IMMUNOMODULATION BY ADIPOKINES IN TYPE 1 DIABETES By Myriam Chimen A thesis submitted to The University of Birmingham For the degree of DOCTOR OF PHILOSOPHY School of Clinical and Experimental Medicine College of Medical and Dental Sciences University of Birmingham Date: February 2012 University of Birmingham Research Archive e-theses repository This unpublished thesis/dissertation is copyright of the author and/or third parties.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 Type 1 diabetes (T1D) is a T cell-mediated autoimmune disease in which the immune system specifically targets and destroys the pancreatic insulin-producing beta-cells. We are interested in defining whether adipose tissue-derived cytokines (adipokines) such as adiponectin (anti-inflammatory) and leptin (pro-inflammatory) could influence T1D progression. We first demonstrate the expression of the leptin receptor (LEPR) on peripheral blood mononuclear cells (PBMC) and observed higher expression of LEPR on PBMC from patients with T1D. However, we found no significant functional relevance for this observation. On the other hand, we show lower expression of the adiponectin receptors on lymphocytes from patients with T1D. This was associated with a reduced capacity of adiponectin to inhibit lymphocyte trans-endothelial migration in T1D. Remarkably, we show that adiponectin strongly inhibited lymphocyte migration either by action on the endothelium or directly on the lymphocytes. We have now established that this effect on lymphocytes is mediated by a newly identified B cell-secreted agent following adiponectin stimulation. The agent stimulates endothelial production of sphingosine-1-phosphate, which in turn is responsible for the inhibition of lymphocyte trans-endothelial migration. These observations were confirmed in vivo using a peritoneal model of inflammation in a B cell knock-out mouse strain. In these animals, T cell recruitment in the peritoneum is increased compared to the wild-type and restored when the agent was administrated. These observations underline the importance of adiponectin in the control of lymphocyte transmigration during an inflammatory response and offer a potential therapeutic agent for T1D as well as other T cell-mediated diseases. Acknowledgements I would firstly like to thank my supervisor Dr Parth Narendran for giving me the opportunity to work on that intringuing project, for your precious advices, guidance and corrections and your patience and kindness. I also would like to thank my “adopted” supervisor Dr Ed Rainger for your help with the transmigration project. Thank you for your “strokes of genius”, your supervision, your enthusiasm with the project and your help with my thesis. I would also like to thank my second supervisor, Dr Peter Lane, for your advices. Then I would like to thank Helen, for introducing me to the world of lymphocyte migration, for contributing to the project and for the help with my thesis. I would like to thank Dr Ashley Martin for your help with the proteomics and for being so clear and patient at explaining the world of mass spectrometry to me. I also thank Dr Francesca Barone, Gemma and Prof Chris Buckley for helping me out with the mice experiments. I would like to thank the other people in the Narendran lab, Terence, Suzy, Ed, Amy and Manita. I would also like to thank everyone in the Samson and Walker labs for their precious advices on proper immunology science and for the exchange of reagents. I would also like to thank all the people in the Link lab, especially Clara and Phil. I also want to thank all the donors who kindly donated their blood. I also thank the people from the Hagedorn Institute and Novo Nordisk who were so welcoming and nice to me over my stay and for the reagents. Then I would like to thank all my desk neighbours, now friends, Ange, Pushpa, Donna, Ana for their support, coffee breaks and always finding the words to make me laugh. Also thanks for coping with my computer moaning. I would also like to thank my family for their great support, love, advices and for always encouraging and pushing me to do what I want in life. Finally but not the last, I would like to thank Phil, my partner, best friend and love for always giving me great support, always pushing me forward and especially putting up with me. Thank you for being there and for your love, I feel very lucky to have you in my life. Table of contents Chapter 1- GENERAL INTRODUCTION .................................................... 1 1. Type 1 diabetes (T1D) disease ........................................................................................... 2 1.1. Clinical aspects of T1D .............................................................................................. 2 1.2. Prevalence of T1D ...................................................................................................... 4 1.3. Immunity and pathogenesis of T1D ........................................................................... 6 1.3.1. Autoimmunity in T1D .......................................................................................... 6 1.3.2. Trafficking and recruitment of lymphocytes during inflammation .................... 13 1.3.3. Vascular function in T1D ................................................................................... 24 2. T1D, obesity and insulin resistance .................................................................................. 26 3. Adipokines ........................................................................................................................ 28 3.1. Leptin ........................................................................................................................ 28 3.1.1. Pleiotropic properties of leptin ........................................................................... 28 3.1.2. Leptin in immunity ............................................................................................. 30 3.1.3. Leptin in T1D ..................................................................................................... 34 3.2. Adiponectin .............................................................................................................. 37 3.2.1. Adiponectin and its receptors ............................................................................. 37 3.2.2. Adiponectin role in metabolism ......................................................................... 39 3.2.3. Anti-inflammatory properties of adiponectin ..................................................... 40 3.2.4. Adiponectin modulates immune cell migration ................................................. 43 3.2.5. Adiponectin in T1D ............................................................................................ 45 4. Hypothesis and aims ........................................................................................................ 46 CHAPTER 2-MATERIAL AND METHODS ........................................... 47 1. Material ............................................................................................................................. 48 1.1. List of main reagents ................................................................................................ 48 1.2. Antibodies ................................................................................................................. 49 2. Methods ............................................................................................................................ 50 2.1. Measurement of gene expression ............................................................................. 50 2.1.1. PBMC isolation .................................................................................................. 50 2.1.2. RNA extraction ................................................................................................... 50 2.1.3. Reverse transcription .......................................................................................... 53 2.1.4. Conventional PCR .............................................................................................. 53 2.1.5. Real-Time PCR .................................................................................................. 54 2.2. Measurement of protein expression .......................................................................... 55 2.2.1. Flow cytometry ................................................................................................... 55 2.2.2. Western Blot ....................................................................................................... 56 2.3. ELISA assay ............................................................................................................. 56 Table of contents 2.4. Cell sorting ............................................................................................................... 57 2.4.1. Positive selection of PBMC subsets ................................................................... 57 2.4.2. Negative selection of PBMC subsets ................................................................. 57 2.5. DCs Phenotyping