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. We show that loss of the glial transcription factor repo, and corresponding removal of the contribution these cells make to apoptotic cell clearance, causes macrophages in the developing embryo to be challenged with large numbers of apoptotic cells. 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.
Introduction
Understanding the interactions between macrophages and apoptotic cells is an important biological question: failures in how immune cells deal with apoptotic cell death can lead to damaging autoimmune conditions [1]. Apoptotic cell clearance also plays a critical role in the resolution of inflammation and reprogramming immune cells during this process [2]. Furthermore, interactions between dying cells and macrophages occur at numerous sites of pathology including at sites of atherosclerosis [3] and in the chronically inflamed lungs of COPD patients [4]. As such, macrophage-apoptotic cell interactions have the potential to impact these diseases and many other damaging human conditions [1].
Drosophila has proven an excellent organism with which to study innate immunity [5], haematopoiesis [6] and blood cell function [7]. Drosophila blood is dominated by macrophage-like cells (plasmatocytes), with these cells making up 95% of the blood cells (hemocytes) in the developing embryo [8]. Embryonic macrophages disperse over the entire embryo during development, phagocytosing apoptotic cells and secreting matrix as they migrate [7]. Alongside their functional and morphological similarities to vertebrate macrophages, Drosophila macrophages are specified through the action of related transcription factors to those used in vertebrate haematopoiesis [9]. Failed dispersal or ablation of these embryonic macrophages leads to developmental abnormalities and failure to hatch to larval stages [10–12]. Drosophila macrophages are also able to respond to injury, mounting inflammatory responses to epithelial wounds [13]. In embryos and pupae, wounding elicits a rapid calcium wave through the epithelium, a process that requires transient receptor potential (Trp) channel function [14,15]. The increase in cytoplasmic calcium drives activation of Dual Oxidase (DUOX) and production of hydrogen peroxide, which is necessary for immune cell recruitment [14], resembling events upon tissue damage in higher organisms such as zebrafish [16,17].
In addition to macrophages, the other main phagocyte population within Drosophila embryos is the glia of the developing nervous system. Drosophila embryonic macrophages interact with the developing ventral nerve cord (VNC) and the glial cells that encase it, as they disperse along the ventral side of the embryo [18]. The VNC contains two populations of glia, the Sim-positive midline glia that help establish the ladder-like structure of neurons early in development and Repo-positive lateral glia [19]. Repo is a homeodomain transcription factor that specifies glial fate [20–22] and is required for expression of at least some phagocytic receptors in these cells [23]. While there are transcription factors in common between blood cells and glia (e.g. Gcm and Gcm2) [24,25], they are derived from distinct progenitors and Repo antagonises haematopoiesis to promote a glial fate [26]. Nonetheless both phagocytic populations express a similar repertoire of receptors for apoptotic cells, including Draper and Simu [27]. The close interactions between these glia and macrophages means that, should one population fail to clear apoptotic cells, it is likely that the other population would be able to both detect this deficiency and be able to compensate. Loss of apoptotic cell receptors such as Simu leads to a build up of apoptotic cells within developing embryos [28] and this impairs macrophage behaviours, including their developmental dispersal and inflammatory responses [29]. However, whilst mutants such as simu enable exposure of macrophages to elevated numbers of apoptotic cells in vivo, this approach is not ideal, as it also perturbs receptors that may be involved in immune cell reprogramming [30,31].
To investigate interactions between macrophages and apoptotic cells in more detail, we stimulated macrophages with enhanced levels of apoptotic cells using a genetic approach that did not alter the macrophages themselves. By impairing glial differentiation using repo mutants, we increased the number of apoptotic cells macrophages face within the developing embryo. This enhanced apoptotic challenge impaired macrophage dispersal, migration and their inflammatory responses to wounds. In this background, clearance of apoptotic cells by macrophages is not perturbed and migration can be rescued by preventing apoptosis. Surprisingly, and in contrast to phagocytic receptor mutants, blocking apoptotic cell death in the presence of defective glia failed to rescue wound responses. Further analysis revealed that injury-induced calcium waves propagate beyond the wounded epithelium and that this process is defective in repo mutants. This suggests that glial cells play an active role in the propagation of even the earliest responses to wounding. Thus, this model provides a unique insight into how macrophage-apoptotic cell interactions dictate macrophage responses to injury and the cell types that contribute to the activation of those responses.
