Elimination of plasmatocytes by targeted apoptosis reveals their role in multiple aspects of the Drosophila immune response Bernard Charroux and Julien Royet1 Institut de Biologie du De´veloppement de Marseille Luminy, Unite´Mixte de Recherche 6216, Centre National de la Recherche Scientifique, Universite´dela Méditerrannée Aix-Marseille II, 13288 Marseille Cedex 9, France Communicated by Jules A. Hoffmann, Centre National de la Recherche Scientifique, Strasbourg, France, April 17, 2009 (received for review January 14, 2009) Drosophila hemocytes have strong phagocytic capacities and pro- generating plasmatocyte-depleted individuals and by analyzing duce antimicrobial peptides (AMPs). However, the precise role of their development and immune response. blood cells during immune responses and developmental processes has only been studied using indirect means. To overcome this Results limitation, we generated plasmatocyte-depleted flies by specifi- To obtain flies devoid of plasmatocytes, we decided to trigger cally overexpressing the proapoptotic protein Hid into plasmato- cell death in blood cells by overexpressing the proapoptotic cytes. Unexpectedly, these plasmatocyte-depleted animals have a protein Hid using the plasmatocyte (and crystal cells)–specific normal larval and pupal development and do not exhibit any hml(⌬)-Gal4 driver (20, 21). A UAS-EGFP transgene was used obvious defect after birth. Remarkably, plasmatocyte-depleted to precisely characterize the spatiotemporal expression of the adults show a strong susceptibility to infections by various micro- hml(⌬)-Gal4 driver. GFP is not expressed during embryogenesis, organisms, although activation of systemic AMP gene transcription even in late embryos in which Croquemort-positive blood cells via the Toll and immune deficiency (IMD) pathways is wild-type. are detected (Fig. 1A). The expression of GFP in plasmatocytes Our data show that this susceptibility, which correlates with appears shortly after hatching in early first-instar larvae (Fig. overproliferation of bacteria, is likely due to the absence of 1B), is maintained throughout the larval stages (Fig. 1F and data IMMUNOLOGY phagocytosis. We also demonstrate that during larval stages, not shown), and remains in the adult (Fig. 1G). GFP-positive plasmatocytes play an essential role in mediating AMP production cells are also detected in the cortical zone of the larval lymph by the fat body after oral bacterial infection. Finally, we show that gland (Fig. 1C) (12, 22). In addition to the circulating and sessile plasmatocytes are involved in immune surveillance during pupal plasmatocytes, we identified 2 yet-uncharacterized blood cell development, because they prevent bacterial infection that causes populations located in close contact to the gut cells. One group pupal lethality. is composed of approximately 50 cells, which are positioned within the larval proventriculus, where they surround the esoph- AMP ͉ blood cells ͉ innate immunity ͉ Toll agus (Fig. 1D). More posteriorly, in the larval ventriculus, few scattered GFP-positive cells are found within the gut epithelium he cellular interactions and pathways involved in the develop- (Fig. 1E). Some of these cells span the entire epithelium and are Tment and function of blood cells are, to a certain extend, therefore in contact with both the gut lumen and the hemolymph conserved between vertebrates and invertebrates (1–3). As in (supporting information Fig. S1). These cells, which are still present vertebrates, Drosophila blood cell development occurs in 2 phases. in adult guts (Fig. S1), are also labeled by the srpHemo-Gal4 driver, A first wave takes place in the embryonic procephalic mesoderm suggesting that these are indeed plasmatocytes (Fig. S1). Interest- and gives rise to both plasmatocytes and crystal cells (4, 5). ingly, both cell groups are present in gut regions competent to Plasmatocytes, the major function of which is phagocytosis, are the produce antimicrobial peptides after oral infection by the Gram- negative bacteria Erwinia carotovora (Fig. 1 D and E). Confident predominant hemocyte population present at all developmental ⌬ stages (6). In the embryo they engulf apoptotic corpses formed that the hml( )-Gal4 driver is blood cell specific, we then asked during developmental processes (4, 7, 8). In larvae and adults they whether it is indeed panhemocytic. We observed that more than 96% (ϩ3 SD) of the circulating hemocytes labeled by DAPI (n ϭ are responsible for phagocytosis of invading bacteria and fungi (9, ⌬ 10). At the pupal stage they play a fundamental role by engulfing 4585) in hml( )-Gal4, UAS-EGFP individuals are GFP positive and recycling doomed cells during metamorphosis (6). In addition (Fig. 1K). The remarkable tissue specificity of this Gal4 driver and to their phagocytic function, plasmatocytes also produce and se- its broad plasmatocyte distribution prompted us to use it as a tool to eliminate blood cells in vivo. crete a number of peptides/proteins, such as antimicrobial peptides (AMPs) (11). The crystal cells, which persist until the onset