gpr40 expression and function in immune cells and experimental arthritis

208 Pages·2017·9.26 MB·English

GPR40 E I
XPRESSION AND FUNCTION IN MMUNE
C E A
ELLS AND XPERIMENTAL RTHRITIS

Mrs Patricia Regina Soares de Souza

Submitted in partial fulfilment of the requirements of the Degree of Doctor of
Philosophy

Centre for Biochemical Pharmacology
William Harvey Research Institute
Barts and the London School of Medicine and Dentistry
Queen Mary University of London
Charterhouse Square, London, EC1M 6BQ

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I, Patricia Regina Soares de Souza, confirm that the research included within this
thesis is my own.

I attest that I have exercised reasonable care to ensure that the work is original, and
does not to the best of my knowledge break any UK law, infringe any third party’s
copyright or other intellectual Property Right, or contain any confidential material.

I accept that the College has the right to use plagiarism detection software to check
the electronic version of the thesis.

I confirm that this thesis has not been previously submitted for the award of a degree
by this or any other university.

The copyright of this thesis rests with the author and no quotation from it or
information derived from it may be published without the prior written consent of the
author.

Signed:

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“The important thing is not to stop questioning.
Curiosity has its own reason for existing.”

Albert Einstein
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ABSTRACT

Omega-3 fatty acids (ω-3 FA, including eicosapentaenoic acid [EPA] and
docosahexaenoic acid [DHA]), are essential polyunsaturated fatty acids which are
correlated with lower incidence of chronic diseases. DHA and EPA can be
enzymatically converted to resolvins, protectins and maresins, which play important
roles in resolution of inflammation. Additionally, ω-3 FA can also directly activate
surface receptors, namely the long-chain free fatty acid receptors GPR40 and
GPR120, two GPCRs with poorly investigated biology.
Using real-time PCR analysis, GPR40 transcript in human neutrophils was
detected; the protein expression was also confirmed by flow cytometry and image
stream analysis. Expression of GPR40 protein was up-regulated after stimulation with
platelet-activating factor (PAF, 10nM) or leukotriene B (LTB , 10nM) for 10 minutes.
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I utilised the selective agonist GW9508 to investigate the biology of GPR40. Tested
on human neutrophils, GW9508 elevated intracellular calcium when applied within
the 0.1-10µM range. The up-regulation of GPR40 expression by pro-inflammatory
stimuli suggested to us potential regulatory roles for this receptor during inflammation.
I then showed that 1 and 10µM GW9508 increased neutrophil chemotaxis in response
to the cytokine IL-8 (30ng/ml). In addition, GPR40 activation by GW9508 enhanced
phagocytosis of E. coli by human neutrophils by approximately 50% when tested at
0.1 and 1µM. Moreover, GW9508-neutrophil stimulation augmented microvesicle
release and delayed apoptosis after stimulation.
Finally, I demonstrated that GPR40 is expressed in inflammatory cells isolated
from murine arthritic joints, such as neutrophils, macrophages and inflammatory
monocytes. KBN-serum induced arthritic mice developed a more severe disease
when treated prophylactically with GW9508 (10mg/kg, i.p. treated from day 0, daily),
characterized by a higher clinical score and increased oedema when compared to
ABSTRACT
vehicle control mice. Therapeutic intervention with GW9508 at the peak of the disease
(day 5) delayed the resolution of arthritis.
In summary, the data suggest that activation of GPR40 by GW9508 enhances
neutrophil activation, up regulating the pro-inflammatory properties of this cell type,
and therefore, exacerbating experimental inflammatory arthritis.

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ACKNOWLEDGEMENTS

I gratefully acknowledge the opportunity, support and guidance of Prof Mauro Perretti.
Without his thoughtful encouragement and careful supervision this thesis would never
have taken shape. I am also grateful to my other supervisor Dr Lucy Norling for her
continuous optimism, enthusiasm, encouragement and support.

My thanks also go out to the financial support provided by the program Science
Without Borders of the Brazilian Government. To all staff members of the William
Harvey Research Institute, especially for Martin Goss and Silvia Ayguade. I am
thankful for the members of the Pathology Department, Flow Cytometry Department
and Genome Center Departments. I would like to thank all members of the BSU in
Chapterhouse Square and Whitechapel. I also would like to thank the midwives of
The Royal London Hospital for kindly help us with the collections of umbilical cords.
In addition, a big thanks to all the blood donors.

I also thank the examiners for accepting the invitation, their time spent analysing
this thesis, and in advance, for the discussion and criticism that will be pointed
during the Viva, I’m sure I’ll learn a lot.

I am forever thankful to my colleagues at Biochemical Pharmacology Center for their
friendship and support, and for creating a cordial and funny working environment,
especially the old and new colleagues in the PhD Student’s office Chiara, Hanna,
Hazem, Hefin, Laura, Louise, Racheal, Sarah and Urszula. I thankfully acknowledge
the contributions of Dr Dianne Cooper in several different situations. I would like to
thank Dr Oliver Haworth for helping me with flow cytometry analysis. I would also like
to thank Hefin Rhys with the help with Image Stream.
ABSTRACT
Finally, I extend my deepest thanks to my husband Simone for the cooked meals
every night, his understanding in spending weekends at home, his patience and love.
Thanks to my parents Valter and Aparecida for the support when their daughter
decided to cross the ocean in search for another challenge. Thanks to my brother
Adriano which inspired me as a scientist. And thank you to all my sisters Adriana, Ana
Elisa and Debora for being always there. “Quando a saudade aperta, é a lembrança
dos bons momentos juntos que me dá forças pra seguir em frente”.

