InteractiveFly: GeneBrief

croquemort: Biological Overview | References

Gene name - croquemort

Synonyms -

Cytological map position - 21C2-21C2

Function - transmembrane receptor

Keywords - a member of the CD36 family of scavenger receptors - required for microbial phagocytosis and efficient bacterial clearance - booster for croquemort interacts with the zinc finger domain of the GATA transcription factor Serpent (Srp), to enhance its direct binding to the crq promoter; thus, Crq and Srp function together in regulating crq expression and efferocytosis, the action of removing apoptotic particles

Symbol - crq

FlyBase ID: FBgn0015924

Genetic map position - chr2L:448,254-453,024

NCBI classification - CD36 family

Cellular location - surface transmembrane

NCBI links: EntrezGene, Nucleotide, Protein

Crq orthologs: Biolitmine

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. This study reports 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, these data demonstrate that Crq plays a key role in maintaining immune and organismal homeostasis (Guillou, 2016).

This study shows that Crq is required for the engulfment of microbes by plasmatocytes and their clearance. The mild immune deficiency due to crq mutation is associated with increased susceptibility to infection, defects in immune homeostasis, gut hyperplasia, and decreased lifespan. This study also re-confirmed a role for crq in apoptotic cell clearance, although the phagocytosis defect of crqko plasmatocytes is less severe than what had been previously observed with two lethal crq deficiency mutants, Df(2L)al and Df(2L)XW88. 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. 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. The current study shows 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. The in vivo data in crqko 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. Previous experiments to test whether crq is required for bacterial phagocytosis in embryos were qualitative rather than quantitative and did not allow identification of a role for crq at that stage. In contrast, the current experiments in adult crqko flies are quantitative and allowed identification of a delay in phagocytosis, followed by a defect in bacterial clearance in crqko 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. 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 (Guillou, 2016).

Because the immune competence of hemocytes varies during development, the potential role for crq in innate immunity was examined by knocking it out. This study shows that Crq is a major plasmatocyte marker at all developmental stages of the fly. crqko flies are homozygous viable, but short-lived, and can hardly be maintained as a homozygous stock in a non-sterile environment; crqko pupae become susceptible to environmental bacteria and their microbiota during pupariation. In a recent study, Arefin (2015) induced the pro-apoptotic genes hid or Grim in plamatocytes and crystal cells using the Hml-Gal4 driver (Hml-apo). A similar pupal lethality was observed but also associated with an induction of lamellocyte differentiation and the apparition of melanotic tumors of hemocyte origin. It was therefore concluded that the death of hemocytes triggered lamellocyte accumulation and melanotic tumor phenotypes. In contrast, no obvious melanotic tumors were observed in crqko flies, despite observing a loss of hemocytes in aging crqko flies and crqko flies subjected to Ecc15 infection. One possible explanation is that hemocytes do not die of apoptosis in crqko 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. Thus the 27.6% of non-Hml plasmatocytes (thus non induced for apoptosis, which is Hml-Gal4 dependent) 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 crqko 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. 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. 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. Accordingly, the rescue of the crqko 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 (Guillou, 2016).

Hosts use both resistance and tolerance mechanisms to withstand infection and survive a specific dose of microbes. crqko flies exhibit a shorter lifespan when compared to control flies, but they are equally tolerant to aseptic wounds and infections. The crqko flies are less resistant to infection, as crq is required to promote efficient microbial phagocytosis. crqko plasmatocytes can still engulf bacteria, albeit at a lower efficiency than their controls. The 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 (Han, 2014). 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 (Guillou, 2016).

'Hemoless', Hml-apo and crqko 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. Hml-apo larvae however, showed an upregulation in Toll-dependent constitutive Drs mRNA levels whereas Dpt expression was suppresse. In contrast, crqko 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 the results argue that phagosome maturation defects in crqko 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 (Guillou, 2016).

This study 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. These data suggest that AMPs act in parallel with hemocytes to fight infections. It was also found that crqko 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, the opposite was observed for infection with other Gram-negative or positive bacteria and fungi. Drosophila mutants for AMP production were more susceptible to infection than crqko 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. The 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 (Guillou, 2016).

Chronic activation of immune pathways can be detrimental to organismal health. In Drosophila, multiple negative regulators of the Imd pathway, including PGRP-LB, act in concert to maintain immune homeostasis. This study has observed that crqko 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 crqko flies than was previously observed in mutants of three negative regulators of the Imd pathway, namely pirkEY, PGRP-SCΔ, and PGRP-LBΔ, and over 1,000-fold higher in PGRP-LBLBΔ, crqko 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, this 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 (Guillou, 2016).

The ability of a host to control microbes decreases with age, a phenomenon called immune senescence. 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. Recent studies have argued that microbial dysbiosis and disruption in gut homeostasis contribute to early aging. In addition, persistent activation of the JAK-STAT pathway in the gut has been linked to age-related decline in gut structure and function. Aging crqko 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 crqko 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. This study also found that chronic upd3 expression in crqko flies triggers premature midgut hyperplasia, which is known to alter host physiology and promote premature aging. It has recently been proposed that plasmatocytes can influence gut homeostasis by secreting dpp ligands and modulating stem cell activity. These 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. Altogether, these results demonstrate that the interaction between hemocytes and the gut tissue are central to host health, and the 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, the following model is proposed: in crqko 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 (Guillou, 2016).

Collectively, these 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. It is speculated 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 (Guillou, 2016).

bfc, a novel serpent co-factor for the expression of croquemort, regulates efferocytosis in Drosophila melanogaster

Efferocytosis is the process by which phagocytes recognize, engulf, and digest (or clear) apoptotic cells during development. Impaired efferocytosis is associated with developmental defects and autoimmune diseases. In Drosophila melanogaster, recognition of apoptotic cells requires phagocyte surface receptors, including the scavenger receptor CD36-related protein, Croquemort (Crq, encoded by crq). In fact, Crq expression is upregulated in the presence of apoptotic cells, as well as in response to excessive apoptosis. This study identified a novel gene bfc (booster for croquemort), which plays a role in efferocytosis, specifically the regulation of the crq expression. Bfc protein interacts with the zinc finger domain of the GATA transcription factor Serpent (Srp), to enhance its direct binding to the crq promoter; thus, they function together in regulating crq expression and efferocytosis. Overall, this study shows that Bfc serves as a Srp co-factor to upregulate the transcription of the crq encoded receptor, and consequently boosts macrophage efferocytosis in response to excessive apoptosis. Therefore, this study clarifies how phagocytes integrate apoptotic cell signals to mediate efferocytosis (Zheng, 2021).

