A PI3K-calcium-Nox axis primes leukocyte Nrf2 to boost immune resilience and limit collateral damage

Macrophages activate the cytoprotective factor Nrf2 upon phagocytic ROS production via PI3K-calcium-Nox signaling during immune surveillance. This protective response is crucial to sustain vital immune functions and limit macrophage acquisition of senescence-like features. Macrophage Nrf2 also acts non-autonomously to limit bystander damage to adjacent healthy tissues.


Introduction
Tissues are constantly exposed to highly reactive and potentially toxic metabolites, such as reactive oxygen species (ROS) derived from cellular metabolism and physiology, or generated upon exposure to radiation or noxious chemicals (Finkel and Holbrook, 2000). Given the uncontrolled accumulation of cellular damage (including oxidative damage to cellular biomolecules) is considered to be a leading cause of tissue damage and aging (Gladyshev, 2014;Liochev, 2013), cells must employ powerful cytoprotective strategies to mitigate indiscriminate, deleterious effects of ROS (and other toxic byproducts) and shield tissues from potential injury (Bise et al., 2019;Burbridge et al., 2021;Telorack et al., 2016;Weavers et al., 2019). Cytoprotective strategies may thus be crucial in vivo to sustain tissue health and delay a progressive decline in tissue function.
Whilst mitochondria-derived ROS have received much attention in relation to tissue damage and aging (Balaban et al., 2005), in fact, NADPH and Dual oxidases (NOX and DUOX, respectively) represent the major source of endogenous ROS (Lambeth, 2004). Dual oxidases are expressed in several cell types including the epithelium, where moderate ROS release following injury steers immune cells toward sites of damage (Niethammer, 2016;Razzell et al., 2013;Yoo et al., 2011) and modulates enzymatic activities involved in healing (Hunter et al., 2018). Nevertheless, uncontrolled oxidative damage could hinder repair, thus damaged tissues upregulate cytoprotective pathways to minimize collateral damage and ensure effective healing (Telorack et al., 2016;Weavers et al., 2019).
NOX enzymes have been best characterized in cells of the innate immune system. Phagocytes voraciously engulf unwanted particles (e.g., dysfunctional "self" or dangerous intruders) and degrade them through a rapid NOX-dependent "burst" of ROS (Weavers and Martin, 2020). This local, sharp increase in ROS is not only an important antimicrobial weapon but can also activate phagosomal proteases to enhance proteolytic digestion (Reeves et al., 2002). Paradoxically, given their highly reactive and indiscriminate nature, ROS could cause substantial collateral damage to both the phagocyte and surrounding tissues. Nevertheless, leukocytes exhibit an unprecedented "resilience" to this hostile environment, suggesting they must be empowered with robust ROS detoxification strategies to preserve immune function, and yet retain sensitivity to low ROS levels for signaling. However, the exact identity of these protective strategies, their molecular regulation, and physiological significance remain largely unclear.
Comprehensive profiling of immune cytoprotective responses will help inform the development of novel strategies to enhance immunological vigor (i.e., the long-term maintenance of immune function; Hirokawa Katsuiku, 2019). Indeed, the ability to effectively buffer ROS is known to decline with age (Meng et al., 2017), suggesting that the activation of protective responses might become compromised. Repeated cycles of oxidative injuries could reduce the effectiveness of cytoprotective mechanisms and eventually trigger immune senescence, which in itself greatly increases the risk of degenerative disorders, neoplasia, and chronic infections (Akbar and Gilroy, 2020;Nikolich-Zugich, 2018;Shaw et al., 2013).
Drosophila has recently emerged as an invaluable in vivo system to explore the pathway(s) conferring "resilience" to oxidative stress, particularly in vulnerable epithelial tissues following injury (Burbridge et al., 2021;Mundorf et al., 2019;Weavers et al., 2019). Here, we characterize the cytoprotective mechanism(s) that increase Drosophila macrophage tolerance to oxidative stress during immune surveillance and inflammatory migration. We find that macrophages activate Nrf2 downstream of a conserved calcium-PI3K-Nox signaling axis to minimize the deleterious effects of phagosomal ROS. Strikingly, the activation of macrophage Nrf2 not only autonomously sustains multiple immune functions and delays the acquisition of cellular features typically associated with early senescence, but also plays a crucial role in minimizing ROS-associated bystander damage to surrounding (otherwise healthy) tissues. Further characterization of the cytoprotective strategies that boost innate immunity will no doubt open novel routes to intervene in immune decline and alleviate the morbidities of age-related disease.