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TABLE OF CONTENTS

Abstract ................................................................................................................... 4
Acknowledgements .................................................................................................. 6
Table of Contents .................................................................................................... 8
List of Figures ........................................................................................................ 12
List of Tables ......................................................................................................... 14
List of Abbreviations .............................................................................................. 15
CHAPTER 1: INTRODUCTION .................................................................................. 21
1.1. The Inflammatory Response .................................................................. 22
1.1.1. General aspects of the inflammatory response ....................................... 22
1.1.2. Mediators of inflammation ...................................................................... 26
1.1.3. Major steps of the inflammatory response .............................................. 30
1.2. Rheumatoid Arthritis ............................................................................... 41
1.2.1. Introduction ............................................................................................ 41
1.2.2. Aetiology of RA ...................................................................................... 42
1.2.3. Pathogenesis of RA ................................................................................ 43
1.2.4. Experimental models of Arthritis ............................................................. 45
1.3. Free Fatty Acid Receptors and Immune System..................................... 50
1.3.1. Introduction ............................................................................................ 50
1.3.2. Overview of FFAs and FFARs ................................................................ 50
1.3.3. A family of GPCRs for Free Fatty Acids .................................................. 52
1.3.4. Fatty acids in Rheumatoid Arthritis ......................................................... 56
1.4. Scope of the thesis ................................................................................. 58
1.4.1. Hypothesis ............................................................................................. 58
CHAPTER 2: MATERIAL AND METHODS ................................................................... 60
Materials ................................................................................................................ 61
2.1. Cell Culture ............................................................................................ 61
TABLE OF CONTENTS
2.2. Flow Cytometry ...................................................................................... 61
2.3. Molecular biology reagents ..................................................................... 63
2.4. Other reagents and materials ................................................................. 63
2.5. Software and equipment ......................................................................... 64
Methods ................................................................................................................. 65
2.6. Isolation and culture of primary human umbilical vein endothelial cells
(HUVEC) 65
2.7. Human blood leukocyte isolation ............................................................ 66
2.8. Collection of Exudated Human PMN ...................................................... 69
2.9. In vitro culture of chondrocytes ............................................................... 70
2.10. Modulation of GPR40 expression in neutrophils and macrophages ........ 71
2.11. Gene expression analyses ..................................................................... 72
2.12. Flow cytometry analysis ......................................................................... 77
2.13. Imaging flow cytometry ........................................................................... 81
2.14. Intracellular Calcium measurement ........................................................ 83
2.15. PMN chemotaxis assay .......................................................................... 83
2.16. Flow chamber assay .............................................................................. 85
2.17. Phagocytosis Assay ............................................................................... 87
2.18. Measurement of apoptosis in neutrophils ............................................... 87
2.19. Mice ....................................................................................................... 91
2.20. K/BxN Serum Transfer Arthritis .............................................................. 91
2.21. Isolation of cells of the paws for flow cytometry analysis ........................ 93
2.22. GPR40 expression in cells from arthritic joints ........................................ 93
2.23. Gene expression in cells isolated from arthritic joints ............................. 94
2.24. Histological staining and analysis ........................................................... 95
2.25. Statistical analyses ................................................................................. 99
CHAPTER 3: RESULTS ......................................................................................... 100
Expression and Modulation of GPR40 ................................................................. 101
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TABLE OF CONTENTS
3.1. Genomic expression of GPR40 ............................................................ 101
3.2. Expression of GPR40 in human neutrophils ......................................... 102
3.3. Expression of GPR40 in macrophages ................................................. 109
Functionalities of GPR40 in Human Leukocytes .................................................. 112
3.4. GPR40-induced intracellular calcium mobilization in neutrophils .......... 112
3.5. Modulation of PMN adhesion molecules by GW9508 ........................... 113
3.6. Effect of GW9508 on neutrophil-endothelial interactions under flow ..... 115
3.7. Effect of GW9508 on neutrophil chemotaxis ......................................... 117
3.8. Effects of GPR40 agonist GW9508 on phagocytosis of E. coli ............. 118
3.9. Release of microvesicles induced by GW9508 in neutrophils ............... 120
3.10. Effects of GW9508 induction on neutrophil apoptosis. .......................... 122
3.11. M1/M2 polarization of macrophages by GW9508 ................................. 125
3.12. Effects of GW9508 stimulation in efferocytosis ..................................... 126
Roles of GPR40 In Vivo ....................................................................................... 130
3.13. Expression of GPR40 in cells from murine arthritic joints ...................... 130
3.14. Effects of GW9508 in the initiation and progression of inflammatory
arthritis 132
3.15. Effects of GW9508 treatment during the resolution phase of inflammatory
arthritis 140
CHAPTER 4: DISCUSSION OF RESULTS ................................................................. 142
CHAPTER 5: CONCLUSION AND FUTURE PLAN ...................................................... 154
CHAPTER 6: REFERENCES .................................................................................. 158
CHAPTER 7: ATTACHMENTS ................................................................................. 173
Attachments ......................................................................................................... 174
7.1. Implications for eicosapentaenoic acid- and docosahexaenoic acid-
derived resolvins as therapeutics for arthritis ....................................................... 175
7.2. Neutrophil-derived microvesicles enter cartilage and protect the joint in
inflammatory arthritis ........................................................................................... 184
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