Drosophila phagocytosis - still many unknowns under the surface

In mammals, phagocytosis coordinates host defence on two levels: It acts both as an effector of the innate immunity, as well as an initiator of the adaptive immunity. The fruit fly Drosophila melanogaster (D. melanogaster) lacks the adaptive immune response, and the role of Drosophila plasmatocytes, cells that resemble phagocytosing mammalian macrophages, is limited to innate immune responses. During the past years, several studies have shed light on the role of phagocytosis in the Drosophila host defence. At least in some infection models, the systemic production of potent antimicrobial peptides (AMPs) does not completely compensate for the need for cellular immune responses. As a model, Drosophila offers powerful tools for studying phagocytosis including, large-scale RNA interference (RNAi) based in vitro screens that can be combined with classical Drosophila genetics. These kinds of approaches have led to important discoveries related especially to microbial recognition by Drosophila plasmatocytes. Events following initial recognition, however, have remained more elusive. This review summarizes the current knowledge on Drosophila phagocytosis focusing on the most recent advancements in the field, and highlighting the benefits the Drosophila system has to offer for research on phagocytosis.     

Infectious non-self recognition in invertebrates: lessons from Drosophila and other insect models

The vertebrate innate immune system recognizes infectious non-self by employing a set of germline-encoded receptors such as nucleotide-binding oligomerisation domain proteins (NODs) or Toll-like receptors (TLRs). These proteins are involved in the recognition of various microbial-derived molecules, including lipopolysaccharide (LPS), peptidoglycan (PGN) and beta1,3-glucan. Drosophila Toll receptors are not directly dedicated to non-self recognition and insect NOD orthologues have not yet been identified. Studies started more than 20 years ago and conducted on different insect models have identified other receptors on which invertebrate innate systems rely to sense invading microorganisms.     

Peptidoglycan recognition proteins (PGRPs)

Innate immune system recognizes microorganisms through a series of pattern recognition receptors that are highly conserved in evolution. Peptidoglycan recognition proteins (PGRPs) are pattern recognition molecules that are conserved from insects to mammals and recognize bacteria and their unique cell wall component, peptidoglycan (PGN). Drosophila, mosquito, and mammals have families of 13, 7, and 4 PGRP genes, respectively, and some of these genes are alternatively spliced. PGRPs are differentially expressed in various cells and tissues, their expression is often upregulated by bacteria, and they mediate host responses to bacterial infections. Insect PGRPs have four known effector functions that are unique for insects: activation of prophenoloxidase cascade, activation of Toll receptor, activation of Imd pathway, and induction of phagocytosis. One function, amidase activity, is shared by some insect and mammalian PGRPs, whereas antibacterial activity of some mammalian PGRPs is unique for mammals.     

Role and activation of type III secretion system genes in Pseudomonas aeruginosa-induced Drosophila killing

Pseudomonas aeruginosa strains PAO1 and CHA showing type III system-dependent cytotoxicity towards macrophages ex vivo are able to induce rapid death of adult fly Drosophila melanogaster accompanied by bacterial multiplication to high-titers. The role of P. aeruginosa type III secretion system in rapid fly killing was demonstrated here by using several isogenic CHA mutants, selectively affected in this system. The activation of P. aeruginosa pexsCBA, the regulatory operon of the type III system, and the activation of the Drosophila gene diptericin, showed the host-pathogen recognition during infection process.     

Macrophage-mediated innate host defense against protozoan parasites

Macrophages are immune cells that play a pivotal role in the detection and elimination of pathogenic microorganisms. Macrophages possess a variety of surface receptors devoted to the recognition of non-self by discriminating between host and pathogen-derived structures. Recognition of foreign microorganisms by the macrophage ultimately results in phagocytosis and the eventual destruction of microorganisms by lysosomal enzymes, toxic reactive oxygen and nitrogen intermediates, and/or nutrient deprivational mechanisms. However, protozoan parasites such as Toxoplasma gondii, Trypanosoma cruzi, and Leishmania spp., parasitize macrophages, utilizing them as a host cell for their growth, replication, and/or maintenance of their life cycles. The protozoan parasites of the genus Leishmania are unique in that their intracellular replication in the host is predominantly restricted to a single cell type, the macrophage. This review focuses on the cellular processes involved in macrophage-mediated host defense against protozoan parasites, from the initial host-parasite interactions that mediate recognition to the mechanisms employed by macrophages to destroy and eliminate the pathogen. As an example model system of experimental study, we describe in more more detail the cellular interactions between macrophages and the obligate intracellular parasite of mammalian macrophages, Leishmania spp.     

