Gábor Csordás

The cell-mediated immunity of Drosophila melanogaster: hemocyte lineages, immune compartments, microanatomy and regulation.

In the animal kingdom, innate immunity is the first line of defense against invading pathogens. The dangers of microbial and parasitic attacks are countered by similar mechanisms, involving the prototypes of the cell-mediated immune responses, the phagocytosis and encapsulation. Work on Drosophila has played an important role in promoting an understanding of the basic mechanisms of phylogenetically conserved modules of innate immunity. The aim of this review is to survey the developments in the identification and functional definition of immune cell types and the immunological compartments of Drosophila melanogaster. We focus on the molecular and developmental aspects of the blood cell types and compartments, as well as the dynamics of blood cell development and the immune response. Further advances in the characterization of the innate immune mechanisms in Drosophila will provide basic clues to the understanding of the importance of the evolutionary conserved mechanisms of innate immune defenses in the animal kingdom.

Cell lineage tracing reveals the plasticity of the hemocyte lineages and of the hematopoietic compartments in Drosophila melanogaster

Much of our knowledge on hematopoiesis, hematopoietic compartments, hematopoietic cell lineages and immunity has been derived from studies on the vertebrate immune system. The sophisticated innate immunity of insects, the phylogenetic conservation and the power of Drosophila genetics allowed the investigation of immune cell (hemocyte) lineage relationships in Drosophila melanogaster. The development of the hemocyte lineages in Drosophila is a result of a precisely regulated succession of intracellular and intercellular events, though the nature and extent of these interactions are not known. We describe here a cell lineage tracing system set up to analyze the development of hemocyte lineages and functionally distinct hemocyte subsets. This system allowed us to distinguish two major embryonic hemocyte lineages, the crq and Dot lineages, in two, physically separated compartments, the embryonic macrophages and the embryonic lymph gland. We followed the fate and development of these lineages in the construction of the larval hematopoietic compartments and during the cell-mediated immune response, the encapsulation reaction. Our results revealed the considerable plasticity and concerted action of the hematopoietic compartments and the hemocyte lineages in the development of the innate immune system and in the course of the cell-mediated immune response in Drosophila.

MiniCORVET is a Vps8-containing early endosomal tether in Drosophila

Yeast studies identified two heterohexameric tethering complexes, which consist of 4 shared (Vps11, Vps16, Vps18 and Vps33) and 2 specific subunits: Vps3 and Vps8 (CORVET) versus Vps39 and Vps41 (HOPS). CORVET is an early and HOPS is a late endosomal tether. The function of HOPS is well known in animal cells, while CORVET is poorly characterized. Here we show that Drosophila Vps8 is highly expressed in hemocytes and nephrocytes, and localizes to early endosomes despite the lack of a clear Vps3 homolog. We find that Vps8 forms a complex and acts together with Vps16A, Dor/Vps18 and Car/Vps33A, and loss of any of these proteins leads to fragmentation of endosomes. Surprisingly, Vps11 deletion causes enlargement of endosomes, similar to loss of the HOPS-specific subunits Vps39 and Lt/Vps41. We thus identify a 4 subunit-containing miniCORVET complex as an unconventional early endosomal tether in Drosophila. DOI: http://dx.doi.org/10.7554/eLife.14226.001

In vivo detection of lamellocytes in Drosophila melanogaster

Drosophila has recently become a powerful model organism for studies of innate immunity. The cellular elements of innate immunity in Drosophila, the hemocytes, have been characterized by morphological criteria, molecular markers, and cell-type-specific immunological markers. Here we suggest that an MiET1 GFP-reporter element insertion in the untranslated region of a gene (l1-atilla) - expressed in a subset of hemocytes, the lamellocytes - allows in vivo investigations of lamellocyte differentiation and facilitates genetic screens.

Multinucleated Giant Hemocytes Are Effector Cells in Cell-Mediated Immune Responses of Drosophila.

We identified and characterized a so far unrecognized cell type, dubbed the multinucleated giant hemocyte (MGH), in the ananassae subgroup of Drosophilidae. Here, we describe the functional and ultrastructural characteristics of this novel blood cell type as well as its characterization with a set of discriminative immunological markers. MGHs are encapsulating cells that isolate and kill the parasite without melanization. They share some properties with but differ considerably from lamellocytes, the encapsulating cells of Drosophila melanogaster, the broadly used model organism in studies of innate immunity. MGHs are nonproliferative effector cells that are derived from phagocytic cells of the sessile tissue and the circulation, but do not exhibit phagocytic activity. In contrast to lamellocytes, MGHs are gigantic cells with filamentous projections and contain many nuclei, which are the result of the fusion of several cells. Although the structure of lamellocytes and MGHs differ remarkably, their function in the elimination of parasites is similar, which is potentially the result of the convergent evolution of interactions between hosts and parasites in different geographic regions. MGHs are highly motile and share several features with mammalian multinucleated giant cells, a syncytium of macrophages formed during granulomatous inflammation.