Results and discussion
Macrophages activate Nrf2 downstream of apoptotic corpse uptake Professional phagocytes, such as neutrophils and macrophages, release ROS during the "respiratory burst" as a potent microbiocidal weapon (Fang, 2011;Herb and Schramm, 2021;Slauch, 2011). Here, microinjection of Drosophila embryos with fluorescent dyes for superoxide (DHE) and hydrogen peroxide (H 2 DCFDA;Soh, 2006) revealed ROS production at the phagosomes of stage 15 embryonic macrophages, even in the absence of bacterial infection or laser-induced tissue damage (Fig. 1,A and B;and Fig. S1 A).
Professional phagocytes not only orchestrate the removal of invading pathogens but also of dying cells (Weavers and Martin, 2020). Therefore, the production of phagosomal ROS (Fig. 1, A and B) might correlate with the intense apoptotic corpse engulfment by macrophages in the early stages of Drosophila embryogenesis (Weavers et al., 2016a). In fact, phagocytes facilitate crucial tissue sculpting by engulfing dying cells from surrounding tissues, a macrophage function well-conserved from flies to mammals (Jacobson et al., 1997;Kerr et al., 1972;Wood et al., 2000). Intriguingly, this corpse clearance "primes" the macrophages for future, robust inflammatory behavior (Weavers et al., 2016a). At embryonic stage 12, a subset of macrophages had already cleared apoptotic corpses as indicated by the presence of cytosolic vacuoles (Fig. 1 C, dashed outlines). Within the same embryo, many macrophages were still "naïve" and yet to phagocytose any corpses ( Fig. 1 C, solid outlines). Strikingly, "primed" macrophages strongly accumulated superoxide, proportionally to the number of phagosomes within the cell body, suggesting that corpse engulfment drove an oxidative burst (Fig. 1, C9 and D). Macrophages extend long cytoplasmic protrusions ("pseudopods") for the long-range uptake of cellular debris (Weavers et al., 2016a). We observed the release of ROS on nascent phagosomes at the very tip of pseudopods ( Fig. 1 E), reinforcing the correlation between corpse uptake and the oxidative burst. We also analyzed embryos homozygous for the chromosomal deletion Df(3L)H99, which removes the proapoptotic genes hid, grim, and reaper, resulting in embryos completely lacking apoptosis (White et al., 1996); H99 macrophages were "naïve," having not phagocytosed apoptotic corpses (Weavers et al., 2016a) and exhibited a marked decrease in ROS production (Fig. 1, F and G).
Given that uncontrolled and prolonged ROS exposure could lead to the progressive accumulation of oxidative damage (Pizzino et al., 2017), we speculated that macrophages could employ cytoprotective pathways to control phagosomal ROS during immune surveillance, even in the absence of infection. The cap-andcollar (CNC) transcription factor nuclear factor erythroid derived-2 (Nrf2) controls the expression of genes involved in redox balance by binding to their antioxidant responsive elements (AREs; Schäfer and Werner, 2015). In Drosophila, two independent transcriptional reporters are available to monitor Nrf2 activation; one reporter expresses GFP under the control of synthetic ARE repeats, providing a general readout of Nrf2 activation (Chatterjee and Bohmann, 2012), whilst a second reporter expresses GFP under the control of the GstD1 promoter, a well-known downstream target of Nrf2 (Sykiotis and Bohmann, 2008). Drosophila embryonic macrophages strongly activated both reporters of Nrf2 ARE binding at midembryogenesis, suggesting robust activation of the Nrf2regulated antioxidant response (Fig. 1, H and I).
Next, we tested whether the Nrf2 signaling pathway was activated in macrophages downstream of corpse uptake. For this, we measured activation of the GstD1, ARE-GFP reporter in H99 mutant macrophages; these naïve macrophages not only exhibited reduced phagosomal ROS production (Fig. 1,F and G) but also significantly reduced activation of Nrf2 compared to controls, as shown by immunofluorescence (Fig. 1, J and J'), flow cytometry (Fig. 1,K and K0), and RT-qPCR ( Fig. 1 L). These data suggest that the phagocytosis-dependent release of ROS is a key trigger of the Nrf2-mediated antioxidant response in macrophages during embryonic immune surveillance in vivo. The Nrf2 response has been recently detected within murine macrophages following bacterial infection (Wang et al., 2019). Nevertheless, the mechanisms controlling Nrf2 activation independently of pathogen encounter and its role in preserving vital immune activities remains largely unclear.
