The role of chloride intracellular channel 1 in immune/inflammatory responses Kanin Salao A thesis submitted in fulfillment of the requirement for the degree of Doctor of Philosophy School of Medicine The University of New South Wales Sydney, Australia December 2016 PLEASE TYPE THE UNIVERSITY OF NEW SOUTH WALES Thesis/Dissertation Sheet Surname or Family name: SALAO First name: KANIN Other name/s· Abbreviation for degree as given in the University calendar: PhD of Medicine School: St Vincent's Clinical School Faculty: Medicine Title. The role of Intracellular chloride channel 1 in immune/inflammatory------ --- responses-- --- Intracellular chloride channel protein Abstract1 (CLIC1) 350 wordsparticipates maximum: in (PLEASE inflammatory TYPE) processes, at least in part by regulating macrophage phagosomal functions such as pH, proteolysis. Here, we sought to determine if CLIC1 can regulate immune/inflammatory responses by actions on dendritic cells (DC), the key professional antigen presenting cell. To study role of CLIC1 in immune/inflammatory responses, I first investigated immune cell phenotype changes in various tissue compartments associated with germline deletion of the gene for CLIC1 and found that both normal physiological conditions and under inflammatory responses, the immune cell composition is altered significantly in CLIC 1·1· mice when compared CLIC 1.,. mice. To further investigate the role of CLIC1 in dendritic cells, I first generated bone marrow-derived DC (BMDCs) from germline CLIC1 gene deleted (CL1c1·'·) mice and examined the effect of CLIC1 gene deletion on dendritic cell migration from peripheral 1 footpad to secondary lymph nodes of mice. I found that more cuc1· · BMDCs migrated from the site of 1 injection in the footpad and homed to popliteal and to inguinal lymph nodes than CUC 1 • ·sMDCs in both 1 1 cuc1• • and cuc1··mice. Subsequently, I identified the subcellular localization of CLIC1 in BMDCs after phagocytosis and found that cytoplasmic CLIC1 translocation to the phagosomal membrane where it regulated phagosomal pH and proteolysis. Phagosomes from cuc1·'· BMDCs displayed impaired acidification and 1 1 proteolysis, which could be reproduced in wild type cuc1· ·, but not cuc1· • cell were treated with IAA94, a CLIC family ion channel blocker. CLIC r'· BMDC displayed reduced in vitro antigen processing and presentation of full-length myelin oligodendrocyte glycoprotein (MOG) reduced MOG induced experimental autoimmune encephalomyelitis. These data suggest that CLIC1 regulates immune/inflammatory responses by means of changing immune cell composition, altering dendritic cell migration, modulating dendritic cell phagosomal pH and facilitating optimal processing of antigen for presentation to antigen specific T-cells. Further, they indicate that CLIC1 is a novel therapeutic tar et for inflammator diseases. ---------------------, Declaration relating to disposition of project thesis/dissertation 11 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 Microfilmsto use the 350 word abstract of my thesis in Dissertation Abstracts International (this is applicable to doctoral theses only) . ......................·.� ··· .................. � ................ Witnes 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 exce tional circumstances and re uire the a roval 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 ii 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 .........................� ... � ............................ 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 ............................................JO/ 08"/ ,R o/6. ............................... iii Acknowledgments I would like to thanks to my supervisor, Prof Samuel N Breit and co-supervisor, Dr Lele Jiang and Prof David A Brown for their advice, guidance, encouragement, understanding, patience and support throughout the study and preparation of this thesis. I especially thanks Sam for always give me a second chance one after the other. I learned a lot from you and all of these will be with me for the rest of my career. For Lele, you are like my Mum who has never given up on me. I couldn’t thanks you more. I would like to express my sincere thanks to the Royal Thai Government for the award of the scholarship and faculty of medicine UNSW for top up scholarship for the financial support and opportunity during my PhD study. I also would like to thanks to our colleges Dr Mohammad Mohammad, Dr Vicky Tsai Dr Xu Wei Luo, Dr Yasmin Husaini, Hui Li, Michelle Lee, Tamara Kuffner, Hong Ping Zhang and other inflammation group members at St Vincent’s for Applied Medical Research (AMR) for their suggestion and assistance throughout these years. My thanks to friends, Jennifer, Kuan, Jod, Yut, A, Nadine and Bird and Pan and other soccer mates for their friendship and support throughout these years. I especially would like to express my thanks to Reeni Donohoe for her English correction one draft after the other. I would like to also sincere thanks Dr Xu Wei Luo for his moral support and also English proof reading. Big thanks to my Dad for his love and support for most of my difficult time. Finally very importantly, I thank Aoom, my wife to be, for her love, support, understanding and encouragement throughout these years. For my Mum and beloved sister, I wish you two here with me to see me hand in this Thesis. Without you both, things were not easy. I just hope that you both realize how important your support were and are. Thank you for having me as brother and son. v Table of contents 1 Literature review ......................................................................................................... 1 1.I Chloride Intracellular Channel PROTEIN (CLICs) ............................................. 1 1.I.A Introduction ................................................................................................... 1 1.I.B Electrophysiology of CLICs ......................................................................... 3 1.I.C Biochemistry of CLICs ............................................................................... 10 1.I.D Structure of CLICs ...................................................................................... 26 1.I.E Tissue and SubCellular distribution of CLICs ............................................ 34 1.I.F Biological functions of clics ........................................................................ 43 1.II Dendritic cells (DCs) .......................................................................................... 68 1.II.A Introduction ................................................................................................. 68 1.II.B DC Subsets .................................................................................................. 69 1.II.C DC Development ........................................................................................ 75 1.III Functions of cDCs .............................................................................................. 82 1.III.A Introduction ................................................................................................. 82 1.III.B Tissue surveillance ...................................................................................... 84 1.III.C Antigen uptake ............................................................................................ 88 1.III.D Antigen processing ..................................................................................... 98 1.III.E Maturation ................................................................................................