Variation of NimC1 expression in Drosophila stocks and transgenic strains

The NimC1 molecule has been described as a phagocytosis receptor, and is being used as a marker for professional phagocytes, the plasmatocytes, in Drosophila melanogaster. In studies including tumor-biology, developmental biology, and cell mediated immunity, monoclonal antibodies (P1a and P1b) to the NimC1 antigen are used. As we observed that these antibodies did not react with plasmatocytes of several strains and genetic combinations, a molecular analysis was performed on the structure of the nimC1 gene. In these strains we found 2 deletions and an insertion within the nimC1 gene, which may result in the production of a truncated NimC1 protein. The NimC1 positivity was regained by recombining the mutation with a wild-type allele or by using nimC1 mutant lines under heterozygous conditions. By means of these procedures or using the recombined stock, NimC1 can be used as a marker for phagocytic cells in the majority of the possible genetic backgrounds.

Drosophila Nimrod proteins bind bacteria

Engulfment of foreign particles by phagocytes is initiated by the engagement of phagocytic receptors. We have previously reported that NimC1 is involved in the phagocytosis of bacteria in Drosophila melanogaster. We have identified a family of genes, the Nimrod gene superfamily, encoding characteristic NIM domain containing structural homologues of NimC1. In this work we studied the bacterium-binding properties of the Nimrod proteins by using a novel immunofluorescencebased flow cytometric assay. This method proved to be highly reproducible and suitable for investigations of the bacteriumbinding capacities of putative phagocytosis receptors. We found that NimC1, NimA, NimB1 and NimB2 bind bacteria significantly but differently. In this respect they are similar to other NIM domain containing receptors Eater and Draper.

In vivo immunostaining of hemocyte compartments in Drosophila for live imaging

In recent years, Drosophila melanogaster has become an attractive model organism in which to study the structure and development of the cellular immune components. The emergence of immunological markers greatly accelerated the identification of the immune cells (hemocytes), while the creation of genetic reporter constructs allowed unique insight into the structural organization of hematopoietic tissues. However, investigation of the hemocyte compartments by the means of immunological markers requires dissection and fixation, which regularly disrupt the delicate structure and hamper the microanatomical characterization. Moreover, the investigation of transgenic reporters alone can be misleading as their expression often differs from the native expression pattern of their respective genes. We describe here a method that combines the reporter constructs and the immunological tools in live imaging, thereby allowing use of the array of available immunological markers while retaining the structural integrity of the hematopoietic compartments. The procedure allows the reversible immobilization of Drosophila larvae for high-resolution confocal imaging and the time-lapse video analysis of in vivo reporters. When combined with our antibody injection-based in situ immunostaining assay, the resulting double labeling of the hemocyte compartments can provide new information on the microanatomy and functional properties of the hematopoietic tissues in an intact state. Although this method was developed to study the immune system of Drosophila melanogaster, we anticipate that such a combination of genetic and immunological markers could become a versatile technique for in vivo studies in other biological systems too.

A novel method for the identification of factors involved in host-pathogen interactions in Drosophila melanogaster.

A new method was established, standardized and validated for screening factors involved in the response to septic injury in Drosophila melanogaster. The method, based on inducing lesion by removing the tarsal segments of the first pair of legs of Drosophila adults and exposing them to different bacteria, imitates injury that often occurs in the natural habitat. The method is easy to perform, highly reproducible and suitable for large-scale genetic screens with the aim of identifying factors involved in host-pathogen interactions. The technique was validated by using mutant variations of different components of the immune response, blood clotting as well as the involvement of a number of genes known to be instrumental in the humoral and cell-mediated immune responses of Drosophila was confirmed. Moreover, the combination of the present method with antibiotic treatment allows the screening of potential antimicrobial drugs in vivo.

Genes encoding cuticular proteins are components of the Nimrod gene cluster in Drosophila

The Nimrod gene cluster, located on the second chromosome of Drosophila melanogaster, is the largest synthenic unit of the Drosophila genome. Nimrod genes show blood cell specific expression and code for phagocytosis receptors that play a major role in fruit fly innate immune functions. We previously identified three homologous genes (vajk-1, vajk-2 and vajk-3) located within the Nimrod cluster, which are unrelated to the Nimrod genes, but are homologous to a fourth gene (vajk-4) located outside the cluster. Here we show that, unlike the Nimrod candidates, the Vajk proteins are expressed in cuticular structures of the late embryo and the late pupa, indicating that they contribute to cuticular barrier functions.