Modulation of cell-mediated immunity by HIV-1 infection of macrophages Lucy Caitríona Kiernan Bell Division of Infection and Immunity University College London PhD Supervisor: Dr Mahdad Noursadeghi A thesis submitted for the degree of Doctor of Philosophy University College London August 2014 Declaration I, Lucy Caitríona Kiernan Bell, confirm that the work presented in this thesis is my own. Where information has been derived from other sources, I confirm that this has been indicated in the thesis. 2 Abstract Cell-mediated immunity (CMI) is central to the host response to intracellular pathogens such as Mycobacterium tuberculosis (Mtb). The function of CMI can be modulated by human immunodeficiency virus (HIV)-1 via its pleiotropic effects on the immune response, including modulation of macrophages, which are parasitized by both HIV-1 and Mtb. HIV-1 infection is associated with increased risk of tuberculosis (TB), and so in this thesis I sought to explore the host/pathogen interactions through which HIV-1 dysregulates CMI, and thus changes the natural history of TB. Using an in vitro model of human monocyte-derived macrophages (MDMs), I characterise a phenotype wherein HIV-1 specifically attenuates production of the immunoregulatory cytokine interleukin (IL)-10 in response to Mtb and other innate immune stimuli. I show that this phenotype requires HIV-1 integration and gene expression, and may result from a function of the HIV-1 accessory proteins. I identify that the phosphoinositide 3-kinase (PI3K) pathway specifically regulates IL-10 production in human MDMs, and thus may be a target for HIV-1 to mediate IL-10 attenuation. I show that HIV-1 may attenuate IL-10 to maximise its own replication, and identify potential consequences of IL-10 attenuation for CMI. By using the tuberculin skin test (TST) as a human challenge model, I evaluate HIV-1 modulation of CMI in vivo in active TB patients, and demonstrate IL-10 attenuation in this context. I identify a role for type I inteferons (IFNs) in HIV-1 anergy, and observe exaggerated T helper 2 responses associated with the immune reconstitution inflammatory syndrome (IRIS). To fully explore CMI in vivo by transcriptional profiling, I utilize the transcriptional heterogeneity of stimulated macrophages to develop a modular analysis strategy for transcriptional profiles, and apply this in the TST model. My results delineate novel modulatory effects of HIV-1 on the function of CMI, and thus provide insights into immunopathogenesis in HIV-1/TB co-infection. 3 Acknowledgements First and foremost I must acknowledge my supervisor, Dr Maddy Noursadeghi, whose zeal, support and advice has seen me through the challenges of the last four years, from scientific conundrums to experimental catastrophes. His unmitigated enthusiasm for discussing experiments and hypotheses has been invaluable for the research presented herein, and for stimulating my own enthusiasm for scientific endeavour. I must thank both Maddy and my secondary supervisor, Professor Benny Chain, for creating a supportive and stimulating environment in the lab, which has helped me learn how to be a scientist (I hope!). I have immensely enjoyed my time working with them, and have learned so much. Secondly I have to thank everybody in the lab who has supported me over the course of this work. I must particularly mention Dr Gillian Tomlinson, whose own work formed the basis for much of mine, and whose patience guided me through my first months of lab work. Special thanks also go to Dr Nandi Simpson, Dr Rhia Kundu and Dr Theres Oakes, for saving me from many inept early moments. I must also thank Dr Elspeth Potton, Dr Jennifer Roe, Dr Rachel Byng-Maddick, Dr Gabriele Pollara and Jamie Heather, each of whom has contributed hugely to this work through our collaboration in the lab over the years – along with everyone else in the Noursadeghi and Chain groups, and in the Division of Infection and Immunity at UCL. I would also like to acknowledge Professor Keertan Dheda and Dr Mellissa Pascoe at the University of Cape Town, Professor Greg Towers and Dr Jane Rasaiyaah at UCL, and Professor Robin May at the University of Birmingham, whose collaboration has been essential for the work I have presented here. A huge thank you goes to the many blood donors who gave up 120ml of their circulating volume in the name of my experiments, and to all of the patients in London and Cape Town who participated in the TST study – this would not have been possible without their goodwill. I would not have had the opportunity to do this work without the support of Professor Gordon Stewart and the UCL MB PhD programme. Professor Stewart has been a source of wisdom, guidance and humour, and I can’t thank him enough for his support and belief in me. I would also like to thank Professor Ashley Moffatt at the University of 4 Cambridge, for being the first person to stimulate my interest in immunology, and for introducing me to the notion of mixing clinical medicine and basic science. Very appreciative thanks go to Elspeth Potton, Ina Schim van der Loeff, Katharine Best and Gabriele Pollara for proof-reading various parts of this thesis in various unkempt forms. I have to thank all of my friends for their support, which has sustained me throughout the last few years. Very special thanks have to go to Bryony Hopkinshaw and Victoria Sampson, who have been through the ups and downs with me, and who have listened to more scientific work crises than any friends should ever have to. Thanks also go to Becca Burrell, Ina Schim van der Loeff and Chris McKinnon, who have been a constant source of advice, coffee and pep talks during the busy final year of labwork and thesis- writing. I would like to thank and acknowledge all of my family, who have been a tremendous source of love and support. My cousin Victoria Lennon has been a huge support in London, and has always been on hand with a G&T, a chat and even a spare bedroom in times of need. My grandparents Finella and David Wilson have provided invaluable support to me throughout my education, and many childhood conversations with Papa David, as well as with my uncle and godfather Thomas McErlean, may have helped ignite my enthusiasm for academia at an early age. Lastly, I would like to thank my sisters Alice and Charlotte, and parents Bronagh and Hugh, whose love and support has been the greatest support and solace of all. Their belief in me has got me to where I am today, and their reassurance has got me through the most difficult times of all – I would like to dedicate this thesis to all of them. 5 Table of Contents Abstract ......................................................................................................................... 3 Acknowledgements ...................................................................................................... 4 Table of Contents ......................................................................................................... 6 Table of figures ........................................................................................................... 11 List of tables ............................................................................................................... 16 Abbreviations .............................................................................................................. 18 Publication .................................................................................................................. 24 Chapter 1. Introduction ........................................................................................ 25 1.1 The cell-mediated immune system ............................................................... 25 1.1.1 Overview: the delayed-type hypersensitivity and cell-mediated immunity paradigm ............................................................................................................... 25 1.1.2 CMI and infectious diseases ...................................................................... 28 1.2 The role of macrophages in CMI ................................................................... 31 1.2.1 The mononuclear phagocyte system ......................................................... 31 1.2.2 Monocytes and monocyte-derived cells ..................................................... 34 1.2.3 Dendritic cells ............................................................................................ 36 1.2.4 Embryologically-derived tissue macrophages ............................................ 37 1.2.5 Functions of macrophages in CMI ............................................................. 41 1.2.6 Innate immune recognition by macrophages ............................................. 41 1.2.7 Macrophage effector functions................................................................... 45 1.2.8 Interactions of macrophages with the adaptive immune system ................ 47 1.2.9 Macrophage functional plasticity ................................................................ 51 1.3 HIV-1 infection and CMI ................................................................................. 53 1.3.1 The human immunodeficiency virus .......................................................... 53 1.3.2 Epidemiology, clinical course and treatment of HIV-1 disease ................... 53 1.3.3 HIV-1 virology ............................................................................................ 54 1.3.4 Interactions of HIV-1 with host cells: the viral lifecycle, host restriction and immune