Materials and methods
Fly lines and husbandry
Flies were reared at 25°C on cornmeal/agar/molasses media (see Supplementary Table 1 for recipe). Srp-GAL4 [32] and crq-GAL4 [13] were used to label Drosophila macrophages in the embryo in combination with UAS-GFP and/or UAS-red stinger [33]; e22c-GAL4 (VNC and epithelium) [34], act5C-GAL4 (ubiquitous) [35], da-GAL4 (ubiquitous) [36], elav-GAL4 (neuronal) [37] and repo-GAL4 (glial cells) [38] were used to drive expression in other tissues. UAS-GCaMP6M [39] was used to image changes in cytoplasmic calcium concentration. Experiments were conducted on a w1118 background and the following mutant alleles were used: Df(3L)H99 [40], repo03702 [20–22], simu2 [28]. See Supplementary Table 2 for a full list of genotypes used in this study and sources of the Drosophila lines used. Embryos were collected from apple juice agar plates on which flies had laid overnight at 22°C. Embryos were washed off plates with distilled water and dechorionated in bleach for 1-2 minutes. Bleach was thoroughly washed away with distilled water ahead of fixation or mounting of embryos for live imaging. The absence of the fluorescent balancers CTG, CyO dfd, TTG and TM6b dfd [41,42] was used to select homozygous mutant embryos after dechorionation.
Fixation and immunostaining
Dechorionated embryos were fixed and stained as per Roddie et al., 2019 [29]. Antibodies were diluted in PATx (0.1% Triton-X100 (Sigma-Aldrich), 1% BSA (Sigma-Aldrich) in PBS (Oxoid, Thermo Fisher, MA, USA)). Rabbit anti-GFP (ab290 1:1000; Abcam, Cambridge, UK) or mouse anti-GFP (ab1218 1:200; Abcam) were used to detect GFP-labelled macrophages. Rabbit anti-cDCP-1 (9578S 1:1000; Cell Signaling Technologies), mouse anti-Repo (concentrate of clone 8D12 used at 1:1000; Developmental Studies Hybridoma Bank, University of Iowa, USA) or mouse anti-Futch (supernatant of clone 22C10 used at 1:200; Developmental Studies Hybridoma Bank) were also used as a primary antibodies. Goat anti-mouse or goat anti-rabbit secondary antibodies conjugated to AlexaFluor568, AlexaFluor488 (Invitrogen, Thermo Fisher) or FITC (Jackson Immunoresearch, Cambridge, UK) were used to detect primary antibodies; these were diluted from stock solutions made according to the recommendations of the supplier (1:400 in PATx). Stained embryos were stored in DABCO mountant (Sigma-Aldrich) and mounted on slides for imaging.
Imaging and analysis
Immunostained embryos were imaged using a 40X objective lens (CFI Super Plan Fluor ELWD 40x, NA 0.6) on a Nikon A1 confocal. Embryos containing GFP-labelled macrophages were stained for GFP and cDCP-1 and the phagocytic index (number of cDCP-1 punctae per macrophage per embryo), a measure of apoptotic cell clearance, was calculated from at least ten macrophages on the ventral midline in each embryo as per Roddie et al., 2019 [29].
Developmental dispersal was assessed by counting numbers of segments lacking GFP-labelled macrophages on the ventral side of the VNC in stage 13/14 embryos. Embryos that had been fixed and stained for GFP were scored using a Leica MZ205 FA fluorescent dissection microscope with a PLANAPO 2X objective lens. The same embryos were orientated ventral-side-up, imaged on a Nikon A1 confocal, and numbers of macrophages between epithelium and VNC counted (segments 4-8 were scored).
