Phd Thesis – Title Page Nayyar Ahmed

PhD Thesis – Title Page Nayyar Ahmed

Identification and Characterisation of a Novel Interleukin-4 Receptor Antagonist for Allergy Treatment ______

Nayyar Ahmed

B.Biomed.Sc. (Honours)

Submitted in fulfilment of the requirements for the degree of

Doctor of Philosophy

Deakin University

February, 2015

PhD Thesis – Acknowledgements Nayyar Ahmed

Acknowledgements

Firstly, I would like to thank Associate Professor Cenk Suphioglu for being such a caring and supportive person throughout my PhD. Your friendliness surpasses your duty as a professor and no matter how many words I spill on this paper, they will never be enough. Thank you for being so patient with my work. Thank you once again, for all your efforts and constant guidance throughout my candidature. I am also grateful to you for giving me the opportunity to be a part of the NARL laboratory. I wasn’t sure if I wanted to pursue a career as a research scientist, however after joining your laboratory, you have inspired me and increased my passion for scientific research. Ever grateful to you!

I would like to thank my co-supervisors Professor Colin Barrow and Professor Leigh

Ackland for serving as my co-supervisors for my candidature. I also want to thank you for your brilliant comments and suggestions. I would also like to thank Professor Andrew

Sinclair and Dr. Philip Taylor for their unprecedented support for my research.

My sincere thanks to Nadia Sadli, who helped me with tissue culture work. Thanks Nadia for being such a caring friend and a wonderful lab mate. And thanks to all the members of

NARL, God Bless you all!

My very special thanks to Pathum (MEYA). You have been like a brother to me, thanks a lot for making my candidature such a joy. I would be privileged, if I ever get the opportunity to work with you again! I’ll miss the good times we’ve spend together. Thanks for all the favours you have done for me. You are nothing less than an angel.

PhD Thesis – Acknowledgements Nayyar Ahmed

My sincere gratitude goes to the Deakin University security guards, Tom, Dayle, Alex, Rohit and Shiv. Thanks for all the good times and late-night chats. Thank you for making me feel safe while working in the lab around the clock. I am ever grateful to you.

I would like to express my appreciation to my beloved wife Mashal Pall who spent sleepless nights with me and was always my support in moments when there was no one to answer my queries. Thank you for your timely support and your words of encouragement. I would like to extend my sincere and heart-filled thanks to my wife’s parents (Waseem Pall & Farhat Pall) and siblings (Mehwish Pall, Sabahat Pall and Zoha Pall). One can only wish for such wonderful in-laws like yourselves. Thanks for making my wedding a success and for all the kind favours you did for me. I am forever grateful to you all.

Last but not the least, I want to extend my gratitude and love to my family members, my father Shams Daud (Abbu), my beloved mother Attiya Shams (Ammi), my brother Nasir

Ahmed and his wife Ajam Ahmed. Thank you Abbu & Ammi for your encouraging words and unconditional love for which I am ever thankful and indebted to you. Your prayer for me was what sustained me thus far. Thanks for your emotional support; you guys have stood by me in my times of weakness. And thank you for waiting so patiently for me to finish my studies. I have been away from you both far too long.

Finally I thank my God Almighty for letting me through all the difficulties. I have experienced Your guidance day by day. You are the one who let me finish my degree. I will keep on trusting You for my future. Thank you, Lord.

PhD Thesis – List of Publications Nayyar Ahmed

List of Publications

Journal Articles: Ahmed N, Dhanapala P, Suphioglu C. Identification and characterization of a novel IL-4 receptor α chain (IL-4Rα) antagonist to inhibit IL-4 signalling. Cell Physiol Biochem 2015; 36:831-842 (Chapter 2 & 3 results published)

Ahmed N, Dhanapala P, Sadli N, Barrow CJ, & Suphioglu C. Mimtags: The use of phage displaytechnology to produce novel protein-specific probes. Journal of Immunological Methods. 2014; 405(0):121-9 (Chapter 2 results published).

Ahmed N, Barrow CJ, Sinclair A, Suphioglu C. Discovering the effects of omega-3 and omega-6 fatty acids on allergy using a HEK-Blue cell-line. International Journal of Molecular Sciences (Chapter 4 results submitted for publication).

Ahmed N, Barrow CJ, Adcock J & Suphioglu C. Discovering the effects of CoQ10 and fatty acid derived resolvins on allergy using a HEK-Blue cell-line. International Journal of Molecular Sciences (Chapter 5 results under preparation).

Taylor PE, Ahmed N, Suphioglu C. Relationship between ryegrass pollen count and weather variables in regional and urban Victoria, Australia. (Chapter 6 results under preparation)

Book Chapters: Ahmed N, Dhanapala P, Suphioglu C., (2014). Nothing to be sneezed at: Th2 cytokines as novel therapeutic targets for the production of anti-allergy agents, Bentham Science Publishers (eds), frontiers in clinical drug research anti-allergy agents (Chapter 1 literature review submitted).

Sadli N, Ahmed N, Ackland M.L, Sinclair A, Barrow C & Suphlioglu, C., (2011). Effect of zinc and DHA on expression levels and post-translational modifications of histones H3 and H4 in human neuronal cells, in Raymond, Chuen-Chung Chang (eds), Neurodegenerative diseases - processes, prevention, protection and monitoring, pp. 141-164, InTech, Rijecka, Croatia.

Conference Papers (abstracts published): Presented at the European Academy of Allergy and Clinical Immunology (EAACI) conference

Ahmed N, Dhanapala P, Sadli N, Barrow CJ, & Suphioglu C, 2014, ‘Mimtags: the use of phage display technology to produce novel protein specific probes’, Allergy, vol. 69, p. 298.

PhD Thesis – List of Publications Nayyar Ahmed

Ahmed N & Suphioglu C, 2014, ‘Discovering the effects of omega-6 fatty acids on allergy using a HEK-Blue cell line’, Allergy, vol. 69, p. 534.

Ahmed N, Ramsbottom K, Carr S & Suphioglu C, 2013, ‘Identification and characterization of IL-4 and IL-4 receptor as novel intervention targets for allergy treatment’, Allergy, vol. 68, p. 470.

Ahmed N, Barrow CJ, Adcock J, Sinclair A & Suphioglu C, 2013, ‘Importance of CoQ10 and omega-3 fatty acids as novel therapeutics for allergy’, Allergy, vol. 68, p. 344.

Ahmed N, Dhanapala P, Carr S & Suphioglu C, 2011, 'Characterisation of a novel IL-4 receptor antagonist for allergy treatment', Allergy, vol. 66, p. 509-10.

Presented at the Australasian Society for Clinical Immunology and Allergy (ASCIA) conference

Ahmed N & Suphioglu C, 2014, ‘Discovering the effects of omega-3 and omega-6 fatty acids on allergy using a HEK-Blue cell line’, Internal Medicine Journal, vol. 44, p. 1.

Ahmed N, Dhanapala P & Suphioglu C, 2013, 'Discovering the effects of Omega-3 fatty acids on allergy using a HEK-Blue cell line', Internal Medicine Journal, vol. 43, p. 1.

Ahmed N, Dhanapala P & Suphioglu C, 2013, 'Identification and characterisation of a novel cytokine IL-13 antagonist for allergy treatment', Internal Medicine Journal, vol. 43, p. 1.

Ahmed N, Dhanapala P & Suphioglu C, 2012, 'Identification and characterization of a novel IL-4 receptor antagonist for allergy treatment', Internal Medicine Journal, vol. 42, p. 1.

vii

PhD Thesis – Table of Contents Nayyar Ahmed

Tables of Contents

Page number

Title page……………………………………………………………………….i

Deakin University – ACCESS TO THESIS- A………………………………..ii

Candidate Declaration Form…………………………………………………...iii

Acknowledgements…………………………………………………………....iv

List of Publications…………………………………………………………….v

Table of Contents……………………………………………………………...viii

Abstract………………………………………………………………………..xviii

List of Abbreviations…………………………………………………………..xxi

Chapter 1 – Literature review………………………………………………….1

1.1 Introduction of Allergy and Allergens…………………………………...2

1.1.1 Sources of Allergens………………………………………………….2

1.1.2 Symptoms of Allergy…………………………………………………4

1.1.3 Rising Incidence/Trends in Allergic Disease…………………………4

1.2 Overview of Allergy as a Disease: Atopy in Allergic Individuals………5

1.2.1 Underlying Causes of Allergy in Atopic individuals…………………5

1.2.2 Diagnostic Tests for Allergic Conditions……………………………..9

1.2.2.1 RAST…………………………………………………………....9

1.2.2.2 Skin Prick Tests…………………………………………………9

1.2.2.3 Blood Specific IgE Testing……………………………………..9

1.2.3 Current Available Treatments………………………………………..10

1.3 Immune Response: Allergic vs. Non-Allergic Response………………..11

1.3.1 Non-Allergic Immune Response……………………………………..11 viii

PhD Thesis – Table of Contents Nayyar Ahmed

1.3.2 Allergic Immune Response…………………………………………..12

1.3.2.1 Humoral Response in Allergic Individuals…………………….12

1.3.2.2 Cell-Mediated Response in Allergic Individuals…………………..14

1.4 Role of Cytokines in Immunity…………………………………………..14

1.4.1 Cytokines IL-4 and IL-13…………………………………………….15

1.4.2 Comparative Roles of IL-4/IL-13 with IL-4Rα chain………………..17

1.5 IL-4Rα as an Important Target for Allergy Treatment………………..19

1.5.1 Anti-IL-4R Monoclonal Antibody…………………………………...19

1.5.2 Soluble IL-4 Receptor (sIL-4R)……………………………………...21

1.5.3 Targeting IL-4R with Hybrid Peptides………………………………21

1.5.4 Further Analysis of Blocking IL-4R………………………………....22

1.5.5 Current Available Treatments for Targeting IL-4

and IL-13 Cytokines……………………………………………………….,22

1.5.5.1 Specific Immunotherapy (SIT)…………………………………23

1.5.5.2 Modified Recombinant Vaccines…………………………….…25

1.5.5.3 Allergoids………………………………………………….……25

1.5.5.4 Adjuvants……………………………………………………….26

1.5.5.5 Peptide Immunotherapy………………………………………...26

1.5.5.6 Plasmid Deoxyribonucleic Acid (DNA) Immunotherapy……...27

1.5.5.7 DNA Vaccines………………………………………………….28

1.5.5.8 Toll-Like Receptor 9 (TLR-9)………………………………….28

1.5.5.9 Anti-IgE………………………………………………………...29

1.5.5.11 Chinese Herbal Medicine……………………………………..30

1.5.5.12 Anti-IL-4 and IL-13 Antagonists……………………………..31

1.5.5.13 Piktrakinra………………………………………………….....33 ix

PhD Thesis – Table of Contents Nayyar Ahmed

1.6 Phage Display Technology…………………………………………….....33

1.6.1 Mimtags the Use of Phage Display Technology

to Produce Novel Protein-Specific Probes………………………………….36

1.6.1.1 Mimtags…………………………………………………………38

1.6.2 Testing the Peptide’s Inhibitory Efficiency in Tissue Culture…...... 39

1.6.3 HEK Cytokine Reporter Cells………………………………………...39

1.6.4 Comparative Role of Ramos B Cells and HEK-Blue Cells…………..40

1.7 Effects of Dietary Antioxidant and Fatty Acids on Allergy:

Connecting the Dots…………………………………………………………...42

1.7.1 Inhibition of Allergy Pathway Using n-3 Fatty Acids

and Vitamin E Antioxidant…………………………………………………42

1.7.2 Discovering the Effects of Omega-6 AA on Allergy………………...45

1.8 Anti-inflammatory Effects of Resolvins and Coenzyme Q10

on Allergy…………………………………………………………………...... 47

1.8.1 Role of CoQ10 in Allergy…………………………………………….47

1.8.2 Role of Resolvins in Allergy………………………………………....48

1.8.3 What is the Resolution Phase of Acute Inflammation?...... 50

1.8.4 Resolving Inflammation…………………………………………...... 51

1.9 Pollen Allergy…………………………………………………………...... 53

1.9.1 Epidemiology of Pollen Allergy…………………………………...... 53

1.9.2 Overview of Ryegrass Pollen Induced Allergy…………………...... 54

1.9.3 Diseases Associated with Ryegrass Pollen Allergy……………….....56

1.9.4 Diagnosis of Grass Pollen Protein Extracts……………………….....57

1.9.5 Grass Pollen Count and Meteorological Data……………………...... 57

1.9.6 Treatment for Pollen Allergy………………………………………...58 x

PhD Thesis – Table of Contents Nayyar Ahmed

1.9.7 Deakin AIRwatch: Pollen Counting and Forecasting Facility…….....60

1.11 Overall Aims and Objectives of PhD Studies………………………….60

1.12 Expected Outcomes of Study………………………………………...... 61

Chapter 2 - Identification and Characterization of a Novel IL-4R

Antagonist for Allergy Treatment…………………………………………...63

2.1 Introduction……………………………………………………………….64

2.2 Materials and Methods…………………………………………………...66

2.2.1 General Description of the M13 Phage Library……………………...66

2.2.2 Biopanning…………………………………………………………...66

2.2.3 Amplification and Identification of Eluted Phage Clones…………....68

2.2.4 Sequencing of Phage DNA: Rapid Purification……………………...68

2.2.5 M13 Binding ELISA………………………………………………....70

2.2.6 Synthesis of the Biotinylated Peptide………………………………...71

2.2.7 Direct ELISA……………………………………………………...... 71

2.2.8 Inhibition ELISA……………………………………………………..72

2.2.9 Statistical Analysis…………………………………………………...73

2.3 Results……………………………………………………………………..73

2.3.1 Phage Display Biopanning…………………………………………...73

2.3.2 M13 ELISA with Three Homologous M13 phage clones………...... 75

2.3.3 Direct ELISA……………………………………………………...... 75

2.3.4 Inhibition ELISA……………………………………………………..79

2.4 Discussion………………………………………………………………….81

2.4.1 Phage Display………………………………………………………...81

2.4.2 M13 Binding ELISA with N4, N5 and N7 Phage Clone…………….82 xi

PhD Thesis – Table of Contents Nayyar Ahmed

2.4.3 Direct ELISA and Inhibition ELISA………………………………...83

2.4.4 Conclusion……………………………………………………………84

Chapter 3 - Expression of HEK-Blue IL-4/IL-13 Reporter Cell-line and Further Characterization of the IL-4Rα, IL-4 and IL-13

Peptide Antagonist in-vitro………………………………………………...... 85

3.1 Introduction………………………………………………………………..86

3.2 Materials and Methods…………………………………………………....88

3.2.1 HEK-Blue™ IL-4/IL-13 Cells.………………………………...... 88

3.2.2 Synthesis and Use of Peptides N1, P9 and K1………………...... 89

3.2.3 HEK-Blue Cell Line Inhibition Assay with N1 Peptide……………..89

3.2.4 HEK-Blue Cell Line Inhibition Assay with P9

and K1 Peptide……………………………………………………………...90

3.3 Results……………………………………………………………………...91

3.3.1 HEK-Blue Cell Line Inhibition Assay with N1 Peptide…...... 91

3.3.2 HEK-Blue Cell Line Inhibition Assay with P9 Peptide……………...93

3.3.3 HEK-Blue Cell Line Inhibition Assay with K1 Peptide……………..95

3.4 Discussion………………………………………………………………….97

3.4.1 Characterization of Novel Peptide Antagonists

Using a HEK-Blue Cell Line……………………………………………….97

3.4.2 Conclusion……………………………………………………...... 98

Chapter 4 - Discovering the Effects of Omega-3 and

Omega-6 Fatty Acids on Allergy Using a HEK-Blue Cell Line……………..100

4.1 Introduction………………………………………………………………101 xii

PhD Thesis – Table of Contents Nayyar Ahmed

4.2 Materials and Methods…………………………………………………..103

4.2.1 Cell Culture, PUFA and Vitamin E Solutions………………………103

4.2.2 HEK-Blue Cell Viability Test Using Cytokines

IL-4 and IL-13……………………………………………………………..105

4.2.3 Time-course Assay Using Cytokine IL-4 and IL-13………………...106

4.2.4 Statistical Analysis…………………………………………………..107

4.3 Results……………………………………………………………………..107

4.3.1 HEK-Blue Cell Viability Test Using Cytokine

IL-4 and IL-13……………………………………………………………..107

4.3.2 HEK-Blue Time-course with Fatty Acid and

Vitamin E Treatment…...... 111

4.4 Discussion…………………………………………………………………114

4.4.1 Fatty Acids and SEAP Secretion…………………………………….114

4.4.2 Importance of Arachidonic Acid in Allergy………………………....115

4.4.3 Conclusion…………………………………………………………...116

Chapter 5 - Discovering the Effects of CoQ10 and

Resolvins on Allergy Using a HEK-Blue Cell Line………………………….117

5.1 Introduction………………………………………………………………118

5.2 Materials and Methods…………………………………………………..120

5.2.1 Cell Culture, Resolvins and CoQ10…………………………………120

5.2.2 HEK-Blue Detection Assay Using Cytokines IL-4 and IL-13……....120

5.2.3 Time-course Assay Using Cytokine IL-4 and IL-13………………...121

5.2.4 Statistical Analysis…………………………………………………..122

5.3 Results…………………………………………………………………….122 xiii

PhD Thesis – Table of Contents Nayyar Ahmed

5.3.1 HEK-Blue Detection Assay Using Cytokines IL-4 and IL-13……...122

5.3.2 HEK-Blue Time-course with Resolvins and CoQ10 Treatment…….126

5.4 Discussion…………………………………………………………………129

5.4.1 CoQ10 and Resolvins………………………………………………..129

5.4.2 Significance of Expression of SEAP/IgE Secretion…………………131

5.4.3 Conclusion…………………………………………………………...131

Chapter 6 - Relationship between Ryegrass Pollen Counts and Weather Variables in Regional Victoria, Australia……………………..133

6.1 Introduction……………………………………………………………....134

6.2 Materials and Methods…………………………………………………..135

6.2.1 Establishment of the Air Sampler…………………………………...135

6.2.2 Preparation of Slides………………………………………………...135

6.2.3 Changing the Burkard Air Sampler Using the 24 hour Drum………138

6.2.4 Preparing Slides for Pollen Counting and Examining

Using an Olympus BX50 Microscope…………………………………….140

6.2.5 Calculations for Pollen per m3 of Air………………………………..141

6.3 Results……………………………………………………………………..143

6.3.1 Ryegrass Pollen……………………………………………………...143

6.3.2 Weather Variables and Grass Pollen Count (2012-2013)…………...145

6.3.3 Weather Variables and Grass Pollen Count (2013-2014)…………...147

6.3.4 Weather Variables and Grass Pollen Count (2014-2015)…………...149

6.4 Discussion…………………………………………………………………151

6.4.1 Seasonal Changes in Grass Pollen Count……………………………151

6.4.2 Thunderstorm Allergy in Victoria, Australia………………………..153 xiv

PhD Thesis – Table of Contents Nayyar Ahmed

6.4.3 Ruptured vs. Intact Ryegrass Pollen…………………………………153

6.4.4 Conclusion…………………………………………………………...155

Chapter 7 – General Discussion……………………………………………...156

7.1 General Discussion……………………………………………………….157

7.2 Overview of IL-4Rα……………………………………………………...157

7.2.1 Phage Display……………………………………………………….158

7.3 Mimtags: The use of Phage Display Technology to

Produce Novel Protein-Specific Probes……………………………...... 160

7.3.1 The use of Phage Display in Isolating Mimtags as

Protein-Specific Probes…………………………………………………...160

7.3.2 Probing of a Conformational Epitope on Target Protein

with Mimtag...... 162

7.3.3 Mimtag Technology………………………………………………...164

7.3.4 Further Applications of Mimtag Technology……………………….165

7.4 Inhibition of STAT6 Phosphorylation Pathway in

HEK-Blue Cells Using Peptides as Antagonists……………………………166

7.4.1 Preferential Use of Peptides as Antagonists………………………...166

7.4.2 Importance of Inhibitory Values at 50% (IC50) for

Inhibition Studies………………………………………………………….167

7.5 Further Analysis of n-3 and n-6 PUFA…………………………………167

7.5.1 Production of Inflammatory Prostaglandins………………………...168

7.6 The Missing Link: Inflammation and Allergy………………………….169

7.7 Deakin AIRwatch Project………………………………………………..171

7.7.1 Limitations of the Deakin AIRwatch Project………………………..172 xv

PhD Thesis – Table of Contents Nayyar Ahmed

7.8 Future Directions…………………………………………………………174

7.8.1 Future Directions for IL-4Rα, IL-4 and IL-13 Inhibition…………...174

7.8.2 Future Directions for Fatty Acid, CoQ10 and Resolvins……………177

7.8.3 Pollen Sampling……………………………………………………..178

References…………………………………………………………………….179

List of Tables

Table 2.1……………………………………………………………………….74

Table 2.2……………………………………………………………………….75

Table 6.1………………………………………………………………………143

List of Figures

Figure 1.1………………………………………………………………………3

Figure 1.2………………………………………………………………………6

Figure 1.3………………………………………………………………………8

Figure 1.4………………………………………………………………………13

Figure 1.5………………………………………………………………………16

Figure 1.6………………………………………………………………………18

Figure 1.7………………………………………………………………………20

Figure 1.8………………………………………………………………………35

Figure 1.9………………………………………………………………………41

Figure 1.11……………………………………………………………………..43

Figure 1.12……………………………………………………………………..49

Figure 2.1………………………………………………………………………67 xvi

PhD Thesis – Table of Contents Nayyar Ahmed

Figure 2.2………………………………………………………………………77

Figure 2.3………………………………………………………………………78

Figure 2.4………………………………………………………………………80

Figure 3.1………………………………………………………………………92

Figure 3.2………………………………………………………………………94

Figure 3.3………………………………………………………………………96

Figure 4.1………………………………………………………………………109

Figure 4.2………………………………………………………………………110

Figure 4.3………………………………………………………………………112

Figure 4.4………………………………………………………………………113

Figure 5.1………………………………………………………………………124

Figure 5.2………………………………………………………………………125

Figure 5.3………………………………………………………………………127

Figure 5.4………………………………………………………………………128

Figure 6.1………………………………………………………………………137

Figure 6.2………………………………………………………………………139

Figure 6.3………………………………………………………………………142

Figure 6.4………………………………………………………………………144

Figure 6.5………………………………………………………………………146

Figure 6.6………………………………………………………………………148

Figure 6.7………………………………………………………………………150

Figure 7.1………………………………………………………………………163

xvii

PhD Thesis – Abstract Nayyar Ahmed

Abstract

In recent times, allergy has become a financial, physical and psychological burden to the society as a whole. Allergic reactions can result in life-threatening situations causing morbidity and high economic cost. Therefore, more effective reagents are needed for allergy treatment. In Chapter 2, IL-4RD was targeted by employing a novel molecular method of phage display technology. A novel synthetic peptide (N1 biopep) was isolated using this technology, and demonstrated a strong affinity to the target IL-4RD. This was followed by extensive immunoassays such as ELISA. Indeed, the N1 biopep has shown to be very promising in its capacity to significantly down-regulate the interactions between IL-4RD and the cytokine IL-4.

In Chapter 3, peptides were commercially synthesized and used for in vitro assays and a

HEK-BlueTM IL-4/IL-13 reporter cell line model, transfected with a gene producing an enzyme SEAP. SEAP acts as a substitute to IgE when cells are stimulated with cytokine IL-4 and IL-13. QUANTI-Blue was added as a substrate that breaks down in the presence of

SEAP producing blue coloration. The blue color confirmed the activation of STAT6 pathway using a spectrophotometer. We have successfully used peptides N1 to IL-4RD, K1 to IL-4 and P9 to IL-13 that demonstrated inhibition of SEAP production in HEK-Blue cells. A colorimetric analysis showed a >50% inhibition, resulting into less blue color with all three peptides. A statistical Student’s t-test revealed the significance of the results. Since IL4 and

IL-13 interaction with IL-4R/IL-13R is a common pathway for many allergies, a prophylactic treatment can be devised by using such novel methods in the future.

In Chapter 4, it was hypothesized that a causal relationship exists between the intake of omega-3/6 fatty acids such as DHA, EPA, DPA and AA and atopic individuals suffering xviii

PhD Thesis – Abstract Nayyar Ahmed from allergies. In allergic cascades, cytokines IL-4 and IL-13 bind to IL-4 receptor and IL-13 receptor (IL-4R and IL-13R), respectively, which activate the STAT6 phosphorylation pathway leading to gene activation of allergen-specific IgE antibody production by B cells.

The overall aim here was to characterize omega-3/6 fatty acids and their effect on IL-4/IL-13 signaling. The fatty acids were tested in vitro with the HEK-Blue IL-4/IL-13 reporter cell line model. We have successfully used DHA, EPA and DPA in our studies that demonstrated a decrease of SEAP secretion as opposed to an increase in SEAP secretion with AA treatment.

A statistical Student’s t-test revealed the significance of the results. We have successfully identified and characterised DHA, EPA, DPA and AA in our allergy model with varying potentials. While AA was a potent stimulator, DHA, EPA and DPA were potential inhibitors of IL-4RD signalling, which regulates the STAT6 induced pathway in allergic cascades in vitro.

As literature suggests, resolvins and CoQ10 have shown promising results with down- regulation of allergy and inflammation. In Chapter 5, we assessed the effect of CoQ10 and resolvins on the HEK-Blue cell model of allergy. Through our series of experiments, we have shown the differential effects of resolvins and CoQ10 on SEAP secretion. A statistical

Student’s t-test revealed the significance of the results. Resolvins, although anti-inflammatory by nature, showed an increase in SEAP secretion as opposed to CoQ10, which stood out as a promising candidate in repressing allergy. SEAP secretion was completely inhibited in the presence of CoQ10 even at lower concentrations, suggesting a novel therapeutic for allergy treatment.

Lastly, we assessed the distribution of ryegrass pollen in the regional city of Geelong

(Victoria, Australia). The purpose of Chapter 6 was to establish a link between weather xix

PhD Thesis – Abstract Nayyar Ahmed variables and the presence of ryegrass pollen (intact vs. ruptured) in the atmosphere. For this purpose, a Burkard volumetric air trap was installed at the Deakin University (Waurn Ponds,

Geelong) for pollen collection (known as Deakin AIRwatch). Our results show that ryegrass pollen distribution is fairly correlated to weather variables such as rain, temperature, humidity and wind. Pollen counting from three years were incorporated as part of this study. We have successfully shown the presence of intact and ruptured pollen in air, which is important for future studies relevant to allergic individuals suffering from pollen allergy.

To sum up, chapters 2-5 have shown the use of different therapeutics to down-regulate allergic pathways which are common to all allergies. The isolated peptides along with n-3 fatty acids and CoQ10 can serve as novel candidates for allergy treatment. On the contrary, avoidance of pro-inflammatory products such as n-6 fatty acids and resolvins can prevent allergic symptoms. In chapter 6, we have conducted a thorough investigation of ryegrass pollen distribution in regional Victoria which can help alleviate allergy through awareness and avoidance of pollen allergy hence, improving quality of life. Our work paves a new and exciting path for the safe and effective treatment of allergies.

PhD Thesis – List of Abbreviations Nayyar Ahmed

List of Abbreviations

7-mer Amino acid chain consisting of 7 amino acids

12-mer Amino acid chain consisting of 12 amino acids

AA Arachidonic Acid

AHR Airway Hyper-Responsiveness

ALX Lipoxin Receptor

APC Antigen Presenting Cells

Ara h 1 Arachis hypogaea 1

Ara h 2 Arachis hypogaea 2

Ara h 3 Arachis hypogaea 3

ASCIA Australasian Society of Clinical Immunology and

Allergy

Bet v 1 Betula verrucosa 1

Biopep Biotinylated Peptide

BSA Bovine Serum Albumin

C1 Control 1

C2 Control 2

CCR5 Chemokine Receptor Type 5

CD4+T Cluster of Differentiation Expressing T-cells

CD23 Cluster of Differentiation-23

CD55 Complement Decay Accelerating Factor

ChemR23 Chemokine-Like Receptor 1

COX Cyclooxygenase

CpG Phosphorothioate Guanisosine

Da Daltons xxi

PhD Thesis – List of Abbreviations Nayyar Ahmed

DC Dendritic Cells

DH2O Distilled Water

DHA Decosahexaenoic Acid

DMEM Dulbecco’s Modified Eagle Medium

DMSO Dimethyl Sulfoxide

DNA Deoxyribonucleic Acid

DPA Docosapentaenoic Acid

EDTA Ethylenediaminetetraacetic Acid

ELISA Enzyme Linked Immunosorbant Assay

EPA Eicosapentanoic Acid

FAHF-2 Food Allergy Herbal Formula

FBS Fetal Bovine Serum

FcεRI Fc Epsilon RI

FEV1 Forced Expiratory Volume in 1 Second

Fmoc Fluorenyl-methyl-oxycarbonyl

FOXP3+ Forkhead box P3

GPR32 G Protein Coupled Receptor

H2SO4 Sulphuric Acid

HBTU O-(Benzotriazol-1-yl)-N,N,N’,N’-tetramethyluronium

hexafluorophosphate

HEK Human Embryonic Kidney

HKE Heat-Killed E. coli

HKE MP123 Heat-Killed E. coli modified peanut protein Ara h 1-3

HOBt Hydroxybenzotriazole hrs Hours xxii

PhD Thesis – List of Abbreviations Nayyar Ahmed hr Hour

HPLC High Performance Liquid Chromatography

HRP Horseradish Peroxidase

Hsp65 Heat-Shock Protein 65

IFN Interferons

IFN-γ Interferon- Gamma

IgA Immunoglobulin A

IgD Immunoglobulin D

IgE Immunoglobulin E

IgG Immunoglobulin G

IgG2a Immunoglobulin G subclass 2a

IgG4 Immunoglobulin G subclass 4

IgM Immunoglobulin M

IL-1 Interleukin-1

IL-2 Interleukin-2

IL-4 Intereukin-4

IL-4DM IL-4 Double Mutant

IL-4R Interleukin-4 Receptor

IL-4Rα Interleukin-4 Receptor-Alpha

IL-5 Interleukin-5

IL-10 Interleukin-10

IL-12 Interleukin-12

IL-13 Interleukin-13

IL-13Rα1 Interleukin-13 Receptor-Alpha1

IMO Immune Modulatory Oligonucleotide xxiii

PhD Thesis – List of Abbreviations Nayyar Ahmed

IPTG Isopropyl β-D-1-thiogalactopyranoside

JAK Janus Kinase kDa Kilo-Daltons

LB Luria Broth

LT Leukotrienes

LTB4 leukotriene B4

LOX Lipoxygenase mAb Monoclonal Antibody

MHC class II Major Histocompatibility Complex class II

Mimtags Mimotopes that can be Tagged

Min Minutes

NaN3 Sodium Azide

NARL NeuroAllergy Research Laboratory

NCBI National Centre for Biotechnology Information

NF-κB Nuclear Factor kappa-light-chain-enhancer of

Activated B cells

NK Natural Killer Cells

OD Optical Density

OPD ortho-Phenyl Diamine

PAF Platelet Activating Factor

PC Positive Control

PCR Polymerase Chain Reaction

PEG Polyethylene Glycol

Pfu Plaque-Forming Units

PG’s Prostaglandins xxiv

PhD Thesis – List of Abbreviations Nayyar Ahmed

PGD2 Prostaglandin-2

PGE2 Prostaglandin E2

PGE3 Prostaglandin E3

Ph.D Phage Display

PKC Protein Kinase C

PMN Polymorphonuclear Leukocyte

PPI Protein-Protein Interactions

PUFA Polyunsaturated Fatty Acids

RAST Radioallergosorbent Test rAlt a 2 Alternaria Alternata Allergen 2

RH Relative Humidity

RhuIL-4R Recombinant Human Interleukin-4 Receptor

RhuIL-4Rα Recombinant Human Interleukin-4 Receptor Alpha rpm Revolutions Per Minute

RT Room Temperature

RvD1 Resolvin D 1

RvD2 Resolvin D2

SCIT Subcutaneous Immunotherapy

SEAP Secreted Embryonic Alkaline Phosphatase shRNA Short-Hairpin Ribonucleic Acid

SIT Specific Immunotherapy sIL-4R Soluble Interleukin-4 Receptor sIL-4Rα Soluble IL-4 Receptor Alpha

STAT Signal Transducer and Activation of Transcription

TBST Tris-buffered Saline with Tween-20 xxv

PhD Thesis – List of Abbreviations Nayyar Ahmed

TetR Tetracycline Resistant

Th T helper

Th1 T helper Type 1

Th2 T helper Type 2

TNF Tumor Necrosis Factor

TNFα Tumor Necrosis Factor Alpha

Tr1 Type 1 Regulatory T Cells

Treg Regulatory T Cells

U.S. FDA United States Food and Drug Administration

X-gal 5-bromo-chloro-indolyl-galactopyranoside

xxvi

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Chapter 1 - Literature Review

PhD Thesis – Chapter 1 Nayyar Ahmed

1.1 Introduction of Allergy and Allergens

The term “allergy” was first coined by Von Pirquet in 1906. He described the word allergy as a ‘changed reactivity’ induced by antigens (1). Nearly a century later, the term allergy has been revised as an exaggerated reaction to foreign substances, which are mostly harmless by nature but injurious to allergic individuals. These foreign substances are known as allergens and cause the immune system to respond aggressively. Allergies can be induced by various allergens such as pollens, dust mite, molds, dander, and food (2). In addition, adjuvant risk factors such as smoking or air pollution can also enhance an individual’s intolerance to allergens.

