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Functional and Transcriptional Characterisation of Synovial Fibroblasts in Early Inflammatory Arthritis and Established Rheumatoid Arthritis

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Functional and Transcriptional Characterisation of Synovial Fibroblasts in Early Inflammatory Arthritis and Established Rheumatoid Arthritis Functional and transcriptional characterisation of synovial fibroblasts in early inflammatory arthritis and established rheumatoid arthritis By María Jesús Juárez Pérez A thesis submitted to The University of Birmingham For the degree of DOCTOR OF PHILOSOPHY School of Immunity and Infection College of Medical and Dental Sciences The University of Birmingham August 2014 University of Birmingham Research Archive e-theses repository This unpublished thesis/dissertation is copyright of the author and/or third parties. The intellectual property rights of the author or third parties in respect of this work are as defined by The Copyright Designs and Patents Act 1988 or as modified by any successor legislation. Any use made of information contained in this thesis/dissertation must be in accordance with that legislation and must be properly acknowledged. Further distribution or reproduction in any format is prohibited without the permission of the copyright holder. ABSTRACT A pathogenic role for synovial fibroblasts has been established based on the study of samples originating from patients with longstanding rheumatoid arthritis (RA). To assess whether these cells also have such role in early RA and whether they play a part in the resolution of inflammatory arthritis, functional and transcriptional characterisation was performed on synovial fibroblasts from patients in five distinct outcome groups: normal joints, resolving arthritis, very early RA, early RA and longstanding RA. Functional characterisation revealed differences in migration rates between groups. Migration rates of very early RA and longstanding RA synovial fibroblasts were significantly slower than those of normal ones. No differences in invasive characteristics were identified. Transcriptional analysis demonstrated differing transcriptional signatures between very early and longstanding RA synovial fibroblasts in both, unstimulated and TNF stimulated samples. No differences in the transcriptomic profile of resolving and very early RA cells were identified. The study of transcriptional responses to TNF stimulation in each outcome group revealed generic changes in response to pro-inflammatory stimuli as well as outcome group- specific responses. These data extend previous observations of the pathogenic role of synovial fibroblasts indicating that they may play a role in early RA and providing clues to potential targets differentiating early and late RA. i To my parents ii “The three great essentials to achieve anything worthwhile are hard work, stick-to-itiviness and common sense” Thomas A Eddison iii ACKNOWLEDGMENTS Many people deserve my thanks for their contribution to this thesis. My supervisor, Andrew Filer, has been crucial to the completion of this project. He has challenged me and allowed me to become an independent researcher whilst providing direction and guidance. He also supported me during very difficult personal times and for that I will always be in his debt. The arrival of Andy Clark to Birmingham marked a step change in my project and enabled it to become what it is. I am very grateful to Andy and his team that took me under their wing and helped me navigate the complexities of molecular biology. My thanks to Chris Buckley who gave me the opportunity of doing a basic science PhD, despite not having any lab experience. Chris has always had time, a smile and words of wisdom for me. Many thanks to Karim Raza who has always had an open door and has been the source of support and inspiration. Past and present members of the Rheumatology Research Group deserve my thanks for their fantastic technical support, stimulating scientific conversations and copious amounts of cake and good humour. In particular, I would like to acknowledge the contributions of Holly Adams, Tim Smallie, Kath Bignal, Tina Tang, Martin Fitzpatrick, Dagmar Scheel-Toellner, Mark Pearson, Lorraine Yeo, Helen McGettrick, Debbie Hardie, Beth Clay, Rachel Bayley, Banesa de Paz, Eva Li and Julia Spengler. My special thanks to Holly, Martin and Beth for filling me with sweets and cups of tea, always answering my questions with a smile and most importantly, making my every day great. My thanks also to my collaborators in this project Mark Cooper, Rowan Hardy, Paul Badenhorst and Steve Kissane. Thanks to Lyn Williamson, Elizabeth Price, David Collins and Robert Marshall for their career guidance and continued support. I would like thank my parents who, amongst many other things, have taught me the value of hard work and things well done. They love me more than I will ever be able to express and have given me the most precious gift: my siblings. Thanks to Dev for being a source of happiness, love and support, driving for miles so that we could live together and making me smile every single day. The patients that contributed to this study deserve special gratitude. I would also like to acknowledge the support of the Wellcome Trust in funding my Clinical Research Training Fellowship. When I embarked on this project, having worked as a clinician for years, some of my good friends and colleagues warned me I might not like this job. The pace is markedly different to clinical work and the challenges posed of a different nature. They need not have worried. I can honestly say that I have loved my time here and have found my career passion. iv TABLE OF CONTENTS 1 Introduction ................................................................................................................................................. 1-1 1.1 Rheumatoid arthritis.................................................................................................................................... 1-1 1.1.1 Classification of RA .......................................................................................................................... 1-1 1.1.2 Pathophysiology ................................................................................................................................ 1-4 1.1.3 Changing concepts: early disease and the window of opportunity .................................................. 1-19 1.1.4 Pre-clinical rheumatoid arthritis and disease chronology ................................................................ 1-23 1.2 The role of synovial fibroblasts in rheumatoid arthritis ............................................................................ 1-24 1.2.1 RA synovial fibroblasts as mediators of persistent inflammation ................................................... 1-27 1.2.2 RA synovial fibroblasts as mediators of cartilage and bone destruction ......................................... 1-29 1.2.3 RA synovial fibroblasts and synovial hyperplasia ........................................................................... 1-32 1.2.4 Signalling pathways ........................................................................................................................ 1-34 1.2.5 Known transcriptional profile.......................................................................................................... 1-36 1.2.6 Unanswered questions: the study of early inflammatory arthritis and the comparison between early and established RA ........................................................................................................................................ 1-37 1.3 Hypothesis and aims ................................................................................................................................. 1-39 2 Patients, materials and methods ................................................................................................................ 2-41 2.1 Patient cohorts, sample collection and selection ....................................................................................... 2-41 2.1.1 Patient and outcome group selection ............................................................................................... 2-41 2.1.2 Patient cohorts ................................................................................................................................. 2-42 2.1.3 Sample collection, coding and validation of phenotype .................................................................. 2-52 2.1.4 Testing of long term cultures for infection ...................................................................................... 2-53 2.2 Cell culture ................................................................................................................................................ 2-54 2.2.1 Tissue culture reagents .................................................................................................................... 2-54 2.2.2 Medium ........................................................................................................................................... 2-55 2.2.3 Primary cell cultures ........................................................................................................................ 2-55 2.2.4 Cell line maintenance in culture ...................................................................................................... 2-56 2.2.5 Fibroblast trypsinisation (splitting) ................................................................................................. 2-56 2.2.6 Freezing, storage and recovery of samples ...................................................................................... 2-56 2.2.7 Passage number of cells used .........................................................................................................
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