Apoptosis is a developmentally programmed cell death process in multicellular organisms essential for the removal of excessive or harmful cells; whereby apoptotic cells (ACs) are swiftly removed by phagocytes to prevent the release of toxins and induction of inflammation, a process crucial for organ formation, tissue development, homeostasis, and normal immunoregulation. In fact, defects in AC clearance (efferocytosis) can lead to the development of various inflammatory and autoimmune diseases. During efferocytosis, the effective clearance of ACs is accomplished through the recognition and binding of engulfment receptors or bridging molecules on the surface of phagocytes to 'eat me' signals exposed on the surface of ACs. After receptor activation, downstream signals trigger actin cytoskeleton rearrangement and membrane extension around the ACs to form phagosomes. Finally, mature phagosomes fuse with lysosomes to form phagolysosomes, where the internalized ACs are ultimately digested and cleared (Zheng, 2021).

Since efferocytosis is conserved throughout evolution, it has been studied not only in mammals but also in Drosophila melanogaster. Of note, in D. melanogaster, ACs are removed by non-professional phagocytes, such as epithelial cells and professional phagocytes, such as macrophages and glial cells. Importantly, Drosophila macrophages perform similar functions to those of mammalian macrophages; they participate in both the phagocytosis of ACs and pathogens. Several engulfment receptors have been identified as key players in the recognition and removal of ACs in Drosophila. Franc and colleagues first characterized Croquemort (Crq), a Drosophila CD36-related receptor required by macrophages to engulf ACs. Additionally, Draper (Drpr, a homolog of CED-1/MEGF10) also mediates AC clearance in both glia and macrophages; JNK signaling plays a role in priming macrophages to rapidly respond to injury or microbial infections. Of note, Drpr and its adapter Dmel\Ced-6 (GULP homolog) also seemed important for axon pruning and the engulfment of degenerating neurons by glial cells. The Src tyrosine kinase Src42A (Frk homolog) promotes Drpr phosphorylation and its association with another soluble tyrosine kinase, Shark (ZAP70 homolog), which in turn activates the Drpr pathway. In addition to Drpr, Six-Microns-Under (SIMU) [10] and integrin αPS3 [21] contribute to efferocytosis. SIMU, a Nimrod family cell surface receptor, functions upstream of Drpr to mediate the recognition and clearance of ACs as well as of non-apoptotic cells at wound sites through the recognition of phosphatidylserine (PS). Importantly, the transcriptional factor Serpent (Srp), a GATA factor homolog, was recently found to be required for the efficient phagocytosis of ACs in the context of Drosophila embryonic macrophages and acted via the regulation of SIMU, Drpr, and Crq (Zheng, 2021).

Searching for other genes required for efferocytosis, this study performed transcriptomic analysis (RNA-seq) and RNAi screening, and discovered 12 genes required for AC clearance in Drosophila S2 cells. In particular, a novel gene, bfc (booster for croquemort), involved in efferocytosis, encodes a specific regulatory factor for the crq transcriptional expression, both in vitro and in vivo. Importantly, this study demonstrated that the GATA factor Srp directly binds to the crq promoter, while Bfc strengthens this binding by interacting with the Srp zinc finger domain. Therefore, a model is proposed in which Bfc cooperates with Srp to enhance crq expression and subsequently induce efferocytosis in D. melanogaster (Zheng, 2021).

In mammals, ACs are recognized by CD36, one of the several phagocyte cell surface receptors, with the AC surface molecules serving as cognate 'eat-me' signals/ligands. ACs also secrete molecules that attract distant phagocytes and modulate the immune response or phagocytic receptor activity. However, the mechanisms underlying this effect remain unclear. Crq is a CD36-related scavenger receptor in Drosophila and is expressed immediately after the onset of apoptosis in embryonic macrophages. The expression of Crq is regulated by the extent of apoptosis, although the regulatory mechanisms by which ACs control the expression of Crq and subsequently induce phagocytosis in embryonic macrophages have not been described (Zheng, 2021).

This study has revealed a novel protein, Bfc (Booster for Crq), that plays a key role in efferocytosis via specifically regulating the expression of crq in a manner dependent on the extent of apoptosis. Bfc interacts with the zinc finger domain of the transcription factor Srp as a cofactor to enhance the binding of Srp to the crq promoter, leading to the upregulation of crq expression and the consequent induction of efferocytosis in Drosophila melanogaster. Importantly, the data reveal the molecular mechanisms by which ACs affect Crq expression, as well as how the phagocytic ability of embryonic macrophages is boosted in the presence of excessive apoptosis (Zheng, 2021).

This study found that in S2 cells, the ACs induced the transcriptional upregulation of crq. In vivo, the macrophages developed as early as the first wave of developmentally programmed apoptosis began at embryogenic stage 11, when the expression of crq was activated and subsequently became widespread throughout the embryo. Importantly, these results are similar to the regulatory mechanisms associated with the expression of other phagocytic receptors, such as Drpr and integrin. For instance, studies showed that AC engulfment rapidly triggers an intracellular calcium burst followed by increased levels of drpr transcripts in Drosophila macrophages; similarly, Draper and integrins become apically enriched soon after the engulfment of apoptotic debris in epithelial follicle cells (Zheng, 2021).

That the expression of crq was elevated early after the co-culture of ACs and S2 cells, but gradually decreased to the basal levels as efferocytosis continued, suggesting that the regulation of AC clearance and crq expression follow a similar pattern. It was demonstrated that most AC samples added to live S2 cells were composed of apoptotic cells rather than necrotic cells. However, the upregulated expression of genes in response to the presence of a few necrotic cells cannot eliminated. Indeed, based on transcriptome analysis, 12 genes were identified that are required for AC clearance, which was confirmed by subsequent efferocytosis assays using their individual knockdown in S2 cells. Interestingly, among the 12 genes, two were related to innate immunity. CecA1, regulated at the transcriptional level encodes an antibacterial peptide, as well as a secreted protein that mediates the activation of the Toll pathway during bacterial infection. This result may contradict the discreet nature of the apoptotic process. However, ATPs released by bacteria are known to mediate inflammation, and the toll-like receptor 4 (TLR4) is activated by ACs to promote dendritic cell maturation and innate immunity in human monocyte-derived dendritic cells. These results indicate that innate immune pathways are activated in the presence of ACs, and may contribute to their recognition or clearance in Drosophila (Zheng, 2021).

