The Drosophila CD36 Homologue croquemort Is Required to Maintain Immune and Gut Homeostasis during Development and Aging

Phagocytosis is an ancient mechanism central to both tissue homeostasis and immune defense. Both the identity of the receptors that mediate bacterial phagocytosis and the nature of the interactions between phagocytosis and other defense mechanisms remain elusive. Here, we report that Croquemort (Crq), a Drosophila member of the CD36 family of scavenger receptors, is required for microbial phagocytosis and efficient bacterial clearance. Flies mutant for crq are susceptible to environmental microbes during development and succumb to a variety of microbial infections as adults. Crq acts parallel to the Toll and Imd pathways to eliminate bacteria via phagocytosis. crq mutant flies exhibit enhanced and prolonged immune and cytokine induction accompanied by premature gut dysplasia and decreased lifespan. The chronic state of immune activation in crq mutant flies is further regulated by negative regulators of the Imd pathway. Altogether, our data demonstrate that Crq plays a key role in maintaining immune and organismal homeostasis.


Introduction
Mounting appropriate immune responses against pathogens is critical for the survival of all animals. Mechanisms to both eliminate microbes and resolve infection by returning the immune system to basal activity are necessary to maintain an adequate and balanced immune response [1,2]. Alterations in these responses can lead to immune deficiency or auto-inflammation [3][4][5]. Yet, to date, how these mechanisms are coordinated upon infection remains unclear.
Drosophila is a prime model to genetically dissect humoral and cellular innate immune responses to a variety of pathogens [6][7][8]. Humoral responses include the pro-phenoloxidase (PO) cascade, which leads to the generation of reactive oxygen species and melanization, and the rapid production of antimicrobial peptides (AMPs) regulated by the Toll and Imd pathways [7]. Upon recognition of microbial lysine (Lys)-type peptidoglycan (PGN), damage-associated molecular patterns (DAMPs), or exogenous protease activity, the Toll pathway promotes the nuclear translocation of the NF-κB-like transcription factor Dorsal-related Immune Factor (Dif) to induce AMP genes, such as Drosomycin [6,9]. In contrast, detection of bacterial meso-diaminopimelic acid (DAP)-type peptidoglycan activates the Imd pathway and leads to the nuclear translocation of the NF-κB-like transcription factor Relish (Rel) to induce transcription of AMP genes, such as Diptericin [10,11]. It has also been shown that proteases, such as Elastase and Mmp2, can activate the Imd pathway through cleavage of the receptor PGRP-LC [12]. As in mammals, chronic activation of immune responses is deleterious to the fly, and negative regulators are required to maintain immune homeostasis [13][14][15]. For instance, amidase PGN recognition proteins (PGRPs), such as PGRP-LB and PGRP-SC, negatively regulate the Imd pathway by enzymatically degrading PGN [14][15][16].
Phagocytosis and encapsulation are key cellular innate immune responses [7]. Phagocytosis allows for the uptake and digestion of microbes and apoptotic cells by phagocytes, including specialized immune cells called plasmatocytes [7,17]. Encapsulation results in the isolation and melanization of large materials, such as wasp eggs or damaged tissues, by dedicated immune cells named lamellocytes [18]. Both phagocytosis and humoral responses are required to fight infection. Indeed, decreasing the phagocytic ability of plasmatocytes by pre-injecting latex beads, which they take up, impairs fly survival upon infection with Gram-positive bacteria [19]. Similarly, inhibiting phagocytosis increases the susceptibility of Imd pathway-deficient flies to Escherichia coli (E. coli) infection, arguing that phagocytosis and the humoral response act in parallel [20]. Plasmatocytes were proposed to activate the production of AMPs by releasing immunostimulatory pathogen-associated molecular patterns (PAMPs) following phagocytosis [21]. They also express cytokines such as Unpaired 3 (Upd3), a ligand of the JAK-STAT pathway, which regulates immune-related genes [22]. Yet, ablation of the majority of plasmatocytes by targeted apoptosis has only a moderate effect on the fly's ability to fight infection [23,24]. Therefore, the role of phagocytosis in the regulation of the humoral response and the resolution of infection remains unclear.
Several plasmatocyte receptors promote the recognition and engulfment of bacteria [25]. The scavenger receptor dSR-CI and a transmembrane protein, Eater, bind to both Gram-negative and -positive bacteria [26,27]. The membrane receptor PGRP-LC binds to and engulfs Gram-negative but not Gram-positive bacteria, and its membrane localization is dependent on the nonaspanin TM9SF4 [28,29]. Draper (Drpr) promotes clearance and degradation of neuronal debris and apoptotic cells via phagosome maturation, as well as phagocytosis of Staphylococcus aureus (S. aureus) together with the integrin βv and PGRP-SC1 [30][31][32]. Nimrod C1, which is related to Eater and Drpr, promotes phagocytosis of both S. aureus and E. coli by Drosophila S2 cells, and suppression of its expression in plasmatocytes inhibits phagocytosis of S. aureus [33]. Peste, a member of the CD36 family of scavenger receptors plays a role in the recognition and uptake of Mycobacterium by S2 cells [34]. Finally, croquemort (crq), another CD36 family member, promotes apoptotic cell clearance by embryonic plasmatocytes [35] and phagosome maturation of neuronal debris by epithelial cells [36].
In mammalian immunity, CD36 promotes the uptake of oxidized low density lipoproteins (oxLDLs) [37,38] and also regulates the host inflammatory response [39,40]. In addition, it is required to fight Mycobacteria and S. aureus infections in mice [41] and to induce pro-inflammatory cytokines in response to Plasmodium falciparum infection [42]. Using two lethal deficiencies that delete crq (as well as other genes), we previously proposed that Crq was specific to apoptotic cell clearance, as crq-deficient embryonic plasmatocytes retained some ability to engulf both E. coli and S. aureus in vivo [35]. However, Crq was subsequently implicated in phagocytosis of S. aureus by S2 cells, a heterogeneous cell line with phagocytic abilities derived from late embryonic stages [41]. Thus, we generated a knock-out of crq and further investigated its role in microbial phagocytosis and its relationship with the humoral response at larval and adult stages in vivo.
