Macrophage Regulation in Atherosclerosis A thesis submitted to the Faculty of Medicine, Imperial College London for the degree of Doctor of Philosophy Mika André Falck-Hansen SUPERVISORS Professor Claudia Monaco Kennedy Institute of Rheumatology NDORMS, University of Oxford Professor Rob Krams Department of Bioengineering Faculty of Engineering Imperial College London London, United Kingdom September 2013 “If you would be a real seeker after truth, it is necessary that at least once in your life you doubt, as far as possible, all things.” – René Descartes “Discovery consists of seeing what everybody has seen and thinking what nobody has thought.” – Albert Szent-Györgyi Abstract Atherosclerosis is the leading cause of cardiovascular disease, topping the mortality list in the United Kingdom and the rest of the world. Macrophage activation and polarisation are key steps in host defence and chronic inflammatory diseases, including atherosclerosis. The myeloid glycoprotein receptor CD200R1 belongs to a family of four isoforms and signals after binding to its cognate ligand, CD200. The CD200/CD200R1 interaction blocks pro-inflammatory cytokines and has never before been studied in atherosclerosis. My work has demonstrated that CD200R is weakly expressed in atherosclerotic lesions during disease progression and significantly down-regulated in secondary lymphoid organs. Moreover, changes in shear stress had no effect on the expression of CD200R in plaques. During the steady state, the expression of CD200R on circulating monocytes was highest on the CD11b+CD115+Ly6Cint subset, revealing a potential mechanism for regulation of maturing monocytes. In vitro studies demonstrated that CD200R expression is sensitive to M1 macrophage polarising cytokine IFN-γ and MyD88-dependent TLR ligands. In contrast, non-MyD88-dependent TLR3 signalling had no effect on its expression, supporting previous findings in the literature. Moreover, CD200R1 mRNA expression was induced on alternatively activated M2a macrophage subsets following IL-4 and IL-13 cytokine treatment, whereas oxLDL had no effect. Immunotherapy with CD200-Fc in ApoE-/- mice exerted no significant changes on lesion size and phenotype compared to controls after nine weeks. My findings indicate that the type of inflammatory stimulus may play a role in dictating the ability of myeloid cells to terminate their own activation via CD200/CD200R1 signalling. Hence, chronic inflammation may be promoted by reduced presence of inhibitory signals leading to sustained, unresolved inflammation. In summary, my work has revealed new insight into the regulation of the inhibitory CD200R pathway in atherosclerosis, the molecular signals that may affect the regulation of inflammation through CD200R and possible future therapeutic strategies. I Declaration This thesis is the result of my own work with the exception of microsurgery implants and injections done in collaboration with M.E.G. and J.E.C. All collaborations have been acknowledged. The work was carried out at the Kennedy Institute of Rheumatology, Faculty of Medicine, and Department of Bioengineering, Faculty of Engineering, Imperial College London and was funded by the Kennedy Trust for Rheumatology Research. The copyright of this thesis rests with the author and is made available under a Creative Commons Attribution Non-Commercial No Derivatives licence. Researchers are free to copy, distribute or transmit the thesis on the condition that they attribute it, that they do not use it for commercial purposes and that they do not alter, transform or build upon it. For any reuse or redistribution, researchers must make clear to others the licence terms of this work. Mika André Falck-Hansen September 2013 II Acknowledgements This thesis would not have been possible without the contribution of numerous friends and colleagues. First and foremost I would like to thank and acknowledge my main supervisor Professor Claudia Monaco for securing an interesting and challenging project and for always being thorough and rigorous on feedback of written and experimental work and finally, for encouraging the dissemination of my work to the wider scientific community. I would also like to thank Professor Rob Krams for hosting me in his laboratory for a period to develop 3-D histology on murine atherosclerotic arteries, for providing advice on the scientific methodology and reviewing my work along the way. A token of gratitude to Dr Ewa Paleolog for always following-up with the Kennedy Institute students and coordinating progress reports through our studies, Dr Kim Midwood for mentoring and giving helpful advice. A special thanks to the members of the Monaco Lab, past and present: Mike, Jennifer, Joe, Louise, Amanda, Nagore, Leena, Ilona, Anusha, Ida, Christina