The effects of the citrullinating enzyme, peptidylarginine deiminase, on the activation of T cells Rita Barreto Duarte Carilho Torrão Doctor of Philosophy Aston University September, 2016 © Rita Barreto Duarte Carilho Torrão, 2016 Rita Barreto Duarte Carilho Torrão asserts her moral right to be identified as the author of this thesis. This copy of the thesis has been supplied on condition that anyone who consults it is understood to recognise that its copyright rests with its author and that no quotation from the report and no information derived from it may be published without proper acknowledgement. 1 Aston University The effects of the citrullinating enzyme, peptidylarginine deiminase, on the activation of T cells Rita Barreto Duarte Carilho Torrão Doctor of Philosophy 2016 Rheumatoid arthritis (RA) and periodontitis (PID) are two chronic inflammatory diseases associated with the modification of self-proteins by citrullinating peptidyl arginine deiminase (PAD) enzymes, leading to a loss of tolerance by the immune system. The main goal of this study was to explore the action of PAD enzyme- mediated citrullination on T cell membrane proteins and gene expression in relation to the T cell phenotype in PID. Effects on cells of the adaptive immune system have been less well studied in PID and the data obtained here shows that citrullination of peripheral blood mononuclear cells (PBMC) by PAD enzymes impairs T cell activation. Microarray studies showed that PAD enzyme treatment led to the dysregulation of genes involved in glucose and amino acid metabolism in PBMC. Real time quantitative polymerase chain reaction (RT-QPCR) in CD4 and CD8 T cells from PID patients showed a trend towards down-regulation of hexokinase 3 and up-regulation of argininosuccinate synthase1. Also, proteomic and genomic studies in PBMC implicated the involvement of the complement system in the impairment of the T cell response by PAD enzymes. Taken together, the results obtained here support a potential link between T cell surface citrullination and metabolic asynchrony in T cells and may offer an explanation for the lack of immune suppression in PID. 2 Dedication I would like to dedicate this thesis to my dear grandma “avó Tina”. Your smile, love and dedication to our family will always live in our hearts. 3 Acknowledgements Firstly I would like to thank the MC-ITN-RAPID project for kindly funding my PhD, in particular to my supervisor Professor Helen Griffiths for all the guidance and support during my PhD. I would also like to thank Professor Iain Chapple and Doctor Paul Weston from the Dental Hospital, University of Birmingham, for all the help in collecting samples. I would like to thank Doctor Andrew Creese and Lorraine Wallace from the Functional Genomics, Proteomics and Metabolomics Facility, University of Birmingham, for all the help and assistance with mass spectrometry and microarray. I would like to thank to my Aston family, present and former members of lab 358 and 360 and office 436, for all the help and support. In particular, I would like to thank Chris and Stuart for all the help in collecting blood, protein analysis and flow cytometry, and to Aisha for all the help and for being so adorable. Lastly, I would like to thank to RAPIDers, for making all the meetings and workshops so much more enjoyable and fun. On a personal note, I would like to thank Shibu for being my person. I would also like to thank to Justin, Rachel, Sabah, Ali, Kiran, Chat, Iru, Aisha, Edyta and Charlie for making the last four years so fun and enjoyable and fulfilled with cake and chocolate. I would like to thank Ana for being such an amazing life mentor. Finally, I would like to thank my lovely family, in special to my parents for all the guidance and love and to my brother for keeping up with me during all these years. Last, but by no means least, I would like to thank to my wonderful husband, Flávio, who had to deal with my ups and downs and who always believed in me, and to my dearest son Manuel, for being the light of my days and for making every moment count. 4 Table of Contents Summary ………………………………………………………………………………………. 2 Dedication .................................................................................................................... 3 Acknowledgements ...................................................................................................... 4 Table of Contents ......................................................................................................... 5 List of figures .............................................................................................................. 10 List of tables ............................................................................................................... 14 List of equations ......................................................................................................... 16 Abbreviations ............................................................................................................. 17 Chapter 1 General introduction ................................................................................ 27 1.1. Inflammation and immunity ........................................................................... 28 1.1.1. Innate immunity ...................................................................................... 28 1.1.2. Adaptive immunity .................................................................................. 29 1.1.3. The complement system ......................................................................... 29 1.1.4. Cell surface proteins and inflammatory response ................................... 32 1.2. T cell biology ................................................................................................. 36 1.2.1. T cell maturation ..................................................................................... 36 1.2.2. T cell activation and T cell receptor signalling ......................................... 38 1.2.3. Interleukin-2 signalling pathway .............................................................. 40 1.2.4. T cell differentiation into different subsets ............................................... 42 1.2.5. T cell metabolism .................................................................................... 46 1.3. Immune tolerance and autoimmunity ............................................................ 50 1.3.1. Citrullination ............................................................................................ 52 1.3.2. Rheumatoid arthritis ............................................................................... 56 1.3.3. Periodontitis ............................................................................................ 59 1.4. Rheumatoid arthritis and periodontitis: a link through citrullination ................ 61 1.5. Hypothesis and aims ..................................................................................... 63 Chapter 2 Materials and methods ............................................................................ 64 2.1. Materials ....................................................................................................... 65 2.1.1. Consumables ......................................................................................... 65 2.1.2. Antibodies............................................................................................... 65 2.2. Fibrinogen citrullination ................................................................................. 66 2.2.1. Reagents ................................................................................................ 66 2.2.2. Background ............................................................................................ 66 2.2.3. Protocol .................................................................................................. 66 2.3. Studies on Jurkat E6.1 T cells ....................................................................... 67 2.3.1. Reagents ................................................................................................ 67 2.3.2. Background ............................................................................................ 67 2.3.3. Protocol: activation of Jurkat E6.1 T cells ............................................... 67 2.4. Studies on primary T cells in peripheral blood mononuclear cells population 68 2.4.1. Reagents ................................................................................................ 68 2.4.2. Volunteers .............................................................................................. 68 2.4.3. Background ............................................................................................ 68 2.4.4. Protocol: optimisation of peripheral blood mononuclear cells isolation .... 69 2.4.5. Protocol: activation of primary T cells from peripheral blood mononuclear cells ............................................................................................................... 71 5 2.4.6. Protocol: citrullination of peripheral blood mononuclear cells .................. 71 2.5. Clinical samples ............................................................................................ 72 2.5.1. Reagents ................................................................................................ 72 2.5.2. Volunteers .............................................................................................. 72 2.5.3. Background ...........................................................................................