For live imaging of macrophage morphology, migration speed and wound responses and calcium dynamics upon injury, live embryos were mounted in voltalef oil (VWR) as per Evans et al., 2010 [18] and imaged on a Perkin Elmer UltraView Spinning Disk system using a 40X objective lens (UplanSApo 40x oil, NA 1.3). Epithelial wounds were made on the ventral side of embryos using a nitrogen-pumped Micropoint ablation laser (Andor, Belfast, UK), as per Evans et al., 2015 [43]. Macrophage inflammatory responses to wounds were imaged and analysed as per Roddie et al., 2019 [29]. Macrophage wound responses (numbers of macrophage at or touching the wound edge at 60 minutes divided by wound area in μm2) were normalised to control levels. The percentage of macrophages responding (% responders) was calculated by counting the proportion of macrophages present immediately following wounding that reached the wound within 60 minutes; macrophages already at the wound or absent from the field of view immediately after wounding were not considered in this analysis. Numbers of macrophages in the image taken prior to wounding (pre-wound) were counted as a measure of macrophages available to respond to the injury. The percentage of macrophages leaving the wound (% leavers) is the proportion of macrophages present at the wound site at any point during a 60-minute movie that leave the wound site; macrophages retaining contacts with the wound site or edge were not scored as leavers.
For analysis of random migration, 60-minute movies of GFP and red stinger double-labelled macrophages were made with macrophage position imaged every 2 minutes on the ventral midline between the overlying epithelium and VNC. Image stacks were despeckled and the movements of macrophages within maximum projections tracked as per Roddie et al., 2019 [29]. The manual tracking and Ibidi chemotaxis plugins were used to calculate speed per macrophage, per embryo, in Fiji [44].
Calcium dynamics were imaged using GCaMP6M expressed using a range of GAL4 lines. Calcium responses were quantified from average projections of z-stacks collected immediately before and after wounding. Initial wound responses of projections corresponding to epithelial z-slices (from da-GAL4,UAS-GCaMP6M embryos) were quantified by measuring the area of the GCaMP6M response and the corresponding mean gray value (MGV) of this region of interest (ROI) in Fiji immediately after wounding (F1). The same ROI was then used to measure the MGV of GCaMP6M fluorescence in the pre-wound image (F0). Both the area of the initial response and the F1/F0 ratio of MGVs were used as measures of the calcium response to injury (Figure 7c-d). For analysis of glial responses and the cells within the VNC more broadly (neurons and glia), repo-GAL4 and e22c-GAL4 were used to drive GCaMP6M expression, respectively. Responding glial cells were manually selected using the freehand selection tool in Fiji (Figure 8h-i), while the outer edge of the vitelline membrane was used to define a ROI to quantify GCaMP6M intensity for projections constructed from deeper volumes of the z-stack (Figure 8c).
Image processing and statistical analyses
Images were despeckled in Fiji before maximum or average projections were made. All images were blinded ahead of quantification and Adobe Photoshop was used to assemble figures. Statistical analyses were performed in GraphPad Prism; legends and text contain details of statistical tests used. N numbers report numbers of individual Drosophila embryos analysed. Immunostained embryos are representative examples taken from batches of pooled embryos collected across multiple days.
Results
Embryonic macrophages disperse in close contact with the developing central nervous system
Drosophila embryonic macrophages migrate out from the presumptive head region to disperse over the entire embryo during development [7]. Migration along the developing ventral nerve cord (VNC) is an essential route for macrophage dispersal, with macrophages contacting the overlying epithelium and glial cells on the surface of the nerve cord (Figure 1a-b). During dispersal, macrophages encounter and clear apoptotic cells (Figure 1c), while VNC glia also phagocytose dying cells [45]. The interaction of macrophages and glia suggested to us that impairing glial-mediated apoptotic cell clearance could increase exposure of macrophages to apoptotic cell death in vivo, thus providing a model with which apoptotic cell-macrophage interactions and their effects on macrophage behaviour could be studied in detail.
