This electronic thesis or dissertation has been downloaded from the King’s Research Portal at https://kclpure.kcl.ac.uk/portal/ IDENTIFYING THE EFFECT OF IRON CHELATION ON HUMAN CD4+ T CELLS Tewari, Damini Awarding institution: King's College London The copyright of this thesis rests with the author and no quotation from it or information derived from it may be published without proper acknowledgement. END USER LICENCE AGREEMENT Unless another licence is stated on the immediately following page this work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International licence. https://creativecommons.org/licenses/by-nc-nd/4.0/ You are free to copy, distribute and transmit the work Under the following conditions: Attribution: You must attribute the work in the manner specified by the author (but not in any way that suggests that they endorse you or your use of the work). Non Commercial: You may not use this work for commercial purposes. No Derivative Works - You may not alter, transform, or build upon this work. Any of these conditions can be waived if you receive permission from the author. Your fair dealings and other rights are in no way affected by the above. Take down policy If you believe that this document breaches copyright please contact [email protected] providing details, and we will remove access to the work immediately and investigate your claim. Download date: 04. Oct. 2021 IDENTIFYING THE EFFECT OF IRON CHELATION ON HUMAN CD4+ T CELLS Damini Tewari A thesis submitted to King’s College London, University of London in part fulfillment of the requirements for the degree of Doctor of Philosophy King’s College London April 2015 ABSTRACT The importance of iron in physiological and metabolic pathways is well established in the literature. However, the literature is equally rife with studies highlighting the association of iron with cellular toxicities such as production of Reactive Oxygen Species and even the growth of intra-cellular pathogens. This study was based on the observation that in conditions of chronic inflammation, such as Rheumatoid Arthritis, cellular sequestration of iron leads to increased cell proliferation and tissue damage. The inability of the human body to physiologically excrete this excess iron highlights the need to develop a cheap yet effective iron chelator. Results indicate that CP655, an HPO iron chelator, specifically targets human CD4+ T cells and causes significant inhibition of proliferation of the cells along with significantly inhibiting production of inflammatory cytokine such as IFN-γ and IL- 17. A microarray conducted on stimulated human CD4+ T cells demonstrated that the genes most significantly modulated by iron chelation were those encoding for cell cycle regulators and inhibitors of DNA replication, hence preventing cell proliferation, as indicated by the in-vitro data. This was validated by cell cycle analysis. Finally, mechanistic experiments revealed that the chelator may be causing an up-regulation of the cell cycle inhibitor protein CDKN1A (p21) as one of its molecular mechanism of action. Since the depletion of iron can cause dysregulation of several cellular pathways, some of the other potential mechanisms suggested in this study include the reduction in STAT5 phosphorylation following CP655 treatment and inhibition of T cell Receptor downstream signaling molecules. In conclusion, this study demonstrates that intracellular iron has the ability to modulate CD4+ T cell responses and indicates that the use of novel iron chelators could prove to have therapeutic potential for RA and other diseases driven by excessive T cell proliferation. 2 ACKNOWLEDGEMENTS This thesis represents the most important accomplishment of my career so far which would not have been accomplished without the help, guidance and encouragement of my supervisors, Dr. Helen Collins and Dr. Stephen Thompson. I am extremely grateful to them for providing me the opportunity to fulfil my dream and for their faith in my abilities. I would like to whole heartedly express my gratitude to them for all their patience, trust and support over the past few years. I thank my committee members, Prof. Andrew Cope, Prof. Andrew McKie and Dr. Stuart Neil for their guidance. Thank you to my lab members specially Ahmad Gazali, Katie-Lloyd Jones and Frank Kaiser for constantly helping me learn and grow as a scientist. I would like to sincerely thank Dr. Susan John and Dr. David Cousins for their help and advice throughout this project. I would also like to express my sincere gratitude to King’s College London for funding my PhD and for providing me an excellent environment to hone my skills and personality, Even though a few lines are not sufficient to express how I feel, I would like to thank my parents and my sister for letting me dream and helping my dreams come true. Their love and support has been my strength throughout the past few years. A special thank you to my grandfather, Prof. P.C.Gururani and grandmother, Indira Gururani. Last but not the least, I would like to thank my fiancé, Ankit Gaur, who has supported me, in every way possible, throughout these past few years. I owe everything I have achieved so far to these people. 3 DECLARATION I declare that I have personally prepared this report and that the work described is my own unless otherwise stated. All sources of information have been acknowledged by means of references. Damini Tewari April 2015 4 TABLE OF CONTENTS ABSTRACT................................................................................................................2 ACKNOWLEDGEMENTS.......................................................................................3 DECLARATION........................................................................................................4 TABLE OF CONTENTS...........................................................................................5 LIST OF FIGURES..................................................................................................10 LIST OF TABLES....................................................................................................13 LIST OF ABBREVIATIONS..................................................................................14 1. INTRODUCTION..............................................................................................18 1.1 General Introduction: Iron in Biological Processes........................................19 1.2 Iron Regulation and Storage...........................................................................21 1.3 Uptake of Dietary Iron....................................................................................24 1.3.1 Non-Haem iron uptake.......................................................................24 1.3.2 Haem - iron uptake.............................................................................25 1.3.3 Regulation of absorbed dietary iron...................................................26 1.4 Regulation via Hepcidin.................................................................................27 1.5 Transport of Iron.............................................................................................28 1.5.1 Transferrin-receptor mediated uptake................................................28 1.5.2 Non-Transferrin bound Iron (NTBI)..................................................29 1.6 Disorders of Iron Transport and Metabolism.................................................30 1.6.1 Iron Deficiency Anaemia (IDA).........................................................30 1.6.2 Anaemia of Chronic Disease..............................................................31 1.7 Iron, Immunity and Inflammation..................................................................34 1.7.1 Effect of Iron deficiency on Immunity...............................................35 1.7.2 Iron and T Lymphocytes....................................................................38 1.8 Cell Cycle and the Role of Iron......................................................................39 1.8.1 Overview of the mammalian cell cycle..............................................40 5 1.8.2 Cell Cycle Regulation.........................................................................44 1.9 Iron Chelation.................................................................................................48 1.9.1 Desferrioxamine (DFO)......................................................................51 1.9.2 HPO Family chelators........................................................................53 1.10 Overall Aims and Objectives..............................................................56 2. MATERIALS AND METHODS.......................................................................58 2.1 Peripheral blood mononuclear cell isolation..................................................59 2.1.1 CD14+ cell isolation from peripheral blood mononuclear cells .......61 2.1.2 CD4+ cell isolation from CD14- cell fraction....................................61 2.2 In vitro stimulation of primary human CD4+ T cells.....................................62 2.3 Culturing Jurkat Cells.....................................................................................63 2.4 Supernatant ELISA.........................................................................................64 2.5 Proliferation Assays........................................................................................65 2.5.1 3H Thymidine Proliferation assay......................................................65