Allergens are a particular type of antigen that are detected by the immune system and translated as a foreign invader. As outlined in Figure 1.1, when atopic individuals come into contact with these antigens, the body reacts by producing very large amounts of immunoglobulin E (IgE) antibodies. These IgE antibodies sensitize effectors cells of allergy

(e.g. basophils and mast cells), which subsequently cross-link with allergens, releasing chemical mediators causing allergy symptoms (2).

1.1.1 Sources of Allergens

There are many different forms of allergens like house dust mite, birch tree pollens and pollens from grass such as timothy grass, to name a few. The Arachis hypogaea 1 (Ara h 1),

Arachis hypogaea 2 (Ara h 2) and Arachis hypogaea (Ara h 3) are highly allergenic peanut proteins, which have become a potential hazard in recent years (3). Peanut allergy is mainly associated with fatal-food induced anaphylaxis (4). IgE antibody production is induced more severely by food allergens like milk, beans, nuts, rice, and wheat (5).

PhD Thesis – Chapter 1 Nayyar Ahmed

The Immune System

Foreign Substance

(cat dander, pollen, virus, bacteria)

Normal Immune Response IgM, Exaggerated Immune Response

IgG, IgA, IgD and various IgE is overproduced in

immune cells respond to attack. response to cat dander, pollens,

and other harmless allergens.

Subsequent exposure results in Foreign substance is eliminated. an allergic reaction.

Non-Allergic Individual Allergic Individual

Figure 1.1: When a foreign substance enters the body, an allergic individual responds by overproducing IgE antibodies, which is the initial sensitizing stage. Secondary exposure to the same antigen results in the release of inflammatory chemicals like histamines that cause the clinical symptoms observed in allergy. Other antibodies like immunoglobulin

M (IgM), immunoglobulin G (IgG), immunoglobulin A (IgA) and immunoglobulin D (IgD) are also produced when a foreign substance is introduced into the body but does not result into an allergic cascade. Figure adapted from (2). 3

PhD Thesis – Chapter 1 Nayyar Ahmed

The presence of allergens in food is mainly due to their high resistance to heating and enzymatic breakdown rendering them unavoidable in food products (6).

Unfortunately, the only remedial method available is to avoid food products that may contain these allergens. For example, accidental ingestion of peanut by-products is on the rise; an inevitable situation at times, especially with children. Thus, the chronic nature of this allergy, its consequences and its availability in a wide range of products has made it a very important field of clinical and laboratory research (7).

1.1.2 Symptoms of Allergy

Although there are several symptoms for allergy, the most important clinical symptoms of allergy are listed below;

(i) Allergic rhinitis: nasal obstruction, sneezing, itching and rhinorrhea (3);

(ii) Asthma: breathlessness due to airway obstruction, which is sometimes partial or

totally reversible (3);

(iii) Anaphylaxis: a severe systemic allergic reaction that causes the release of

inflammatory histamine. This causes symptoms like laryngeal edema and asthma (3);

(iv) Atopic eczema: characterized by a red itchy rash (3).

1.1.3 Rising Incidence/Trends in Allergic Disease

There has been a marked increase in prevalence of atopic disease in regions such as Western

Europe, the US and Australasia during recent years, especially in industrialized nations. A

20-fold increase has been experienced by western countries and as much as 40% of the population suffers from this disease (8). A recent statistical study carried out (9) has shown that a rise in allergy trend has taken place over the last 20 years. A rise in trend has also been 4

PhD Thesis – Chapter 1 Nayyar Ahmed studied by the Australasian Society of Clinical Immunology and Allergy (ASCIA) shown in

Figure 1.2 (10). These studies indicate an alarming situation and require a more thorough and aggressive approach to resolve this disease.

1.2 Overview of Allergy as a Disease: Atopy in Allergic Individuals

To understand allergic diseases, it is important to know the difference between atopic and non-atopic individuals. Atopy is an inherited predisposition that renders susceptible individuals to allergic conditions such as rhinitis, asthma and atopic dermatitis, which is caused by IgE-mediated responses (11, 12). Atopic diseases can further be sub-divided into extrinsic allergy, which exhibits high levels of serum IgE antibody, and an intrinsic atopic response, which exhibits low level of IgE antibody (12).

1.2.1 Underlying Causes of Allergy in Atopic Individuals

In atopic individuals, an immune response is mounted by T cells that are activated by allergens, promoting T helper Type 2 (Th2) variant of cells. Once stimulated, these cells subsequently produce cytokines such as Interleukin-4 (IL-4), Interleukin-5 (IL-5) and

Interleukin-13 (IL-13). Unlike the cytokines produced by T helper Type 1 (Th1) cells in non- atopic individuals (detail in the following section), an excessive production of cytokines takes place from Th2 cells (13). Figure 1.3 illustrates the predisposition factors that can trigger atopy in individuals. Briefly, allergen exposure to patients with an already existing genetic predisposition can become a risk factor for sensitization (more detail in the later sections).

Acknowledgment of this risk is essential (14). An example of genetic factors is polymorphism in the IL-4 family of cytokine genes.

PhD Thesis – Chapter 1 Nayyar Ahmed

Figure 1.2: Rise in trend of allergies over the next four decades in both sexes. ‘Std’ in the figure denotes using one standard prevalence rate, rather than a different rate every year -

‘trend’. Figure adapted from (10).

PhD Thesis – Chapter 1 Nayyar Ahmed

The two most important reasons of allergy prevalence in the western world are high level of hygiene maintenance and excessive use of antibiotics at a younger age (Figure 1.3). Both factors impede the immune system from acclimatizing to environmental conditions, which causes a release of a Th2 cells cytokine profile in allergic patients (15, 16). This is referred to as the hygiene hypothesis.

A number of studies have shown that children who are raised on farms have a lower predisposition to hay fever and atopic sensitization, as opposed to children who are raised in urban areas. The important environmental changes recognized are animal sheds (exposure to diverse microbes) and consumption of unpasteurized milk (17).

PhD Thesis – Chapter 1 Nayyar Ahmed

Figure 1.3. Genetic and environmental triggers of atopic diseases and its effect on various parts of the body. Environmental factors such as allergens and genetic factors (as mutations and polymorphisms) are responsible for a combined atopic effect. Figure adapted from (11).

PhD Thesis – Chapter 1 Nayyar Ahmed

1.2.2 Diagnostic Tests for Allergic Conditions

Allergy testing is an important component of allergy and asthma management. It enables the identification of avoidable environmental triggers for asthma and allows appropriate avoidance advice to be provided.

1.2.2.1 RAST

RAST or radio-allergosorbent immunological test system is only useful if the immune reaction is IgE antibody-mediated (18). It uses an in vitro immunological measuring device to determine allergen-specific antibodies. The precise measurement of allergen antibodies helps to verify the diagnosis of asthma, allergies and other pulmonary disorders.

1.2.2.2 Skin Prick Tests

The more popular among the two different methods of skin prick test is the skin prick test.

The allergen is applied directly on skin and the skin is pricked in order to absorb the allergen through the epidermal surface (19). Measurements are recorded at regular intervals of 10-15 minutes (min) for swollen skin (in mm).

A similar but alternative method is to inject the allergen directly into the skin and monitor for an inflammatory reaction at the site. This method is known as the intradermal skin test (20).

However, skin prick tests pose some risks (e.g. anaphylactic food allergens such as peanuts) and appropriate precautions need to be taken in order to perform the necessary tests (21).

1.2.2.3 Blood Specific IgE Testing

Blood specific IgE testing helps to quantify a broad spectrum of allergens that induce allergen-specific IgE antibodies. In contrast to skin prick test, it is less sensitive to allergens 9

PhD Thesis – Chapter 1 Nayyar Ahmed but more useful when anaphylaxis is being investigated. Blood specific IgE testing can be performed on sensitive patients who carry risks of allergy or those taking antihistamines. This allows an alternative to skin prick test, which can otherwise cause adverse reactions in patients (21).

1.2.3 Current Available Treatments

Current allergy treatment focuses mainly on counteracting the symptoms of allergic reactions.

Treatment of allergy principally involves three different approaches:

i) To know what the person is sensitive to and avoid exposure to that particular

substance. This is usually called ‘environmental control’. For example, if an

individual is allergic to peanuts, avoiding them would be sufficient to overcome

symptoms (22);

ii) The second approach involves the administration of drugs such as decongestants,

steroid nasal sprays, antihistamines, adrenergic agonists, anti-inflammatory drugs,

leukotrienes (LT) and bronchodilators to relieve allergic symptoms (23);

iii) The third most important therapy involves desensitizing the patient by repeated

administration of a specific allergen in small doses (24). This approach helps the

patient to develop immunity against the allergen; the treatment is known as

specific immunotherapy (SIT). SIT has many functions including, the induction of

Th1 cells in the production of IgG immunoglobulin rather than IgE (24, 25). Th2

cytokines, principally IL-4 and IL-13, are reduced considerably, further alleviating

the patient from the symptoms (25). However, it has been argued that SIT has its

disadvantages such as unwanted effects, poor efficacy and specificity as a result of

poor quality of allergy vaccines; comprised of poor quality of allergen extracts

PhD Thesis – Chapter 1 Nayyar Ahmed

(26). Moreover, it is of high risk for patients with anaphylactic allergy (e.g. peanut

allergy).

Although these methods are the only available remedy for allergy, they offer very little resistance to the growing problem at hand, with problems of efficacy and safety. These therapies are yet to undergo further development before they can offer any significant change.

Hence new, safer and more reliable methods are required to combat allergic diseases.

1.3 Immune Response: Allergic vs. Non-Allergic Response

This section will demonstrate the involvement of allergen, antigen presenting cells (APC), lymphocytes, plasma cells, mast cells and cytokines in the allergic immune response (27).

1.3.1 Non-Allergic Immune Response

During a non-allergic immune response, an allergen is engulfed by an APC, which processes the internalized allergen and subsequently presents a fragment of the allergen on its surface, via major histocompatibility complex (MHC) Class II proteins (28). The APCs migrate to the lymph nodes to present the processed antigens to MHC Class II proteins, which further attach themselves to T-cell receptors (29). This process is called priming. This causes differentiation in naïve T helper cells (Th cells) also known as cluster of differentiated (CD4+) T cells into

Th1 cells (29). The Th1 cells produce cytokines interleukin-2 (IL-2) and interferon-gamma

(IFN-γ), which evoke cell-mediated immunity and phagocyte-dependent inflammation. As a result, B-cell proliferation and IgG antibody production takes place (30, 31).

PhD Thesis – Chapter 1 Nayyar Ahmed

1.3.2 Allergic Immune Response

The allergic response can be divided into two separate reaction phases. The initial phase is known as the ‘humoral response’ and occurs as a result of initial sensitization of an allergen which triggers B cells or B lymphocytes to release IgE antibodies (11). The second phase,

‘cell-mediated response’, involves an exposure to the same allergen but elicits a response by effector cells, such as mast cells and basophils, that release chemical mediators. These chemical mediators, namely histamine and heparin, are responsible for allergy symptoms

(32). Figure 1.4 below illustrates a brief summary of these events.

1.3.2.1 Humoral Response in Allergic Individuals

Allergic individuals respond to antigens by differentiating Th cells into Th2 cells, rather than

Th1. This occurs in the presence of the cytokines IL-4 and IL-13, which are also associated with the production of IgE and eosinophilic response in atopic disease (11, 33). The primary source of IL-4 in atopy is Th2 lymphocytes and mast cells (Figure 1.4) (11), while the primary source of IL-13 is activated Th2 cells (34).

In atopic individuals, the APC engulf the allergen and cross link the exposed antigen to naïve

T cells via the MHC class II molecule. The T cells then differentiate into Th2 variant enhancing cytokines IL-4/IL-13, ensuing production and proliferation of B lymphocytes to plasma cells via the CD40 ligand expressed on B cells (35-38). The Th2 cells further release cytokines IL-4 and IL-13 that bind to B lymphocytes, activating them which releases more

IgE into the system (36). These IgE antibodies then bind to high affinity IgE receptor Fc epsilon RI (FcεRI) on the surface of basophils (not shown) and mast cells. This phase, shown in Figure 1.4 is alternatively known as the ‘sensitization phase’ (24, 39, 40).

PhD Thesis – Chapter 1 Nayyar Ahmed

Figure 1.4. Cellular interaction in the allergy phase of immune response and its consequences. When allergens come into contact with APC, the allergens are processed and presented to naïve T cells, which mature into Th2 cells and release cytokines such as IL-4/IL-

13. The cytokines bind to receptors on B cells, which then differentiate into plasma cells and trigger the release of IgE antibodies (A-F). Secondary exposure to the same allergen cross- links neighboring membrane-bound IgE antibodies on mast cells (as well as circulating basophils) and releases the chemical mediators, which cause symptoms like sneezing, wheezing, eczema etc. (G-H). Figure adapted from (41).

PhD Thesis – Chapter 1 Nayyar Ahmed

1.3.2.2 Cell-Mediated Response in Allergic Individuals

This phase, also known as the ‘effector phase’ is initiated by subsequent exposure to the same allergen, which was the previous sensitizing agent (36). As demonstrated in Figure 1.4, the allergen cross-links with the IgE on the surface of basophils and mast cells, which leads to degranulation. As a result, chemical mediators are released, such as proteoglycans, LT’s, proteases, chemokines, histamine and most importantly cytokines, such as tumor necrosis factor-alpha (TNF-α), IL-13 and IL-4 (42). This results in a variety of symptoms, such as asthma (caused by excess mucous production), hay fever, anaphylaxis, itchy rash, dermatitis etc.

The shift in cytokine production from Th1 cells (IFN-γ and IL-2) to Th2 (IL-4 and IL-13) variant forms the hallmark of allergic response in atopic individuals. Therefore, the aim of my project was to reduce the interaction of IL-4 and IL-13 with their common interleukin-4 receptor (IL-4R) (discussed in the later sections). Hence, this will down-regulate the allergic cascade from progressing to the ‘sensitizing phase’ of the immune response.

1.4 Role of Cytokines in Immunity

Cytokines are secreted proteins that orchestrate a range of innate, adaptive and inflammatory responses in the body (43). They are important for the development of hematopoietic and nervous systems. The major cytokine producing cells are macrophages, lymphocytes and monocytes (43). They stimulate target cells by affecting gene expression (44), subsequently causing cell proliferation and cell differentiation (43).

PhD Thesis – Chapter 1 Nayyar Ahmed

Cytokines play this role by binding to specific receptors on cell surfaces known as Type-1 membrane proteins (45). There are a variety of cytokines, namely chemokines, colony stimulating factors (also known as transforming growth factors), interferons and interleukins

(45). Interleukin family of cytokines is involved in immune responses and associated diseases. IL-4 and IL-13 play a more dominant role in the progression of allergic disease.

Studies carried out in the previous decade suggest that IL-4 and IL-13 are extensively used as therapeutic targets for alleviating allergy (46-48).

1.4.1 Cytokines IL-4 and IL-13

IL-4 and IL-13 are examples of interleukins involved in allergic responses and play fundamental roles in causing allergic diseases (49). Th2 cells are responsible for the production of IL-4 and IL-13 (49) and both IL-4 and IL-13 are capable of initiating the Janus kinase (JAK) signaling pathway and activate signal transducer and transcription factor known as signal transfer and activation of transcription-6 (STAT6), which triggers gene expression in the nucleus and results in the production of IgE antibodies (refer to Figure 1.6) (50, 51).

IL-13 (Figure 1.5A) and IL-4 (Figure 1.5B) are pleiotropic cytokines (having multiple effects), secreted by many cells such as Th2 cells, activated B cells basophils and mast cells, as well as dendrites (52). Both cytokines share many similar characteristics like inducing the production of IgE class of immunoglobulins and have 30% amino acid homology with similar chromosomal locations (52). They share many common functional characteristics as well, but the most significant difference lies in the ability of IL-4 cytokine to develop CD4+

Th2 cells as opposed to IL-13 which has no effect on T cells (53). IL-4 is also involved in the differentiation of monocytes to dendritic cells (DC) and is a pioneer of allergic diseases associated with IgE-mediated response (54). In a study carried out on IL-4 knockout mice, 15

PhD Thesis – Chapter 1 Nayyar Ahmed the Th2 cell differentiation did not take place and as expected, the production of eosinophils and IgE antibodies were significantly decreased or abrogated altogether. 1.5A) 1.5B)

1.5C)

Figure 1.5. Crystal structures of cytokines IL-13 (1.5A), IL-4 (1.5B) and IL-4Rα (1.5C).

Reducing the interaction between IL-4/IL-13 and IL-4R complex can ultimately reduce the

IgE concentration in sera, hence, minimizing the allergic threshold. Figure adapted from (55-

57).

PhD Thesis – Chapter 1 Nayyar Ahmed

IL-4 and IL-13 are potent inducers of allergic diseases and symptoms of allergy (58).

Therefore, the focus of this study is to reduce the interaction of cytokines IL-4/IL-13 with their common IL-4Rα (Figure 1.5). This would consequently reduce the production of IgE antibodies to a level of a non-atopic individual. This approach is particularly appealing, as it may provide a treatment to all common allergies.

1.4.2 Comparative Roles of IL-4/IL-13 with IL-4Rα Chain

IL-4 and IL-13 play key roles in Th2 immunity and asthma pathogenesis. The function of these cytokines is partially linked through their shared use of the IL-4Rα chain. The Type I receptor comprising IL-4Rα and the common γ-chain is expressed by hemopoietic cells and is exclusively responsive to IL-4. In contrast, the Type II receptor comprising IL-4Rα and interleukin-13 receptor-Alpha 1 (IL-13Rα1) is responsive to both IL-4 and IL-13 (Figure 1.6)

(59).

The binding of IL-13 to the IL-13Rα1 induces heterodimerization with the IL-4Rα. STAT6 then dimerizes and travels through cell cytoplasm until it reaches the cell nucleus, which activates genes that encode for another cluster of differentiation protein called CD23 and

MHC Class II (60). Dephosphorylation of STAT6 can result into gene repression and deactivation. In lymphocytes, STAT6 plays a primary role of proliferation and defense against apoptosis (60).

PhD Thesis – Chapter 1 Nayyar Ahmed

Figure 1.6. Signaling pathways of IL-4 and IL-13, along with their corresponding receptors. The IL-4 cytokine is not only capable of activating Type I but Type II receptors as well, since both receptors share a common IL-4Rα chain. By reducing this interaction, it is predicted to suppress the effect of IL-4 on cell signaling mechanisms. The JAK signaling molecules activate STAT6 transcription factor that subsequently activates gene expression in the cell nucleus. Figure adapted from (47).

PhD Thesis – Chapter 1 Nayyar Ahmed

In IL-4 and STAT6 knockout mice, it was found that IgE production diminished by a factor of 100 and the mice lacked T cell development (61). The cytokines appear to have a link in regulating allergic conditions and in some cases also improve the effects of tissue-damaging autoimmunity (61). These physiological roles lay down the importance of cytokines IL-4 and

IL-13 with IL-4R signaling mechanisms. For this reason, targeting the IL-4R complex along with its cytokines would make tangible targets.

Therefore, the concept that IL-4 and IL-13 can be targeted simultaneously is of great importance because of their shared dependence on the IL-4Rα chain of the receptor. Hence, this research targets the common receptor with a novel synthetic antagonist (Figure 1.7).

1.5 IL-4Rα as an Important Target for Allergy Treatment

Various researches have been conducted using a range of techniques to down regulate the interaction between IL-4/IL-13 with IL-4R, namely monoclonal antibodies, antagonists and soluble receptors, but the efficacy of these procedures still remains in doubt and under a lot of scrutiny. Therefore, an alternative technology, known as phage display, will be used to carry out the task of identifying a novel peptide inhibitor as an intervention target for IL-4R.

1.5.1 Anti-IL-4R Monoclonal Antibody

Over the years, monoclonal antibodies (mAb) have been developed and designed for more complicated procedures for use as therapeutics. Mouse models have been designed to improve mouse monoclonal antibodies against human IL-4R to study the effects of T and B cell differentiation (62, 63). 19

PhD Thesis – Chapter 1 Nayyar Ahmed

Figure 1.7. Hypothetical representation of a synthetic peptide inhibitor reducing the interaction between IL-4 and IL-4R on B cells. The reduction of this binding can ultimately reduce the IgE concentration in sera, hence, minimizing the allergic threshold.

Process leading from G-H (effector phase) may be blocked (red crosses); alleviating symptoms of allergy. Figure adapted from (41).

PhD Thesis – Chapter 1 Nayyar Ahmed

In one study, an M1 mAb had down-regulating effects on cell secretion of IgG1 and IgE, whereas another mAb M2 showed no effects. The research in this area is still incomplete and demands a more thorough and complete investigation to make it a reality (63-65).

1.5.2 Soluble IL-4 Receptor (sIL-4R)

One of the therapeutic applications of soluble receptors was the development of recombinant human IL-4 receptor (RhuIL-4R). RhuIL-4R only has the extracellular domain of the IL-4R which has the ability to bind to cytokine IL-4 but simultaneously acts as an antagonist to block the function of the cytokine (66). As we know that allergic symptoms occur from combined effects of IL-4 and IL-13 cytokines, the disadvantage of this system is that it does not affect IL-13 but only IL-4. Therefore, this remains an unreliable method of curing allergy

(67). In a separate study carried out by Holtzman (2003), it was found that administration of soluble IL-4 receptor alpha (sIL-4Rα) had no clinical efficacy in treating asthma and the development of this project has been stopped altogether (67).

These results are disappointing and raise concerns regarding therapies for allergy. Therefore, a need arises to redirect strategies in the favour of phage display technology, in the quest to identify novel peptide inhibitors for allergy treatment.

1.5.3 Targeting IL-4R with Hybrid Peptides

In a study by Yang et al., (2012), it was found that using L-amino acid peptides as a remedy to cancer showed detrimental effects in vivo. A hybrid peptide, composed of IL-4Rα binding moiety, killed normal cell lines at low concentrations in vitro and failed to show antitumor activity in vivo. However, when the peptide was modified to a DL-amino acid, it was able to

PhD Thesis – Chapter 1 Nayyar Ahmed induce a modest cancer cell death (68). Here, it is important to note that peptides are now becoming an important study to counter diseases at the cellular level.

1.5.4 Further Analysis of Blocking IL-4R

In an experiment carried out by Konig et al., (1995), IL-4 induced antigen CD23 fragments were suppressed by sIL-4R and a partial antagonistic mutant IL-4 protein, Y124D was produced. The target cells showed an initial suppression of IgE synthesis but 4 days later, IgE synthesis occurred (69). In a study carried out by Lai et al., (1991), blockade of IL-2R and

IL-4R both resulted in the increase of IL-4 (70).

In yet another study, IL-2R and IL-4R were blocked with an MR6 (mAb against IL-4R) and showed a reduction in interferon gamma production in T cells. The blocking of IL-2R with an antagonist suppressed the full expression of IL-4R (70).

All of these studies indicate that a new strategy needs to be devised to fight allergy and overcome allergic symptoms. Such a new strategy includes the innovative method of phage display technology, where novel peptide inhibitors of a target can be readily and successfully identified.

1.5.5 Current Available Treatments for Targeting IL-4 and IL-13 Cytokines

Treatment of cytokines IL-4 and IL-13 both result into a decrease of IL-4R signaling. The

NeuroAllergy Research Laboratory (NARL) research group at Deakin University has conducted experiments using both cytokines and IL-4R as novel targets for allergy intervention. Hence, it is highly important to highlight the current treatments available for not only targeting IL-4R but it’s cytokines as well. 22

PhD Thesis – Chapter 1 Nayyar Ahmed

1.5.5.1 Specific Immunotherapy (SIT)

The third most important therapy involves desensitizing the patient by repeated administration of a specific allergen in small doses (24). This approach helps the patient to develop immunity against the allergen by temporary loss of responsiveness because of continuous exposure; the treatment is known as specific SIT. SIT has many functions including the induction of Th1 cells in the production of IgG immunoglobulin rather than IgE

(24, 71). Th2 cytokines, principally IL-4 and IL-13, are reduced considerably, further alleviating the patient from the symptoms (71-73). SIT is commonly used for respiratory allergy but the underlying mechanism is not well understood. Initial desensitization occurs in mast cells and basophils. Effector cells may also include allergen specific T and B cells. The aim of allergen SIT is to induce peripheral T cell tolerance, suppress the activation level of mast cells and basophils and inhibit the secretion of histamines in response to IgE antibodies

(74). The production of peripheral T cell tolerance during allergen SIT plays a vital role in the generation of allergen-specific T regulatory cells (Treg) that are able to produce anti- inflammatory cytokines such as interleukin-10 (IL-10) and transforming growth factor-β

(TGF-β). Treg cells regulate allergen-specific IgE and are capable of recruiting immunoglobulin G 4 (IgG4) and IgA production. The two subsets of inducible Treg cells known as forkhead box P3 (FOXP3+) and the IL-10-positive type 1 regulatory T cells (Tr1) play important roles in allergen tolerance. These cells can be induced by allergen SIT in humans (75-77).

Subcutaneous immunotherapy (SCIT) is being use for well over a century and has been proved to be very effective in patients. However, the use of SCIT for food allergens has proved to be lethal for patients suffering from severe allergic symptoms such as an anaphylactic shock (78). Alternative methods for administering immunotherapy have been 23

PhD Thesis – Chapter 1 Nayyar Ahmed devised over the last few decades such as oral immunotherapy (OIT). This technique has proved to be more effective than SCIT with various food allergens (79, 80). The initial aim for this type of immunotherapy is to increase desensitization and to increase the threshold dose for exposure to peanut. This reduces the risk of severe allergic symptoms in response to accidental intake of peanut (80). The risk of such therapies has to be weighed against their benefits. A study carried out by Longo et al., (2008) showed that highly milk-allergic population with repeated therapy using OIT reduced the risk of allergy in almost half the population. 36% of the population became completely desensitized and 54% had become tolerant to milk-derived products (81). Although some of the patients showed adverse reaction to OIT, the benefits outweigh the risks since a large number of the population was cured (80). Overall, OIT has been found to reduce peanut-specific IgE levels and increase peanut-specific IgG4 levels, while basophil activation is reduced. Down-regulation of Th2 cytokines did not occur with OIT or the up-regulation of Th1 cytokines (80). Sublingual immunotherapy (SLIT) is another potential treatment for allergic rhinitis and asthma. A randomized double-blind, placebo-controlled study investigating SLIT for hazelnut was carried out by Enrique et al., (2005, 2008), in which 12 patients were treated with SLIT for a period of five months. Significant changes in sensitivity to hazelnut were observed following

SLIT treatment. There was an increase in hazelnut-specific IgG4 and IL-10 cytokine post- treatment. A decrease in specific IgE antibodies was also observed in patients with interrupted SLIT treatments (82-84).

However, it has been argued that SIT has its disadvantages such as unwanted effects, poor efficacy and specificity as a result of low quality of allergy vaccines, comprised of poor quality of allergen extracts. Moreover, it is of high risk for patients with anaphylactic allergy

PhD Thesis – Chapter 1 Nayyar Ahmed

(e.g. peanuts). Additional randomized, placebo-controlled clinical trials need to be carried out to determine the true efficacy and potential use of these therapies as novel allergy treatments.

1.5.5.2 Modified Recombinant Vaccines

Modified recombinant food proteins are developed to decrease IgE binding capacity but retain dominant T-cell epitope regions. Currently, many recombinant proteins have been developed such as peanut, apple and fish allergens. A study carried out by Ferreira et al.,

(1996) used polymerase chain reaction (PCR) to prepare nine isoforms of Betula verrucosa 1

(Bet v 1) from birch pollen. Two isoforms showed low IgE reactivity, however induced similar T-cell proliferation when compared to other seven IgE-reactive isoforms (85, 86).

Modified peanut allergens using site-directed mutagenesis can stimulate T cells but have greatly reduced IgE binding capacity (87, 88). A study carried out by Li et al., (2003) showed the effects of heat-killed E. coli (HKE) producing recombinant peanut proteins on a murine model of peanut anaphylaxis. The group treated with high doses of HKE modified peanut protein Ara h 1-3 (HKE MP123) demonstrated reduced clinical symptoms when compared to the placebo-treated group. A significant decrease in IgE levels and cytokines IL-4, IL-5, IL-

13 and IL-10 were also observed as opposed to an increase in IFN-γ. The study demonstrated that HKE-MP123 can induce long-term down-regulation of peanut hypersensitivity by targeting Th2 cytokine production and up-regulating Th1 cytokines (89).

1.5.5.3 Allergoids

Allergoids are extracts of allergen polymerized to form larger aggregates that exhibit reduced allergenicity but retain their immunogenicity properties. Formaldehyde is commonly used to polymerize allergenic proteins to produce allergoids and poses less threat of inducing systemic reactions during immunotherapy. Other polymerizing agents such as gluteraldehyde 25

PhD Thesis – Chapter 1 Nayyar Ahmed and polyethylene glycol conjugated allergens have been used in clinical studies with comparable effectiveness to other allergoids (90).

1.5.5.4 Adjuvants

To reduce the risk of IgE-mediated side effects such as anaphylaxis during treatment, modified allergoids have been used to treat allergic response. A study carried out by

Haydenreich et al., (2014) analysed that adsorbance of allergens to aluminum hydroxide

(alum) had no consistent adjuvant effect but decreased allergenicity in vitro, while showing an adjuvant effect with regards to IgG production in vivo (91, 92).

1.5.5.5 Peptide Immunotherapy

Peptide fragments containing T-cell epitopes which lack tertiary conformational structure to bind with IgE antibody can be used to decrease adverse reactions caused by immunotherapy

(84). In a murine model of peanut allergy, mice were treated with immunotherapy using peptides that contained IgE epitopes to the major peanut allergen Ara h 2. This was done prior to allergen challenge. Mice only experienced mild allergic reactions as compared to placebo-treated mice showing severe anaphylaxis (84). Several murine models of allergy have demonstrated a down-regulation of immune reactions in atopic individuals using peptides which are designed with T cell epitopes (93). Allergen peptide immunotherapy has been used in many clinical applications to date. In one of the clinical trials carried out by

Norman et al., (1996), a peptide vaccine for cat allergy showed variable effects. Some patients suffered a local reaction a few hours (hrs) after administering the peptide (94). Large peptides can sometimes cause physiological reactions in patients, which is a common phenomenon. However, many reasons cast a shadow of doubt on the use of peptides as novel

PhD Thesis – Chapter 1 Nayyar Ahmed therapeutics such as peptide stability, susceptibility to degradation, size (usually > 500 Da) and methods of delivery (95).