Nrf2 induces resilience to oxidative stress in Drosophila macrophages We next sought to test whether Nrf2 fine-tunes macrophage ROS levels to minimize potentially debilitating oxidative damage. Given that the release of phagosomal ROS was dependent on corpse uptake during developmental dispersal (Fig. 1, F and G), we examined whether wild-type macrophages accumulated progressively higher levels of oxidative damage as they  Fig. 2 A), a known biomarker of mutagenesis caused by oxidative stress (Ock et al., 2012). Intriguingly, accumulation of oxidative damage correlated with macrophage maturation even in wild-type embryos (Fig. 2, A and A9). This stagedependent increase in macrophage oxidation was paralleled by marked activation of Nrf2 by stage 15 (Fig. 2, B and B9; and Fig. S1 B). Intriguingly, oxidative damage did not increase significantly from stage 13 to 15 ( Fig. 2 A9), which perhaps reflects the robust Nrf2 activation at this time.
To further explore the role of Nrf2 in buffering intracellular ROS, we inhibited macrophage Nrf2 using multiple independent RNAi lines and the macrophage-specific driver serpent-Gal4. Genetic inhibition of Nrf2 attenuated the macrophage antioxidant response, as shown by reduced expression of GstD1 and gclM, key Nrf2 targets ( To explore whether the elevated oxidative damage in Nrf2 RNAi macrophages was directly caused by phagosomal ROS, we analyzed DNA oxidation in Nrf2 RNAi macrophages from H99 mutant embryos. Here, genetic inhibition of corpse uptake (via H99) rescued the accumulation of oxidative damage caused by macrophage Nrf2 RNAi (Fig. 2, F and F9). These data suggest that Nrf2-dependent resilience counteracts the harmful consequences of ROS accumulation downstream of corpse uptake in Drosophila macrophages (Fig. 2 G). Interestingly, monocytes isolated from blood donors are more sensitive to oxidative DNA lesions than their more mature macrophage descendants (Ponath and Kaina, 2017); this differential vulnerability to ROS is consistent with our findings that redox-stress responses are activated upon differentiation to maximize self-protection and boost the fitness of these long-lived cells.
Macrophages activate Nrf2 downstream of calcium and PI3Kdependent Nox activity We next explored the molecular mechanisms underlying macrophage ROS release and the downstream activation of Nrf2. NADPH oxidases are well-conserved enzymes dedicated to the production of superoxide (Taylor and Tse, 2021). Although both classes of NADPH oxidases, Nox and Duox, were expressed within Drosophila macrophages, corpse uptake transcriptionally upregulated Nox expression, leaving levels of Duox relatively unchanged ( Fig. 3 A). Moreover, downregulation of macrophage Nox, using multiple independent RNAi lines, drastically reduced the release of phagosomal superoxide (Fig. 3 Phagosomal NADPH oxidases are tightly controlled to protect resting cells from dangerous exposure to unwanted ROS (Nathan and Cunningham-Bussel, 2013). A well-known marker of phagosome maturation is the progressive change in phospholipid composition (Swanson, 2014). Using a GFP-Pleckstrin homology (GPH) reporter, we monitored the accumulation of phosphoinositides PtdIns (3,4,5)P3 (PIP 3 ) on the membrane of newly-formed phagosomes (Fig. 3, D and D9 and Video 1). Macrophage-specific expression of the mutated (catalytically inactive) phosphoinositide 3-kinase (PI3K) subunit Dp110 D954A (previously shown to act as an inhibitor of PI3K signaling [Wood et al., 2006]) not only reduced PIP 3 accumulation at the cell body (srp > GPH, Fig , suggesting that accumulation of PIP 3 is required for the phagocytosis-dependent activation of Nox. Interestingly, inhibition of PI3K resulted in a hypervacuolation phenotype ( Fig. 3 F), similar to that observed following Nox RNAi , again suggesting a role for ROS in the timely digestion of phagosomal content.

