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Eater and NimC1 are transmembrane receptors of the Drosophila Nimrod family, specifically expressed in haemocytes, the insect blood cells. Previous ex vivo and in vivo RNAi studies have pointed to their role in the phagocytosis of bacteria. Here, we have created a novel NimC1 null mutant to re‐evaluate the role of NimC1, alone or in combination with Eater, in the cellular immune response. We show that NimC1 functions as an adhesion molecule ex vivo, but in contrast to Eater it is not required for haemocyte sessility in vivo. Ex vivo phagocytosis assays and electron microscopy experiments confirmed that Eater is the main phagocytic receptor for Gram‐positive, but not Gram‐negative bacteria, and contributes to microbe tethering to haemocytes. Surprisingly, NimC1 deletion did not impair phagocytosis of bacteria, nor their adhesion to the haemocytes. However, phagocytosis of both types of bacteria was almost abolished in NimC1 1 ;eater 1 haemocytes. This indicates that both receptors contribute synergistically to the phagocytosis of bacteria, but that Eater can bypass the requirement for NimC1. Finally, we uncovered that NimC1, but not Eater, is essential for uptake of latex beads and zymosan particles. We conclude that Eater and NimC1 are the two main receptors for phagocytosis of bacteria in Drosophila, and that each receptor likely plays distinct roles in microbial uptake., Eater and NimC1 are transmembrane receptors belonging to the Drosophila Nimrod family. These receptors are expressed in insect blood cells (haemocytes) and have been implicated in bacterial phagocytosis. In this study, Bruno Lemaitre and colleagues created a novel NimC1 null mutant and used this to characterize the role of NimC1 in cellular immune responses, alone and in combination with Eater. The authors reveal that Eater and NimC1 contribute synergistically to the initial step of phagocytosis, notably adhesion to bacteria, and that the receptors play distinct roles in this process.

In animals, growth is regulated by the complex interplay between paracrine and endocrine signals. When food is scarce, tissues compete for nutrients, leading to critical resource allocation and prioritization. Little is known about how the immune system maturation is coordinated with the growth of other tissues. Here, we describe a signaling mechanism that regulates the number of hemocytes (blood cells) according to the nutritional state of the Drosophila larva. Specifically, we found that a secreted protein, NimB5, is produced in the fat body upon nutrient scarcity downstream of metabolic sensors and ecdysone signaling. NimB5 is then secreted and binds to hemocytes to down-regulate their proliferation and adhesion. Blocking this signaling loop results in conditional lethality when larvae are raised on a poor diet, due to excessive hemocyte numbers and insufficient energy storage. Similar regulatory mechanisms shaping the immune system in response to nutrient availability are likely to be widespread in animals.

In animals, growth is regulated by the complex interplay between paracrine and endocrine signals. When food is scarce, tissues compete for nutrients, leading to critical resource allocation and prioritization. Little is known about how the immune system maturation is coordinated with the growth of other tissues. Here, we describe a signaling mechanism that regulates the number of hemocytes (blood cells) according to the nutritional state of theDrosophilalarva. Specifically, we found that the adipokine NimB5 is produced in the fat body upon nutrient scarcity downstream of metabolic sensors. NimB5 is then secreted and bind to hemocytes to down-regulate their proliferation and adhesion. Blocking this signaling loop results in conditional lethality when larvae are raised on a poor diet, due to excessive hemocyte numbers and insufficient energy storage. Similar regulatory mechanisms shaping the immune system in response to nutrient availability are likely to be widespread in animals.Author summaryDrosophilalarval hemocytes (blood cells) are found in two compartments: the lymph gland considered as a reservoir, and the peripheral compartment. Peripheral hemocytes form sessile patches attached to the internal surface of the larval body wall or are found freely circulating in the hemolymph. Little is known about the signals that regulate hemocytes proliferation and localization in the peripheral compartment. In this study, we have identified a new gene,NimrodB5, coding for the NimB5 protein, which is secreted by the fat body and binds to hemocytes. NimB5 inhibits hemocyte proliferation while promoting sessility, leading to an increased number of circulating hemocytes and adhesion defects inNimB5mutant. We show thatnimrodB5expression by the fat body is controlled by metabolic cues to adjust hemocyte number to the physiological state of the larvae.Interestingly, deregulation ofNimB5causes lethality when larvae are raised on a poor diet due to a defect in regulating hemocytes proliferation. In conclusion, we have identified a new adipokine that optimizes hemocytes number to the physiological state of larvae. Our study also reveals a major role of the fat body in peripheral hematopoiesis regulation and outline how it can be costly to maintain a basal immune defense.