1.5.5.6 Plasmid Deoxyribonucleic acid (DNA) Immunotherapy

DNA nanoparticles containing the gene for allergens can be synthesized and administered orally for immunotherapy. A study carried out by Fronseca et al., (2012) showed

Mycobacterium leprae heat-shock protein 65 (Hsp65) expressed by recombinant DNA plasmid could reduce earlier established allergic response. Mice used were experimental models of airway allergic inflammation to test the effects of plasmid DNA immunotherapy.

Allergic mice were previously sensitized and challenged using ovalbumin. Post-challenge, mice were administered intramuscular doses of recombinant DNA encoding Hsp65. After treatment, mice were administered a second allergen challenge and the allergic response was recorded. It was shown that immunotherapy reduced airway inflammation by reducing Th2 cytokine and mucus production. Another important discovery was that cells transferred from

DNA-immunized mice to allergic mice travelled to allergic infected sites and attenuated Th2 response (96). Another study using a murine model demonstrated the use of DNA nanoparticles containing the gene for Ara h 2, which was administered orally to mice.

Immunized mice displayed less severe anaphylactic response following allergen challenge compared to unimmunized mice. This was accompanied by an attenuated production of IgE antibody, histamine and vascular leakage (97).

Hence new, safer and more reliable methods are required to combat allergic diseases. 27

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1.5.5.7 DNA Vaccines

DNA-based vaccines can be used to stimulate immune responses against foreign antigens.

The DNA, once injected, can be incorporate into local cells and synthesize the coded protein, which induces an immune response. The fungus Alternaria was found to cause asthma with severity, which prompted Sanchez et al., (2009) to experimentally test a rat model of

Alternaria-induced asthma. The major alternaria alternata allergen 2 (rAlt a 2), was targeted using the DNA-based vaccine with remarkable results. Post-administration, decreased production of IgE antibody was detected, along with a reduction in IL-13 cytokine secretion and reduced airway inflammation. Treatment of sensitized animals with DNA-based vaccines leads to a more balanced production of Th1 and Th2 cytokines, which reduces allergic symptoms (90, 98).

1.5.5.8 Toll-Like Receptor-9 (TLR-9)

TLR-9s are found on cells of the innate immune system such as DC. TLR-9s are activated by non-coding sequence of DNA referred to as phosphorothioate guanisosine (CpG), found in bacteria. Once activated, they lead to the synthesis of cytokines such as IFN-γ, IL-10 and interleukin-12 (IL-12) and increased expression of CD40 ligands. These responses shift the balance from a Th2 profile towards Th1. In murine models of asthma, it was found that treatment with CpG DNA reduced eosinophilic airway inflammation, mucus secretion, airway remodeling and airway hyper-reactivity (90, 99). A study published by Zhu et al.,

(2007) used a synthetic agonist called immune modulatory oligonucleotide (IMO) for TLR-9 to modulate peanut-induced allergy in mice. Peanut allergen was orally administered to the mice with or without IMO in a prevention model and in a treatment protocol, mice were sensitized with peanut allergen at days 0 and 14 and treated 4 times with administration of

IMO starting on 21 days. In the prevention model, mice that were administered with 28

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IMO/peanut showed decreased IgE and increased immunoglobulin G subclass 2a (IgG2a) levels in serum and intestinal tissue. In the treatment model, mice treated with IMO showed attenuation of IgE, IL-5 and IL-13 levels and increased IgG2a and IFN-γ in serum (100).

Subsequently, the experiments showed that IMOs can switch peanut-induced Th2 response toward a Th1 response.

1.5.5.9 Anti-IgE

Recombinant monoclonal humanized anti-IgE treatment reduces circulating IgE, suppresses inflammation and provides an enhanced control for allergic diseases (101). 84 patients with a history of peanut allergy were treated in a double-blind, randomized, dose-dependent trial for managing food allergy. Patients were randomly given 150, 300 or 450mg of anti-IgE antibodies or placebo for 4 months. As hypothesized, the patient given the highest dose of anti-IgE antibody, experienced a significant drop in clinical symptoms for allergy when the patient was challenged with peanut allergens. The placebo group showed no significant change to the peanut challenge. Although the anti-IgE treatment was successful in 25% of the patients, the other 25% showed zero tolerance of allergen challenge, which indicated that this treatment was effective but variable (102).

Combination therapy of anti-IgE mAb and allergen immunotherapy was recently investigated by Kuehr et al., (2002). This was a randomized, double-blinded, placebo-controlled, parallel- group study with a total of 225 patients treated with combination therapy using anti-IgE and

SIT for birch and grass pollen. The results show a reduction of 48% in clinical symptoms for the entire combined pollen season (average length, 84 days) for SIT + anti-IgE-treated patients when compared to patients treated with SIT + placebo. The study showed that

PhD Thesis – Chapter 1 Nayyar Ahmed combination therapy with monoclonal anti-IgE antibody and SIT was more effective than using immunotherapy alone for allergy treatment (103).

1.5.5.11 Chinese Herbal Medicine

A 9-herb formula derived from traditional Chinese medicine is under investigation as a potential treatment of food allergy. The medicine is referred to as the food allergy herbal formula (FAHF-2) and is effective in a murine model of peanut-induced anaphylaxis (104).

FAHF-2 treatment protected mice against allergic reactions when peanuts were administered to mice. These pre-clinical studies showed that clinical effects were persistent for 6 months after discontinuation of treatment. The underlying effects of these treatments showed a decrease of peanut-specific IgE antibody and an enhancement in peanut-specific IgG2a levels. More importantly, Th2 cytokines such as IL-4 and IL-13 were suppressed as well. The protective effect of FAHF-2 lasted for 6 months post-therapy, which makes it a very effective treatment in the murine model. Furthermore, the treatment had successfully treated allergic response in the murine model of multiple food allergies making it a prophylactic treatment for multiple allergic diseases (105).

Preliminary studies with a human peripheral blood mononuclear cell line taken from peanut- allergic individuals showed that when cells were treated with peanut extracts in the presence of FAHF-2, a decrease in antigen-dependent T-cell proliferation was observed. A dose- dependent reduction in IL-4 and IL-13 cytokines was also noted which indicates that FAHF-2 inhibits the Th2 response in atopic individuals (106).

With the studies mentioned above, it was determined that FAHF-2 was highly effective and safe to use in a murine model. This led the United States food and drug administration (U.S. 30

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FDA) to approve FAHF-2 as an investigational drug to be used in clinical trials in 2007. The first human study was an acute phase 1 trials, which investigated the safety of using FAHF-2 to treat food allergies in atopic individuals. A total of 18 subjects were used in this study between the age group of 12 and 45 years. Patients suffered from different allergies such as peanut, tree nut, fish and shellfish. The patients were part of a double-blind, placebo- controlled, dose-dependent trial. Accordingly, the subjects were administered 4, 6 or 12 tablets three times a day for a week. It was found that all doses for FAHF-2 were tolerated by the individuals and the patients showed no signs of side-effects. While no adverse effect of

FAHF-2 was observed in the individuals, no suppression or inhibition of IgE antibody and

Th2 cytokines was detected which raises doubts about the efficacy of this drug in a human model (107).

Studies have also been performed to demonstrate the synergistic effect of all the 9-herbs that make up the FAHF-2 formula. In a series of experiments it was understood that using individual herbs did not show the same level of inhibition of peanut-induced anaphylaxis as

FAHF-2. In addition, none of the 9 individual herbs were able to suppress or inhibit Th2 responses and/or enhancing Th1 response (106, 107).

1.5.5.12 Anti-IL-4 and IL-13 Antagonists

A humanized anti-IL-4 neutralizing antibody (pascolizumbab) was designed and used in a study by Hart et al., (2002), which inhibited IL-4 cytokine from binding to human-derived cell lines and was tolerated in monkeys (108). Pharmacokinetic studies involving cynomolgus

(Southeast Asian) monkey models have found two humanized anti-IL-13 mAbs, named Ab-

01 and Ab-02, to be effective at suppressing acute airway hypersensitivity (109). Ab-01 blocks contact between IL-13 and IL-4Rα whereas Ab-02 blocks contact between IL-13 and 31

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IL-13Rα1. Additionally, murine studies have confirmed the efficacy of two unnamed anti-IL-

13-mAbs at not only preventing asthmatic inflammation, but also at reversing mucus hyperplasia to basal levels under persistent allergen challenge (110, 111). Interestingly, the

IL-13-specific mAb lebrikizumab is more effective for treatment of asthma. Molecular observations suggest that high-affinity interaction between lebrikizumab and IL-13 sterically hinder the binding of IL-13 to IL-4Rα, which down-regulates receptor signalling (112, 113).

IL-13 cytokines can be selectively targeted using a naturally occurring soluble IL-13Rα2-Fc fusion protein. This receptor silences the effect of IL-13 cytokine when administered to mice post-allergen challenge. This treatment suppressed mucus hyper-secretion and completely down-regulated airway hyper-responsiveness (AHR) (114). The efficacy of these mAbs on humans is yet to be elucidated. However, mAbs are large molecules that are not as stable and as cheaply produced as synthetic peptide inhibitors, as employed in our current studies.

Antagonists are substances that block the signaling action of agonists (i.e. signaling molecules). IL-13 antagonists disrupt the interaction between IL-13 and IL-13R by binding to either of the molecules. An example is the IL-13 binding protein, named IL-13BP, binds IL-

13 with 100-300 times higher affinity than IL-13Rα chains in vitro (115). IL-13E13K is another antagonist, which binds IL-13R with 8-fold affinity than IL-13 in vitro (116). Murine studies have identified IL-4 double mutant (IL-4DM) as a potent IL-13 and IL-4 inhibitor due to its ability to bind to IL-4Rα with high affinity and blocks signal transduction (117). IL-

4DM treatment effectively controlled the dermatitis, reduced mast cell and eosinophil numbers, and inhibited plasma IgE and histamine. In addition, splenocytes produced an increased level of IFN-γ (117).

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1.5.5.13 Piktrakinra

It has been previously established that IL-4 and IL-13 cytokines have a shared dependency on the IL-4Rα chain, which makes it important to target the receptor, hence, a prophylactic treatment can prove to be more useful if it can target the IL-4Rα chain. Piktrakinra is an example of such a treatment. Piktrakinra is a recombinant form of IL-4 cytokine, which is a potent inhibitor of cytokine IL-4 and IL-13 binding to the IL-4Rα/IL-13Rα1 complex.

Administration of piktrakinra protected allergic monkeys from AHR when challenged with allergen (118). In a randomized, double-blind, placebo-controlled experiment carried out by

Wenzel et al., (2007), it was shown that, when compared with placebo, decrease in forced expiratory volume in 1 second (FEV1) after allergen challenge was significantly reduced after inhalation of piktrakinra for 4 weeks, which showed that inhibition of IL-4 and IL-13 cytokine can affect late asthmatic response after allergen challenge in subjects. The incidence of spontaneous asthma attacks were also reduced with piktrakinra administration suggesting a novel method in controlling asthma symptoms (119).

However, all of these findings require clinical trials prior to application on human allergic conditions, and their efficacy is questionable. Therefore, more stable synthetic peptide inhibitors promise to be important alternatives.

1.6 Phage Display Technology

A current strategy in improving allergic conditions involves pharmacotherapy, immunotherapy and avoidance of exposure to allergens. It is a hope that the strategy of phage display technology will provide a more swift and efficient method of improving antibody related diseases with the identification of novel synthetic peptide antagonists/inhibitors. In a nutshell, this technology allows one to perform protein-ligand interactions (120). Phage 33

PhD Thesis – Chapter 1 Nayyar Ahmed display is a selection technique in which a library of peptide variants are expressed on the surface of a phage virion, in this case, an M13 phage (121). Minor coat proteins pIII are expressed on the M13 phage vector surface. These proteins have 12-mer peptides (amino acid chain consisting of 12 amino acids) fused to their N-terminal. The library has 2.7 x 109 variants in peptide display, which gives a high probability of peptides showing affinity to the target molecule. This peptide-displaying clone is then identified, isolated, amplified and sequenced (Figure 1.8).

Biopanning is carried out by incubating a library of phage-displayed peptides on a micro-titer plate with an immobilized target. The unbound phage is washed away while eluting the target-bound phage. The eluted phage is then taken through additional binding/amplification cycles to get a consensus in binding sequences (Figure 1.8). The amplification of phage is achieved in an E. coli strain ER2738. The phage is then grown on agar plates containing isopropyl β-D-1-thiogalactopyranoside (IPTG) and 5-bromo-chloro-indolyl- galactopyranoside (X-gal). The library cloning vector M13 is a derivative of cloning vector

M13mp19, which carries the lacZα gene. This gives a blue color to the plaques that successfully grow in this medium. The blue plaques are hand-picked, isolated and sequenced for further analyses (122, 123).

An affinity selection in this way allows the selection of peptides that mimic IL-4 cytokine, and hence, will most likely block the interaction between IL-4 and IL-4R. If this is successfully achieved, production of IgE antibodies by B cells may be effectively reduced, resulting in a down-regulation of the allergic cascade.

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Figure 1.8. The biopanning strategy of phage display technology. Step 1: A library of phage (M13), each displaying a different peptide sequence is exposed to a 96-well plate coated with recombinant human IL-4Rα (RhuIL-4Rα) target molecule. Step 2: Phage M13, which is unable to bind to the target molecule, is washed away with Tris-buffered saline containing Tween-20 (TBST). Step 3: Phage M13, which was able to bind to the target molecule, are eluted using Glycine/HCl by lowering the pH. Step 4: The eluted phage is immediately neutralized, amplified and taken through additional binding/amplification cycles to enrich the pool in favor of binding sequences.

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1.6.1 Mimtags the Use of Phage Display Technology to Produce Novel Protein-Specific

Probes

The NARL research group at Deakin University has published an article based on the results of Chapter 2 (“Identification and characterization of a novel IL-4R antagonist for allergy treatment”). Although not directly relevant to our research for peptide antagonist, it is essential to highlight the importance of phage display technology as a useful tool for epitope mapping and protein tagging. Our paper highlights the use of this technology to detect different proteins such as IL-4R.

Protein-specific probes used for epitope mapping and protein-tagging studies have been the focal point of research during recent years employing various methods of detection of proteins, such as antibodies and labelled proteins. Currently, protein analysis consists of immunoassay techniques such as enzyme-linked immunosorbant assay (ELISA), polyacrylamide gel electrophoresis and related blotting techniques that use antibodies for detection and interactions with antigens (124). The advent of monoclonal antibodies with the development of hybridoma technology by Kohler and Milstein in 1975 was another remarkable milestone, which has revolutionized the way we conduct research today (125-

128). Monoclonal antibodies are generally specific to the target antigen and are often used for epitope mapping of proteins for exploring the human proteome for diagnostics and therapeutic purposes (129). The epitope region can either be linear or conformational in their structure and the amino acid sequence can be continuous or discontinuous (130). The ability of monoclonal antibodies to detect a single antigenic determinant (epitope) has earned it a major advantage over the use of polyclonal antisera. However, there are disadvantages of using antibodies where precise recognition of epitopes is seldom accurate (131) and there are limitations and disadvantages with these techniques (132). The hybridoma technology 36

PhD Thesis – Chapter 1 Nayyar Ahmed involves many phases from the fusion of the primed B cells with myeloma cell line to the large-scale production of monoclonal antibodies from these highly selective hybrid cell lines

(125, 133). For researchers working in the field, the use of this available in vitro technique is often problematic and has found to be very labour intensive and expensive (133). Animal ethics is a major concern when it comes to development of monoclonal antibodies, which causes unnecessary suffering of animals (133, 134). Another disadvantage of larger protein- based-probes, such as biotin binding proteins, is the size of these antibodies that are tagged with a detectable label. Larger probes present a challenge of inefficiently penetrating the conformational structures of target proteins as opposed to short peptides. A larger protein, such as an antibody, can also interact with a random functional groups on a single protein to the exclusion of thousands of other competing cytoplasmic proteins, nucleic proteins, carbohydrates and small molecules, which poses an enormous chemical problem (135). It is therefore imperative to find new techniques that are particularly important for quantitative proteomics, less tedious and cost-effective.

A more contemporary form of research has brought short peptides sequences under the spotlight as target specific probes for in vitro and in vivo analysis of protein ligands such as transmembrane proteins. This type of epitope tagging has led to very successful epitope mapping of target antigens. The methodology has been used for protein localization, immunoprecipitation, and protein-protein interaction (136). Briefly, an epitope is a sequence of amino acids in any structure that is recognized by an antibody. A single protein may carry multiple epitopes that can be recognized by antibodies. Two decades ago it was realized that using peptide phage libraries, mimotopes (short peptide sequences) could be isolated which mimic the epitope on a protein in their physical and chemical properties. Using this technique, the 3-dimensional structure of the epitope can be determined along with the 37

PhD Thesis – Chapter 1 Nayyar Ahmed critical residues that make up the epitope (137). Hence, in this case the mimotope acts as a probe against the desired epitope on a protein or antibody as a therapeutic agonist or antagonist.

1.6.1.1 Mimtags

A new term introduced in this paper (138) was ‘Mimtags’, which are the same short sequences of amino acids that play a critical role in the advancement of phage technology from a very different perspective. These short sequences of peptides are a part of fused proteins on the surfaces of phage used in phage display libraries. These epitope-mimics or mimotopes (139) that can be tagged (mimtags) on to protein sequence, can effectively identify the epitope region of a target antigen and used as protein-specific probes. Since the methods and results for this paper are part of Chapter 1, the term “mimtag” will now be used for discussion purposes in section 7.3.

The technique has been used in various studies to prove its effectiveness. A recent study conducted by the NARL research group at Deakin University has shown remarkable results

(138, 140-142). In our study, random peptide library on M13 phage was used to screen an IL-

4R and IL-13. An affinity selection in this way allows the identification of mimtags that bind to the cytokine and its receptor, and hence, will most likely target epitopes more effectively due to the small size and nature of the mimtag (12-mer peptide). The mimtag also has the ability to conform 3-Dimensional configuration increasing its binding efficiency (143). The bound peptide was sequenced and synthesized for further characterization by immunoassay techniques. The study shows the potential use of biotinylated peptide mimtags as examples and their advantage over complex antigens and antibodies being applied as novel protein specific probes. 38

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1.6.2 Testing the Peptide’s Inhibitory Efficiency in Tissue Culture

Once the novel peptide inhibitor is isolated by phage display, the next logical step is to test its inhibition properties in tissue culture. Human embryonic kidney cells-blue (HEK-Blue) will be used as model systems in this study to characterize the capacity of the synthetic peptide inhibitor to down-regulate IL-4 signaling pathway in vitro. This will allow us to establish the profundity of the peptide in more detail.

1.6.3 HEK Cytokine Reporter Cells

HEK-Blue™ IL-4/IL-13 cells allow the detection of bioactive IL-4 and IL-13 through the activation of the STAT6 pathway. These cells are derived from a stable transfection of

HEK293 cells with human STAT6 gene to obtain a fully active STAT6 pathway. The cells are also transfected with another stable STAT6 inducible secreted embryonic alkaline phosphatase (SEAP) reporter gene. Upon stimulation from either IL-4 or IL-13 cytokine, instead of producing IgE antibodies (as is the case with human B cells), the cells secrete

SEAP. The levels of SEAP production can be detected using QUANTI-Blue™, which turns purple/blue in the presence of SEAP (Figure 1.9 (A)). These cells, though very new, have turned out to be very robust in research activity. Publications as recent as 2011 suggest they are being extensively used for allergy research (144). The overlapping biological functions of

IL-4 and IL-13 have been thoroughly discussed in section 1.5.2 above with their common receptor chains; IL-4Rα and IL-13Rα1. The HEK-Blue cells are transfected with recombinant

IL-4Rα/ IL-13Rα1 receptor which can be activated by IL-4 and IL-13.

Using HEK-cells for tissue culture research carries many benefits. To name a few, the cells have been used as an expression tool for recombinant IL-4R for over 25 years. They remain highly stable and efficient when transfected with foreign genes. Also, once transfected, the 39

PhD Thesis – Chapter 1 Nayyar Ahmed translation and processing of the gene product (proteins) is performed with high proficiency.

1.6.4 Comparative Role of Ramos B Cells and HEK-Blue Cells

Ramos, a B lymphocyte cell line derived from Burkitt’s lymphoma, is a very suitable and optimized model system used to study B lymphocyte behavior and apoptosis (146). The cell line carries the model system for STAT6 phosphorylation upon induction of IL-4/IL-13 cytokines through their respective receptors. This triggering mechanism leads to the production of IgE antibodies (147) (Figure 1.9 (B)).

In this project, the identified novel peptide inhibitor from earlier studies will be tested on

HEK-Blue cells to test the peptide’s efficacy in reducing the IL-4/IL-4R signaling pathway.

This method has not been used in previous literature. A dose-dependent relationship will be established where a higher concentration of peptide will deliver a powerful antagonizing effect, hence, reducing SEAP in HEK-Blue cells (Figure 1.9). The visual spectrum of color in HEK-Blue cells will ultimately form the basis of future testing of the peptide in an animal model system. The transfected model mimics the production of IgE antibody from B lymphocytes where IgE is replaced with SEAP secretion.

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Figure 1.9A

Figure 1.9B

Figure 1.9. Comparison between the JAK-STAT6 signaling in transfected HEK-Blue cells that lead to SEAP production (1.9 A) as opposed to Ramos B cells that leads to IgE production (1.9 B). The HEK-Blue cells mimic the internal mechanism of STAT6 phosphorylation found in Ramos B cells. Figure adapted from (147).

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1.7 Effects of Dietary Antioxidant and Fatty Acids on Allergy: Connecting the Dots

Over recent years, the etiology of allergy has implicated several factors that are responsible for an increase in prevalence of this disease. However, the most important factor is diet. It has been suggested that low intake of omega-3 fatty acids along with antioxidants such as vitamins have resulted in a rapid rise in atopic diseases, especially in children (148, 149).

Hence, a thorough study of this underlying cause will perhaps reveal a more reliable method of therapy for allergy treatment.

1.7.1 Inhibition of Allergy Pathway Using n-3 Fatty Acids and Vitamin E Antioxidant

Hence, a thorough study of this underlying cause will perhaps reveal a more reliable method of therapy for allergy treatment.

Omega-3 or n-3 fatty acids are polyunsaturated fatty acids (PUFA) with a double bond (C=C) at the third carbon atom from the end of the carbon chain, hence the name (150). Recently, it has been shown that n-3 and n-6 fatty acids (which have a double bond (C=C) at the sixth carbon atom from the end of the carbon chain) (150) shown in Figure 1.11. They have opposite effects on allergic conditions (151). More than a decade ago, this phenomena was suggested by Black and Sharpe (1997), who indicated that an increase in consumption of dietary n-6 fatty acids and a decrease in fish oil derived n-3 or omega-3 PUFA leads to an increase in asthma and atopic disease in allergic individuals (152).

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Docosahexaenoic acid (DHA), 22:6ω3 (22:6n-3) contains 22 carbon atoms and 6 double bonds.

Eicosapentaenoic acid (EPA), 20:5ω3 (20:5n-3) contains 20 carbon atoms and 5 double bonds.

Docosapentaenoic acid (DPA), 22:5ω3 (22:5n-3) contains 22 carbon atoms and 5 double bonds.

Arachidonic acid (AA), 20:4ω6 (20:4n-6) contains 20 carbon atoms and 4 double bonds.

α -tocopherol (Vitamin E) ((2R)-2, 5, 7, 8-Tetramethyl-2-[(4R, 8R)-(4, 8, 12- trimethyltridecyl)]-6-chromanol).

Figure 1.11. The chemical structures of DHA, EPA, DPA and vitamin E (153).

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A decrease in fish oil derived n-3 or omega-3 PUFA leads to an increase in asthma and atopic disease in allergic individuals (152).

It has been shown that n-3 and n-6 fatty acids have opposite effects on allergic conditions

(151). More than a decade ago, this phenomena was suggested by Black and Sharpe (1997), who indicated the increase in consumption of dietary n-6 fatty acids and a decrease in fish oil derived n-3 or omega-3 PUFA leads to an increase in asthma and atopic disease in allergic individuals (152).

The use of fatty acid supplementation in diets has increased and gained much attention in the recent years, particularly its effect in developing a strong immune system in early stages of life (154, 155). Pregnant women are given dietary supplements of fatty acids to maintain a steady supply of development in the child’s immune system. Current studies have suggested that such supplements have shown to effect IL-4/IL-13 levels and reduce asthma and other related allergic disorders during childhood. The three main fatty acids included in the studies were DHA, EPA and DPA (Figure 1.11). The supplementation was started at 20 weeks of gestation period (156). No credible studies have been carried out to suggest that DPA has a link to allergic diseases (157).

Furthermore, a study undertaken recently with the effects of DHA on IL-4/IL-4R signaling mechanisms in human B cells, which forms the precursor to IgE production, has suggested that incubating DHA with Ramos B cells resulted in a dose-dependent inhibition of IgE by hampering the IL-4 signaling pathway via STAT6 dimerization (158).

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In addition to this, vitamin E (Figure 1.11) has been found to dampen IgE levels in animal models. This suggests a protective role of vitamin E in atopic disease and hence can be used as a model system for challenging allergy “head on” (151, 159). However, a study carried out in Germany, in a population of over a thousand people, suggested that IgE serum levels had no significant change after administration of vitamin E (106). These conflicting results suggest that the underlying mechanism is still unclear on whether vitamin E is anti- inflammatory in nature or vice-versa. We will deal with the already existing hypothesis that an increase in dose of vitamin E and PUFA is inversely proportional to serum IgE levels. In other words, both of these nutrients will affect the STAT6 signaling pathway in HEK-Blue cell line, as planned in this study.

The aim of this part of the project is to reduce the IL-4/IL-13 with IL-4R and IL-13R signaling pathway in HEK-Blue cells using colorimetric analyses. This would open new vistas to our project and help alleviate allergy in atopic individuals by using natural dietary remedies such as DHA and EPA coupled with the vitamin E antioxidant.

1.7.2 Discovering the Effects of Omega-6 AA on Allergy

Over recent years, Omega-6 fatty acids have gained much attention because of their effects on the etiology of allergic disease. Industrialized nations have increased their dietary intake of Omega-6 fatty acids over the past few years. As a result of these increased consumption of

Omega-6 rich vegetables and oils, the ratio between Omega-3:Omega-6 fatty acids has increased many fold from 1:1 to 10:1 (160). Eicosanoids have important role in inflammation. Omega-6 oils are precursors to short-lived but very powerful enzymes known as eicosanoids which play an important role in the pathophysiology of atopic disease (160).

Eicosanoids derived from n-6 fatty acids are generally regarded as pro-inflammatory. 45

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Eicosanoids such as LT, thromboxanes (TX) and prostaglandins are all pro-inflammatory with the latter two causing bronchoconstriction in asthmatic patients. Cyclooxygenase (COX) and lipoxygenase (LOX) can convert AA to the 2-series of prostaglandins, the 2-series of TX, and the 4-series of LT. In individuals with asthma, a bronchial allergen leads to enhanced prostaglandin-2 (PGD2) production. PGD2 production begins with the liberation of AA from membrane phospholipids by phospholipase A in response to inflammatory stimuli (Th2 cytokines). Thus, during asthmatic attacks in humans, PGD2 is released in large amounts by mast cells (161). PGD2 is a major product from COX-catalyzed reactions in a variety of tissues and cells such as T cells, dendritic cells, macrophages, mast cells and basophils and a major cause of bronchial asthma (162, 163).

A study showed that early supplementation of Omega-6 fatty acids did not prevent the expression of atopy as suggested by serum IgE levels (164). However, conflicting evidence suggests that Omega-6 fatty acids do not alleviate allergy, although it did reduce serum IgE levels in 12 months old infants (165). Although researches on Omega-6 fatty acids and prevention of allergic diseases are on the rise, there is no systematic evidence to prove that

Omega-6 fatty acids can prevent allergic disease.

Chemoattractant receptor homologous molecule is expressed on the surface of Th2 cells. This receptor is also known as DP2. This receptor binds PGD2 and stimulates the activation and recruitment process of Th2 lymphocytes leading to production of Th2 cytokines IL-4, IL-13 and IL-5 (166, 167). Antigen-induced cross-linking of cell-surface IgE on mast cells leads to the rapid abundant production of PGD2. In addition, PGD2 interacts with another receptor known as DP1 on DC to activate polarization of Th1 cells to Th2 variant. These agonistic

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effects of AA derived PGD2 prostanoids on Th2 cells make it an important target for research.

1.8 Anti-inflammatory Effects of Resolvins and Coenzyme Q10 on Allergy

Recently, it was revealed that certain PUFA derived lipid mediators had astonishing effects on inflammation. The lipid mediators, namely E series resolvins (derived from EPA) and D series resolvins (derived from DHA) (Figure 1.12), were found to be very specific in their effects on allergy. Instead of causing immunosuppression, the resolvins use pin-point targeting mechanism to resolve inflammation and therefore, help to main homeostasis (168).

The effects of coenzyme Q10 (CoQ10) in allergy have also been recognized in recent studies.

The studies are associated with symptoms of allergy such as bronchial asthma and acquired deficiency in CoQ10 intake (169, 170). However, none of these reports were able to conclusively demonstrate the inhibitory effects of CoQ10 (Figure 1.12) and resolvins on allergic models.

1.8.1 Role of CoQ10 in Allergy

Coenzyme Q is well defined as an important component of the oxidative phosphorylation pathway in mitochondria and converts carbohydrates and fatty acids into ATP (171). Another important substrate called CoQ10, also known as ubiquinone, plays decisive roles in the production of cell energy and prevents free oxygen radicals from causing cellular damage.

CoQ10 is normally found in tissues and organs which require high energy consumption

(169). The primary role of CoQ10 is its role as an intermediate in the electron transport chain system found in all mitochondria. Hence, it is ubiquitous in nature and forms an important substance in cellular respiration. Due to its antioxidant properties, it has been speculated that

CoQ10 is beneficial for allergic patients and asthmatics. CoQ10 has been implicated in 47

PhD Thesis – Chapter 1 Nayyar Ahmed allergic conditions for more than two decades (172). Bronchial asthma is a medical condition where, as a result of CoQ10 deficiency, bronchial mucosal inflammation arises (169, 173).

Elaborating on the asthma condition, it was documented by Gazdik et al., (2002) that asthmatics had reduced levels of CoQ10, causing disruption in antioxidative processes at the cellular level. This oxidative imbalance is connected to chronic inflammation of the airway.

In yet another study carried out in 2007, it was found that food intolerance and allergies in children were due to an acquired CoQ10 deficiency (170). With all the overwhelming evidence that is available, one can hypothesize that CoQ10 may play an important role in allergic diseases. Hence, dietary supplementation of asthmatics with CoQ10 may result in a decrease in allergic prevalence.

1.8.2 Role of Resolvins in Allergy

Resolvins are a family of lipid mediators that are derived from EPA and DHA. The term resolvins (resolution-phase interaction products) was first coined to signify that these products were synthesized during the resolution phase of inflammation, possessing very potent anti-inflammatory and immuno-regulatory actions. These actions include reducing neutrophil infiltration, regulating cytokine and reactive oxygen species, and lowering the extent of the reaction (174). Resolvins have been studied and understood as anti- inflammatory and moreover, promote the resolution of the inflammatory response to a non- inflamed state (175). Resolvin D1 (RvD1) is produced from the oxygenation of DHA by lipoxygenase. RvD1 reduces human polymorphonuclear leukocyte (PMN) trans-endothelial migration, which is the initial phase of acute inflammation.

PhD Thesis – Chapter 1 Nayyar Ahmed

Resolvin D1 - (4Z,7S,9E,11E,13Z,15E,17S,19Z)-7,8,17-Trihydroxy-4,9,11,13,15,19- docosahexaenoic acid.

Resolvin D2 - (4Z,7R,8E,10Z,12E,14E,17S,19Z)-7,16,17-Trihydroxy-4,8,10,12,14,19- docosahexaenoic acid.

Resolvin E1 - Sodium (6Z,8E,10E,14Z,16E)-5,12,18-trihydroxy-6,8,10,14,16- icosapentaenoate.

