Drosophila Immunity Research Articles

PGRP-LB Minds the Fort

Peptidoglycan recognition proteins (PGRPs) are a class of molecules that play a critical role in Drosophila immunity. In this issue of Immunity, Zaidman-Rémy et al. (2006) show that PGRP-LB controls systemic immune responses as well as homeostasis at the barrier surfaces.

A Spätzle-Processing Enzyme Required for Toll Signaling Activation in Drosophila Innate Immunity

The Toll receptor was originally identified as an indispensable molecule for Drosophila embryonic development and subsequently as an essential component of innate immunity from insects to humans. Although in Drosophila the Easter protease processes the pro-Spätzle protein to generate the Toll ligand during development, the identification of the protease responsible for pro-Spätzle processing during the immune response has remained elusive for a decade. Here, we report a protease, called Spätzle-processing enzyme (SPE), required for Toll-dependent antimicrobial response. Flies with reduced SPE expression show no noticeable pro-Spätzle processing and become highly susceptible to microbial infection. Furthermore, activated SPE can rescue ventral and lateral development in embryos lacking Easter, showing the functional homology between SPE and Easter. These results imply that a single ligand/receptor-mediated signaling event can be utilized for different biological processes, such as immunity and development, by recruiting similar ligand-processing proteases with distinct activation modes.

WntD is a feedback inhibitor of Dorsal/NF-κB in Drosophila development and immunity

Regulating the nuclear factor-kappaB (NF-kappaB) family of transcription factors is of critical importance to animals, with consequences of misregulation that include cancer, chronic inflammatory diseases and developmental defects. Studies in Drosophila melanogaster have proved fruitful in determining the signals used to control NF-kappaB proteins, beginning with the discovery that the Toll/NF-kappaB pathway, in addition to patterning the dorsal-ventral axis of the fly embryo, defines a major component of the innate immune response in both Drosophila and mammals. Here, we characterize the Drosophila wntD (Wnt inhibitor of Dorsal) gene. We show that WntD acts as a feedback inhibitor of the NF-kappaB homologue Dorsal during both embryonic patterning and the innate immune response to infection. wntD expression is under the control of Toll/Dorsal signalling, and increased levels of WntD block Dorsal nuclear accumulation, even in the absence of the IkappaB homologue Cactus. The WntD signal is independent of the common Wnt signalling component Armadillo (beta-catenin). By engineering a gene knockout, we show that wntD loss-of-function mutants have immune defects and exhibit increased levels of Toll/Dorsal signalling. Furthermore, the wntD mutant phenotype is suppressed by loss of zygotic dorsal. These results describe the first secreted feedback antagonist of Toll signalling, and demonstrate a novel Wnt activity in the fly.

Overview of Innate Immunity in Drosophila

Drosophila protects itself from infection by microbial organisms by means of its pivotal defense, the so-called innate immunity system. This is its sole defense as it lacks an adaptive immunity system such as is found in mammals. The strong conservation of innate immunity systems in organisms from Drosophila to mammals, and the ease with which Drosophila can be manipulated genetically, makes this fly a good model system for investigating the mechanisms of virulence of a number of medically important pathogens. Potentially damaging endogenous and/or exogenous challenges sensed by specific receptors initiate signals via the Toll and/or Imd signaling pathways. These in turn activate the transcription factors Dorsal, Dorsal-related immune factor (Dif) and Relish, culminating in transcription of genes involved in the production of antimicrobial peptides, melanization, phagocytosis, and the cytoskeletal rearrangement required for appropriate responses. Clarifying the regulatory interactions between the various pathways involved is very important for understanding the specificity and termination mechanism of the immune response.

RETRACTED: Expression and function of Toll-like receptor on T cells

Toll is the founder of a group of pattern recognition receptors, which play a critical role in the innate immunity in Drosophila. At least 13 distinct Toll-like receptors (TLRs), recognising pathogen-associated molecular pattern (PAMPs), have now been identified in humans. Most investigations on TLRs have focused on cells of the innate system. We report here that naïve human T cells expressed high levels of cell surface TLR2 after activation by anti-T cell receptor (TCR) antibody and interferon-alpha. Activated cells produced elevated levels of cytokines in response to the TLR2 ligand, bacterial lipopeptide (BLP). Furthermore, CD4(+)CD45RO(+) memory T cells from peripheral blood constitutively expressed TLR2 and produced IFNgamma in response to BLP. BLP also markedly enhanced the proliferation and IFNgamma production by CD45RO(+) T cells in the presence of IL-2 or IL-15. Thus, TLR2 serves as a co-stimulatory receptor for antigen-specific T cell development and participates in the maintenance of T cell memory. This suggests that pathogens, via their PAMPs, may contribute directly to the perpetuation and activation of long term T cell memory in both antigen dependent and independent manner.