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Journal of Cell  (Sievers et al., 2011). In Drosophila, the uptake of apoptotic corpses triggers a rapid rise in intracellular calcium (Weavers et al., 2016a), likely mediated by release from intracellular stores via Ryanodine ER/SR Ca 2+ channels (RyR receptors [Cuttell et al., 2008]). Given that Drosophila Nox possesses a conserved EF-hand domain, we tested for calcium-dependent regulation of Nox activity. Blocking the release of Ca 2+ from the ER by macrophagespecific knockdown of RyR, or inhibitory doses of the drug Ryanodine, significantly decreased phagosomal ROS (Fig. 3, G and G9;and Fig. S2,F and G9), suggesting that Ca 2+ positively regulated Nox activity. We also exposed embryos to 1 μM Thapsigargin to force Ca 2+ release from the ER (Fig. S2, H and H9); however, the treatment of H99 mutant embryos with Thapsigargin did not rescue the production of intracellular ROS (Fig. S3, I and I9), suggesting that Ca 2+ was required but not sufficient to stimulate Nox activity. Our data suggest that the activation of fully functional NADPH oxidases, such as Drosophila Nox, are under strict control, providing protection from potential exposure to unwanted ROS. However, there is a significant lack of knowledge on how NADPH oxidase activity modulates cytoprotection in vivo. Strikingly, multiple methods that dampened macrophage ROS levels (inhibition of Ca 2+ release via Ryr RNAi , knockdown of Nox, or inhibition of Dp110) significantly decreased macrophage Nrf2 activation (Fig. 3 H and Fig. S2, J-L), suggesting that macrophages fine-tune their Nrf2-mediated antioxidant response according to the levels of phagosomal ROS production downstream of a conserved calcium-PI3K-Nox signaling axis (Fig. 3 I).
Macrophage Nrf2 sustains proinflammatory behavior in vivo Whilst our data suggest that macrophages activate Nrf2mediated cytoprotection during immune surveillance to limit potentially deleterious oxidative damage, the importance of this protection for macrophage behavior in vivo remains unclear. Therefore, we investigated whether loss of Nrf2 affected the migratory behavior of leukocytes by live-imaging their dynamics in vivo. The developmental migration of Drosophila macrophages along the ventral nerve cord and their subsequent lateral movement away from the midline have been previously well characterized (Tepass et al., 1994;Wood et al., 2006). Inhibition of macrophage Nrf2 did not alter the speed and directionality of these early developmental migrations (Fig. 4, A and B), so by stage 15, the macrophages had reached their stereotypical positions (data not shown).
From stage 15, macrophage migration becomes more randomized and contact inhibition of locomotion (CIL) maintains their even distribution during immune surveillance (Davis et al., 2015). Macrophages lacking Nrf2 at this stage moved slower than controls (Fig. 4 C) and remained in contact for longer periods of time ( Fig. 4 D), suggesting that they were less efficient in repolarizing their movement after contacting a neighboring cell. Mature stage 15 macrophages might rely more on Nrf2-mediated protection to sustain effective migration to counteract oxidative damage that has progressively accumulated during immune surveillance. Indeed, ROS are a known permissive signal for cell migration (Hurd et al., 2012), and through the oxidation of redox-sensitive enzymes, may act as a functional switch that allows fine control of migration. Robust ROS detoxification strategies might be required to maintain key regulators of cell migration in a functional, responsive state. Excessive ROS has also been linked to the inactivation of redox-sensitive phagosomal enzymes, such as cathepsins, dedicated to the digestion of engulfed materials (Rybicka et al., 2010). Intriguingly, macrophages lacking Nrf2 also accumulated cytosolic vacuoles, accompanied by increased cell body size (Fig. 4, E-G), which might reflect either a defect in phagosome digestion or precocious phagocytosis of corpses from the environment. Given that hypervacuolation was previously linked to defective cell migration , it might also contribute to the inability of Nrf2 RNAi macrophages to move effectively.