Among these 12 genes, CG9129 (bfc) and CG30172 regulated the expression of crq and hence, efferocytosis. Further studies must be performed to elucidate the role of CG30172 in efferocytosis. On the other hand, The role played by bfc in efferocytosis as well as the underlying mechanism was clearly dissected. Using several different experimental approaches, this study demonstrated that bfc regulates crq expression in response to excessive apoptosis. First, bfc RNAi treatment decreased the crq expression levels in S2 cells exposed to ACs, but not in the absence of ACs. Second, the increase in crq transcription was proportional to the extent of apoptosis in embryos, which was blocked by the loss of bfc. Notably, other phagocytic receptors have been reported to be activated by dying cells. The integrin heterodimer αPS3/βPS can be enriched in epithelial follicle cells after the engulfment of dying germline cells. In addition, Drpr expression increases in follicle and glial cells, which activates the downstream JNK signaling during the clearance of apoptotic germline cells and neurons, respectively. Collectively, the available scientific literature suggests that the expression of phagocytic receptors can be stimulated by the presence of excessive ACs to improve the phagocytic activity of macrophages or epithelial cells in different tissues (Zheng, 2021).

Bioinformatics analysis of the conserved domains and gene structure indicated that Bfc does not likely function directly as a transcription factor. This study identified Srp as a Bfc interaction partner using yeast two-hybrid and Co-IP analyses. Shlyakhover (2018) reported that Srp is required for the expression of SIMU, Drpr, and Crq receptors in embryonic macrophages; however, the current results demonstrated that bfc only affects the expression of crq expression through interaction with Srp, with no impact on the expression of several other genes. A plausible hypothesis for this phenotype is that Bfc assistance for Srp binding to the promoters of simu and drpr, may have limited effects. Thus, the results suggest that Bfc may regulate the Crq expression levels in the first wave of AC recognition via binding to Srp, whereas other regulatory factors participate in the Srp-mediated regulation of Drpr and SIMU (Zheng, 2021).

Srp directly binds to the DNA consensus sequence GATA of the crq promoter via its highly conserved Cys-X2-Cys-X17-Cys-X2-Cys zinc finger binding domain (C4 motif). Meanwhile, Srp also interacts with Bfc through its zinc finger domain; curiously, while the mutation of the C4 motif did not affect the latter interaction, it completely blocked the former. Importantly, it was also shown that mutation in the GATA site abolished the expression of the crq in Drosophila embryo macrophages. As a potential Srp cofactor, Bfc increased the ability of Srp to bind to the crq promoter, while bfc knockdown inhibited the crq transcriptional activity. Ush (homolog of FOG-2 in Drosophila), a cofactor of GATA transcriptional factors, can bind Srp and limit crystal cell production during Drosophila blood cell development. Interestingly, genetic studies have demonstrated that Ush acts with Srp to maintain the pluripotency of hemocyte progenitors and suppresses their differentiation. Ush was reported to repress crq expression by interacting with the isoform of Srp, SrpNC (with two GATA zinc finger) while the other isoform of Srp, SrpC (with one GATA zinc finger) induced crq expression, which may indicate Bfc and Ush act on different isoforms of Srp to regulate crq expression by opposite mechanisms (Zheng, 2021).

Although the results elucidate several factors that contribute to efferocytosis in Drosophila embryos, some mechanistic details remain unresolved; for instance, how ACs induce Bfc-mediated regulation of crq expression in macrophages remains unclear. Bfc regulates Crq expression and efferocytosis, but not macrophage development. Moreover, this study found that Bfc-mediated activation of crq transcription and Crq accumulation leads to positive feedback to promote increased Bfc expression, which is required for engulfment. As expected, the upregulation of Bfc expression occurred earlier than that of crq in S2 cells after incubation with ACs. Therefore, further studies are required to elucidate the upstream signals in the context of the crq-mediated regulation of bfc expression. As previous studies have shown that Crq is required for phagosome maturation during the clearance of neuronal debris by epithelial cells and bacterial clearance, further studies should be conducted to determine whether Bfc is involved in the clearance of neuronal debris (Zheng, 2021).

This study is not without limitations. For instance, other potential regulators of efferocytosis, whose expression is not affected by ACs could not be detected in this study. In mammals, CD36 is involved in the clearance of ACs and regulates the host inflammatory response. As a CD36 family homolog, Crq promotes the clearance of ACs and bacterial uptake via efferocytosis. Researchers have reported that the GATA factor Srp is required for Crq expression; this study confirmed this finding and showed that Srp directly binds to the crq promoter via its GATA binding site, which is enhanced by Bfc. However, no apparent Bfc homologs exist in vertebrates, and whether GATA factors regulate the CD36 family in a mechanism similar to that in flies remains unclear. Nevertheless, it is predicted that one or more functional homologs of Bfc may exist in mammals and are likely involved in apoptotic cell clearance. Unraveling them as well as determining whether and how bfc participates in eliminating pathogens and innate immunity is essential (Zheng, 2021).

In summary, this study has shown that the expression of the engulfment receptor Crq is transcriptionally regulated by the presence of ACs, via Srp, and its newly identified cofactor, Bfc. Altogether, these findings imply that macrophages adopt a precise mechanism to increase the expression of engulfment receptors to boost their phagocytic activity, in the presence of excessive ACs. A similar role and mechanism is anticipated in the context of mammalian engulfment receptors in response to excessive ACs. Therefore, the findings of this study have significant implications for a wide range of human diseases, including those associated with aberrant apoptotic cell death and efferocytosis, such as tumor progression, neurodegenerative disorders, and other severe inflammatory conditions (Zheng, 2021).

Drosophila GATA factor serpent establishes phagocytic ability of embryonic macrophages

During Drosophila embryogenesis, a large number of apoptotic cells are efficiently engulfed and degraded by professional phagocytes, macrophages. Phagocytic receptors Six-Microns-Under (SIMU), Draper (Drpr) and Croquemort (Crq) are specifically expressed in embryonic macrophages and required for their phagocytic function. However, how this function is established during development remains unclear. This study demonstrates that the key regulator of Drosophila embryonic hemocyte differentiation, the transcription factor Serpent (Srp), plays a central role in establishing macrophage phagocytic competence. Srp, a homolog of the mammalian GATA factors, is required and sufficient for the specific expression of SIMU, Drpr and Crq receptors in embryonic macrophages. Moreover, each of these receptors can significantly rescue phagocytosis defects of macrophages in srp mutants, including their distribution in the embryo and engulfment of apoptotic cells. This reveals that the proficiency of macrophages to remove apoptotic cells relies on the expression of SIMU, Crq and/or Drpr. However, Glial Cells Missing (GCM) acting downstream of Srp in the differentiation of hemocytes, is dispensable for their phagocytic function during embryogenesis. Taken together, this study discloses the molecular mechanism underlying the development of macrophages as skilled phagocytes of apoptotic cells (Shlyakhover, 2018).