Blood cells of Drosophila: cell lineages and role in host defence

Drosophila haemopoiesis gives rise to three independent cell lineages: plasmatocytes, crystal cells and lamellocytes. The regulation of Drosophila stem cell proliferation and lineage specification involves transactivators and signalling pathways, many of which have mammalian counterparts that control haemopoietic processes. Drosophila plasmatocytes are professional phagocytes that resemble the monocyte/macrophage lineage, crystal cells play a critical role in defence-related melanisation, and lamellocytes encapsulate large invaders. Crystal cells and lamellocytes have no clear mammalian homologues. Research into the molecular mechanisms that underlie the various immune functions of Drosophila blood cells, such as non-self recognition, is now taking wing.     

Drosophila miR-14 regulates insulin production and metabolism through its target, sugarbabe

Energy homeostasis depends on insulin signaling in metazoans. Insulin levels reflect the nutritional status of the animal to control levels of circulating sugar and regulate storage of resources in the form of glycogen and fat. Over the past several years, evidence has begun to accumulate that insulin production and secretion, as well as cellular responsiveness to insulin, are subject to regulation by microRNAs. Here we present evidence that miR-14 acts in the insulin-producing neurosecretory cells in the adult Drosophila brain to control metabolism. miR-14 acts in these cells through its direct target, sugarbabe. sugarbabe encodes a predicted zinc finger protein that regulates insulin gene expression in the neurosecretory cells. Regulation of sugarbabe levels by nutrients and by miR-14 combines to allow the fly to manage resource mobilization in a nutritionally variable environment.     

Macrophage-Mediated Innate Host Defense Against Protozoan Parasites

Macrophages are immune cells that play a pivotal role in the detection and elimination of pathogenic microorganisms. Macrophages possess a variety of surface receptors devoted to the recognition of non-self by discriminating between host and pathogen-derived structures. Recognition of foreign microorganisms by the macrophage ultimately results in phagocytosis and the eventual destruction of microorganisms by lysosomal enzymes, toxic reactive oxygen and nitrogen intermediates, and/or nutrient deprivational mechanisms. However, protozoan parasites such as Toxoplasma gondii, Trypanosoma cruzi, and Leishmania spp., parasitize macrophages, utilizing them as a host cell for their growth, replication, and/or maintenance of their life cycles. The protozoan parasites of the genus Leishmania are unique in that their intracellular replication in the host is predominantly restricted to a single cell type, the macrophage. This review focuses on the cellular processes involved in macrophage-mediated host defense against protozoan parasites, from the initial host-parasite interactions that mediate recognition to the mechanisms employed by macrophages to destroy and eliminate the pathogen. As an example model system of experimental study, we describe in more more detail the cellular interactions between macrophages and the obligate intracellular parasite of mammalian macrophages, Leishmania spp.     

The intracellular accumulation of polymeric neuroserpin explains the severity of the dementia FENIB

Familial encephalopathy with neuroserpin inclusion bodies (FENIB) is an autosomal dominant dementia that is characterized by the retention of polymers of neuroserpin as inclusions within the endoplasmic reticulum (ER) of neurons. We have developed monoclonal antibodies that detect polymerized neuroserpin and have used COS-7 cells, stably transfected PC12 cell lines and transgenic Drosophila melanogaster to characterize the cellular handling of all four mutant forms of neuroserpin that cause FENIB. We show a direct correlation between the severity of the disease-causing mutation and the accumulation of neuroserpin polymers in cell and fly models of the disease. Moreover, mutant neuroserpin causes locomotor deficits in the fly allowing us to demonstrate a direct link between polymer accumulation and neuronal toxicity.     

Making tubes in the Drosophila embryo.

Epithelial and endothelial tubes come in various shapes and sizes and form the basic units of many tubular organs. During embryonic development, single unbranched tubes as well as highly branched networks of tubes form from simple sheets of cells by several morphogenic movements. Studies of tube formation in the Drosophila embryo have greatly advanced our understanding of the cellular and molecular mechanisms by which tubes are formed. This review highlights recent progress on formation of the hindgut, Malpighian tubules, proventriculus, salivary gland, and trachea of the Drosophila embryo, focusing on the cellular events that form each tube and their genetic requirements.     

Sensing microorganisms in the gut triggers the immune response in Eisenia andrei earthworms

The tube-within-tube body plan of earthworms is appropriate for studying the interactions of microorganisms with the immune system of body cavities such as the digestive tract and coelom. This study aims to describe the immune response on the molecular and cellular level in the coelomic cavity and the gut of the earthworm Eisenia andrei after experimental microbial challenge by administering two bacterial strains (Escherichia coli and Bacillus subtilis) or yeast Saccharomyces cerevisiae to the environment. The changes in mRNA levels of defense molecules (pattern recognition receptor CCF, lysozyme, fetidin/lysenins) in the coelomocytes and gut tissue were determined by quantitative PCR. The immune response at a cellular level was captured in histological sections, and the expression of CCF was localized using in situ hybridization. Coelomocytes respond to the presence of bacteria in the coelomic cavity by increasing the mRNA levels of defense molecules, especially CCF. The immune response in gut tissue is less affected by microbial stimulation because the epithelial cells of gut exhibit basically strong mRNA synthesis of ccf as a defense against the continuous microbial load in the gut lumen. The cellular immune response is mediated by coelomocytes released from the mesenchymal lining of the coelomic cavity. These combined immune mechanisms are necessary for the survival of earthworms in the microbially rich environment of soil.     

