. n=13-21. Mann-

Whitney U-test. *p<0.05 Wildtype Saline sensitised compared to Wildtype Ovalbumin sensitised

groups. #p<0.05 CD4-/- Ovalbumin sensitised compared to Wildtype Ovalbumin sensitised groups.

No significant difference between groups is denoted ns.

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4.3.3 The Role of CD8+ Cells in the OVA Driven Mouse Model of LAR

OVA-sensitised wildtype controls displayed a distinct LAR compared with Saline sensitised wildtype

mice. Saline sensitised CD8-/- mice exhibited a baseline response comparable to Saline sensitised

wildtype mice following OVA challenge. CD8-/- OVA sensitised and challenged mice presented an

enhanced response compared to the wildtype OVA sensitised and challenged group. This difference

was significant with regards to Penh AUC and Penh Peak response (figure 4.5).

Figure 4.5: The Role of CD8+ Cells in the OVA-driven Mouse Model of LAR. Immediately following





groups. #p<0.05 CD8-/- Ovalbumin sensitised compared to Wildtype Ovalbumin sensitised groups.

No significant difference between groups is denoted ns.

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4.3.4 The Role of B-Cells in the OVA Driven Mouse Model of LAR

The wildtype OVA sensitised and challenged group again demonstrated an LAR peaking at 3 hours and

resolving at around 8 hours after challenge. Interestingly, the B-cell-/- saline sensitised controls

exhibited a significantly increased Penh AUC and Peak Penh compared with the equivalent saline

sensitised wildtype group. No significant differences were observed between the OVA-sensitised and

challenged B-cell-/- and wildtype groups (figure 4.6).

Figure 4.6: The Role of B-Cells in the OVA-driven Mouse Model of LAR. Immediately following





groups. #p<0.05 Wildtype Saline sensitised compared to B-cell-/-Saline sensitised groups. No

significant difference between groups is denoted ns.

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4.3.5 The Role of Mast Cells in the OVA Driven Mouse Model of LAR

The wildtype controls responded as previously described, with a distinct LAR in the OVA sensitised

group compared with the saline sensitised wildtype group as a result of OVA challenge. Similarly to

the B-cell-/- mice, the saline sensitised mast cell-/- mice had a significant enhancement in Penh AUC and

Peak Penh in response to OVA challenge compared to the saline sensitised wildtype mice. The OVA-

sensitised mast cell-/- group displayed a response to OVA challenge which was not significantly

different to that seen in the OVA sensitised wildtype group (figure 4.7).

Figure 4.7: The Role of Mast Cells in the OVA-driven Mouse Model of LAR. Immediately following





groups. #p<0.05 Wildtype Saline sensitised compared to Mast cell-/-Saline sensitised groups. No

significant difference between groups is denoted ns.

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4.3.6 The Role of IgE in the OVA Driven Mouse Model of LAR

An LAR was exhibited by the wildtype OVA sensitised group compared to the wildtype saline sensitised

group. Again, similarly to the B-cell-/- and mast cell-/- mice, the saline sensitised IgE-/- group exhibited a

significantly enhanced Penh AUC in response to OVA challenge compared to the saline sensitised

wildtype mice. No significant differences were observed between the OVA-sensitised and challenged

IgE-/- and wildtype groups (figure 4.8).

Figure 4.8: The Role of IgE in the OVA-driven Mouse Model of LAR. Immediately following recovery

from allergen challenge, mice were placed in WBP chambers and Penh recorded for 12 hours. Data

is expressed as A) Penh average over the recording time period; B) Penh Area Under Curve; and C)

Penh average at peak response. Data expressed as mean ± s.e.m. n=6-9. Mann-Whitney U-test.

*p<0.05 Wildtype Saline sensitised compared to Wildtype Ovalbumin sensitised groups. #p<0.05

Wildtype Saline sensitised compared to IgE-/-Saline sensitised groups. No significant difference

between groups is denoted ns.

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4.3.7 Plasma IgE Levels in the OVA Driven Mouse Model of LAR

After attaining Penh recordings, mice were culled and blood samples obtained which were assessed

for the presence of OVA-specific IgE. This was to confirm allergen specific allergic sensitisation in the

wildtype mice, to assess the effect of the various genetic modifications on plasma IgE levels in an

allergic environment and to observe IgE levels in relation to the functional LAR.

In all experiments assessing the various cell deficient mice in the LAR model, wildtype mice were

confirmed to have been allergically sensitised by a significant increase in plasma OVA-specific IgE in

the OVA sensitised groups compared with the saline sensitised groups. All saline sensitised GM strains

were confirmed to be deficient of plasma OVA-specific IgE. OVA sensitised CD4-/-and B-cell-/-mice had

significantly decreased plasma OVA-specific IgE compared with wildtype OVA-sensitised mice. No

significant differences were found between the OVA sensitised wildtype controls and corresponding

CD8-/- and mast cell-/-groups (figure 4.10).

In the IgE knockout mice, plasma total IgE levels and OVA-specific IgE levels were assessed in order to

validate that the mice were deficient of IgE. Negligible levels of total and OVA-specific IgE was

measured in the saline sensitised and OVA sensitised IgE-/- mice in comparison to the wildtype groups

(figure 4.9).

Figure 4.9: Plasma IgE Levels in IgE Deficient Mice. Following Penh recordings, blood samples were

attained via cardiac puncture and plasma samples assessed for A) Total IgE and B) OVA-Specific IgE

via ELISA. IgE knockout mice in comparison to wildtype groups were assessed. Data is expressed as

raw absorbance values taken at 450nm ±s.e.m. n=6-9.Mann-Whitney U-test. *p<0.05 Wildtype

Saline sensitised compared to Wildtype Ovalbumin sensitised groups. #p<0.05 IgE-/- Ovalbumin

sensitised compared to Wildtype Ovalbumin sensitised groups.

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Figure 4.10: Plasma OVA-Specific IgE Levels in Cell Deficient Mice. Following Penh recordings, blood

samples were attained via cardiac puncture and plasma samples assessed for OVA-Specific IgE via

ELISA. Genetically modified strains in comparison to wildtype groups assessed were: A) CD4-/-; B)

CD8-/-; C) B-cell-/- and D) Mast cell-/-. Data is expressed as raw absorbance values taken at 450nm

±s.e.m. n=6-11.Mann-Whitney U-test. *p<0.05 Wildtype Saline sensitised compared to Wildtype

Ovalbumin sensitised groups. #p<0.05 Knockout Ovalbumin sensitised compared to Wildtype

Ovalbumin sensitised groups. No significant difference between groups is denoted ns.

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4.3.8 Further Exploration of the Enhanced Baseline Response

No increases in Penh were observed in the naive OVA challenged wildtype mice compared with naive

saline challenged mice. The naive mast cell-/- and IgE-/- mice did not display an enhanced response

compared with wildtype controls as a result of OVA challenge (figure 4.11).

Figure 4.11: Further Exploration of the Enhanced Baseline Response. A) Naive Wildtype (C57BL/6)

mice were challenged with either Saline or OVA (25 µl; 2% OVA, i.t.) before being placed in WBP

chambers upon recovery and Penh recorded for 12 hours. B) Naive Wildtype (C57BL/6), Mast cell-/-

and IgE-/- mice were challenged with OVA (25 µl, 2% OVA, i.t.) before being placed into WBP

chambers and Penh recorded for 12 hours. Data is expressed as i.) Penh average over the recording

time period and ii.) Penh Area Under Curve. Data is expressed as mean ± s.e.m. n=4.

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4.4 Discussion

The overall aim of this chapter was to investigate the relevance of key allergic effector cells in the LAR

by applying GM mice to the OVA-driven mouse model of LAR. Before these investigations could take

place it was considered necessary to characterise the phenotype of the mice with respect to the cell

populations within the lung tissue by flow cytometry and histology.

All GM strains were found to be deficient of their relevant knockout cell type. For example Mast cell

knockout mice were found to be deficient of mast cells. No subsequent effects in terms of the CD4+,

CD8+, B-cell and Mast cell lung populations were observed in the non-relevant strains. For example

mast cell deficient mice had no significant up regulation or down regulation of lung tissue CD4+ cells,

CD8+ cells or B-cells. Baseline levels of eosinophils, neutrophils, alveolar macrophages and

monocytes/tissue macrophages were found to be comparable to those in wildtype mice across all GM

strains. It could be concluded that knocking out a particular cell type via genetic modification had no

resulting effects on other lung tissue cell populations. The results of these studies confirmed that the

GM mice were suitable for application to the OVA-driven mouse model of the LAR.

The GM strains were then applied to the OVA-driven mouse model of the LAR. The CD4-/- mice

displayed a much attenuated response compared with wildtype mice, suggesting a role for CD4+ cells

in the driving mechanisms behind the LAR.

Clinical studies have also explored the importance of CD4+ cells within the LAR. Researchers have

identified increased levels of CD4+ cells in asthmatic bronchial biopsies compared with healthy

controls (Mueller et al., 1996). Allergen challenge has been associated with less circulating CD4+ T-

lymphocytes in isolated early responders compared with dual responders (those displaying an EAR

and LAR) (Muratov et al., 2005). Inhibition of the LAR by Cyclosporin A in steroid dependant asthmatics

was linked with a decrease in the numbers of BAL CD4+ T-lymphocytes (Khan et al., 2000). Animal

models have also demonstrated the importance of CD4+ cells within the driving mechanisms behind

the LAR. Increased levels of CD4+ cells have been found in the BAL fluid of dogs with ragweed allergy

at 4 hours post allergen challenge (Out et al., 2002). Enhanced levels of CD4+ cells in murine models

of asthma have also been shown to correlate with the LAR (Nabe et al., 2005). Depletion and adoptive

transfer studies in rats and mice have also deduced a central role for CD4+ cells within this response

(Meyts et al., 2008; Nabe et al., 2011b; Ohtomo et al., 2009; Watanabe et al., 1995).

In this chapter, the CD8-/- mice displayed a significantly enhanced response compared to wildtype

mice, implicating a protective role for CD8+ cells within the LAR. The literature on the subject indicates

that these cells have also been explored with respect to the LAR in animal models by other

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researchers. Increased numbers of CD8+ T-cells have been found in the airway submucosa of sensitised

rats exposed to OVA and depletion of these cells using a monoclonal antibody has been shown to

enhance the LAR (Haczku et al., 1995a; Watanabe et al., 1995; Olivenstein et al., 1993a; Allakhverdi et

al., 2000). Adoptive transfer studies have shown that the application of sensitised CD8+ cells into rat

models of the LAR resulted in suppression of the response upon allergen re-exposure (Suzuki et al.,

1999). This LAR suppression has been shown to correlate with enhanced levels of the cytokine IFN-γ

in the BAL fluid and the administration of this cytokine to rat models can reduce the LAR (Isogai et al.,

2000, 2003). CD8+ T-cells are known producers of IFN-γ and so could elicit a protective effect in the

allergic airway via this cytokine (Kemeny et al., 1994; Li et al., 1995). Indeed, adoptive transfer studies

have shown that application of CD8+γδ T-cells into rat models of allergic asthma resulted in a

diminished LAR. Pre-treatment of CD8+γδ T-cells with an antisense oligonucleotide to inhibit IFN-γ

before transfer into sensitised recipients resulted in complete recovery of the LAR (Isogai et al., 2007).

Relationships between CD4+ cells and CD8+ cells with respect to the LAR have been explored by

researchers using animal models. As previously mentioned, CD8+ T-cells are known producers of IFN-

γ. It has been shown in numerous investigations that IFN-γ can suppress the proliferation of Th2 type

CD4+ T-cells and it has been shown to inhibit the infiltration of CD4+ cells into the airways in a mouse

allergic asthma model (Gajewski, Goldwasser & Fitch, 1988; Gajewski, Joyce & Fitch, 1989; Young &

Hardy, 1995; Nakamura et al., 1997; Iwamoto et al., 1993). Isogai and colleagues showed that

transferring CD4+ cells into recipient rats resulted in an LAR upon allergen exposure; and in the same

study showed the depletion of CD8+ cells using monoclonal antibodies resulted in an enhanced LAR

(Isogai et al., 2005).

Levels of CD4+ and CD8+ cells have been explored clinically in the asthmatic lung. Human subjects

experiencing an isolated EAR after undergoing allergen challenge have been shown to have relative

increases in CD8+ cells in the BAL fluid compared to patients experiencing a dual response who have

increased levels of CD4+ cells in their BAL fluid (Gonzalez et al., 1987). Studies assessing the effects of

intradermal challenge in humans showed that a higher proportion of CD4+ T-lymphocytes and a lower

proportion of CD8+ T-lymphocytes were accumulated in skin chamber fluids during the late phase

response (Werfel et al., 1995). Allergen inhalation challenge in atopic asthmatic subjects is associated

with decreased IFN-γ positive CD8+ T-lymphocytes in peripheral blood and sputum (Matsumoto et al.,

2002; Yoshida et al., 2005).

The studies undergone so far exhibit CD4+ T-cells as drivers of the LAR, with CD8+ T-cells providing an

inhibitory input. These studies have mainly involved the utilisation of monoclonal antibodies targeting

specific cell types, or adoptive transfer models. So far, the application of GM mice deficient in CD4+

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and CD8+ cell types when exploring the specific mechanisms behind the LAR is a missing determinant

in this field. This application is advantageous as there is the argument that the previous models

applying monoclonal antibodies to allergic asthma models may target other cell types non-specifically.

The results obtained in this chapter correspond with the findings of CD4+ T-cells being LAR drivers and

CD8+ T-cells having a protective role in this response and so further supports the existing literature on

the subject.