Critical Roles of Threonine 187 Phosphorylation in Cellular Stress-induced Rapid and Transient Activation of Transforming Growth Factor-β-activated Kinase 1 (TAK1) in a Signaling Complex Containing TAK1-binding Protein TAB1 and TAB2

Transforming growth factor-beta-activated kinase 1 (TAK1) mitogen-activated protein kinase kinase kinase has been shown to be activated by cellular stresses including tumor necrosis factor-alpha (TNF-alpha). Here, we characterized the molecular mechanisms of cellular stress-induced TAK1 activation, focusing mainly on the phosphorylation of TAK1 at Thr-187 and Ser-192 in the activation loop. Thr-187 and Ser-192 are conserved among species from Caenorhabditis elegans to human, and their replacement with Ala resulted in inactivation of TAK1. Immunoblotting with a novel phospho-TAK1 antibody revealed that TNF-alpha significantly induced the phosphorylation of endogenous TAK1 at Thr-187, and subsequently the phosphorylated forms of TAK1 rapidly disappeared. Intermolecular autophosphorylation of Thr-187 was essential for TAK1 activation. RNA interference and overexpression experiments demonstrated that TAK1-binding protein TAB1 and TAB2 were involved in the phosphorylation of TAK1, but they regulated TAK1 phosphorylation differentially. Furthermore, SB203580 and p38alpha small interfering RNA enhanced TNF-alpha-induced Thr-187 phosphorylation as well as TAK1 kinase activity, indicating that the phosphorylation is affected by p38alpha/TAB1/TAB2-mediated feedback control of TAK1. These results indicate critical roles of Thr-187 phosphorylation in the stress-induced rapid and transient activation of TAK1 in a signaling complex containing TAB1 and TAB2.

Peptidoglycan recognition protein (PGRP)-LE and PGRP-LC act synergistically in Drosophila immunity

In innate immunity, pattern recognition molecules recognize cell wall components of microorganisms and activate subsequent immune responses, such as the induction of antimicrobial peptides and melanization in Drosophila. The diaminopimelic acid (DAP)-type peptidoglycan potently activates imd-dependent induction of antibacterial peptides. Peptidoglycan recognition protein (PGRP) family members act as pattern recognition molecules. PGRP-LC loss-of-function mutations affect the imd-dependent induction of antibacterial peptides and resistance to Gram-negative bacteria, whereas PGRP-LE binds to the DAP-type peptidoglycan, and a gain-of-function mutation induces constitutive activation of both the imd pathway and melanization. Here, we generated PGRP-LE null mutants and report that PGRP-LE functions synergistically with PGRP-LC in producing resistance to Escherichia coli and Bacillus megaterium infections, which have the DAP-type peptidoglycan. Consistent with this, PGRP-LE acts both upstream and in parallel with PGRP-LC in the imd pathway, and is required for infection-dependent activation of melanization in Drosophila. A role for PGRP-LE in the epithelial induction of antimicrobial peptides is also suggested.

Drosophila: the genetics of innate immune recognition and response.

Because of the evolutionary conservation of innate mechanisms of host defense, Drosophila has emerged as an ideal animal in which to study the genetic control of immune recognition and responses. The discovery that the Toll pathway is required for defense against fungal infection in Drosophila was pivotal in studies of both mammalian and Drosophila immunity. Subsequent genetic screens in Drosophila to isolate additional mutants unable to induce humoral responses to infection have identified and ordered the function of components of two signaling cascades, the Toll and Imd pathways, that activate responses to infection. Drosophila blood cells also contribute to host defense through phagocytosis and signaling, and may carry out a form of self-nonself recognition that is independent of microbial pattern recognition. Recent work suggests that Drosophila will be a useful model for dissecting virulence mechanisms of several medically important pathogens.

Drosophila melanogaster innate immunity: an emerging role for peptidoglycan recognition proteins in bacteria detection.

Over the past years, parallel studies conducted in mammals and flies have emphasized the existence of common mechanisms regulating the vertebrate and invertebrate innate immune systems. This culminated in the discovery of the central role of the Toll pathway in Drosophila immunity and in the implication of Toll-like receptors (TLRs)/interleukin-1(IL-1) in the mammalian innate immune response. In spite of clear similarities, such as shared intracellular pathway components, important divergences are expected between the two groups, whose last common ancestor lived more than half a billion years ago. The most obvious discrepancies lie in the mode of activation of the signalling receptors by microorganisms. In mammals, TLRs are part of protein complexes which directly recognize microbe-associated patterns, whereas Drosophila Toll functions like a classical cytokine receptor rather than a pattern recognition receptor. Recent studies demonstrate that members of the evolutionarily conserved peptidoglycan recognition protein family play an essential role in microbial sensing during immune response of Drosophila.

