Two Independent Functions of Collier/Early B Cell Factor in the Control of Drosophila Blood Cell Homeostasis

Blood cell production in the Drosophila hematopoietic organ, the lymph gland, is controlled by intrinsic factors and extrinsic signals. Initial analysis of Collier/Early B Cell Factor function in the lymph gland revealed the role of the Posterior Signaling Center (PSC) in mounting a dedicated cellular immune response to wasp parasitism. Further, premature blood cell differentiation when PSC specification or signaling was impaired, led to assigning the PSC a role equivalent to the vertebrate hematopoietic niche. We report here that Collier is expressed in a core population of lymph gland progenitors and cell autonomously maintains this population. The PSC contributes to lymph gland homeostasis by regulating blood cell differentiation, rather than by maintaining core progenitors. In addition to PSC signaling, switching off Collier expression in progenitors is required for efficient immune response to parasitism. Our data show that two independent sites of Collier/Early B Cell Factor expression, hematopoietic progenitors and the PSC, achieve control of hematopoiesis.     

Cellular immune response to parasitization in Drosophila requires the EBF orthologue collier

Drosophila immune response involves three types of hemocytes (‘blood cells’). One cell type, the lamellocyte, is induced to differentiate only under particular conditions, such as parasitization by wasps. Here, we have investigated the mechanisms underlying the specification of lamellocytes. We first show that collier (col), the Drosophila orthologue of the vertebrate gene encoding early B-cell factor (EBF), is expressed very early during ontogeny of the lymph gland, the larval hematopoietic organ. In this organ, Col expression prefigures a specific posterior region recently proposed to act as a signalling centre, the posterior signalling centre (PSC). The complete lack of lamellocytes in parasitized col mutant larvae revealed the critical requirement for Col activity in specification of this cell type. In wild-type larvae, Col expression remains restricted to the PSC following parasitization, despite the massive production of lamellocytes. We therefore propose that Col endows PSC cells with the capacity to relay an instructive signal that orients hematopoietic precursors towards the lamellocyte fate in response to parasitization. Considered together with the role of EBF in lymphopoiesis, these findings suggest new parallels in cellular immunity between Drosophila and vertebrates. Further investigations on Col/EBF expression and function in other phyla should provide fresh insight into the evolutionary origin of lymphoid cells.     

Mechanisms of JAK/STAT pathway negative regulation by the short coreceptor Eye Transformer/Latran

Transmembrane receptors interact with extracellular ligands to transduce intracellular signaling cascades, modulate target gene expression, and regulate processes such as proliferation, apoptosis, differentiation, and homeostasis. As a consequence, aberrant signaling events often underlie human disease. Whereas the vertebrate JAK/STAT signaling cascade is transduced via multiple receptor combinations, the Drosophila pathway has only one full-length signaling receptor, Domeless (Dome), and a single negatively acting receptor, Eye Transformer/Latran (Et/Lat). Here we investigate the molecular mechanisms underlying Et/Lat activity. We demonstrate that Et/Lat negatively regulates the JAK/STAT pathway activity and can bind to Dome, thus reducing Dome:Dome homodimerization by creating signaling-incompetent Dome:Et/Lat heterodimers. Surprisingly, we find that Et/Lat is able to bind to both JAK and STAT92E but, despite the presence of putative cytokine-binding motifs, does not detectably interact with pathway ligands. We find that Et/Lat is trafficked through the endocytic machinery for lysosomal degradation but at a much slower rate than Dome, a difference that may enhance its ability to sequester Dome into signaling-incompetent complexes. Our data offer new insights into the molecular mechanism and regulation of Et/Lat in Drosophila that may inform our understanding of how short receptors function in other organisms.     

A parasitoid wasp of Drosophila employs preemptive and reactive strategies to deplete its host’s blood cells