Plasma IgE levels were also measured in the mice from these experiments in order to observe this

parameter in relationship to the presence of an LAR. IgE-/- mice were confirmed to be deficient of total

and OVA-specific IgE as were all saline sensitised mice. OVA-sensitised Wildtype mice were confirmed

for allergic sensitisation by enhanced levels of plasma OVA-specific IgE which also corresponded with

the presence of an LAR. The CD4-/-mice had significantly decreased levels of plasma OVA-specific IgE

which mirrored the diminished LAR seen in this strain. The B-cell deficient mice had negligible levels

of Plasma allergen specific IgE, which one may expect due to the fact that B-cells are the IgE producing

cells (Barnes, 2008; Holgate, 2008a). The mast cell deficient plasma IgE levels were found to be

comparable to the corresponding wildtype groups, as were those seen in the CD8-/- mice. It is difficult

to interpret in the B-cell, mast cell and IgE deficient strains whether their plasma IgE levels

corresponded with an LAR due to the interesting responses exhibited by the non-sensitised controls

in these experiments. In the experiments where this wasn’t an issue, the CD4 deficient mice, which

exhibited a decreased LAR, were also found to have diminished levels of plasma OVA-specific IgE.

However, the CD8 deficient mice, which exhibited an enhanced LAR response, did not exhibit raised

levels of allergen specific IgE. Overall, further investigations into the relationship between IgE and the

LAR are warranted as from these results it is still unclear as to what this link is, if in fact there is one.

In this chapter the roles of B-cells, Mast cells and IgE in the driving mechanisms behind the LAR were

also explored. GM mice deficient in B-cells, Mast cells and IgE were applied to the mouse LAR model.

Upon application, OVA sensitised and challenged B-cell-/-, Mast cell-/-and IgE-/-mice displayed a

response which was comparable to that seen in wildtype mice, suggesting that these cell types and

mediators are not involved in the driving mechanisms behind the LAR. Interestingly, however, these

particular GM mouse strains also exhibited an enhanced baseline response even though they were

not sensitised to allergen, making these results difficult to draw conclusions from. When the mast cell

deficient mice were applied to the mouse model of the LAR, the negative control saline sensitised

mast cell-/- mice also displayed enhanced bronchoconstriction. Similar results were also obtained

when the IgE-/- and B-cell-/- mice were applied to the mouse OVA-driven LAR model, making the results

difficult to interpret. Upon further investigation into the nature of this finding in naive mice, it was

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established that the chamber environment, the anaesthesia (isofluorane), the intratracheal challenge

method nor the innate presence of OVA resulted in enhanced Penh. From this we can thus far

conclude that the responses seen in these particular strains are associated with the sensitisation

protocol and warrants further investigation. Thus far, we know that when Alum is administered

intraperitoneally, even in the absence of the allergen, a response was exhibited in response to

intratracheal instillation of OVA in the mast cell/IgE/B-cell deficient mice but not in wildtype mice. It

could therefore be considered that these cell types exert possible protective effects against inhalation

of non-specific stimuli. Indeed, OVA contains LPS and mast cells have been shown to be stimulated by

LPS via TLR4 to release anti-inflammatory cytokines, reactive oxygen species and antimicrobial

peptides (REF). The Alum adjuvant requirement for this response to occur is certainly interesting.

There is little evidence within the literature of Alum interactions with these cell types. It could be

postulated that Alum boosts the response to innate stimuli and this is usually downplayed by the mast

cell/IgE/B-cell axis. Further investigation into this area could provide conclusions relating to the

mystery surrounding the mechanism of action of Alum in boosting immune responses. Overall, for

the purposes of this investigation into the role of these cell types in the LAR, we can unfortunately

draw no conclusions as yet.

The existing literature on the involvement of the B-cell – Mast cell – IgE products axis in the LAR is

varied and conflicting. Different authors state contrasting conclusions on whether the LAR occurs

dependently or independently of B-cells, Mast cells and IgE.

Some clinical studies have indicated a prominent role for these cell types and mediators in the LAR.

Increased numbers of mast cells and increased levels of IgE in BAL fluid has been associated with the

magnitude of the LAR (Peebles et al., 2001; Crimi et al., 1991). Patients being treated with

corticosteroids (which inhibit the LAR) have been shown to have decreased numbers of mast cells in

the smooth muscle and epithelium (James et al., 2012). DSCG, a known mast cell stabiliser, has been

shown to inhibit the LAR in asthmatic patients (Cockcroft & Murdock, 1987; Pepys et al., 1968, 1974;

Booij-Noord, Orie & De Vries, 1971; Cieslewicz et al., 1999b). Anti-IgE monoclonal antibodies have also

been shown to suppress allergen induced late phase bronchoconstriction (Busse et al., 2001; Holgate

et al., 2004, 2005; Fahy et al., 1997).

In contrast, other clinical studies have alluded to mechanisms independent of B-cells, Mast cells and

IgE to be important in driving the LAR. In asthmatic patients, Durham et al(1984) failed to see an

association between total or allergen specific IgE and the LAR. Total IgE has been shown to be a poor

indicator of the LAR (Tschopp et al., 1998). In the African population, serum levels of IgE have even

been reported to be higher in non-asthmatics compared to asthmatics (Scrivener & Britton, 2000).

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Studies involving the inhalation of T-cell peptides derived from the feline allergen Fel d 1demonstrated

the induction of an isolated LAR without an accompanying EAR, implying the LAR to be T-cell

dependent and IgE independent (Ali et al., 2004a). Khan et al (2000) demonstrated that while

Cyclosporin A decreased the LAR in patients, no effects were seen on the EAR indicating that the LAR

occurs independently of the B-cell-Mast cell- IgE products axis.

Animal models of the LAR have also resulted in conflicting conclusions being drawn on the role of B-

cells, Mast cells and IgE in the LAR. Mizutani et al (2012) demonstrated that mice sensitised with OVA-

specific IgE displayed an LAR upon OVA challenge. Inhibitors of mast cell products such as Cysteinyl

Leukotrienes have been shown to inhibit the LAR at least partially (Nabe et al., 2002; Roquet et al.,

1997). Nabe et al (2005) demonstrated a correlation between the presence of mast cells and the ability

to display an LAR in a mouse asthma model. However, the authors in this investigation also exhibited

that the LAR was not associated with IgE as Dexamethasone was shown to suppress the LAR but not

plasma levels of IgE(Nabe et al., 2005).The same authors also more recently showed that applying an

antibody targeting mast cells did not attenuate the LAR (Nabe et al., 2013). Investigators adoptively

transferring CD4+ and CD8+ cells into rat models of the LAR noted that there were no changes in the

EAR and serum IgE levels where there were effects upon the LAR (Watanabe et al., 1995; Isogai et al.,

2003). Interestingly, McMenamin et al. (1995) showed that CD8+γδ T-cells suppress plasma IgE levels,

although the authors did not demonstrate a functional link to the LAR.

Overall, the existing literature on the subject of the involvement of B-cells, Mast cells and IgE in the

LAR is sparse and conflicting. This could be due to the fact that at present there are limited tools for

examining the roles of these cells and mediators with respect to asthmatic responses such as the LAR.

Specificity issues concerning depleting antibodies in asthma investigations have been raised. For

example, the antibody MAR-1, targeting the high affinity receptor FcεRI has not only been shown to

inhibit mast cell driven processes, but has been shown to activate mast cells (Kojima et al., 2007;

Ohnmacht et al., 2010; Voehringer, 2013). Due to this, the utilisation of cell deficient mice was

desirable for this investigation when attempting to delineate the mechanisms behind the LAR.

Cell deficient strains have been used by others investigating other features of asthma such as AHR and

airway inflammation. These investigations have yielded conflicting results and this variation seen in

experimental results has been attributed to differences in genetic background, differences in

sensitisation and challenge protocols and also the use of adjuvants (Lei et al., 2013).More prominently,

questions have also been raised as to the suitability of mouse asthma models and the fact that there

is a discrepancy between the results obtained in these models and those obtained in clinical trials

(Krug & Rabe, 2008). When assessing the roles particularly of mast cells in murine models, it should

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be noted that there are differences between mice and humans with respect to lung mast cell

populations. In terms of location, in humans mast cells are found around the airways and vessels in

both the large and small airways and in the parenchyma, whereas in mice, mast cells are mainly

located in the trachea and larger airways only (Andersson et al., 2009; Xing et al., 2011). Mast cells in

murine lungs preferentially secrete 5-HT, compared to human mast cells which secrete histamine and

other preformed mediators, in response to IgE activation (Szelenyi, 2000). In mice, mast cell driven

responses such as the EAR are driven by 5-HT being released by mast cells which causes the release

of neuronal ACh which in turn binds to muscarinic M3 receptors to cause

bronchoconstriction(Fernandez-Rodriguez et al., 2010; Moffatt, Cocks & Page, 2004). This is in

contrast to humans whereby mast cells release histamine, Cysteinyl Leukotrienes and prostaglandins

which directly bind to receptors in ASM to cause bronchoconstriction (Roquet et al., 1997; Curzen,

Rafferty & Holgate, 1987).It could be that when investigating mast cell responses (and possibly other

linked cells and mediators such as B-cells and IgE) the mouse model is not the best model to employ.

More favourable models in terms of investigating mast cells are considered to be Guinea-Pig or Rat

models. These models have a wider distribution of mast cells throughout the lung which is more

similar to humans (Bachelet, Bernaudin & Fleury-Feith, 1988). Rat models in particular display more

similar features of pulmonary reactivity to humans such as a more obvious observable biphasic

asthmatic response(Stevenson & Birrell, 2011; Lei et al., 2013). Overall, for the purposes of this

investigation, when further pursuing the role of mast cells within the LAR, it could be concluded that

it would be worthwhile using an alternative model. The Brown Norway Rat model of the LAR has

already been established and characterised within the Respiratory Pharmacology group and so would

be an ideal model to apply to the purpose of further investigating the role of the B-cell – Mast cell –

IgE products axis in the LAR.

In summary, this chapter explored the possible roles of CD4+ cells, CD8+ cells, Mast cells, B-cells and

IgE in the LAR. This was done by applying GM mice to an OVA-driven mouse model of the LAR which

is currently a missing determinant in this field. The data obtained highlighted a driving role for CD4+

cells and an inhibitory role for CD8+ cells in the LAR which is consistent with the existing literature on

the subject. Unfortunately, no conclusions with respect to the roles of Mast cells, B-cells and IgE could

be drawn with respect to the LAR. However, it was concluded that this particular mouse model is

unsuitable to investigate the roles of these particular effector cells and mediators in this response.

The existing literature on the subject is difficult to draw conclusions from due to its conflicting nature.

Therefore, further work is required to delineate the role of the B-cell -Mast cell – IgE axis in the LAR.

The next chapter explores this further.

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Chapter 5

Utilising Compound 48/80 and

Disodium Cromoglycate as tools to

further explore the LAR

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5 Utilising Compound 48/80 and Disodium Cromoglycate as tools

to further explore the LAR

5.1 Rationale

In the previous chapter it was concluded that further work was necessary to delineate the role of the

B-cell – Mast cell – IgE products axis within the LAR. Therefore, the aim of this chapter was to further

investigate this via the application of pharmacological tools to the animal models of the LAR. In the

previous chapter, the mouse model was utilised due to the availability of knockout mice. However, as

it is possible to apply pharmacological compounds to both the rat and mouse models of the LAR, both

models were considered within this chapter.

Compound 48/80 is described as a Mast cell degranulating agent and when administered acutely, mast

cells degranulate to release their preformed mediators such as histamine, 5-HT, Leukotrienes and

prostaglandins (Hannon et al., 2001). A number of investigations have also used this compound to

deplete mast cells of their mediator stores via repeated administration in sub-chronic treatment

schedules (Das, Flower & Perretti, 1997; Vergnolle, Hollenberg & Wallace, 1999; Hannon et al., 2001;

Haddad et al., 2002). The first aim of this chapter was to deplete mast cells in naive rats and mice via

sub-chronic treatment with compound 48/80 as done in Vergnolle et al. 1999 and Haddad et al. 2002.

The success of the mast cell depletion was evaluated via histologically staining mast cell contents in

lung tissue samples. The second aim was to apply the sub-chronic compound 48/80 treatment

schedule to the OVA-driven models of LAR in order to ascertain the importance of mast cells within

this response. The species this was carried out in was determined by the success of the mast cell

depletion protocol.

DSCG is described as a mast cell stabilising compound (Sheard & Blair, 1970; Storms & Kaliner, 2005).

A number of studies have applied this compound to animal models when investigating the role of mast

cells in features of disease (Das, Flower & Perretti, 1997; Hannon et al., 2001; Haddad et al., 2002;

Schemann et al., 2012). The third aim of this chapter was to apply DSCG to the OVA driven animal

models of the LAR in order to further explore the role of Mast cells in this response. The species this

was carried out in was determined from the results of the first two aims in this chapter in order to

ensure experimental continuity within the investigation.

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5.2 Methods


described below.