Mass transit: epithelial morphogenesis in the Drosophila egg chamber.

Epithelial cells use a striking array of morphogenetic behaviors to sculpt organs and body plans during development. Although it is clear that epithelial morphogenesis is largely driven by cytoskeletal rearrangements and changes in cell adhesion, little is known about how these processes are coordinated to construct complex biological structures from simple sheets of cells. The follicle cell epithelium of the Drosophila egg chamber exhibits a diverse range of epithelial movements in a genetically accessible tissue, making it an outstanding system for the study of epithelial morphogenesis. In this review, we move chronologically through the process of oogenesis, highlighting the dynamic movements of the follicle cells. We discuss the cellular architecture and patterning events that set the stage for morphogenesis, detail individual cellular movements, and focus on current knowledge of the cellular processes that drive follicle cell behavior.     

Muscle stem cells and model systems for their investigation.

Stem cells are characterized by their clonal ability both to generate differentiated progeny and to undergo self-renewal. Studies of adult mammalian organs have revealed stem cells in practically every tissue. In the adult skeletal muscle, satellite cells are the primary muscle stem cells, responsible for postnatal muscle growth, hypertrophy, and regeneration. In the past decade, several molecular markers have been found that identify satellite cells in quiescent and activated states. However, despite their prime importance, surprisingly little is known about the biology of satellite cells, as their analysis was for a long time hampered by a lack of genetically amenable experimental models where their properties can be dissected. Here, we review how the embryonic origin of satellite cells was discovered using chick and mouse model systems and discuss how cells from other sources can contribute to muscle regeneration. We present evidence for evolutionarily conserved properties of muscle stem cells and their identification in lower vertebrates and in the fruit fly. In Drosophila, muscle stem cells called adult muscle precursors (AMP) can be identified in embryos and in larvae by persistent expression of a myogenic basic helix-loop-helix factor Twist. AMP cells play a crucial role in the Drosophila life cycle, allowing de novo formation and regeneration of adult musculature during metamorphosis. Based on the premise that AMPs represent satellite-like cells of the fruit fly, important insight into the biology of vertebrate muscle stem cells can be gained from genetic analysis in Drosophila.     

Mononuclear phagocyte proliferation, maturation and function.

The mononuclear phagocytic system is a continuum of cells beginning with the bone marrow monoblast and promonocyte, through the monocyte to the larger tissue macrophages and multinucleate giant cells. This system of cells is widely distributed throughout the body in the blood and bone marrow; the pleural, peritoneal, and alveolar spaces; the lymph nodes, spleen, liver, and other parenchymal organs. The activity and composition of the cell varies with the level of maturation, changes in cellular environment, and with various cellular activities. The monocyte-macrophage group of cells plays an active role in defense reactions against certain microorganisms, and in the removal of dying cells and cell debris. They are an integral part of both the inductive phase of the immune response, and of cell-mediated immune reactions. In addition, they probably play a role in the defence against spontaneously arising tumours, in the control of granulopoiesis, and possibly in erythropoiesis.     

Characterization and mutant analysis of the Drosophila sema 5c gene.

Class V semaphorins are transmembrane glycoproteins characterised by the presence of thrombospondin type I (Tsp) repeats linked to their extracellular semaphorin domain. Sema 5C is the only class V semaphorin found in Drosophila. Dsema 5C RNA is maternally provided and its embryonic expression is prominent in the mesoderm and muscle attachment sites. Here, we show that DSema 5C exists in two protein isoforms as a result of alternative splicing and that both protein and RNA have similar expression patterns. Using a combination of various molecular markers, we show that the DSema 5C protein becomes enriched in mesodermal cells that would normally give rise to fat body and visceral structures. In late embryos, DSema 5C is expressed in segment boundary cells that would constitute subsets of muscle attachment sites. Both RNA and protein are excluded from the somatic precursors and the mature muscles. The expression data suggest DSema 5C localised to the epidermal component of muscle attachment sites. Mutations in Dsema 5C were isolated from a P-element excision screen and by blotting analysis. The Dsema 5C mutants are homozygous viable and show no obvious embryonic phenotypes, suggesting that the maternal and zygotic components of Dsema 5C are not essential for fly development.     