of hml(⌬)-Gal4, UAS-EGFP, UAS-hid Individuals Can Reach the Adult metamorphosis, mainly contribute to larval melanization reactions Stage. We first characterized the phenotype of Drosophila of the (5). The second wave of hematopoiesis occurs at the larval stage in hml(⌬)-Gal4, UAS-EGFP, UAS-hid/ϩ (later called hml-hid) the lymph gland. The precursors of the lymph gland derive from the genotype. As expected from the hml(⌬)-Gal4 expression pattern, dorsal thoracic mesoderm and subsequently grow during the first embryonic plasmatocytes were not ablated in these embryos and second larval instars (12–15). At the third-instar larval stage, (Fig. S2). However, third-instar larval plasmatocytes of the same the lymph gland is the main site of hemocyte production. The lymph genotype were strongly affected by Hid overexpression. First, these gland is also the production site of lamellocytes, which are cells devoted to the encapsulation of foreign bodies too large to be phagocytosed (6, 13, 16). The contribution of hemocytes to Dro- Author contributions: B.C. designed research; B.C. performed research; B.C. and J.R. ana- sophila development and immunity has so far been addressed by lyzed data; and B.C. and J.R. wrote the paper. blocking phagocytosis after bead injection or by using mutants with The authors declare no conflict of interest. a reduced number of hemocytes (17–19). Because these mutants 1To whom correspondence should be addressed. E-mail: [email protected]. show pleiotropic phenotypes and affect tissues other than hemo- This article contains supporting information online at www.pnas.org/cgi/content/full/ cytes, we decided to re-evaluate the function of plasmatocytes by 0903971106/DCSupplemental. www.pnas.org͞cgi͞doi͞10.1073͞pnas.0903971106 PNAS Early Edition ͉ 1of6 Downloaded by guest on September 29, 2021 A B C D E F G H I J K LM N O P Q Fig. 1. Obtainment of plasmatocyte-depleted Drosophila. Although numerous Croquemort-positive cells are present in hml(⌬)-Gal4, UAS-EGFP embryos (A), these cells do not express GFP. Gal4 expression, visualized through GFP signal, is present in hml(⌬)-Gal4, UAS-EGFP first- (B) and third-instar larvae (F) and in adults (G). A magnification of a hml(⌬)-Gal4, UAS-EGFP/dome-MESO–LacZ (labeling the medullary zone) third-instar larval lymph gland (C) shows that only peripheral differentiated cells from the cortical zone express Gal4.(D and E) Two populations of GFP-positive cells are found in the larval gut. One is located in the proventriculus and surrounds the digestive tract (dashed lines) (D). The second is found in the most posterior region of the ventriculus (E). Some blood cells (arrow) span the entire gut epithelium and therefore contact both the gut lumen and the hemolymph. Ectopic expression of the proapoptotic protein Hid using the hml(⌬)-Gal4 driver gives rise to larvae (H) and adults (I) that are completely devoid of GFP-positive plasmatocytes. (J–M) When bled onto a coverslip, hemolymph from hml(⌬)-Gal4, UAS-EGFP, UAS-hid/ϩ larvae (L) contain approximately 40% of the number of DAPI-positive cells observed in hml(⌬)-Gal4, UAS-EGFP/ϩ control larvae (J). These cells are GFP negative (compare K with M), suggesting that they are dying. This is confirmed by the fact that more than 96% of these DAPI-positive cells are TUNEL positive (P and Q). This number is less than 5% in hml(⌬)-Gal4, UAS-EGFP/ϩ control larvae (N and O). larvae were almost totally devoid of GFP-positive cells (Fig. 1 H and role of blood cells during immune response using our model of M). Second, when bled onto a coverslip, we observed that hml-hid plasmatocyte-depleted Drosophila. larval hemolymph contains 40% (Ϯ4 SD; n ϭ 1,702) DAPI-positive cells when compared with controls (n ϭ 4,263; Fig. 1 J and L). Plasmatocytes Are Dispensable for Immune Deficiency (IMD) and Toll Third, more than 96% (Ϯ2 SD) of these remaining DAPI-positive Pathways Activation in Adults. To test the role of plasmatocytes cells were undergoing apoptosis, as indicated by TUNEL staining during the immune response, we compared the ability of control (Fig. 1 P and Q). The number of TUNEL-positive cells in controls and hml-hid adults to survive infection. Flies devoid of plas- was much lower (5%, Ϯ2 SD; Fig. 1 N and O). Thus, we estimated matocytes showed a clear increased susceptibility to infection by that hml-hid larvae possess less than 2% of nonapoptotic plas- both Gram-positive and Gram-negative bacteria (Fig. 2). These matocytes. Because plasmatocytes have been described as par- observed susceptibilities were weaker in plasmatocyte-depleted ticipating in tissue remodeling by phagocytosing larval tissues adults than in mutant for genes in either the Toll (spz)orthe rm7 during pupariation, we were surprised to note that a significant IMD (PGRP-LC) pathways. Whereas all spz mutant flies percentage (45%, Ϯ12 SD; n ϭ 432) of the hml-hid larvae could succumbed to infection by Enterococcus faecalis within 28 h, only reach the pharate adult stage and emerge as phenotypically 67% of the plasmatocyte-depleted adults were dead 68 h after normal adults (Fig.