Amino Acid Metabolism in the Inflammatory Niche
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AMINO ACID METABOLISM IN THE INFLAMMATORY NICHE Alice Chun-yin Wang Department of Haematology Faculty of Medicine Imperial College London A thesis submitted for the degree of Doctor of Philosophy in Imperial College London 2015 將這份榮耀獻給我的爸爸媽媽 我愛你們 2 DECLARATION OF ORIGINALITY The research described in this thesis is the sole and original work of the author. Contributions by other persons have been acknowledged where necessary. COPYRIGHT DECLARATION ‘The copyright of this thesis rests with the author and is made available under a Creative Commons Attribution Non-Commercial No Derivatives licence. Researchers are free to copy, distribute or transmit the thesis on the condition that they attribute it, that they do not use it for commercial purposes and that they do not alter, transform or build upon it. For any reuse or redistribution, researchers must make clear to others the licence terms of this work’ 3 ACKNOWLEDGEMENTS First and foremost, I would like to thank my supervisor Prof. Francesco Dazzi for his guidance to expand my scientific knowledge and make my PhD experience stimulating. He has taught me, both consciously and unconsciously, how to grow as a research scientist. The members of the Dazzi group have contributed immensely to my personal and professional time at Imperial. Special thanks to Ilaria Marigo and Cristina Trento, my Italian sisters, who offer endless support and have faith in me even during the tough times in my PhD pursuit. I would also like to thank Antonio Galleu, Yuan-Tsung Li and Luigi Dolcetti, for sharing their enthusiasm for both science and life with me. Other past and present colleagues that I have had pleasure to work with are Ling Weng, Andrea Guerra, Luciene Lopes and Monica Beato Coelho. I am also grateful to George Nteliopoulos for his support and technical advices. I owe a dept of appreciation to Dr. Mark Birrell, Prof. Vincenzo Bronte, Prof. Jorge Caamano, Dr. Neil Rogers, for providing me important research tools. I am hugely grateful for a few friends who encourage me all the time and their love have constructed a second home for me in London. I thank Kevin, Chien-nien, Wei Wei, Ellen, Carolyn, Isabella, Rebecca, Vincent and Jack for this. Last, but not least, I would like to thank my parents for all their unconditional love, support and encouragement. Thank you. 4 ABSTRACT Stroma and parenchyma represent the supportive and functional components in every organ of the body, respectively. Beyond their ability to produce structural support and to differentiate into tissues of mesodermal origin, mesenchymal stromal cells (MSC) exhibit potent immunomodulatory properties. Such a function requires an activation step (‘licensing signal’) provided by the inflammatory microenvironment to which MSC are exposed. My results have attributed immunosuppressive effects of MSC to essential amino acid (EAA) deprivation. Amongst the EAA consuming enzymes examined, blocking nitric oxide synthase 2 (NOS2) and histidine decarboxylase (HDC) both resulted in impaired anti-proliferative activity of MSC, while NOS2 appeared to be a more prominent effector. My results have also demonstrated that TNF-α and IFN- γ differently account for NOS2 and HDC up-regulation, respectively. Furthermore, MyD88 and NF-ĸB were identified as upstream mediators for initiating NOS2 production. The role of TNF-α and NOS2 in MSC-mediated immunosuppression was assessed in vivo using a murine model of peritonitis. MSC treatment remarkably reduced the local inflammatory response during acute peritoneal inflammation. Nevertheless, both Nos2-/- and Tnfr1/r2-/- MSC delivered similar effects compared to WT MSC, indicating the presence of other complementary mechanisms in MSC- mediated immunosuppression in vivo. In addition to their immunomodulatory properties, MSC are fundamental in regulating self-renewal and differentiation of haematopoietic stem cells (HSC). MSC protect HSC from potential damage by maintaining their quiescence. My results have revealed that the ability of MSC to enhance the quiescence of HSC was associated with cell-cycle arrest induced by NOS2. As striking parallels exist between the normal and malignant stem cell niche, I investigated the ability of MSC to protect haematopoietic malignant cells from chemotherapy-induced apoptosis. MSC were observed to confer protection from etoposide- induced necrosis of EL4 cells, possibly due to their ability to suppress EL4 proliferation. Collectively, my results have demonstrated the role of MSC across the fields of immunomodulation, niche-supporting and anti-apoptotic effects. 5 TABLE OF CONTENTS AMINO ACID IN THE INFLAMMATORY NICHE…………………………………. 1 DECLARATION OF ORIGINALITY…………………………………………………. 