Drosophila plasmatocytes derive from pro-hemocytes originating either in the procephalic mesoderm of the embryo, with some further expanding by self-renewal in larval hematopoietic pockets, or from a second hematopoietic organ, the larval lymph glands, that persist to adulthood, or finally from adult hematopoietic hubs [43][44][45][46][47][48][49][50][51]. Here, we show that Crq is a major marker of plasmatocytes that is not required for hematopoiesis. The survival to adulthood of crq knock-out (crq ko ) mutants allowed us to quantitatively demonstrate that crq is required for pupae to survive environmental microbe infections and for adults to resist infection against Gram-negative and Gram-positive bacteria and fungi. crq ko flies tolerate infections as well as control flies, but are unable to efficiently eliminate microbes. Indeed, crq ko plasmatocytes are poorly phagocytic and defective in phagosome maturation. Crq acts parallel to the Imd and Toll pathways in eliminating pathogens, and crq ko flies display elevated and persistent Dpt and upd3 expression, demonstrating that mutating crq promotes a state of chronic immune activation. As a consequence, crq ko flies die prematurely with early signs of gut dysplasia and premature intestinal stem cell hyperproliferation. Therefore, we propose a model wherein crq is central to immune and organismal homeostasis. Overall, our results shed new light on the links between phagocytes, commensal microbes, gut homeostasis, and host lifespan. 1C). Crq immunostaining of hemocytes bled from eater-nls::GFP flies revealed that 85.2±2.6% of them were Crq and eater-dsRed positive, while 14.8±2.6% were Crq-positive but did not express eater-dsRed (Fig 1C). From this, we extrapolated that about 72.4% of circulating hemocytes are positive for all three markers, 12.8% are double positive for Crq and Eater, and 14.8% solely express Crq (Fig 1C). Therefore, crq is expressed in all Eater and Hml-positive hemocytes and marks the majority, if not all, adult plasmatocytes.
To study its role in vivo, we generated a knock-out allele of crq (crq ko ) by homologous recombination [36]. This mutant deletes the entire crq open reading frame (S1A Fig), and thus abolishes its expression [36]. As previously reported [35], crq was not required for embryonic hematopoiesis. As for crq deletion mutants, crq ko embryonic plasmatocytes were less efficient at clearing apoptotic cells, having a phagocytic index of 1.6±0.2 versus 2.45±0.3 apoptotic cells/ plasmatocyte for wild-type embryos (p<0.05, S1B Fig). Homozygous crq ko flies were viable and appeared morphologically normal. To ask whether crq is required for hematopoiesis at later developmental stages, we recombined an eater-nls::GFP transgene (i.e., the broadest plasmatocyte reporter after Crq) (Fig 1C) into the crq ko mutants, bled larvae and adults, and semiautomatically scored their eater-nls::GFP positive plasmatocytes by microscopy (S1C Fig and  Fig 1D). As previously reported for wild-type [56,57], adult crq ko flies had about 5-fold less plasmatocytes than larvae, and their number of eater-nls::GFP-positive plasmatocytes at both larval and adult stages were similar to that of wild-type flies (Fig 1D). Pro-hemocytes that differentiate into plasmatocytes can also differentiate into crystal cells, which are involved in melanization [58]. Furthermore, self-renewing plasmatocytes of the embryonic lineage can also differentiate into crystal cells by trans-differentiation [59,60]. Thus, we tested whether crq ko flies have differentiated crystal cells by scoring the melanotic dots formed following heatinduced crystal cell lysis. We found no significant difference between crq ko and wild-type larvae (S1D Fig). Therefore, Crq is a major plasmatocyte marker that is not required for hematopoiesis or hemocyte differentiation. croquemort mutant flies are susceptible to environmental microbes While crq ko homozygous flies were viable to adulthood, we could not maintain a homozygous stock on conventional fly food. We found that 36±3.2% homozygous crq ko larvae arose from crosses between crq ko heterozygous flies over GFP-marked CyO balancer chromosome, indicating full viability of the homozygous larvae (Fig 1E). However, only 18±1.7% of emerging adults were homozygous crq ko flies, indicating that half of the crq ko homozygous progeny died during pupariation. Because flies with decreased plasmatocyte counts undergo pupal death associated with the presence of otherwise innocuous environmental microbes [23], we asked whether supplementing the food with antibiotics could rescue crq ko lethality. With this treatment, we recovered 29±3.6% of crq ko homozygous adults (Fig 1E), indicating a partial rescue of pupal lethality (homozygous vs balanced adults, p = 0.021). These results suggest that crq ko pupae are susceptible to environmental microbes.
No adult progeny could be recovered from crq ko homozygous crosses on conventional fly food, but crq ko adults emerged in the presence of antibiotics that gave rise to a second adult progeny (Fig 1E and 1F). Maintaining a homozygous viable stock with antibiotics, however, remained difficult. We next bleached homozygous crq ko embryos and raised them on sterile food. Under these axenic conditions, we successfully cultured a homozygous crq ko line ( Fig  1F). Therefore, environmental microbes represent a health constraint for crq ko homozygous flies.

croquemort mutant flies are broadly susceptible to infection
The susceptibility of crq ko pupae to environmental microbes suggested that crq is required to mount an appropriate immune response. We next asked whether crq was up-regulated in flies injected with the Gram-negative bacterium Pectinobacterium (previously known as Erwinia) carotovora 15 (Ecc15) or the Gram-positive Enterococcus faecalis (E. faecalis). As anticipated, there was no crq expression in unchallenged (UC) or infected crq ko flies as detected by RT-qPCR (Fig 2A). While crq was expressed in both UC pXH87-crq transgenic (the parental transgenic strain used for the generation of crq ko flies, hereafter referred to as PXH87) and Canton S (Cs) control flies, it was not up-regulated within the first 24hrs of infection with Ecc15 or E. faecalis (Fig 2A). However, we cannot exclude the possibility that crq may be up-regulated in plasmatocytes specifically at these early time points after infection. Its expression was also not altered in mutant flies for the NF-κB-like transcription factor Relish (Rel E20 ) downstream of the Imd pathway, or in flies mutant for the Toll ligand spz (spz rm7 ), upstream of the Toll pathway during that time-frame [9]. Surprisingly, we did observe an increase in crq mRNA levels at ±6.5% of PXH87 and Cs flies had died by that time-point. crq ko flies were, however, less susceptible than Rel E20 mutants (p<0.0001), which are defective in the production of AMPs downstream of Imd [61]. All Rel E20 flies died within 72 hpi, while only 56±7.7% of crq ko flies had succumbed by that same time-point (Fig 2B). To verify that the susceptibility to Ecc15 infection was due to the crq ko mutation and not to a background mutation, we infected trans-heterozygous flies for crq ko and Df(2L)BSC16, which deletes crq, with Ecc15 ( S2D Fig). These flies were as susceptible to Ecc15 infection as the crq ko homozygous flies; they all died within 288 hpi, indicating that the crq mutation is responsible for this phenotype (S2D Fig). crq ko flies also succumbed to infection with E. coli (39±8.1% survival at 336 hpi), a Gram-negative bacterium that does not kill Cs (97±2.5% survival) or PXH87 (86±5.6% survival) flies. However, crq ko flies were less susceptible to E. coli infection than Rel E20 flies, which all died within 312 hpi (p<0.0001) (Fig 2C and S2E Fig). Therefore, crq ko flies are susceptible to various Gram-negative bacterial infections.