and Inhye for helping with experimental work and the daily routine in the lab. All the friends and colleagues from other labs: Jonny, Adam, Leandro, Salvo, Simone, Eugénie, Barbara, Helene, Phil, Anna, Szymon, Tim, David, Sunil, Sara, James, Felix, Parisa, Emily, Bernard, Trish (Kennedy Institute) and Zoltán, Vikram, Sandra, Ryan, Nico, Jennifer, Taka (Bioengineering), Bob (NIMR), Brian (Minnesota), Bjørn (Stockholm) and Kim (NTNU). Without the love and support from my fiancé Ania, it would have been quite a different experience to get through this research project and I am forever grateful for having you by my side during this journey. Thank you to my brother for moving to London so we could be closer to each other after many years apart, and my grandparents, mother and wider family for their unfailing support. A big thanks to my father for always being there for me since my first day of school in 1989 and for visiting me at every place I have lived since moving from home. Finally, this thesis is dedicated to Mr Kjell Arnljot Wig for inspiration to conduct research, to dream and to believe in myself. III List of Abbreviations AF Alexa fluor ANOVA Analysis of variance AP-1 Activator protein 1 APC Allophycocyanin APCs Antigen presenting cells ApoE Apolipoprotein E ARE Antioxidant response element ARG Arginase ASC Apoptosis-associated speck-like protein containing a CARD BMDM Bone marrow derived-macrophage BMI Body Mass Index C57BL/6 Black 6 inbred mice CARD Caspase recruitment domain CCL Chemokine ligand CCR Chemokine receptor CD Cluster of differentiation CD11b MAC-1 antigen CD14 TLR4 co-receptor CD25 IL-2 receptor CD68 Macrosialin, Scavenger Receptor CD68 CD115 MCSF receptor CD154 CD40 ligand (CD40L) CD163 Scavenger receptor CD163 CD200 CD200 ligand CD200R1 CD200 receptor 1 CD206 Mannose receptor CD36 Scavenger receptor CD36 CD45RO Protein tyrosine phosphatase, receptor type C cDNA Complementary DNA IV CHD Coronary heart disease CIA Collagen induced arthritis COX-2 Cyclooxygenase 2 CRP C-reactive protein CVD Cardiovascular disease CX3CR1 Fractalkine receptor CXCL Chemokine CXC motif ligand DAMP Damage-associated molecular pattern DC Dendritic cell DMEM Dulbecco’s modified eagle medium DOK Downstream of tyrosine kinase EAE Experimental autoimmune encephalomyelitis EC Endothelial cell ER Endoplasmic reticulum ESL-1 E-Selectin ligand 1 EVG Elastin van Gieson FACS Fluorescence-activated cell sorting FBS Foetal bovine serum Fc Fragment crystallisable FcγRIII Fc receptor/CD16 FITC Fluorescein isothiocyanate Fizz-1 Found in inflammatory zone 1 FOXP3 Forkhead box P3 FSL-1 Synthetic diacylated lipoprotein GCSF Granulocyte colony-stimulating factor GMCSF Granulocyte-macrophage colony-stimulating factor GR Galactose receptor GR1 Granulocyte differentiation antigen 1 HDL High density lipoprotein HLA-DR MHC class II cell surface receptor HLH Haemophagocytic lymphohistiocytosis HO-1 Heme-oxygenase 1 V HODE 9- and 13-hydroxyoctadecadienoic acid HSS High shear stress I-A/I-E Murine MHC class II ICAM-1 Intracellular adhesion molecule 1 IFN-γ Interferon gamma Ig Immunoglobulin IHC Immunohistochemistry IkBa Nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha IKK IκB kinase IL Interleukin IL-1Ra IL-1 receptor antagonist IL-1R IL-1 receptor type I ILR2 Interleukin 1 receptor type II, decoy receptor IMR Intima/media ratio iNOS/NOS2 inducible nitric oxide synthase IRAK Interleukin-1 receptor-associated kinase IVUS Intravascular ultrasound JUPITER Justification for the Use of Statins in Primary Prevention: An Intervention Trial Evaluating Rosuvastatin LDL Low density lipoprotein LDLR Low density lipoprotein receptor LOX-1 Lectin-like oxLDL receptor LPS Lipopolysaccharide LSS Low shear stress Ly6C Lymphocyte antigen 6 complex M1 Classically activated macrophage M2 Alternatively activated macrophage MAL MyD88-adaptor-like MCP-1 Monocyte chemotactic protein-1 MCSF Macrophage colony-stimulating factor MD2 Lymphocyte antigen 96 VI MFI Mean fluorescence intensity MHC II Major histocompatibility complex, class two MI Myocardial Infarction MMLV-RT Moloney murine leukaemia virus reverse transcriptase MMP Matrix metalloproteinase MOX Mox macrophage mRNA Messenger RNA MS Multiple sclerosis MTHR Methylene tetrahydrofolate reductase MyD88 Myeloid differentiation primary response gene 88 Mφ Macrophage NALP3 NACHT, LRR and PYD domains-containing protein 3 NF-κB Nuclear factor kappa B NK Natural killer cell NO Nitric oxide NRF2 NF-E2-related factor-2 OCT Optimal cutting temperature 6-OHDA 6-hydroxydopamine OSS Oscillatory shear stress OxLDL Oxidised low-density lipoprotein PAMP Pathogen associated molecular pattern PAPC 1-palmitoyl-2-arachidonoyl-sn-3-phosphorylcholine PBS Phosphate buffered saline PDGF Platelet-derived