Increased efferocytosis by embryonic macrophages in the absence of functional glial cells
Both macrophages and glial cells use phagocytic receptors such as Draper and Simu to clear apoptotic cells [28,46,47]. The absence of these receptors perturbs clearance of apoptotic cells, which in turn disrupts macrophage functions, including migration speed and inflammatory responses [29,43]. While this suggests that apoptotic cells modulate macrophage behaviour in flies, these interventions remove genes with key roles in pathways that control macrophage fate [30]. Therefore, in order to expose macrophages to increased amounts of apoptotic cell death without directly impacting their own specification mechanisms or removing important regulators of phagocytosis, we targeted the glia of the VNC. We hypothesised that blocking glial-mediated clearance would lead to macrophages becoming exposed to increased numbers of apoptotic cells due to the inability of glial cells to contribute to this process.
The homeodomain transcription factor Repo is expressed by all glial cells within the developing VNC [20–22], except the midline glia, which are specified via the action of sim [48]. Repo is absolutely required for normal specification of these glial cells and absence of repo leads to the failure to express a variety of phagocytic receptors required for apoptotic cell clearance [23]. Repo is not expressed by macrophages in the developing embryo (Figure 1d; [38]) and repo03702 mutants lack detectable protein expression in the VNC (Figure 1e).
To test whether failed glial specification would lead to macrophages encountering increased numbers of apoptotic cells in the developing embryo, we analysed macrophage morphology. Macrophages in repo mutants are highly vacuolated compared to controls (Figure 2a-b); vacuoles within Drosophila embryonic macrophages typically contain previously engulfed apoptotic cells [49]. To test whether apoptotic cell clearance by macrophages is increased in repo mutants, control and repo mutant embryos were immunostained for a marker of apoptotic cell death (cleaved DCP-1 immunostaining; DCP-1 is cleaved by caspases during apoptosis [50]). Macrophages in repo mutants contain far higher numbers of cDCP-1 positive inclusions compared to controls (Figure 2c-e). Furthermore, macrophages can be labelled using crq-GAL4 or srp-GAL4 in a repo mutant background and efficiently engulf apoptotic cells (Figure 2), behaviour consistent with their normal specification and physiology. These results therefore suggest that loss of repo function is a suitable tool via which the effects of increased macrophage-apoptotic cell contact can be analysed in vivo.
An increased burden of apoptotic cell clearance is associated with impaired developmental dispersal of macrophages
Apoptotic cells represent the top priority for Drosophila macrophages to respond to within developing embryos [51]. As a result, increased numbers of apoptotic cells have the potential to disrupt macrophage behaviours, such as their dispersal and recruitment to sites of tissue injury. repo mutants lack gross dispersal defects, with macrophages present along both sides of the VNC at stage 13 (Figure 3a-b). However, reduced numbers of macrophages are present on the ventral midline in repo mutants (Figure 3c-e), suggesting an impairment in dispersal. Further increasing apoptotic burden in a repo mutant background (via removal of the apoptotic cell clearance receptor Simu) causes large dispersal defects (Figure 3a-b), comparable to those observed when critical regulators of migration are absent, e.g. SCAR/WAVE [49]. Taken together, these results indicate that challenge of macrophages with excessive amounts of apoptosis can impair developmental dispersal of macrophages.
Apoptotic cells are responsible for attenuation of macrophage migration in repo mutants
To test the effects of increased apoptotic cell exposure on macrophage migration, we tracked the movements of macrophages on the ventral midline at stage 15 after completion of developmental dispersal, a point at which macrophages exhibit wandering/“random migration”. In repo mutant embryos, macrophages move at significantly slower speeds compared to those in controls (Figure 4). In order to test whether this attenuation of migration speed is dependent on interactions with apoptotic cells, we removed all developmental apoptosis from a repo mutant background using the Df(3L)H99 deficiency, which deletes the pro-apoptotic genes Hid, reaper and grim [40]. In these repo mutants that lack apoptosis there is a significant rescue of macrophage migration speed (Figure 4c-e), suggesting that it is interactions with apoptotic cells that impair macrophage migration in vivo, rather than defective glial specification per se.