Coenzyme Q10 - 2-[(2E,6E,10E,14E,18E,22E,26E,30E,34E)-3,7,11,15,19,23,27,31,35,39- Decamethyl-2,6,10,14,18,22,26,30,34,38-tetracontadecaen-1-yl]-5,6-dimethoxy-3-methyl- 1,4-benzoquinone.

Figure 1.12. The Chemical Structures of Resolvins D1-2, E1 and CoQ10 (153). 49

PhD Thesis – Chapter 1 Nayyar Ahmed

In a study carried out by Sun et al., (2007), it was shown that the 17(R) epimer of RvD1, which is triggered by aspirin, reduces leukocyte infiltration in a mouse-model of peritonitis with maximal inhibition of 35% at 100 ng dose (176). The 17(R) and 17(S) D series of resolvins exhibit potent anti-inflammatory action in vivo as described by Song et al., (2003).

Trout brain cells also produce RvD1 from endogenous stores of DHA which also indicates that RvD1 carries a conserved evolutionary structure (175). Resolvin D2 (RvD2) is produced physiologically from the oxygenation of DHA by lipoxygenase and shares the same function as RvD1 in dampening excessive neutrophil trafficking during acute inflammation. It reduces zymosan-stimualted PMN infiltration by 70% at doses as low as 10 pg per mouse and significantly reduces platelet activating factor (PAF) stimulated leukocyte adherence and migration at 1nM. It also stimulates Nitric Oxide (NO) production, which decreases leukocyte-endothelial interactions (177).

1.8.3 What is the Resolution Phase of Acute Inflammation?

Inflammation is an important part of the immune system. It is triggered by many different stimuli that pose a threat to the overall homeostasis of tissue structure. In the acute inflammatory process neutrophils, eosinophils and basophils rapidly mobilize to the site of inflammation. This is vital for the removal of harmful agents such as bacteria, fungi, viral infection and parasitic invasion. Allergic response is another example of such inflammation that result into severe clinical manifestations in the form of inflammation. Resolution of inflammation is understood to be a process by which granulocytes (mentioned above) are eliminated from the site of inflammation whereby reducing the mononuclear cell numbers to basal levels (178, 179). During spontaneous resolution, neutrophils undergo cell apoptosis, which is a highly regulated process. Phagocytes travel to the site of inflammation and mediate effective clearance of dying cells (180). An acceptable theory is that resolution is an 50

PhD Thesis – Chapter 1 Nayyar Ahmed active process and is controlled by a range of cellular activities. Acute inflammatory phase is self-limiting and results into tissue restoration and homeostasis. In cases where tissue restoration or reduction in acute inflammation does not occur, the condition is said to be chronic inflammation (178, 181).

1.8.4 Resolving Inflammation

Resolvins D1 and aspirin-triggered RvD1 have both been shown to be potent regulators of neutrophils in a human and mouse model. In microglial cells, RvD1 blocks tumor necrosis factor (TNF) induced activation of pro-inflammatory cytokine interleukin-1 (IL-1), which is expressed in high quantities during a neuronal injury. In addition, human macrophages have shown to be more active in the presence of RvD1, which interacts with the lipoxin receptor

(ALX) and G protein coupled receptor (GPR32) while short-hairpin ribonucleic acid

(shRNA) knockdown of each receptor resulted into decreased rate of phagocytosis of neutrophils.

RvD2 reduced peritoneal neutrophil infiltration by 70% after the mice were challenged with zymosan at ultra-low doses (in picograms). In a study carried out by Gaboury et al., (1993), it was found that 1 nM of RvD2 was sufficient to reduce platelet activating factor-stimulated leukocyte adherence and reduced cell interaction between neutrophils and endothelial cells.

Additionally, RvD2 was also found to reduce local production of NO, which is a well-known anti-adhesive mediator (182). In another study carried out by Spite et al., (2009), mice treated with RvD2 shows lower levels of peritoneal neutrophil numbers and the inflammatory cytokines such as IL-6, IL-23 and IL-17 were all reduced (177). The inflammatory lipid mediators’ prostaglandin E2 (PGE2) and leukotriene B4 (LTB4) were both reduced by the antagonistic effect of RvD2 treatment. In vitro RvD2 showed increase phagocytosis by 51

PhD Thesis – Chapter 1 Nayyar Ahmed macrophages and treatment of neutrophils showed an increase phagocytosis of E. coli (177,

183).

Resolvin E1 (RvE1) and RvD1 upregulate chemokine receptor type 5 (CCR5) expression in dying neutrophils. CCR5 expression on apoptotic neutrophils acts as a terminator for chemokine signaling. In addition to this, RvE1 specifically induces the expression of complement decay accelerating factor (CD55), which is an anti-adhesion molecule expressed on the mucosal surface of epithelial cells and therefore promotes the clearance of mucosal neutrophils from mucosal surfaces (184, 185).

RvE1 is a trihydroxy EPA metabolite that has been identified in murine inflammatory exudates and in human plasma when subjects are treated with both aspirin and supplemental

EPA. RvE1 is a very potent anti-inflammatory EPA metabolite and has been shown to protect against experimental colitis in a murine model (186-188). In 2010, a publication suggested that RvE1, an anti-inflammatory bioactive lipid mediator, reduced airway inflammation and asthma in a murine model (189). In addition to this finding, ovalbumin-specific IgE antibodies were also shown to decrease with the administration of RvE1 (189) and hence, airway inflammation. The Resolvins E1 and D1 have been found to attack the resolution of inflammation, a vital process that helps to shut off the inflammation cascade that can eventually lead to tissue scarring and damage (190).

RvE1 also down-regulates the production of neutrophils by interacting with two receptors known as chemokine-like receptor 1 (ChemR23) and the leukotriene B4 receptor (BLT1).

Upon binding with the ChemR23 receptor, the RvE1 attenuates TNF-stimulated nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), which results into counter- 52

PhD Thesis – Chapter 1 Nayyar Ahmed regulatory action (186). This is a critical step since TNF is a key mediator during the early phases of acute inflammation. In the case of BLT1, the RvE1 resolvin interacts in a stereospecific manner and acts as a potent antagonist to neutrophils. In a similar fashion,

RvE1 selectively interacts with ChemR23 also found on mononuclear and DC leading to resolution of inflammation (191). The anti-inflammatory action of RvE1 is markedly reduced in a model of mice deficient in BLT1 (191).

The NARL research group at Deakin University, in collaboration of colleagues at Deakin, will evaluate the potential usefulness of Resolvins E1 and D1-2 along with CoQ10, in allergy treatment by using our HEK-Blue cell line. This will determine the underlying mechanisms of resolvins and CoQ10 on cytokine stimulated allergic response.

1.9 Pollen Allergy

Pollen allergy is caused by pollen grains. The pollen grain is the male part of a flowering plant, which is necessary to propagate seeds. It is released by one plant and transported by an insect or wind. Plants that use insects for pollinating their seeds are brightly colored to attract insects. These plants however pose no threat to allergic individuals. However, plants that pollinate using wind as transport factors are the ones that cause the real trouble. Windblown pollen is lifted into the air and in enormous amounts. In particular, green plants such as grass are the main culprits that cause allergy symptoms such as hay-fever and asthma (192).

1.9.1 Epidemiology of Pollen Allergy

Pollen was first recognized by Charles Harrison Blackley in 1873 which was a major cause of allergic symptoms in patients and performed a skin prick test on himself to test the theory

(193). Aerobiology is the field of science that includes the dispersion of bacteria, fungal 53

PhD Thesis – Chapter 1 Nayyar Ahmed spores and pollen in air which are studied by trapping pollen using a volumetric spore trap

(194). The method employed to perform grass pollen counts using a microscope was addressed by Hirst for the first time in 1952 (195).

Aerobiologists have played a very important role in establishing the correlation between pollen dispersion in air and allergy symptoms. The most important carriers of allergen in the air are found within pollen grains, which can range between 15 and 60 μm. However, these pollen are large in size and cannot penetrate the lower airways, which are smaller in diameter.

Hence, it is important to note that pollen have to be water-soluble in order to penetrate lower airways, which results in an IgE-specific allergic reaction among sensitized individuals (196).

1.9.2 Overview of Ryegrass Pollen Induced Allergy

Generally, it is found that grass pollen season starts with the release of pollen into the air when the pollen dehisce at the time of flowering. Plants are considered important if the yield of pollen grains is high. Proteins and glycoproteins which are derived from pollen and spores can function as allergens (197). As explained in the previous sections, IgE mediated Type 1 allergy is an exaggerated response by the immune system to substances or particles that are normally deemed un-harmful by nature (198). In our current study, we have considered pollens released by ryegrass. It is important to note that almost 20% of the world population now suffers from symptoms of hay fever or allergic asthma and the prevalence has risen to more than double in the past few decades (199). In Europe, Australia and America, around

70% of the population is allergic to grass pollen when tested for IgE reactivity (200). The patient is sensitized upon secondary exposure to the allergens when exposed to airways or skin. Also, the allergic response in each individual varies in its severity (201). One individual

PhD Thesis – Chapter 1 Nayyar Ahmed may suffer from mild symptoms such as sneezing or hay fever as opposed to anaphylaxis in a few others.

The final deposition site for allergens within the pollen reveals the mechanism involved in their reaction in the human body. To study allergens, a fixation method was employed in order to localize the allergens found within mature pollen. Ryegrasses (Lolium spp.) is the most dominant source of allergenic pollen because of its abundance during the flowering season. Ryegrass pollens were first reported by Johnson and Marsh in 1965 (202). Ryegrass pollen grains have a diameter of 25-30 μm and have one pore. The pollen wall consists of a multi-layered exine traversed by micro-channels (203). A mature ryegrass pollen typically contains 700 starch granules. Each amyloplast found inside pollen grains contains one starch granule with a diameter of 1 μm (197, 204). A range of different allergens have been identified to date of ryegrass pollen from the perennial family. The total number of ryegrass pollen allergens identified are 17. The most important proteins identified to date are Lol p 1-5

(197). However, the most allergenic proteins are Lol p 1 (35 kDa glycoprotein) (205) and Lol p 5 (28-33 kDa glycoprotein) (206) that elicited IgE-mediated reactions when tested in patients using skin prick tests. Although less abundant than Lol p 1, Lol p 5 showed 90% of patients with IgE antibody against this allergen (207). Both of these proteins have been extensively studied and characterized. The amino acid sequences of Lol p 1 and Lol p 5 share non resemblance to other know grass allergens. Another important allergen from ryegrass identified by Suphioglu et al., (1992) was Lol p 5 (previously known as Lol p IX), which is located within the starch granules located inside the pollen (208). Detailed molecular knowledge of the allergenic epitopes of pollen allergens is very important to design mutants of these proteins resulting into altered allergenicity (209). These mutants can then be used as therapeutic agents (209). The IgE binding epitope of Lol p 1 has been characterized and 55

PhD Thesis – Chapter 1 Nayyar Ahmed described by Esch and Klapper, 1989 (210). The characterization and identification of IgE binding epitopes for Lol p 5 was done by Suphioglu et al., in 1998 (211).

1.9.3 Diseases Associated with Ryegrass Pollen Allergy

Climate change is increasing the prevalence of atmospheric pollen exposure, which results into greater incidence of allergic disease in Australia. The incidence of allergic disease in

Australia is one of the highest in the world compared to Europe and North America.

Although asthma incidence doubled in the 1980’s and 1990’s, it plummeted for the next 10 years due to a below average rainfall in the south-eastern region of Australia (Melbourne,

Victoria). While pollen grains are large enough to remain at bay without penetrating the airway system of the lungs, occurrence of rainfall during pollen season releases starch granules and allergens, which can penetrate into the lower airways where they can trigger asthma. Asthma is a chronic inflammatory disease which is marked by symptoms such as wheezing, chest tightness and repeated coughing (212). A study carried out by Pollart et al.,

(1988) demonstrated a positive correlation between IgE antibody produced in allergic patients and ryegrass pollen. It also supports the fact that patients suffering from a developed

IgE Ab to an environmental allergen is exacerbated by continued exposure to the same allergen, which results in acute attacks of asthma (213). Allergic rhinitis, also known as hay fever, is another pollen induced disease characterized by an inflamed nose in response to IgE- mediated inflammation. Symptoms of rhinitis include sneezing, nasal obstruction and nasal itching. Conjunctivitis is also considered a symptom of rhinitis (214, 215). In a study carried out by Annesi-Maesano et al., (2012) showed a positive correlation between grass pollen count per m3 of air and allergic rhinitis among allergy sufferers (216). Another study carried out by Kemp et al., (2009) reported that children at a tender age of 8 years, who were born during the pollen season, had an increased risk of developing allergic rhinitis but not for 56

PhD Thesis – Chapter 1 Nayyar Ahmed children born outside the pollen season. This birth cohort study was carried out in the state of

Tasmania, Australia (217).

1.9.4 Diagnosis of Grass Pollen Allergy using Protein Extracts

Clinical tests take into account the total grass pollen protein extracts to establish whether a patient is suffering from allergy. RAST and skin prick tests are used to determine if the allergens are binding to IgE antibody, which establishes the basis of primary cause of allergy in individuals. Detail of these tests has been outlined in sections 1.2.2.1 and 1.2.2.2

1.9.5 Grass Pollen Count and Meteorological Data

Grass pollen counts are highly variable from one pollen season to another. In Melbourne, seasonal total grass pollen ranged from 1000 in 1980 and 1981 to greater than 8000 grains/m3 in 1993 and 1994. Under suitable conditions, Melbourne experiences high Northerly winds with high temperatures, which translates to a high count of ryegrass pollen per m3 of air.

Ryegrass is commonly grown in Victoria as a pasture grass (218). Pollen grains are ruptured in rainwater by osmotic shock, which releases starch granules into the air. A growing body of evidence suggests that relative humidity (RH) and temperature effect the dispersion of ryegrass pollen in air. A study carried out by Glovsky et al., (2007) reported that peaks in either RH values or rainfall is sufficient to rupture or fragment pollen grains (219). As for wind speed, a minimum of 6 mph was required to release ryegrass pollen from their anthers.

In another study reported by Grote et al., (2000), it was found that ryegrass pollen grains when exposed to isotonic solutions, remained intact. However, upon exposure to distilled water or rainwater, they were able to expel starch grains and cytoplasmic debris by pollen rupture (220). It is also reported by Suphioglu in 1998 that ryegrass pollen ruptures upon contact with water, which releases 700 starch granules. Evidently, starch granules have been 57

PhD Thesis – Chapter 1 Nayyar Ahmed captured from air in Melbourne, Australia with an increase of 50-fold after an episode of rain

(204). Another phenomenon associated with meteorological data and pollen dispersion is thunderstorm asthma. Epidemics of acute asthma association with thunderstorms have been reported worldwide, such as 1987/1989 in Melbourne and 1994 in London. During springtime thunderstorms, asthma and the marked elevation of ryegrass pollen counts display a positive correlation, since ryegrass pollen grains rupture upon contact with water, which releases starch granules. A thunderstorm is usually accompanied with sudden atmospheric changes such as fall in temperature and air pressure along with rainfall and humidity, all of which act as contributing factors for the dispersion of pollen (221, 222). Another thunderstorm event that took the Canadians by surprise took place on the 31st of July 2000 in

Calgary, Alberta. As reported by Calgary Health Region, 157 people reported symptoms of respiratory distress as opposed to expected number of people reporting such symptoms over a period of 48 hrs. A sudden onset of high winds or gusts was attributed to the increase in pollen into the atmosphere, which caused the epidemic (223). Another thunderstorm- associated asthma study was carried out by Grundstein et al., (2008), which was conducted over a period of 10 years between from 1993-2004. Emergency department visits from 41 hospitals in Atlanta, Georgia were taken into account. A total number of thunderstorm days of 564 was included as part of this study to find the risk associated with asthma. Rainfall and wind gust levels had the strongest association with emergency department visits confirming a positive correlation (224).

1.9.6 Treatment for Pollen Allergy

SIT has been discussed thoroughly in the previous sections of this chapter. However, there is a growing body of evidence that suggests the use of SIT to treat pollen allergy. Although therapeutic by nature, this treatment has disadvantages as described in section 1.5.5.1. 58

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A study carried out by Jutel et al., (2005) employed a randomized, double-blind, placebo- controlled study of SCIT therapy in patients suffering from allergic rhino-conjunctivitis, with or without asthma. The symptom medication score showed significant reduction of symptoms in patients receiving recombinant allergens as opposed to placebo (225). In the largest clinical programme conducted with allergen-specific immunotherapy used Grazax for sublingual administration to a total of 75,000 patients. Results from this trial showed substantial inhibition of allergic symptoms in individuals suffering from rhino-conjunctivitis. The trial was conducted throughout the grass pollen season. Subjects that were treated with Grazax showed an improved quality of life. Although it was declared safe to use Grazax for treatment of pollen allergy, there were a number of side-effects associated such as puritis, edema mouth, ear puritis, throat irritation and sneezing (226). In a similar study carried out by

Winther et al., (2000), it was concluded that allergen-specific immunotherapy involving grass-pollen extract was associated with a higher number of systemic reactions in atopic individuals (227). In a separate study, it was reported that SIT prevents the onset of new sensitizations to other allergens and reduces the development of asthma in patients suffering from allergic rhinitis. It also reports the development and improvement of SIT by reducing

IgE-mediated reactions in atopic individuals (228, 229). The use of recombinant and genetically engineered allergens is also being explored by many researchers. The clinical efficacy of SLIT and SCIT against birch-pollen allergy was also determined by a randomized, placebo-controlled, double-blind, double-dummy study and showed comparable effects on 71 patients suffering from allergic rhinitis. The study also suggested the safe method of SLIT over SCIT (230). Many other studies have suggested similar treatments for pollen allergy but are in the initial stages of development and have yet to undergo several clinical trials to prove their efficacy (228, 229, 231-234).

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1.9.7 Deakin AIRwatch: Pollen Counting and Forecasting Facility

The Deakin AIRwatch project was launched in October of 2012 with a Burkard volumetric air trap installed on the roof of the library at the premises of Deakin University (Waurn Ponds and Burwood campuses). The ryegrass pollen season typically runs from October till January of the following year. Currently, the only operational counting station in Victoria is housed at the University of Melbourne and provided to the public by the Asthma Foundation of

Victoria (see: http://www.asthma.org.au/Home.aspx). Since there is currently no measure of atmospheric pollen and spore concentrations in regional Victoria (e.g. Geelong) and eastern

Melbourne (e.g. Burwood), we have established Deakin AIRwatch, incorporating pollen and spore counting stations at both the Waurn Ponds and Burwood campuses of Deakin

University. This is timely due to ever-increasing allergy and asthma epidemics. Deakin

AIRwatch network will not only directly benefit the public with pollen and spore counting service to assist in their allergen avoidance programs but also contribute to significant research and clinical studies, which is lacking for the greater Geelong area. The term Deakin

AIRwatch was coined by Associate Professor Cenk Suphioglu (PhD supervisor).

1.11 Overall Aims and Objectives of PhD Studies

Primarily, the objective of this study is to identify a novel antagonist that can bind to the cytokine IL-4RD and inhibit/down-regulate the interaction between the cytokines IL-4 and

IL-13 with IL-4Rα to potentially reduce the Th2 allergic response. The second aim is to use

PUFA’s, resolvins and CoQ10 as natural dietary remedies for allergic diseases. The third aim of this project was to evaluate and quantify ryegrass pollen count in regional Victoria

(Australia) with regards to meteorological factors. Below is a list of specific aims and objectives for this project.

PhD Thesis – Chapter 1 Nayyar Ahmed

In a nutshell, this project aims:

1. To identify a human IL-4R inhibitor by screening a 12-mer phage displayed random

peptide library (Chapter 2);

2. To investigate the ability of the novel synthetic peptide inhibitor to bind to the IL-4R

(Chapter 2);

3. To analyse the effectiveness of the identified novel inhibitor in reducing the

interaction between IL-4/IL-13 and IL-4R by performing quantitative immunoassay

techniques (Chapter 2);

4. To test the peptide antagonist in vitro by using a recombinant cell line (HEK-Blue)

(Chapter 3);

5. To utilise fatty acids to inhibit cytokines IL-4 and IL-13 interaction with their

receptors, subsequently blocking STAT6 phosphorylation in a HEK-Blue model of

allergy (Chapter 4);

6. To utilise resolvins and CoQ10 and their effects on IL-4R and IL-13R signalling in a

HEK-Blue model of allergy (Chapter 5);

7. To study and model atmospheric concentration of grass pollen count and its

correlation with weather variables in regional Victoria, which may improve quality of

life (Chapter 6).

1.12 Expected Outcomes of Study

• Identification of a novel synthetic peptide will pave the way to develop a prophylactic

treatment for allergy in the future.

• Cytokine IL-4 and IL-13 interaction with IL4-R is a common pathway for many

allergies, blocking this process may cure allergies in general.

PhD Thesis – Chapter 1 Nayyar Ahmed

• The use of fatty acids, resolvins and CoQ10 is expected to suppress allergic symptoms

and offer an alternative dietary route to reduce STAT6 phosphorylation induced by

cytokines IL-4 and IL-13 to tackle the allergic disease.

• The evaluation of ryegrass pollen distribution in regional Victoria will help improve

quality of life in individuals suffering from allergy and also the overall understanding

of pollen distribution and its relationship to changing climate variables.

PhD Thesis – Chapter 2 Nayyar Ahmed

Chapter 2 – Identification and Characterization of a Novel IL-4R Antagonist for Allergy Treatment

63 PhD Thesis – Chapter 2 Nayyar Ahmed

2.1 Introduction

There has been a marked increase in prevalence of atopic disease in regions such as Western

Europe, the US, Australasia and Asia Pacific during recent years, especially in industrialized nations. A 20-fold increase has been experienced by western countries and as much as 40% of the population suffers from this disease (8, 235-239). A recent statistical study carried out has shown that a rise in allergy trend has taken place over the last 20 years (9). A rise in trend has also been studied by ASCIA (18). These studies indicate an alarming situation and require a more thorough and aggressive approach to resolve this disease.

In atopic individuals, an immune response is mounted by T cells that are activated by allergens, promoting Th2 variant of cells. Once stimulated, these cells subsequently produce cytokines such as IL-4 and IL-13. Unlike the cytokines produced by Th1 cells in non-atopic individuals, an excessive production of cytokines takes place from Th2 cells (13, 240-243).

When the allergen comes into contact with antigen APC, the allergens are processed and presented to naïve T cells, which mature into Th2 cells and release cytokines such as IL-4, causing IgE-mediated immune response. Chemical mediators that are released as a result cause symptoms like sneezing, wheezing and eczema (13, 244-246).

13Rα1 is responsive to both IL-4 and IL-13 (59, 243, 247-249). Therefore, the concept that

IL-4 and IL-13 can be targeted simultaneously is of great importance because of their shared

64 PhD Thesis – Chapter 2 Nayyar Ahmed dependence on the IL-4Rα chain of the receptor. Hence, this research targets the common IL-

4Rα with a novel synthetic antagonist.

Current strategies to alleviate or avoid allergic symptoms include avoiding the allergens altogether, administration of drugs such as antihistamines and anti-inflammatory drugs and

SIT to relieve the symptoms (22, 23). SIT has many functions including, the induction of Th1 cells in the production of IgG immunoglobulin rather than IgE (24, 25). Th2 cytokines, principally IL-4Rα and IL-13, are reduced considerably, further alleviating the patient from the symptoms (25). However, it has been argued that SIT has its disadvantages such as unwanted effects of acute allergic reactions (250), poor efficacy and specificity as a result of poor quality of allergy vaccines, comprised of poor quality of allergen extracts (26, 251-254).

Moreover, it is of high risk for patients with anaphylactic allergy (e.g. peanut allergy).

Current available treatments and results from various researches are disappointing and raise concerns regarding therapies for allergy. Therefore, a need arises to redirect strategies in the favour of phage display technology, in the quest to identify novel peptide inhibitors for allergy treatment, which we report for the first time for human IL-4Rα receptor. Following successful identification of the novel peptide, a series of immunoassays were carried out to test the efficacy of the peptide in its binding to IL-4R. Our aim is to identify and characterize a novel IL-4R antagonist which can successfully inhibit the interaction of IL-4 cytokine with

IL-4R hence, inhibiting the allergic cascade.

65 PhD Thesis – Chapter 2 Nayyar Ahmed

2.2 Materials and Methods

2.2.1 General description of the M13 Phage library

Phage display libraries, Ph.D. 12, in which 12-mer random peptide are expressed at the amino terminus of protein pIII of the ﬁlamentous bacteriophage were purchased from New England

BioLabs Inc. as a kit. The peptide is followed by a short spacer sequence Gly-Gly-Gly-Ser, followed by the wild-type pIII sequence. Each of the libraries has a complexity of 2x109 virions and was ampliﬁed to give a ﬁnal titer of 2x1011 plaque-forming units (pfu). All experiments were carried out under strict sterile conditions using a Class II laminar flow cabinet (Figure 2.1).

2.2.2 Biopanning

Aliquots of the phage libraries were screened with RhIL-4Rα (Abcam®); coated on flat- bottomed 96 microtiter microplates (Thermo Scientific Co.). In the first and second rounds of panning, the coating concentration of protein was 50 nM, which was prepared using

NaHCO3. To increase the stringency of the panning, the wells were coated with decreasing concentrations of protein and increasing doses of Tris-buffered saline and tween 20 (TBST)

(TBS; 50 mM Tris-HCl pH 7.5, 150 mM NaCl + 0.1% [v/v] Tween-20). M13 phage was selected for further amplification from the wells that tightly bound to the target antigen. In the fifth round of panning, the concentration of the protein was 10 nM. A binding disruption buffer 0.2 M glycine HCl (pH 2.2) was used to elute the bound phage along with 1mg/ml of bovine serum albumin (BSA). 100 μl of this buffer was added to the wells of the microtiter plate and was rocked gently for 10 min. The eluate was transferred into a microcentrifuge tube, and immediately neutralized with 150 μl of 1 M Tris-HCl, pH 9.1.

66 PhD Thesis – Chapter 2 Nayyar Ahmed

A library of phage (M13), each displaying a different peptide

sequence were exposed to IL-4 receptor

M13 phage unable to bind to the target molecule was washed away

with TBST

M13 phage which were able to

bind to the target molecule were eluted

The eluted phage were amplified and taken through additional binding/amplification cycles

Figure 2.1. Schematic diagram of the steps taken to elute the phage and for perform biopanning. Purified recombinant human IL-4Rα was used as the target antigen.

67 PhD Thesis – Chapter 2 Nayyar Ahmed

2.2.3 Amplification and Identification of Eluted Phage Clones

The titer of the unamplified eluate was mixed with a freshly prepared culture of Escherichia coli (E. coli) ER2738 (Phage Display Kit; New England Biolabs) host strain which is tetracycline resistant (TetR) and incubated in a rotator shaker at 37°C and 240 revolutions per minute (rpm), for 4.5 h. The culture was then transferred to a centrifuge tube and spun for 10 min at 10,000xg at 4°C, to pellet bacterial cells. The supernatant (containing the amplified phage) was transferred to a fresh tube and re-spun for 5 min. Pellet was then discarded. Next,

80% of the upper supernatant was transferred to another fresh centrifuge tube. The phage was precipitated by using 20% (w/v) polyethylene glycol (PEG)/2.5 M NaCl, or roughly 1/6th of the total volume of eluate recovered from amplification. Dilutions of the phage were prepared and grown on plates containing Luria broth (LB) along with IPTG and X-gal (LB medium +

15g/L agar + 1.25g of IPTG + 1g X-gal in 25 ml of dimethyl formamide; Sigma Aldrich). The library-cloning vector M13 is taken from the common cloning vector M13mp19, which carries the lacZα gene. This resulted into phage plaques appearing blue when plated on media containing X-gal and IPTG (255).

2.2.4 Sequencing of Phage DNA: Rapid Purification

Phage DNA was sequenced from randomly selected clones, as described in the phage manual provided by New England Biolabs Inc. (256-260), using the Micromon DNA Sequencing

Facility at Monash University (Melbourne, Australia). After the plaque is amplified, as described in the section above, 500 μl of the supernatant containing phage was transferred to a fresh microfuge tube. 200 μl of 20% PEG/2.5 M NaCl was added to the phage for precipitation. The mixture was inverted several times and let to stand for 20 min at room temperature (RT). A centrifugation was carried out on this mixture at 13,000 rpm for 10 min at 4°C. The supernatant was discarded and the mixture was once again re-spun for 5 min. The

68 PhD Thesis – Chapter 2 Nayyar Ahmed supernatant was carefully discarded using a pipette. The pellet, which was left behind, was suspended in 100 μl of iodide buffer (10 mM Tris-HCl at pH 8.0 + 1 mM

Ethylenediaminetetraacetic acid (EDTA) + 4 M NaI) by vigorously tapping the tube, to generate single-stranded phage DNA for sequencing. Furthermore, 250 μl of 100% ethanol was added to the mixture and incubated for 10-20 min at RT. This allowed precipitation of single-stranded phage DNA and isolation of phage proteins in solution. The solution was spun in a microfuge at 13,000 rpm for 10 min at 4°C and the supernatant discarded. The pellet was subjected to another wash with 0.5 ml of 70% ethanol, re-spun briefly, the supernatant discarded. The pellet was allowed to dry in a biological class II cabinet. The pellet was finally suspended in 30 μl of TE buffer (10 mM Tris-HCl at pH 8.0 + 1 mM

EDTA) followed by quantification of DNA using a Nanodrop 1000 Nano-spectrophotomer

(Thermo Scientific Co.). Once quantified, 5 μl aliquot was taken from each DNA sample and transferred to smaller 600 μl microfuge tubes and stored at -20°C. These 5 μl DNA samples

(ten in total) along with the -96 gIII sequencing primer (phage display kit) were sent to

Micromon DNA Sequencing Facility at Monash University for DNA sequencing analysis.

Once the DNA sequences for all 10 phage clones were obtained, they were further analyzed using the FINCH TV sequence analysis software. This helped ascertain which sequences out of the 10 were most suitable to carry out further immunoassays. The flanking regions were used to locate the 36 base pairs of DNA of interest, which encoded the 12-mer peptide sequence displayed on the M13 phage surface. The hybridization positions of the -28 and -96 sequencing primers were indicated as well. Short peptide designated N1 (Primary Sequence:

XXXXXXXXXXXX) along with the biotinylated version were chemically synthesized by

AUSPEP (Melbourne, Australia) and received in a lyophilized condition with >90% purity.

The biotin was added to the N-terminal of the peptide. The quality of all the peptide was assessed by high performance liquid chromatography (HPLC) and confirmed by mass

69 PhD Thesis – Chapter 2 Nayyar Ahmed spectrometry analysis (purity >90%). Further analysis included alignment of 12-mer sequences with IL-4 and IL-13 cytokines using ClustalW2 multiple alignment software. The sequence of the peptide has not been shown due to intellectual property reasons with Deakin

University (Waurn Ponds, VIC, Australia).

2.2.5 M13 Binding ELISA

Four rows of a 96-well microtiter plate were coated with 90 μl of the target IL-4Rα (10 nM) protein. The plate was incubated at 4°C with gentle agitation. Following day, the plate was taken out of the box and excess target solution was removed by inverting the plate facedown onto a paper towel. The wells containing the target were blocked using blocking solution (5 mg/ml BSA and 0.02% sodium azide (NaN3)). Simultaneously, a second microtiter plate was also blocked with the blocking solution. Both plates were sealed with a parafilm and incubated at 4°C for 1-2 hrs. The excess blocking solution was removed by slapping the plate face down on a paper towel. Each plate was washed 5 times using 0.3% TBST. A second microtiter plate, without the target, was used to make 4-fold serial dilutions of the phage clone in 200 μl of 0.3% TBST; starting with 1012 pfu in the first well, down to 2 x 105 pfu in the 12th well. Phage dilutions from 4th, 5th, 6th, 7th and 8th wells were transferred to the plate with the target. This was incubated for 1-2 hrs at RT, with agitation. Once again, the plate was washed 5 times with TBST. 200 μl of primary antibody, namely mouse anti-M13 antibody (Sigma Aldrich) (1:1000 dilution in blocking buffer) was added to each well and incubated for 1 hour (hr) at RT. Subsequently, the plate was washed 5 times with TBST.