Similarly, crq ko flies were more susceptible to infection with the Gram-positive bacterium E. faecalis than controls (p = 0.0006) (Fig 2D and S2F Fig) and died in 312 hpi. However, they were less susceptible than spz rm7 flies (p<0.0001), which are defective in the production of AMPs downstream of Toll and died within 72 hpi (Fig 2D and S2F Fig). crq ko flies also died with intermediate susceptibility between that of control and spz rm7 flies (p<0.0001 for both) after septic injury with the pathogenic yeast Candida albicans (Fig 2E and S2G Fig). Similarly, crq ko flies were significantly more susceptible to exposure to spores of the entomopathogenic fungus Beauveria bassiana than Cs and PXH87 flies (p<0.0001), but less susceptible than spz rm7 flies (p<0.0001) (Fig 2F and S2H Fig). Finally, crq ko flies were more susceptible to S. aureus infection than spz rm7 flies (p = 0.0073) (Fig 2G and S2I Fig), and spz rm7 flies were only slightly more susceptible than Cs and PXH87 flies (p = 0.0006 and p<0.0001 respectively). Therefore, crq ko flies are susceptible to Gram-positive bacteria and fungal infections and strongly susceptible to infection with S. aureus, a bacterium specifically cleared by phagocytosis [19,62,63].
These results argue that crq is required to fight infection. To further confirm this, we drove the expression of a UAS-crq transgene under the control of a crq promoter-Gal4 driver in the crq ko flies (crq ko ; crq-Gal4>UAS-crq). These rescue flies were no longer susceptible to Ecc15 We did not observe any significant differences between rescue experiments with the crq-Gal4 or srp-Gal4 drivers after infection with Ecc15, E. coli, or E. faecalis (p>0.05). The hemocyte-specific rescue of crq ko flies infected with C. albicans, however, was slightly less efficient than the rescue with the crq-Gal4 driver (p = 0.0269). Thus, crq appears to be required mostly in phagocytes to fight infection by both Gram negative and Gram positive bacteria, although it appears to also be required in other tissues to fight C. albicans infection.

croquemort mutant flies are tolerant but poorly resistant to infection
Multi-cellular organisms use two complementary strategies to fight infection: resistance, to eliminate microbes, and tolerance, to allow them to endure the infection and/or its deleterious effects [64,65]. Compared to controls, crq ko flies die prematurely at around 552 hours even in the absence of infection (S4A Fig), suggesting these flies could be generally unfit or susceptible to damage. To test their response to abiotic damage, we pricked crq ko flies with sterile needles at two separate thoracic sites. These flies did not die any earlier than non-pricked crq ko flies (S4A Fig). Thus, despite their decreased lifespan, crq ko flies are not susceptible to aseptic wounds.
To date, few studies have quantified the tolerance of immune-deficient flies [66,67]. Tolerance can be measured as the dose response curve relating health to microbe load. This curve takes the shape of a sigmoid; life expectancy in unchallenged conditions is considered as vigor, and the slope of the response curve (the portion of the health/load curve which is linear) estimates the ability to tolerate infection (S4B Fig) [67]. crq ko flies have shortened lifespan and therefore an altered vigor (S4A Fig). We further aimed to estimate whether crq ko flies show a decrease in tolerance by measuring the relationship (statistical interaction) between microbial load and the corresponding health of the host [64,67]. We used three approximations to relate health to microbe load of crq ko flies and focused on the linear part for each regression. First, we estimated the regression between the LT50 (time at which 50% of the flies are dead) of Ecc15 or E. faecalis-infected flies and the number of bacteria injected (measured as colony forming units or CFUs) (S4C and S4D Fig). We did not detect any significant LT50~Time interaction between PXH87 and crq ko flies (p = 0.21782 for E. faecalis, p = 0.55800 for Ecc15) (S4C and  S4D Fig). However, this measure of bacterial load does not take into account the growth of the pathogen within the host. We therefore also quantified the regression between LT50 and the number of bacteria in the flies at 24 hpi (Fig 3A and 3B). We detected significant LT50~Time interaction between PXH87 and crq ko flies (p = 0.008486 for E. faecalis, p = 0.018965 for Ecc15), with PXH87 flies having lower tolerance than crq ko flies (Fig 3A and 3B). Finally, to get another estimate of the health of the flies, we plotted the health/bacterial load curve using survival at 3 time-points post Ecc15 infection and their corresponding bacterial load (S4E Fig).
