A Study of Classical and Novel Markers of Disease in Multiple Sclerosis By Ghaniah Z. Hassan-Smith A thesis submitted to the University of Birmingham for the degree of DOCTOR OF PHILOSOPHY (PhD) IN MEDICINE School of Infection and Immunity College of Medical and Dental Sciences University of Birmingham February 2015 University of Birmingham Research Archive e-theses repository This unpublished thesis/dissertation is copyright of the author and/or third parties.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 Multiple sclerosis (MS) is a chronic inflammatory and degenerative condition of the Central Nervous System. Focal demyelinating lesions are its neuropathological hallmark, but widespread abnormalities found in otherwise “normal-appearing” tissue are better associated with disability outcomes. HMGB1 is a promiscuous sensor of cellular stress, acting as a link between sterile damage and innate immune mechanisms, with its extra-nuclear release producing diverse outcomes. We report novel findings of significantly increased HMGB1 expression throughout the brain tissue of MS vs. non-MS patients, particularly in macrophages/microglia and oligodendrocytes (OGD). In addition, cerebrospinal fluid HMGB1 levels were increased in early-stage MS patients compared to non-inflammatory control patients. HMGB1 stimulation in-vitro upregulates expression of its receptors in an OGD cell line, potentially propagating chronic inflammation. Expression of the Leucine Rich Repeat and Ig-domain-containing molecules, AMIGO-3 and LINGO-1 is also significantly increased by HMGB1 stimulation in-vitro. These molecules demonstrate particularly intense immunoreactivity in human brain tissue taken at biopsy, at an early disease stage. Thus, exogenous HMGB1 may influence neurodegenerative processes via AMIGO-3 and LINGO-1 and blocking their function could have therapeutic value. Increased expression of HMGB1 in OGD, however, may highlight endogenous neuroprotective mechanisms in response to an unknown trigger. ii For Zaki and Levizah iii Acknowledgements I extend sincere thanks to my supervisors, Dr Mike Douglas and Dr John Curnow, for their guidance and support over the period of study. The project would not have been possible without John’s involvement and I am deeply indebted to him for this, and his mentorship. I am also grateful to Mike who, along with Dr Gordon Mazibrada, created this research post and provided an excellent learning environment. In addition, I benefitted greatly from Mike’s breadth of knowledge in both scientific technique and clinical neurology and his respect for intellectual freedom was much appreciated. I am also most grateful to Dr Gordon Mazibrada and Dr John Woolmore for the excellent training in clinical MS. Gordon was incredibly supportive in all aspects of the project, in particular pursuing all educational opportunities. Heartfelt thanks also to Dr Martyn Carey and Dr Santhosh Nagaraju for their patience in dealing with my (many) neuropathological queries, despite ever-present clinical commitments. Thanks also to Alex Sinclair for her support and mentorship. Many thanks to the UK MS Brain Bank, Hammersmith Hospital and in particular to Professor Richard Reynolds and Dr. Djordje Gveric. Professor Margaret Esiri provided additional control tissue from the Thomas Willis Oxford Brain Collection, and I am most grateful to her for that. Many thanks also to Dr Peter Nightingale who was most helpful in reviewing statistical aspects of the project. Sincere thanks to Jane Steele’s fantastic team at the HBRC; I have immense respect for Souad Messahel and Johanna Dieguez Navas, who went out of their way to help with the project. In addition, thanks to Lindsay Durant, Matt Edmunds, Marie Voice, Robert iv Barry, Paul Tomlins and Seema Kalra from the Curnow Lab for their much- appreciated input on our collective ‘journey’. The project was only written up due to the help of a stellar supporting cast. Many thanks to my mother and father-in-law who were so generous with their time and help. My wonderful sister and her family offered unwavering support for which we are truly grateful. I would not have produced this document at all without the practical help of my parents and I sincerely thank them for this. More importantly, I thank them for their wisdom, kindness and inspiration, which have influenced me throughout my life. This thesis is wholly dedicated to Zaki and to Levizah, for being the light of our lives. v Publications arising from the thesis 1. High sensitivity and specificity of elevated cerebrospinal fluid kappa free light chains in suspected multiple sclerosis. Hassan-Smith G, Durant L, Tsentemeidou A, Assi LK, Faint JM, Kalra S, Douglas MR, Curnow SJ. J Neuroimmunol. 