Functional glia are required for normal macrophage migration to wounds
Given the impaired migration of macrophages in repo mutant embryos, we analysed their inflammatory responses to sterile, laser-induced wounds. Wounding of repo mutant embryos revealed that reduced numbers of macrophages reached wound sites by 60-minutes post-wounding compared to controls (Figure 5a-b,d). Since there are fewer macrophages present locally at this developmental stage in repo mutants (Figure 5c), the percentage of cells responding to the injury was also quantified, revealing a significant decrease in the ability of cells to respond to wounds (Figure 5e; Supplementary Movie 1). Early migration away from wounds does not appear to underlie these defects (Figure 5f), suggesting that excessive amounts of apoptotic cells turn off wound responses in repo mutants. To further evidence this conclusion, we removed developmental apoptosis, comparing wound responses in Df(3L)H99 mutants and Df(3L)H99,repo double mutant embryos. In contrast to the rescue of migration speed, removing apoptosis from a repo mutant background failed to rescue either the numbers of cells responding to the injury or the percentage of cells able to respond (Figure 6). Therefore, the underlying defect in repo mutants that hinders macrophage inflammatory responses does not appear to be contact with apoptotic cells (Figure 6), in contrast to the effect seen for migration defects (Figure 4).
Neurons and glia respond to injury through changes in their calcium dynamics
Since removing the apoptotic cell burden from macrophages in repo mutants failed to improve their inflammatory responses to wounds (Figure 6), we investigated whether upstream signalling mechanisms that form part of the normal response to wounds remain intact in this mutant background. Laser ablation of embryos triggers the rapid spread of a calcium wave through the epithelium, away from the site of damage (Figure 7a; Supplementary Movie 2; [14]). This wave of calcium activates hydrogen peroxide production via DUOX, which is itself required for migration of macrophages to wound sites [14,43,52]. Imaging calcium dynamics after wounding using the cytoplasmic calcium sensor GCaMP6M [39], we were unable to identify a difference immediately post-wounding in the epithelial calcium responses of repo mutant embryos compared to controls (Figure 7a-d; Supplementary Movie 2). However, in performing these experiments we noticed that the calcium response was not limited to the epithelium, with this signal visible deeper within the embryo including within the neurons and glia of the VNC (Figure 7e-g) – a structure located immediately underneath the epithelium on the ventral side of the embryo (Figure 1a-b). These changes in intracellular calcium were not limited to the damaged tissue and extended away from the necrotic core of the wound (Figure 7e-g; Supplementary Movie 3), with changes in calcium levels particularly striking within the axons of the CNS (Figure 7g). Strikingly, quantification of calcium responses in sub-epithelial regions upon wounding showed a reduced calcium response in repo mutants compared to controls (Figure 8a-c), suggesting that glial cells are responsive to injury and contribute to damage-induced signalling.
repo is required for normal calcium responses to injury in the developing embryo
Using the tissue-specific drivers elav-GAL4 (neurons) and repo-GAL4 (glia) to express GCaMP6M confirmed that both neurons and glial cells within the VNC respond to injury by transiently increasing their cytosolic calcium levels (Figure 8d-e; Supplementary Movies 4 and 5). Changes in cytoplasmic calcium are transmitted beyond the confines of the wound (Figure 8d,g) and remain elevated at wound sites several hours post-injury (Supplementary Figure 1). These responses suggest that these cells communicate damage signals away from sites of tissue damage, potentially contributing to inflammatory recruitment of macrophages and regenerative processes. Additionally, the transmission of calcium signals distal to the site of physical injury suggests these responses are not simply limited to those cells damaged during the wounding process.