Eater and NimC1 are transmembrane receptors of theDrosophilaNimrod family, specifically expressed in hemocytes, the insect blood cells. Previousex vivoandin vivoRNAi studies have pointed to their role in the phagocytosis of bacteria. Here, we have created a novel null mutant in NimC1 to re-evaluate the role of NimC1, alone or in combination with Eater, in the cellular immune response. We show that NimC1 functions as an adhesion moleculeex vivo, but in contrast to Eater is not required for hemocyte sessilityin vivo.Ex vivophagocytosis assays and electron microscopy experiments confirmed that Eater is the main phagocytic receptor for Gram-positive, but not Gram-negative bacteria, and contributes to microbe tethering to hemocytes. Surprisingly, theNimC1deletion did not impair phagocytosis of bacteria, nor their adhesion to the hemocytes. However, phagocytosis of both types of bacteria was almost abolished inNimC11;eater1hemocytes. This indicates that both receptors contribute synergistically to the phagocytosis of bacteria, but that Eater can bypass the requirement for NimC1. Finally, we uncovered that NimC1, but not Eater, is essential for uptake of latex beads and zymosan particles. We conclude that Eater and NimC1 are the two main receptors for phagocytosis of bacteria inDrosophila,and that each receptor likely plays distinct roles in microbial uptake.

The melanization response is a rapid and important defense mechanism in arthropods. Melanin is produced around wound sites and invading microorganisms by phenoloxidases (POs), which need to be activated by the sequential activation of an extracellular serine protease (SP) cascade.Drosophila melanogasterhas been a useful genetic model for dissecting insect immune signaling, but understanding these proteolytic cascades has been complicated by the large number of SP genes, possibly with redundant function. Taking advantage of recently-generated null and compound mutants, we re-investigated the role of SPs involved in the melanization response inD. melanogasterand discovered phenotypes previously concealed in single mutant analysis. We found that two of them, Hayan and Sp7, can activate the melanization response in two different manners: Hayan is required in the local blackening of wound sites, while Sp7 regulates an alternate melanization reaction responsible for the clearance of septic infections withStaphylococcus aureus. We present evidence that both Sp7 and Hayan regulate the Toll NF-kB pathway. Sp7 is regulated by canonical Toll signaling downstream of PGRP-SA, ModSP, and Grass, leading to control of septic infections via a Sp7-dependent melanization response. Additionally, we found that Hayan and the Toll-regulating SP Psh are the result of a recent gene duplication. Using genetic manipulations, we reveal the hidden role for Hayan, alongside Psh, in propagating Toll signaling downstream of pattern recognition receptors. Thus, we describe the existence of two pathways leading to the melanization response and reveal previously unknown dynamics in the activation of the Toll pathway.

The JAK/STAT pathway is a key signaling pathway in the regulation of development and immunity in metazoans. In contrast to the multiple combinatorial JAK/STAT pathways in mammals, only one canonical JAK/STAT pathway exists in Drosophila. It is activated by three secreted proteins of the Unpaired family (Upd): Upd1, Upd2 and Upd3. Although many studies have established a link between JAK/STAT activation and tissue damage, the mode of activation and the precise function of this pathway in the Drosophila systemic immune response remain unclear. In this study, we used mutations in upd2 and upd3 to investigate the role of the JAK/STAT pathway in the systemic immune response. Our study shows that haemocytes express the three upd genes and that injury markedly induces the expression of upd3 by the JNK pathway in haemocytes, which in turn activates the JAK/STAT pathway in the fat body and the gut. Surprisingly, release of Upd3 from haemocytes upon injury can remotely stimulate stem cell proliferation and the expression of Drosomycin-like genes in the intestine. Our results also suggest that a certain level of intestinal epithelium renewal is required for optimal survival to septic injury. While haemocyte-derived Upd promotes intestinal stem cell activation and survival upon septic injury, haemocytes are dispensable for epithelium renewal upon oral bacterial infection. Our study also indicates that intestinal epithelium renewal is sensitive to insults from both the lumen and the haemocoel. It also reveals that release of Upds by haemocytes coordinates the wound-healing program in multiple tissues, including the gut, an organ whose integrity is critical to fly survival., Author Summary Innate immunity acts as the primary line of defense to overcome invading organisms. This response starts through sensing of microbe-specific molecules such as lipopolysaccharide and peptidoglycan by host receptors. Metazoans can also recognize signals that are associated with tissue damage and wounding, to then activate specific pathways involved in tissue repair or inflammation. Previous studies have suggested a link between JAK/STAT activation and tissue repair in Drosophila, but the mode of activation and the precise function of this pathway remain unclear. In this article, we analyze the role of the JAK/STAT pathway in the resistance to wounding in Drosophila. We show that this pathway contributes to fly resistance to wounding and bacterial infection. Mechanistically, injury induces the production of secreted molecules called Unpaireds by blood cells, which then activate the JAK/STAT pathway in the fat body (an organ equivalent to the vertebrate liver) and in the gut. One of the most surprising findings of our study is that Unpaireds released from blood cells can remotely stimulate intestinal stem cell proliferation. Thus, we uncover an unexpected interaction between circulating blood cells and intestinal stem cell proliferation that contributes to fly survival after injury.