Post-incubation with anti-M13 antibody, 200 μl of secondary antibody, namely anti-mouse horseradish peroxidise (HRP) conjugated antibody (Sigma Aldrich) (1:2000 in blocking buffer) was added to each well and incubated for 1 hr with agitation on a rocker. This was followed by another round of 5 washes with 0.3% TBST. A developing solution was

70 PhD Thesis – Chapter 2 Nayyar Ahmed prepared, by dissolving a single tablet of phospho-citrate buffer with sodium perborate in 100 ml of distilled water (DH2O), then 1 tablet of HRP substrate ortho-phenyl diamine (OPD;

Sigma Aldrich,) was dissolved in 7.5 ml of this solution. 200 μl of this solution was added to each well; the plates were wrapped in foil and incubated for 30 min at RT with agitation. The reaction was stopped by adding 50 μl of 4M sulphuric acid (H2SO4). The color development on the plates was read by using a multi-scan plate reader (Thermo Labsystems Inc.), set at 492 nm of wavelength. Three phage clones were used for this experiment namely N4, N5 and N7.

Hence, the method shown was repeated in the same exact manner for all three clones.

2.2.6 Synthesis of the Biotinylated Peptide

All 10 phage clones isolated in this study gave a strong amino acid sequence consensus, only one 12-mer peptide was sent for synthesis, denoted as N1 (AUSPEP, Australia). A biotinylated version of the peptide was also synthesized (N1 biopep). The purity of the peptides was >90% and the total peptide synthesized was 5 mg. Dilution of biotinylated peptide: 1 mg of the N1 biopep was taken from the 5 mg stock and dissolved in 500 μl of

DH20 (filter-sterilized). The peptide dissolved immediately; hence, another 500 μl DH20 was added to make up to the required 1mg/ml of peptide concentration.

2.2.7 Direct ELISA

Immobilization of 90 μl target IL-4Rα (10 nM) was carried out on a 96-well plate overnight.

The plate was sealed with a parafilm and placed in a humidified container with agitation.

Following day, the plate was slapped face down on a paper towel to get purged of target solution. 220 μl of blocking buffer (BSA + NaN3 blocking solution) was added to the wells.

The plate was incubated at 4°C for 1-2 h. The blocking buffer was removed by slapping the plate face down on the paper towel, and gently washed 3-4 times with 280 μl/well of 0.3%

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TBST. Next, 100 μl of biotinylated N1 peptide was added to each designated well of the

ELISA plate. The plate was incubated for 1-2 hrs at RT with agitation on a rocker. Negative controls were setup in 3 separate wells without the addition of N1 peptide. The wells were subjected to DH20 only, with the exception of the peptide. This was followed by 3-4 washes with 280 μl/well of 0.3% TBST. Finally, 150 μl HRP-conjugated streptavidin (Sigma

Aldrich) (1:500 dilution in blocking buffer) was added to the wells and incubated for 1 h with agitation on a rocker. Once again, the plate was gently washed 3-4 times with 280 μl/well of

0.3% TBST. A developing solution was prepared, by dissolving a single tablet of phospho- citrate buffer with sodium-perborate in 100 ml of dH2O, then 1 tablet of HRP substrate OPD was dissolved in 7.5 ml of this solution. 200 μl of this solution was added to each well; the plates were wrapped in foil and incubated for 30 min at RT, with agitation on a rocker. The reaction was stopped by adding 50 μl of 4 M H2SO4. The color development on the plates was read by using a multi-scan plate reader, set at 492 nm of wavelength. Statistical analysis was performed on the data obtained from the ELISA.

2.2.8 Inhibition ELISA

Initially, the same protocol was followed, as described in the direct ELISA protocol above.

After the blocking of the target IL-4Rα, IL-4 cytokine (Abcam®; diluted in 1:500 filter- sterilized dH2O) was pipetted into the wells and was incubated for 1-2 hrs at RT, with agitation on a rocker, followed by 3-4 washes with 250 μl 0.3% TBST. Following this, 120 μl of N1 biopep (0.2 mg/ml) was added to each designated well of the ELISA plate. This was then developed as described in the section above. Further, statistical tests were performed on the results obtained from the ELISA.

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2.2.9 Statistical Analysis

The data were analysed using Microsoft Excel 2013 (Microsoft Inc.). The results were analysed by Student’s t-test to determine any statistically significant differences between the positive control (PC) and treatment wells in all of the above experiments. The statistical significance was set at P < 0.05.

2.3 Results

Selected peptides from 12-mer libraries were analyzed by taking 3 random peptides from the

9 individual phage clones isolated from the 5th round of biopanning, demonstrating identical sequence motifs (see next section below). Each sample was diluted, plated and individual clones were isolated, amplified, sequenced and tested for reactivity with the target antigen IL-

4Rα using ELISA immunoassays.

2.3.1 Phage Display Biopanning

A 12-mer phage-displayed random peptide library was screened through a process known as

‘biopanning’. The procedure was performed with five cycles, repeated consecutively.

Initially, the first round of biopanning was completed and the amplified phage was titered onto IPTG/X-gal plates. A total of 5 rounds of biopanning were repeated. Table 2.1 shows the calculations done to obtain input volumes for subsequent panning rounds.

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Table 2.1 Input phage titers for subsequent rounds of biopanning and appropriate calculations. Initial pfu Input titer of phage Input volume Biopanning rounds for next round

Round 1 2.25 x 1011 2 x 1011/2.25 x 1011 0.88 μl Round 2 2.3 x 1010 2 x 1011/2.3 x 1010 8.8 μl Round 3 5.8 x 1011 2 x 1011/5.8 x 1011 0.344μl Round 4 1.3 x 1011 2 x 1011/1.3 x 1011 1.3 μl Round 5 Last round, hence no further calculations required

From the final 5th round of biopanning, 10 clones were collected by stabbing plaques on the agar plates and amplified as described in the phage display manual. The plaques were sequenced to reveal the DNA sequence of the single-stranded phage DNA. Upon return, 9 of the 10 sequences had identical consensus in binding sequence, N1 to N9. Since the peptides from the clones N1 to N9 showed a consensus in sequence, N10 was discarded and was no longer used for further analysis. The sequence of the peptide has not been shown due to intellectual property reasons with Deakin University.

Further, ClustalW2 was used to perform sequence analysis of N1 amino acids with the IL-4 cytokine to observe the similarities between their sequences. This was performed to evaluate the binding capacity of the peptide to IL-4Rα, and as a result, “mimic” the cytokine IL-4 in its amino acid sequence. This also revealed the epitope region of specific amino acid sequence of IL-4Rα that would hybridize with the peptide. Table 2.2 shows a comparison between IL-4 and the N1 peptide with 6 amino acid residues showing identical residues, and also, three weakly conserved residues.

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Table 2.2 Results obtained from ClustalW2 alignments, performed using N1 peptide.

Percentage amino acid similarity With IL-4 With N1 peptide with 12-mer N1 peptide Identical sequences 6 amino acids (50%) 100%

Strongly conserved None 100%

Weakly conserved 3 amino acids (25%) 100%

2.3.2 M13 ELISA with Three Homologous M13 phage clones

M13 ELISA was performed using 3 selected phage clones N4, N5 and N7 from the 9 clones with identical sequences selected for mimtag synthesis. This step was performed to further enhance results showing specific binding of the phage mimtag to the target IL-4R.

Irrespective of which phage clones were used for the M13 ELISA, since all of them displayed the same amino acid sequence, the ELISA was carried out using an anti-M13 antibody, followed by an anti-mouse HRP antibody to detect antibody-binding and OPD substrate for

HRP, to develop the solution for absorbance detection (Figure 2.2). The detection was based on a strong affinity between the displayed mimtag by the phage clones and the target IL-4R.

Overall, all of the three selected peptides (e.g. N4, N5 and N7) showed a binding to the IL-4R target. With comparable error bars overlapping between the 3 different clones, shown in the graph (Figure 2.2), the phage clones displaying the mimtag gave higher absorbance readings when compared to the control. This is an indication of the accuracy and specificity of the

M13 phage binding to its target antigen with in vitro analysis.

2.3.3 Direct ELISA

In view of the fact that N1 amino acid sequence shared a 50% homology when aligned with cytokine IL-4, it was necessary to conduct further tests on the N1 biopep, to determine its avidity with IL-4Rα (Figure 2.3). This was performed by using direct ELISA, in which the

75 PhD Thesis – Chapter 2 Nayyar Ahmed peptide was allowed to interact with the IL-4RΑin the absence of the cytokines to determine its avidity to the target antigen. In a similar fashion as other ELISA assays, this was carried out using a HRP conjugated streptavidin, to detect the biotin on the N1 peptide, and OPD substrate to develop the solution for absorbance detection as described in earlier sections.

Evidently, the differences in absorbance readings obtained showed that N1 biopep had specifically bound IL-4Rα, when compared to the control. A trend was seen with increasing concentration of the peptide, where higher absorbance readings were recorded on the spectrophotometer. This indicated that the streptavidin-HRP had indeed recognized the biotin on peptide N1, which in turn interacted with the IL-4Rα target antigen.

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Figure 2.2. M13 ELISA performed using 3 selected phage clones of IL-4Rα. The phage clones were randomly selected from the 9 clones with identical sequences to verify the binding affinity and efficacy of the mimtag to the target IL-4Rα protein. Figure illustrates the absorbance readings that were obtained when a range of phage dilutions were used to perform the experiment. As a result, with increasing phage clone titers, a higher absorbance reading was obtained at 492 nm, and vice-versa. The results are representing 3 replicates (mean values) for each bar graph; hence the error bars denote a standard deviation. A statistical

Student’s t-test revealed a significant difference between the mean absorbance values of the

1.5 x 1010 phage dilution and the negative control (P < 0.05).

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* *

* = P < 0.05

* *

Figure 2.3. Direct ELISA of biotinylated N1 peptide interaction with IL-4Rα. This assay was conducted to verify the affinity and efficacy of the newly synthesised N1 peptide, which showed considerable binding with the IL-4Rα target. The graph illustrates the absorption readings that were obtained when a range of N1 peptide concentrations were used to carry out the experiment in vitro. As a result, with increasing peptide concentrations, a higher absorbance reading was obtained at 492 nm, and vice-versa. The results represent 3 replicates

(mean values) for each bar graph; hence the error bars denote a standard deviation. A statistical Student's t-test revealed a significant difference between the mean absorbance values of the peptide at 0.2 mg/ml concentration and the negative control (P < 0.05).

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2.3.4 Inhibition ELISA

Shortly after the results were achieved from the direct ELISA, the next step was to verify whether the synthesized N1 biopep was capable of inhibiting the interaction between IL-4 and its receptor IL-4Rα. In this case, the IL-4Rα was immobilized on a 96-well plate, followed by the addition of the IL-4 cytokine. The ELISA was carried out using a HRP conjugated streptavidin, to detect the biotin on the N1 biopep and OPD substrate for the development and absorbance. The inhibition was found to be ~73% with respect to the biopep. These results show that the biopep N1 is able to successfully inhibit IL-4 cytokine from binding to its receptor, as shown in Figure 2.4.

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* * = P < 0.05 *

Figure 2.4. Inhibition ELISA showed that the biotinylated peptide N1 successfully fastened to the target IL-4Rα in the presence of IL-4, as the inhibitor. With the pre- incubation of cytokine IL-4, the peptide showed lower absorbance reading as opposed to in its absence. However, the peptide was still able to compete with the cytokine and record significant readings when observed using a spectrophotometer. The negative control had no biotinylated peptide N1 or cytokine IL-4 (graph bar on far left). The results represent mean value of 3 replicates for each bar graph; hence the error bars denote a standard deviation. A statistical Student’s t-test revealed a significant difference between the mean absorbance values when compared to the control (P < 0.05).

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2.4 Discussion

We report here for the very first time a peptide antagonist identified and characterized against

IL-4Rα which successfully inhibits the interaction of cytokine IL-4 with the receptor. The peptide antagonist identified is 12 amino acid long and binds with strong affinity to the IL-

4Rα. Hence, rendering it as a useful tool for future allergy therapy.

2.4.1 Phage Display

IL-4Rα plays a vital role in allergic diseases since it is able to bind IL-4 and IL-13 cytokines.

Activation of Th cells by APC leads to the production of these cytokines that cause B cells to produce IgE. Once synthesized, IgE antibodies circulate in the blood before binding to the high-affinity IgE receptor FcεRI that is present on mast cells in tissue or on peripheral blood basophils. After re-exposure, allergens cross-link to mast cell-bound specific IgE, thus causing the release of preformed mediators, such as histamine, the synthesis of prostaglandins

(PGs) and LT’s (261). Thus, it is necessary to find a novel antagonist to IL-4Rα, to inhibit the interaction of the cytokines with their receptor, hence down-regulating the excess production of IgE and thus the allergic cascade, as a prophylactic treatment.

Current clinical strategies for allergy treatment involve allergen avoidance by patients, pharmacotherapy and specific immunotherapy. Only the third strategy has provided a long- term solution but with little success, since the allergen extracts used are poorly characterized and suffers from batch to batch variation (262). In addition, natural allergen extracts exhibit high allergenic activity that often causes side effects and it has been reported that SIT with allergen extracts can induce IgE sensitization to new allergens (262, 263). The display of foreign proteins on the surface of bacteriophage M13 has been used to understand protein- protein interactions at the molecular level. For this reason, phage display technology has been

81 PhD Thesis – Chapter 2 Nayyar Ahmed used as a novel approach to identify peptide antagonists (264, 265). This technology has evolved over the last two decades and has been used for drug discovery and identifying novel synthetic peptides, which can be used as antagonists to many protein-ligand interactions

(266-269). Our findings indicate that a novel synthetic peptide N1 has been identified and characterized as an antagonist to IL-4α which inhibits the interaction between the cytokine

IL-4 and its receptor. IL-4, as discussed earlier, plays an important role in the allergic cascade, eventually leading to the release of inflammatory histamines from mast cells and basophils that lead to allergic symptoms. Hence, targeting the receptor was the main objective of this study.

2.4.2 M13 Binding ELISA with N4, N5 and N7 Phage Clone

Since N1-N9 sequences were chosen for further analysis, and they all shared an identical sequence, only N1 peptide was chosen for synthesis. The peptide sent for synthesis was biotinylated, on the N-Terminal end of the peptide. While this was being carried out, an M13 binding ELISA was performed by randomly choosing 3 phage clones N4, N5 and N7 from the 9 identical peptides. ELISA protocol is sufficient for rapidly determining whether the selected phage clones bind to the target IL-4R or not. The method was useful to determine qualitatively whether the selected clones in parallel have a relative binding to the target when compared to binding to the plastic support. Hence, this ELISA does not determine whether the clones are binding with high or low affinity to the target receptor, but rather indicate a positive or negative response. As can be seen in the results, phage clones N4 and N5 had overlapping error bars which suggested that they had similar binding affinity towards the IL-

4R. The N7 had shown a slightly higher affinity towards the IL-4Rα. The subtle changes might have resulted from the phage titer that was determined by growing the phage clones on

X-gal/IPTG plates, similar to biopanning. This preliminary study carried out with the phage

82 PhD Thesis – Chapter 2 Nayyar Ahmed clones N4, N5 and N7 laid down the hypothesis that the selected phage clones can indeed bind to the IL-4Rα target, and may therefore, act as a potential inhibitor for interaction with the cytokines IL-4 and IL-13. A statistical Student’s t-test analysis showed significant difference between the experimental values and controls (P < 0.05).

2.4.3 Direct ELISA and Inhibition ELISA

A direct ELISA was performed in our recent study to test the ability of the synthesized peptide to bind with the IL-4Rα target. The biotin on the peptide was detected by a HRP- conjugated streptavidin protein. This immunoassay was specifically done to test the efficacy and affinity of the newly synthesized biopep N1 to the IL-4Rα. The trend was clearly visible via the graphed readings where higher absorption readings are observed with increasing peptide concentrations. This was an indication that the IL-4Rα was bound to the bottom of the well and the N1 biopep was interacting with the target. A final aim of this study was to test the ability of the identified antagonist to not only bind with the target but down-regulate or inhibit its interaction with the target IL-4Rα. Hence, an inhibition ELISA was performed using the N1 biopep as the inhibitor. The inhibition ELISA is a highly sensitive and specific immunoassay method that uses an antigen capture method to bind the analytes present in the solution. Results are shown in the graph and a Student’s t-test analysis of the values compared to the control was highly significant (P < 0.05). This proved that the affinity of N1 biopep for the IL-4Rα was much higher than the cytokine itself. Although the IL-4 cytokine concentration used was lower than the concentration of the biopep N1, it was consistent with the recommendation of the manufacturer. The results were comparable to many other researches where peptide-ligand interactions have proved to be very successful (270, 271). As a result of the small size of the peptide, they tend to show a higher target-to-background ratios compared to macromolecular compounds.

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2.4.4 Conclusion

We have sucessfully identified and characterized a novel antagonist to IL-4R which down- regulates the interaction of the IL-4R with the cytokine IL-4. Using a phage display random peptide library, the biopanning procedure established in identifying and isolating the synthetic peptide antagonist N1. Furthermore, the second aim was to test the identified antagonist using immunoassay techniques and tissue culture assays to justify that the novel peptide was indeed inhibiting the IL-4R from interacting with IL-4. Results from ELISA immunoassays suggested that the peptide was proficient in binding to the IL-4R with a higher affinity and hence, was able to retard the overall protein-ligand interaction between the cytokines and IL-4R. Furthermore, using HEK-Blue IL-4/IL-13 cells a >50% inhibition was achieved with the peptide with a simple colorimetric analysis. As this peptide targets the two most clinically important cytokines in allergy, it promises to provide for the future treatment for all IgE-mediated allergies.

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Chapter 3 - Expression of HEK-Blue IL-4/IL-13

Reporter Cell-line and Further Characterization of the

IL-4Rα, IL-4 and IL-13 Peptide Antagonist in vitro

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3.1 Introduction

The cells of the immune system are peripatetic and therefore require a complex, but efficient, communication system in order to regulate prompt and integrated immune responses. Such sophisticated communication networks exploit a diversity of soluble and membrane bound signaling molecules, including cytokines (272, 273). These cytokines, being non-enzymatic mediators synthesized by multiple regulatory levels, function by interacting with membrane spanning receptors on target cells to send secondary intracellular signals, which in turn stimulate target cell differentiation, proliferation, migration or further production of cytokines

(272-274). There are six types of cytokines involved in immune responses: interleukins (IL), chemokines, interferons (IFN), transforming growth factor, hemopoietic colony-stimulating factors, and TNF (272, 275, 276). Of all the cytokines, interleukins play a pivotal role in allergy by acting as lymphocyte mediators (277).

Interleukins function via type I or type II cytokine receptors that possess different types of sub units (e.g. γ chain), which are shared by groups of interleukins (278, 279). This provides an explanation for the superfluous nature of interleukins during inflammation, including allergy. IL-4 and IL-13, the foremost mediators of atopic and allergic diseases, as well as their receptors (IL-4R and IL-13R) share molecular features and function cooperatively during an allergic reaction (280, 281). IL-4 and IL-13 are type I pleiotropic cytokines synthesized by activated T-cells and are significant allergic mediator that share structural and functional properties (282).

IL-4 and IL-13 have various pathological effects on a range of hematopoietic and non- hematopoietic cells during pathogenesis of allergy. In B cells, the cytokines induces B-cell proliferation, differentiation and class switching of immunoglobulin to produce IgE (283).

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Cytokines IL-4 and IL-13 are known to down-regulate the expression of immune mediators including IL-1α, IL-1β, IL-6, IL-8, IL-12 and TNF-α by moderately suppressing the DNA transcription regulator NF-κB (53, 284). The cytokines also contribute to IgE priming of mast cells, further influencing increased IgE production (53, 284). However, unlike IL-4, IL-13 does not affect T-cell function, differentiation or proliferation (285), therefore IL-13 can be classified as a mediator of the effector phase (involving B-cells, mast cells and basophils) of allergy.

IL-4 and IL-13 play key roles in Th2 immunity and asthma pathogenesis. The function of these cytokines is partially linked through their shared use of the IL-4Rα chain. The Type I receptor comprising IL-4Rα and the common γ-chain is expressed by hematopoietic cells and is exclusively responsive to IL-4. In contrast, the Type II receptor comprising IL-4Rα and IL-

13Rα1 is responsive to both IL-4 and IL-13 (Figure 1.6) (59).

A practical approach to treating allergy is the neutralization of the activities of interleukins. It is imperative to conduct studies on neutralizing IL-4 and IL-13 and their corresponding receptors. We have previously reported (138) the identification of a peptide antagonist potentially capable of inhibiting IL-13 interaction with its receptor which is explained in chapter 2. Here we have conducted in vitro studies to test the ability of the isolated peptides to inhibit the IL-4 and IL-13 interaction with the receptor type II IL-4Rα/IL-13αR1 using a

HEK-Blue cell-line. This will elucidate the effects of the peptide antagonist on JAK-STAT6 phosphorylation which leads to IgE synthesis in allergic patients via IL-4 and IL-13 signaling. Hence, blocking the interaction of the cytokines with their receptors using the isolated peptide antagonists is the specific aim of this chapter.

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3.2 Materials and Methods

3.2.1 HEK-Blue IL-4/IL-13 Cells

HEK-Blue™ IL-4/IL-13 (InvivoGen) cells allow the detection of bioactive IL-4 and IL-13 by monitoring the activation of the JAK-STAT6 phosphorylation pathway. The cells are a result of a stably transfected human JAK-STAT6 gene. Other pathways linked to the STAT6 pathway are naturally expressed by the cells. The cells are further transfected with a STAT6- inducible SEAP reporter gene as an alternative to IgE secretion in B lymphocytes when signaled by cytokines IL-4 and IL-13. Finally, the cells are transfected with a stably expressed recombinant type II IL-4Rα/IL-13αR1 receptor (Figure 1.9).

Growth medium: HEK-Blue cells were cultured in Dulbecco’s Modified Eagle Medium

(DMEM) (GIBCO), supplemented with 10% fetal bovine serum (FBS), in a humidified incubator at 37°C with 5% CO2. Antibiotics were added to the medium to avoid contamination (50 U/ml of penicillin, 50 μg/ml of streptomycin, 100 μg/ml of normocin, 10

μg/ml of blasticidin and 100 μg/ml of zeocin - GIBCO), Trypsin/EDTA (0.05%; GIBCO) was used for trypsinization.

Experimental medium: Growth medium was replaced with 2mM L-Glutamine and heat inactivated 10% FBS.

Three peptides N1, P9 and K1 were used to perform these assays. Peptide P9 and K1 were both characterized by fellow colleagues Pathum Dhanapala and Kelley Ramsbottom at the

NARL laboratory at Deakin University. The projects were undertaken as part of their

Honours year project as an extension to their undergraduate degrees. Pathum Dhanapala performed phage display using cytokine IL-13 as a potential target to identify novel peptide

88 PhD Thesis – Chapter 3 Nayyar Ahmed antagonist P9. Kelley Ramsbottom used cytokine IL-4 as a potential target to identify novel peptide antagonist K1. Both colleagues identified and characterized the peptides in a similar fashion as in the case of peptide N1 for IL-4R. However, the peptides were not tested in vitro and needed further characterization in vitro.

3.2.2 Synthesis and Use of Peptides N1, P9 and K1

All 10 phage clones isolated in this study gave a strong amino acid sequence consensus, only one 12-mer peptide was sent for synthesis, denoted as N1 for IL-4Rα, P9 for IL-13 cytokine and K1 for IL-4 cytokine (AUSPEP, Australia). The purity of the peptides was >90% and the total peptide synthesized was 10 mg of each. Dilution of the peptides was prepared by dissolving 1 mg in 500 μl of DH20 (sterilized). The peptide dissolved immediately; hence, another 500 μl DH20 was added to make up to the required 1 mg/ml of peptide concentration.

However, in the case of peptide K1, 5 mg was dissolved in 1 ml of DH20. Subsequent dilutions for all peptides were prepared to be used for experiments with HEK-Blue cells as shown below.

3.2.3 HEK-Blue Cell line Inhibition Assay with N1 Peptide

The protocol used was taken from the datasheet provided with the HEK-Blue IL-4/IL-13 kit.

A few adjustments were made in the protocol to perform the assay. Cells were grown in a T-

75 flask and were used for the detection assay. The cells were detached using 3 ml trypsin which was neutralized with 7 ml of media (FBS - neutralising agent) to give a total volume of

10 ml. The average cells were counted using a TC10 automated cell counter (Bio-Rad

Laboratories Inc.) and cultured at a density of 50,000 cells per well of a 96-well plate. Six treatment wells were used in total; 4 wells treated with peptide N1 (MW 1354Da) and 2 wells without peptide as positive and negative controls (shown in results section). Cells in the

89 PhD Thesis – Chapter 3 Nayyar Ahmed treatment wells were incubated with 50 μl of N1 peptide (75, 150 and 225 μM corresponding to 0.1, 0.2 and 0.3 mg/ml) at 37°C in a rotator shaker for 1 hr, whereas the control wells were incubated with filter-sterilized DH2O. Post-incubation, 20 μl of IL-4 cytokine (100 ng/ml) was added to the PC and three treatment wells. The 96-well plate was sealed using a parafilm and incubated at 37°C with 5% CO2 for a period of 24 hrs. Post-incubation, QUANTI-Blue substrate was prepared using the instructions in the HEK-Blue kit and 180 μl of this solution was added to 6 wells in a fresh 96-well plate. 20 μl of induced HEK-Blue IL-4 cells supernatant from each of the treatment wells was added to the QUANTI-Blue solution. The subsequent results were read using an xMark microplate absorbance spectrophotometer (Bio-

Rad Laboratories Inc.) at a wavelength of 640 nm.

3.2.4 HEK-Blue Cell Line Inhibition Assay with P9 and K1 Peptide

The protocol used for this experiment was slightly changed without affecting the overall nature of the experiment. Concentrations used for P9 (MW 1322Da) peptide were 0.1, 0.2 and 0.3 mg/ml which corresponds to 75, 150 and 225 μM. However, in the case of peptide

K1 (MW 1238 Da) the concentrations used were 0.5, 1 and 1.5 mg/ml which corresponds to

400, 800 and 1200 μM. The following changes made to the protocol are shown below:

- 100 μl of the cytokines IL-4 and IL-13 at a concentration of 100 ng/ml were incubated

with 50 μl of peptides P9 and K1 and incubated for 1 hr;

- Post-incubation, 30 μl of this solution was added to the HEK-Blue cells to maintain

cytokine concentration at 100 ng/ml.

The protocol from here onwards was the same as in the case of peptide N1 inhibition studies.

Absorption readings were taken after 24 hrs of incubation with HEK-Blue cells. The average cells were counted using a TC10 automated cell counter.

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3.3 Results

3.3.1 HEK-Blue Cell Line Inhibition Assay with N1 Peptide

Once all necessary immunoassays had been carried out to determine the efficacy of the N1 peptide, further analysis was required to confirm the specificity of the peptide in vitro in inhibiting the IL-4 signaling pathway. As shown in Figure 3.1A, with the addition of the peptide N1, a dose-dependent relationship was observed with the increasing concentration of the peptide, which means less SEAP was secreted by the cells, hence less color produced in the presence of the QUANTI-Blue substrate. All positive and negative controls behaved as expected with high and low peaks as suggested by the HEK-Blue IL-4/IL-13 kit. As hypothesized, >50% inhibition of the IL-4 signaling pathway was achieved with the highest concentration of peptide at 225 μM (Figure 3.1B). Consistency was also observed with cell viability when tested for all the treatments and compared to the PC.

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3.1(A)

3.1(B)

Figure 3.1. In the presence of N1 peptide, the receptor was successfully antagonized and hence, denoted lower SEAP levels. A dose-dependent relationship can be observed with the three treatment wells containing peptide N1 (Figure 3.1A). PC included cells treated with IL-4 cytokine alone. Control 1 (C1) included cells incubated with peptide only and Control 2 (C2) included cells without any treatment. P < 0.05 denotes significance of optical density (OD) readings compared to PC. % Inhibition of HEK-Blue cells with

N1 peptide. Inhibitory concentration was obtained with 225 μM of peptide N1, which inhibited 58.8% of

SEAP secretion. Hence, IC50 concentration was determined as 178.1 μM to achieve 50% inhibition. PC is denoted at 0% which is used as a referral point for the standard value at which inhibition begins (Figure

3.1B).

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3.3.2 HEK-Blue Cell line Inhibition Assay with P9 Peptide

Similar to the previous experiment, the efficacy of peptide P9 was further tested in a HEK-

Blue cell line. As shown in Figure 3.2A, with the addition of the peptide P9, a dose- dependent relationship was observed with increasing concentration of the peptide, hence less color produced in the presence of the QUANTI-Blue substrate. As hypothesized, >50% inhibition of the IL-4/IL-13 receptor signaling pathway was achieved with the highest concentration of peptide at 225 μM (Figure 3.2B). Peptide had no negative impact on cell viability.

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3.2(A)

3.2(B)

Figure 3.2. In the presence of P9 peptide, the cytokine IL-13 was successfully antagonized and hence, denoted lower SEAP levels. A dose-dependent relationship can be observed with the three treatment wells containing peptide P9 (Figure 3.2A). PC included cells treated with IL-13 cytokine. C1 included cells incubated with peptide only and C2 included cells without any treatment. P < 0.05 denotes significance of

OD readings compared to PC. % Inhibition of HEK-Blue cells with P9 peptide. Inhibitory concentration was obtained with 225 μM of peptide P9 which inhibited 51.8% of SEAP secretion. Hence, IC50 concentration was determined as 213.2 μM to achieve 50% inhibition. PC is denoted at 0% which is used as a referral point for the standard value at which inhibition begins (Figure 3.2B).

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3.3.3 HEK-Blue Cell line Inhibition Assay with K1 Peptide

Similar to the previous experiment, the efficacy of peptide K1 was further tested in a HEK-

Blue cell line. As shown in Figure 3.3A, with the addition of the peptide K1, a dose- dependent relationship was observed with increasing concentration of the peptide, hence less color produced in the presence of the QUANTI-Blue substrate. >50% inhibition of the IL-

4/IL-13 receptor signaling pathway was achieved with the highest concentration of peptide at

1200 μM (Figure 3.3B). Peptide had no negative impact on cell viability.

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3.3(A)

3.3(B)

Figure 3.3. In the presence of K1 peptide, the cytokine IL-4 was successfully antagonized and hence, denoted lower SEAP levels. A dose-dependent relationship can be observed with the three treatment wells containing peptide P9 (Figure 3.3A). PC included cells treated with IL-13 cytokine. C1 included cells incubated with peptide only and C2 included cells without any treatment. P < 0.05 denotes significance of

OD readings compared to PC. % Inhibition of HEK-Blue cells with K1 peptide. Inhibitory concentration was obtained with 1200 μM of peptide P9 which inhibited 51.8% of SEAP secretion. Hence, IC50 concentration was determined as 995 μM to achieve 50% inhibition. PC is denoted at 0% which is used as a referral point for the standard value at which inhibition begins (Figure 3.3B).

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3.4 Discussion

3.4.1 Characterization of Novel Peptide Antagonists Using a HEK-Blue Cell Line

We have successfully identified and characterized a novel antagonist to IL-4Rα (N1 peptide),

IL-4 (K1 peptide) and IL-13 (P9 peptide) which down-regulates the interaction of the cytokines with their receptors. Using a phage display random peptide library, the biopanning procedure established in identifying and isolating the synthetic peptide antagonist N1, K1 and

P9 (explained in Chapter 2). Furthermore, the second aim was to test the identified antagonists using immunoassay techniques and tissue culture assays to justify that the novel peptides were indeed inhibiting the cytokines from interacting with IL-4. Using HEK-Blue

IL-4/IL-13 cells a >50% inhibition was achieved with the peptides using a simple colorimetric analysis. IL-4 and IL-13 cytokines signal via the type II heterodimeric IL-

4Rα/IL-13αR1 and IL-13αR2 receptors. IL-13αR1 and IL-13Rα2 bind IL-13 with sequence specificity. However, the latter acts as a high affinity decoy receptor which does not initiate signal transduction (285). Although IL-13Rα1 binds IL-13 with low affinity, pairing with IL-

4Rα increases the affinity by over 40 times which results in the activation of JAK-STAT

(signal transducer and activator of transcription) signaling pathway responsible for B-cell differentiation (285). Since HEK-Blue cells expresses the heterodimeric receptor IL-4Rα/IL-

13αR1, blocking or inhibiting one of the receptor chains or the cytokine itself may eliminate the response of STAT6 signalling. As these peptides targets the two most clinically important cytokines in allergy, it promises to provide for the future treatment for all IgE-mediated allergies.