Another physiological task performed by macrophages in vivo is their inflammatory migration toward sites of epithelial damage. Live-imaging revealed that Nrf2 RNAi macrophages were desensitized to sterile epithelial wounds; unlike their wild-type counterparts, Nrf2 RNAi macrophages often ignored an adjacent wound (Fig. 4, H and I; Fig. S3 A; and Video 2). Although the persistence of Nrf2 RNAi macrophage migration was not altered (Fig. S3, B-D), Nrf2 RNAi macrophages migrated with a significantly reduced bias toward the wound (Fig. 4 J; and Fig. S3, B and E; directionality calculated as in Franz et al., 2018;Weavers et al., 2016b). Consequently, significantly fewer Nrf2 RNAi macrophages were recruited to the site of damage (Fig. 4 I and Fig. S3 F), although these macrophages moved at similar speeds to controls (Fig. S3, G and H). Our data suggest that Nrf2 activation not only sustains macrophage homeostatic migration but also boosts efficient inflammatory migration. To demonstrate that these migration defects were due to a specific requirement for Nrf2 within macrophages (rather than non-specific effects on the epithelium itself), we confirmed that Nrf2 mRNA levels were not altered in cells other than macrophages (Fig. S3 I). Moreover, the level of Nrf2 mRNA was not altered in embryos carrying the Nrf2 RNAi construct alone in the absence of a tissue-specific Gal4 driver (Fig. S3 J). Extracellular ROS produced by epithelial DUOX are thought to serve as important damage signals that promote immune cell recruitment to epithelial injury (Evans et al., 2015;Razzell et al., 2013;Yoo et al., 2011), although whether these ROS function as permissive cues or chemotactic attractants remains unclear. Interestingly, recent work in Drosophila suggests that woundinduced hydrogen peroxide diffuses into adult macrophages through Prip aquaporin-like channels (Chakrabarti and Visweswariah, 2020); Prip channels are also expressed in embryonic macrophages (Fig. S3 K), however, their role in directing macrophages to sites of injury remains unexplored. The ability of cells to sense elevated ROS in response to injury could rely on differential levels of the signal between the intracellular and extracellular compartments. Perhaps the inability of Nrf2 RNAi macrophages to properly detoxify intracellular ROS significantly reduces the ROS gradient between the intracellular Clemente  and extracellular space and thus compromises their "orienteering" skills.
As well as clearing corpses during immune surveillance, macrophages play important roles in phagocytosing cellular debris at sites of tissue injury, and this is associated with further phagosomal ROS production (Weavers et al., 2019). Moreover, epithelial damage is also associated with elevated (Duox-dependent) ROS production at the wound margin (Razzell et al., 2013). We thus speculated that leukocytes recruited to wounds might require elevated cytoprotection and could further upregulate Nrf2. Analysis of macrophage Nrf2 (using GFP-tagged Nrf2) revealed a significant increase in macrophage Nrf2 levels following epithelial injury (Fig. 4, K and K9; and Video 3). We occasionally observed that macrophages lacking Nrf2 showed signs of DNA fragmentation following corpse uptake at wounds, indicative of apoptotic cell death (Video 4), suggesting Nrf2 RNAi macrophages might be more vulnerable to the build-up of ROS encountered during wound healing. These results suggest that macrophages may need to boost their Nrf2-mediated resilience above homeostatic levels to achieve protection from the elevated oxidative insult they might experience during an inflammatory response.
Leukocyte Nrf2 limits early senescence and minimizes nonautonomous collateral damage Cellular ROS have well-characterized roles regulating MAPK signaling, particularly through the c-Jun N-terminal kinase (JNK) pathway (Finkel, 2011). Since JNK signaling is highly redox-sensitive, we explored whether Nrf2-deficient macrophages exhibited altered JNK activity using a TRE-GFP reporter that consists of AP-1 binding sites upstream of GFP (Chatterjee and Bohmann, 2012). Nrf2 RNAi macrophages exhibited increased JNK activation compared with controls (Fig. 5, A and A9). Given that sustained and excessive levels of JNK signaling have been linked to apoptosis (Liu and Lin, 2005), we explored potential effects on macrophage viability. However, the level of the proapoptotic marker Cleaved Caspase-3 (CC3; Fig. 5 B) as well as the number of ventrally localized macrophages (Fig. 5 C) were indistinguishable from controls, arguing against apoptotic cell death.
Persistent oxidative stress and DNA damage, as well as excessive JNK activity, are well-known triggers of premature stress-induced cellular senescence (d'Adda di Fagagna, 2008;Passos and Von Zglinicki, 2006). Interestingly, Nrf2 RNAi macrophages showed elevated expression of the cyclin-dependent kinase (CDK) inhibitor Dacapo (Fig. 5, D and E), a Drosophila p21/p27 homolog previously associated with an early senescent state in flies (Ito and Igaki, 2016;Nakamura et al., 2014). p21 elevation triggers cell cycle arrest during senescence in Drosophila (Ito and Igaki, 2016) and is implicated in the acquisition of senescent-like phenotypes during mammalian embryonic development (Storer et al., 2013) and in murine postmitotic neurons (Jurk et al., 2012). As well as cellular hypertrophy, cells in the early stages of senescence normally exhibit increased nuclear size and a marked decrease in the levels of Lamin B1 (Lamin Dm0 in flies; Lin et al., 2022;Wang et al., 2017;Freund et al., 2012), all of which we observed in Nrf2-deficient macrophages (Fig. 4 E;and Fig. 5,F and G). Our data thus suggest that depletion of macrophage Nrf2 not only leads to the uncontrolled accumulation of key triggers of senescence (ROS, oxidative, and DNA damage) but alteration of cellular markers typically associated with early senescence.
