Abstract
Apoptosis and efferocytosis are central to maintaining homeostasis and implicated in numerous pathological conditions. Elucidation of these processes during development and disease will help further define mechanism, cellular identity and dynamics in vivo. To date, the identification and characterization of apoptotic cell death and clearance of apoptotic bodies is limited to histological markers, co-localization imaging, synthetic dyes, and antibodies. The lack of tools to monitor apoptosis and phagocytosis in vivo highlight the need for development of genetically encoded reporters.
To meet this need, we developed a genetically encoded dual fluorescent reporter, denoted ‘CharON’ after the Greek mythical character who ferries the recently deceased across the river separating the living from the dead, that can concurrently track both emerging apoptotic cells and their efferocytic clearance by phagocytes. Through use of CharON transgenic Drosophila, we track and validate the key steps of efferocytosis in vivo, and for the first time directly measure coordinated clearance of apoptotic corpses in a living organism. Using CharON, we are able to discern novel challenges macrophages face during efferocytosis in vivo. For instance, when macrophages are confronted with a dense field of apoptotic corpses in vivo, macrophages adopt a strategy which prioritizes debris uptake over corpse acidification and degradation. As a result of this expedited clearance, individual macrophages display huge variation in corpse burden, with additional consequences. When challenged with a necrotic wound, macrophages with high apoptotic corpse burden are impaired in necrotic debris uptake, despite migrating to the wound site. Furthering this idea, enforcing phagocyte ‘eating limits’ via modeling suggests that macrophages benefit from ‘unrestrained’ uptake and variable corpse burden, as the alternative of ‘equal distribution’ drastically increases clearance time. These findings suggest that macrophages ‘gamble’ or make a functional compromise, wherein they maximize clearance of developmental / homeostatic apoptosis that is occurring immediately, while potentially jeopardizing the ability to resolve future tissue damage. Collectively, these live tracking studies advance and unveil new concepts in macrophage efferocytosis in vivo.
As a follow up to CharON, we develop a second reporter, termed ‘CharOFF’, which is tailored for both time-lapse imaging and flow cytometry-based analysis of efferocytosis. This novel probe can detect internalization and phagosome acidification, permitting us to dissect these steps at a cellular and population level. Using the CharOFF probe, we develop a ‘One-Dish’ co-culture efferocytosis assay, which features an inducible death system for on-demand induction of apoptotic targets. This system can report efferocytosis of several target cell types, demonstrating the wide applicability of CharOFF. Finally, we develop a ‘One-Dish, Multi-Target’ system termed ‘Dubbel’ which is able to track single and multi-corpse engulfment events. Using Dubbel we uncover interesting trends with regards to macrophage ‘priming’, whereby engulfment of a corpse enhances the ability of a macrophage to eat a subsequent corpse. Findings from these studies provide a foundation for elucidating the response of efferocytes during single and multi-corpse engulfment. Collectively, these data provide novel insights into apoptosis and efferocytosis in vivo based on new genetically encoded probes.