We did not detect any significant survival-time interaction between PXH87 and crq ko flies (p = 0.335111). Thus, while crq ko flies die prematurely in the absence of infection, they do not show any decreased tolerance to infection when compared to control flies. These data suggest that the increased susceptibility of crq ko flies to infection is due to their inability to control bacterial growth. In order to test this hypothesis, we monitored bacterial load during the course of Ecc15 and E. faecalis infections. In PXH87 flies, Ecc15 is eliminated within the first 48hrs of infection to reach an apparent plateau of low number of CFUs that persist at 72hrs post-infection (Fig 3C). crq ko flies were less able to clear Ecc15 than controls with higher bacterial loads throughout the infection (p<0.001 for 24, 48 and 72hrs) (Fig 3C). In contrast, despite an initial decline of CFUs at 48hrs, E. faecalis grew within control flies at 96 and 168hrs (Fig 3D). During the whole course of infection with E. faecalis, the bacterial loads were significantly lower in wild-type control flies than in the crq ko flies (p<0.001 at 48, 96 and 168hrs) (Fig 3D). These data indicate that crq is required for efficient elimination of both Ecc15 and E. faecalis. croquemort is required for engulfment of bacteria and phagosome maturation crq is required for efficient phagocytosis of apoptotic cells (also known as efferocytosis) in vivo, and phagocytosis of S. aureus by S2 cells (S1B Fig and [ 35,41]). In addition, rescue of crq expression in hemocytes improved survival to various infections (S3D- S3G Fig), suggesting that crq could alter microbial phagocytosis. To test this hypothesis, we first compared the susceptibility of crq ko flies to infection with that of mutants for two phagocytic receptors, Eater and Drpr [26,31]. crq ko flies succumbed to Ecc15 infection significantly faster than eater-deficient (p = 0.0002) and drpr rec8Δ5 loss-of-function flies (p<0.0001). 90±3.58% of crq ko flies died within 192 hpi, while only 60±6.77% of drpr and eater mutants died in that same time ( Fig  4A). However, the crq ko flies were significantly less susceptible to Ecc15 than Rel E20 flies (p<0.0001), which all died within 48 hpi (Fig 4A). In contrast, crq ko flies succumbed to E. faecalis infection at a similar pace to that of both eater-deficient and drpr rec8Δ5 flies with 80-90% of all strains dying within 240 hpi (Fig 4B). However, all mutants were significantly less susceptible than spz rm7 flies, which died within 48 hrs of E. faecalis infection (p<0.0001) (Fig 4B).
To examine the precise role of crq in phagocytosis, we compared the amount of bacteria engulfed within 45min of thoracic injections of dead, Alexa 480-labeled E. coli and S. aureus in Cs, PXH87, and crq ko flies as previously described [20,26] (Fig 4C and 4D). The crq ko flies engulfed both E. coli and S. aureus bacteria with on average 66% less efficiency than control flies (Fig 4C-4F, respectively). This phenotype was completely rescued in crq ko flies expressing a UAS-crq transgene under a crq-Gal4 driver (crq ko , crq-Gal4>UAS-crq), which appeared to engulf more efficiently than control PXH87 flies (S5A Fig and S5B Fig). We speculate that this difference was due to the overexpression of crq in those flies.
To further assess the phagocytosis phenotype, wild-type and crq ko flies carrying the eaternls::GFP plasmatocyte-reporter were injected with dead rhodamine-labeled E. coli, bled, and their plasmatocytes were analyzed by confocal microscopy for internalized bacteria (Fig 4G). The rhodamine-fluorescence per eater-nls::GFP plasmatocyte was quantified at 45min, 3hrs, and 5hrs post-injection and normalized to that of WT plasmatocytes at 3hrs post-injection (Fig 4H). In WT plasmatocytes, the relative rhodamine-fluorescence increased as early as 45min, peaked at 3hrs, and decreased after 5hrs, as bacteria were presumably digested in mature phagosomes (Fig 4H). In contrast, crq ko plasmatocytes accumulated about 2-fold fewer bacteria than controls at 45min and 3hrs post-injection, but accumulated 1.7-fold more bacteria by 5hrs post-injection. In addition, at 45min post-injection, most bacteria were internalized within wild-type plasmatocytes (S5C Fig), whereas bacteria were often bound to the cell surface of crq ko plasmatocytes without being internalized (S5D Fig). Thus, crq ko plasmatocytes can engulf bacteria but are less efficient at it than controls at early time-points; they also appear to accumulate internalized bacteria over time. These results are consistent with a role for crq in promoting efficient uptake of bacteria. Moreover, the observed accumulation of bacteria in crq ko plasmatocytes at 5hrs post-injection suggested that crq could also be required for phagosome maturation and digestion of bacteria. To test this, we injected control, crq ko , and rescue flies with pH-sensitive pHrodo E. coli and S. aureus. pHrodo bacteria fluoresce when engulfed into a fully mature, acidified phagosome [68] (S5E and S5F Fig). After quantification, we observed about 50% less fluorescence in crq ko when compared to controls at 1, 3, and 5hrs post-injection (S5G and S5H Fig, p<0.5 when comparing PXH87 and crq ko flies). This phenotype was again completely rescued in crq ko , crq-Gal4>UAS-crq flies (S5E and S5F Fig and S5G  and S5H Fig, p>0.5 when comparing PXH87 and rescue flies). At the single cell level, crq ko plasmatocytes had up to 63±5.66% and 55±7.46% less pHrodo E. coli than controls at 3 and 5hrs, respectively (Fig 4I).
Finally, to ask whether mutating crq resulted in persistence of pathogenic bacteria, we injected live GFP-labeled Ecc15 in control and crq ko flies carrying the eater-dsred plasmatocyte reporter (Fig 4J and S5I Fig). Control PXH87 plasmatocytes had little to no GFP signal at 4 days post-infection, indicating that most bacteria had been engulfed and digested (Fig 4J and  S5I Fig). In contrast, crq ko plasmatocytes had a 6-fold higher GFP signal, demonstrating that live Ecc15 accumulate in crq ko plasmatocytes (Fig 4J and S5I Fig). Taken together, these results show that crq is required for efficient microbial phagocytosis by playing a role in bacterial uptake and phagosome maturation.

