Kelly, Christopher James (2019) Profiling chemokine signalling bias of CCR4, CCR7 and CCR10. PhD thesis https://theses.gla.ac.uk/41049/ Copyright and moral rights for this work are retained by the author A copy can be downloaded for personal non-commercial research or study, without prior permission or charge This work cannot be reproduced or quoted extensively from without first obtaining permission in writing from the author The content must not be changed in any way or sold commercially in any format or medium without the formal permission of the author When referring to this work, full bibliographic details including the author, title, awarding institution and date of the thesis must be given Enlighten: Theses https://theses.gla.ac.uk/ [email protected] 1 Profiling chemokine signalling bias of CCR4, CCR7 and CCR10 Christopher James Kelly A thesis submitted to the College of Medicine, Veterinary and Life Sciences, University of Glasgow in fulfillment of the requirements for the degree of Doctor of Philosophy October 2018 Institute of Infection, Immunity and Inflammation University of Glasgow 120 University Place G12 8TA 2 Author’s declaration I declare that, except where explicit reference is made to the contribution of others, that this thesis is the result of my own work and has not been submitted for any other degree at the University of Glasgow or any other institution. Signature .................................................... Printed name: Christopher James Kelly 3 List of abbreviations 7 transmembrane domains (7TM) Adult t-cell leukaemia/lymphomas (ATLL) Angiotensin 2 type 1 receptor (AT1R) Atypical chemokine receptors (ACKRs) Bioluminescence resonance energy transferred (BRET) Bone marrow derived dendritic cell (BMDC) Calcitonin gene-related peptide (CGRP) Cells from 4 days into the BMDC differentiation protocol (D4) Cells from 7 days into the BMDC differentiation protocol (D7) Central memory T-cells (Tcm) Central nervous system (CNS) Cutaneous lymphocyte antigen positive (CLA+) CXCL12-abundant reticular (CAR) cells Dendritic cells (DCs) Dopamine D2 receptor (D2R) Double negative thymocytes (DNTs) Double positive thymocytes (DPT) Doxycycline (DOX) Dulbecco’s minimal essential medium (DMEM) Dulbecco’s phosphate buffered saline, calcium and magnesium free (DPBS) Effector memory T-cells (Tem) Endoneuraminidase-N (endo-N) Foetal calf serum (FCS), G-protein coupled receptor kinases (GRKs) G-protein coupled receptors (GPCRs) Gene/genes of interest (GOI) Granulocyte-macrophage colony stimulating factor (GM-CSF) Guanidine diphosphate (GDP) 4 Guanidine nucleotide exchange factor (GEF) Guanidine triphosphate (GTP) Hanks balanced salt solution (HBSS, Gibco) Hematopoietic stem cells (HSC) High endothelial venules (HEVs) High fidelity (HF) Human immune deficiency virus (HIV) Human T-lymphotropic virus 1 (HTLV-1) Imiquimod treated (Imq) Immunoprecipitation (IP) Interferon alpha (INFa) Interleukin 4 IL-4 Knockout mouse strain (KO) Lipopolysaccharide (LPS) -Logarithm of 50% maximal response (pEC50) -Logarithm of dissociation constant (pKi) Lymph nodes (LNs) Mean fluorescence intensity (MFI) Medullary thymic epithelial cells (mTECs) Mesenchymal stem cells (MSCs) Mitogen-activated protein kinase (MAPK) Molecular weight (MW) Nanoluciferase (nLuc) Natural killer (NK) Neural cell adhesion molecule (NCAM) Neuropilin 2 (NRP2) No-chemokine control (NC) Percentage staining/band density compared to WT (%WT) Pertussis toxin (PTX) 5 Polyethylenimine (PEI) Polymerase chain reaction (PCR) Polysialic acid (pSia) Post-translational modifications (PTMs) Rat corticotropin releasing factor receptor (CRF1R) Regulatory T-cell (Treg) Relative efficacy (RA) Renilla luciferase (rLuc) Reverse transcription quantitative PCR (qPCR) Rheumatoid arthritis (RA) Secondary lymphoid organs (SLOs), Single positive (i.e. CD4+ or CD8+) thymocytes (SPTs) Sphingosine-1-phosphate 1 receptor (S1P1R) Standard deviation (SD) Streptavidin-BV421 (SABV421) Streptavidin-PE (SAPE) Systematic protein affinity strength modulation (SPASM) T-helper 2 (Th2) TATA binding protein (TBP) Tertiary lymphoid organs (TLOs) Tissue culture (TC) Toll like receptor (TLR) Toll-like receptor 7 (TLR7) Tris-acetate-EDTA (TAE) Tumour associated macrophages (TAMs) Tumour necrosis factor alpha (TNFa, Miltenyi) Unnatural amino acids (UAA) Unstimulated bone marrow (D0) Vascular endothelial growth factor D (VEGF-D) 6 Vehicle treated (Veh) Wildtype (WT) Yellow fluorescent protein (YFP) 7 Acknowledgements I would like to thank my supervisors Gerard Graham and Graeme Milligan for their support and advice over the years, and for giving me the freedom to follow my own path. I would also like to thank the GLAZgo discovery centre for funding and supporting me throughout my time at the university. Thanks to everyone on level 3 (and beyond), past and present. It’s been a fantastic experience getting to know everyone and I really couldn’t have asked for a better or friendlier environment to spend the last 4 years in. I think it’s fair to say I’ve made a number of lifelong friends here. I would especially like to thank Sam and Robin, who did take on the slightly onerous task of living with me. I’m not easy to live with at the best of times, and don’t imagine I improved during the final few stages. I definitely wouldn’t have made over the finish line without you guys, and I owe you a lot for that. Lastly, I’d like to thank my entire family, but in particular my parents, without whom I both literally and figuratively wouldn’t be here. It’s not always been easy, but you helped turn impossible in to not-easy on more than one occasion, and I don’t think I can thank you enough for that. I promise, this time I will actually stop being a student once I’ve finished here. 8 Table of Contents List of abbreviations ......................................................................................................................................... 3 Acknowledgements ........................................................................................................................................... 7 List of figures/tables ..................................................................................................................................... 11 Abstract ............................................................................................................................................................ 16 1 Introduction ........................................................................................................................................... 18 1.1 Overview ........................................................................................................................................ 18 1.2 The chemokines .............................................................................................................................. 19 1.2.1 Homeostatic chemokines .......................................................................................................... 21 1.2.2 Inflammatory chemokines ........................................................................................................ 22 1.3 The chemokine receptors ............................................................................................................... 23 1.3.1 Chemokine receptor signalling ................................................................................................. 23 1.3.2 Atypical chemokine receptors ................................................................................................... 25 1.3.3 Chemokine/receptor interaction ................................................................................................ 25 1.3.4 Pharmacological targeting of the chemokine system ................................................................ 27 1.3.5 Biased agonism in chemokine biology ..................................................................................... 28 1.4 CCR4 in health and disease ........................................................................................................... 31 1.5 CCR7 in health and disease ........................................................................................................... 34 1.6 CCR10 in health and disease ......................................................................................................... 36 1.7 Aims of the project ......................................................................................................................... 38 2 Materials and Methods ......................................................................................................................... 45 2.1 RNA extraction and cDNA conversion .......................................................................................... 45 2.2 PCR, DNA digest and purification ................................................................................................ 45 2.2.1 PCR ........................................................................................................................................... 45 2.2.2 DNA digests .............................................................................................................................. 46 2.3 Cloning and sub-cloning, mutagenesis programme, bacterial transformation and culture, and plasmid purification ..................................................................................................................................... 47 2.3.1 Cloning and sub-cloning ..........................................................................................................