5.2.1 Validation of the Compound 48/80 Dosing Protocol

Naive male Brown Norway rats (200-225g) and C57BL/6 mice (20-25g) were dosed with either vehicle

(saline, 5 ml/kg i.p.) or Compound 48/80 (0.6 mg/kg; 5 ml/kg i.p.) twice daily for 3 days. On day 4, the

animals received a final higher dose of either Compound 48/80 (1.2 mg/kg; 5 ml/kg i.p.) or vehicle

(saline, 5 ml/kg i.p.) in the morning. In the evening, the animals were culled via overdose with Sodium

Pentobarbitone. The Compound 48/80 dosing regimen was based on the methodology used in

Vergnolle et al. 1999 and Haddad et al. 2002. A BAL fluid sample was obtained and leukocyte

population analysed by differential cell count as detailed in general methods section 2.6.3. Lung tissue

was isolated, fixed and stained for mast cells using a standard Toluidine Blue stain. The tissue slices

were mounted on slides and assessed for the presence of mast cells using light microscopy by a

blinded observer as detailed in general methods section 2.3.4.

Figure 5.1: Timeline demonstrating protocol for drug administration of Compound 48/80

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5.2.2.1 Compound 48/80 Protocol

Male Brown Norway rats (200-225g) were sensitised as detailed in general methods section 2.4.2. On

days25-28, rats were dosed as previously described with Compound 48/80. On day 28, the rats

underwent allergen challenge such that the final dose of Compound 48/80 was administered 60

minutes before challenge (see figure 5.2). Penh was recorded from 60 minutes after the end of

allergen challenge for a period of 5 hours as detailed in general methods section 2.5.3.

Figure 5.2: Timeline demonstrating protocol for final administration of Compound 48/80 in OVA

driven rat model of LAR

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5.2.2.2 DSCG Protocol

For the DSCG experiments, male Brown Norway rats were sensitised and challenged as detailed in

general methods section 2.4.2. On the day of allergen challenge, rats were dosed with DSCG (10

mg/kg; 1 ml/kg; i.t.) or vehicle (0.5% ethanol in saline, 1 ml/kg; i.t.) 60 minutes before allergen

challenge and 30 minutes following the end of allergen challenge (see figure 5.3). Dosing regimens

were based on those used in Das et al. 1997 and Hannon et al. 2001. Penh was recorded from 30

minutes after the final drug administration for a period of 5 hours as detailed in general methods

section 2.5.3.

Figure 5.3: Timeline demonstrating protocol for DSCG administration in OVA driven rat model of

LAR





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5.3 Results

5.3.1 Validation of the Compound 48/80 Dosing Protocol

In order to validate that the dosing protocol had successfully depleted the contents of lung tissue mast

cells, a pilot study was performed. In these experiments animals were culled following completion of

the dosing protocol at the time when, if the dosing was successful, the LAR would be measured in the

follow up investigation. The lung tissue mast cell population was assessed as well as BAL leukocyte

populations in order to observe the effects of Compound 48/80.

In both the rat and mouse, Compound 48/80 conferred no significant increases or decreases,

compared to vehicle, in the BAL lymphocyte, macrophage, neutrophil or eosinophil populations (See

figures 5.4 and 5.5).

Figure 5.4: Assessment of BAL fluid following treatment with compound 48/80 in naive (C57BL/6)

mice. BAL fluid was obtained from naive (C57BL/6) mice following completion of the Compound

48/80 treatment protocol and was assessed via differential cell count for numbers of: A)

Lymphocytes; B) Macrophages; C) Neutrophils and D) Eosinophils. Data is expressed as mean cell

number per ml of BAL fluid ± s.e.m. n=4. Mann-Whitney U-test. *p<0.05 compared to vehicle (saline)

treated control group. No significant difference between groups is denoted ns.

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Figure 5.5: Assessment of BAL fluid following treatment with compound 48/80 in naive Brown

Norway Rats. BAL fluid was obtained from naive rats following completion of the Compound 48/80

treatment protocol and was assessed via differential cell count for numbers of: A) Lymphocytes; B)

Macrophages; C) Neutrophils and D) Eosinophils. Data is expressed as mean cell number per ml of

BAL fluid ± s.e.m. n=4. Mann-Whitney U-test. *p<0.05 compared to vehicle (saline) treated control

group. No significant difference between groups is denoted ns.

117

In the mouse model, the numbers of undepleted lung tissue mast cells in the compound 48/80 dosed

animals were found to be comparable to the vehicle group (Compound 48/80 75.17 ±11.62 mast

cells/tissue slice vs. Vehicle 101.2±7.32 mast cells/tissue slice). In the rat model, the stained lung tissue

mast cells were significantly decreased in the Compound 48/80 group compared with the vehicle

group (Compound 48/80 281.8 ± 36.38 mast cells/tissue slice vs. 722.9 ± 21.17 mast cells/tissue slice).

Therefore, the dosing protocol successfully depleted lung tissue mast cell contents in the rat but not

the mouse model. Due to this, the OVA-driven Brown Norway rat model was used in order to assess

the effects of depleting mast cells using Compound 48/80 on the LAR (See figure 5.6).

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Figure 5.6: Validation of Mast Cell depletion by Compound 48/80. Lung tissue slices were obtained

from A) Wildtype C57BL/6 mice and B) Brown Norway Rats following treatment protocol with (i)

Vehicle (saline) or (ii) Compound 48/80. They were stained utilising a standard Toluidine Blue

histological stain and numbers of mast cells per slide were counted (x40 magnification) by a blinded

observer. Data is expressed as mean cell number per slide ±s.e.m. n=4. Mann-Whitney U-test.

*p<0.05 compared to vehicle (saline) treated control group. No significant difference between

groups is denoted ns.

A.(i)

A.(ii)

B.(i)

B.(ii)

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5.3.2 The Effect of Compound 48/80 on the LAR

OVA challenge resulted in a marked LAR in OVA sensitised rats. This response peaked at 2-3 hours and

lasted for up to 6 hours after challenge. The saline sensitised rats did not display a response following

OVA challenge.

Compound 48/80 treatment in the OVA sensitised/OVA challenged rats resulted in a significant

enhancement of the LAR in terms of Penh Peak response but not Area Under Curve response when

compared with vehicle treated rats. Compound 48/80 treatment in the control saline sensitised/OVA

challenged rats did not have any impact on baseline Penh recordings when compared with vehicle

treated animals (See figure 5.7).

Figure 5.7: Effect of Compound 48/80 treatment in the rat OVA-driven LAR. 1 hour following allergen



Penh average at peak response. Data expressed as mean ± s.e.m. n=8. Mann-Whitney U-test.

*p<0.05 Vehicle/Saline compared to Vehicle/Ovalbumin. #p<0.05 Compound 48/80

treated/Ovalbumin compared to Vehicle/Ovalbumin. No significant difference between groups is

denoted ns.

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5.3.3 The Effect of DSCG on the LAR

Again, OVA challenge resulted in a distinct LAR in the OVA sensitised rats, peaking at 2-3 hours and

lasting for up to 6 hours after challenge. The saline sensitised rats did not display a response following

OVA challenge.

DSCG treatment resulted in a significantly decreased LAR in the OVA sensitised/OVA challenged rats

in terms of Penh Peak response when compared with vehicle treated rats (figure 5.8).

Figure 5.8: Effect of DSCG treatment in the rat OVA-driven LAR. 1 hour following allergen challenge

rats were placed in WBP chambers and Penh recorded for a further 5 hours. Data is expressed as A)

Penh average over the recording time period; B) Penh Area Under Curve; and C) Penh average at

peak response. Data expressed as mean ± s.e.m. n=8. Mann-Whitney U-test. *p<0.05 Vehicle/Saline

compared to Vehicle/Ovalbumin. #p<0.05 DSCG treated/Ovalbumin compared to

Vehicle/Ovalbumin. No significant difference between groups is denoted ns.

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5.4 Discussion

The overall aim of this chapter was to further investigate the role of the B-cell – Mast cell – IgE products

axis in the LAR via the application of pharmacological tools to the OVA-driven rat and mouse models

of the LAR. This was to be done by applying the Mast cell degranulating agent, Compound 48/80, and

the Mast cell stabilising compound, DSCG, to the rat and mouse LAR models.

Before these investigations could take place, it was considered necessary to validate the Compound

48/80 treatment schedule in order to determine its success at depleting the contents of the lung tissue

mast cell population. This was done as a pilot study in naive rats and mice where lung tissue samples

were assessed for mast cells using the standard Toluidine blue histological stain. BAL leukocytes were

also evaluated in order to ascertain that the dosing regimen conferred no additional effects in terms

of the inflammatory cell population. In both species, no significant differences were observed in

vehicle treated animals compared with Compound 48/80 treated animals in terms of the BAL

macrophage, lymphocyte, neutrophil and eosinophil populations. The treatment protocol resulted in

a significant decrease in the number of intact lung tissue mast cells in rats but not mice. From this it

could be concluded that the Compound 48/80 treatment schedule was successful in the rat, but not

the mouse model and the treatment conferred no additional effects in terms of other inflammatory

cell populations. Literature on the subject of lung mast cells in mouse models suggests that in wildtype

(C57BL/6) mice there is a distinct lack of a prominent mast cell response compared with other

species(Lei et al., 2013). The distribution of mast cells is different in the mouse lung compared with

humans, with the majority of cells located in the larger airways compared with the smaller airways

and parenchyma respectively (Andersson et al., 2009; Xing et al., 2011). Recent investigations have

shown more distinctive mast cell responses in mast cell deficient mice having undergone mast cell

engraftment, and authors have postulated that this is due to the mast cells being present in greater

numbers and located in the more peripheral areas of the lung (Gersch et al., 2002; Fuchs et al., 2012;

Wolters et al., 2005). Taking this into consideration, the observation of a lack of Mast cell content

depletion in the mouse lung by Compound 48/80 in this chapter could be explained by the fact that

there is a lack of mast cell presence in the lungs of wildtype (C57BL/6) mice to begin with. This is

interesting, however, as even though mast cell numbers within the mouse lung are low, the mast cell

deficient mice within Chapter 4 of this thesis did exhibit an alternative LAR. Overall, it was not

considered suitable to proceed with the mouse model of the LAR to investigate the role of mast cells

within the response. Due to this the rat OVA-driven LAR model, and not the mouse model, was used

to assess the effect of depleting mast cells in the LAR.

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Compound 48/80 is defined as a secretagogue compound, activating mast cells to degranulate and

release their preformed mediators (Hannon et al., 2001). Its mechanism of action is receptor

independent and is thought to occur via a hydrophobic moiety of the compound penetrating the

membrane of cells and directly activating the Gi/o class of G-proteins to result in degranulation (Aridor,

Traub & Sagi-Eisenberg, 1990; Mousli et al., 1990; Chahdi et al., 1998; Chahdi, Fraundorfer & Beaven,

2000; Palomäki & Laitinen, 2006). In this chapter, we applied the compound to chronically deplete

mast cell contents in order to investigate their importance within the LAR. Overall, no abrogation of

LAR was observed with mast cell content depletion. It is noteworthy that though compound 48/80

was seen to significantly deplete the lung tissue mast cell population of their content, it was not

completely abolished. Therefore, if mast cells were central to an LAR, it is possible that only small

numbers are required to elicit factors contributing to the response. Another possibility is that even

though the stores of preformed mediators were depleted, the Mast cells’ ability to synthesise and

excrete mediators de novo was not. This means that it is still feasible that a mediator produced by

mast cells is involved in the driving mechanisms behind the LAR. While no difference in the response

was found in terms of the Area Under Curve analysis between the vehicle treated and Compound

48/80 treated OVA-sensitised rats upon OVA challenge, a significant increase in Peak Penh was

observed in the OVA sensitised Compound 48/80 treated animals. The saline sensitised compound

48/80 treated control animals did not exhibit a response, ruling out an effect of the dosing protocol in

itself. Thus, on an allergic background compound 48/80 conferred additional effects, enhancing the

LAR response in this experimental setting. Due to experimental caveats such as this, it was considered

desirable to investigate the role of mast cells in the OVA-driven rat model further using additional

pharmacological tools.

Further investigation into the role of mast cells in the LAR was carried out via the application of DSCG,

a mast cell stabiliser, to the rat model. In this model, the LAR was attenuated with DSCG treatment

compared to vehicle treated animals. This decrease in response was significant in terms of Peak Penh.

DSCG has been shown to be effective in the treatment of features of asthma, but due to the fact that

it has to be taken 4 times daily, its use is limited, alluding to a poor pharmacokinetic profile(Patel et

al., 1986; Yahav et al., 1988; Kato et al., 1999). This could be why at the beginning of the LAR in the

rat model (i.e. the Peak Penh response) the LAR was diminished significantly in the DSCG treated

group, but later in the recording period, the Penh average was again enhanced, resulting in the overall

AUC appearing to be diminished in the DSCG group, though not significantly.

It is not surprising that DSCG was effective in the treatment of the LAR in this model. Indeed, this

compound has previously been shown to be an effective treatment in this particular feature of

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asthma(Booij-Noord, Orie & De Vries, 1971; Cockcroft & Murdock, 1987). It is, however, of interest

that although this compound is described as a mast cell stabiliser and is effective in modulating the

LAR; the previous experiments depleting mast cell contents conferred no diminishing effects in terms

of the response. This alludes to a mechanism of action of DSCG in the attenuation of the LAR which is

unrelated to the stabilisation of mast cells. Literature on the subject also implies DSCG to confer

activity outside of the mast cell degranulation paradigm. It has been shown to be effective in inhibiting

bronchoconstriction against stimuli such as exercise, SO2, cold air and distilled water; all of which are

considered unrelated to mast cell degranulation. In addition to this, newly developed mast cell

stabilisers have been shown to have little effect in clinic (Poppius et al., 1970; Breslin, McFadden &

Ingram, 1980; Robuschi et al., 1989; Allistone et al., 1985; Dixon & Barnes, 1989).