2014 Nov 15;276(1-2):175-9. Conference proceedings 1. High-mobility Group Box 1 (HMGB1) Expression Is Increased In The Normal-appearing Brain Tissue Of Multiple Sclerosis (MS) Patients Vs. Controls. Ghaniah Z. Hassan-Smith et al. E-poster oral presentation at ACTRIMS/ ECTRIMS, Boston, September 2014 2. The expression pattern of High-mobility group box 1 (HMGB1) in the normal-appearing brain tissue of Multiple Sclerosis (MS) patients and controls. Ghaniah Z. Hassan-Smith, Martyn Carey, Santhosh Nagaraju, Souad Messahel, Gordon Mazibrada Ana-Marie Gonzalez, S. John Curnow and Michael R. Douglas. Association of British Neurologists, May 2014 vi Table of Contents Chapter 1. General Introduction ............................................................................................................... 1 vii Chapter 2. General Methods ...................................................................................................................... 58 2.1.1 MO3.13 cell line ................................................................................................................. 59 2.1.2 Cell proliferation ............................................................................................................... 60 2.1.3 Freezing down cells ......................................................................................................... 61 2.2.1 RNA quantification ........................................................................................................... 61 viii Chapter 3. Characterisation of early active lesional changes in a patient with clinically isolated syndrome, using classical and novel markers. ........................................ 72 ..................................................................... 77 3.4.2 Analysis ................................................................................................................................ 77 .......................................................................................................................... 78 .......................................... 80 ............................................................................................................................ 83 3.5.4 HMGB1 immunoreactivity is evident in peri-lesional regions ……….....……83 3.5.5 HMGB1 immunoreactivity persists into non-lesional region …...…………....86 3.5.6 Cortical regions distal to white matter lesions demonstrate specific HMGB1-positive expression patterns .................................................................................... 88 ix Chapter 4. Post-mortem study of HMGB1 expression patterns in MS and non-MS control patients…..………………………………………………………................………........105 4.1 Introduction….…………………………………………………………………………………….......…106 4.2 Aims……………………………………………………………………………………………..……….……108 4.3 Hypothesis.. .................................................................................................................................... 108 4.5 Statistical analysis……………………………………………………………………………………....116 4.6 Results………………………………………………………………………………………………………..117 4.6.1 HMGB1 expression is increased in active lesions from patients with progressive MS compared to non-MS controls……………………………………….……..117 4.6.2 HMGB1 expression is increased in non-lesional white matter compared to non-MS controls…………………………………………………….……………………………………118 4.6.3 HMGB1 expression is increased in non-lesional grey matter compared to non-MS controls………………………………………………………………………………………….121 4.6.4 HMGB1 expression is decreased in demyelinated vs. myelinated regions of the brain……………………………………………………………………………………………………..126 4.6.5 CD68-positive cells are increased in normal-appearing white matter (NAWM) and active lesional tissue blocks in MS patients……………………………...128 x 5.1 Introduction ................................................................................................................................... 145 5.1.1 CSF biomarkers in MS .................................................................................................... 146 5.5 Statistical analysis………………………………………………………………………………………158 xi Chapter 6. Exploration of the relationship between LRRIg molecules and HMGB1 in MS……….………………………………….…………………………………………....………………….…………….181 6.1 Introduction…..…………………………………………….………………………………….………….182 6.6.1 HMGB1 is expressed in the MO3.13 cell line………………….192 6.6.2 Stimulation of MO3.13