3 ACKNOWLEDGMENTS……………………………………………………………… 4 ABSTRACT……………………………………………………………………………. 5 TABLE OF CONTENTS………………………………………………….……………. 6 LIST OF FIGURES AND TABLES………………………………………………….… 8 ABBREVIATIONS…………………………………………………………………….. 10 1 INTRODUCTION…………………………………………………………………... 14 1.1 Mesenchymal stromal cells (MSC)………………………………………............. 16 1.1.1 Source and ontogeny of MSC…………………………………………........... 17 1.1.2 Phenotype of MSC…………………………………………………………… 18 1.2 Role of MSC in inflammation………………………………………………….… 20 1.2.1 Inflammation determines MSC function…………………………………….. 21 1.2.2 Regulation of immune cells by MSC………………………………………… 22 1.2.2.1 Recruitment of immune cells………………………………………….. 22 1.2.2.2 Re-education of immune cell functions……………………………...... 25 1.2.2.3 Inhibition of immune cell proliferation……………………………….. 31 1.2.3 EAA deprivation in the regulation of immune responses…………………… 34 1.2.3.1 Arginine metabolism by ARG and NOS…………..………………….. 36 1.2.3.2 Tryptophan metabolism by IDO………………………………………. 45 1.2.3.3 Histidine metabolism by histidine decarboxylase (HDC)…………….. 47 1.2.3.4 Phenylalanine metabolism by Interleukin-4 Induced Gene 1 (IL4I1)… 50 1.3 Role of MSC in haematopoiesis………………………………………………….. 51 1.3.1 The endosteal niche………………………………………………………….. 54 1.3.2 The vascular niche…………………………………………………………… 55 1.3.3 MSC in the haematopoietic niche……………………………………………. 57 1.3.4 Role of MSC in HSC transplantation………………………………………... 59 1.3.5 Contribution of MSC to haematopoietic malignancies……………………… 61 1.4 Hypothesis and aims………………………………………………………...….... 63 2 MATERIALS AND METHODS…………………………………………………... 64 2.1 Chemicals and Reagents………………………………………………………….. 64 2.2 Animals…………………………………………………………………………… 64 2.3 Cells and cell culture……………………………………………………………... 65 2.3.1 Generation of MSC…………………………………………………………... 65 2.3.2 Preparation of splenocytes…………………………………………………… 65 2.3.3 Cell culture………………………………………………………..………….. 65 2.3.4. Co-culture………………………………………………………..………….. 66 2.4 PCR………………………………………………………..……………………… 67 2.5 Real-time PCR ………………………………………………………..………….. 68 2.6 Western blot………………………………………………………..……………... 68 2.7 Confocal……………………………………………………..……………………. 69 2.8 Immunosuppressive assay………………………………………………………... 70 6 2.9 NO quantification………………………………………………………..……….. 70 2.10 Cell viability………………………………………………………..…………… 70 2.11 Quantification of HY-primed T cells stimulated in vivo………………………... 71 2.12 Thioglycollate (TG)-induced peritonitis ………………………………………... 72 2.13 FACS analysis ………………………………………………………………….. 72 2.14 Statistical analysis……………………………………………………………….. 73 3 THE ROLE OF EAA METABOLISM IN MSC-MEDIATED IMMUNOSUPPRESSION………………………………………………………………. 74 3.1 Introduction……………………………………………………………………….. 74 3.2 Results…………………………………………………………………………….. 76 3.2.1 Characterisation of isolated MSC……………………………………………. 76 3.2.2 MSC express a selective pattern of EAA-consuming enzymes in response to activated splenocytes……………………………………………………….... 78 3.2.3 Regulation of MSC immunosuppressive functions by inhibitors of EAA consuming enzymes……………………………………………………......... 81 3.2.4 Cytokine priming selectively skews EAA-consuming enzyme expression in MSC………………………………………………………………………….. 87 3.2.5 NOS2 deficiency leads to the impairment of the immunosuppressive functions of MSC………………………………………................................. 93 3.2.6 Eliminating the effect of TNF-α partially impaired the immunosuppressive activities of MSC…………………………………………………….............. 99 3.2.7 The role of NF-κB as a key upstream mediator to induce NOS2 expression in MSC............................................................................................................. 105 3.2.8 The effect of MyD88 in regulating NOS2 expression in MSC…………….... 110 3.3 Discussion…………………………………………................................................ 114 4 THE EFFECT OF MSC ON HAEMATOPOIETIC CELLS: CELL CYCLE REGULATION AND APOPTOSIS……………………………………………….. 122 4.1 Introduction……………………………………………………...………............... 122 4.2 Results……………………………………………………...………....................... 123 4.2.1. NOS2 plays a fundamental role in the MSC mediated cell-cycle arrest of activated splenocytes…………....................................................................... 123 4.2.2 MSC induced haematopoietic stem/progenitor cells (HSPC) cell-cycle arrest in vitro………………………………………………………………………... 126 4.2.3 Effect of MSC on tumour cell proliferation………………………………….. 132 4.3 Discussion………………………………………………………………………… 138 5 THE FUNCTION OF NOS IN THE MSC-MEDIATED IMMUNE RESPONSE DURING INFLAMMATION ……………………………………………………... 142 5.1 Introduction…………………………………...………........................................... 142 5.2 Results……………………………...………........................................................... 144 5.2.1 HY responses in mice immunised with male splenocytes …………………... 144 5.2.2 MSC markedly reduced the TG-induced inflammatory infiltration………….