István Andó

Relish, a central factor in the control of humoral but not cellular immunity in Drosophila.

The NF-kappa B-like Relish gene is complex, with four transcripts that are all located within an intron of the Nmdmc gene. Using deletion mutants, we show that Relish is specifically required for the induction of the humoral immune response, including both antibacterial and antifungal peptides. As a result, the Relish mutants are very sensitive to infection. A single cell of E. cloacae is sufficient to kill a mutant fly, and the mutants show increased susceptibility to fungal infection. In contrast, the blood cell population, the hematopoietic organs, and the phagocytic, encapsulation, and melanization responses are normal. Our results illustrate the importance of the humoral response in Drosophila immunity and demonstrate that Relish plays a key role in this response.

Activation of the Drosophila NF‐κB factor Relish by rapid endoproteolytic cleavage

The Rel/NF‐κB transcription factor Relish plays a key role in the humoral immune response in Drosophila. We now find that activation of this innate immune response is preceded by rapid proteolytic cleavage of Relish into two parts. An N‐terminal fragment, containing the DNA‐binding Rel homology domain, translocates to the nucleus where it binds to the promoter of the Cecropin A1 gene and probably to the promoters of other antimicrobial peptide genes. The C‐terminal IκB‐like fragment remains in the cytoplasm. This endoproteolytic cleavage does not involve the proteasome, requires the DREDD caspase, and is different from previously described mechanisms for Rel factor activation.

Nimrod, a Putative Phagocytosis Receptor with EGF Repeats in Drosophila Plasmatocytes

The hemocytes, the blood cells of Drosophila, participate in the humoral and cellular immune defense reactions against microbes and parasites [1-8]. The plasmatocytes, one class of hemocytes, are phagocytically active and play an important role in immunity and development by removing microorganisms as well as apoptotic cells. On the surface of circulating and sessile plasmatocytes, we have now identified a protein, Nimrod C1 (NimC1), which is involved in the phagocytosis of bacteria. Suppression of NimC1 expression in plasmatocytes inhibited the phagocytosis of Staphylococcus aureus. Conversely, overexpression of NimC1 in S2 cells stimulated the phagocytosis of both S. aureus and Escherichia coli. NimC1 is a 90-100 kDa single-pass transmembrane protein with ten characteristic EGF-like repeats (NIM repeats). The nimC1 gene is part of a cluster of ten related nimrod genes at 34E on chromosome 2, and similar clusters of nimrod-like genes are conserved in other insects such as Anopheles and Apis. The Nimrod proteins are related to other putative phagocytosis receptors such as Eater and Draper from D. melanogaster and CED-1 from C. elegans. Together, they form a superfamily that also includes proteins that are encoded in the human genome.

Sessile hemocytes as a hematopoietic compartment in Drosophila melanogaster

The blood cells, or hemocytes, in Drosophila participate in the immune response through the production of antimicrobial peptides, the phagocytosis of bacteria, and the encapsulation of larger foreign particles such as parasitic eggs; these immune reactions are mediated by phylogenetically conserved mechanisms. The encapsulation reaction is analogous to the formation of granuloma in vertebrates, and is mediated by large specialized cells, the lamellocytes. The origin of the lamellocytes has not been formally established, although it has been suggested that they are derived from the lymph gland, which is generally considered to be the main hematopoietic organ in the Drosophila larva. However, it was recently observed that a subepidermal population of sessile blood cells is released into the circulation in response to a parasitoid wasp infection. We set out to analyze this phenomenon systematically. As a result, we define the sessile hemocytes as a novel hematopoietic compartment, and the main source of lamellocytes.

Hemese, a hemocyte-specific transmembrane protein, affects the cellular immune response in Drosophila

We have identified a previously undescribed transmembrane protein, Hemese, from Drosophila melanogaster blood cells (hemocytes), by using a monoclonal pan-hemocyte antibody. Heavy glycosylation is suggested by the heterogeneous size distribution, ranging between 37 and 70 kDa. Hemese expression is restricted to the cell surfaces of hemocytes of all classes, and to the hematopoietic organs. The sequence of the corresponding gene, Hemese (He), predicts a glycophorin-like protein of 15 kDa, excluding an N-terminal signal peptide, with a single hydrophobic transmembrane region. The extracellular region consists mainly of Ser/Thr-rich sequence of low complexity, with several potential O-glycosylation sites. Hemese contains phosphotyrosine and the cytoplasmic region has potential phosphorylation sites, suggesting an involvement in signal transduction. Depletion of Hemese by RNA interference has no obvious effect under normal conditions, but the cellular response to parasitic wasps is much enhanced. This finding indicates that Hemese plays a modulatory role in the activation or recruitment of the hemocytes.

