Iwan Evans

Calcium Flashes Orchestrate the Wound Inflammatory Response through DUOX Activation and Hydrogen Peroxide Release

Summary A crucial early wound response is the recruitment of inflammatory cells drawn by danger cues released by the damaged tissue. Hydrogen peroxide (H2O2) has recently been identified as the earliest wound attractant in Drosophila embryos and zebrafish larvae [1, 2]. The H2O2 signal is generated by activation of an NADPH oxidase, DUOX, and as a consequence, the first inflammatory cells are recruited to the wound within minutes. To date, nothing is known about how wounding activates DUOX. Here, we show that laser wounding of the Drosophila embryo epidermis triggers an instantaneous calcium flash, which travels as a wave via gap junctions several cell rows back from the wound edge. Blocking this calcium flash inhibits H2O2 release at the wound site and leads to a reduction in the number of immune cells migrating to the wound. We suggest that the wound-induced calcium flash activates DUOX via an EF hand calcium-binding motif and thus triggers the production of the attractant damage cue H2O2. Therefore, calcium represents the earliest signal in the wound inflammatory response.

Prioritization of Competing Damage and Developmental Signals by Migrating Macrophages in the Drosophila Embryo

The function of immune cells is critically dependent on their capacity to respond to a complex series of navigational cues that enable them to home to various organ sites in the body or to respond to inflammatory cues such as those released at sites of tissue damage. From early embryonic stages, immune cells are faced with a barrage of signals that will not all be directing the cell to do the same thing. Here we use the Drosophila embryo to investigate how hemocytes (Drosophila macrophages), are able to prioritize key guidance signals and ignore others so that they are not pulled every which way. We identify the immediate wound attractant signal as H(2)O(2) and investigate how Drosophila macrophages respond to competing guidance cues-those emanating from a wound-versus standard developmental guidance cues, as well as those signals drawing cells toward neighboring dying cells. We reveal a hierarchy of responsiveness to attractant cues that varies over time and we identify why there is a wound refractile period early in embryonic development when macrophages cannot be distracted from their developmental migratory pathway to a site of tissue damage.

Clasp-mediated microtubule bundling regulates persistent motility and contact repulsion in Drosophila macrophages in vivo

A microtubule arm regulates cell–cell repulsion, pointing hemocytes in opposite directions when they contact each other in Drosophila embryos.

Genetic Ablation of Drosophila Phagocytes Reveals Their Contribution to Both Development and Resistance to Bacterial Infection

Drosophila phagocytes participate in development and immune responses through their abilities to perform phagocytosis and/or secrete extra-cellular matrix components, antimicrobial peptides, clotting factors and signalling molecules. However, our knowledge of their functional impact on development and host resistance to infection is limited. To address this, we have used a genetic cell ablation strategy to generate Drosophila individuals lacking functional phagocytes. Our results highlight the essential contribution of phagocytes to embryonic development including central nervous system morphogenesis. Phagocytes also ensure optimal viability during post-embryonic development through immune functions. The use of phagocyte-depleted flies reveals the contribution of phagocytes in the resistance of Drosophila adults upon systemic infections with specific bacteria. Phagocytes were not involved in the expression of antimicrobial peptides by the fat body indicating a clear separation between cellular and humoral immune responses at this stage. Finally, we confirm that phagocytosis is a critical effector mechanism of the cellular arm by demonstrating that phagocytosis contributes to resistance to infection with Staphylococcus aureus in adults. Our results highlight the power of this cell ablation strategy to reveal the contribution of phagocytes to specific biological processes. We now provide a blueprint of phagocyte importance during both development and innate immune responses in Drosophila.

Drosophila Embryos as Model Systems for Monitoring Bacterial Infection in Real Time

Drosophila embryos are well studied developmental microcosms that have been used extensively as models for early development and more recently wound repair. Here we extend this work by looking at embryos as model systems for following bacterial infection in real time. We examine the behaviour of injected pathogenic (Photorhabdus asymbiotica) and non-pathogenic (Escherichia coli) bacteria and their interaction with embryonic hemocytes using time-lapse confocal microscopy. We find that embryonic hemocytes both recognise and phagocytose injected wild type, non-pathogenic E. coli in a Dscam independent manner, proving that embryonic hemocytes are phagocytically competent. In contrast, injection of bacterial cells of the insect pathogen Photorhabdus leads to a rapid ‘freezing’ phenotype of the hemocytes associated with significant rearrangement of the actin cytoskeleton. This freezing phenotype can be phenocopied by either injection of the purified insecticidal toxin Makes Caterpillars Floppy 1 (Mcf1) or by recombinant E. coli expressing the mcf1 gene. Mcf1 mediated hemocyte freezing is shibire dependent, suggesting that endocytosis is required for Mcf1 toxicity and can be modulated by dominant negative or constitutively active Rac expression, suggesting early and unexpected effects of Mcf1 on the actin cytoskeleton. Together these data show how Drosophila embryos can be used to track bacterial infection in real time and how mutant analysis can be used to genetically dissect the effects of specific bacterial virulence factors.