When matured to a full senescence state, senescent cells can release secretory components such as inflammatory cytokines, matrix remodeling factors (e.g., MMP1), and growth factors in a phenomenon known as senescence-associated secretory phenotype (SASP; Ito and Igaki, 2016); in fact, JNK regulates expression of SASP factors in mammals and Drosophila (Ito and Igaki, 2016). However, levels of the Drosophila-secreted MMP, MMP1 were not elevated in stage 15 Nrf2 RNAi macrophages (data not shown), suggesting that Nrf2 depletion alone is insufficient for the induction of a fully senescent state associated with SASP at this stage in Drosophila macrophages.
Given the close proximity of phagocytes to non-immune tissues in vivo, a dysregulated immune system could compromise the health of remote organs. Indeed, recent work has shown that murine immune cells that lack the DNA repair protein Ercc1 not only undergo premature senescence but exert detrimental systemic effects on non-lymphoid tissues (Yousefzadeh et al., 2021). Senescent immune cells might release proinflammatory cytokines (and/or SASP mediators) that could contribute to the onset of a chronic inflammatory state, fueling the accumulation of collateral damage. Here, the inability of Nrf2-deficient macrophages to properly detoxify ROS could have marked nonautonomous effects on surrounding tissues, perhaps through the release of ROS (e.g., hydrogen peroxide) that diffuse readily across membranes through aquaporin-like channels (Bienert et al., 2006;Chakrabarti and Visweswariah, 2020). Indeed, we found that loss of macrophage Nrf2 was associated with significant, non-autonomous accumulation of hydrogen peroxide in the adjacent epithelium (Fig. 5 H; also see Fig. S1 G). Interestingly, epithelial ROS levels (but not Nrf2 activity) were reduced in H99 mutant embryos compared with controls (Fig. 5, I and J), suggesting that even wild-type macrophages contribute to the (E-G) Nrf2 knockdown (srp > Nrf2 RNAi ) increased the number of phagosomes per macrophage and caused a modest increase in cell body size (hypertrophy). (H and I) Wound recruitment defect upon macrophage-specific Nrf2 RNAi analyzed at stage 15 of embryonic development. (J) Macrophage Nrf2 RNAi decreased the migrational bias toward sites of epithelial damage, as measured by the angle α relative to the center of the wound. (K) 18 h APF pupal macrophages strongly upregulated Nrf2 upon wounding. Cell bodies (E), site of epithelial wounds (H), and insets (K) are indicated by white dashed outlines. Macrophages labeled in green (srp > GFP, E, H) or magenta (srp-mcherry, K). Macrophage nuclei labeled in red (srp > H2A::mcherry, H), Nrf2 in green (Nrf2-GFP, K). All Nrf2 RNAi data were generated using Nrf2 RNAi Flybase ID FBtp0069370. PW: post-wounding. ns: not significant, *, P < 0.05; **P < 0.01, ****P < 0.0001 via Mann-Whitney test (A, B, C, F, G, J) or unpaired t-test (I), one-way ANOVA followed by Dunn's comparison analysis (K). Images were collected from 5 control and 5 srp > Nrf2 RNAi embryos (A, B); 5 control and 5 srp > Nrf2 RNAi embryos (C); stage 13: 5 control and 12 srp > Nrf2 RNAi embryos and stage 15: 24 control and 33 srp > Nrf2 RNAi embryos (F); 44 control, 58 srp > Nrf2 RNAi embryos (G); 7 control and 7 srp > Nrf2 RNAi embryos (I, I9); 4 pupae (K).

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Journal of Cell Biology 9 of 16 Nrf2 curbs phagosomal ROS to sustain immune health https://doi.org/10.1083/jcb.202203062 . These data suggest that epithelial oxidative damage occurs downstream of macrophagemediated corpse clearance, but that this is normally restrained by macrophage Nrf2. Moreover, levels of epithelial damage were significantly increased in srp > Nrf2 RNAi animals compared with those carrying the Nrf2 RNAi construct alone, excluding leaky non-specific expression of the RNAi construct (Fig. 5 N). The epithelium of embryos lacking macrophage Nrf2 also exhibited elevated levels of the Drosophila p21/p27 homolog (Dacapo; Fig. 5, O and P), suggesting that persistent ROS-induced epithelial damage might trigger the acquisition of cellular features typically associated with early senescence, despite non-immune tissues retaining normal levels of Nrf2 (Fig. S3 I).
