Find me and eat me: understanding how signals from dying cells regulate macrophage behaviour

How multifunctional cells such as macrophages interpret the different cues within their environment and undertake an appropriate response is a key question in developmental biology. Understanding how cues are prioritised is critical to answering this – both the clearance of apoptotic cells (efferocytosis) and the migration towards damaged tissue is dependent on macrophages being able to interpret and prioritise multiple chemoattractants, polarise, and then undertake an appropriate migratory response. Herein, I have used Drosophila melanogaster as a model to assess the dynamics of efferocytosis in vivo using live-caspase fluorophores and examine the role of Spitz, the cardinal Drosophila epidermal growth factor ligand, in regulation of macrophage behaviour in the developing fly embryo. In addition, I have also used ex vivo human macrophages to assay the chemotactic potential of apoptotic find-me cues in a novel adaption of the Ibidi -slide migration system. The results show that misexpression of activated Spitz can impact embryonic macrophage polarity, distribution, migration and perturb apoptotic cell clearance. Imaging of apoptosis and efferocytosis during Drosophila embryogenesis has revealed that caspase activity and clearance by immune cells is not an exclusive pathway, and that apoptotic recruitment appears to be more context and range dependent than previously thought. I have also been able to show that monocyte derived macrophages can be successfully used in the 2D Ibidi -slide assay and that signals derived from apoptotic cells are able to interfere with normal chemotactic responses in a localised fashion. Taken together, these results present several important conclusions: Spitz regulates macrophage migration and may operate as a chemoattractant in certain context, information which may help us to understand the role EGF ligands play in immune cell recruitment during development and at sites of disease pathology; exposure to apoptotic-cell derived find-me cues are able to inhibit macrophage chemotaxis towards the pro-inflammatory chemokine C5a; and efferocytosis in vivo occurs in a more rapid fashion than that observed in vitro, with clearance being only one fate for apoptotic cells.