Leishmania amazonensis engages CD36 to drive parasitophorous vacuole maturation

Leishmania amastigotes manipulate the activity of macrophages to favor their own success. However, very little is known about the role of innate recognition and signaling triggered by amastigotes in this host-parasite interaction. This work developed a new infection model in adult Drosophila to take advantage of its superior genetic resources to identify novel host factors limiting Leishmania amazonensis infection. The model is based on the capacity of macrophage-like cells, plasmatocytes, to phagocytose and control the proliferation of parasites injected into adult flies. Using this model, a collection of RNAi-expressing flies were screened for anti-Leishmania defense factors. Notably, three CD36-like scavenger receptors (croquemort, CG31741, and CG10345) were found that were important for defending against Leishmania infection. Mechanistic studies in mouse macrophages showed that CD36 accumulates specifically at sites where the parasite contacts the parasitophorous vacuole membrane. Furthermore, CD36-deficient macrophages were defective in the formation of the large parasitophorous vacuole typical of L. amazonensis infection, a phenotype caused by inefficient fusion with late endosomes and/or lysosomes. These data identify an unprecedented role for CD36 in the biogenesis of the parasitophorous vacuole and further highlight the utility of Drosophila as a model system for dissecting innate immune responses to infection (Okuda, 2016).

Epidermal cells are the primary phagocytes in the fragmentation and clearance of degenerating dendrites in Drosophila

During developmental remodeling, neurites destined for pruning often degenerate on-site. Physical injury also induces degeneration of neurites distal to the injury site. Prompt clearance of degenerating neurites is important for maintaining tissue homeostasis and preventing inflammatory responses. This study shows that in both dendrite pruning and dendrite injury of Drosophila sensory neurons, epidermal cells rather than hemocytes are the primary phagocytes in clearing degenerating dendrites. Epidermal cells act via Draper-mediated recognition to facilitate dendrite degeneration and to engulf and degrade degenerating dendrites. Using multiple dendritic membrane markers to trace phagocytosis, it was shown that two members of the CD36 family, croquemort (crq) and debris buster (dsb), act at distinct stages of phagosome maturation for dendrite clearance. These findings reveals the physiological importance of coordination between neurons and their surrounding epidermis, for both dendrite fragmentation and clearance (Han, 2014).

Removal of nonfunctional or damaged tissues is an important biological process during tissue remodeling or repair. This study shows that, for Drosophila class IV da neurons in the periphery, degenerating dendrites in both dendrite pruning and injury models are removed by neighboring epithelial cells rather than professional phagocytes. By developing multiple dendritic markers that label phagosomes differentially, the clearance of degenerating dendrites was established as an in vivo model to study phagocytosis. With these tools, key players in engulfment and phagosome maturation were analyzed, and roles of the CD36 family members Crq and Dsb were elucidated. This study further reveals that, as phagocytes, epidermal cells actively participate in not only the removal but also the fragmentation of degenerating dendrites (Han, 2014).

Professional phagocytes such as macrophages in vertebrates and plasmatocytes in Drosophila dispose the majority of apoptotic cells in development, as well as invading microorganisms during infection. However, nonprofessional phagocytes may take charge when macrophages or other professional phagocytes are absent or cannot easily access cell corpses, as in apoptosis of rat lens cells, follicular atresia, and degeneration of Drosophila egg chambers induced by protein deprivation. This scenario does not apply to Drosophila da neuronal dendrites, which are exposed to circulating plasmatocytes in the hemolymph and sessile plasmatocytes clustered around da neuron somas. Indeed, previous observation of dendrite debris engulfment by plasmatocytes during dendrite pruning has led to the conclusion that plasmatocytes clear pruned dendrites. The current finding that clearance of degenerating dendrites is mainly carried out by epidermal epithelial cells demonstrates that nonprofessional phagocytes are not just a substitute for professional phagocytes in their absence. Rather, plasmatocytes and epidermal cells probably carry out different functions reflecting specialization of cellular functions. The removal of pruned dendrites by Drosophila epidermal cells perhaps can be seen as a parallel to the clearance of photoreceptor outer segments by retinal pigment epithelial cells; in both cases epithelial cells maintain homeostasis of the nervous system as part of their physiological functions. The observation that epidermal cells are also responsible for clearing injured dendrites indicates that the same cellular mechanism is also used to cope with perturbations in the peripheral nervous system (Han, 2014).

Epithelial cells may profoundly influence the development of dendritic arbors of da neurons. During larval development, growing epithelial cells signal to the dendritic arbors so they can grow proportionally to epithelial cells in order to maintain the same coverage of receptive fields of the sensory neurons, a phenomenon known as dendritic scaling. Epithelial cells also contribute to the patterning of dendritic arbors of da neurons by tethering dendrites to the 2D space of the extracellular matrix so that dendrites have to avoid sister dendrites from the same neuron (self-avoidance) or dendrites from neighboring like-neurons (tiling). The finding that epithelial cells mediate the clearance of degenerating dendrites substantially adds to the growing list of dendrite properties regulated by epithelial cells (Han, 2014).

The vertebrate CD36 family members CD36 and scavenger receptor class B type I (SR-BI) mediate phagocytosis of apoptotic cells and microbial pathogens in vitro. The Drosophila CD36 family member Crq is required for efficient phagocytosis of cell corpses in embryos and mediates binding of apoptotic cells by in vitro cultured cells, leading to its proposed role as a receptor for apoptotic cells. This study shows that in epithelial cells crq is required for phagosome maturation but not for the engulfment of degenerating dendrites. As loss of crq does not completely abolish the engulfment of apoptotic cells in the embryo, it is possible that the cell-corpse clearance defect in crq mutant embryos may be a consequence of blocked phagosome maturation. An alternative possibility is that Crq may be required for engulfment and/or phagosome maturation of apoptotic cells by embryonic macrophages but only required for phagosome maturation of pruned or injured dendrites by epithelial cells. This could be due to the fact that macrophages have to actively search for and bind apoptotic cells, while epithelial cells engulf neighboring debris. Further experiments will be needed to determine whether Crq also plays a role in phagosome maturation during phagocytosis by macrophages (Han, 2014).

Loss of Crq function resulted in the fusion of dendrite-derived phagosomes accompanied with a failure of degradation of phagosome contents, most likely due to inefficient delivery of degradation machineries to late phagosomes. As phagosomes normally acquire hydrolases and other phagolysosomal components by fusing with endosomes and lysosomes, it is hypothesized that Crq suppresses homotypic phagosome fusion to promote fusion between phagosomes and late endosomes/lysosomes. Homotypic phagosome fusion rarely happens during normal phagocytosis but is induced by infection of bacterial pathogens such as Helicobacter pylori and Chlamydia trachomatis; the ability of different strains of H. pylori to induce phagosome fusion correlates with the virulence and intracellular survival of these bacteria. Therefore, regulation of the balance between homotypic phagosome fusion and heterotypic fusion between phagosomes and late endosomes/lysosomes is probably critical for the degradation of internalized materials (Han, 2014).