Using HEK-cells for tissue culture research carries many benefits. To name a few, the cells have been used as an expression tool for recombinant IL-4Rα for over 25 years. They remain highly stable and efficient when transfected with foreign genes. Also, once transfected, the

97 PhD Thesis – Chapter 3 Nayyar Ahmed translation and processing of the gene product (proteins) is performed with high efficiency.

Finally, their effortless reproduction and maintenance adds to the benefits of using this cell line as a model system (145). HEK-Blue cells on the other hand, allow a colorimetric analysis, which carries simplicity, high sensitivity and low cost to conduct the required IL-4 signaling experiments. HEK-Blue detection assay was performed using a HEK-Blue transfected cell line with the N1, K1 and P9 peptides as inhibitors. As the results suggest, an increase in peptide concentration reduced SEAP levels that were detected through a colorimetric analysis. Although some minor changes have been made to the overall protocol of the detection assay, the absorption readings observed were at peak values in the PC. The aim of this experiment was to achieve a >50% inhibition of the IL-4/IL-13 signaling pathway that would allow the severity of allergic symptoms to be lowered in atopic individuals (286-

289). Completely blocking the production of IgE antibodies can lead to other illnesses since

IgE plays an important role in parasitic immunity such as helminthes infections (290-293).

Although it has been criticized that peptides selected against purified recombinant protein may not be able to access their targets on living cells, it has proved otherwise in our case

(explained in Chapter 7). The positive and negative controls were setup to pinpoint any discrepancy in the results. More importantly, the detrimental effects of peptide concentrations were also tested by performing a cell viability test for all the parameters. Consistency in cell count suggested that even at higher doses, the peptide antagonist did not affect cell viability.

A Student’s t-test analysis of the results from the graph revealed a significant difference between the PC and the treatments performed (P < 0.05).

3.4.2 Conclusion

To conclude, we have successfully tested three peptide antagonists N1, P9 and K1 as potential inhibitors of allergy pathway in a recombinant HEK-Blue cell line. The STAT6

98 PhD Thesis – Chapter 3 Nayyar Ahmed phosphorylation pathway is activated by bioactive IL-4 and IL-13 via the IL-4Rα and IL-

13Rα1 receptors. This pathway leads to a production of SEAP which mimics the production of IgE antibody from B lymphocytes. Hence, a decrease in SEAP can be translated as a decrease in IgE antibody. All three peptides were able to successfully bind to their targets and inhibit the signaling of STAT6 phosphorylation hence reducing SEAP secretion from cells.

However, K1 peptide was used in higher concentrations as opposed to N1 and P9 to achieve

>50% inhibition. Since IL-4 and IL-13 interaction with IL-4Rα is a common pathway for many allergies, a prophylactic treatment can be devised by inhibiting this interaction for future treatment of allergies.

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Chapter 4 - Discovering the Effects of Omega-3 and

Omega-6 Fatty Acids on Allergy Using a HEK-Blue

Cell Line

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4.1 Introduction

Over the past few decades, there has been a dramatic rise in allergic individuals worldwide.

New cases are sprouting in almost every continent and the trend appears to be on a steady rise. This is becoming increasingly difficult for worldwide governments and healthcare organizations to cope with the financial burden (294, 295). Allergic reactions can be as mild as a sneeze or in some cases even life-threatening such as an anaphylactic shock (296). Many studies have been carried out to investigate the causal relationship between allergy and various factors that result in atopic disease, such as genetic predisposition, lifestyle or environment (297, 298). The occurrence of allergic disease has become more prevalent in the industrialized world. There has been a marked increase in prevalence of atopic disease in regions such as Western Europe, the US and Australasia during recent years. A 20-fold increase has been experienced by western countries and as much as 40% of the population suffers from this disease (8). However, the results are skewed and more research is needed at this stage to enhance a clear image and understanding to exactly how fatty acids interact with different cells to stimulate or inhibit the effect of allergy. Although it is known that allergies run in families and that genetic predisposition is the biggest factor, dietary factors have also become very important in recent times (296). The intake of certain fatty acids at a young age and their role in determining the outcome of atopic disease in later life has been thoroughly investigated by many scientists throughout the world, clinical and non-clinical (156, 299-

303).

It has been suggested that an imbalance in dietary intake of essential fatty acids (EFA), such as omega-3 (n-3 PUFA) and omega-6 (n-6 PUFA), may lead to a predisposition to allergic disease. This is usually caused by an increased intake of n-6 fatty acids such as AA (C20:4) with a simultaneously decreased intake of omega-3 fatty acids such as DHA (C22:6), EPA

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(C20:5) and DPA (C22:5) (304). Such an imbalanced diet leads to an insufficiently balanced

T helper cell Type 1 and 2 pathways. Evidently, concentrations of dietary n-6 PUFA stimulates a Th2 differentiation of naïve T cells whereas the n-3 PUFA stimulates the Th1 variant (304). In atopic individuals, an immune response is mounted by T cells that are activated by allergens, promoting Th2 variant of cells. Once stimulated, these cells subsequently produce cytokines such as IL-4, IL-5 and IL-13 that stimulate B lymphocytes to produce IgE antibody. Subsequently, this reaction triggers the release of histamines and LT that result in allergic symptoms (296, 305). Unlike the cytokines produced by Th1 cells, such as IFN-γ and IL-2 in non-atopic individuals, an excessive production of cytokines takes place from Th2 cells (13). The shift in cytokine production from Th1 cells (IFN-γ and IL-2) to Th2

(IL-4 and IL-13) variant forms the hallmark of allergic response in atopic individuals.

It was recently reported in dietary intervention studies that during pregnancy, n-3 PUFA show a strong tendency to reduce serum IgE antibody levels. In a different study carried out by Weise et al., (2011) showed that B cells treated with DHA inhibited IgE production by inhibiting the STAT6 phosphorylation pathway. It has also been suggested that DHA can reduce IgE production by human B cells by affecting the IL-4 pathway via an effect on CD40 ligand (158).

These studies along with many others have prompted many researchers across the world to revisit the effects of fatty acid supplements on allergic individuals. The main objective of this study was to investigate the effect of n-3 and n-6 fatty acids on STAT6 phosphorylation pathway that leads to the production of IgE antibodies when stimulated by bioactive IL-4 and

IL-13 cytokines (Figure 1.9).

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The specific aim of this chapter was to test the effects of omega-3 and omega-6 fatty acid on

STAT6 phosphorylation pathway using a HEK-Blue cell model. This will enable us to understand the direct effect of these fatty acids on SEAP secretion which is triggered by bioactive IL-4 and IL-13 cytokines. The HEK-Blue transfected model of cells produce SEAP as an alternative to IgE antibody produced by B cells when signaled by cytokines IL-4 and

IL-13. Since omega-3 fatty acids have been found to inhibit allergy, we hypothesized that omega-3 fatty acids will inhibit STAT6 signaling in HEK-Blue cells.

4.2 Materials and Methods

4.2.1 Cell Culture, PUFA and Vitamin E Solutions

HEK-Blue cells have been described in detail in Section 3.2.1 (Figure 1.9).

Growth medium: as described in Section 3.2.1.

Experimental medium: Growth medium was replaced with 2 mM L-glutamine and heat inactivated 10% FBS along with omega-3 and omega-6 fatty acids namely; DHA (MW

328Da), EPA (MW 302Da), DPA (MW 330.5Da) and AA (MW 304.46Da), all of which were commercially purchased from InvitrogenTM at a concentration of 10 μg/ml. The final concentrations prepared were 30, 60, 90, 120 and 150 μM using 100% ethanol as solvent.

Vitamin E (InvitrogenTM) was prepared as an antioxidant to prevent oxidation of fatty acids using growth medium. The final concentrations prepared were 0.05, 0.1, 0.15, 0.2 and 0.25

μM to all corresponding treatments as outlined above.

Initially, cells were tested in the presence of ethanol and vitamin E separately to confirm whether cell viability and/or STAT6 phosphorylation pathway was affected or whether it

103 PhD Thesis – Chapter 4 Nayyar Ahmed caused apoptosis in cells. Cells were incubated with experimental medium containing ethanol and vitamin E over a period of 24 hrs and cell viability was tested at 5 intervals (0 hrs, 6 hrs,

12 hrs, 18 hrs and 24 hrs). Our results confirmed that ethanol and vitamin E had no effect on cell viability and/or STAT6 phosphorylation pathway with or without the addition of ethanol and vitamin E (results not shown). Cells were now ready to be tested with fatty acids.

Stock solution of DHA, EPA, DPA and AA in ethanol were stored at -20°C and pre- incubated in complete growth medium at 37°C overnight to allow protein conjugation. We tested a range of concentrations on HEK-Blue cells to test the STAT6 phosphorylation pathway that leads to a production of serum embryonic alkaline phosphatase (SEAP). This is an alternative product to IgE antibody that is produced by B lymphocytes when triggered by bioactive IL-4 and IL-13. SEAP breaks down Quanti-Blue which gives a blue coloration. The blue color can then be detected using a spectrophotometer. Vitamin E stock was stored at 4°C and was freshly prepared in ethanol when preparing experimental media with fatty acids.

Two sets of experiments were performed to determine the effects of fatty acids on the STAT6 phosphorylation pathway. In the preliminary experiments, a range of different concentrations of fatty acids and vitamin E were studied to confirm their effect on SEAP production. Once confirmed, the most optimum concentration of fatty acid was used to perform a time course experiment. Cell viability and SEAP production were detected at 5 different time points over a period of 24 hrs. These experiments allowed us to study two different variables; time and concentration of fatty acid needed to suppress SEAP secretion by HEK-Blue cells by using a very simple colorimetric analysis.

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4.2.2 HEK-Blue Cell Viability Test Using Cytokines IL-4 and IL-13

The protocol used was taken from the datasheet provided with the HEK-Blue IL-4/IL-13 kit.

A few adjustments were made in the protocol to perform the assay without compromising the signalling in cells. Cells were grown in a T-75 flask and were used for detection assay. The cells were detached using 3 ml trypsin, which was neutralized with 7 ml of media containing

FBS to give a total volume of 10 ml. The average cells were counted using a TC10 automated cell counter (Bio-Rad Laboratories, Inc.). HEK-Blue cells were pipetted into 15 mL tubes, which were centrifuged at 1200 RPM for 5 min. The supernatant was discarded and experimental media was added to suspend the cell pellet. If required, cell concentration was reduced by adding growth medium to achieve optimum cell density. The cells were pipetted into 24-well plates (Corning, Sigma-Aldrich) at a density of ~200,000 cells per well for a total volume of 720 μl. A total of 12 treatment wells were setup in triplicates. Cells were treated with a combination of fatty acids (30, 60, 90, 120 and 150 μM) with and without vitamin E (0.05, 0.1, 0.15, 0.2, 0.25 μM). 80 μl of cytokine IL-4 (Integrated Sciences) and

IL-13 (Integrated Sciences) were pipetted to each treatment well at a concentration of 100 ng/ml. PC (with cytokine stimulation) and negative controls (without cytokine stimulation) were included to provide a comparative study. Cells were incubated with the treatments for a period of 24 hrs. Post-incubation, 20 μl of induced HEK-Blue IL-4/IL-13 cells supernatant from each treatment well was added to a corresponding plate with 180 μl of Quanti-Blue in each well. This is incubated for a total of 3 hrs to detect SEAP levels. The blue color was detected using a xMark microplate absorbance spectrophotometer (Bio-Rad Laboratories

Inc.) at a wavelength of 640 nm. Cell viability was recorded by using the TC10 automated cell counter.

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4.2.3 Time-course Assay Using Cytokine IL-4 and IL-13

A time course assay was performed with an optimum concentration of n-3 and n-6 fatty acids.

Once again, the cells were cultured in five (one for each time interval) 24-well plates at a density of ~200,000 cells per well. Cells were counted using the TC10 automated cell counter. HEK-Blue cells were pipetted into 15 ml tubes, which were centrifuged at 1200 rpm for 5 min. The supernatant was discarded and experimental media was added to suspend the cell pellet. If required, cell concentration was reduced by adding growth medium to achieve optimum cell density. The cells were pipetted into 24-well plates (Corning, Sigma-Aldrich) at a density of ~200,000 cells per well for a total volume of 720 μl. Concentration of fatty acids used at this stage was 90 μM, which is within high physiological levels and the most optimum concentration of fatty acid to inhibit SEAP secretion (established from previous section). Cells were incubated in a humidified incubator at 37°C with 5% CO2 for a period of

24 hrs. A total of five time points were used to measure the effect of fatty acids on SEAP production over a period of 24 hrs; 0 hrs, 6 hrs, 12 hrs, 18 hrs and 24 hrs, respectively. PC’s were also setup for each time interval which was without fatty acid treatment. Post- incubation, 80 μl of cytokine IL-4 and IL-13 were pipetted to each treatment well at a concentration of 100 ng/ml and the plates were incubated for 24 hrs to stimulate the STAT6 phosphorylation pathway. Simultaneously, the experimental media in the wells was replaced with a fresh supply of growth medium to ensure a steady supply of nutrients to the cells. 20

μl of induced HEK-Blue IL-4/IL-13 cell supernatant from each treatment well was added to a corresponding plate with 180 μl of Quanti-Blue in each well. This was incubated for a total of

3 hrs to detect SEAP levels. The blue color was detected using a spectrophotometer at 640 nm. Cell viability was recorded by using the TC10 automated cell counter.

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4.2.4 Statistical Analysis

The data were analysed using Microsoft Excel 2013 (Microsoft Inc.). The results were analysed by Student’s t-test using a two-tailed method to determine any statistically significant difference between the controls and treatment wells in all of the above experiments. The statistical significance was set at P < 0.05.

4.3 Results

4.3.1 HEK-Blue Cell Viability Test Using Cytokine IL-4 and IL-13

As the results suggest, a total of two sets with 4 assays in each set were performed using cytokines IL-4 and IL-13 (Figures 4.1 and 4.2). A dose-dependent relationship was observed between fatty acid concentration and SEAP secretion from HEK-Blue cells. A gradual increase in fatty acid concentration meant that SEAP levels would decrease hence, reducing blue coloration once the HEK-Blue cells supernatant was added to Quanti-Blue. All positive and negative controls behaved as expected with high and low peaks as suggested by the

HEK-Blue IL-4/IL-13 kit. Statistically significant results were achieved with an optimum concentration of fatty acids at 90 μM for n-3 PUFA. All inhibitions achieved with n-3 fatty acid treatments were statistically significant with P values being less than 0.05 with a two- tailed Student’s t-test. While DHA (Figures 4.1A and 4.2A) and EPA (Figures 4.1B and

4.2B) showed gradual decrease in SEAP secretion with both IL-4 and IL-13, DPA (Figures

4.1C and 4.2C) showed a sudden drop in SEAP levels suggesting a more potent inhibition.

However, the treatment of HEK-Blue cells with n-6 PUFA AA showed no significant change in the IL-4 cytokine assay (Figure 4.1D). However, an increase in SEAP secretion was observed with AA treatment when cells were tested with the IL-13 cytokine (Figure 4.2D).

OD readings showed no inconsistencies when n-3 fatty acids were incubated with HEK-Blue cells without the vitamin E. However, in the case of AA, it was noted that SEAP secretions

107 PhD Thesis – Chapter 4 Nayyar Ahmed were higher than the PC. Consistency was also observed with cell viability when tested for all the treatments and compared to the PC.

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4.1(A) 4.1(B)

4.1(C) 4.1(D)

Figure 4.1. IL-4 detection assay with n-3 and n-6 fatty acids (μM) with HEK-Blue cells using Quanti-Blue. 4.1A and 4.1B shows the detection assay performed with DHA and EPA with a dose-dependent inhibition. However, in Figure 4.1C, a dose-dependent inhibition does not occur with DPA but rather a dose-response inhibition occurs. N-3 fatty acids showed no abnormal effects on SEAP secretion without vitamin E. However, AA without vitamin E caused higher than normal SEAP secretion. P < 0.05 denotes significance of OD readings compared to PC.

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4.2(A) 4.2(B)

4.2(C) 4.2(D)

Figure 4.2. IL-13 detection assay with n-3 and n-6 fatty acids (μM) with HEK-Blue cells using Quanti-Blue. Figure 4.2A shows a dose-response inhibition of SEAP by DHA. 4.2B and 4.2C depict the detection assay performed with EPA and DPA with a dose-dependent inhibition of SEAP secretion from HEK-Blue cells. However, in Figure 4.2D with higher concentrations of AA, there is a rise in SEAP levels hence, a higher OD reading. N-3 fatty acids showed no abnormal effects on SEAP secretion without vitamin E. However, AA without vitamin E caused higher than SEAP secretion than the PC. P < 0.05 denotes significance of OD readings compared to PC.

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4.3.2 HEK-Blue Time-course with Fatty Acid and Vitamin E Treatment

As shown in Figures 4.3 and 4.4 a total of 8 assays were performed with 4 assays in each set.

Figure 4.3 shows results with cytokine IL-4 whereas Figure 4.4 shows results with cytokine

IL-13. DHA (Figures 4.3A and 4.4A), EPA (Figures 4.3B and 4.4B) and DPA (Figures

4.3C and 4.4C) all showed a decrease in SEAP secretion and hence produced less blue coloration at 18 and 24 hr time points when compared to the PC values. All inhibitions achieved with n-3 fatty acid treatments were statistically significant with P values being less than 0.05 with a two-tailed Student’s t-test. Since the concentration of fatty acid was constant at 90 μM, this experiment was a time-dependent inhibition of STAT6 phosphorylation in

HEK-Blue cells. However, as seen in Figures 4.3D and 4.4D, AA has shown to increase

SEAP secretion hence, it shows a higher absorption reading at 18 and 24 hr time points when compared to the PC values. No significant change was observed at 0 hrs, 6 hrs or 12 hr time points. Cell viability was performed at the end of the 24 hr incubation to check for any anomalies. It was found that AA showed a higher cell count when compared to PC’s in both experiments; with cytokine IL-4 and IL-13. However, the cell viability count was well within the range of 105 cells.

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4.3(A) 4.3(B)

4.3(C) 4.3(D)

Figure 4.3. HEK-Blue detection assay with 90 μM of omega-3 and omega-6 fatty acids with 0.15μM vitamin E using cytokine IL-4 bioactive agent. Figures 4.3A, 4.3B and 4.3C depict a time-dependent inhibition of SEAP secretion from HEK-Blue cells when treated with

DHA, EPA and DPA, respectively. Figure 4.3D shows an increase in SEAP secretion from

HEK-Blue cells when treated with AA at the 24 hr time point. P < 0.05 denotes significance of OD readings compared to PC depicted in blue color. C1 depicts the OD reading obtained when HEK-Blue cells are treated with fatty acid only without the cytokine stimulation.

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4.4(A) 4.4(B)

4.4(C) 4.4(D)

Figure 4.4. HEK-Blue detection assay with 90 μM of omega-3 and omega-6 fatty acids with 0.15 μM vitamin E using cytokine IL-13 bioactive agent. Figures 4.4A, 4.4B and

4.4C depict a time-dependent inhibition of SEAP secretion from HEK-Blue cells when treated with DHA, EPA and DPA, respectively. Once again, Figure 4.4D shows an increase in SEAP secretion from HEK-Blue cells when treated with AA at the 24 hr time point. P <

0.05 denotes significance of OD readings compared to PC depicted in blue color. C1 depicts the OD reading obtained when HEK-Blue cells are treated with fatty acid only without cytokine stimulation.

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4.4 Discussion

4.4.1 Fatty Acids and SEAP Secretion

Intake of pro-inflammatory omega-6 fatty acid such as AA has increased in the past few decades along with a decrease in omega-3 fatty acid diet. This has led to an increase in allergy prevalence as well as clinical manifestations of allergic disease (306-308). A diet comprising n-3 PUFA is mainly found in fatty fish as opposed to n-6 PUFA, which are mainly found in margarine and vegetable oils. N-3 PUFA, specially DHA, EPA and DPA, exert anti-inflammatory effect, whereas n-6 fatty acids do not (309). The purpose of this study was to investigate the changes that n-3 and n-6 fatty acids have on STAT6 phosphorylation pathway which leads to IgE production in B lymphocytes. The rationale of the present study was to investigate the authenticity and general observations made in previous observations. In this study we have shown for the first time that n-3 fatty acids

DHA, EPA and DPA all suppress and down-regulate SEAP secretion from transfected HEK-

Blue cells. The HEK-Blue model uses a very simple colorimetric analysis as an alternative method to measuring IgE antibody production from B cells. Incubation with all three n-3

PUFA resulted in a dose-dependent decrease in SEAP secretion which in turn gave less blue color when added to Quant-Blue substrate. This decrease in SEAP secretion was not due to a higher cell proliferation as demonstrated by trypan blue cell viability count. The reason for choosing DHA, EPA, DPA and AA for n-3 fatty acid was due to their abundant availability.

Breast milk contains all three fatty acids and they are also found in a variety of meat products such as beef, lamb and fish (310-312). Secondly, it is well established and documented through many clinical studies that DHA, EPA, DPA and AA have a strong link with allergy

(157). Recent publications have reported that lower incidence of eczema was observed in children who consumed fish and whose mothers had higher intake of fish diet during the gestation period (313, 314). In the case of AA, several papers have reported the rise of

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317). All of these studies lead to a possible explanation of reduced SEAP secretion from

HEK-Blue cells, which in turn avoids clinical symptoms in allergic individuals since SEAP secretion occurs as a result of JAK-STAT6 phosphorylation. Although many clinical studies of allergic individuals have been carried out over the past decade, there is still a lack of in vitro studies to look at the cellular effects of fatty acids.

4.4.2 Importance of Arachidonic Acid in Allergy

The purpose of choosing AA for these studies was due to the abundance of this fatty acid in body tissues. It is a major component of mammalian cell membranes and accounts up to a quarter of all phospholipid fatty acid (311, 312), hence making it a very important target for allergy studies because of its ubiquitous nature. Previous studies on the relationship between allergy markers such as IgE and AA have not been carried out. No significant change was observed in the case of AA when using a range of concentrations in our experiments.

However, in the time course experiment an increase in SEAP production was observed and slightly elevated levels of cell viability count. This evidence is in agreement with the fact that

AA causes cell proliferation in endothelial cells and HEK-Blue cells are derived from endothelial cells found in kidneys (318). It was also observed that AA incubated with HEK-

Blue cells without vitamin E supplementation gave higher SEAP secretion than the PC. This is consistent with literature which suggests that vitamin E suppresses the production of IgE antibody from B cells hence, it can suppress SEAP secretion by the same mechanism. Three decades ago, in a study carried out by Inagaki et al., (1984), vitamin E was supplemented to mice in diets and oral administration with sesame oil which resulted in suppression of IgE antibody production (319). Furthermore, AA showed inhibition of SEAP secretion with IL-4 detection assay which is an anomaly we found in the overall effects of AA. This may be

115 PhD Thesis – Chapter 4 Nayyar Ahmed explained by the fact that AA incorporated into cell membranes can effect membrane fluidity, which may consequently effect cell function (309, 320, 321).

4.4.3 Conclusion

In conclusion, treatment of HEK-Blue cells with n-3 fatty acids lead to a repression of SEAP production through inhibition of IL-4 and IL-13 signaling pathways. These results comprise a great biological relevance to the possible development of a prophylactic treatment for allergic diseases. With n-6 fatty acid AA, an increase in SEAP output was recorded which is coherent with previous studies. Our studies, presented here, opens a new corridor to research with n-3 fatty acids using a transfected model for allergy research. DHA, EPA and DPA supplementation may offer a molecular basis for a well-developed anti-allergic effect and therefore, must be an essential part of our regular diet. Although further investigation will reveal the best allergy markers that can be used for suppression, it is clear that n-3 fatty acids offer a great opportunity as potential natural inhibitors of allergy.

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Chapter 5 - Discovering the Effects of CoQ10 and

Resolvins on Allergy Using a HEK-Blue Cell Line

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5.1 Introduction

In recent years, it has become very clear that the burden of allergic disorders reflect long-term consequences of chronic allergic inflammation at sites that face repetitive exposure to allergens. This alone has led many scientists to test different therapeutic drugs to down- regulate the effects of allergic disease such as corticosteroids, which increases tolerance in allergic individuals to a specific allergen (11, 322). IgE-mediated responses in allergic disorders are the most important ingredients of allergic inflammation (323). Since allergic reaction can lead to acute inflammation, no such link has been established so far on the effects of pro-resolving metabolites such as resolvins in allergy. It may be not far-fetched to hypothesize that resolvins may have a significant effect on allergy since they down-regulate inflammation or vice-versa. This theory has not been tested on an IgE model of cells such as on B lymphocytes, which are primarily responsible for the production of IgE antibodies during an allergic cascade. The NARL research group at Deakin University has taken the initiative to conduct a string of experiments using CoQ10 and resolvins on a HEK-Blue cell model to establish the effects of resolvins on SEAP secretion in response to IL-4 cytokine signaling, as a substitute to IgE production by B cells. The experimental model is based on our well-characterized model of HEK-Blue cells that mimic the same IL-4 and IL-13 signaling that is used for the production of IgE antibodies by B lymphocytes.

Asthma is characterized by chronic inflammation of the airways with additional episodes of acute inflammation (324). These exacerbations can be life threatening and are associated with high health care costs (325). Allergens are mainly responsible for exacerbations of these responses. During an acute inflammation phase, the recruitment of a significant number of neutrophils as well as eosinophils can lead to an obstruction of airflow to lungs (326).

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Treatments for asthma symptoms include standard therapies such as SIT (262, 327).

However, they have not been able to show satisfactory results as novel therapeutics.

Resolution of inflammation is now understood to be an actively regulated process (179).

Lipid derived mediators such as resolvins have demonstrated to be anti-inflammatory and pro-resolving in their ability to reduce allergic symptoms (183). Resolvins such as resolvin

D1-D2 (RvD1 & RvD2) and RvE1 derived from their precursor fatty acids DHA and EPA via the action of cyclooxygenase enzyme, have been found to inhibit inflammation in several models of diseases as well as reducing neutrophil infiltration during inflammation (328).

Similarly, it was found in a study that low dietary intake of CoQ10 resulted in an increased risk of allergy among children (329). These studies has prompted the NARL research group to conduct experiments using resolvins and CoQ10 as novel therapeutics for treatment of allergy.

For this chapter, we hypothesized that since resolvins and CoQ10 suppress allergy and inflammation, the HEK-Blue model will serve best to test this theory. In the present study, we first assessed the effect of the administration of resolvins and CoQ10 in HEK-Blue cells using a range of concentrations over a period of 24 hrs. Secondly, a time-course experiment with the most optimum concentration was performed to assess the effect of treatments using time as variable. Therefore, we assessed the overall effects of resolvins and CoQ10 on SEAP secretion from HEK-Blue cells as an alternative to IgE antibody secretion from B lymphocytes.

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5.2 Materials and Methods

5.2.1 Cell Culture, Resolvins and CoQ10

HEK-Blue cells have been described in detail in section 3.2.1 (Figure 1.9).

Growth medium: as described in section 3.2.1.

Experimental media: as described in section 4.2.1. Resolvin stocks were purchased from

Cayman Chemicals Company (Ann Arbor, Michigan, USA) in a solution of 100% ethanol

(RvD1 376Da & RvD2 372Da; 10 μg/ml for each). However, RvE1 was in a lyophilized powder form and the solution was prepared using 100% methanol (250 μg/ml) as suggested in the datasheet provided by the supplier. A subsequent stock of RvE1 was prepared by diluting the stock solution to 10 μg/ml using methanol. Experimental solutions were prepared by mixing 1 μl of the stock solution with 1 ml of experimental media to get a final concentration of 30 μM. Subsequently, 2 μl of the stock was added to 1 ml of experimental media to get 60 μM, so on and so forth. CoQ10 was generously provided by Professor

Andrew Sinclair and Professor Colin Barrow at a concentration of 100 μM (50 ml), which was subsequently diluted down to the required concentrations for experimental purposes. The

CoQ10 solution was prepared in dimethyl sulfoxide (DMSO).

5.2.2 HEK-Blue Detection Assay Using Cytokines IL-4 and IL-13

The protocol used for performing cell-assays with resolvins has already been described in detail in section 4.2.2. HEK-Blue cells were used to perform this set of experiments without making any changes to the protocol listed in the datasheet provided by InvitrogenTM. Cells were treated with a combination of resolvins (30, 60, 90, 120 and 150 μM). 80 μl of cytokine

IL-4 (Integrated Sciences) and IL-13 (Integrated Sciences) were pipetted to each treatment

120 PhD Thesis – Chapter 5 Nayyar Ahmed well at a concentration of 100 ng/ml. The concentrations used for resolvins were consistent with the concentrations used for the fatty acids to maintain an overall standard of the experiments. However, the concentrations prepared for resolvins were not incubated with vitamin E as in the case for fatty acids in the previous chapter. CoQ10 was initially tested at

10 and 20 μM concentrations which showed significant inhibition of both IL-4 and IL-13 signaling. Further experiments were performed with concentrations of CoQ10 which showed

100% inhibition of STAT6 phosphorylation pathway in HEK-Blue cells (20 μM, 40 μM, 60

μM, 80 μM and 100 μM). While referring to the above sections, cells were incubated with the treatments for a period of 24 hrs. Post-incubation, 20 μl of induced HEK-Blue IL-4/IL-13 cells supernatant from each treatment well was added to a corresponding plate with 180 μl of

Quanti-Blue in each well. This is incubated for a total of 3 hrs to detect SEAP levels. The blue color was detected using an xMark microplate absorbance spectrophotometer (Bio-Rad

Laboratories Inc.) at a wavelength of 640 nm. Cell viability was recorded by using a TC10 automated cell counter.

5.2.3 Time-course Assay Using Cytokine IL-4 and IL-13

Time-course assay was performed in a similar fashion as in the case of fatty acids (Section

4.2.3). However, the highest possible concentration with the resovlins at 150 μM was used for experiments. In the case of CoQ10, 50 μM was used to perform the time-course. This value was used as a median value and was used in both IL-4 and IL-13 experiments. A total of five time points were used to measure the effect of resolvins on SEAP production over a period of 24 hrs; 0 hrs, 6 hrs, 12 hrs, 18 hrs and 24 hrs, respectively. PC’s were also included for each time interval which was without fatty acid treatment. Post-incubation, 80 μl of cytokine IL-4 and IL-13 were pipetted to each treatment well at a concentration of 100 ng/ml and the plates were incubated for 24 hrs to stimulate the STAT6 phosphorylation pathway.

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20 μl of induced HEK-Blue IL-4/IL-13 cells supernatant from each treatment well was added to a corresponding plate with 180 μl of Quanti-Blue in each well. This was incubated for a total of 3 hrs to detect SEAP levels. The blue color was detected using a spectrophotometer at

640 nm. Cell viability was recorded by using the TC10 automated cell counter.

5.2.4 Statistical analysis

The data were analyzed using Microsoft Excel 2013 (Microsoft Inc.) The results were analyzed by Student’s t-test using a two-tailed method to determine any statistically significant difference between the controls and treatment wells in all of the above experiments. The statistical significance was set at P < 0.05.