Definition of Drosophila hemocyte subsets by cell-type specific antigens.

We analyzed the heterogeneity of Drosophila hemocytes on the basis of the expression of cell-type specific antigens. The antigens characterize distinct subsets which partially overlap with those defined by morphological criteria. On the basis of the expression or the lack of expression of blood cell antigens the following hemocyte populations have been defined: crystal cells, plasmatocytes, lamellocytes and precursor cells. The expression of the antigens and thus the different cell types are developmentally regulated. The hemocytes are arranged in four main compartments: the circulating blood cells, the sessile tissue, the lymph glands and the posterior hematopoietic tissue. Each hemocyte compartment has a specific and characteristic composition of the various cell types. The described markers represent the first successful attempt to define hemocyte lineages by immunological markers in Drosophila and help to define morphologically, functionally, spatially and developmentally distinct subsets of hemocytes.

Drosophila melanogaster Rac2 is necessary for a proper cellular immune response.

It has been reported that during Drosophila embryonic development, and in cell culture, that the Rac GTPases are redundant. To better elucidate Rac function in Drosophila, we decided to study the role of Rac2 in larval cellular defense reactions against the parasitiod Leptopilina boulardi. Here we show a dramatic effect in the context of cellular immunity where, unlike embryonic development, Rac2 appears to have a non‐redundant function. When an invading parasitoid is recognized as foreign, circulating hemocytes (blood cells) should recognize and attach to the egg chorion. After attachment the hemocytes should then spread to form a multilayered capsule surrounding the invader. In Rac2 mutants this process is disrupted. Immune surveillance cells, known as plasmatocytes, adhere to the parasitoid egg but fail to spread, and septate junctions do not assemble, possibly due to mislocalization of the Protein 4.1 homolog Coracle. Finally, larger cells known as lamellocytes attach to the capsule but also fail to spread, and there is a lack of melanization. From these results it appears that Rac2 is necessary for the larval cellular immune response.

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.

Deletion of proteasomal subunit S5a/Rpn10/p54 causes lethality, multiple mitotic defects and overexpression of proteasomal genes in Drosophila melanogaster

The regulatory complex of the 26S proteasome is responsible for the selective recognition and binding of multiubiquitinated proteins. It was earlier shown that the subunit S5a/Rpn10/p54 of the regulatory complex is the only cellular protein capable of binding multiubiquitin chains in an in vitro overlay assay. The role of this subunit in substrate selection, however, is a subject of debate, following the observation that its deletion in Saccharomyces cerevisiae is not lethal and instead causes only a mild phenotype. To study the function of this subunit in higher eukaryotes, a mutant Drosophila strain was constructed by deleting the single copy gene encoding subunit S5a/Rpn10/p54. This deletion caused larval-pupal polyphasic lethality, multiple mitotic defects, the accumulation of higher multimers of ubiquitinated proteins and a huge accumulation of defective 26S proteasome particles. Deletion of the subunit S5a/Rpn10/p54 does not destabilise the regulatory complex and does not disturb the assembly of the regulatory complex and the catalytic core. The pupal lethality is a consequence of the depletion of the maternally provided 26S proteasome during the larval stages and a sudden increase in the proteasomal activity demands during the first few hours of pupal development. The huge accumulation of the fully assembled 26S proteasome in the deletion mutant and the lack of free subunits or partially assembled particles indicate that there is a highly coordinated accumulation of all the subunits of the 26S proteasome. This suggests that in higher eukaryotes, as with yeast, a feedback circuit coordinately regulates the expression of the proteasomal genes, and this adjusts the actual proteasome concentration in the cells according to the temporal and/or spatial proteolytic demands.