Croquemort acts in parallel to the Imd and Toll pathways
Phagocytosis has been proposed as a key step to initiate AMP production [21]. To assess the effect of mutating crq on AMP production downstream of both the Imd and Toll pathways, we next quantified the expression of Diptericin (Dpt) and Drosomycin (Drs)-encoding genes by RT-qPCR after Ecc15 or E. faecalis infections (Fig 5A and 5B). As previously reported, septic injury of control flies with Ecc15 induced Dpt expression, which peaked at 10 hpi and returned to near-basal levels within 48 hpi (Fig 5A) [7]. In crq ko flies, Dpt expression was 2-fold higher than in control flies at 10hrs post-infection and failed to return to basal levels within 48hrs (Fig  5A). In contrast, there was no significant difference in Drs induction between control and crq ko flies after E. faecalis inoculation (Fig 5B). Survival curves indicated that crq ko flies were less susceptible to a non-pathogenic E. coli infection than Rel E20 flies, while double mutants for crq ko and Rel E20 were statistically more susceptible than Rel E20 or crq ko mutants alone (Fig 5C). The extreme sensitivity of Rel E20 flies to infection with pathogenic bacteria prevented us from carrying out these experiments with Ecc15. Instead, we inoculated the flies with 20 times fewer E. coli than previously used in Fig 2. Similarly, crq ko and spz rm7 double mutants were also statistically more susceptible to C. albicans infection than the spz rm7 or crq ko mutants alone (Fig 5D). Therefore, crq is not required for the induction of AMPs and acts in parallel to the Toll and Imd pathways.
These results suggested that aberrant phagocytosis in crq ko flies can result in enhanced and persistent Imd pathway activation. Multiple negative regulators of the Imd pathway help maintain immune homeostasis. For example, Peptidoglycan Recognition Proteins (PGRPs) with amidase activity, such as PGRP-LB, degrade immunostimulatory molecules [15]. Thus, we next assessed Dpt expression levels by RT-qPCR in single and double PGRP-LB Δ and crq ko mutants upon Ecc15 infection. Single crq ko and PGRP-LB Δ mutants expressed statistically higher levels of Dpt than Cs and PXH87 controls at 48hrs (Fig 5E). The Dpt expression resolved back to basal levels within 72hrs post infection in control flies, but remained high in single PGRP-LB Δ or crq ko mutants despite a steady decline in its expression (Fig 5E). Moreover, double mutants for crq ko and PGRP-LB Δ expressed Dpt at levels 5-fold higher than controls at 24hrs post-infection, and levels remained high at 48 and 72hrs (Fig 5E). These results demonstrate the critical interplay between phagocytosis and negative regulators of the immune system to achieve proper resolution of AMP expression upon systemic infection.
Plasmatocytes are also a major source of cytokine production upon systemic infection. Upd3, the Drosophila analogue of IL-6, can induce the JAK-STAT pathway, which regulates the systemic immune response and metabolic homeostasis in the fat body, as well as gut homeostasis [6,22,69,70]. Using RT-qPCR, we asked whether crq is required for upd3 expression upon Ecc15 infection. Control flies displayed a small and temporary induction of upd3 expression that resolved within 72hrs (Fig 5F). In contrast, UC and Ecc15-challenged crq ko flies showed a 1.5-fold stronger induction of upd3 expression, which further increased over 72hrs (Fig 5F). Thus crq is not required to induce upd3 expression, but crq mutation results in enhanced and continuously increasing upd3 expression. Altogether, these results demonstrate that crq is required for bacterial clearance and mutation of crq alters the resolution of AMPs and Upd3 cytokine production. croquemort mutant flies have a short lifespan with early gut dysplasia PGRP-LB Δ and Rel E20 mutants all die prematurely, within about 696 hrs (29 days) of age, when compared to wild-type (p<0.0001) and PXH87 (p<0.0001) control flies, which die after about 912 hrs (37 days) on conventional food at 29°C [15] (Fig 6A). crq ko flies died on average within 552 hrs (23 days), considerably earlier than Rel E20 and PGRP-LB Δ mutants (p<0.0001). Double crq ko and PGRP-LB Δ or crq ko and Rel E20 mutants died within about 480 hrs (20 days) and 408 hrs (17 days) of age, respectively (Fig 6A). Antibiotic treatment partially rescued these phenotypes, as the lifespan of crq ko flies and the double mutants increased significantly (p<0.0001) (Fig 6B). To ask whether the premature aging of crq ko flies might correlate with a loss of immune cells or their function, we estimated the number of plasmatocytes present in control and crq ko flies using the eater-nlsGFP reporter (Fig 6C). As previously reported [56,57], the number of plasmatocytes was decreased by about 40% in 16-day-old control flies (Fig 6C), while similarly aged crq ko flies had lost 80% of their plasmatocytes (Fig 6C). Treatment with antibiotics rescued this crq ko phenotype but had no effect on the plasmatocyte counts of control flies. crq ko flies also lost about 40% of their plasmatocytes at 4 days post-E. faecalis infection when compared to similarly challenged wild-type controls (Fig 6D). This loss of crq ko hemocytes may be a consequence of accumulation of undigested bacteria inside their phagosomes. Thus, crq is required for plasmatocytes to survive innocuous or pathogenic bacterial infection.
Dpt expression in wild-type and PXH87 flies is relatively low and stable over the first 8 days of their lives and increases as flies age [71] (Fig 6E). Strikingly, Dpt expression was 70-fold higher in 8-day-old crq ko flies and nearly 1,100-fold higher in the double mutants for crq ko and PGRP-LB Δ compared to controls (Fig 6E). Thus, the Imd pathway is strongly up-regulated early on in the life of these mutant flies, even in the absence of infection. This points to a role for Crq in phagocytosis and in maintaining immune homeostasis. Likewise, upd3 expression steadily increased as PXH87 flies aged, and it was further enhanced by nearly 10-fold in 8-and 16-day-old crq ko flies (Fig 6F). Antibiotic treatment partially rescued the levels of Dpt expression in crq ko flies (S6A Fig), arguing that the hyper-activation of the Imd pathway in these flies results from their inability to control environmental microbes. To address this, we plated fly extracts on both LB (on which most pathogens can grow) and MRS (on which most Drosophila microbiota can grow) agar plates and quantified the resulting CFUs (S6B Fig). In line with previous studies, the CFUs obtained from 2 week-old control flies were in the range of 2,000 per fly (S6B Fig) [72][73][74]. Significantly fewer CFUs were recovered from PGRP-LB Δ mutants, while both crq ko and Rel E20 extracts showed a 10-fold increase. Double mutants for crq ko and Rel E20 had 50-fold more CFUs than controls (S6B Fig). Altogether, these results demonstrate that Crq and the Imd pathway act in parallel and are required for the management of environmental microbes.