The Drosophila genome encodes fourteen CD36 family members. Besides the involvement of Crq in phagocytosis, another member Pes mediates mycobacteria infection. This study found that the CD36 family member Dsb regulates late stages of phagosome maturation. Interestingly, LIMP-2, the mammalian CD36 family member with the highest homology to Dsb, is an intrinsic lysosomal protein required for the degradation of Listeria in phagosomes. Dsb and LIMP-2 thus appear to have evolutionarily conserved functions in phagosome maturation (Han, 2014).

Phagocytes not only clear cell corpses but may also engulf still-living cells and promote cellular degeneration in many contexts. This study shows that efficient degeneration of dendrites requires the coordination with phagocytic epithelial cells. One mechanism for such coordination is the Drpr-mediated recognition of degenerating dendrites by epidermal phagocytes that form actin-rich membrane structures wrapping around the dendrites to facilitate their fragmentation. In the postnatal mouse brain, microglia actively induce apoptosis of Purkinje cells by producing superoxide ions. It remains to be determined whether nonprofessional phagocytes such as epidermal cells also promote neurite degeneration by emitting diffusible agents (Han, 2014).

Autophagy and phagocytosis-like cell cannibalism exert opposing effects on cellular survival during metabolic stress

Understanding mechanisms controlling neuronal cell death and survival under conditions of altered energy supply (e.g., during stroke) is fundamentally important for the development of therapeutic strategies. The function of autophagy herein is unclear, as both its beneficial and detrimental roles have been described. Previous work demonstrated that loss of AMP-activated protein kinase (AMPK), an evolutionarily conserved enzyme that maintains cellular energy balance, leads to activity-dependent degeneration in neuronal tissue. This study shows that energy depletion in Drosophila AMPK mutants results in increased autophagy that convincingly promotes, rather than rescues, neurodegeneration. The generated excessive autophagic response is accompanied by increased TOR and S6K activity in the absence of an AMPK-mediated negative regulatory feedback loop. Moreover, energy-depleted neurons use a phagocytic-like process as a means to cellular survival at the expense of surrounding cells. Consequently, phagocytosis stimulation by expression of the scavenger receptor Croquemort significantly delays neurodegeneration. This study thus reveals a potentially novel strategy for cellular survival during conditions of extreme energy depletion, resembling xeno-cannibalistic events seen in metastatic tumors. This study provides new insights into the roles of autophagy and phagocytosis in the neuronal metabolic stress response and open new avenues into understanding of human disease and development of therapeutic strategies (Poels, 2012).

The SNMP/CD36 gene family in Diptera, Hymenoptera and Coleoptera: Drosophila melanogaster, D. pseudoobscura, Anopheles gambiae, Aedes aegypti, Apis mellifera, and Tribolium castaneum

Sensory neuron membrane proteins (SNMPs) are membrane bound proteins initially identified in olfactory receptor neurons of Lepidoptera and are thought to play a role in odor detection; SNMPs belong to a larger gene family characterized by the human protein CD36. This study has identified 12-14 candidate SNMP/CD36 homologs from each of the genomes of Drosophila melanogaster, D. pseudoobscura, Anopheles gambiae and Aedes aegypti (Diptera), eight candidate homologs from Apis mellifera (Hymenoptera), and 15 from Tribolium castaneum (Coleoptera). Analysis (sequence similarity and intron locations) suggests that the insect SNMP/CD36 genes fall into three major groups. Group 1 includes the previously characterized D. melanogaster emp (epithelial membrane protein). Group 2 includes the previously characterized D. melanogaster croquemort, ninaD, santa maria, and peste. Group 3 genes include the SNMPs, which fall into two subgroups referred to as SNMP1 and SNMP2. D. melanogaster SNMP1 (CG7000) shares both significant sequence similarity and five of its six intron insertion sites with the lepidopteran Bombyx mori SNMP1. The topological conservation of this gene family within the three major holometabolous lineages indicates that it predates the coleopteran and hymenoptera/dipera/lepidoptera split 300+ million years ago. The current state of knowledge of the characterized insect members of this gene family is discussed (Nichols, 2008).

Macrophage-mediated corpse engulfment is required for normal Drosophila CNS morphogenesis

Cell death plays an essential role in development, and the removal of cell corpses presents an important challenge for the developing organism. Macrophages are largely responsible for the clearance of cell corpses in Drosophila melanogaster and mammalian systems. This study examined the developmental requirement for macrophages in Drosophila and found that macrophage function is essential for central nervous system (CNS) morphogenesis. Mutations in the Pvr locus, which encodes a receptor tyrosine kinase of the PDGF/VEGF family that is required for hemocyte migration, were generated and analyzed. Loss of Pvr function causes the mispositioning of glia within the CNS and the disruption of the CNS axon scaffold. This study further found that inhibition of hemocyte development or of Croquemort, a receptor required for macrophage-mediated corpse engulfment, causes similar CNS defects. These data indicate that macrophage-mediated clearance of cell corpses is required for proper morphogenesis of the Drosophila CNS (Sears, 2003).

Two isoforms of Serpent containing either one or two GATA zinc fingers have different roles in Drosophila haematopoiesis

serpent encodes a GATA transcription factor essential for hematopoiesis in Drosophila. Previously, Srp was shown to contain a single GATA zinc finger of C-terminal type. srp encodes different isoforms, generated by alternative splicing, that contain either only a C-finger (SrpC) or both a C- and an N-finger (SrpNC). The presence of the N-finger stabilizes the interaction of Srp with palindromic GATA sites and allows interaction with the Friend of GATA factor U-shaped (Ush). The respective functions of SrpC and SrpNC during embryonic hematopoiesis were examined. Both isoforms individually rescue blood cell formation, which is lacking in a srp null mutation. Interestingly, while SrpC and SrpNC activate some genes in a similar manner, they regulate others differently. Interaction between SrpNC and Ush is responsible for some but not all aspects of the distinct activities of SrpC and SrpNC. These results suggest that the inclusion or exclusion of the N-finger in the naturally occurring isoforms of Srp can provide an effective means of extending the versatility of srp function during development (Waltzer, 2002).