Whilst, as a result of the experiments in this chapter and in chapter 3 we cannot substantiate a

definitive role for mast cells within the driving mechanisms behind the LAR, we can conclude that

there is the possibility of DSCG modulating the LAR via a mechanism independent of mast cells. Further

exploration into this mechanism of action could lead to the development of novel therapeutics

without the poor pharmacokinetic profile of DSCG. Other investigators have suggested that DSCG acts

independently of mast cells, with studies pointing towards the modulation of sensory nerve

responses(Dixon & Barnes, 1989; Barnes, 1993; Jackson, Pollard & Roberts, 1992; Norris & Alton, 1996;

Dixon, Jackson & Richards, 1980) . Within the Respiratory Pharmacology group, a role for airway

sensory nerves and the ion channel TRPA1 has been suggested to be involved in the driving

mechanisms behind the LAR (Raemdonck et al., 2012). It could be hypothesised that DSCG could be

inhibiting the LAR via its actions on airway sensory nerves.

In summary, this chapter further explored the possible roles for the B-cell – Mast cell – IgE axis in the

driving mechanisms behind the LAR. This was achieved by pharmacologically targeting Mast cells for

depletion and stabilisation in the OVA-driven rat model of the LAR, as the mouse model was deemed

unsuitable for this purpose. Interestingly the mast cell stabiliser DSCG conferred suppressive effects

on the LAR, whilst depleting mast cell contents using compound 48/80 did not inhibit the response.

Although from this a role for mast cells within the driving mechanisms behind the LAR could neither

be confirmed not refuted, the possibility of an alternative mechanism of action for DSCG, independent

of mast cells, could be proposed. Literature on the subject has implied DSCG to interact with sensory

nerves (Dixon, Jackson & Richards, 1980; Dixon & Barnes, 1989; Barnes, 1993; Jackson, Pollard &

Roberts, 1992; Norris & Alton, 1996). Within the Respiratory Pharmacology group a role for airway

sensory nerves and the ion channel TRPA1 has previously been proposed to be central to the driving

mechanisms behind the LAR(Raemdonck et al., 2012). It could therefore be hypothesised that DSCG

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attenuates the LAR via modulation of airway sensory nerve responses. This possibility will be further

investigated in chapters 6 and 7 of this thesis.

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Chapter 6

Further Exploration into the Role of

Airway Sensory Nerves in the LAR

126

6 Further exploration into the role of airway sensory nerves in the

LAR

6.1 Rationale

It has been proposed that airway sensory nerves are central to the driving mechanisms behind the

LAR. Within the Respiratory Pharmacology group, animal models of the LAR initially demonstrated

that the LAR, but not the EAR was lost under general anaesthesia indicating the involvement of the

CNS. Application of the non-selective ion channel inhibitor, Ruthenium Red, abolished the LAR,

highlighting the possibility of sensory nerve involvement in this response. Further investigation

utilising selective ion channel blockers highlighted a role for the TRPA1 channel within the LAR. The

LAMA, Tiotropium Bromide, was found to inhibit the LAR, demonstrating the possibility of cholinergic

constriction within this response. This lead to the overall hypothesis that airway sensory nerves, a

central reflex arc and a parasympathetic cholinergic effector arm are involved in the aetiology behind

the LAR (see figure 6.1) (Raemdonck et al., 2012).

The involvement of a cholinergic constrictor response in the LAR was initially concluded based upon

the application of the LAMA Tiotropium, to the rat LAR model. However, it has since been shown that

Tiotropium has off target effects involving the modulation of TRPV1 responses on airway sensory

nerves (Birrell et al., 2014). In light of this new data, it was deemed desirable to further characterise

the role of the parasympathetic cholinergic effector response and the roles of TRPA1 and TRPV1 in the

LAR. The first aim of this chapter was to investigate if cholinergic constrictor responses are central to

the LAR via the application of a second LAMA, Glycopyrrolate, to the OVA-driven rat model of the LAR.

The second aim of this chapter was to test new TRPA1 and TRPV1 inhibitors, Janssen130 and

XEND0501 respectively, in the rat model of the LAR. The rat model was used in order to ensure

experimental continuity from the previous chapters within this thesis.

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Figure 6.1: The Proposed Mechanisms involved in the LAR. Allergen challenge is thought to lead to

a sequence of events resulting in 1.) The activation of airway sensory nerves via TRPA1 channels

(blocked by Ruthenium Red and selective ion channel blockers); 2.) Central reflex events (blocked

via anaesthesia); 3.) Parasympathetic Cholinergic response (blocked by Tiotropium).

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6.2 Methods


described below.


6.2.1.1 Tiotropium and Glycopyrrolate Protocol

Male Brown Norway rats were sensitised and challenged as detailed in general methods section 2.4.2.

On day 28, rats were dosed with; Vehicle (0.5% ethanol in saline, 1 ml/kg; i.t.), Tiotropium (1 mg/kg, 1

ml/kg; i.t.) or Glycopyrrolate (1 mg/kg, 1 ml/kg; i.t.) 60 minutes before and 30 minutes following the

end of allergen challenge (see figure 6.2). Penh was recorded from 30 minutes after the final drug

administration for a period of 5 hours as detailed in general methods section 2.5.3.

Figure 6.2: Timeline demonstrating protocol for Tiotropium and Glycopyrrolate administration in

the OVA driven rat model of LAR

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6.2.1.2 Janssen130 and XEND0501 Protocol

For Janssen130 (TRPA1 inhibitor) and XEND0501 (TRPV1 inhibitor) experiments, male Brown Norway

rats were sensitised and challenged as previously described in general methods section 2.4.2. On the

day of allergen challenge, rats were dosed with either Vehicle (0.5% Methylcellulose + 0.2% Tween80

in saline, 10 ml/kg; i.p. across 2 sites) or Janssen 130 (100 mg/kg or 300 mg/kg, 10 ml/kg; i.p. across 2

sites) or XEND0501 (10 mg/kg; i.p. across 2 sites) 60 minutes before allergen challenge and 30 minutes

following the end of challenge (see figure 6.3). Dosing regimens were decided based upon

pharmacokinetic profiling of these compounds. Penh was recorded from 30 minutes after the final

drug administration for a period of 5 hours.

Figure 6.3: Timeline demonstrating protocol for Janssen130 administration in OVA driven rat model

of LAR





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6.3 Results

6.3.1 The Effect of Tiotropium and Glycopyrrolate on the LAR

OVA sensitised rats displayed a marked LAR upon OVA challenge. This response peaked at 2-3 hours

and lasted for up to 6 hours following allergen challenge. Saline sensitised rats did not display a

response following OVA challenge. Treatment with Tiotropium and Glycopyrrolate resulted in a

significant attenuation of the LAR in terms of both Penh AUC and Peak Penh responses in the OVA

sensitised and challenged rats (figure 6.4).

Figure 6.4: Effect of Tiotropium and Glycopyrrolate treatment in the rat OVA-driven LAR. 1 hour

following allergen challenge rats were placed in WBP chambers and Penh recorded for a further 5


Curve; and C) Penh average at peak response. Data expressed as mean ± s.e.m. n=8. Mann-Whitney

U-test. *p<0.05 Vehicle/Saline compared to Vehicle/Ovalbumin. #p<0.05 Drug treated/Ovalbumin

compared to Vehicle/Ovalbumin.

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6.3.2 The Effect of Janssen130 on the LAR

OVA challenge resulted in a marked LAR in OVA sensitised rats, peaking at 2-3 hours and lasting for up

to 6 hours after challenge. The saline sensitised rats did not display a response following OVA

challenge. Application of Janssen130 resulted in a diminished LAR in the OVA sensitised/OVA

challenged rats at both doses tested. This decrease in response was significant at the 300 mg/kg

concentration in terms of both Penh AUC and Peak Penh response (figure 6.5).

Figure 6.5: Effect of Janssen130 treatment in the rat OVA-driven LAR. 1 hour following allergen




*p<0.05 Vehicle/Saline compared to Vehicle/Ovalbumin. #p<0.05 Janssen130 (various

concentrations) treated/Ovalbumin compared to Vehicle/Ovalbumin.

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6.3.3 The Effect of XEND0501 on the LAR

The OVA sensitised rats, again, displayed a marked LAR upon OVA challenge. The response peaked at

2-3 hours and lasted for up to 6 hours after challenge. The XEND0501 treated rats displayed a

diminished response compared to the OVA sensitised/Vehicle treated group. This difference,

however, was not significant in terms of either Penh AUC or Peak Penh response (figure 6.6). However,

it could be deemed as approaching significance (p=0.0792) and as such a non-significant result could

simply be due to low n numbers within the experiments.

Figure 6.6: Effect of XEND0501 treatment in the rat OVA-driven LAR. 1 hour following allergen




*p<0.05 Vehicle/Saline compared to Vehicle/Ovalbumin. #p<0.05 XEND0501 treated/Ovalbumin


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6.4 Discussion

It has previously been proposed that airway sensory nerves, a central reflex arc and parasympathetic

cholinergic constrictor responses are involved in the aetiology behind the LAR. In these investigations,

a parasympathetic cholinergic constrictor response within the driving mechanisms behind the LAR was

deduced based upon the observation that the LAR was diminished in animals treated with Tiotropium,

a LAMA (Raemdonck et al., 2012). Since then, Tiotropium has been shown to have off target effects

involving the modulation of TRPV1 responses on airway sensory nerves (Birrell et al., 2014). This is

interesting as the study by Raemdonck et al (2012) also deduced that TRPA1 and not TRPV1 were

central to the driving mechanisms behind the LAR using pharmacological intervention. The original

conclusions were made based upon results from single tool compounds in animal models of the LAR.

Therefore, when further pharmacological tools became available it was deemed desirable to further

investigate the possible roles of cholinergic constrictor responses, and the ion channels TRPA1 and

TRPV1 in the LAR. The aim of this chapter was to further validate the roles of these factors in the LAR

via the application of newly available pharmacological tools to the OVA-driven rat model.

Application of the LAMA, Glycopyrrolate, to the OVA-driven rat model resulted in a diminished LAR

similar to that previously seen with Tiotropium. Whereas Tiotropium has more recently been shown

to have off target effects involving the modulation of TRPV1 on airway sensory nerves, the same

investigation also showed that these effects were not applicable to Glycopyrrolate (Birrell et al., 2014).

Therefore, it could be validated that a parasympathetic cholinergic effector arm is central to the

aetiology behind the LAR.

In order to further validate a role for TRPA1 in the mechanisms behind the LAR, the TRPA1 inhibitor

Janssen130 was applied to the OVA-driven rat model. In these experiments an attenuated LAR was

observed in a dose-dependent manner with Janssen130 treatment, highlighting a role for TRPA1 in

the driving mechanisms behind the LAR. TRPA1 is thought to be mainly expressed in neurons such as

airway sensory nerves (Bautista et al., 2006a; Story et al., 2003; Bandell et al., 2004; Jang et al., 2012;

Nassenstein et al., 2008). As the functional relevance of TRPA1 on airway sensory nerves has been

well characterised, it could be deduced that TRPA1 present on airway sensory nerves triggers a

neuronal response resulting in the bronchoconstriction that is the LAR (Grace & Belvisi, 2011;

Raemdonck et al., 2012). However, a non-neuronal role for TRPA1 in the LAR cannot be ruled out as

expression of TRPA1 has also been reported in non-neuronal cell types such as CD4+ T-cells, CD8+ T-

cells, B-cells and Mast cells (Banner, Igney & Poll, 2011b; Prasad et al., 2008). The role of TRPA1 in

other features of asthma such as inflammation and AHR has been explored by researchers. It was

reported that TRPA1 deficient mice have reduced inflammation and AHR when applied to mouse

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models of allergic asthma (Caceres et al., 2009). Conversely, it has also been shown that application

of acrolein (a known TRPA1 agonist) resulted in a decreased inflammatory response in a murine

allergic asthma model (Spiess et al., 2013). Other studies have indicated a role for TRPA1 in the direct

control of airway tone. It has been reported that activation of TRPA1 channels on isolated guinea-pig

bronchus results in constriction which is secondary to the release of neuropeptides via local axons

(Andre et al., 2008). Further investigation into possible functions of non-neuronal TRPA1 is warranted.

Overall, with respect to the LAR, it can be concluded that TRPA1, possibly on airway sensory nerves,

is central to the driving mechanisms behind the LAR. However, it also cannot be ruled out that TRPA1

on a non-neuronal cell type is central to the aetiology behind the LAR.

Application of the TRPV1 selective inhibitor, XEND0501, to the rat model of LAR did not result in

significant attenuation of the response. However, it must be noted that although not significant a

slightly diminished response in terms of Penh AUC and Peak Penh was observed with XEND0501

treatment in OVA sensitised and challenged rats. Similarly to TRPA1, TRPV1 has been reported to be

mainly expressed in airway sensory nerves but expression is also present in lung tissue (Kunert-Keil et

al., 2006; Banner, Igney & Poll, 2011b; Jang et al., 2012). Studies investigating the role of TRPV1 in the

control of airway tone in diseases such as asthma have yielded conflicting results. Agonists of TRPV1

have been shown to contract isolated guinea-pig ASM via mediating the release of sensory

neuropeptides from finite stores in nerve endings which consequently act upon neurokinin receptors

on ASM (Belvisi et al., 1992). Investigations utilising animal models have reported TRPV1 to have a

proinflammatory role in allergic asthma (Rehman et al., 2013b), whilst others have shown TRPV1 to

be protective against allergic inflammation (Mori et al., 2011). Other researchers have reported that

TRPV1 is not involved at all in features of allergic asthma (Raemdonck et al., 2012; Caceres et al.,

2009). Overall, studies investigating the role of TRPV1 in allergic asthma have produced conflicting

results. With respect to the role of TRPV1 in the aetiology behind the LAR, Raemdonck et al (2012)

previously reported that no attenuation of the LAR was observed with the application of the TRPV1

selective inhibitor, JNJ-17203212. The results in this chapter also denote that although a slight

attenuation of response was observed with the application of the TRPV1 inhibitor XEND0501, this

reduction was not significant. It should be noted that as this particular TRPV1 inhibitor is a recent

development and extensive pharmacological profiling is ongoing. As such off-target effects cannot be

ruled out. Thus far, it can be concluded that TRPV1 is not involved in the aetiology behind the LAR.