Elevated levels of Upd3 are associated with midgut hyperplasia in aging flies [72,75]. In addition, loss of gut barrier integrity leads to early death in a microbiota-dependent manner [76,77]. Because 8-day-old crq ko flies expressed high levels of upd3, we asked whether they also displayed premature gut hyperplasia by looking at the number of mitotic PH3-positive intestinal stem cells of their midgut. While PXH87 and crq ko flies did not show any signs of midgut hyperplasia at day 7, midguts of 16 day-old crq ko flies had a 2-fold increase in PH3-positive cells compared to that of similarly aged controls (p = 0.0109) (Fig 6G). This phenotype was completely rescued in crq ko ; crq-Gal4>UAS-crq flies (Fig 6G). The double mutants for crq ko and PGRP-LB Δ or for crq ko and Rel E20 showed even higher levels of intestinal stem cell proliferation than controls (p = 0.03) and did so more prematurely (already in 7-day-old flies) (Fig  6G). The premature increase in midgut stem cell proliferation was partially dependent on Upd3, as upd3;crq ko double mutant flies had significantly less mitotic cells (p = 0.04) and lived longer than crq ko flies (p<0.0001) (Fig 6H and 6I). However, the lifespan of upd3;crq ko double mutants flies was still shorter than that of PXH87 flies (p<0.0001), suggesting that additional mechanisms play a role in the shortened lifespan of crq ko flies. We further asked whether crq is required in hemocytes to maintain intestinal homeostasis. Hemocyte-specific re-expression of crq led to a strong rescue of lifespan compared to crq ko flies (p<0.0001) but not to the levels of PXH87 flies (p = 0.0462) and to a partial rescue of midgut hyperplasia in 16-day-old flies (p = 0.003 for crq ko vs rescue and p = 0.0123 for rescue vs PXH87) (Fig 6H and 6I). Altogether, these results indicate that flies lacking crq display chronically elevated expression of upd3 that triggers early midgut hyperplasia and promotes premature death.

Discussion
Our study shows that Crq is required for the engulfment of microbes by plasmatocytes and their clearance, and that the mild immune deficiency due to crq mutation is associated with increased susceptibility to infection, defects in immune homeostasis, gut hyperplasia, and decreased lifespan (S7 Fig). We have also re-confirmed a role for crq in apoptotic cell clearance, although the phagocytosis defect of crq ko plasmatocytes is less severe than what had been previously observed with two lethal crq deficiency mutants, Df(2L)al and Df(2L)XW88 [35]. A possible explanation is that these deficiencies may have deleted at least one other gene required for apoptotic cell clearance. Additionally, morphological defects associated with secondary mutations could have exacerbated the crq phagocytosis defect by preventing efficient plasmatocyte migration to apoptotic cells. These same deficiency mutants had been assessed qualitatively for phagocytosis of bacteria by injecting embryos with E. coli or S. aureus; their plasmatocytes had no obvious defect in their ability to engulf these bacteria [35]. However, a role for crq in phagocytosis of S. aureus, but not that of E. coli, was subsequently proposed based on S2 cell phagocytosis assays following knock-down of crq by RNAi [41]. Here, we show that crq is required in vivo for uptake and phagosome maturation of both S. aureus and E. coli. A simple explanation of this discrepancy with E. coli could be that knocking down crq by RNAi is not sufficient to affect its role in E. coli phagocytosis (but sufficient to affect its role in S. aureus phagocytosis), and that completely abrogating crq expression by in vivo knock-out leads to a stronger phenotype with both bacteria. Our in vivo data in crq ko flies further demonstrate that crq is required to resist multiple microbial infections, such as Ecc15, E. faecalis, B. bassiana, and C. albicans. These data therefore argue that crq plays a more general role in microbial phagocytosis than was previously anticipated. Our previous experiments to test whether crq is required for bacterial phagocytosis in embryos were qualitative rather than quantitative, and did not allow us to identify a role for crq at that stage [53]. In contrast, the experiments we now report in adult crq ko flies are quantitative and allowed us to identify a delay in phagocytosis, followed by a defect in bacterial clearance in crq ko hemocytes. A possible explanation for this discrepancy would be that hemocytes may differ in their expression profile, behavior, and phagocytic ability at various developmental stages due to differences in their microenvironment and/or sensitivity to stimuli. Accordingly, it has recently been shown that the phagocytic activity of embryonic hemocytes acts as a priming mechanism, increasing the ability of primed cells to phagocytose bacteria at later stages [78]. It is therefore possible that embryonic, larval and adult hemocytes display very different levels of priming and bacterial phagocytic activity, and that crq is required mostly in larval/adult bacterial phagocytosis. Alternatively, a potential defect in phagocytosis of bacteria by embryonic hemocytes of the crq deficiencies may have been suppressed by the deletion of (an)other gene(s) in that genomic region.
Because the immune competence of hemocytes varies during development [50,79,80], we were prompted to re-examine the potential role for crq in innate immunity by knocking it out. Here, we show that Crq is a major plasmatocyte marker at all developmental stages of the fly. We have found that crq ko flies are homozygous viable, but short-lived, and can hardly be maintained as a homozygous stock in a non-sterile environment; crq ko pupae become susceptible to environmental bacteria and their microbiota during pupariation. In a recent study, Arefin and colleagues induced the pro-apoptotic genes hid or Grim in plamatocytes and crystal cells using the hml-gal4 driver (Hml-apo) and observed a similar pupal lethality, but also associated with an induction of lamellocyte differentiation, and the apparition of melanotic tumors of hemocyte origin [81]. The authors therefore concluded that the death of hemocytes triggered lamellocyte accumulation and melanotic tumor phenotypes [81]. In contrast, we did not observe any obvious melanotic tumors in crq ko flies, despite observing a loss of hemocytes in aging crq ko flies (Fig 6C) and crq ko flies subjected to Ecc15 infection (Fig 6D). One possible explanation is that hemocytes do not die of apoptosis in crq ko flies, but of a distinct mechanism. Alternatively, crq mutation could affect more hemocytes than Hml-apo flies, as crq is expressed in all plasmatocytes, while Hml is only expressed in 72.4% of all plasmatocytes expressing crq (from Fig  1C). Thus the 27.6% of non-Hml plasmatocytes (thus non induced for apoptosis, which is hml-Gal4 dependent [81]) may respond to the death of the other plasmatocytes by inducing a signal that triggers the induction of lamellocytes and the subsequent formation of melanotic tumors. Considering the role of crq in apoptotic cell clearance, this signal may require a functional crq, which could explain why crq ko flies do not develop melanotic tumors. Strikingly, in the Arefin study, as well as in previous studies, targeted ablation of plasmatocytes also made resulting 'hemoless' pupae more susceptible to environmental microbes [23,24,81]. Extensive tissue remodeling takes place at pupariation, and plasmatocytes are essential to remove dying cells, debris, and bacteria. Thus, it was argued that this increased susceptibility was likely due to environmental bacteria invading the body cavity after disruption of the gut [82]. In addition, it was found that the gut microbiome of Hml-apo flies could influence pupal lethality, as the eclosure rate of Hml-apo flies varied depending on the quality of the food they were reared on [81]. Accordingly, our rescue of the crq ko pupal lethality with antibiotics demonstrates that their premature aging and death are indeed due to infection by normally innocuous environmental bacteria. Altogether, these data suggest that phagocytes and crq are important actors regulating the interaction between a host and its microbiome.