5.3 Results

5.3.1 HEK-Blue Detection Assay Using Cytokines IL-4 and IL-13

As the results indicate, a total of two sets with 4 assays in each set were performed using cytokines IL-4 and IL-13 (Figures 5.1 and 5.2). A differential effect of resolvins and CoQ10 was observed at different concentrations and SEAP secretion from HEK-Blue cells. A gradual increase in resolvins and CoQ10 concentration meant that SEAP levels would decrease or increase hence, reducing or producing blue coloration once the HEK-Blue cells supernatant was added to Quanti-Blue. All positive and negative controls behaved as expected with high and low peaks as suggested by the HEK-Blue IL-4/IL-13 kit. All inhibitions and agonistic results achieved with resolvins and CoQ10 treatments were statistically significant with P values being less than 0.05 with a two-tailed Student’s t-test using standard deviations. While RvD1 (Figures 5.1A and 5.2A) and RvD2 (Figures 5.1B and 5.2B) showed gradual increase in SEAP secretion with both IL-4 and IL-13, RvE2

(Figures 5.1C and 5.2C) showed no change at all, even at 150 μM concentration. However,

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CoQ10 (Figures 5.1D and 5.2D) showed a sudden drop in SEAP levels suggesting a more potent inhibition at 40 μM for IL-4 signaling and 60 μM for IL-13. OD readings showed no inconsistencies when resolvins and CoQ10 were incubated with HEK-Blue cells without cytokine stimulation. Consistency was also observed with cell viability when tested for all the treatments and compared to the PC.

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5.1(A) 5.1(B)

5.1(C) 5.1(D)

Figure 5.1. IL-4 detection assay using resolvins and CoQ10 (μM) with HEK-Blue cells and Quanti-

Blue. Figures 5.1A and 5.1B shows the detection assay performed with RvD1 and RvD2 with a dose- dependent increase in SEAP. However, in Figure 5.1C, no inhibition occurs with RvE1 while CoQ10 shows a rather potent dose-dependent inhibition (Figure 5.1D). No significant change was observed when resolvins and/or CoQ10 was used in the absence of cytokines IL-4. P < 0.05 denotes significance of OD readings compared to PC.

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5.2(A) 5.2(B)

5.2(C) 5.2(D)

Figure 5.2. IL-13 detection assay using resolvins and CoQ10 (μM) with HEK-Blue cells and Quanti-

Blue. Figure 5.2A showed no change when RvD1 was used as a treatment in the presence of IL-13 cytokine whereas 5.2B shows the detection assay performed with RvD2 with a dose-dependent increase in SEAP secretion. However, in Figure 5.2C, no inhibition occurs with RvE1 while CoQ10 shows a rather potent dose-dependent inhibition of IL-13 signaling (Figure 5.2D). No significant change was observed when resolvins and/or CoQ10 was used in the absence of cytokine IL-13. P < 0.05 denotes significance of OD readings compared to PC.

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5.3.2 HEK-Blue Time-course with Resolvins and CoQ10 Treatment

As shown in Figures 5.3 and 5.4, a total of 8 assays were performed with 4 assays in each set. Figure 5.3 shows results with cytokine IL-4 whereas Figure 5.4 shows results with cytokine IL-13. RvD1 (Figures 5.3A and 5.4A), RvD2 (Figures 5.3B and 5.4B) and RvE1

(Figures 5.3C and 5.4C) all showed a significant increase in SEAP secretion and hence produced more blue coloration at 18 and 24 hrs’ time points when compared to the PC values. However, CoQ10 once again showed significant decrease in SEAP secretion when treated with 50 μM concentration. At 24 hrs, an almost 100% inhibition of SEAP signaling was achieved in the case of both cytokines IL-4 and IL-13 (Figures 5.3D and 5.4D). All inhibitions and agonistic effects achieved with resolvins and CoQ10 treatments were statistically significant with P values being less than 0.05 with a two-tailed Student’s t-test using standard deviation values. Since the concentration of resolvins was constant at 150 μM, this experiment was a time-dependent assay. No significant change was observed at 0 hr, 6 hr or 12 hr time points. Cell viability was performed at the end of the 24 hr incubation to check for any anomalies. However, the cell viability count was well within the range of x105 cells.

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Figure 5.3. HEK-Blue detection assay with 150 μM of resolvins and 50 μM of CoQ10 in the presence of IL-4 cytokine as a bioactive agent. Figures 5.3A, 5.3B and 5.3C depict a time-dependent increase of

SEAP secretion from HEK-Blue cells when treated with RvD1, RvD2 and RvE1, respectively. Figure 5.4D shows a decrease in SEAP secretion from HEK-Blue cells when treated with CoQ10 at 12, 18 and 24 hr time points. P < 0.05 denotes significance of OD readings compared to PC depicted in blue color. C1 depicts the

OD reading obtained when HEK-Blue cells are treated with resolvins and CoQ10 only, without the cytokine stimulation.

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Figure 5.4. HEK-Blue detection assay with 150 μM of resolvins and 50 μM of CoQ10 in the presence of IL-13 cytokine as a bioactive agent. Figures 5.4A, 5.4B and 5.4C depict a time-dependent increase of

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5.4 Discussion

5.4.1 CoQ10 and Resolvins

Resolvins and CoQ10 have not been previously tested on a HEK-Blue model of cells, which mimics IL-4 and IL-13 signaling for IgE production by B lymphocytes. No previous study to date has tested the effects of resolvins and CoQ10 on an allergy cell model. In this chapter, we report for the first time that resolvins exacerbate the production of SEAP production from

HEK-Blue cells when stimulated by bioactive IL-4 and IL-13. On the other hand, CoQ10 showed very promising results as a potential antagonist to IL-4 signaling for allergy treatment, showing statistically significant inhibition of SEAP secretion with results achieving up to 100% inhibition.

Although it was hypothesized by Ye et al., (1988), approximately 3 decades ago that CoQ10 can play an important role in patients suffering from autoimmune conditions such as allergy

(172), CoQ10 has gained much attention in recent years for its potential inhibitory effects on inflammation (330). In a study carried out by Lee et al., (2013), it was reported that CoQ10 showed anti-inflammatory activity in coronary artery disease when human subjects were supplemented with 300 mg/day of CoQ10 (330). Inflammatory cytokines such as TNF-α were significantly reduced as well when patients were supplemented with CoQ10 (330). In another study, Schmelzer et al., (2008), demonstrated that CoQ10 wielded anti-inflammation effects during inflammation via the inhibition of NF-κB dependent gene expression (331).

When cytokines IL-4 and IL-13 bind to its receptor IL-4R, this activates protein tyrosine kinases JAK1/JAK3 which results in STAT6 phosphorylation. Following this, dimerized

STAT6 modulates the transcription of IL-4 responsive genes. NFκB and STAT6 work in collaboration for IgE transcription and IgE production in B cells. Although not tested in our current study, it has been previously established by Weise et al., (2011) that NF-κB gene

129 PhD Thesis – Chapter 5 Nayyar Ahmed expression is suppressed by supplementing n-3 fatty acids, which significantly reduces IgE production of B lymphocytes (158) and forms a critical step in the allergic cascade. This in turn influences TNF-α gene expression (331). Since IL-4 and IL-13 cytokines mediate the same response via JAK-STAT signaling pathway, it can be concluded that CoQ10 inhibits

SEAP secretion via this route without effecting cell viability. This helps to preserve homeostasis in cells, which is key to various biochemical pathways.

The recent discovery of resolvins has exposed molecular and cellular mechanisms for the resolution of acute inflammation. In response to acute inflammation, these pro-resolving mediators inhibit the inflammatory response by reducing neutrophil infiltration at the site of inflammation. During an allergic response, resolvins recruit natural killer (NK) cells to enable

T cells and macrophages in the removal of allergen deposits in the lung (332). The resolution of acute inflammation is an active process which is crucial for the establishment of homeostasis and tissue protection. ChemR23 was the first receptor identified which interacts with RvE1. ChemR23 is expressed on a range of white blood cells (WBC’s), such as monocytes, macrophages and dendritic cells. Treatment with RvE1 inhibits the prominent transcription factor NF-κB (333). Although this is in sharp contrast to the effects of CoQ10 on NF-κB inhibition, the results achieved with HEK-Blue suggest otherwise. SEAP secretion remained unchanged with RvE1 when cells were treated with a range of different concentrations. However, during the time-course experiment, RvE1 showed to increase

SEAP production. Although no previous study has tested this model of allergy, more information needs to be gathered to form an overall picture of its effects at the molecular level. RvD1 and RvD2, in contrast to RvE1, have shown to increase SEAP from HEK-Blue cells in all the assays performed. This is consistent with published data by Li et al., (2014) where RvD1 potentiated the effects of IL-4 on STAT6 phosphorylation pathway, which leads

130 PhD Thesis – Chapter 5 Nayyar Ahmed to activation of gene expression of SEAP (334). RvD2 shares the same distinct features of resolvins in its ability to resolve acute inflammation. In a mouse-model study of sepsis, RvD2 sharply decreased bacterial burden, cytokine production and neutrophil recruitment while increasing macrophage phagocytosis (335). The differential results obtained with resolvins and their effects on SEAP secretion are evidently surprising. Although literature very strongly supports resolvins to be anti-inflammatory by nature, their effects on IgE secretion appear to be profound and agonistic.

5.4.2 Significance of Expression of SEAP/IgE Secretion

Generally, a negative notion exists with an over-expression of SEAP secretion from HEK-

Blue cells which in turn means a more severe allergic reaction, however it must be noted that

IgE antibody secretion is part of a normal response by the immune system to invading hosts.

Ige is an important component of host-protective immune responses against the helminthic parasites in the majority of the world population, specifically in undeveloped countries (336).

Hence, we speculate that an increase in IgE response by resolvins may be considered as a normal response by tissues to fight against helminthe infection and maintain homeostasis.

5.4.3 Conclusion

In conclusion, treatment of HEK-Blue cells with CoQ10 lead to a repression of SEAP production through inhibition of IL-4 and IL-13 signaling pathways. These results comprise a great biological relevance to the possible development of a prophylactic treatment for allergic diseases. With resolvins, an increase in SEAP output was recorded which is not coherent with the anti-inflammatory properties of resolvins. Our studies, presented here, opens a new corridor to research with the effects of resolvins on a transfected model for allergy research.

CoQ10 supplementation may offer a molecular basis for a well-developed anti-allergic effect

131 PhD Thesis – Chapter 5 Nayyar Ahmed and therefore, must be an essential part of our regular diet. Although further investigation will reveal the best allergy markers that can be used for suppression, it is clear that CoQ10 as opposed to resolvins offer a great opportunity as potential natural inhibitors of allergy

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Chapter 6 - Relationship Between Ryegrass Pollen

Counts and Weather Variables in Regional Victoria,

Australia

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6.1 Introduction

Allergic diseases such as asthma, rhinitis and atopic dermatitis induced by ryegrass pollen are on the rise all over the world. Developed countries are facing a rise in prevalence of allergic diseases which has an additional effect on healthcare costs (337). This trend is clearly evident in south-eastern state of Victoria in Australia (204, 338-341). Awareness and thus avoidance of pollen exposure is the first line of defense in the treatment of allergic disease.

For diagnosis and treatment of allergic disease, the pollen map in an area plays an important role in helping clinicians to perform the appropriate tests and treatments on allergic patients such as immunotherapy (342, 343). High prevalence of perennial ryegrass (Lolium perenne) pollen in south Victoria with a characteristic climate of sunny days, high humidity and thunderstorms (344) in summer make it important to map pollen distribution in regional

Victoria (Geelong) (211). The coastal city of Geelong is located 75km (47 miles) south-west of the state capital, Melbourne.

In the last 30 years or so, aerobiological studies in Victoria have been developed rapidly in the city of Melbourne, Australia, which is well known as the capital of thunderstorm allergy

(340, 341). In the region of Geelong, ryegrass pollen distribution and its relationship to climatic changes have not been studied previously. In our current study the NARL research group has investigated the study of ryegrass pollen counts and their changes during the summer months (October-January). Secondly, the relationship of meteorological variables and pollen distribution were also studied and examined. Thirdly, we examined the presence of ruptured pollen which evidently releases allergens such as Lol p 1 and Lol p 5 that are responsible for allergic reactions in atopic individuals (204, 211).

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In this chapter, we hypothesized that ryegrass pollen distribution in air is positively correlated to weather variables such as rain, temperature, humidity etc. we aim to establish a positive correlation of weather variables with ryegrass pollen and the presence of ruptured pollen in the atmosphere by using a Burkard volumetric air trap as our sampling method. Once this is established, it will open new exciting pathways for future treatment of pollen allergy improving quality of life.

6.2 Methods and Materials

6.2.1 Establishment of the Air Sampler

The Burkard volumetric spore trap (Burkard Agronomics Inc.) purchased from Burkard

Scientific Ltd. was installed on the rooftop of the Deakin University library in Waurn Ponds,

Geelong, Australia (Figure 6.1). The trap was positioned in a way to not be obstructed by any other university building surrounding the pollen trap and no trees that would obstruct wind or act as a bias to the trap. This was primarily done to avoid missing any pollen released from grass and trees that are mixed by the turbulent boundary layer. As a result, all pollen released will be dispersed considerably in air before being sampled by the volumetric trap.

6.2.2 Preparation of Slides

Microscopic slides were prepared using the following solution:

1. Pollen collecting adhesive (10 g gelatine + 155 ml filter-sterilised water + 40 ml

glycerol + 0.5 g phenol).

500 ml glass bottle: Gelatine and water were mixed and warmed at 60°C (hot plate

with gentle stirrer). Once cooled down to RT, glycerol was added along with phenol

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(anti-bacterial agent). 50 ml was decanted into smaller bottles. Bottles were stored at

RT.

2. Coverslip adhesive (35 g Mowiol + 100 ml distilled water + 50 ml glycerol + 0.5 g

phenol).

500 ml glass bottle: Mowiol (gelvatol Burkard Agronomics Inc.) was added to water

and was slowly heated to 95°C. A stirring rod on a magnetic heater/stirrer plate was

used to heat the mixture and then allowed to cool for a few hrs. Once cooled, glycerol

and phenol were added to prepare the coverslip adhesive. The adhesive prepared was

in a volume of 500 ml. Aliquots were prepared in separate bottles of 50 ml and stored

at -20°C for 12 months.

Microscope slides were prepared with the date written on one end of the slide using a marker.

A brush was dipped in the pollen collecting adhesive and the bristles were applied gently to the slides from end to end. To provide a more uniform application of pollen adhesive, a razor blade was gently passed over the slide to even out the adhesive and get rid of any bubbles.

However, care was taken not to apply the adhesive on the date written on the slide to avoid the mixing of marker ink with the adhesive. The slides were placed into the slot of the 24 hr drum and ready to be placed in the sampler.

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Figure 6.1. An aerial view of the spore trap on the roof of the library at Deakin

University, Waurn Ponds, Geelong. The area immediately surrounding the university premises is covered with green pastures that allow the dispersal of grass pollen hence making it a very suitable site for the installation of Burkard volumetric trap to sample air. The photo was taken using Google Maps (Google Inc.). The Figure on top is an actual photo of the sampler installed on the rooftop.

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6.2.3 Changing the Burkard Air Sampler Using the 24 hour Drum

1. Stop the trap from rotating with restraining pin on the chain to avoid injury;

2. The locking arm was pressed down and rotated to open the hatch of the trap. The

drum was pulled out straight from the body of the sampler.

3. The slide from the previous day was removed and replaced with a freshly prepared

slide (Figure 6.2). During the 24 hrs sampling period, the clock moves the slide up 48

mm across the orifice of the trap. Used the knurled ring to reorient the carrier;

4. New slide inserted for the next 24 hrs. Slides were prepared using a brush dipped in

pollen adhesive and applied on the slide to ensure uniform distribution;

5. The clock was wound (anti-clockwise) using a key provided in the kit;

6. The 24 hr drum was placed on the grooves located on the inside of the sampler and

the locking arm was rotated back into position to secure the head of the drum;

7. Check the flow rate with the flowmeter; adjust if necessary. It is advisable to check

the flow rate once a week. To change the flow rate, remove the sampling head and

adjust the screw in the white valve at the base of the chamber;

8. Remove the restraining pin on the chain to allow the trap to rotate freely with caution

to avoid injury.

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At start position After 24 hrs

Figure 6.2. The orientation of the slide before and after a period of 24 hrs during which the slide moves along the orifice of the air sampler for collection of pollen.

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6.2.4 Preparing Slides for Pollen Counting and Examining Using an Olympus BX50

Microscope:

1. The slide was removed from the sampler and coverslip adhesive was applied down

the entire length of the slide (avoiding the date written on one end of the slide) using a

micropipette. Care was taken not to allow the adhesive to dry (Figure 6.3);

2. Immediately, a coverslip was placed at a 45° angle and dropped carefully so as to fall

on the coverslip adhesive covering the entire width of the slide. Care was taken not to

move the coverslip;

3. Gentle pressure was applied using forceps to evenly spread the adhesive;

4. The slides were allowed to dry for a period of 24 hrs;

5. Any access adhesive that may have oozed out from the edges of the coverslip was

carefully removed using a razor blade;

6. Slides were stored away in boxes specifically designed for storing microscope slides

for long-term storage;

7. Slides were examined using an Olympus BX50 microscope (Olympus Australia) at

40x magnification and gradually increasing magnification to 200x. To confirm the

identification of ryegrass pollen, the magnification used was 400x;

8. Pollen were counted using a line scan method which is counting the pollen in one

longitudinal transect of the slide (345). It must be acknowledged that the Burkard

volumetric trap tends to show an instrumental variation of 25% shown in Table 6.1

(345).

9. Photos of the pollen were taken using a Canon DS126291 camera (Canon Inc.) which

was connected to a computer;

10. Pollen data was uploaded on a daily basis on the Deakin AIRwatch webpage which

also included a thunderstorm associated asthma risk;

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11. All weather parameters including, temperature, humidity, precipitation and wind

speed were recorded courtesy of the Geelong Weather website. The weather station

installed is Davis Vantage Pro2 which collects weather information every 5 seconds.

The station comprises of an anemometer, rain gauge and a thermo-hydro sensor

situated in optimal positions for highest accuracy possible.

6.2.5 Calculations for Pollen per m3 of Air

An Olympus BX50 microscope has a field of view of approximately 1 mm at 200x magnification (micro-meter was used for measurement). The Burkard volumetric trap has a vacuum pump which takes in air at a rate of 10 litres/min. Hence, total air sampled over a period of 24 hrs was 14.4 m3. The width of the orifice on the air sampler is 14 mm. Since a line scan method was used to count daily pollen, the entire length of the slide is taken into account at 48 mm since the 24 hr drum moves 2mm/hr hence 48 mm over a period of 24 hrs.

Formula applied = Field of view/width of slide x (Total volume of air)

= 1/14 x (14.4) = 1 m3

Correcting count per m3 of air

Formula = final count of pollen per line scan / 1 m3 of air = Total pollen count per m3 of air

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Coverslip

Coverslip adhesive

Glass slide Coverslip adhesive

Figure 6.3. Diagrammatic representation of the coverslip adhesive placed on the slide surface where the pollen is captured using the pollen adhesive.

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6.3 Results

6.3.1 Ryegrass Pollen

Pollen captured on the surface of microscope slides was prepared as shown in methods section. This was followed by an examination of the slides using an Olympus BX50 microscope that allowed us to take photos of ryegrass pollen. It should be elucidated that pollen are much more visible without the use of stain. The photos taken were at random from slides to show the distinct shape and rupturing of ryegrass pollen. All pollen was counted using a line-scan method along the longitudinal traverses of the slide. For results purposes, all pollen, whether partial or fully ruptured, were taken into the same category. Intact pollen showed granules within the shell and were identified by the presence of a single distinct pore on the wall of the pollen (Figure 6.4).

Table 6.1. Weather Variables and Grass Pollen Count. Annual Season 2012-2013 (Oct-Jan) 2013-2014 (Oct-Jan) 2014-2015 (Oct-Jan) Mean maximum 23.7202381 22.004 21.09°C temperature °C Average daily 17.38°C 16.35°C 17.36°C temperature °C Average high wind 31.5 km/hr 32.25 km/hr 32.14 km/hr speed km/hr Average wind 6.7 km/hr 4.41 km/hr 4.58 km/hr speed km/hr Average RH Max. 82 % 77.70 % 78 % Values (%) Total precipitation 60.4 mm 117.1 mm 145.4 mm in mm Intact pollen per 1,066 per m3 of air 1,397 per m3 of air 1,027 per m3 of air m3 of air (+/- 25%) Ruptured pollen 249 per m3 of air 364 per m3 of air 295 per m3 of air per m3 of air (+/- 25%) Total pollen for 1,315 per m3 of air 1,761 per m3 of air 1,322 per m3 of air season per m3 of air (+/- 25%)

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A) B) C) D) E)

Figure 6.4. Micrographs of ryegrass pollen identified by using an Olympus BX50 microscope at magnification of 400x. Figure 6.4A shows the ryegrass pollen when stained with safranin. Figure 6.4B shows intact ryegrass pollen. Figure 6.4C shows an empty shell of ryegrass pollen which has released all starch granules and allergens. Figure 6.4D shows partially ruptured ryegrass pollen with starch granules released from the pore. Figure 6.4E also shows partially ruptured ryegrass pollen.

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6.3.2 Weather Variables and Grass Pollen Count (2012-2013)

For the purpose of determining the effects of weather variables on grass pollen count, we considered 4 parameters: temperature, wind speed, RH values and total precipitation during the whole season (Figure 6.5). The start of the pollen season was determined by the appearance of ryegrass pollen when examining slides. The first day with pollen appearing for

2012-2013 took place on the 18th of October, 2012 and the last day was the 10th of January

2013. Looking at Figure 6.5, three prominent peaks can be found when considering intact and ruptured grass pollen count. For results purposes, these three peaks will be considered as significantly higher than other peaks hence showing important events during the season. The first day denoted 19th of November of 2012 showed the first high peak after an episode of rain a day earlier and an elevated temperature high in the 20’s. The second most important peak of the season fell on the 29th of November 2012 followed by smaller peaks, where an episode of rain along with higher average temperatures immediately translated into a higher pollen count and ruptured pollen as well. The third most important peak of the season fell on the 13th of December 2012 when higher than average temperature and wind gave a higher pollen count. It is worthy to note that although the intact grass pollen count on this day was equally high to other peaks, very few pollen resulted in rupture due to a dip in RH on that particular day. The initial peaks also showed lower intact grass pollen counts but had a higher ratio of ruptured pollen when compared to other peaks.

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Figure 6.5. Weather variables for the grass pollen season 2012-2013 (Oct-Jan).

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6.3.3 Weather Variables and Grass Pollen Count (2013-2014)

The first day with pollen appearing for 2013 took place on the 18th of October, 2014 and the last day was the 31st of December 2013. Looking at Figure 6.6, three prominent peaks can be seen when looking at intact and ruptured grass pollen count. For results purposes, these three peaks will be considered as significantly higher than other peaks hence showing important events during the season. The first day denoted 8th of November of 2013 did not show a higher than average temperature, however high gusts of wind in excess of 30km/hr may also have played an important role in the dispersal of pollen on this particular day. The second most important peak of the season fell on the 18th of November 2013 followed by a sudden drop in pollen counts which coincides with an episode of rain. The sudden rise can be attributed to a rise in average daily temperature which may have resulted into the release of pollen and rupturing. Despite of the high peak, this day can be regarded as an anomaly when looking at weather parameters and ruptured pollen. There was no rain prior to this peak and

RH values were within normal range, which are the main triggers of pollen rupturing. The third most important peak of the season fell on the 8th of December 2013 when higher than average temperature and an episode of rain a day earlier resulted in a higher pollen count.

Referring to data in Table 6.1, a higher level of precipitation was found when compared to the previous year. This explains a higher grass pollen count. All rainy days coincided with no release of pollen. However, the peaks of ruptured pollen between the 23rd and 25th of

December 2013 showed a higher ratio of ruptured pollen, which coincides with a rainy day.

However, with less precipitation, the pollen were able to dehisce since a higher temperature peak was attained and dispersed by wind.

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Figure 6.6. Weather variables for the grass pollen season 2013-2014 (Oct-Dec).

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6.3.4 Weather Variables and Grass Pollen Count (2014-2015)

The first day with pollen appearing for 2014 took place on the 12th of October, 2014 and ended on the 31st of December 2014. Looking at Figure 6.7, two prominent peaks and a cluster of peaks can be found when looking at intact and ruptured grass pollen count. For results purposes, these peaks will be considered as significantly higher than other peaks hence showing important events during the season. The first day denoted 22nd October of 2014, which appears to be an anomaly. With lower average temperature and no rain episodes on previous days and lower wind speed all indicate a lower grass pollen count. The spike may have been a result of a sudden gust of wind which causes pollen to be airborne. The second most important peak of the season fell on the 29th of October 2014, which was followed by an episode of rain on the previous day. Also to be noted, a higher ratio of ruptured pollen can also be seen on this day because of a rise in RH max values. The other important peaks of the season fell in the first week of November of 2014 when an episode of rain along with higher wind gusts resulted into higher pollen count. However, it can be noticed that the ruptured pollen may have resulted from the rain on the 1st of November. Referring to data in Table 6.1, a higher level of precipitation was found when compared to the previous year. This explains a higher grass pollen count. With the exception of the first week of November, all rainy days coincided with very few or no release of pollen. However, the peak of pollen on the 25th of

November 2014 showed a higher ratio of ruptured pollen, which coincides with a rainy episode on the prior day and a higher average daily temperature. The season of 2014-2015 experienced the most amount of precipitation but this cannot be correlated with the total amount of pollen released and ruptured since a lower mean maximum temperature for the season was recorded (Table 6.1).

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Figure 6.7. Weather variables for the grass pollen season 2014-2015 (Oct-Dec).

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6.4 Discussion

6.4.1 Seasonal Changes in Grass Pollen Count

In our study of ryegrass pollen distribution in Waurn Ponds, Geelong we report for the first time the collection of daily pollen counts and their relationship to meteorological conditions prevailing in regional Victoria.

Ryegrass is grown in pastures for livestock grazing and can be viewed in Figure 6.1 around the Waurn Ponds campus of Deakin University. Analyzing the overall collection of pollen samples over a three-year seasonal period shows a differential correlation between weather parameters and grass pollen albeit with exceptions. Referring to Table 6.1, it was revealed that the season 2012-2013 has received the least amount of precipitation which coincides with the theory of fewer grass pollen being released and ruptured. In contrast, the following seasons had higher pollen counts due to a significant amount of precipitation and higher temperatures. However, it must be stressed that high temperature can have negative effects on the maturing and development of pollen grains (346). Although the highest precipitation occurred in the season of 2014-2015, fewer pollen counts were reported when compared to

2013-2014. This may be a result of a lower average peaks in temperature which is a contributing factor for the dehiscence of pollen (346). Also, it must be emphasized that grass pollen count fluctuations were not significantly correlated with changes in weather variables in some instances.

The pollen peaks achieved during 2012-2013 coincide with high temperature peaks which cause anthesis. Following anthesis, pollen dispersal is dependent on a variety of physiological and meteorological factors such as wind speed and wind turbulence, which causes dehydration of the anther that results in dehiscence. Hence, the dispersal of pollen may

151 PhD Thesis – Chapter 6 Nayyar Ahmed continue a few days after anthesis (347). Also, it can be observed from Figure 6.5 that the three major peaks in 2012-2013 occurred in the month of November and December. This may have been a result of rain episodes that peaked in these months along with higher than average temperature peaks when compared to October. Rain episodes seemed to have clustered during this season rather than frequent episodes. The peaks in the initial part of

December of 2012-2013 also coincides with higher temperatures and stronger winds that disperse pollen. Rainfall is the single most important factor in determining plant growth, hence making the observations important. Peaks in RH max values and rainfall are sufficient to cause ryegrass pollen fragmentation.

In the following season of 2013-2014, higher than average temperature along with relatively higher wind speeds resulted into the highest pollen count. Referring to Table 6.1, it was revealed that the season 2013-2014 has received the second highest rainfall, which coincides with the theory of higher grass pollen being released and ruptured. The rain distribution during this season is more evenly distributed throughout the season with higher than average rain peaks in October, November and December. This explains a more concomitant release of pollen when temperatures reached high into the 30’s. This resulted into the drying of the anthers and thus releasing pollen on the days that followed.

In the latest season of 2014-2015, it was determined that despite of receiving the heaviest rainfall out of the three seasons, the pollen count failed to achieve higher numbers.

Temperature played an important role in this regard. Referring to Table 6.1, it can be noted that a lower mean maximum temperature for the season inhibits anthesis and less pollination of grass by wind and other factors. Another observation can be seen for the month of October which received the least amount of rainfall and yet showed significant peaks. They may have

152 PhD Thesis – Chapter 6 Nayyar Ahmed been a result of higher than average wind speeds and temperatures recorded at the initial days of the season. The highest peaks were recorded in the last week of October and first week of

November, which also coincides with high wind speeds and a hot day prior to these peaks.

6.4.2 Thunderstorm Allergy in Victoria, Australia

Although severe thunderstorm events have been reported previously in the city of Melbourne,

Victoria, such as the thunderstorm event in the spring of 2010 and the 1987/1989 thunderstorms (204, 339-341, 348), no such event has been reported in the three consecutive seasons we have reported so far. Although some rainfall episodes were accompanied by few thunders, we are yet to report any unusual increase in pollen count and pollen rupture associated with thunderstorms.

6.4.3 Ruptured vs. Intact Ryegrass Grass Pollen

Grass pollen develops inside the anther sac. With high temperatures and dry conditions, the anther splits open releasing approximately 5000 pollen (dehiscence). However, the dispersal of pollen from the anther sac depends on other variables such as wind. This has been reported by Taylor et al., (2002) that pollen remained on the anthers despite the fact that anther sacs were open on wind-pollinated plants such as ryegrass. Hence, it is important to recognize that anther dehiscence and pollen release are two different events that do not occur simultaneously (349).

It is a well-documented fact that allergens within pollen grains cause allergy rather than the pollen itself. Since pollen can’t penetrate respiratory airways, it is the allergens such as Lol p

1 and Lol p 5 which are located on and within the micro-particles found in a pollen grain that are small enough in size to penetrate lower airways and trigger the allergic symptoms (350,

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351). It has been reported by several papers that ryegrass pollen, upon contact with water molecules, results in the bursting of the intine layer of the aperture pore. This results in the release of ~700 starch grain particles (0.6 to 2.5 μm in length) that form the part of respirable allergen aerosols (208, 338, 349). An experiment designed by Taylor et al., (2002) describes the harvesting of flowering plants such as ryegrass in a plexiglass chamber. The plants had their anthers open which releases pollen inside the chamber. A particle counter then collects the pollen and cytoplasmic debris to give an overall number of particles released by each flowering plant. Repeated drying and wetting of the chamber did result into the rupturing of pollen. Gentle airflow inside the chamber also resulted into the dispersal of the cytoplasmic debris and micronic particles released from the dehisced anthers. It is established that these micronic particles are responsible for triggering asthma responses in individuals (204, 349).

Hence, a few important observations with regards to pollen rupture were made during the entire pollen seasons as can be seen in Figures 6.5, 6.6 and 6.7. A typical example can be seen in the recent pollen season on the 1st of November of 2014. A prior sunny day with a high temperature of >30°C, followed by rain on the 1st of November that causes rupturing of pollen. Although rainfall can result into the washing off of pollen grains and micronic particles (352), we report that ruptured ryegrass pollen incidence increased with rainfall episodes. Wind speed was also reported to be higher than average on the 1st of November.

These events make it a ‘perfect storm’ for the release of ruptured pollen from anthers that may have dehisced on the previous sunny day and ruptured upon contact with water during the rainfall episode followed by wind. Hence, it is important to note that although some days may appear to have higher than average wind speeds, the pollen counts are reported as low since anther dehiscence may not have taken place due to low temperatures and lack of rain on previous days. Similarly, it is not necessary that a rainy day during the pollen season may

154 PhD Thesis – Chapter 6 Nayyar Ahmed result into a high ruptured pollen count since exposure to water must take place after dehiscence of the anthers.

6.4.4 Conclusion

To conclude, in the regional city of Victoria (Geelong), pollen distribution and its relationship with weather variables have not been studied before. In our study, we investigated the distribution of ryegrass pollen and their changes during the spring/summer season in Victoria. It is important to establish a pollen concentration in this region where atopic individuals suffer from pollen allergy. In this three year study, it was observed that there was a significant increase of pollen counts on days that had high temperatures and wind gusts. High pollen count days were preceded by rainy days in some instances. Pollen rupturing was also added into the mix as a more rational approach to understanding pollen debris that carries allergens. However, more seasons of pollen counting will need to be carried out in the future which will increase our understanding of the effects of weather variables on ryegrass pollen distribution.