However, due to the conflicting nature of current literature on the subject, it would be desirable to

further investigate the role of this ion channel in the LAR (and other aspects of asthma) should new

investigative tools become available in the future.

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In summary, this chapter has further explored the role of cholinergic constrictor responses, TRPA1 and

TRPV1 within the LAR via the application of pharmacological tools to the OVA-driven rat model of the

LAR. The LAMA Glycopyrrolate attenuated the LAR, confirming a role for cholinergic constrictor

responses within the response. Selective inhibitors of TRPA1 (Janssen130) and TRPV1 (XEND0501)

confirmed a role for TRPA1 but not TRPV1 within the LAR. In Chapter 5, DSCG was found to inhibit the

LAR and the possibility of an alternative mechanism of action for DSCG involving airway sensory nerve

modulation was raised. Chapter 6 has provided further evidence for the hypothesis that TRP channels,

airway sensory nerves, a central reflex arc and parasympathetic cholinergic responses are central to

the driving mechanisms behind the LAR. Therefore, it could be hypothesised that DSCG attenuates the

LAR via modulation of airway sensory nerve responses involving TRP channels such as TRPA1. This will

be investigated in Chapter 7.

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Chapter 7

The Role of Disodium Cromoglycate on

Airway Sensory Nerves in the LAR

137

7 The Role of Disodium Cromoglycate on airway sensory nerves in

the LAR

7.1 Rationale

It was observed in Chapter 5 that the mast cell stabiliser DSCG modulated the LAR; however, depleting

mast cell contents had no effect on the response. This led to the consideration that DSCG could be

attenuating the LAR via a mechanism that is independent of mast cells. Literature on the subject

suggested that DSCG interacts with airway sensory nerves and a role for airway sensory nerves within

the LAR has previously been shown (Dixon, Jackson & Richards, 1980; Dixon & Barnes, 1989; Barnes,

1993; Jackson, Pollard & Roberts, 1992; Norris & Alton, 1996; Raemdonck et al., 2012). This has led us

to postulate that DSCG could attenuate the LAR via its action on airway sensory nerves. The overall

purpose of this chapter is to further investigate this possibility and explore possible mechanisms of

action associated with this.

The first aim of this chapter was to investigate the possible direct inhibitory actions of DSCG on TRP

channel mediated airway sensory nerve depolarisation utilising an ex vivo isolated vagus nerve setup.

It was then considered desirable to further explore the mechanism of action of DSCG on inhibiting

TRPA1 mediated sensory nerve depolarisation. The literature on the subject suggested that DSCG is

an agonist at the G-protein coupled receptor 35 (GPR35) and agonists of this receptor modulate

calcium signalling in neurons (Guo et al., 2008; Yang et al., 2010). Therefore, the second aim was to

investigate the possible role of the GPR35 receptor in DSCG mediated inhibition of TRPA1 driven

depolarisation in the isolated vagus nerve utilising pharmacological tools. The third aim of this chapter

was to investigate if the findings in the isolated vagus nerve were applicable to the LAR. This was done

via the application of DSCG and a GPR35 receptor antagonist to the in vivo rat model of the LAR.

Finally, it was deemed necessary to corroborate these findings by observing the effects of DSCG and

its possible mechanism of action on airway sensory nerve firing in vivo. Therefore, the fourth aim of

this chapter was to investigate the effect of DSCG and a GPR35 receptor antagonist on TRPA1 induced

firing of C-fibre afferents in whole animal single afferent recording studies.

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7.2 Methods


described below.

7.2.1 Isolated Vagus Nerve Experiments

7.2.1.1 Effect of DSCG on TRPA1 and TRPV1 mediated depolarisation of the isolated

vagus nerve

The animal and human tissue was attained, harvested and mounted in a ‘grease gap’ recording

chamber as detailed in general methods section 2.7.

The nerve was allowed to equilibrate for 10 minutes in the ‘grease gap’ recording chamber. Following

this, 2-3 stimulations of consistent magnitude with either acrolein (300 µM) or capsaicin (1 µM) were

achieved, washing between stimulations with KH in order to return to baseline depolarisation. The

nerve was then incubated with either vehicle (0.1% H2O v/v) or DSCG (1, 10 or 100 M) for 10 minutes

and the stimulant then reapplied in the presence of the drug. The nerve was then washed as previously

described and a final application of stimulating agent applied in order to ascertain the viability of the

nerve. A change in the level of depolarisation as a result of drug application was reported as

percentage of the average of the two initial basal responses (general methods section 2.7).

It is of note that slightly higher concentrations of DSCG were used than might be deemed

physiologically relevant. This is because historically the vagus preparation requires higher

concentrations of ligands possibly due to the fact that the nerve trunk is used rather than the actual

nerve ending. Nevertheless, as part of this preparation, concentration responses are performed as

standard, always ensuring that the “effect” is modulated by a specific antagonist across numerous

species including human tissue.

7.2.1.2 The role of GPR35 Receptor in DSCG mediated inhibition of TRPA1 driven

depolarisation in the isolated vagus nerve

Male Brown Norway rats or Dunkin Hartley guinea-pigs were euthanized, the vagal tissue harvested

and mounted in a ‘grease gap’ recording chamber as detailed in general methods section 2.7.

Following a 10 minute equilibration period, 2-3 stimulations of consistent magnitude were attained

via the application of acrolein (300 µM) as previously described. The nerve was then incubated with

either vehicle (0.1% v/v DMSO) or the GPR35 inhibitor ML145 (1 µM) for 5 minutes. Following this the

nerve was then incubated with either DSCG (10 µM) or a known GPR35 agonist – Pamoic Acid (10 µM),

Kynurenic Acid (10 µM) or Zaprinast (10 µM) for 10 minutes. The stimulant (acrolein, 300 µM) was

139

then applied in the presence of the drug(s), the percentage inhibition reported as previously

described. The nerve was then finally assessed for viability (see general methods section 2.7).

Concentrations of TRPA1 and TRPV1 agonists were chosen based on previous experiments within the

Respiratory Pharmacology group (Birrell et al., 2009b). Concentrations of antagonists were selected

that were approximately 100-fold the PA2 value (defined as the negative logarithm of the molar

concentration of an antagonist that would produce a 2-fold shift in the concentration response curve

for an agonist) as is common practice.


7.2.2.1 The effect of ML145 on DSCG mediated inhibition of the LAR

Male Brown Norway rats were sensitised and challenged as detailed in general methods section 2.4.2.

On day 28, rats were dosed with ML145 (30 mg/kg, i.p) or Vehicle (0.5% MC + 0.2% tween80 in saline;

10 ml/kg, i.p.) 90 minutes before and 5 minutes after allergen challenge. Rats were also dosed with

DSCG (10 mg/kg, i.t.) or Vehicle (0.5% ethanol in saline; 1 ml/kg, i.t) 30 minutes prior to and 20 minutes

following the end of allergen challenge. Penh was recorded from 30 minutes after end of allergen

challenge for a period of 5 hours as detailed in general methods section 2.5.3.

Figure 7.1: Timeline demonstrating protocol for ML145 and DSCG administration in the OVA driven

rat model of LAR

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7.2.3 Whole Animal Single Afferent Recordings

7.2.3.1 The effect of DSCG on TRPA1 induced firing of C-fibre afferents

Surgery, setup and identification of a C-fibre were performed as detailed in general methods section

2.8. Animals were allowed to stabilise for at least 30 minutes before a 2 minute baseline recording

took place. Capsaicin (100 µM) was then administered by aerosol for 15 seconds and changes in nerve

fibre firing, intratracheal pressure and blood pressure recorded until baseline levels were re-

established. Two reproducible responses to acrolein aerosol (10 mM, 1.0 min) were attained before

administration of DSCG (10 mg/kg; i.t.) or Vehicle (0.5% ethanol in saline; 1 ml/kg, i.t). Following a 30

minute incubation period, a response to acrolein was attained as previously described. A final

capsaicin response took place at the end of the experiment in order to confirm nerve viability (see

figure 7.2). Data was recorded and expressed as peak impulses.S-1 as detailed in general methods

section 2.8.

Figure 7.2: Recording protocol for in vivo single afferent recording. Briefly, following a 2 minute

baseline recording, capsaicin (100 M aerosol, 15 s) was administered. After baseline was re-

established, two reproducible responses to acrolein (10 mM aerosol, 1.0 min) were attained. After

a suitable interval, DSCG (10 mg/kg, i.t.) or vehicle (i.t.) was administered and an incubation time of

30 mins took place. A response to acrolein (10 mM aerosol, 1.0 min) was then obtained before a

final capsaicin (100 µM aerosol, 15 s) response was recorded.

141

7.2.3.2 The effect of ML145 on DSCG mediated inhibition of C-fibre afferent firing

Surgery and setup were performed as detailed in general methods section 2.8. The dosing and

recording protocol was performed as previously described (section 7.2.3.1) with the application of the

following step for the antagonist experiments. Vehicle (0.5% MC + 0.2% tween80 in saline; 10 ml/kg,

i.p.) or GPR35 receptor antagonist, ML145 (30 mg/kg, i.p) were administered 30 minutes before the

Vehicle or DSCG (1hour before the final acrolein challenge).

7.2.4 Statistical Analyses


Mann Whitney-U tests with two-tailed p-values or Paired t-tests were carried out in order to assess

statistical significance between 2 groups (details for individual experiments found in figure legends).

Differences were considered statistically significant if p<0.05.

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7.3 Results

7.3.1 Effect of DSCG on TRPA1 and TRPV1 mediated depolarisation of the

isolated vagus nerve

In the Brown Norway isolated vagus nerve experiments, DSCG elicited a concentration dependent

inhibition of TRPA1-mediated depolarisation induced by acrolein (See figure 7.3). Similar inhibition

was not observed with respect to TRPV1-mediated depolarisation induced by capsaicin.

Figure 7.3: Effect of DSCG on TRPA1 and TRPV1 mediated depolarisation of Brown Norway rat vagus

nerve. Vagal tissue was harvested and mounted in a ‘grease-gap’ recording chamber. Two basal

responses to stimulant were obtained after which the tissue was incubated with vehicle (H20, 0.1%

v/v) or DSCG (1, 10 or 100uM) for 10 minutes. Stimulant was reapplied and changes in the level of

depolarisation reported as percentage of the average of the two basal responses. Data is expressed

as A) Percentage inhibition of TRPA1 (acrolein; 300 µM) mediated responses and B) Percentage

inhibition of TRPV1 (capsaicin; 1 µM) mediated responses. Data expressed as mean ± s.e.m. n=4.

*p<0.05 comparing depolarisation levels before and after treatment using a paired t-test.

143

In order to translate this finding into human vagal tissue, a sub-maximal concentration of DSCG (10

µM) was chosen to test against TRPA1 (acrolein) and TRPV1 (capsaicin) mediated depolarisation. DSCG

inhibited TRPA1 mediated depolarisation by approximately 81% which was not observed with respect

to TRPV1 mediated depolarisation (approximately 6% inhibition) (See figure 7.4).

Figure 7.4: Effect of DSCG on TRPA1 and TRPV1 mediated depolarisation of human vagus nerve.

Vagal tissue was harvested and mounted in a ‘grease-gap’ recording chamber. Two basal responses

to stimulant were obtained after which the tissue was incubated with vehicle (H2O, 0.1% v/v) or

DSCG (10uM) for 10 minutes. Stimulant was reapplied and changes in the level of depolarisation

reported as percentage of the average of the two basal responses. Data is expressed as Percentage

inhibition of TRPA1 (acrolein; 300 µM) mediated responses and TRPV1 (capsaicin; 1 µM) mediated

responses. N=4 (4 donor patients; 3 males, 36-57 years old and 1 female, 45 years old).

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7.3.2 The role of GPR35 Receptor in DSCG mediated inhibition of TRPA1 driven

depolarisation in the isolated vagus nerve

In order to test the hypothesis that DSCG modulated TRPA1-driven sensory nerve effects via the

activation of GPR35, a range of GPR35 agonists were applied to the isolated vagus nerve in parallel to

DSCG. The GPR35 agonists elicited inhibition against acrolein induced depolarisation (mean

inhibitions: Pamoic Acid 51%; Kynurenic Acid 65% and Zaprinast 74%) as did DSCG (mean inhibition:

68%). In order to confirm that the inhibition observed was via the GPR35 receptor, the GPR35

antagonist ML145 was utilised. The application of ML145 abolished the ability of DSCG and the other

GPR35 agonists to inhibit TRPA1-induced depolarisation (mean inhibitions: DSCG 7%, Pamoic Acid 8%,

Kynurenic Acid 19% and Zaprinast 7%) (See figure 7.5).