Hosts use both resistance and tolerance mechanisms to withstand infection and survive a specific dose of microbes [65,83]. crq ko flies exhibit a shorter lifespan when compared to control flies, but they are equally tolerant to aseptic wounds and infections. The crq ko flies are less resistant to infection, as crq is required to promote efficient microbial phagocytosis. crq ko plasmatocytes can still engulf bacteria, albeit at a lower efficiency than their controls. Our data also demonstrate that crq plays a major role in phagosome maturation during bacterial clearance. This is in agreement with a recent study showing that crq promotes phagosome maturation during the clearance of neuronal debris by epithelial cells [36]. Thus, crq is required at several stages of phagocytosis. Similar observations have been made for the C. elegans Ced-1 receptor and for Drpr, as both promote engulfment of apoptotic corpses and their degradation in mature phagosomes [84,85].
'Hemoless' , Hml-apo and crq ko flies are all more susceptible to environmental microbes and their microbiota. While it is not known whether mutants of eater, which encodes a phagocytic receptor for bacteria but does not play a role in phagosome maturation, are more susceptible to environmental microbes during pupariation, both eater mutants and 'hemoless' flies showed either decreased or unaffected systemic responses [23,24,26]. Hml-apo larvae however, showed an upregulation in Toll-dependent constitutive Drs mRNA levels whereas Dpt expression was suppressed [81]. In contrast, crq ko flies showed no significant difference in constitutive or infection induced expression of Drs, but showed an increased expression of Dpt with age, and infection induced an increased and chronic expression of Dpt. Altogether our results argue that phagosome maturation defects in crq ko flies lead to persistence of bacteria and thus to an increased and persistent systemic immune response via the Imd pathway. Such defects in phagosome maturation are not present in hemocyte ablation experiments, which could explain different outcomes for the host immunity and survival.
We have found that Crq acts in parallel to the Toll and Imd pathways. In the mealworm Tenebrio molitor, hemocytes and cytotoxic enzymatic cascades eliminate most bacteria early during infection, and AMPs are required to eliminate persisting bacteria [86]. These data suggest that AMPs act in parallel with hemocytes to fight infections. We have also found that crq ko flies are more susceptible to infection with S. aureus than wild-type and Toll pathway-deficient flies. These results are consistent with S. aureus infection being mainly resolved via phagocytosis and Crq having a major role in this process. Surprisingly, we have observed the opposite for infection with other Gram-negative or positive bacteria and fungi. Drosophila mutants for AMP production were more susceptible to infection than crq ko flies, arguing that AMPs are critical to eliminate the bulk of pathogens. Indeed, crq (thus phagocytosis) is not essential for Ecc15 elimination, but accelerates bacterial clearance. Our results also suggest that the defects in phagosome maturation may allow some bacteria to persist and grow within hemocytes, where they are hidden from systemic AMPs. Thus, depending on the microbe, humoral and cellular immune responses can act at distinct stages of infection. In this context, phagocytosis acts as a main defense mechanism against pathogens that may escape AMPs or modulate their production.
Chronic activation of immune pathways can be detrimental to organismal health [13][14][15]. In Drosophila, multiple negative regulators of the Imd pathway, including PGRP-LB, act in concert to maintain immune homeostasis [14][15][16]. We have observed that crq ko flies sustain high production levels of the AMP Dpt and the cytokine Upd3, demonstrating that defects in phagocyte function can lead to chronic immune activation. Notably, the level of Dpt expression induced by activation of the Imd pathway in unchallenged conditions is stronger in crq ko flies than was previously observed in mutants of three negative regulators of the Imd pathway, namely pirk EY , PGRP-SC Δ , and PGRP-LB Δ [15], and over 1,000-fold higher in PGRP-LB Δ , crq ko double mutants. This is despite the persistence of only a few hundred bacteria in these mutants. This phenotype may be due solely to the accumulation of these persistent bacteria, or Crq may also function in plasmatocytes to remove immunostimulatory molecules from the hemolymph. Nonetheless, our study shows that plasmatocytes, Crq, and phagocytosis are all key factors in the immune response, and that losing crq induces a state of chronic immune induction.