Corpse Engulfment Generates a Molecular Memory that Primes the Macrophage Inflammatory Response

Summary Macrophages are multifunctional cells that perform diverse roles in health and disease. Emerging evidence has suggested that these innate immune cells might also be capable of developing immunological memory, a trait previously associated with the adaptive system alone. While recent studies have focused on the dramatic macrophage reprogramming that follows infection and protects against secondary microbial attack, can macrophages also develop memory in response to other cues? Here, we show that apoptotic corpse engulfment by Drosophila macrophages is an essential primer for their inflammatory response to tissue damage and infection in vivo. Priming is triggered via calcium-induced JNK signaling, which leads to upregulation of the damage receptor Draper, thus providing a molecular memory that allows the cell to rapidly respond to subsequent injury or infection. This remarkable plasticity and capacity for memory places macrophages as key therapeutic targets for treatment of inflammatory disorders.

Enabled negatively regulates diaphanous-driven actin dynamics in vitro and in vivo.

Summary Actin regulators facilitate cell migration by controlling cell protrusion architecture and dynamics. As the behavior of individual actin regulators becomes clear, we must address why cells require multiple regulators with similar functions and how they cooperate to create diverse protrusions. We characterized Diaphanous (Dia) and Enabled (Ena) as a model, using complementary approaches: cell culture, biophysical analysis, and Drosophila morphogenesis. We found that Dia and Ena have distinct biochemical properties that contribute to the different protrusion morphologies each induces. Dia is a more processive, faster elongator, paralleling the long, stable filopodia it induces in vivo, while Ena promotes filopodia with more dynamic changes in number, length, and lifetime. Acting together, Ena and Dia induce protrusions distinct from those induced by either alone, with Ena reducing Dia-driven protrusion length and number. Consistent with this, EnaEVH1 binds Dia directly and inhibits DiaFH1FH2-mediated nucleation in vitro. Finally, Ena rescues hemocyte migration defects caused by activated Dia.

Ena drives invasive macrophage migration in Drosophila embryos

SUMMARY It is seldom the primary tumour that proves fatal in cancer, with metastasis the fundamental pathological process for disease progression. Upregulation of Mena, a member of the evolutionarily conserved Ena/VASP family of actin cytoskeletal regulators, promotes metastasis and invasive motility of breast cancer cells in vivo. To complement in vitro studies of Ena/VASP function in fibroblasts, we manipulated levels of Ena, the Drosophila homologue of Mena, in migrating embryonic macrophages (haemocytes). Consistent with data from fibroblasts in vitro, Ena localises to regions of actin dynamics within migrating haemocytes, stimulates lamellipodial dynamics and positively regulates the number and length of filopodia. However, whereas Ena overexpression in fibroblasts reduces migration speeds, overexpressing Ena in haemocytes leads to a dramatic increase in migration speeds, more closely resembling the increased motility of breast cancer cells that overexpress Mena. We provide evidence that this key difference is due to spatial constraints imposed on cells within the three-dimensional environment of the embryo; this might explain how Mena can be used to promote aggressive migratory behaviour during cancer progression.

Interdependence of macrophage migration and ventral nerve cord development in Drosophila embryos.

During embryonic development, Drosophila macrophages (haemocytes) undergo a series of stereotypical migrations to disperse throughout the embryo. One major migratory route is along the ventral nerve cord (VNC), where haemocytes are required for the correct development of this tissue. We show, for the first time, that a reciprocal relationship exists between haemocytes and the VNC and that defects in nerve cord development prevent haemocyte migration along this structure. Using live imaging, we demonstrate that the axonal guidance cue Slit and its receptor Robo are both required for haemocyte migration, but signalling is not autonomously required in haemocytes. We show that the failure of haemocyte migration along the VNC in slit mutants is not due to a lack of chemotactic signals within this structure, but rather to a failure in its detachment from the overlying epithelium, creating a physical barrier to haemocyte migration. This block of haemocyte migration in turn disrupts the formation of the dorsoventral channels within the VNC, further highlighting the importance of haemocyte migration for correct neural development. This study illustrates the important role played by the three-dimensional environment in directing cell migration in vivo and reveals an intriguing interplay between the developing nervous system and the blood cells within the fly, demonstrating that their development is both closely coupled and interdependent.

Draper/CED-1 Mediates an Ancient Damage Response to Control Inflammatory Blood Cell Migration In Vivo

Summary Tissue damage leads to a robust and rapid inflammatory response whereby leukocytes are actively drawn toward the wound. Hydrogen peroxide (H2O2) has been shown to be an immediate damage signal essential for the recruitment of these inflammatory blood cells to wound sites in both Drosophila and vertebrates [1, 2]. Recent studies in zebrafish have shown that wound-induced H2O2 is detected by the redox-sensitive Src family kinase (SFK) Lyn within the responding blood cells [3]. Here, we show the same signaling occurs in Drosophila inflammatory cells in response to wound-induced H2O2 with mutants for the Lyn homolog Src42A displaying impaired inflammatory migration to wounds. We go on to show that activation of Src42A is necessary to trigger a signaling cascade within the inflammatory cells involving the ITAM domain-containing protein Draper-I (a member of the CED-1 family of apoptotic cell clearance receptors) and a downstream kinase, Shark, that is required for migration to wounds. The Src42A-Draper-Shark-mediated signaling axis is homologous to the well-established SFK-ITAM-Syk-signaling pathway used in vertebrate adaptive immune responses. Consequently, our results suggest that adaptive immunoreceptor-signaling pathways important in distinguishing self from non-self appear to have evolved from a more-ancient damage response. Furthermore, this changes the role of H2O2 from an inflammatory chemoattractant to an activator signal that primes immune cells to respond to damage cues via the activation of damage receptors such as Draper.