Figure 7.5: The role of the GPR35 Receptor in DSCG mediated inhibition of TRPA1 driven

depolarisation in the Brown Norway rat isolated vagus nerve. The effect of various GPR35 agonists

(PA - pamoic acid, KY - Kynurenic acid, ZA Zaprinast and DSCG, 10uM) on acrolein induced

depolarisation of rat vagal nerves in the presence of vehicle (0.1% v/v, DMSO) or GPR35 antagonist

ML145 (1 µM). Data expressed as mean ± s.e.m. n=4. ).* = p<0.05 using a paired t-test to compare

the response to acrolein in absence and presence of GPR35 agonist.

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7.3.3 Effect of ML145 on DSCG mediated inhibition of the LAR

OVA sensitised rats displayed a marked LAR upon OVA challenge. This response peaked at 2-3 hours

and lasted for up to 6 hours following allergen challenge. Saline sensitised rats did not display a

response following OVA challenge. Treatment with DSCG resulted in an attenuation of the LAR which

was significant in terms of Peak Penh response. This attenuation of the LAR was blocked with the

application of the GPR35 inhibitor, ML145. No effect on the LAR was observed with the application of

ML145 alone (See figure 7.6).

Figure 7.6: Effect of ML145 on DSCG mediated inhibition of the LAR. 1 hour following allergen




*p<0.05 Vehicle/Saline compared to Vehicle/Ovalbumin. #p<0.05 Drug treated/Ovalbumin


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7.3.4 The role of TRPA1 and the GPR35 Receptor in DSCG mediated inhibition

of depolarisation in the Guinea-Pig isolated vagus nerve

It was previously shown in this chapter that DSCG modulated TRPA1-driven sensory nerve effects via

the activation of GPR35 in Brown Norway Rat Vagal tissue. This was useful as the in vivo LAR model is

in this species. The final aims of this chapter involve assessing the modulation of airway sensory nerve

activation by DSCG in vivo. These investigations utilise a whole animal single afferent recording model

which is in the Dunkin-Hartley guinea-pig species. Therefore, prior to these investigations taking place,

it was deemed necessary to validate that DSCG modulates TRPA1-driven sensory nerve responses in

the guinea-pig and if so assess that the same mechanisms are applicable in this species as was found

in the Brown Norway rat.

In the Dunkin-Hartley isolated vagus nerve experiments, DSCG elicited an inhibition of TRPA1-

mediated depolarisation induced by acrolein. This inhibition was not observed with respect to TRPV1-

mediated depolarisation induced by capsaicin (see figure 7.7 (A)).

In order to investigate that DSCG modulates TRPA1-driven sensory nerve effects via GPR35 (as seen

in the rat), GPR35 agonists were applied to the isolated vagus nerve in parallel to DSCG. The GPR35

agonists elicited an inhibition of the acrolein induced depolarisation similar to that observed with

DSCG (mean inhibitions: Kynurenic Acid 66%; Zaprinast 70% and DSCG 67%). In order to confirm that

the inhibition observed was via the GPR35 receptor, ML145 was applied. This resulted in a reduction

in the ability of DSCG and the other GPR35 agonists to inhibit TRPA1-induced depolarisation (mean

inhibitions: DSCG 23%, Kynurenic Acid 42% and Zaprinast 30%) (see figure 7.7 (B)).

Overall, it was shown in the Dunkin-Hartley guinea-pig vagal tissue that DSCG inhibits TRPA1 mediated

responses and the mechanism of action involves GPR35. This is consistent with observations in the

Brown-Norway rat. Therefore, it was considered acceptable to use the guinea-pig whole animal single

afferent recording model to further these investigations.

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Figure 7.7: The role of TRPA1 and the GPR35 Receptor in DSCG mediated inhibition of depolarisation

in the Guinea-Pig isolated vagus nerve. A) The effect of DSCG on either TRPA1 (acrolein; 300 µM) or

TRPV1 (capsaicin; 1 µM) mediated responses. B) The effect of various GPR35 agonists (KY - Kynurenic

acid, ZA Zaprinast and DSCG, 10 µM) on acrolein induced depolarisation of guinea-pig vagal nerves

in the presence of vehicle (0.1% v/v, DMSO) or GPR35 antagonist ML145 (1 µM). Data expressed as

percentage inhibition of stimulant induced response; mean ± s.e.m. n=4. ).* = p<0.05 using a paired

t-test to compare the response to acrolein in absence and presence of GPR35 agonist.

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7.3.5 Effect of DSCG on TRPA1 induced firing of C-fibre afferents

In order to assess that DSCG was modulating airway sensory nerve activation in vivo, the effect of

DSCG on acrolein-evoked firing of airway-terminating, single vagal afferent chemosensitive C-fibres

was examined. DSCG significantly reduced the peak firing rate (16.7±2.2 to 4.3±0.9 imp.s-1) evoked by

acrolein. Little baseline firing was observed (see figure 7.8).

Figure 7.8: Effect of DSCG on TRPA1 induced firing of C-fibre afferents. All animals were firstly

exposed to aerosolised capsaicin (15 s; 100 µM), twice to acrolein (1 min; 10 mM) before incubating

with Vehicle (0.5% EtoH in Saline, i.t.) or DSCG (10 mg/kg, i.t.). Following this a final response to

acrolein was obtained prior to subsequent capsaicin stimulation. Panels A and B show the peak imp

s-1 before and after vehicle or DSCG application: Baseline firing (BF), capsaicin response (C) and

acrolein response (A). Data is expressed as mean ±s.e.m. n=3. Paired t-test. *p<0.05 acrolein

responses before and after treatment.

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7.3.6 Effect of ML145 on DSCG mediated inhibition of C-fibre afferent firing

In order to ascertain that the inhibition of acrolein-evoked firing of airway-terminating, single vagal

afferent chemosensitive C-fibres was via the GPR35 receptor, ML145 was applied to this experimental

protocol. After administration of Vehicle, DSCG significantly inhibited the peak firing rate (12.0±2.3 to

3.0±0.6 imp.S-1) evoked by acrolein. Following administration of ML145, DSCG did not impact on the

firing rate (9.7±1.8 to 14±4.2 imp.S-1) evoked by acrolein (see figure 7.9).

Figure 7.9: Effect of ML145 on DSCG mediated inhibition of C-fibre afferent firing. All animals were

firstly exposed to aerosolised capsaicin (15 s; 100 µM), twice to acrolein (1 min; 10 mM) before

incubating with Vehicle DSCG (10 mg/kg, i.t.) in the presence of either ML145 (30 mg/kg, i.p.) or

Vehicle (0.5% Methylcellulose + 0.2% Tween 80 in saline, i.p.). Following this a final response to

acrolein was obtained prior to subsequent capsaicin stimulation. Data is expressed as peak imp s-1.

Panels A and B show the effect of either Vehicle or ML145 on DSCG mediated inhibition of acrolein

driven C-fibre firing. Data is expressed as mean ±s.e.m. n=3. Paired t-test. *p<0.05 acrolein

responses before and after treatment.

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7.4 Discussion

The overall aim of this chapter was to investigate if DSCG attenuates the LAR via an action on airway

sensory nerves, and to further explore possible mechanisms of action associated with this.

In Chapter 5 of this thesis, DSCG was shown to inhibit the LAR via a mechanism of action independent

of mast cells. Other studies have suggested that DSCG interacts with airway sensory nerves (Dixon,

Jackson & Richards, 1980; Dixon & Barnes, 1989; Barnes, 1993; Jackson, Pollard & Roberts, 1992;

Norris & Alton, 1996). Previously within the Respiratory Pharmacology group, a role for airway sensory

nerves within the aetiology behind the LAR was deduced (Raemdonck et al., 2012). In chapter 6 of

this thesis, TRPA1 but not TRPV1 was confirmed to be central to the driving mechanisms behind the

LAR. The first aim of this chapter was to investigate the action of DSCG on TRP channel mediated

airway sensory nerve depolarisation utilising isolated vagus nerve experiments. As the LAR

observations were in the Brown Norway Rat model, this species was initially chosen for the isolated

vagus nerve experiments. Acrolein (TRPA1 agonist) or capsaicin (TRPV1) was used to evoke

depolarisation of the isolated nerve and DSCG was applied in order to observe any attenuation of

depolarisation. DSCG was found to inhibit TRPA1, but not TRPV1, mediated depolarisation in a

concentration dependant manner. It should be noted that at the highest concentration of DSCG

tested, around a 40% inhibition was exhibited to TRPV1 stimuli. However, as the sub-maximal dose

was taken forward for use in further experiments and this dose was not effective in inhibiting TRPV1

mediated effects it was concluded in this instance that DSCG inhibited TRPA1 mediated depolarisation

only. These findings were translated into human vagal tissue, where a sub-maximal dose of DSCG was

also shown to inhibit TRPA1, but not TRPV1, mediated depolarisation. Other studies have also

reported that DSCG attenuates sensory nerve activity in pre-clinical cough models (Lai & Lin, 2005;

Jackson, 1988; Dixon, Jackson & Richards, 1980) and also in translational human investigations

(Moroni et al., 1996; Zielen et al., 2006). The results from this chapter concur with the literature and

further the understanding in this field via the suggestion of the modulation of specifically TRPA1 driven

responses on airway sensory nerves.

After concluding that DSCG inhibits TRPA1 mediated depolarisation of airway sensory nerves, it was

considered desirable to explore the possible mechanisms of action associated with this. Other

investigations have shown DSCG to be an agonist of a G-protein coupled receptor known as GPR35

(Yang et al., 2010). Agonists of this receptor have also been shown to modulate calcium signalling in

neurons (Guo et al., 2008). Therefore, it was postulated that DSCG could modulate TRPA1 driven

sensory nerve activity via activation of GPR35. In order to investigate this possibility, experiments were

performed where agonists of GPR35 were applied to the isolated vagus nerve preparation in parallel

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to DSCG and their effect on TRPA1 driven nerve depolarisation examined. The GPR35 agonists were

found to elicit a similar degree of inhibition of TRPA1 mediated nerve depolarisation to that observed

with DSCG. In order to confirm that the inhibition observed was indeed via GPR35, an antagonist of

this receptor, ML145, was also applied in this line of investigation. The GPR35 antagonist was observed

to block the inhibition of sensory nerve responses by DSCG (and the GPR35 agonists), thus leading to

the conclusion that GPR35 is central to the effect of DSCG on airway sensory nerve responses.

The next aim of this chapter was to examine if the findings in the isolated vagus nerve were applicable

to the LAR. In order to achieve this, DSCG was applied to the OVA-driven rat model of the LAR with

either vehicle or the GPR35 antagonist, ML145. DSCG was shown to inhibit the LAR and ML145

successfully blocked this inhibition. It has previously been postulated that sensory nerves are central

to the driving mechanisms behind the LAR (Raemdonck et al., 2012) and DSCG is able to interact with

airway sensory nerves (Dixon & Barnes, 1989; Barnes, 1993; Jackson, Pollard & Roberts, 1992; Norris

& Alton, 1996; Dixon, Jackson & Richards, 1980). In this chapter, the mechanisms previously elucidated

in the isolated vagus nerve have been shown to apply to the LAR in vivo. Therefore, there is strong

evidence to suggest that DSCG attenuates the LAR via acting on GPR35. However, it must be noted

that while applying pharmacological tools to the in vivo LAR model supports the role of GPR35 in the

DSCG mediated inhibition of response it does not demonstrate that the action is on airway sensory

nerves. Due to this, the final aim of this chapter was to establish the role of GPR35 in DSCG mediated

inhibition of airway sensory nerve firing in vivo.

This final aim of this chapter was achieved via the use of an in vivo single afferent recording model. In

this set of experiments, DSCG was found to significantly attenuate TRPA1 induced firing of C-fibre

afferents and the application of ML145 resulted in the modulation of this effect. This demonstrates

that DSCG attenuates TRPA1 driven airway sensory nerve responses via GPR35. It is of note that this

model is in the Dunkin Hartley guinea-pig, not the Brown Norway rat. This is because at present in the

Respiratory Pharmacology group, the whole animal single afferent recording model has been

characterised only in the guinea-pig. Due to the time-constraints of this thesis it wasn’t deemed

feasible to set-up, characterise and validate the model in the Brown Norway rat, although this is a

desirable aim for the future. This would allow us to further investigate the role of airway sensory nerve

responses on backgrounds of allergic disease more specifically. However, for the purposes of this

thesis, the use of the guinea-pig model was validated as isolated vagus nerve depolarisation studies

were paralleled in this species and the results were found to be concordant with those found in the

rat.

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DSCG has long been shown to be effective in the treatment of features of asthma, but due to the fact

that it has to be taken 4 times daily, its use is limited, alluding to a poor pharmacokinetic profile (Patel

et al., 1986; Yahav et al., 1988; Kato et al., 1999). In the literature, DSCG has been widely shown to be

effective in inhibiting the LAR (Booij-Noord, Orie & De Vries, 1971; Cockcroft & Murdock, 1987; Nabe

et al., 2004; Koyama et al., 2005). It was originally postulated that this efficacy is due to its action as a

mast cell stabiliser, however, newly developed mast cell stabilisers have shown little efficacy in clinic

(Sheard & Blair, 1970). In this chapter, it was shown that DSCG inhibits the LAR via activation of the G-

protein coupled receptor GPR35, likely via its actions on airway sensory nerves. This data is intriguing

as not only does it explain why in clinic previously developed mast cell stabilisers have been found

ineffective in the treatment of asthma, it also presents a novel target for asthma therapy. If a ligand

with an appropriate pharmacokinetic profile was developed that targeted GPR35, in theory the

beneficial effects of DSCG would be maintained without the need for repeated dosing regimens. This

is an extremely exciting prospect for the future which could lead to the development of novel asthma

therapies.

