Overexposure to apoptosis via disrupted glial specification perturbs Drosophila macrophage function and reveals roles of the CNS during injury

Abstract

Apoptotic cell clearance by phagocytes is a fundamental process during development, homeostasis and the resolution of inflammation. However, the demands placed on phagocytic cells such as macrophages by this process, and the limitations these interactions impose on subsequent cellular behaviours are not yet clear. Here, we seek to understand how apoptotic cells affect macrophage function in the context of a genetically tractable Drosophila model in which macrophages encounter excessive amounts of apoptotic cells. Loss of the glial-specific transcription factor Repo prevents glia from contributing to apoptotic cell clearance in the developing embryo. We show that this leads to the challenge of macrophages with large numbers of apoptotic cells in vivo. As a consequence, macrophages become highly vacuolated with cleared apoptotic cells, and their developmental dispersal and migration is perturbed. We also show that the requirement to deal with excess apoptosis caused by a loss of repo function leads to impaired inflammatory responses to injury. However, in contrast to migratory phenotypes, defects in wound responses cannot be rescued by preventing apoptosis from occurring within a repo mutant background. In investigating the underlying cause of these impaired inflammatory responses, we demonstrate that wound-induced calcium waves propagate into surrounding tissues, including neurons and glia of the ventral nerve cord, which exhibit striking calcium waves on wounding, revealing a previously unanticipated contribution of these cells during responses to injury. Taken together, these results demonstrate important insights into macrophage biology and how repo mutants can be used to study macrophage-apoptotic cell interactions in the fly embryo. Furthermore, this work shows how these multipurpose cells can be 'overtasked' to the detriment of their other functions, alongside providing new insights into which cells govern macrophage responses to injury in vivo.

Live cell tracking of macrophage efferocytosis during Drosophila embryo development in vivo

“…Next, using DRAQ7 dye to label the laser-induced necrotic corpses, we visualized inflammation in wild-type and repo embryos by means of three-color imaging. Although repo mutant macrophages have impaired inflammatory chemotaxis, sufficient macrophages are recruited to analyze efferocytosis ( 22 ). Wild-type macrophages cleared all DRAQ7-labeled necrotic corpses within 30 to 60 min (Fig.…”

“…4, E and F, and fig. S10B) (20)(21)(22). This resulted in more macrophages with high and extreme corpse burdens and a near absence of macrophages with low burden.…”

Macrophage subpopulation identity inDrosophilais modulated by apoptotic cell clearance and related signalling pathways

“…Loss of Simu, an apoptotic cell receptor, drives expansion of specific plasmatocyte subpopulations We previously described how numbers of haemocytes in subpopulations defined by the VT17559, VT32897, VT57089 and VT62766 enhancers are reduced in a genetic background containing excess apoptotic cells (repo mutants; Armitage et al, 2020;Coates et al, 2021). To explore the mechanistic basis for this effect, we examined the effect of loss of simu (six microns under); simu encodes a receptor used by macrophages to recognise phosphatidylserine present on the surface of apoptotic cells (Kurant et al, 2008).…”

“…To investigate the functional impact of amo mutations in the presence of elevated apoptotic challenge – i.e., in a repo mutant background (Armitage et al, 2020; Shklyar et al, 2014) – lysotracker staining was utilised to visualise defects in phagosome acidification. Phagosome acidification occurs during the later stages of efferocytosis, downstream of engulfment (Kinchen & Ravichandran, 2008), and is a calcium-dependent process (Westman et al, 2019).…”

Understanding the diversity and dynamics of in vivo efferocytosis: Insights from the fly embryo

“…The macrophages and phagocytic glia of the Drosophila embryo co‐operate to clear apoptosis within the developing CNS (Figure 2). This is underscored by the overburdening of macrophages in embryos that lack all glia 96 . However, the macrophages and the embryonic glia are strikingly different phagocytes.…”