Activation and control of complement, inflammation, and infection associated with the use of biomedical polymers

It is generally acknowledged that artificial biomaterials are much less immunologically active than transplants or tissue derived biomaterials. However, activation of both the coagulation cascade and the complement system is a common occurrence when human blood is exposed to biomaterial surfaces during extracorporeal procedures, such as renal hemodialysis or cardiopulmonary bypass. Both individual and collective activation of these cascades often produce local and systemic effects. A number of complement activation products function as the mediators of inflammation. They serve as ligands for specific receptors on polymorphonuclear leukocytes, monocytes, macrophages, mast cells, and other cells. Such an interaction leads to induction of cellular responses in adhered cells, including release of oxidative products, lysosomal enzymes, or both, which often contribute to a number of pathologic conditions. Most pathogens invading the human body are attacked by the immune system directly following entry, especially when they are in contact with blood. However, bacteria and parasites have developed a large number of specific strategies to overcome immune defense among others by avoiding either recognition or eradication by complement. In this aspect, of concern are several microorganisms responsible for formation of antibiotic resistant biofilms on biomaterial surfaces, namely Staphylococcus epidermidis, Staphylococcus aureus, and Pseudomonas aeruginosa.     

Drosophila C-type lectins enhance cellular encapsulation

C-type lectins are calcium-dependent carbohydrate binding proteins, and animal C-type lectins participate in innate immunity and cell-cell interactions. In the fruit fly Drosophila melanogaster, more than 30 genes encode C-type lectin domains. However, functions of Drosophila C-type lectins in innate immunity are not well understood. This study is to investigate whether two Drosophila C-type lectins, CG33532 and CG33533 (designated as DL2 and DL3, respectively), are involved in innate immune responses. Recombinant DL2 and DL3 were expressed and purified. Both DL2 and DL3 agglutinated Gram-negative Escherichia coli in a calcium-dependent manner. Though DL2 and DL3 are predicted to be secreted proteins, they were detected on the surface of Drosophila hemocytes, and recombinant DL2 and DL3 also directly bound to hemocytes. Coating of agarose beads with recombinant DL2 and DL3 enhanced their encapsulation and melanization by Drosophila hemocytes in vitro. However, hemocyte encapsulation was blocked when the lectin-coated beads were pre-incubated with rat polyclonal antibody specific for DL2 or DL3. Our results suggest that DL2 and DL3 may act as pattern recognition receptors to mediate hemocyte encapsulation and melanization by directly recruiting hemocytes to the lectin-coated surface.     

Evolutionary conservation and divergence of the segmentation process in arthropods.

A fundamental characteristic of the arthropod body plan is its organization in metameric units along the anterior-posterior axis. The segmental organization is laid down during early embryogenesis. Our view on arthropod segmentation is still strongly influenced by the huge amount of data available from the fruit fly Drosophila melanogaster (the Drosophila paradigm). However, the simultaneous formation of the segments in Drosophila is a derived mode of segmentation. Successive terminal addition of segments from a posteriorly localized presegmental zone is the ancestral mode of arthropod segmentation. This review focuses on the evolutionary conservation and divergence of the genetic mechanisms of segmentation within arthropods. The more downstream levels of the segmentation gene network (e.g., segment polarity genes) appear to be more conserved than the more upstream levels (gap genes, Notch/Delta signaling). Surprisingly, the basally branched arthropod groups also show similarities to mechanisms used in vertebrate somitogenesis. Furthermore, it has become clear that the activation of pair rule gene orthologs is a key step in the segmentation of all arthropods. Important findings of conserved and diverged aspects of segmentation from the last few years now allow us to draw an evolutionary scenario on how the mechanisms of segmentation could have evolved and led to the present mechanisms seen in various insect groups including dipterans like Drosophila.     

Planar polarity of hair cells in the chick inner ear is correlated with polarized distribution of c-flamingo-1 protein.

Hair cells of the vertebrate inner ear are directional mechanosensors: they have a polarity, defined by a vector in the plane of the sensory epithelium. It has been suggested that this polarity might be controlled by genes homologous to those that control planar cell polarity (PCP) in Drosophila, and vertebrate homologues of the Drosophila PCP genes Van Gogh/strabismus and flamingo/starry night are indeed essential for normal hair cell PCP. The underlying molecular mechanism is unclear, however. Although the PCP protein Flamingo shows a polarized intracellular distribution in the fly, it is unknown whether this is necessary for its function. Here, we describe the expression pattern of a flamingo homologue, c-flamingo-1 (c-fmi-1), in the developing chick ear and show that its protein product, like that of flamingo in the fly, has a polarized distribution in each hair cell, defining an axis that corresponds to the structural PCP axis. This conservation between fly and vertebrate suggests that the polarized protein localization is functionally important. In the basilar papilla, the same localization is seen in supporting cells also, suggesting that supporting cells are cryptically polarized, despite having no overt structural polarity; they may thus participate in PCP signal transmission across the sensory patch.