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Chapter 8

Summary and Future Studies

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8 Summary and Future Studies

8.1 Summary

The hypothesis for this body of work was “mast cells and airway sensory nerves are central to the

driving mechanisms behind the LAR”. The various experiments undertaken addressing this hypothesis

are detailed below.

The LAR is a key feature of allergic asthma and refers to a prolonged bronchoconstriction event taking

place approximately 3-8 hours following contact with allergen. It is experienced by approximately 50%

of asthma sufferers and is often used as an endpoint in clinical studies exploring new therapeutic

options (Booij-Noord, Orie & De Vries, 1971; Booij-Noord et al., 1972). Despite this, the specific

mechanisms driving the LAR have yet to be elucidated. Studies exploring this particular feature of

asthma have concluded that it is likely that the LAR is at least in part driven by inflammation. This is

largely based on the fact that the response is accompanied by cellular inflammatory influx into the

lung and that steroid treatment impacts on the response (Bousquet et al., 2000; Barnes, 2008). Others

have suggested other driving mechanisms behind the LAR such as possible roles for airway sensory

nerves (Raemdonck et al., 2012). The overall aim of this thesis was to further explore the driving

mechanisms behind the LAR using in vivo models.

Prior to commencing mechanistic investigations of the LAR it was considered necessary to validate the

clinical relevance of the in vivo models available. In asthmatic patients the LAR occurs approximately

3-8 hours following allergen exposure (Raemdonck et al., 2012) and can be attenuated using

glucocorticoid steroids. Therefore, OVA driven Brown Norway Rat and C57BL/6 mouse models of the

LAR were characterised in terms of response times and also via application of the clinically relevant

glucocorticoid steroid Budesonide. Both models demonstrated responses at time points comparable

to those seen in human disease. The application of Budesonide was successful in significantly

attenuating the responses in both model species. Overall, these studies demonstrated that both

models were displaying responses consistent with the LAR as seen in clinic. As a result, it was decided

to take both these models forward for further investigating the driving mechanisms behind the LAR.

Previous studies exploring the LAR have concluded that the response is at least in part driven by

inflammation due to the fact that the response is accompanied by cellular inflammatory influx into

the lung and that steroid treatment impacts on the response (Bousquet et al., 2000; Barnes, 2008).

The aim of this thesis was to further explore the driving mechanisms behind the LAR; therefore, it was

considered desirable to further explore the roles of key allergic effector cell types within this response.

Previous investigations exploring the roles of inflammatory cell populations in animal models of the

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LAR have yielded conflicting results. This is thought to be due to the fact that monoclonal antibodies

targeting key effector cells were employed in these studies and issues concerning the target specificity

of these antibodies exist. In order to circumvent these issues in the studies as part of this thesis, it was

decided to instead apply GM mice deficient in key allergic effector cell populations to the OVA driven

mouse model of the LAR to determine the role of these cell types within the response.

Before these investigations could commence, the phenotype of the knockout mice (CD4-/-, CD8-/-, B-

cell-/- and mast-cell-/-) was assessed with respect to lung tissue cell populations by flow cytometry and

histology. All GM strains were found to be deficient of their relevant knockout cell type and no

subsequent effects in terms of the CD4+, CD8+, B-cell and Mast cell lung populations were observed in

the non-relevant strains. For example mast cell deficient mice were found to be deficient of lung tissue

mast cells and had no significant up regulation or down regulation of lung tissue CD4+ cells, CD8+ cells

or B-cells. Baseline levels of other inflammatory cell populations (eosinophils, neutrophils, alveolar

macrophages and monocytes/tissue macrophages) were found to be comparable to those in wildtype

mice across all GM strains. Overall, it was concluded that knocking out a particular cell type via genetic

modification had no resulting effects on other lung tissue cell populations. The results of these studies

confirmed that the GM mice were suitable for application to the OVA-driven mouse model of the LAR.

Following application to the OVA-driven mouse model of the LAR the CD4-/- mice displayed a much

attenuated response and the CD8-/- animals a significantly enhanced response compared to wildtype

mice. This implicated CD4+ cells in the driving mechanisms behind the LAR and suggested CD8+ cells to

have a protective role with respect to this response. These findings were consistent with current

literature on the subject. The role of B-cells, Mast cells and IgE in the LAR were also investigated in

this manner. These knockout mice displayed responses comparable to those seen in wildtype mice,

suggesting that these cells and mediators are not involved in the driving mechanisms behind the LAR.

However, enhanced baseline responses were displayed in all three GM strains (B-cell-/-, Mast cell-/- and

IgE-/-) even though they were not sensitised to allergen which made the results from these

experiments difficult to draw conclusions from. Further investigation into the experimental protocol

suggested the phenomenon was likely attributed to the sensitisation protocol. However, for the

purpose of the investigation into the role of B-cells, Mast cells and IgE in the LAR, we could

unfortunately draw no conclusions from this particular set of experiments. It was concluded that

further work was required to delineate the role of the B-cell-Mast cell-IgE products axis in the LAR.

Further investigation into the role of the B-cell - Mast cell - IgE products axis was conducted via

application of the pharmacological tools; Compound 48/80 (a mast cell degranulating agent) and DSCG

(a mast cell stabilising compound) to the in vivo models of the LAR. Prior to this it was considered

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necessary to validate the Compound 48/80 mast cell degranulation protocol in the Brown Norway Rat

and C57BL/6 mouse. This was assessed via histologically staining mast cell contents in lung tissue

samples. The results of this showed that the treatment protocol was successful in significantly

depleting mast cell contents in the Brown Norway rat but not the C57BL/6 mouse. Due to this, it was

considered unsuitable to utilise the mouse model of the LAR to ascertain the importance of mast cells

within the response and the rat model was taken forward into the next phase of the investigation.

The mast cell depletion protocol was then applied the OVA-driven rat model of the LAR and no

attenuation of response was observed. DSCG was also applied to the rat LAR model. This resulted in

an abrogation of response. This was not surprising as DSCG has been shown to be an effective

treatment in this particular feature of asthma (Booij-Noord, Orie & De Vries, 1971; Cockcroft &

Murdock, 1987). However, the fact that depleting mast cell contents conferred no effect on the LAR

lead us to question the mechanism of action of DSCG which is traditionally referred to as a mast cell

stabilising compound. Overall, as a result of these investigations we were unable to substantiate a

definitive role for mast cells in the driving mechanisms behind the LAR; however, the possibility of

DSCG modulating the LAR via a mechanism independent of mast cells was raised. Literature on the

subject has implied DSCG to interact with sensory nerves and within the Respiratory Pharmacology

group a role for airway sensory nerves has been proposed to be central to the driving mechanisms

behind the LAR (Dixon, Jackson & Richards, 1980; Dixon & Barnes, 1989; Barnes, 1993; Jackson,

Pollard & Roberts, 1992; Norris & Alton, 1996; Raemdonck et al., 2012). It could therefore be

hypothesised that DSCG attenuates the LAR via modulation of airway sensory nerve responses. This

possibility was investigated further in the subsequent chapters of this thesis.

Previous work conducted within the Respiratory Pharmacology group suggested a role for airway

sensory nerves, the CNS and a parasympathetic cholinergic constrictor response within the driving

mechanisms behind the LAR (Raemdonck et al., 2012). A role for TRPA1 and not TRPV1 within the

response was also postulated. Within this body of work, the involvement of a cholinergic constrictor

response was concluded based upon the application of the LAMA Tiotropium to the rat model of the

LAR. However, Tiotropium has since been shown to have off target effects involving the modulation

of TRPV1 responses on airway sensory nerves (Birrell et al., 2014). Due to this it was considered

necessary to further investigate the role of the parasympathetic cholinergic constrictor response and

the roles of TRPA1 and TRPV1 in the LAR within this thesis. This was done via the application of a

second LAMA, Glycopyrrolate, and newly available inhibitors of TRPA1 and TRPV1 to the OVA-driven

rat model of the LAR.

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The application of Glycopyrrolate to the OVA-driven rat model resulted in a diminished LAR. This effect

was comparable to that previously seen with Tiotropium, and whereas Tiotropium was previously

shown to have off target effects upon TRPV1, this was not the case with Glycopyrrolate (Birrell et al.,

2014). Therefore, a role for parasympathetic cholinergic constrictor responses within the LAR could

be corroborated as a result of this investigation. The application of the TRPA1 and TRPV1 inhibitors to

the OVA-driven rat model of the LAR concluded a role for TRPA1 and not TRPV1 in the driving

mechanisms behind the response. Overall, these studies further validated the hypothesis that TRP

channels, airway sensory nerves, a central reflex arc and parasympathetic cholinergic responses are

central to the driving mechanisms behind the LAR.

The investigations as part of this thesis so far showed that DSCG (a mast cell stabiliser) modulated the

LAR but Compound 48/80 (a mast cell degranulating tool) failed to impact upon the response. This led

to the consideration that DSCG was attenuating the LAR via mechanisms which were independent of

mast cells. In the literature DSCG has been shown to interact with airway sensory nerves. A role for

airway sensory nerves in the driving mechanisms behind the LAR has previously been shown by the

Respiratory Pharmacology group and this work was further validated within this thesis. Overall this

led us to hypothesise that DSCG attenuates the LAR via actions upon airway sensory nerves. The final

part of this thesis was to investigate this hypothesis.

The direct action of DSCG on airway sensory nerves was investigated utilising isolated vagus nerve

experiments. As TRP channels were previously implicated in the LAR, the effects of DSCG on TRP

channel mediated airway sensory nerve depolarisation were assessed. DSCG was found to inhibit

TRPA1, but not TRPV1, mediated depolarisation and these findings were translated into human vagal

tissue. This allowed us to conclude that DSCG inhibits TRPA1 mediated depolarisation of airway

sensory nerves. The next part of the investigation was to explore possible mechanisms of action

associated with this.

The literature on the subject of DSCG receptor interactions suggested DSCG to be an agonist of the G-

protein coupled receptor GPR35 (Yang et al., 2010; Guo et al., 2008). Therefore, it was hypothesised

that DSCG could modulate TRPA1 driven sensory nerve activity via the activation of GPR35. This was

investigated by applying GPR35 agonists to the isolated vagus nerve in parallel with DSCG and their

effect on TRPA1 driven depolarisation examined. The results from these experiments showed GPR35

agonists to confer comparable effects to DSCG. The application of a GPR35 antagonist successfully

blocked the inhibition of depolarisation by DSCG, confirming that GPR35 is central to the effect of

DSCG on airway sensory nerve responses.

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The next study was to examine if the findings in the isolated vagus nerve were applicable to the LAR.

DSCG was applied to the OVA-driven rat model of the LAR in parallel with a GPR35 antagonist or

vehicle. The results of this study showed that the DSCG mediated attenuation of the LAR was blocked

by the GPR35 inhibitor, thus providing evidence that DSCG inhibits the LAR via its action on GPR35.

The previous results showed that DSCG conferred effects on the LAR via an action on GPR35; however,

they did not directly demonstrate that these effects were occurring via an action on airway sensory

nerves. Due to this, the final investigation as part of this thesis was to establish the role of GPR35 in

DSCG mediated inhibition of airway sensory nerve firing in vivo using a single fibre afferent recording

model. The results from these studies showed that DSCG attenuated TRPA1 induced firing of C-fibre

afferents and this could be blocked by a GPR35 antagonist, thus demonstrating that DSCG attenuates

TRPA1 driven airway sensory nerve responses via the G-protein coupled receptor, GPR35.

DSCG is traditionally referred to as a mast cell stabiliser and has been shown to be effective in the

treatment of features of asthma such as the LAR. However, due to the fact that it has to be

administered 4 times daily, its use is limited and alludes to a poor pharmacokinetic profile (Patel et

al., 1986; Yahav et al., 1988; Kato et al., 1999). Other mast cell stabilisers have also shown little efficacy

in clinic (Sheard & Blair, 1970). The results obtained as part of this thesis shown DSCG to confer actions

on airway sensory nerves via the GPR35 receptor to impact upon the LAR. This data is exciting as not

only does it suggest why previously developed mast cell stabilisers have been found to be ineffective

in clinic, it also presents a novel target for future asthma therapy.

Overall, this body of work provides new insights into the driving mechanisms behind the LAR and may

have uncovered the mystery of the mechanism of action of DSCG. From this, new therapeutic options

for asthmatics may become available in the future. In addition, the hypothesis of “mast cells and

airway sensory nerves are central to the driving mechanisms behind the LAR” was addressed. Central

roles for both mast cells and airway sensory nerves within the LAR were concluded and so the

hypothesis could be accepted. Further work surrounding this hypothesis is detailed below (see section

8.3).

8.2 Thesis Limitations

Within this thesis there are a number of limitations associated with the experimental techniques

employed, some of which have been outlined previously.

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The use of OVA driven models of allergic asthma has received criticism in the past for several reasons.

Firstly, OVA as a clinically relevant allergen prevalent in human disease is questionable. Secondly,

tolerance to OVA as an inducer of allergic asthmatic responses has been widely reported as a practical

disadvantage to the model (Van Hove et al., 2007; Swirski et al., 2002). Due to this, difficulties arise

when using OVA to model chronic models of allergic asthma. Thirdly, the model requires systemic

sensitisation along with AlumTM adjuvant. Atopy in asthma is associated with the lungs and so systemic

sensitisation is considered to be less clinically relevant than other topical routes. The use of an

adjuvant is also not considered to be appropriate with respect to clinical asthma (See Introduction

section 1.10.2). Models utilising more disease relevant allergens such as HDM are currently under

development. These models have the added advantage that adjuvants are not required to induce

features of allergic asthma and topical application protocols are employed. So far success has been

achieved in modelling features of asthma such as AHR, airway inflammation and remodelling events,

however, none of these models display the LAR (Verheijden et al., 2015; Sivapalan et al., 2014;

Mariñas-Pardo et al., 2014). Due to this, the use of a HDM driven model in this thesis was not a

possibility.