Given the profound cell-autonomous and systemic alterations caused by the loss of macrophage Nrf2, the ability to boost leukocyte cytoprotection could have enormous clinical benefits. Since wild-type macrophages even elevate ROS levels nonautonomously in adjacent tissues during immune surveillance, perhaps further activation of Nrf2-mediated ROS detoxification in the innate immune system could benefit host fitness. Therapeutic augmentation of phagocyte cytoprotective activity could particularly help individuals suffering from inflammatory conditions associated with elevated ROS production or those patients with a prematurely aged immune system. Nevertheless, given that recent work suggests constitutive elevation of cytoprotection in many cell types can have detrimental effects (Hiebert et al., 2018;Kucinski et al., 2017), any therapeutic augmentation of cytoprotection will need to be carefully controlled. Indeed, there is increasing evidence that basal levels of cellular stress even have beneficial hormetic effects by triggering adaptive responses that promote stress resistance (Fischer and Ristow, 2020).
Our work highlights the urgent need to define the complex networks of immune "resilience" pathways and gain an in-depth mechanistic understanding of their systemic impact on organismal aging. Phagocytes generate significant ROS throughout development, homeostasis, and inflammation, but to resist the costly consequences on cell health, immune cells simultaneously activate powerful protective machinery to mitigate the damage. However, protection from indiscriminate ROS is likely only one arm of this complex immune cytoprotective network; indeed, the recent targeted deletion of the DNA repair protein Ercc1 in hematopoietic cells accelerated immune and systemic aging (Yousefzadeh et al., 2021). The unrivaled potential for in vivo large-scale screening in Drosophila could allow the design of novel therapies to finely tune (or even reactivate) immune cytoprotection to mitigate deleterious effects of an aging immune system and tackle age-related multimorbidities.

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Journal of Cell Dissection and imaging of Drosophila pupae Pupae were staged (18 h APF) in regular vials at 25°C. Animals were carefully dissected out of their pupal case using forceps as described before (Weavers et al., 2018) and mounted on glassbottomed dishes (Cat. #P35G-0-10-C; MatTek) for live imaging.
For wounding experiments, wounds were generated on pupal wings by using a Nitrogen-pumped laser point ablation laser tuned at 435 nm (Andor Technologies). Samples were imaged on a TSC SP8 confocal microscope using a 40/1.3 oil immersion objective.
Live imaging of embryos and wounding Embryos of an overnight collection were dechorionated in 50% bleach for 1 min and extensively washed in water. Embryos at stage 15 of embryonic development (600-800 min AEL [Vässin et al., 1985]) were mounted ventral-side up on double-sided scotch tape on a glass slide in 10S Voltalef Oil (Cat. #24627.188; VWR). For wounding experiments, wounds were generated by using a Nitrogen-pumped Laser point ablation laser tuned at 435 nm (Andor Technologies). Samples were imaged on a TSC SP8 confocal microscope using a 63/1.4 oil immersion objective. Imaging processing was performed using ImageJ (NIH) and Adobe Illustrator software.

Injection of fluorescent dyes and drug treatment
Embryos of an overnight collection were dechorionated in 50% bleach for 1 min and extensively washed in water. Stage 15 embryos were mounted on double-sided scotch tape on a glass slide and dehydrated for a minimum of 10 min before being covered in Voltalef Oil. Embryos were microinjected with the desired fluorescent probe for the detection of ROS Imaging processing was performed using ImageJ (NIH) and Adobe Illustrator software.