The ability of a host to control microbes decreases with age, a phenomenon called immune senescence [71]. The causes of immune senescence remain elusive, but the loss of immune cells with age and a decline in their ability to phagocytose have been suggested [56,57]. Recent studies have argued that microbial dysbiosis and disruption in gut homeostasis contribute to early aging [76,77,87]. In addition, persistent activation of the JAK-STAT pathway in the gut has been linked to age-related decline in gut structure and function [88]. Aging crq ko flies lose a greater number of hemocytes than wild-type flies after infection, which may be the result of accumulating bacteria in these hemocytes in which phagosomes fail to mature. The premature death of crq ko flies could be partially rescued by the presence of antibiotics. This demonstrates that phagocytosis, and phagosome maturation in particular, plays a crucial role in managing the response to environmental microbes and potentially, the gut microbiota directly to promote normal aging. We have also found that chronic upd3 expression in crq ko flies triggers premature midgut hyperplasia, which is known to alter host physiology and promote premature aging [72,76,89]. It has recently been proposed that plasmatocytes can influence gut homeostasis by secreting dpp ligands and modulating stem cell activity [90]. Our results reinforce the possibility of an interaction between plasmatocyte function and gut homeostasis, and suggests that cytokines derived from hemocytes can trigger cell responses in the gut. These results are also in agreement with a recent publication showing that Upd3 from hemocytes can trigger intestinal stem cell proliferation [69]. Altogether, these results demonstrate that the interaction between hemocytes and the gut tissue are central to host health, and our data demonstrate that phagocytic defects can be associated with chronic gut inflammation and aberrant intestinal stem cell turn-over. As gut aging and barrier integrity are in turn important to maintain bodily immune homeostasis [76], we propose the following model: in crq ko flies, plasmatocyte-derived cytokines accelerate gut aging promoting loss of gut homeostasis and microbial dysbiosis, with immune and plasmatocyte activation acting in a positive feedback loop (S7 Fig). Collectively, our data show that Crq is essential in development and aging to protect against environmental microbes. Interestingly, the impact of mutating crq on host physiology is strikingly different from previously reported phagocytic receptor mutations. We speculate that this could be due to its dual role in uptake and phagosome maturation during phagocytosis. Crq is required for microbial elimination in parallel to the Toll and Imd pathways and acts to maintain immune homeostasis. This situation is surprisingly reminiscent of inflammatory disorders, such as Crohn's disease, that result from primary defects in bacterial elimination and trigger chronic immune activation and disruption of gut homeostasis. Further characterization of the crq mutation in Drosophila will provide an interesting conceptual framework to understand auto-inflammatory diseases and their repercussions on immune homeostasis and host health.

Materials and Methods
Fly rearing, stocks, and mutant generation All stocks were raised at 22°C on standard medium, unless otherwise specified. Rel E20 , spz rm7 , and PGRP-LB Δ stocks were described in [15,61,91]. The crq ko stock was generated by homologous recombination, which removed the majority of the crq open reading frame [36] and (S1B Fig).  For B. bassiana infection, flies were shaken in a petri dish with mature germinating Beauveria for spore coating. All infections and aging experiments were performed at 29°C. In antibiotic treatments, a cocktail of kanamycin, ampicillin, rifampicin, streptomycin, and spectinomycin (5mg/mL each) was added to the fly medium. Axenic stocks were generated as described in [72,73]. Survival experiments represent at least 3 independent repeats with 20 flies (60-100 flies tested). Survival was analyzed by a Log-rank test using the statistical programs R and Prism.

Quantification of bacterial CFUs
Flies were individually homogenized in 500 μl of sterile PBS using bead beating with a tissue homogenizer (OPS Diagnostics). Dilutions of the homogenate were plated onto LB agar or MRS agar with a WASP II autoplate spiral plater (Microbiology International), incubated at 29°C, and the CFUs counted. Results were analyzed using a Krustal-Wallis test in R.

Phagocytosis assays and plasmatocyte immunostaining
Flies were injected in their thorax with 69nl of pHrodo red or Alexa 488 bacteria (Life Technologies Inc.) using a nanoject injector (Drummond). The fluorescence within the abdomen of the flies was then imaged at 45min, 3hrs, and 5hrs post-injection with a Leica MZFLIII fluorescent microscope and DFC300 FX camera and quantified using Image J 2.0.0-rc-30/1.49s (NIH).
For ex vivo imaging, flies were injected with 46nl of PBS at 45min, 3hrs and 5hrs after infection to release all hemocytes, and 10 flies were bled on a lysine-coated slide by mechanically scraping their hemocytes onto a drop of PBS. Once settled for 10min on the slide, hemocytes were quickly dried and mounted with AF1 mounting solution (Citifluor Ltd). Slides were automatically scanned using a Zeiss LSM 700 confocal microscope, and the number of plasmatocytes and average fluorescence signal per plasmatocyte quantified.
For immunostaining, flies were bled as described above and the hemocytes fixed in a solution of PBS, Tween 0.1%, PFA 4% for 30min. The samples were incubated in PBT with 1% normal goat serum and Crq [53] and GFP antibodies (Roche) at 1:500 overnight at 4°C. Samples were washed at RT three times for 5min in PBS, incubated with the appropriate secondary antibodies at 1:1000 in PBT for 2hrs at RT, and washed three additional times in PBT. Samples were imaged with a Zeiss LSM 700 confocal microscope.

RT-qPCR
Total RNA was extracted from pools of 20 flies per time point using TRIzol (Invitrogen). RNA was reverse-transcribed using Superscript II (Invitrogen), and the qPCR was performed using SYBR green (Quanta) in a Biorad instrument. Data represent the ratio or relative ratio (in %) of mRNA levels of the target gene (crq, Dpt, Drs or upd3) and that of a reference gene (RpL32 also known as rp49). The primer sequences used in this study are provided in the supplementary material. All experiments were performed at least 3 times.  1. (B) The relationship between health and bacterial load (tolerance curve) is depicted here. A tolerance curve adopts a sigmoid shape, and we focus on the linear part of the relationship, where tolerance is represented by the slope of the regression health/load. (C, D) Tolerance graph of PXH87 and crq ko flies given as the plot of regression between LT50 and the log number of injected bacteria for Ecc15 (C) or E. faecalis (D) septic injury. (E) Tolerance graph for PXH87 and crq ko flies given as the plot of regression of their survival at 3 timepoints post infection against the log number of Ecc15 CFUs present at the same timepoint. (TIF) S5 Fig. crq is required for bacterial phagocytosis. (A, B) Fluorescent images of abdomen of control pXH87, crq ko and crq ko ; crq-Gal4 > UAS-crq rescue flies at 3hrs after injection of Alexa488 E. coli and Alexa488 S. aureus, respectively. (C, D) 3D reconstruction and sections of confocal zeta-stacks scans of eater-nls::GFP hemocytes. In WT most hemocytes internalize rhodamine E. coli at 45min post injection. In crq KO  In absence of crq, phagocytic function is decreased and absence of phagosome maturation is associated with a defect in bacterial clearance. This mild immune-deficiency in turns triggers a chronic activation of immune pathways and cytokine production, potentially secondary to the decreased bacterial clearance. This hyperactive immune response includes the activation of the Toll and Imd pathways, and the induction of the cytokine Upd3. This chronic immune activation results in the induction of early midgut hyperplasia and promotes a decrease in lifespan. (TIF)