In repo mutants, while there is less glial proliferation, residual glial cells are present, but lack late glial markers [20,21] and normal patterns of phagocytic receptor expression [23]. In contrast to the wild-type situation, wounding of repo mutants with glial-specific expression of GCaMP6M showed a reduction in the calcium response on injury (Figure 8e-f,h-i). Taken together, these data indicate that, in contrast to our present understanding, tissues other than the epithelium undergo alterations in calcium signalling upon injury. Furthermore, the reduced calcium responses observed in repo mutants on wounding may therefore contribute to the reduced inflammatory responses undertaken by macrophages.
Discussion
Clearance of apoptotic cells is associated with reprogramming of phagocytes such as macrophages towards anti-inflammatory states and is part of the process of resolution of inflammation [2]. Here we show that by impairing glial specification using repo mutants, macrophages can be challenged with increased levels of apoptotic cell death in vivo. Challenging ‘wild-type’ macrophages in this way causes them to become engorged with phagocytosed apoptotic cells. The excessive amounts of apoptosis are associated with impaired developmental dispersal, slowed migration speeds and attenuated inflammatory responses. While migration speeds can be rescued by removing apoptosis from a repo mutant background, wound responses are not improved, suggesting interactions with apoptotic cells do not represent the primary cause of this phenotype. Instead, in contrast to our current understanding, tissues in addition to the wounded epithelium are wound-responsive and glia and the cells they support are likely to contribute to immune cell recruitment and repair mechanisms in Drosophila.
Macrophages in repo mutants exhibit impaired dispersal and reduced migration speeds, phenotypes consistent with other Drosophila mutants that perturb apoptotic cell clearance, including SCAR/WAVE [49], draper [43] and simu [29]. Similarly, removal of the apoptotic clearance burden, via ablation of the apoptotic machinery, improves migration speeds and suggests that apoptotic cells are responsible for these phenotypes. Excessive production of find-me cues – chemoattractants released from apoptotic cells to alert phagocytes to their presence [53] – represent one potential explanation, with such cues distracting or overwhelming macrophages. Consistent with this, apoptotic cells have been inferred to be the most highly-prioritised migratory cue for macrophages in the fly embryo [51]. Currently, this remains hard to investigate further, since the nature and identity of find-me cues remains to be discovered in this organism. Alternatively, reprogramming of macrophages to a different transcriptional or activation state may also explain these phenotypes [54], although little evidence for macrophage polarisation comparable to that observed in vertebrate myeloid cells currently exists for Drosophila.
Phagocytes such as Drosophila macrophages may have to decide whether to engulf or move: for instance both dendritic cells and Dictyostelium amoebae pause during macropinocytosis [55,56], a process related to phagocytosis. Similarly, lysosomal storage disorders that lead to vacuolation are associated with perturbed immune cell migration in patient-derived cells [57–59] and experimental models [60]. Sequestration of regulators of both phagocytosis and motility, such as the lysosomal Trp channel Trpml (the fly homologue of TRPML1/MCOLN1) [61], by excessive phagocytic cup formation in the face of elevated apoptotic cell burdens, could antagonise the ability of phagocytes to migrate. repo mutants represent a model to further understand how apoptotic cells regulate changes in macrophage behaviour in vivo, such as their migration and phagocytic capacities, especially since initial specification of macrophages does not appear compromised in this background and repo plays no direct functional role in these cells.