In order to analyse SrpC and SrpNC activities, their capacities to activate gene expression in vivo were tested during Drosophila embryonic hematopoiesis. Using the UAS-GAL4 system, they were ectopically expressed in the mesoderm and the expression pattern of various hematopoietic markers was assessed. The two genes ush and gcm play critical roles in embryonic hematopoiesis. Their expression in the hematopoietic primordium occurs early and appears to depend on srp activity. Therefore, it was determined whether they are transcriptional targets of SrpC and/or SrpNC. Whereas in a wild-type early embryo, ush expression is restricted to the anterior mesoderm, twist-driven expression of SrpC (twist-SrpC) or SrpNC (twist-SrpNC) induces strong expression of ush throughout the mesoderm. In contrast, twist-SrpC induces gcm expression poorly and in a limited number of mesodermal cells of stage 5 embryos, whereas twist-SrpNC strongly activates gcm expression segmentally from stage 5 to 9 (Waltzer, 2002).

The expression of hematopoietic lineage-specific markers was examined. As plasmatocyte markers, peroxidasin (pxn) and croquemort (crq) were used. Since, crystal cells are the only source of prophenoloxidase (pro-PO) in Drosophila, expression of this gene was used to monitor crystal cell formation. pro-PO transcripts were indeed detected in these cells from early stage 11 to the end of embryogenesis. Analysing these markers, two situations were observed. twist-SrpC and twist- SrpNC have similar abilities to induce expression of the plasmatocyte marker pxn and of the crystal cell marker pro-PO, however expression of crq was induced by twist-SrpC and not by twist-SrpNC. Note that pxn and crq were induced through most of the mesoderm, while pro-PO activation was restricted to the head region (Waltzer, 2002). Taken together, these data show that SrpC and SrpNC have both common and different activities during hematopoiesis. Indeed, both isoforms activate the expression of ush, pxn and pro-PO in a similar manner. However, SrpC and SrpNC differentially stimulate the expression of crq and gcm, respectively, in the mesoderm (Waltzer, 2002).

It is remarkable that srp encodes both single and dual zinc finger-containing products. The results provide strong evidence that this alternative splicing allows production of transcription factors with specific activities. The two isoforms activate the expression of ush and pxn with similar efficiency, suggesting that SrpC and SrpNC have similar transactivating properties in vivo, yet, SrpC (but not SrpNC) activates crq expression, while SrpNC is a much stronger activator of gcm expression than SrpC. The domain coded by exon 4B that is present only in SrpC has no known motif and it is not known if and how it participates in SrpC-specific function. However, the presence of the N-terminal zinc finger encoded by exon 4A may explain some of the distinct features of SrpNC as discussed below (Waltzer, 2002).

As in the case of vertebrate GATA-1, the presence of the N-finger in Srp stabilizes binding to double palindromic GATA sites. Although the N-finger of GATA-1 modulates the binding and the transactivating properties of GATA-1 on synthetic promoters, the functional importance of these effects has remained elusive, particularly since no GATA-1 isoform contains only the C-finger. In the case of srp, these distinct binding properties may have direct functional consequences. For instance, the fact that SrpC and SrpNC activate a common target, ush, whereas only SrpNC strongly activates a specific target, gcm, could be related to the DNA-binding specificity of the two isoforms. A scan of the ush upstream regulatory region shows that it contains several GATA consensus sequences, nine of which are clustered in <1 kb and are organized as three repetitions of three sites. In contrast, GATA sites are far less frequent in gcm regulatory regions and are often organized in palindromes. Considering that ush and gcm are likely to be direct target genes for srp, the different organization of their regulatory regions may explain the differential effect observed (Waltzer, 2002).

The lack of plasmatocyte and crystal cell formation due to an srp null mutation can be rescued by expressing SrpC or SrpNC in the mesoderm. No difference between the two isoforms was seen in this assay, suggesting that the N-finger is not absolutely required for srp function in embryonic blood cell formation. However, in the absence of a functional test, to what extent the formation of embryonic blood cells is fully rescued cannot be determined. Interestingly, rescue experiments with the mouse GATA-1 mutant indicate that the GATA-1 N-finger is dispensable for primitive erythropoiesis but is required for definitive erythopoiesis. In Drosophila, a second wave of hematopoiesis, occurring at the larval stage, gives rise to four different lineages: plasmatocytes, crystal cells, secretory cells and lamellocytes. srp is expressed in the dorsal lymph gland (i.e. the main larval hematopoietic organ) and it probably controls larval hematopoiesis. By analogy to vertebrate GATA-1, the Srp N-finger may provide an additional function for larval hematopoiesis, perhaps during formation of the new cell types (Waltzer, 2002).

In the assay used, the expression of the transgene was limited to the mesoderm but it still rescued blood cell formation. This finding suggests that the early expression of srp in the hematopoietic primordium is sufficient to initiate the genetic program that controls hemocyte formation and differentiation. Interestingly, in the wild-type embryo, srp transcripts are not expressed detectably in hemocytes after stage 11, but Srp protein is detected in plasmatocytes and crystal cells throughout most of embryogenesis. Persistence of srp products in hemocytes might be critical for srp function, and control of srp products at the post-translational level may play a crucial role in the correct regulation of blood cell differentiation. Rescue of crystal cell formation by mesodermal expression of SrpC and SrpNC contrasts with the observation that later expression driven by lz-Gal4 in crystal cells represses their development. Srp levels are reduced in crystal cells compared with surrounding plasmatocytes. Therefore, the results are consistent with a two-step model in which Srp expression is first necessary to induce lz expression and subsequently is downregulated to allow crystal cell differentiation (Waltzer, 2002).

One of the best characterized features of GATA N-fingers is their dimerization with cofactors of the FOG family. Consistent with this feature, it was found that SrpNC interacts with the Drosophila FOG Ush, but SrpC does not. Previous analysis showed that ush regulates the number of crystal cells. It was proposed that this function of ush could be mediated by a putative isoform of Srp containing an N-finger. The current findings strongly support this hypothesis. However, it was not possible to address this issue directly, since both SrpC and SrpNC display a strong Ush-independent repressive effect on crystal cell formation and differentiation (Waltzer, 2002).

A new function of ush revealed here is the regulation of the level of expression of the macrophage receptor crq, suggesting that ush displays a broader function in hematopoiesis than previously assumed. Notably, evidence is provided that Ush modulates SrpNC transactivation of crq. Since Ush interacts with the corepressor dCtBP in vitro, the Ush–SrpNC complex could repress crq expression. However, it is not known whether crq is a direct target of srp, so the possibility that the Ush–SrpNC complex activates a transcriptional repressor that regulates crq cannot be ruled out. Vertebrate FOGs can act as either a coactivator or a corepressor of GATA factors. In Drosophila, Ush is a repressor of Pannier-induced activation in cell culture, and it probably also represses the expression of achaete in the dorso-central proneural cluster in vivo. Furthermore, in a heterologous assay in Drosophila, the CtBP-binding region of mFOG2 is required for repressing the formation of crystal cells but not cardiac cells. Thus several mechanisms seem to regulate the function of the GATA–FOG complex (Waltzer, 2002).