The wasps Leptopilina heterotoma parasitize and ingest their Drosophila hosts. They produce extracellular vesicles (EVs) in the venom that are packed with proteins, some of which perform immune suppressive functions. EV interactions with blood cells of host larvae are linked to hematopoietic depletion, immune suppression, and parasite success. But how EVs disperse within the host, enter and kill hematopoietic cells is not well understood. Using an antibody marker for L. heterotoma EVs, we show that these parasite-derived structures are readily distributed within the hosts’ hemolymphatic system. EVs converge around the tightly clustered cells of the posterior signaling center (PSC) of the larval lymph gland, a small hematopoietic organ in Drosophila. The PSC serves as a source of developmental signals in naïve animals. In wasp-infected animals, the PSC directs the differentiation of lymph gland progenitors into lamellocytes. These lamellocytes are needed to encapsulate the wasp egg and block parasite development. We found that L. heterotoma infection disassembles the PSC and PSC cells disperse into the disintegrating lymph gland lobes. Genetically manipulated PSC-less lymph glands remain non-responsive and largely intact in the face of L. heterotoma infection. We also show that the larval lymph gland progenitors use the endocytic machinery to internalize EVs. Once inside, L. heterotoma EVs damage the Rab7- and LAMP-positive late endocytic and phagolysosomal compartments. Rab5 maintains hematopoietic and immune quiescence as Rab5 knockdown results in hematopoietic over-proliferation and ectopic lamellocyte differentiation. Thus, both aspects of anti-parasite immunity, i.e., (a) phagocytosis of the wasp’s immune-suppressive EVs, and (b) progenitor differentiation for wasp egg encapsulation reside in the lymph gland. These results help explain why the lymph gland is specifically and precisely targeted for destruction. The parasite’s simultaneous and multipronged approach to block cellular immunity not only eliminates blood cells, but also tactically blocks the genetic programming needed for supplementary hematopoietic differentiation necessary for host success. In addition to its known functions in hematopoiesis, our results highlight a previously unrecognized phagocytic role of the lymph gland in cellular immunity. EV-mediated virulence strategies described for L. heterotoma are likely to be shared by other parasitoid wasps; their understanding can improve the design and development of novel therapeutics and biopesticides as well as help protect biodiversity.     

Tissue communication in a systemic immune response of Drosophila

Several signaling pathways, including the JAK/STAT and Toll pathways, are known to activate blood cells (hemocytes) in Drosophila melanogaster larvae. They are believed to regulate the immune response against infections by parasitoid wasps, such as Leptopilina boulardi, but how these pathways control the hemocytes is not well understood. Here, we discuss the recent discovery that both muscles and fat body take an active part in this response. Parasitoid wasp infection induces Upd2 and Upd3 secretion from hemocytes, leading to JAK/STAT activation mainly in hemocytes and in skeletal muscles. JAK/STAT activation in muscles, but not in hemocytes, is required for an efficient encapsulation of wasp eggs. This suggests that Upd2 and Upd3 are important cytokines, coordinating different tissues for the cellular immune response in Drosophila. In the fat body, Toll signaling initiates a systemic response in which hemocytes are mobilized and activated hemocytes (lamellocytes) are generated. However, the contribution of Toll signaling to the defense against wasps is limited, probably because the wasps inject inhibitors that prevent the activation of the Toll pathway. In conclusion, parasite infection induces a systemic response in Drosophila larvae involving major organ systems and probably the physiology of the entire organism.     

JAK/STAT signaling in Drosophila muscles controls the cellular immune response against parasitoid infection

The role of JAK/STAT signaling in the cellular immune response of Drosophila is not well understood. Here, we show that parasitoid wasp infection activates JAK/STAT signaling in somatic muscles of the Drosophila larva, triggered by secretion of the cytokines Upd2 and Upd3 from circulating hemocytes. Deletion of upd2 or upd3, but not the related os (upd1) gene, reduced the cellular immune response, and suppression of the JAK/STAT pathway in muscle cells reduced the encapsulation of wasp eggs and the number of circulating lamellocyte effector cells. These results suggest that JAK/STAT signaling in muscles participates in a systemic immune defense against wasp infection.     

Differential activities of the Drosophila JAK/STAT pathway ligands Upd, Upd2 and Upd3

JAK/STAT signalling in vertebrates is activated by multiple cytokines and growth factors. By contrast, the Drosophila genome encodes for only three related JAK/STAT ligands, Upd, Upd2 and Upd3. Identifying the differences between these three ligands will ultimately lead to a greater understanding of this disease-related signalling pathway and its roles in development. Here, we describe the analysis of the least well characterised of the Upd-like ligands, Upd3. We show that in tissue culture-based assays Upd3-GFP is secreted from cells and appears to interact with the extracellular matrix (ECM) in a similar manner to Upd, while still non-autonomously activating JAK/STAT signalling. Quantification of each of the Upd-like ligands in conditioned media has allowed us to determine the activity of equal amounts of each ligand on JAK/STAT ex vivo and reveals that Upd is the most potent ligand in this system. Finally, investigations into the effects of ectopic expression of Upd3 in vivo have confirmed its ability to activate pathway signalling at long-distance.     