Drosophila melanogaster’s blood cells (hemocytes) play essential roles in wound healing and are involved in clearing microbial infections. Here, we report the transcriptional changes of larval plasmatocytes after clean injury or infection with the Gram-negative bacteriumEscherichia colior the Gram-positive bacteriumStaphylococcus aureuscompared to hemocytes recovered from unchallenged larvae via RNA-Sequencing. This study reveals 676 differentially expressed genes (DEGs) in hemocytes from clean injury samples compared to unchallenged samples, and 235 and 184 DEGs inE. coliandS. aureussamples respectively compared to clean injury samples. The clean injury samples showed enriched DEGs for immunity, clotting, cytoskeleton, cell migration, hemocyte differentiation, and indicated a metabolic reprogramming to aerobic glycolysis, a well-defined metabolic adaptation observed in mammalian macrophages. Microbial infections trigger significant transcription of immune genes, with significant differences between theE. coliandS. aureussamples suggesting that hemocytes have the ability to engage various programs upon infection. Collectively, our data bring new insights onDrosophilahemocyte function and open the route to post-genomic functional analysis of the cellular immune response.

Background The diversification of immune systems during evolution involves the expansion of particular gene families in given phyla. A better understanding of the metazoan immune system requires an analysis of the logic underlying such immune gene amplification. This analysis is now within reach due to the ease with which we can generate multiple mutations in an organism. In this paper, we analyze the contribution of the three Drosophila prophenoloxidases (PPOs) to host defense by generating single, double and triple mutants. PPOs are enzymes that catalyze the production of melanin at the site of infection and around parasites. They are the rate-limiting enzymes that contribute to the melanization reaction, a major immune mechanism of arthropods. The number of PPO-encoding genes is variable among insects, ranging from one in the bee to ten in the mosquito. Results By analyzing mutations alone and in combination, we ascribe a specific function to each of the three PPOs of Drosophila. Our study confirms that two PPOs produced by crystal cells, PPO1 and PPO2, contribute to the bulk of melanization in the hemolymph, upon septic or clean injury. In contrast, PPO3, a PPO restricted to the D. melanogaster group, is expressed in lamellocytes and contributes to melanization during the encapsulation process. Interestingly, another overlapping set of PPOs, PPO2 and PPO3, achieve melanization of the capsule upon parasitoid wasp infection. Conclusions The use of single or combined mutations allowed us to show that each PPO mutant has a specific phenotype, and that knocking out two of three genes is required to abolish fully a particular function. Thus, Drosophila PPOs have partially overlapping functions to optimize melanization in at least two conditions: following injury or during encapsulation. Since PPO3 is restricted to the D. melanogaster group, this suggests that production of PPO by lamellocytes emerged as a recent defense mechanism against parasitoid wasps. We conclude that differences in spatial localization, immediate or late availability, and mode of activation underlie the functional diversification of the three Drosophila PPOs, with each of them having non-redundant but overlapping functions. Electronic supplementary material The online version of this article (doi:10.1186/s12915-015-0193-6) contains supplementary material, which is available to authorized users.

Summary The melanization response is an important defense mechanism in arthropods. This reaction is mediated by phenoloxidases (POs), which are activated by complex extracellular serine protease (SP) cascades. Here, we investigate the role of SPs in the melanization response using compound mutants in D. melanogaster and discover phenotypes previously concealed in single-mutant analyses. We find that two SPs, Hayan and Sp7, activate the melanization response in different manners: Hayan is required for blackening wound sites, whereas Sp7 regulates an alternate melanization reaction responsible for the clearance of Staphylococcus aureus. We present evidence that Sp7 is regulated by SPs activating the Toll NF-κB pathway, namely ModSP and Grass. Additionally, we reveal a role for the combined action of Hayan and Psh in propagating Toll signaling downstream of pattern recognition receptors activating either Toll signaling or the melanization response.