TLR2 is expressed on activated T cells as a costimulatory receptor.

Toll is the founder of a group of pattern recognition receptors that play a critical role in the innate immunity in Drosophila. At least 10 distinct Toll-like receptors (TLRs), recognizing pathogen-associated molecular patterns, have now been identified in humans. Most investigations on TLRs have focused on cells of the innate system. We report here that naïve human T cells expressed high levels of cell-surface TLR2 after activation by anti-T cell receptor antibody and IFN-alpha. Activated cells produced elevated levels of cytokines in response to the TLR2 ligand, bacterial lipopeptide. Furthermore, CD4(+)CD45RO(+) memory T cells from peripheral blood constitutively expressed TLR2 and produced IFN-gamma in response to bacterial lipopeptide, which also markedly enhanced the proliferation and IFN-gamma production by CD45RO(+) T cells in the presence of IL-2 or IL-15. Thus, TLR2 serves as a costimulatory receptor for antigen-specific T cell development and participates in the maintenance of T cell memory. This suggests that pathogens, via their pathogen-associated molecular patterns, may contribute directly to the perpetuation and activation of long-term T cell memory in both antigen-dependent and independent manner.

Activation of the innate immunity in Drosophila by endogenous chromosomal DNA that escaped apoptotic degradation.

Apoptotic cell death is accompanied by degradation of chromosomal DNA. Here, we established in Drosophila a null mutation in the gene for inhibitor of caspase-activated DNase (ICAD) by P-element insertion. We also identified a loss-of-function mutant in Drosophila for DNase II-like acid DNase. The flies deficient in the ICAD gene did not express CAD, and did not undergo apoptotic DNA fragmentation during embryogenesis and oogenesis. In contrast, the deficiency of DNase II enhanced the apoptotic DNA fragmentation in the embryos and ovary, but paradoxically, the mutant flies accumulated a large amount of DNA, particularly in the ovary. This accumulation of DNA in the DNase II mutants caused the constitutive expression of the antibacterial genes for diptericin and attacin, which are usually activated during bacterial infection. The expression of these genes was further enhanced in flies lacking both dICAD and DNase II. These results indicated that CAD and DNase II work independently to degrade chromosomal DNA during apoptosis, and if the DNA is left undigested, it can activate the innate immunity in Drosophila.

Drosophila immunity: genes on the third chromosome required for the response to bacterial infection.

We have screened the third chromosome of Drosophila melanogaster for mutations that prevent the normal immune response. We identified mutant lines on the basis of their failure to induce transcription of an antibacterial peptide gene in response to infection or their failure to form melanized clots at the site of wounding. These mutations define 14 genes [immune response deficient (ird) genes] that have distinct roles in the immune response. We have identified the molecular basis of several ird phenotypes. Two genes, scribble and kurtz/modulo, affect the cellular organization of the fat body, the tissue responsible for antimicrobial peptide production. Two ird genes encode components of the signaling pathways that mediate responses to bacterial infection, a Drosophila gene encoding a homolog of I kappa B kinase (DmIkk beta) and Relish, a Rel-family transcription factor. These genetic studies should provide a basis for a comprehensive understanding of the genetic control of immune responses in Drosophila.

The GATA factor Serpent is required for the onset of the humoral immune response in Drosophila embryos.

Innate immunity in Drosophila is characterized by the inducible expression of antimicrobial peptides. We have investigated the development and regulation of immune responsiveness in Drosophila embryos after infection. Immune competence, as monitored by the induction of Cecropin A1-lacZ constructs, was observed first in the embryonic yolk. This observation suggests that the yolk plays an important role in the humoral immune response of the developing embryo by synthesizing antimicrobial peptides. Around midembryogenesis, the response in the yolk was diminished. Simultaneously, Cecropin expression became inducible in a large number of cells in the epidermis, demonstrating that late-stage embryos can synthesize their own antibiotics in the epidermis. This production likely serves to provide the hatching larva with an active antimicrobial barrier and protection against systemic infections. Cecropin expression in the yolk required the presence of a GATA site in the promoter as well as the involvement of the GATA-binding transcription factor Serpent (dGATAb). In contrast, neither the GATA site nor Serpent were necessary for Cecropin expression in the epidermis. Thus, the inducible immune responses in the yolk and in the epidermis can be uncoupled and call for distinct sets of transcription factors. Our data suggest that Serpent is involved in the distinction between a systemic response in the yolk/fat body and a local immune response in epithelial cells. In addition, the present study shows that signal transduction pathways controlling innate and epithelial defense reactions can be dissected genetically in Drosophila embryos.

