Regulation of monocyte/macrophage activation by Leukocyte Immunoglobulin-Like Receptor B4 (LILRB4) Mijeong (May) Park A thesis in fulfilment of the requirements for the degree of Doctor of Philosophy School of Medical Sciences Faculty of Medicine April 2016 THE UNIVERSITY OF NEW SOUTH WALES Thesis/Dissertation Sheet Surname or Family name: Park First name: Mijeong Other name/s: May Abbreviation for degree as given in the University calendar: PhD School: Medical Sciences Faculty: Medicine Title: Modulation of monocyte/macrophage activation by leukocyte immunoglobulin-like receptor B4 (LILRB4) Abstract The leukocyte immunoglobulin-like receptor B4 (LILRB4) belongs to a family of cell surface receptors, primarily expressed on mono-myeloid cells. LILRB4 has been shown to inhibit FcγRI- mediated pro-inflammatory cytokine production by monocytes and induce tolerogenic dendritic cells in vitro. It is believed that LILRB4 regulates monocyte/macrophage activation through its three intracellular immunoreceptor tyrosine-based inhibitory motifs (ITIMs) by paring with activating receptor, bearing ITAM, and by dephosphorylation of non-receptor tyrosine kinases via recruitment of Src homology phosphatase-1 (SHP-1). However, the exact mechanism and the functions depending on its structure and stimuli are still unclear. In addition, regulatory functions of LILRB4 paring with non- ITAM associated activating receptors, including TLR4 is less researched. Thus, this thesis investigates, for the first time, the functions of LILRB4 in regulation of monocyte/macrophage activation depending on the position of the tyrosine residues of its ITIMs, and stimuli. Here it is shown for the first time that LILRB4 is a complex immuno-regulatory receptor that exerts dual inhibitory and activating functions in FcγRI and/or TLR4-mediated monocyte/macrophage activation including receptor-ligand internalisation, endocytosis, cytokine production, phagocytosis and bactericidal activity. Most importantly, each ITIM of LILRB4 has a different function in monocyte/macrophage activation depending on the position of its tyrosine residue. Thus, the tight regulation of these ITAM-containing and non-ITAM receptors by each LILRB4 ITIM may play a key role in fine-tuning immune responses. In addition, the results for the regulatory function of LILRB4 using various ITIM mutants in response to different stimuli provide better understanding of the signalling pathways of LILRB4 that may contribute to use LILRB4 as a potential therapeutic agent for an appropriate immune response using selective LILRB4 ITIM/s. Although no LILRB4 ligand is identified, new methodologies and the potential candidate ligands of LILRB4 which are provided here may contribute to identifying the nature of LILRB4 ligand/s in the near future. Declaration relating to disposition of project thesis/dissertation I hereby grant to the University of New South Wales or its agents the right to archive and to make available my thesis or dissertation in whole or in part in the University libraries in all forms of media, now or here after known, subject to the provisions of the Copyright Act 1968. I retain all property rights, such as patent rights. I also retain the right to use in future works (such as articles or books) all or part of this thesis or dissertation. I also authorise University Microfilms to use the 350 word abstract of my thesis in Dissertation Abstracts International (this is applicable to doctoral theses only). ……………………………………… ……………………………………..…… ……….… Signature Witness Date he University recognises that there may be exceptional circumstances requiring restrictions on copying or conditions on use. Requests for restriction for a period of up to 2 years must be made in writing. Requests for a longer period of restriction may be considered in exceptional circumstances and require the approval of the Dean of Graduate Research. FOR OFFICE USE ONLY Date of completion of requirements for Award: ORIGINALITY STATEMENT ‘I hereby declare that this submission is my own work and to the best of my knowledge it contains no materials previously published or written by another person, or substantial proportions of material which have been accepted for the award of any other degree or diploma at UNSW or any other educational institution, except where due acknowledgement is made in the thesis. Any contribution made to the research by others, with whom I have worked at UNSW or elsewhere, is explicitly acknowledged in the thesis. I also declare that the intellectual content of this thesis is the product of my own work, except to the extent that assistance from others in the project's design and conception or in style, presentation and linguistic expression is acknowledged.’ Signed …………………………………………….......... Date …………………………………………….............. COPYRIGHT STATEMENT ‘I hereby grant the University of New South Wales or its agents the right to archive and to make available my thesis or dissertation in whole or part in the University libraries in all forms of media, now or here after known, subject to the provisions of the Copyright Act 1968. I retain all proprietary rights, such as patent rights. I also retain the right to use in future works (such as articles or books) all or part of this thesis or dissertation. I also authorise University Microfilms to use the 350 word abstract of my thesis in Dissertation Abstract International (this is applicable to doctoral theses only). I have either used no substantial portions of copyright material in my thesis or I have obtained permission to use copyright material; where permission has not been granted I have applied/will apply for a partial restriction of the digital copy of my thesis or dissertation.' Signed …………………………………………….......... Date …………………………………………….............. AUTHENTICITY STATEMENT ‘I certify that the Library deposit digital copy is a direct equivalent of the final officially approved version of my thesis. No emendation of content has occurred and if there are any minor variations in formatting, they are the result of the conversion to digital format.’ Signed …………………………………………….......... Date …………………………………………….............. i TABLE OF CONTENTS TABLE OF CONTENTS ............................................................................................................ i LIST OF TABLES ..................................................................................................................... vi ABBREVIATIONS ..................................................................................................................... x PUBLISHED WORK FROM THIS THESIS ....................................................................... xvi PUBLISHED WORK DURING PHD .................................................................................... xvi ABSTRACTS (AS PRESENTING AUTHOR) ..................................................................... xvi ACKNOWLEDGEMENTS ................................................................................................... xvii ABSTRACT .......................................................................................................................... xix CHAPTER 1. INTRODUCTION ............................................................................................. 1 1.1 Leukocyte immunoglobulin-like receptors (LILRs) ............................................................. 4 1.1.1 Nomenclature of leukocyte immunoglobulin-like receptors ......................................... 5 1.1.2 Genetic organisation of LILRs and related receptors .................................................... 6 1.1.3 LILR homologues and orthologues ............................................................................... 8 1.1.4 Classification of LILRs ................................................................................................. 9 1.1.5 Ligands for LILRs ....................................................................................................... 17 1.1.6 Rodent orthologues of LILRB1, B3 and B4 ................................................................ 23 1.1.7 LILR expression, gene regulation and functions ......................................................... 25 1.1.1 Clinical association between disease and LILR expression ........................................ 30 1.1.2 Expression, functions and clinical significance of LILRB4 ........................................ 31 1.1.3 The role of LILRs in the regulation of innate immune responses ............................... 33 1.1.4 Coupling of activating and inhibitory signals by receptors containing ITAMs and ITIMs ........................................................................................................................... 47 1.2 Statement of aims ................................................................................................................ 60 CHAPTER 2. GENERAL METHODS .................................................................................. 62 2.1 Antibodies, buffers and reagents ......................................................................................... 62 2.2 Isolation of Peripheral blood mononuclear cells (PBMCs) ................................................. 66 2.3 Cell lines and tissue culture ................................................................................................. 67 2.4 Quality controlling of cell lines and antibodies for endotoxin contamination .................... 68 2.5 Quality controlling of cell surface receptor expression of