Extracellular Adenosine Mediates a Systemic Metabolic Switch during Immune Response

Immune defense is energetically costly, and thus an effective response requires metabolic adaptation of the organism to reallocate energy from storage, growth, and development towards the immune system. We employ the natural infection of Drosophila with a parasitoid wasp to study energy regulation during immune response. To combat the invasion, the host must produce specialized immune cells (lamellocytes) that destroy the parasitoid egg. We show that a significant portion of nutrients are allocated to differentiating lamellocytes when they would otherwise be used for development. This systemic metabolic switch is mediated by extracellular adenosine released from immune cells. The switch is crucial for an effective immune response. Preventing adenosine transport from immune cells or blocking adenosine receptor precludes the metabolic switch and the deceleration of development, dramatically reducing host resistance. Adenosine thus serves as a signal that the “selfish” immune cells send during infection to secure more energy at the expense of other tissues.     

Remote Control of Intestinal Stem Cell Activity by Haemocytes in Drosophila

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.     

Drosophila haematopoiesis

Like in vertebrates, Drosophila haematopoiesis occurs in two waves. It gives rise to three types of haemocytes: plasmatocytes (phagocytosis), crystal cells (melanization) and lamellocytes (encapsulation of parasites). A first population of haemocytes, specified during embryogenesis, gives rise to an invariant number of plasmatocytes and crystal cells. A second population of haemocytes is specified during larval development in a specialized haematopoietic organ, the lymph gland. All three types of haemocytes can be specified in this organ, but lamellocytes only differentiate in response to parasitism. Thus, larval in contrast to embryonic haematopoiesis can be modulated by physiological constraints. Molecular cascades controlling embryonic haematopoiesis are relatively well established and require transactivators such as GATA, FOG and Runx factors, which are also co-opted in mammalian haematopoiesis. Mechanisms involved during larval haematopoiesis are less well understood although a number of chromatin remodelling factors and signalling pathways (JAK/STAT, Toll, Hedgehog, Notch) are required. In healthy larvae a pool of progenitors is maintained within the lymph gland, under the control of a signalling centre which expresses Collier, Serrate, Antennapedia and Hedgehog, and controls haemocyte homeostasis. Its key role in haemocyte homeostasis is reminiscent of interactions described in vertebrates between haematopoietic stem cells and their microenvironment (niche).     

Larval Defense against Attack from Parasitoid Wasps Requires Nociceptive Neurons

Parasitoid wasps are a fierce predator of Drosophila larvae. Female Leptopilina boulardi (LB) wasps use a sharp ovipositor to inject eggs into the bodies of Drosophila melanogaster larvae. The wasp then eats the Drosophila larva alive from the inside, and an adult wasp ecloses from the Drosophila pupal case instead of a fly. However, the Drosophila larvae are not defenseless as they may resist the attack of the wasps through somatosensory-triggered behavioral responses. Here we describe the full range of behaviors performed by the larval prey in immediate response to attacks by the wasps. Our results suggest that Drosophila larvae primarily sense the wasps using their mechanosensory systems. The range of behavioral responses included both “gentle touch” like responses as well as nociceptive responses. We found that the precise larval response depended on both the somatotopic location of the attack, and whether or not the larval cuticle was successfully penetrated during the course of the attack. Interestingly, nociceptive responses are more likely to be triggered by attacks in which the cuticle had been successfully penetrated by the wasp. Finally, we found that the class IV neurons, which are necessary for mechanical nociception, were also necessary for a nociceptive response to wasp attacks. Thus, the class IV neurons allow for a nociceptive behavioral response to a naturally occurring predator of Drosophila.     

Postembryonic Hematopoiesis in Drosophila

We have investigated the blood cell types present in Drosophila at postembryonic stages and have analysed their modifications during development and under immune conditions. The anterior lobes of the larval hematopoietic organ or lymph gland contain numerous active secretory cells, plasmatocytes, few crystal cells, and a number of undifferentiated prohemocytes. The posterior lobes contain essentially prohemocytes. The blood cell population in larval hemolymph differs and consists mainly of plasmatocytes which are phagocytes, and of a low percentage of crystal cells which reportedly play a role in humoral melanisation. We show that the cells in the lymph gland can differentiate into a given blood cell lineage when solicited. Under normal nonimmune conditions, we observe a massive differentiation into active macrophages at the onset of metamorphosis in all lobes. Simultaneously, circulating plasmatocytes modify their adhesion and phagocytic properties to become pupal macrophages. All phagocytic cells participate in metamorphosis by ingesting doomed larval tissues. The most dramatic effect on larval hematopoiesis was observed following infestation by a parasitoid wasp. Cells within all lymph gland lobes, including prohemocytes from posterior lobes, massively differentiate into a new cell type specifically devoted to encapsulation, the lamellocyte.