Remarkably, some functions of SrpNC appear to be independent of Ush. Thus, gcm-specific activation by SrpNC is not affected in an ush mutant embryo. Moreover, SrpNC still represses crystal cell formation in the absence of ush. This is reminiscent of mouse erythropoiesis, where both FOG-dependent and FOG-independent regulation of gene expression by GATA-1 have been observed. The molecular mechanisms underlying the regulation by Ush/FOG-1 of SrpNC/GATA-1 activity on some specific targets remain to be elucidated. It is tempting to speculate that the N-finger of SrpNC is involved in the recognition of promoter sequences, on gcm for example, and thus is not available to recruit Ush. Alternatively, other cofactors already localized to the promoter or bound to SrpNC might prevent Ush binding to the N-finger (Waltzer, 2002).

This study has focussed on hematopoiesis, but srp also participates in other developmental processes, such as germ band retraction, midgut differentiation, fat body formation, induction of the immune response and the ecdysone response. It will be interesting to determine the respective roles of SrpC and SrpNC in these different phenomena. Phylogenetic analysis shows that SrpNC is closely related to vertebrate GATA factors. It has been suggested that srp is a functional homolog of the entire vertebrate GATA family, since srp is required in Drosophila for hematopoiesis, like GATA-1/2/3 in mice, and for endodermal development, like GATA-4/5/6. Nevertheless, this hypothesis was at odds with the fact that Srp seemingly had a single zinc finger while all the vertebrate GATAs have two. The present identification of Srp isoforms with two fingers gives new force to this hypothesis. Further, the expression of isoforms of Srp with distinct activities helps to account for the broad range of functions ensured by this gene (Waltzer, 2002).

It is worth noting that alternative splicing eliminating the N-finger has also been described in Bombyx mori GATAß and in chicken GATA-5 genes. Moreover, a BLAST search analysis revealed alternatively spliced human expressed sequence tags coding for two isoforms of a potential GATA factor with either one or two zinc fingers. This suggests that alternative splicing of GATA genes could be more general than previously thought, and as yet unnoticed splice variants of GATA vertebrate genes may generate proteins with only a C-finger (Waltzer, 2002).

In conclusion, these results shed further light on the molecular control of hematopoiesis by the GATA factor Srp. The alternative splicing of srp gives rise to different Ush-interacting and non-interacting Srp proteins with different target gene specificities, thereby contributing to the exquisite control of Drosophila blood cell formation. It is speculated that alternative splicing of the GATA N-finger might be an important mechanism regulating the activity of other GATA genes from insects to man (Waltzer, 2002).

gcm2 promotes glial cell differentiation and is required with glial cells missing for macrophage development in Drosophila

glial cells missing (gcm) is the primary regulator of glial cell fate in Drosophila. In addition, gcm has a role in the differentiation of the plasmatocyte/macrophage lineage of hemocytes. Since mutation of gcm causes only a decrease in plasmatocyte numbers without changing their ability to convert into macrophages, gcm cannot be the sole determinant of plasmatocyte/macrophage differentiation. This study has characterized a gcm homolog, gcm2. gcm2 is expressed at low levels in glial cells and hemocyte precursors. This study shows that gcm2 has redundant functions with gcm and has a minor role promoting glial cell differentiation. More significant, like gcm, mutation of gcm2 leads to reduced plasmatocyte numbers. A deletion removing both genes has allowed clarification of the role of these redundant genes in plasmatocyte development. Animals deficient for both gcm and gcm2 fail to express the macrophage receptor Croquemort. Plasmatocytes are reduced in number, but still express the early marker Peroxidasin. These Peroxidasin-expressing hemocytes fail to migrate to their normal locations and do not complete their conversion into macrophages. These results suggest that both gcm and gcm2 are required together for the proliferation of plasmatocyte precursors, the expression of Croquemort protein, and the ability of plasmatocytes to convert into macrophages (Alfonso, 2002).

Requirement for croquemort in phagocytosis of apoptotic cells in Drosophila

Macrophages in the Drosophila embryo are responsible for the phagocytosis of apoptotic cells and are competent to engulf bacteria. Croquemort (CRQ) is a CD36-related receptor expressed exclusively on these macrophages. Genetic evidence showed that crq was essential for efficient phagocytosis of apoptotic corpses but was not required for the engulfment of bacteria. The expression of CRQ was regulated by the amount of apoptosis. These data define distinct pathways for the phagocytosis of corpses and bacteria in Drosophila (Franc, 1999).

Croquemort, a novel Drosophila hemocyte/macrophage receptor that recognizes apoptotic cells

Programmed cell death is first observed at stage 11 of embryogenesis in Drosophila. The systematic removal of apoptotic cells is mediated by cells that are derived from the procephalic mesoderm and differentiate into macrophages. This study describes a macrophage receptor for apoptotic cells. This receptor, croquemort (catcher of death), is a member of the CD36 superfamily. Croquemort-mediated phagocytosis represents the concept that phagocytosis evolved primarily as a cellular process for the removal of effete cells. These findings support the idea that the primordial function of macrophages may have been in tissue modeling and that their adapted role is in host defense (Franc, 1996).

Functions of Croquemort orthologs in other species

CD36, a signaling receptor and fatty acid transporter that regulates immune cell metabolism and fate

CD36 is a type 2 cell surface scavenger receptor widely expressed in many immune and non-immune cells. It functions as both a signaling receptor responding to DAMPs and PAMPs, as well as a long chain free fatty acid transporter. Recent studies have indicated that CD36 can integrate cell signaling and metabolic pathways through its dual functions and thereby influence immune cell differentiation and activation, and ultimately help determine cell fate. Its expression along with its dual functions in both innate and adaptive immune cells contribute to pathogenesis of common diseases, including atherosclerosis and tumor progression, which makes CD36 and its downstream effectors potential therapeutic targets. This review comprehensively examines the dual functions of CD36 in a variety of immune cells, especially macrophages and T cells. CD36 function in non-immune cells such as adipocytes and platelets, which impact the immune system via intercellular communication, is briefly discussed. Finally, outstanding questions in this field are provided for potential directions of future studies (Chen, 2022).