Macrophage inflammatory responses were not improved by blocking apoptosis in repo mutants. This was surprising, since preventing apoptosis in simu mutants, another background in which macrophages face large numbers of apoptotic cells, did improve macrophage responses to injury [29]. Thus it is unlikely that apoptotic cell-macrophage interactions represent the primary cause of wound recruitment defects in repo mutants. Faster and more sensitive imaging technologies, alongside the use of alternative drivers to express genetically-encoded calcium reporters enabled capture of larger volumes of the embryo during wounding in comparison to previous studies [14]. This revealed that those tissues surrounding the damaged epithelium, such as the neurons and glia of the VNC, were also responsive to injury. Loss of repo function causes defects in glial specification, proliferation [20–22] and also leads to a perturbed calcium response in the VNC and glia. Potentially, repo drives a transcriptional program enabling glia to respond to injury. Failed glial specification may have a “knock-on” effect and hinder the damage responses of neurons, which are supported by glial cells. Alternatively, the decreased number or mispositioning of glial cells in repo mutants [20] may decrease the amplitude or spread of wound signals activated by injury, such as via defective cell-cell contacts between glia. Impairing the ability of surrounding tissues to respond to injury may therefore perturb the generation of wound cues required for normal macrophage migration to sites of tissue damage and could potentially impact evolutionarily-conserved CNS repair processes [62], especially since calcium waves in the developing Drosophila wing disc are required for regeneration following mechanical injury [63].
The use of calcium as a regulator of wound responses is conserved across evolution with calcium waves visible upon transection of zebrafish larval fins [17], and immediately after wounding Xenopus embryos [64] and the C. elegans epidermis [65]. Release of calcium from internal stores during wave propagation is also a commonality in these models. The mechanism of activation in the Drosophila CNS remains to be established, but in zebrafish tailfin wounds the release of ATP from damaged cells may activate P2Y receptors, leading to subsequent release of calcium from internal stores via PKC signalling [66]. Calcium waves can also be observed within the developing zebrafish brain upon wounding, with these dependent upon glutamate-mediated activation of NMDA receptors. NMDA receptors in turn regulate ATP-dependent recruitment of microglial cells to sites of injury [67]. Traumatic brain injury has long been associated with rapid decreases in extracellular calcium (e.g. Young et al., 1982 [68]; for review see Weber, 2005 [69]) and laser-wounding has been proposed as a useful technique to model this [70]. Indeed, buffering calcium dynamics within damaged neurons in flies can be neuroprotective [71], suggesting this system could uncover novel therapeutic strategies for traumatic brain injury.
In summary, we have shown that increasing the apoptotic cell burden placed on macrophages impairs their normal behaviour, dampening their inflammatory responses to tissue damage. Furthermore, we have uncovered that wound-induced calcium waves spread beyond the epithelium into neighbouring tissues, potentially enabling them to contribute to recruitment of macrophages and subsequent repair processes. How these waves spread and how epithelial and non-epithelial tissues integrate these responses to coordinate inflammation and repair remain key questions for future work. Since specification of macrophages is not perturbed in repo mutants, this model will prove useful to examine macrophage-apoptotic cell interactions in vivo in more detail and shed light on cellular interactions that are fundamental for normal development and homeostasis.
Conflict of interest
The authors have no competing interests to declare.
Availability of Data and Materials
Fly lines and raw data are available on request from I.R.E.
Acknowledgements
We thank Will Wood (University of Edinburgh) for advice and support, Frederico Rodrigues (University of Bristol) for performing preliminary experiments and Karen Plant (University of Sheffield) for technical support. Imaging work was performed in the Wolfson Light Microscopy Facility, using the Perkin Elmer spinning disk (MRC grant G0700091 and Wellcome grant 077544/Z/05/Z) and Nikon A1 confocal/TIRF (Wellcome grant WT093134AIA) microscopes. This work would not be possible without the Bloomington Drosophila Stock Centre (NIH P40OD018537) and Flybase (NIH and MRC grants U41 HG000739 and MR/N030117/1, respectively). We thank the Drosophila community for sharing Drosophila reagents (see Supplementary Table 2). We are grateful to Darren Robinson (Wolfson LMF) and the Fly Facility staff (University of Sheffield) for their assistance and Phil Elks, Simon Johnston, Steve Renshaw and Martin Zeidler (University of Sheffield) for critical reading and feedback on the manuscript. This work was supported by a Sir Henry Dale Fellowship awarded to I.R.E. by Wellcome and The Royal Society (102503/Z/13/Z) and a University of Sheffield Medical School PhD position awarded to I.R.E. and H.G.R.