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Chapter 7 – General Discussion

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7.1 General Discussion

This chapter entails an overall discussion of Chapters 2-6 with more emphasis on IL-4RD inhibition for allergy treatment. However, in latter sections the inhibition of cytokine signaling is discussed with respect to fatty acids, resolvins and CoQ10 as novel therapeutics for allergy. Lastly, we have discussed the mapping of ryegrass pollen distribution in regional

Victoria (Geelong) with links to weather variables.

7.2 Overview of IL-4RD

The IL-4RD plays a vital role in allergic diseases since it is able to bind IL-4 and IL-13 cytokines. Activation of Th2 cells by APC leads to the production of cytokines that cause B cells to produce IgE. Once synthesized, IgE antibodies circulate in the blood before binding to the high-affinity IgE receptor FcHRI that is present on mast cells in tissue or on peripheral blood basophils. After re-exposure, allergens cross-link to mast cell- and basophil-bound specific IgE, thus causing the release of preformed mediators, such as histamine, the synthesis of prostaglandins (PGs) and LT’s (353).

Thus, it is necessary to find a novel antagonist to IL-4RD, to block the interaction of the cytokines with their receptor, hence blocking or down-regulating the allergic cascade as a prophylactic treatment.

157 PhD Thesis – Chapter 7 Nayyar Ahmed level. For this reason, phage display technology has been used as a novel approach to identify peptide antagonists (354).

Also, we have discovered the effect of n-3 and n-6 PUFA on a HEK-Blue model of allergy.

This model is versatile and mimics a simple colorimetric analysis of IgE antibody production from B cells in response to cytokine signaling. Another important finding discussed in this thesis is the effect of resolvins and CoQ10 on IL-4 and IL-13 signaling in the same HEK-

Blue cell-line. As our results suggest, resolvins although anti-inflammatory by nature, have been found to be pro-inflammatory with respect to JAK-STAT6 signaling.

In addition, we have investigated the distribution of ryegrass pollen during three spring/summer seasons in regional Victoria. This will help us evaluate a more clear understanding of allergy prevalence in Victoria. A broader concept to allergy will be to understand the cause of allergy before tackling the problem on a molecular basis. Grass pollen has been implicated in allergy by various scientists all over the world. Hence, it is highly important to map pollen distribution in a given area and conduct aerobiological analysis of the pollen data.

7.2.1 Phage Display

In Chapter 2, it was shown that five successful rounds of biopanning were completed using

IL-4RD as the potential target. It should be stressed here that effective aseptic techniques are required to achieve more than 3-4 biopanning rounds as wild-type M13 phage contamination can easily take over the peptide-displaying recombinant M13 phage. The recombinant phage has reduced infectivity when compared with wild-type M13. The repeated 5 cycles of biopanning meant, selecting phage that binds with the highest affinity to the target IL-4Rα.

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Maintaining a constant input phage concentration in each round, incurred a consensus in binding sequences.

The exposed peptides which showed higher binding affinity to the IL-4Rα target were collected and washed using TBST. The detergent Tween-20 added to the binding and washing buffers reduced non-specific hydrophobic interactions between the phage and blocking/target proteins. Lower concentrations of TBST were used in the earlier rounds, which resulted into lower stringency but higher eluate titers, and the stringency was gradually increased from 0.1% to 0.5% in the final round, selecting for highly specific interactions.

Another purpose of using TBST was to favour ionic interactions rather than hydrophobic ones between the target receptor and the peptide. Target concentration was also reduced during subsequent rounds of biopanning, as this would allow a higher stringency. By decreasing the concentration from 50 nM to 10 nM allowed for selection of ligands with high binding affinity.

Phage-display has become a method of choice for epitope mapping and has been successfully used in numerous published studies (131, 355-357). For this reason, it was suggested that sequences N1-N9 that showed consensus in binding represented an epitope that strongly bound with the IL-4Rα target. This was determined from the ClustalW2 alignments that were conducted using the IL-4 and IL-13 amino acid sequences with the N1-N9 amino acid sequence. 6 amino acid residues were exactly identical with the IL-4 (153 amino acids) along with 3 weakly conserved regions. In essence there was a 50% similarity between the selected peptide sequence and IL-4 cytokine. When the same 12-mer sequence was aligned with IL-13

(146 amino acids), only two amino acids were identical while the strongly and weakly conserved amino acids had 3 each. This confirmed that the isolated peptide mimicked the IL-

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4 cytokine more than it did IL-13. This is consistent with the study that IL-4Rα subunit binds both IL-4 as well as IL-13. Homologous sequences have blocks of alignments separated by gaps (not shown for intellectual property reasons). The gaps usually indicate loop regions with no conserved core secondary structure and hence, explain that the epitope of the peptide is conformational in shape rather than linear (358). Further, a nucleotide BLAST search was conducted on national centre for biotechnology information (NCBI) using the amino acid sequence of the identified peptide. It was used to compare the peptide sequence with those contained in nucleotide databases by aligning the sequence with previously characterized proteins, therefore assisting to identify homologues that are linked with the IL-4RD protein.

However, an analysis of the homologues revealed no relevant protein that might be linked to the interleukin family of proteins.

7.3 Mimtags: The Use of Phage Display Technology to Produce Novel Protein-Specific

Probes

As part of our overall discussion for Chapter 2, we have demonstrated that phage display can also be used as an effective tool to study protein structures and the recognition of epitope regions. The aim of this work was to isolate phage-displayed mimtags (section 1.6.1) against

IL-4RD and to investigate the ability of mimtags to bind target proteins and act as protein- specific probes. High-quality mimtags were isolated during the study, which recognized IL-

4RD. Hence, the term peptide will be replaced with mimtag for discussion purposes.

7.3.1 The Use of Phage Display in Isolating Mimtags as Protein-Specific Probes

Once again, for discussion purposes, only mimtag N1 will be emphasized throughout this section.

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New strategies have been devised in the last few years to characterize regions with epitope binding capacity. Phage display has shown to be a powerful tool to discover and unravel conformational mimic epitopes or mimotopes from random peptide libraries (359, 360).

In this study, phage display was successfully used to identify mimtags for target proteins IL-

4RD. 12-mers are long enough to fold into short structural elements, which may be useful when panning against targets that require structured ligands. The exposed random peptides were allowed to interact with the sequences of the IL-4RD. The purity and efficacy of the target was determined by the company (Sigma-Aldrich), which certified the RhIL-4RD to be pure and biologically active. A 12-mer library was used in the assay because of it renders peptides that include highly reactive mimotopes as opposed to 7-mer libraries (260). The additional 4th and 5th round of selective biopanning discarded almost completely the low affinity binders and 9 out of the 10 mimtags isolated had a consensus in their amino acid sequences.

The use of phage display has many advantages over the conventional system of using antibodies for the detection of epitopes. Many studies over the years have shown the usefulness of phage display in the field of research. The possibility of obtaining mimtags of interest without learning the three-dimensional structures of proteins avoids the need for complicated cloning, crystallizing, and modelling procedures, which had to be carried out first (361-364). The in vitro analysis using a 12-mer phage displayed peptide library eliminates the use of animals for the production of antibody libraries, which subsequently eliminates any issues relating to animal ethics and keeps animals out of harm’s way. Mimtags are also smaller in size when compared to antibodies, which increases their effectiveness and

161 PhD Thesis – Chapter 7 Nayyar Ahmed efficiency to penetrate large protein fragments or antigens with conformational epitopes

(139).

7.3.2 Probing of a Conformational Epitope on Target Protein with Mimtag

The mimtag sequence N1 had shown to share a homology with the cytokine IL-4 at specific amino acid sequences. Before stepping into further analysis of the synthesized mimtag, it was important to understand the interaction between IL-4 and its receptor IL-4RD relative to the binding of mimtag N1 to IL-4RD. The multiple sequence alignment in ClustalW2 showed a

50% identity of amino acid sequences of N1 mimtag with amino acid sequence of IL-4 cytokine. There were gaps in the sequence alignments, which indicated a loop region with no secondary structure conserved. Based on hydrogen bonding, there are three clusters that form ligand receptor interface. In the IL-4RD, two out of three clusters have been identified as the main binding determinants (Figure 7.1), Glu9 (IL-4) and Arg88 (IL-4). The Glu9 makes several hydrogen bonds with IL-4RD at positions Tyr13, Ser70 and Tyr183. The second cluster involves Arg88 of IL-4, which binds with Asp72 of IL-4R (365). The critical binding residues are distant in the primary sequence but close in the folded native conformation of the protein hence forming a discontinuous chain of amino acid residues (269). However, the N1 mimtag showed binding to IL-4RD in the binding ELISA assay which did not overlap the binding sequence of IL-4 cytokine and its receptor when cross-checked with ClustalW2 alignment software. Hence, it may be possible that the N1 mimtag may have identified a novel epitope of IL-4RD, which remains unreported (55, 366).

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Figure 7.1. Cluster domains of the IL-4R indicated by the dotted Y shape. These clusters are responsible for binding with the IL-4 cytokine. Two clusters denoted Cluster I and Cluster

II have shown to bind to IL-4 cytokine with about 80% of the total binding energy. At amino acid positions 13, 70 and 183 lays the first binding region with amino acid at position 9

(glutamate residue). At amino acid position D72 lays an aspartic acid that binds with arginine at position number 88 of the IL-4 cytokine. Figure adapted from (365).

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7.3.3 Mimtag Technology

Mimotope tagging is a very versatile procedure to identify and tag epitope regions of proteins using biotinylated versions of the mimotopes. Mimotopes isolated from phage display libraries enable alignments with the target antigen and subsequent localization of structural epitope regions (367). Another useful outcome of such a study is the use of such mimotopes to tag target proteins, which allows them to be protein-specific probes as in the case of mimtag N1.

There are several advantages of this system over the conventional use of antibodies as protein specific probes. Mimtags can be stored over long periods of time without the fear of deteriorating and are cheaper to produce compared to monoclonal antibodies. They are relatively free of contaminants, such as toxic substances from expression systems.

Application based advantages include the high-throughput of peptide mimtags, which allows the simultaneous bombardment of the target antigen with several billion mimtags and this allows the interaction of the target protein with the best possible sequence of mimtag. No re- probing is required as in the case of antibodies because all immunoassays can be performed with the new mimtag as demonstrated with the M13 phage ELISA and direct ELISA.

Antibodies are difficult to produce employing cell-based and in vivo methods, not to mention, the purification of antibodies (368, 369), which are highly expensive methods and difficult to handle leaving very little margin for errors. The specificity is another factor that most scientists fail to report when using antibodies, which has to be determined first, making it ever more difficult to use in immunoassays and detection systems (370, 371). However, mimtags can conform 3-dimensional structures and can specifically bind and localize an epitope. Hence, we can propose that mimtags may eventually replace antibodies for their use in immunoassays and detection systems.

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Another important feature of using biotinylated mimtags is the spacer between the mimtag and biotin, which reduces steric hindrance to binding with the target epitope as opposed to a labelled antibody where conjugation reaction has shown to cause deleterious effects on antibody avidity (372) and in some cases, labelling index in antibody was negatively correlated with the binding affinity for its target antigen (373). A HRP-conjugated streptavidin protein can detect biotin on the mimtag with high affinity and avidity. This immunoassay was specifically conducted to test the efficacy and affinity of the newly synthesized biotinylated mimtag N1 to the IL-4RD (direct ELISA in Figure 2.3). The trend is clearly visible via the graphed readings where higher absorption readings are observed with increasing peptide concentrations. A Student’s t-test analysis of the results from the graph revealed a significant result when compared to the control (P < 0.05). This was an indication that the IL-4RD was bound at the bottom of the well and the N1 mimtag was interacting with the target.

7.3.4 Further Applications of Mimtag Technology

Apart from their ability to probe and map protein sequences, mimtags can potentially be used for probing and identifying gene sequences and clusters; using a phage library of exon-sized inserts (374-376). Mimtags can also be used for the detection of antibodies in a given sera which is by far an inexpensive, rapid, sensitive, specific and reusable method to detect a humanized therapeutic antibody (377). Phage displayed synthetic mimtags have also been used for biosensor analysis by fluorescently labelling the mimtags that could be detected onto a surface of a sensor chip designed for ligand interaction and replacing larger antibodies as probes (378, 379). Single point substitution in mimtag sequences can be performed to improve the avidity of the mimtag enhancing its bioactivity and interacting capability with the target epitope (380, 381). All of these applications are just a start into replacing larger

165 PhD Thesis – Chapter 7 Nayyar Ahmed antibodies with smaller mimtag protein-specific probes for the detection and labelling of key epitope regions on a given protein with higher specificity and low costs making it a more sophisticated and effective method.

7.4 Inhibition of STAT6 Phosphorylation Pathway in HEK-Blue Cells Using Peptides as

Antagonists

In Chapter 3, it was shown that peptides isolated from phage display showed profound inhibition of protein-ligand interaction. In the case of peptide N1, there was downregulation of IL-4 cytokine signaling of SEAP secretion from HEK-Blue cells which can be translated to be analogous to a decrease of IgE antibody production by B cells. Similarly, peptide K1 binding to cytokine IL-4 showed inhibition of IL-4 signaling suggesting a profound inhibition of SEAP secretion. Finally, P9 peptide isolated from phage display was used as an antagonist to IL-13 and reduced cytokine signaling and hence SEAP secretion. All of the above experiments conducted showed a >50% inhibition of signalling (discussed below in section

7.4.2). Peptide inhibition of biological factors has been studied previously with much success.

Many clinical studies involving peptide inhibition have been previously conducted at the cellular level as therapeutic agent of tumour growth (382-384). Hence, it is worthy to note that inhibition studies of allergy have not been conducted previously using a peptide antagonist for allergy treatment.

7.4.1 Preferential Use of Peptides as Antagonists

Many pharmacological companies believe that peptides are a menace because of their poor agile properties like poor tissue penetration, serum resistance and quick elimination.

However, recent studies suggest that such problems can be tackled with modification of the peptide while other proteins or molecules are more difficult to deal with, due to their larger

166 PhD Thesis – Chapter 7 Nayyar Ahmed sizes. There are potential benefits of using peptides as drugs, which come into play when dealing with protein-protein interactions (PPI’s). Binding pockets of proteins are sometimes small and unable to fit an antagonist large in size (antibodies or molecules) and hence, using peptides is a more viable option; ranging from two to fifty amino acids in length. Second advantage of using peptides is that it’s unlikely to cause any adverse immunological reactions in the subject since they are too small to render any physiological reaction. Thirdly, the chemical diversity found in peptides, as opposed to large molecules, allows more specific interaction with the protein, increasing the antagonist’s efficiency to penetrate the conformational structure of the protein (385, 386).

7.4.2 Importance of Inhibitory Values at 50% (IC50) for Inhibition Studies

It is highly important to elucidate the IC50 values in biochemical reactions and inhibitory studies. Many studies involve inhibition of regulatory receptors with antagonists achieving a

50% inhibition in in vitro and ex vivo applications (387-391). IgE antibody plays an important role in parasite immunity. Human platelets and eosinophils use IgE in vitro to kill blood fluke Schistosoma mansoni (290, 392). Infection with the parasite Trichinella spiralis is supplemented by intestinal mastocytosis and increased IgE responses. T. spiralis requires expulsion of adult worms and destruction of larval cysts that are deposited in muscle tissue. It was found that IgE accelerated the removal of worms from the intestine and reduced viability of larval parasites (393). These are some important functions of IgE antibody in the immune system (392). Hence, it is vital not to inhibit the IgE response in atopic individuals completely and down-regulate this vital antibody in immune reactions.

7.5 Further Analysis of n-3 and n-6 PUFA

In Chapter 4 of the thesis, we have investigated the effects of omega-3 and omega-6 fatty

167 PhD Thesis – Chapter 7 Nayyar Ahmed acids on IL-4 signaling in HEK-Blue cells. It is important to further elucidate and verify our results with previously published literature. In a study carried out by Weise et al., (2011), it was found that DHA inhibited IgE production by B lymphocytes, which are derived from human peripheral blood mononuclear cells. So far, the underlying mechanism is unknown.

Since this study was already established in the case of DHA, we tested other n-3 PUFA in addition to DHA to improve the overall picture of omega-3 and omega-6 fatty acids. It is also known that IL-4RD (CD124) is suppressed when B lymphocytes are incubated with n-3 fatty acids, which leads to a suppression of STAT6 phosphorylation pathway (158, 309). DHA,

EPA and DPA all behaved very similarly in the time course experiments in suppressing

STAT6 signaling in HEK-Blue cells. The JAK-STAT6 transcription factor subsequently activates gene expression in cell nucleus expression. Activity of protein kinases can also be altered by different fatty acids. A study carried out by VanMeter et al., (1994) suggested that

EPA and DHA can suppress the activity of protein kinase C (PKC) which leads to inhibition of STAT6 dimerization and gene suppression (394, 395). This is a possible explanation for less SEAP production by HEK-Blue cells. Furthermore, DHA did not exhibit a dose- dependent inhibition in the IL-13 detection assay but rather a dose-response inhibition. With the highest concentration of DHA, it was observed that inhibition was the highest. Literature suggests that high dose of DHA supplementation changes the outcome of IgE production from B cells in response to cytokine stimulation which may explain the sudden drop in SEAP secretion at 150 μM (158, 396).

7.5.1 Production of Inflammatory Prostaglandins

An important explanation of pro-inflammatory effects of AA can be the production of inflammatory prostaglandins from HEK-Blue cells. AA is found esterified at the sn-2 positions of membrane phospholipids. AA can be released by cytoplasmic phospholipase A2

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and converted by COX 1 and 2 to prostaglandin intermediates known as PGH2. Prostaglandin synthases then convert PGH2 to 2-series prostanoids known as PGE2, which are inflammatory. AA can also be converted to TX of the 2-series for e.g. PGD2) or by 5-, 12-, and 15-LOX which yield inflammatory LT of the 4-series. For the same reason, it was found that AA was suppressing IL-13 cytokine stimulation of HEK-Blue cells when tested with higher doses of AA. A study suggests that IL-13 cytokine suppresses COX-2 gene expression

(397). By contrast n-3 PUFA such as DHA and EPA can replace AA in cell membranes and can be converted by the same enzymes to anti-inflammatory intermediates such as the 3- series prostanoids known as prostaglandin E3 (PGE3) (309, 398, 399). In fact, EPA and DHA are poor COX substrates and are potent antagonist to COX activity. Also, gene expression of proteins involved in AA metabolism can be suppressed by the action of EPA and DHA (399).

All kidney derived cells are capable of producing the COX series of enzymes hence, incubating cells with fatty acids can alter gene expression through modification of transcription factor activity (400, 401). This may effect SEAP secretion from HEK-Blue cells. Although not part of this study, it is important to mention that PGE2 receptors regulate activation and differentiation of mouse B lymphocytes to IgE production (402).

7.6 The Missing Link: Inflammation and Allergy

169 PhD Thesis – Chapter 7 Nayyar Ahmed effects of pro-resolving metabolites such as resolvins in allergy. It is not far-fetched to hypothesize that resolvins may have a significant effect on allergy since they down-regulate inflammation or vice-versa. For this purpose, in Chapter 5 we have established that although resolvins are anti-inflammatory in their ability to suppress acute inflammation, it is worthy to note that they act as agonists in the case of IL-4 and IL-13 signaling in HEK-Blue cells with

SEAP secretion. Evidently, in the case of CoQ10 no previous studies have been conducted at the cellular level to establish a direct link of CoQ10 and allergy.

Omega-3 fatty acids have been implicated in allergy in the past few decades. Higher intake of omega-6 fatty acid combined with a low intake of omega-3 diet can subsequently result into allergy development in children and adults alike (160) (403). Resolvins are precursors of fatty acid that are produced as a result of acute inflammation (404). Resolvins derived from DHA and EPA have been shown to signal through specific receptors such as ChemR23, BLT1 and in the case of RvD1, with PMN transendoethilial migration (186). This consequently reduces neutrophil infiltration on the site of inflammation (177). Hence, it is important to not only assess PUFA in allergy but also the bi-products, which are produced as a result of allergic inflammation. For this purpose, we used the same model of HEK-Blue cells, which showed remarkable inhibition of STAT6 phosphorylation when using omega-3 fatty acids. It was hypothesized that since omega-3 fatty acids were able to reduce SEAP secretion in a remarkable fashion, resolvins would behave in a similar way. However, in the case of resolvins, it was shown that SEAP production from HEK-Blue cells was increased as a consequence of the treatments performed. However, in the case of CoQ10 it was shown that

SEAP was reduced even at lower concentrations, which have been established in previous studies (170, 172).

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7.7 Deakin AIRwatch Project

Grass pollen count is a highly effective technique to investigate the distribution of pollen in air per m3 of air (216, 227, 338, 405). In Chapter 6 of the thesis, we have used aerobiology to study and map distribution of ryegrass pollen in regional Victoria (Geelong) using a Burkard volumetric air trap. In addition, we have studied weather variables linked to grass pollen count. The purpose of this project was three-fold; firstly to get an accurate measure of ryegrass pollen per m3 of air, secondly to evaluate ruptured pollen which is caused by water contact and thirdly to analyze meteorological data in relation to intact and ruptured ryegrass pollen. Although this project is in the initial stages, it opens new vistas to study pollen distribution in regional Victoria where grass pastures are a common sight, suggesting a higher prevalence of pollen in the air (218, 219, 406). A growing body of evidence suggests that weather variables such as temperature affect the grass pollen in air. Wind gusts pick up pollen from dehisced anthers and carry them great distances, which results in allergic symptoms such as hay fever and asthma in genetically predisposed individuals. As the results in Chapter

6 indicate, we were able to show some correlation between weather variables and ryegrass pollen distribution. Higher temperatures, wind and rain are very important factors (219).

Higher temperatures lead to dehiscence of pollen anthers, which may remain intact on the anthers. However, wind causes the pollen to be lifted from the anthers and distributed in air

(349). Although rain has a wash-off effect on flowering plants (352), it is shown that water causes rupturing of pollen (349). The subsequent release of pollen cytoplasm and debris can become airborne due to wind and can be inhaled by allergic individuals (349). Since the size of the pollen is large enough not to penetrate lower airways of atopic individuals, the cytoplasmic debris particles are small enough to penetrate lower airways and cause severe allergic reactions. Hence, it is important to map pollen distribution to establish a link between pollen distribution and allergy. The overall aim of our project was to map pollen data that has

171 PhD Thesis – Chapter 7 Nayyar Ahmed been conducted for 3 consecutive years so far. However, a link between allergy and pollen distribution in regional Victoria remains to be established.

7.7.1 Limitations of the Deakin AIRwatch Project

Chapter 6 of the thesis discussed the importance of the Deakin AIRwatch project. However, it is important to note the disadvantages of our current system, which can be improved for future work and improve the baseline of our project. The disadvantages and future work is listed below:

a. Several disadvantages of Burkard volumetric trap are associated with poor

collection of data. The trap is sensitive to the size of the particles that are being

inhaled through the orifice of the trap. Some of the pollen collected can be adhesive-

sensitive and are unable to bind themselves to the adhesive being used. The method

used for pollen counting is time-consuming and subject to error during observation.

Also, since grass pollen appear similar when observing a slide under microscope, a

qualified person is needed to identify the pollen and spores. During rain periods, it

becomes increasing difficult to get an accurate idea of pollen distribution since they

are unable to be dispersed by wind. Hydrated anthers of pollen are inhibited from

anthesis or washing off effect (407). The static position of the pollen trap also adds to

the list of disadvantages since a primary location can only be setup without moving

the pollen trap to other surrounding areas to allow slight differences between

microclimates and topography (discussed below). Thus, making it important to install

multiple pollen traps at relevant distances to map out the variation between pollen

counts (408).

172 PhD Thesis – Chapter 7 Nayyar Ahmed

b. Another limitation of using daily counts is the study of hourly structures associated

with a 24 hr pollen count. The days can be broken into hours and observed on a slide

that would allow the study of weather variables and pollen distribution. This will

improve the correlation between meteorological data and grass pollen count

dispersion.

c. Perhaps the most important factor in increasing our understanding of relationship

between grass pollen distribution and weather variables is the length of time. Multiple

years and accumulated data are necessary to have an overall picture of the complex

variables and effects of meteorological conditions on pollen dispersal.

d. Difference in microclimates and ecozones can exist in a particular city causing

minor variations in weather variables such as temperature, wind speed, moisture and

rainfall (409). In our study, the location of the pollen trap was in Waurn Ponds,

Geelong. However, the location of the weather station is in Norlane, Geelong at a

distance of approximately 18 km from the location of the pollen trap. Also, the suburb

of Norlane is located 20 m above sea level as opposed to Waurn Ponds which is 75 m

above sea level. These minor differences in geography may have profound effects on

weather parameters. Perhaps this may reduce the chances of any anomalies that may

have resulted due to weather differences at these sites. Hence, it is important to install

a local weather station at the site of pollen trap.

e. Asthma and hay fever studies have been associated with the rise in grass pollen

count in Melbourne (Australia) during the pollen season (208, 213, 222, 410, 411).

Clinical data to measure the full extent of ryegrass pollen count correlated to allergy

173 PhD Thesis – Chapter 7 Nayyar Ahmed

sufferers can also be incorporated for future studies. This will allow in understanding

the effect of ryegrass pollen on public health and the costs associated with it on the

population of Geelong.

f. Another important investigation can be the correlation of ruptured ryegrass pollen

with clinical data. A rise in ruptured grass pollen may result in an increase in asthma

sufferers.

7.8 Future Directions

Future directions entail work that can be undertaken after the completion of all the experiments shown in Chapters 2-6. A list of all future directions can be found in the topics listed below.

7.8.1 Future Directions for IL-4Rα, IL-4 and IL-13 Inhibition

Chapter 2 and 3 discuss the possibility of using peptide antagonists as a novel method to down-regulate IgE secretion for allergy intervention. However, more work needs to be done to establish a thorough understanding of the complex nature of using phage display. A list of future work is itemized below:

a. The Ph.D. 12 library of phage display has proven to be very successful in a range

of experiments carried out. The NARL research group has used this technique to

identify 3 important peptides that bind to IL-4RD, IL-4 and IL-13 cytokines. We

have successfully established the effects of the peptide antagonists in vitro.

However, for future studies, the use of Ph.D. 7 library to identify peptide

analogues that can bind to proteins as antagonists. Ph.D 7 library has M13 phage

174 PhD Thesis – Chapter 7 Nayyar Ahmed

that display a hepta-peptide (7-mer) fused to the N-terminus of PIII sequence. The

length of the peptide is shorter than Ph.D. 12 (12-mer) library making it more

suitable to penetrate protein structures (explained in section 7.4.1).

b. A combination of peptide antagonists: for future studies, a combination of peptide

analogues can be used to look at the overall effect of inhibition assays on a HEK-

Blue cell-line.

c. Testing peptide analogues for future studies: peptide analogues can be produced

using Fluorenyl-methyl-oxycarbonyl (Fmoc) chemistry (412, 413), a routinely

performed technique, on PEG polysterene resin. Couplings can be performed

using O-(Benzotriazol-1-yl)-N,N,N’,N’-tetramethyluronium hexafluorophosphate

(HBTU) and Hydroxybenzotriazole (HOBt) peptide coupling reagents. Further,

peptide purity can be established using HPLC and identity can be confirmed using

mass spectrometry and amino acid analysis. Preparing peptide analogues involves:

d. Alanine scan: amino acid residues can be sequentially replaced in the peptide

inhibitor with Alanine to identify specific amino acid residues responsible for the

peptide’s activity and has been previously performed by our lab (211). This

molecular dissection technique will allow identification of residues with the

highest binding ability to the target IL-4Rα.

e. Truncation studies: This involves systematically removing flanking amino acid

residues from the N- and C-terminus of the peptide, one amino acid residue at a

time (414). This method allows the successful identification of the shortest

sequence needed for activity of the peptide that will bind to the IL-4Rα/IL-13Rα1

and inhibit its interaction with IL-4 and IL-13.

175 PhD Thesis – Chapter 7 Nayyar Ahmed

Analogues prepared using these techniques can allow the development of a very refined form of the peptide inhibitor, which will bind with the target with the minimum amino acid residues but maximum binding capacity.

f. Further analysis using a biosensor: The NARL research group has recently

purchased a biosensor (General Electric Co.). However, due to time-

constraints we were unable to perform experiments using the biosensor. For

future studies, cytokine-receptor interaction experiments can be conducted

either with or without pre-incubation of the chip-immobilized IL-4Rα with the

different concentrations of the peptide analogues, to assess the inhibitory

capacity of the peptides. Kinetic parameters can be calculated using the

Biacore evaluation software.

g. Ramos.2G6.4C10 or Burkitt’s lymphoma B cell line: In a series of

experiments carried out with cell-lines, post-completion of experiments with

HEK-Blue cell model, another cell model can be used to carry out the same set

of experiments. Study with Ramos B cells will give a comparison between

recombinant IL-4RD found in HEK-Blue cells as opposed to natural IL-4RD

found in human Ramos B cells.

h. In vivo testing: The peptide inhibitors can finally be tested in an animal model

system. Tissue and blood serum samples will be taken from a mouse-model

post-allergen challenge and tested for the peptide’s ability to block the allergic

pathway. Following this, the peptide’s ability to inhibit cytokine signaling can

be tested among human subjects in clinical trials.

176 PhD Thesis – Chapter 7 Nayyar Ahmed

7.8.2 Future Directions for Fatty acid, CoQ10 and Resolvins

Currently, our work with omega-3 and omega-6 fatty acids was to evaluate their effects on

STAT6 signaling pathway in HEK-Blue cells. We established our results based on a range of concentrations of DHA, EPA, DPA and AA, along with vitamin E, to test the hypothesis that fatty acids reduce IgE production by inhibiting IL-4/IL-4R interaction in HEK-Blue cells. In the time-course experiment, the most optimum concentration of fatty acid was used to establish the effects of fatty acid on STAT6 signaling using time as a variable. At the indicated time points, different concentrations of DHA and EPA will be incubated with the cells. The results generated as a result of these experiments opens new vistas for research of fatty acids and their correlation to allergy. A study carried out by Kitz et al., (2010) demonstrated the effect of erythrocyte membrane n-3 fatty acid (DHA) in response to pollen challenge among asthmatic patients. It was shown that ratios of n-3/n-6 fatty acid were significantly lower in patients suffering from bronchial asthma. Hence it was concluded that pharmacological use of n-3 PUFA as an intervention for allergy studies could be established for the treatment of allergy (415). Hence, for our future studies, it can incorporate the effects of various fatty acids such as EPA, DPA and AA in response to allergen challenge in patients or perhaps look at the effects on IgE production from B cells. A combination of fatty acids can also be used as treatments in a HEK-Blue model. A study carried out by Weise et al.,

(2011) showed that treatment of ovalbumin sensitive mice with a combination of dietary

DHA and AA diminished the symptoms of allergen-induced dermatitis (416). Thus, it can be concluded that although fatty acids behave differently when used individually for research, they may have synergistic effects when used in combination with other fatty acids. Perhaps our future study can include a range of different combinations of fatty acids and their effect on HEK-Blue cells. Although our study showed that AA induces SEAP secretion in HEK-

Blue cells, further study will elucidate the combinatorial effect of fatty acid at the cellular

177 PhD Thesis – Chapter 7 Nayyar Ahmed level. All of the above strategies can be used in the case of CoQ10 and resolvins as novel antagonists for allergy treatment. For example, since CoQ10 had inhibitory effect on SEAP secretion, it can be used in combination with n-3 fatty acids as a dietary supplement to reduce allergy. A similar pathway for fatty acid, resolvins and CoQ10 can be performed as shown in section 7.8.1. In vivo testing can be undertaken with the range of concentrations using an animal model followed by testing in human subjects.

7.8.3 Pollen Sampling

For pollen sampling, all of the limitations listed in section 7.7.1 must be rectified to enhance the results for aerobiological studies.

------X------X------

THE END

178 PhD Thesis – References Nayyar Ahmed

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