Expression and Regulation of Leucocyte Immunoglobulin-Like Receptors in the Human Colonic Environment A thesis submitted in fulfilment of the requirements for the degree of Doctor of Philosophy by Greta Shao Chu LEE St George and Sutherland Clinical School Faculty of Medicine University of New South Wales 2015 PLEASE TYPE THE UNIVERSITY OF NEW SOUTH WALES Thesis/Dissertation Sheet Surname or Family name: Lee First name: Greta Other name/s: Shao Chu Abbreviation for degree as given in the University calendar: PhD School: St George and Sutherland Clinical School Faculty: Medicine Title: Expression and Regulation of Leucocyte Immunoglobulin-Like Receptors in the Human Colonic Environment Abstract 350 words maximum: (PLEASE TYPE) Immune homeostasis in the healthy gastrointestinal tract is characterised by active suppression of inflammation and immune tolerance and, conversely, chronic inflammatory bowel disease [IBD] results from an imbalance of these tightly regulated signals. Macrophages are abundant immune cells in the colon that protect against foreign antigens and their down-regulated phenotype is influenced by the local stromal milieu. As leukocyte immunoglobulin-like receptors [LILRs] are primarily expressed on cells of the monocytic-macrophage lineage, they are likely important immunoregulatory candidates in the colon. The experiments in this thesis aimed to describe the pattern of distribution of LILRs in the colon and the changes that occur in IBD, and to develop a cell culture model of colonic-like macrophages that enables further characterisation of these receptors. To determine the presence of LILRs, tissue was obtained from subjects undergoing a colonic resection. Immunohistochemistry, immunofluorescence, flow cytometry, Western blotting and qRT-PCR experiments were performed on tissue sections or isolated colonic lamina propria mononuclear cells, protein or RNA. There was universal expression of LILRB1 and LILRA5, predominantly on macrophages, but also on T and B lymphocytes. The majority of subjects also expressed LILRA1 and LILRA3. Interestingly, LILRB1 and LILRB4 were increased in chronic IBD. The presence of both inhibitory and activating LILRs on colonic macrophages suggests that a balance between positive and negative signals is important in the colon. Colonic stromal-derived conditioned media [SCM] generated from the lamina propria of 12 control subjects was used to differentiate peripheral blood monocytes in vitro. Cytokine production by the resulting colonic-like macrophages was suppressed; minimal amounts of TNF-α, IL-1, IL-2, IL-4, IFN-γ, TGF-β and IL-10 were produced, similar to colonic macrophages. Importantly, as assessed by ELISA, multiplex and qRT-PCR, although TNF-α was produced following LPS- stimulation, this was down-regulated by SCM in a dose- and time- dependent manner. SCM also inhibited their ability to produce TGF-β in response to stimulation. Compared with classically-differentiated macrophages, inhibitory LILRs were predominantly up-regulated and, in particular, elevated LILRB1 corresponded with the increase seen on colonic macrophages. This novel in vitro model of colonic-like macrophages allows further delineation of the role of LILRs in the colon. 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 The 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: THIS SHEET IS TO BE GLUED TO THE INSIDE FRONT COVER OF THE THESIS i 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 .............................................................................. ii 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 ................................................................. iii Acknowledgements I wish to express my gratitude to all who have made significant contributions to this project, without whom this PhD thesis would not have been written. First and foremost to my supervisor, Professor Michael Grimm, who has guided me through the numerous components of this project and helped see this thesis through to completion. Thank you for your advice, patience, continued support and belief in my abilities; thank you also for the detailed reviewing of this thesis. Thank you to my co-supervisor, Associate Professor Nicodemus Tedla, for providing access to the leucocyte immunoglobulin-like receptor antibodies, for advice in the early experimental stages of this study, and for performing the arduous task of enumerating positive LILR staining on multiple immunohistochemistry slides. Without the collaboration of the colorectal surgeons this project would not have been possible. Thank you to the following surgeons for allowing access to your patients, assistance with recruitment of subjects and provision of suitable tissue specimens: the late Dr Philip Douglas, Dr Shing Wong, Dr Graham Newstead, Professor Denis King, Dr Shevy Perera, Dr Mark Muhlmann, Dr Sanjay Kariappa, Dr Steven Gan and Dr Carina Chow. A special thank you to all the patients who willingly agreed to participate in this study. Thank you to all the members of the Inflammation and Infection Research Group, UNSW, who have helped me, a novice in the lab, become experienced in a range of experimental techniques. There are too many of you to name individually; you know who you are. Thank you for the countless tips and suggestions, words of wisdom and practical help with the various lab experiments and procedures. Thank you to the National Health and Medical Research Council (NHMRC) for providing a Medical Postgraduate Research Scholarship. And to all the family, friends and colleagues near and far who have supported, encouraged, provided insights into research and tried to keep me sane throughout these years; without your support I would not be at the end of this chapter in my life. Thank you. iv Abstract Immune homeostasis in the healthy gastrointestinal tract is characterised by active suppression of inflammation and immune tolerance and, conversely, chronic inflammatory bowel disease [IBD] results from an imbalance of these tightly regulated signals. Macrophages are abundant immune cells in the colon that protect against foreign antigens and their down-regulated phenotype is influenced by the local stromal milieu. As leukocyte immunoglobulin-like receptors [LILRs] are primarily