부산대학교 도서관

Macrophages dynamically polarise into specialised phenotypes to perform an array of functions as part of the innate immune response. Bone-marrow macrophages interact with erythroblasts (red blood cell precursors) through a complex array of cell-cell interactions forming erythroblastic islands (EBIs). EBIs allow erythropoiesis to occur at an efficiency and scale that cannot be recapitulated in vitro. Understanding macrophage polarisation and manipulating their phenotypes provides an essential tool; to modulate macrophages within inflammatory disease, to determine their role in processes such as erythropoiesis, and for their exploitation as cellular therapeutics. This thesis uses in-depth comparative proteomics, alongside flow cytometry analyses and functional assays to compare macrophage subtypes (MØ, M1, M2a, M2c) generated from CD14⁺ monocytes. Interestingly, >50 % of subtype proteomes differ from each other and have specific functional characteristics illustrating the potential of this system for exploring polarisation dynamics. Treatment of interferon stimulated M1 pro-inflammatory macrophages with dexamethasone induced a partial repolarisation towards an M2c state highlighting the retention of plasticity within this in vitro model. Further, investigations into the re-polarisation of macrophages highlighted the rapid alteration of cell phenotype through targeting of the pro-inflammatory microRNA miR223, which holds potential in cancer therapeutics. A genetically tractable in vitro EBI model was established to explore the mechanisms by which macrophages support erythropoiesis. In combination with a high-throughput quantitative image analysis pipeline, this model determined the contribution of αV and β2 integrins, identified through surface proteomics, to EBIs in humans for the first time and identified a novel role for the P2X₄ mechanoreceptor in macrophage motility. The work presented here provides a comprehensive characterisation of human cultured macrophages, highlighting how subtypes facilitate differing functions and the potential for modulating their phenotype. The development of a model system for identification of proteins crucial for EBI formation provides a potential tool for optimising erythroblast culture methodology. Ultimately assisting in the development of protocols to recapitulate the efficiency and scale of human erythropoiesis.