The use of the isolated vagus nerve recording technique allowed for a cost effective, relatively high

throughout pharmacological evaluation of sensory nerve activity. Another added advantage to this

technique is that findings can be replicated in human donor tissue allowing for translational data.

However, there are limitations associated with this technique. The trunk of the vagus nerve carries all

types of afferent nerve fibres in addition to parasympathetic nerves and fibres innervating other organ

systems such as the heart and the GI tract. This means that assessment of individual fibre types and

the activity of specifically airway terminating nerves is not a possibility. The effect on the isolated

nerve trunk does not necessarily represent what is happening at the nerve terminals.

The in vivo single fibre afferent recording model was advantageous in that it did allow for

discrimination between airway and non-airway terminating nerve responses. However, this technique

is relatively labour intensive and costly as only one nerve fibre can be examined in a single animal.

Another major disadvantage to the use of this technique in this thesis is that at present the model is

established in the Guinea-Pig and not the Brown Norway rat where the initial findings in this thesis

were made. Unfortunately, the time constraints of the thesis did not allow for us to set up,

characterise and validate the model in the Brown Norway Rat. As part of this thesis, key isolated vagus

nerve recording experiments were paralleled in the Guinea-Pig in order to allow for the transition of

species, however, it would have been more desirable to progress straight into a Brown Norway Rat in

vivo single afferent recording model.

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8.3 Future Studies

8.3.1 Further investigation into the Mechanism of Action of DSCG

8.3.1.1 Further Work to Assess the Translational Nature of the Mechanism of Action

of DSCG

Within this thesis DSCG was shown to confer actions on airway sensory nerves via the GPR35 receptor

to impact upon the LAR. This was deduced using in vivo models of allergic asthma. The initial in vitro

sensory nerve depolarisation experiments were, however, conducted on the vagus nerve tissue from

naive animals and non-asthmatic donors. In order to further show that the results translate into

human disease it would be desirable to repeat key experiments in vagus nerve tissue from allergic

asthmatic patients. If the tissue was not available, as is often the case, it would at least be considered

appropriate to repeat key experiments on tissue from models of allergic asthma.

8.3.1.2 Explore the downstream signalling pathways associated with GPR35

Having established that DSCG inhibits TRPA1-driven sensory nerve activation through GPR35, it would

be desirable to investigate the downstream signalling pathways associated with this. It has been

reported that activation of GPR35 triggers a Gi dependent inhibition of N-type calcium channels in rat

neurons (Guo et al., 2008). Other studies have reported that Gi can be inactivated by dosing rats with

Pertussis toxin (Paur et al., 2012). Therefore, it would be proposed that Brown Norway rats would be

dosed with Pertussis toxin by adapting the protocol used in Paur et al (2012). The animals would then

be culled, their vagus nerves harvested and the ability of DSCG to inhibit TRPA1 induced nerve

depolarisation assessed using isolated vagus nerve experiments.

8.3.1.3 Profile the effects of DSCG and GPR35 on mast cells

In this thesis we highlighted the possibility that DSCG could exert its actions on airway sensory nerves

via GPR35. DSCG is traditionally considered to be a mast cell stabiliser and we did not rule out this

possibility within this body of work. Therefore, it would be desirable to consider if DSCG stabilises

mast cells via GPR35. Mast cells would be extracted from the lungs of human donors, cultured and

gene and protein expression levels of TRPA1 and GPR35 assessed. If present on mast cells then the

functional impact of DSCG and GPR35 agonists and antagonists would be tested using mast cell

degranulation assays.

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8.3.2 Further investigate and validate in vivo models

8.3.2.1 Develop the Brown Norway rat in vivo single afferent recording model

Within this thesis a disadvantage was that the in vivo single afferent recording model was in the

Guinea-Pig despite the LAR results being attained using the Brown Norway rat. The time constraints

of the thesis did not allow for us to set up, characterise and validate the model in the Brown Norway

Rat. It would therefore be desirable to do this in the future. It would then be possible to assess the

mechanism of action of DSCG and GPR35 in single fibre afferents using a model species consistent

with that of the LAR.

8.3.2.2 Use an alternative double chamber WBP to further validate the LAR data

Throughout the studies within this thesis single chamber WBP and Penh was used as a measure of

lung function. As previously described (general methods section 2.5.1) there are currently no

alternative methods to measure lung function in a conscious modelling system without using restraint.

The main disadvantage to the single chamber WBP is that the nasal inspiration and thoracic

displacement due to inspiration are measured within the same chamber. Double chamber WBP allows

for the measurement of thoracic and nasal flow simultaneously. This technique can be used to

measure specific airway resistance (sRaw) which is thought to be a good indicator and a more accurate

measure of lung function. A disadvantage is that the animal must be restrained using this method and

so snapshots through time would be attainable as opposed to continuous recording. Nevertheless,

confirmation of Penh measurements by sRaw would further strengthen the LAR data.

8.3.2.3 Investigate the role of other TRP channels in the LAR models

Within this thesis the roles of TRPA1 and TRPV1 in the LAR were investigated. Within the literature

further subtypes of TRP channel are being discovered and roles within allergic disease postulated.

TRPV4 has been shown to have roles in thermosensitisation, mechanosensitisation and osmotic stress

(Watanabe et al. 2003; Strotmann et al. 2000; Suzuki et al. 2003; Liedtke & Friedman 2003). The

bronchial fluid of asthmatics has been reported to be hypotonic due to airway remodelling and this

implies a role for TRPV4 in asthma (Liedtke & Friedman, 2003b). TRPM8 is a thermosensor of

temperatures approximately 15-28 °C (McKemy, Neuhausser & Julius, 2002; Zhou et al., 2011).

Inhalation of cold air has been shown to trigger symptoms of asthma such as bronchoconstriction,

cough, plasma extravasation and mucosal secretion (Carlsen & Carlsen, 2002). TRPM8, being

a thermosensor, has been linked to this (Li et al., 2011a; Fisher, 2011). It has also been reported to be

involved in cold triggered mast cell activation and so further study could implicate it in inflammatory

processes in diseases such as asthma (Cho et al., 2010a). It would be interesting to investigate the

162

roles of these TRP channels in the LAR by applying antagonists of these channels to in vivo models of

the LAR should they become available in the future.

8.3.3 Further Exploration of TRPA1

8.3.3.1 The Role of TRPA1 in Airway Smooth Muscle Contraction

Published data suggests that TRPA1 agonists can cause direct constriction of guinea pig ASM (Andre

et al., 2008). It would therefore be appropriate to assess the effect of TRPA1 ligands on isolated airway

tissue by performing organ bath experiments as done by Buckley et al 2011 (Buckley et al., 2011).

Translational data could also be attained using human tracheal and bronchial strips.

8.3.3.2 Assess if TRPA1 channels are involved in the release of

acetylcholine from parasympathetic ganglia

TRPA1 ligands have been shown to modulate sensory nerve activity. However, it is currently unknown

whether they also interact with other populations of nerve fibres, specifically, the parasympathetic

cholinergic constrictor nerves that innervate the airways. There is a possibility that TRPA1 has a role

in the activation of parasympathetic nerves, the release of ACh or a role in the actions of ACh on M3

receptors on smooth muscle. It would be desirable to assess the role of TRPA1 on ACh release using

TRPA1 ligands and isolated airway tissue in an organ bath setting. The contractile responses of the

isolated airway tissue in response to endogenously released ACh initiated by electrical field

stimulation (EFS) to would be measured and compared to contractile responses to exogenous ACh.

EFS induced ACh release would also be measured using a tritiated radiolabelling technique as

described in Patel et al (1995) (Patel et al., 1995b).

163

8.3.4 Investigate whether the same mechanisms are involved in the LAR on a

background of cigarette smoke exposure under conditions where

steroids have limited efficacy

Preliminary data within the Respiratory Pharmacology group has suggested that allergen induced LAR

in Cigarette Smoke exposed mice is slightly increased in size and resistant to steroid treatment

compared with air exposed mice (See figure 8.1). This could be a good model with which to study

cigarette smoke induced steroid resistance in allergic asthma. The results obtained in this thesis

provided further evidence for airway sensory nerves in the driving mechanisms behind the LAR. Key

experiments could be carried out in order to asses if the same mechanisms are involved in driving the

LAR on a background of cigarette smoke. If the steroid resistant LAR observed in the Cigarette Smoke

exposed animals is mediated by the same mechanisms then targeting airway sensory nerves in these

patients could confer therapeutic effects where steroids have limited efficacy.

Figure 8.1: Steroid resistance in a murine model of asthma. Mice were sensitised to OVA plus alum

i.p, days 0 and 14. On days 26-28, mice were exposed to air or cigarette. 1 hour after final CS

challenge, mice received vehicle or budesonide (3 mg/kg, p.o.), followed by (1 hour later) a single

intratracheal challenge with saline or ovalbumin and Penh recorded (overnight). (Work carried out

by MA Birrell, KRG Raemdonck and N Dale)

164

8.4 Concluding Remarks

In summary, the data presented in this thesis provides insight into the driving mechanisms behind the

LAR. CD4+ cells were found to be central to the driving mechanisms behind the response, with CD8+

cells conferring protective effects. Further investigation into the role of the mast cell – B-cell – IgE axis

using pharmacological tools alluded to the fact that DSCG may work via mechanisms independent of

mast cells. When exploring this further, a central role for airway sensory nerves within the LAR was

concluded, with DSCG exerting effects on sensory nerve channels. Further pharmacological

investigation showed that DSCG confers effects on airway sensory nerves via the G-protein coupled

receptor, GPR35. Overall, it could be concluded that DSCG inhibits the LAR via GPR35 present on

airway sensory nerves. This is an exciting finding as not only does it suggest why previously developed

mast cell stabilisers have been found to be ineffective in clinic, it also presents a novel target for future

asthma therapy. Future investigations highlighted in section 8.3 will hopefully provide further insight

into the mechanisms behind the LAR and further targets for future asthma therapy.

165

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9 Appendix

9.1 Chemicals and Reagents

Drug/Reagent Source

2 N H2SO4 VWR (UK)

Acrolein Sigma-Aldrich (UK)

Albumin from Chicken Egg White (OVA) Sigma-Aldrich (UK)

AlumTM ThermoFisher Scientific (US)

BD OptEIATM set for mouse immunoglobulin E BD Biosciences (US)

Budesonide Sigma-Aldrich (UK)

Capsaicin Sigma-Aldrich (UK)

Collagenase Roche Diagnostics (Germany)

Compound 48/80 Sigma-Aldrich (UK)

Disodium Cromoglycate Sigma-Aldrich (UK)

DMSO Sigma-Aldrich (UK)

Dnase Roche Diagnostics (Germany)

Eosin-Phloxine solution Sigma-Aldrich (UK)

Ethanol VWR (UK)

FBS Invitrogen (UK)

Fixable near-IR dead cell stain kit for 633/635nm

excitation Invitrogen (UK)

Formaldehyde Sigma-Aldrich (UK)

Glycopyrrolate Sigma-Aldrich (UK)

Isoflurane Abbott laboratories (UK)

Janssen130 Almirall (Spain)

Krebs-Henseleit (KH) solution (mM: NaCl 118; KCl 5.9;

MgSO4 1.2; CaCl2 2.5; NaH2PO4 1.2; NaHCO3 25.5

and glucose 5.6; pH 7.4) Sigma-Aldrich (UK)

Kynurenic Acid Sigma-Aldrich (UK)

Medical Oxygen BOC Industrial Gases (UK)

methycellulose Sigma-Aldrich (UK)

ML145 (GPR35 inhibitor) Sun Pharma Ltd (India)

Pamoic Acid Sigma-Aldrich (UK)

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PBA (PBS containing 0.1% (wt/vol) sodium azide and

1% (wt/vol) BSA) Sigma-Aldrich (UK)

PBS Sigma-Aldrich (UK)

Rat anti-mouse CD11b (PerCP) conjugated mAb eBioscience (UK)

Rat anti-mouse CD11c (APC) conjugated mAb BD Pharmingen (UK)

Rat anti-mouse CD19 (FITC) conjugated mAb BD Pharmingen (UK)

Rat anti-mouse CD4 (PerCP) conjugated mAb BD Pharmingen (UK)

Rat anti-mouse CD8 (APC) conjugated mAb BD Pharmingen (UK)

Rat anti-mouse F4/80 (PE) conjugated mAb eBioscience (UK)

Rat anti-mouse Ly6G (FITC) conjugated mAb BD Pharmingen (UK)

Rat anti-mouse SiglecF (PE) conjugated mAb BD Pharmingen (UK)

RPMI Invitrogen (UK)

Saline Fresenius Kabi (UK)

Sodium pentobarbitone Merial Animal Health (UK)

Substrate solution (1:1 mixture of H2O2 and

tetramethylbenzidine) R&D Systems (US)

Tiotropium Bromide Sigma-Aldrich (UK)

Toluidine blue histological stain Sigma-Aldrich (UK)

Tween80 Sigma-Aldrich (UK)

Urethane Sigma-Aldrich (UK)

Vecuronium Bromide Sigma-Aldrich (UK)

Wright-Giemsa stain Sigma-Aldrich (UK)

XEND0501 Ario Pharma Ltd (UK)

Zaprinast Sigma-Aldrich (UK)

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