CD36-mediated metabolic crosstalk between tumor cells and macrophages affects liver metastasis

Liver metastasis is highly aggressive and treatment-refractory, partly due to macrophage-mediated immune suppression. Understanding the mechanisms leading to functional reprogramming of macrophages in the tumor microenvironment (TME) will benefit cancer immunotherapy. This study found that the scavenger receptor CD36 is upregulated in metastasis-associated macrophages (MAMs) and deletion of CD36 in MAMs attenuates liver metastasis in mice. MAMs contain more lipid droplets and have the unique capability in engulfing tumor cell-derived long-chain fatty acids, which are carried by extracellular vesicles. The lipid-enriched vesicles are preferentially partitioned into macrophages via CD36, that fuel macrophages and trigger their tumor-promoting activities. In patients with liver metastases, high expression of CD36 correlates with protumoral M2-type MAMs infiltration, creating a highly immunosuppressive TME. Collectively, these findings uncover a mechanism by which tumor cells metabolically interact with macrophages in TME, and suggest a therapeutic potential of targeting CD36 as immunotherapy for liver metastasis (Yang, 2022).

Response to Staphylococcus aureus requires CD36-mediated phagocytosis triggered by the COOH-terminal cytoplasmic domain

Phagocyte recognition and clearance of bacteria play essential roles in the host response to infection. In an on-going forward genetic screen, this study identified the Drosophila melanogaster scavenger receptor Croquemort as a receptor for Staphylococcus aureus, implicating for the first time the CD36 family as phagocytic receptors for bacteria. In transfection assays, the mammalian Croquemort paralogue CD36 confers binding and internalization of Gram-positive and, to a lesser extent, Gram-negative bacteria. By mutational analysis, it was shown that internalization of S. aureus and its component lipoteichoic acid requires the COOH-terminal cytoplasmic portion of CD36, specifically Y463 and C464, which activates Toll-like receptor (TLR) 2/6 signaling. Macrophages lacking CD36 demonstrate reduced internalization of S. aureus and its component lipoteichoic acid, accompanied by a marked defect in tumor necrosis factor-alpha and IL-12 production. As a result, Cd36-/- mice fail to efficiently clear S. aureus in vivo resulting in profound bacteraemia. Thus, response to S. aureus requires CD36-mediated phagocytosis triggered by the COOH-terminal cytoplasmic domain, which initiates TLR2/6 signaling (Stuart, 2005).


Search PubMed for articles about Drosophila

Alfonso, T. B. and Jones, B. W. (2002). gcm2 promotes glial cell differentiation and is required with glial cells missing for macrophage development in Drosophila. Dev Biol 248(2): 369-383. PubMed ID: 12167411

Arefin, B., Kucerova, L., Krautz, R., Kranenburg, H., Parvin, F. and Theopold, U. (2015). Apoptosis in Hemocytes Induces a Shift in Effector Mechanisms in the Drosophila Immune System and Leads to a Pro-Inflammatory State. PLoS One 10(8): e0136593. PubMed ID: 26322507

Chen, Y., Zhang, J., Cui, W. and Silverstein, R. L. (2022). CD36, a signaling receptor and fatty acid transporter that regulates immune cell metabolism and fate. J Exp Med 219(6). PubMed ID: 35438721

Franc, N. C., Heitzler, P., Ezekowitz, R. A. and White, K. (1999). Requirement for croquemort in phagocytosis of apoptotic cells in Drosophila. Science 284(5422): 1991-1994. PubMed ID: 10373118

Franc, N. C., et al. (1996). Croquemort, a novel Drosophila hemocyte/macrophage receptor that recognizes apoptotic cells. Immunity 4: 431-443. PubMed ID: 8630729

Guillou, A., Troha, K., Wang, H., Franc, N. C. and Buchon, N. (2016). The Drosophila CD36 Homologue croquemort Is Required to Maintain Immune and Gut Homeostasis during Development and Aging. PLoS Pathog 12(10): e1005961. PubMed ID: 27780230

Han, C., Song, Y., Xiao, H., Wang, D., Franc, N. C., Jan, L. Y. and Jan, Y. N. (2014). Epidermal Cells Are the Primary Phagocytes in the Fragmentation and Clearance of Degenerating Dendrites in Drosophila. Neuron. PubMed ID: 24412417

Okuda, K., Tong, M., Dempsey, B., Moore, K. J., Gazzinelli, R. T. and Silverman, N. (2016). Leishmania amazonensis engages CD36 to drive parasitophorous vacuole maturation. PLoS Pathog 12: e1005669. PubMed ID: 27280707

Nichols, Z. and Vogt, R. G. (2008). The SNMP/CD36 gene family in Diptera, Hymenoptera and Coleoptera: Drosophila melanogaster, D. pseudoobscura, Anopheles gambiae, Aedes aegypti, Apis mellifera, and Tribolium castaneum. Insect Biochem Mol Biol 38(4): 398-415. PubMed ID: 18342246

Poels, J., Spasic, M. R., Gistelinck, M., Mutert, J., Schellens, A., Callaerts, P. and Norga, K. K. (2012). Autophagy and phagocytosis-like cell cannibalism exert opposing effects on cellular survival during metabolic stress. Cell Death Differ 19(10): 1590-1601. PubMed ID: 22498699

Sears, H. C., Kennedy, C. J. and Garrity, P. A. (2003). Macrophage-mediated corpse engulfment is required for normal Drosophila CNS morphogenesis. Development 130(15): 3557-3565. PubMed ID: 12810602

Shlyakhover, E., Shklyar, B., Hakim-Mishnaevski, K., Levy-Adam, F. and Kurant, E. (2018). Drosophila GATA factor serpent establishes phagocytic ability of embryonic macrophages. Front Immunol 9: 266. PubMed ID: 29568295

Stuart, L. M., Deng, J., Silver, J. M., Takahashi, K., Tseng, A. A., Hennessy, E. J., Ezekowitz, R. A. and Moore, K. J. (2005). Response to Staphylococcus aureus requires CD36-mediated phagocytosis triggered by the COOH-terminal cytoplasmic domain. J Cell Biol 170(3): 477-485. PubMed ID: 16061696

Waltzer, L., et al. (2002). Two isoforms of Serpent containing either one or two GATA zinc fingers have different roles in Drosophila haematopoiesis. EMBO J. 21: 5477-5486. 12374748

Yang, P., Qin, H., Li, Y., Xiao, A., Zheng, E., Zeng, H., Su, C., Luo, X., Lu, Q., Liao, M., Zhao, L., Wei, L., Varghese, Z., Moorhead, J. F., Chen, Y. and Ruan, X. Z. (2022). CD36-mediated metabolic crosstalk between tumor cells and macrophages affects liver metastasis. Nat Commun 13(1): 5782. PubMed ID: 36184646

Zheng, Q., Gao, N., Sun, Q., Li, X., Wang, Y. and Xiao, H. (2021). bfc, a novel serpent co-factor for the expression of croquemort, regulates efferocytosis in Drosophila melanogaster. PLoS Genet 17(12): e1009947. PubMed ID: 34860835

Biological Overview

date revised: 27 October 2022

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