Nitric Oxide Involvement in Drosophila Immunity

The augmented production of nitric oxide (NO) was observed during the hemocyte-mediated melanotic encapsulation responses of Drosophila melanogaster and D. teissieri. When introduced into the hemocoel of D. melanogaster larvae, NO activated the gene encoding the antimicrobial peptide Diptericin. These observations, together with previous studies documenting the production of superoxide anion (O•−2) and H2O2 in immune-challenged Drosophila, provide evidence that reactive intermediates of both oxygen (ROI) and nitrogen (RNI) constitute a part of the cytotoxic arsenal employed by Drosophila in defense against both microbial pathogens and eukaryotic parasites. These ROI and RNI appear to represent an evolutionarily conserved innate immune response that is mediated by regulatory proteins that are homologous to those of mammalian species.

Relish, a Central Factor in the Control of Humoral but Not Cellular Immunity in Drosophila

The NF-κB-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.

Toll and immunity in Drosophila

Toll and immunity in Drosophila

Toll and immunity in Drosophila

In Drosophila, the Toll signaling pathway regulates the antifungal host defense. In this immune response, when the ligand Spaetzle (Spz) binds to the Toll receptor, the antimicrobial peptide drosomycin is up-regulated. Levashina et al. now add Spn43Ac, a member of the serpin family of serine protease inhibitors, to the Toll cascade. When Spn43Ac is eliminated, Spz is continuously cleaved, which leads to the constitutive activation of Toll signaling; Spn43Ac negatively regulates Toll. In bacterial infection, the lipopolysaccharide (LPS) component of the Gram-negative bacterial cell wall is sensed by the host cells, which triggers an immune response involving the Toll pathway. Toll has been reported to act as a pattern recognition receptor in mammals for LPS. Because the Spn43Ac is located upstream of Toll in Drosophila, Toll does not appear to be its pattern recognition signal. The actual pattern recognition signal in Drosophila is still unknown. Levashina, E.A., Langley, E., Green, C., Gubb, D., Ashburner, M., Hoffman, J.A., Reichart, J-M. (1999) Constitutive activation of Toll-mediated antifungal defense in serpin-deficient Drosophila. Science 285 : 1917-1919. [Abstract] [Full Text]

Toll and Immunity in Drosophila

Toll and Immunity in Drosophila

Serpent regulates Drosophila immunity genes in the larval fat body through an essential GATA motif.

Insects possess a powerful immune system, which in response to infection leads to a vast production of different antimicrobial peptides. The regulatory regions of many immunity genes contain a GATA motif in proximity to a kappaB motif. Upon infection, Rel proteins enter the nucleus and activate transcription of the immunity genes. High levels of Rel protein-mediated Cecropin A1 expression previously have been shown to require the GATA site along with the kappaB site. We provide evidence demonstrating that the GATA motif is needed for expression of the Cecropin A1 gene in larval fat body, but is dispensable in adult fat body. A nuclear DNA-binding activity interacts with the Cecropin A1 GATA motif with the same properties as the Drosophila GATA factor Serpent. The GATA-binding activity is recognized by Serpent-specific antibodies, demonstrating their identity. We show that Serpent is nuclear in larval fat body cells and haemocytes both before and after infection. After overexpression, Serpent increases Cecropin A1 transcription in a GATA-dependent manner. We propose that Serpent plays a key role in tissue-specific expression of immunity genes, by priming them for inducible activation by Rel proteins in response to infection.

Interaction and Specificity of Rel-related Proteins in Regulating Drosophila Immunity Gene Expression

NF-kappaB/Rel family proteins regulate genes that are critical for many cellular processes including apoptosis, inflammation, immune response, and development. NF-kappaB/Rel proteins function as homodimers or heterodimers, which recognize specific DNA sequences within target promoters. We examined the activity of different Drosophila Rel-related proteins in modulating Drosophila immunity genes by expressing the Rel proteins in stably transfected cell lines. We also compared how different combinations of these transcriptional regulators control the activity of various immunity genes. The results show that Rel proteins are directly involved in regulating the Drosophila antimicrobial response. Furthermore, the drosomycin and defensin expression is best induced by the Relish/Dif and the Relish/Dorsal heterodimers, respectively, whereas the attacin activity can be efficiently up-regulated by the Relish homodimer and heterodimers. These results illustrate how the formation of Rel protein dimers differentially regulate target gene expression.