Flow cytometry
Embryos were collected overnight at 25°C from the desired fly lines. w 1118 embryos served as a negative control. Dissociation of embryos was performed using a protocol adapted from Gyoergy et al. (2018). Briefly, embryos were dechorionated in 50% bleach for 1 min and extensively washed in water. For each genotype, a minimum of 100 embryos at stage 14-15 of embryonic development were selected under a dissecting scope and gently transferred into a dounce homogenizer containing 500 μl of freshly prepared, ice-cold Seecof buffer (6 mM Na2HPO 4 , 3.67 mM KH2PO 4 , 106 mM NaCl, 26.8 mM KCl, 6.4 mM MgCl 2 , and 2.25 mM CaCl 2 at a pH of 6.8). All the subsequent steps were performed on ice or at 4°C. Embryos were homogenized in Seecof buffer with seven gentle vertical strokes of a loose pestle. The resulting cell suspension was collected in a fresh tube and spun down at 500 rpm for 5 min to remove debris. Subsequently, the supernatant was spun down at 1,250 rpm for 10 min. After this centrifugation step, the pellet was resuspended in 500 μl Schneider's medium (S0146; Sigma-Aldrich) supplemented with 8% fetal bovine serum (FBS, F7524; Sigma-Aldrich) and filtered through a 40-μm nylon mesh. Cell sorting was performed on BD FACS Aria II (Becton Dickinson) fitted with a 100 μm nozzle. Cells were gated according to forward and side scatters, and dead cells were excluded based on the staining with the LIVE/DEAD permeable dye Draq7 (ab109202; Abcam). GFP and mCherry signals were detected through 530-40 and 610-20 filters, respectively. Cells were sorted in 1.5 ml Eppendorf tubes and immediately resuspended in the appropriate buffer for subsequent use. FCS files generated from sorting were analyzed using FlowJo_v10.6.2 software (Becton, Dickinson & Company).
RNA extraction and RT-qPCR RNA was extracted from sorted cells using the RNeasy Plus Micro Kit (74034; Qiagen). Briefly, cells were resuspended in 350 μl of Lysis buffer and the suspension was passed through a 30 G needle to facilitate lysis. Recovery of RNA was performed following the manufacturer's instructions. RNA was eluted in 100 μl of RNase-free warm water. RNA was precipitated by adding 0.10 vols of 3 M sodium acetate and 2 vol of 100% of icecold ethanol to each sample and incubating overnight at −80°C.

Data analysis
All images and movies were processed and analyzed using Im-ageJ (NIH). For quantification and comparison of fluorescent intensity of immunostaining and DHE injection, images were opened in ImageJ and converted into 16-bit images. For each Z-stack, sum-intensity projections were created and a line was drawn around each cell body manually, using the free-hand tool. For each region of interest, integrated pixel intensity was measured using the Analyse/Measure tool in the channel of interest. Pixel intensities were normalized to background values taken from cell-free regions. Data were normalized to negative controls (uninjected embryos, non-transgenic wild-type w 1118 embryos, or staining without secondary antibody) and values were plotted on Prism as fold-change over the negative control. To quantify the accumulation of the PIP3 (GPH) reporter on the membrane of newly formed phagosomes, sum-projections of macrophages from live-imaging movies were made from Z-stacks using ImageJ. The free-hand tool was used to draw a line around the newly formed phagosome and the Analyse/ Measure function tool was used to quantify pixel intensity along the line. The width of the line was set at 2. Background fluorescence was calculated by measuring pixel intensity from an area of the lamellipodium where no phagosomes were present. Data on the graphs are therefore shown as fold-change over background levels. Expression of cytosolic GFP (cytoGFP) was used as a control. For analysis of macrophage motility, cells were tracked using the automated cell tracking protocol of Imaris or using manual tracking on ImageJ. To quantify the number of macrophages responding to wounds at a specific time point, the total number of cells in contact with the wound margin or within the wound area was calculated from z-stack projections.
All statistical comparisons and graphical representations were generated using Prism8 for Mac (Graphpad). Data in the graphs are represented as mean ± SD, unless otherwise stated. Statistical analysis was performed as specified in each figure legend and the appropriate statistical test was selected after testing samples for normality. P values less than 0.05 were considered significant. n numbers and exact P values are reported on each graph. Fig. S1 shows the activation of Nrf2 protects embryonic macrophages from excessive ROS accumulation. Related to Fig. 1. Fig. S2: A PI3K, Calcium, and NOX axis drive macrophage Nrf2 activation. Related to Fig. 3. Fig. S3: Macrophage Nrf2 is required for the timely detection of epithelial wounds in vivo. Related to Fig. 4. Video 1: PIP3 accumulates on the membrane of a nascent phagosome. Related to Fig. 3. Video 2: Control macrophages, but not Nrf2-RNAi macrophages, migrate toward sites of epithelial damage. Related to Fig. 4. Video 3: Nrf2 is upregulated in 18 h pupal macrophages in response to tissue damage. Related to Fig. 4. Video 4: Zoom into an epithelial wound, related to Video 2.

Data availability
The data underlying Figs. 1, 2, 3, 4, and 5 are available from the corresponding author upon reasonable request.