Florentina Rus

Hormonal regulation of the humoral innate immune response in Drosophila melanogaster.

SUMMARY Juvenile hormone (JH) and 20-hydroxy-ecdysone (20E) are highly versatile hormones, coordinating development, growth, reproduction and aging in insects. Pulses of 20E provide key signals for initiating developmental and physiological transitions, while JH promotes or inhibits these signals in a stage-specific manner. Previous evidence suggests that JH and 20E might modulate innate immunity, but whether and how these hormones interact to regulate the immune response remains unclear. Here we show that JH and 20E have antagonistic effects on the induction of antimicrobial peptide (AMP) genes in Drosophila melanogaster. 20E pretreatment of Schneider S2* cells promoted the robust induction of AMP genes, following immune stimulation. On the other hand, JH III, and its synthetic analogs (JHa) methoprene and pyriproxyfen, strongly interfered with this 20E-dependent immune potentiation, although these hormones did not inhibit other 20E-induced cellular changes. Similarly, in vivo analyses in adult flies confirmed that JH is a hormonal immuno-suppressor. RNA silencing of either partner of the ecdysone receptor heterodimer (EcR or Usp) in S2* cells prevented the 20E-induced immune potentiation. In contrast, silencing methoprene-tolerant (Met), a candidate JH receptor, did not impair immuno-suppression by JH III and JHa, indicating that in this context MET is not a necessary JH receptor. Our results suggest that 20E and JH play major roles in the regulation of gene expression in response to immune challenge.

Rudra interrupts receptor signaling complexes to negatively regulate the IMD pathway

Insects rely primarily on innate immune responses to fight pathogens. In Drosophila, antimicrobial peptides are key contributors to host defense. Antimicrobial peptide gene expression is regulated by the IMD and Toll pathways. Bacterial peptidoglycans trigger these pathways, through recognition by peptidoglycan recognition proteins (PGRPs). DAP-type peptidoglycan triggers the IMD pathway via PGRP-LC and PGRP-LE, while lysine-type peptidoglycan is an agonist for the Toll pathway through PGRP-SA and PGRP-SD. Recent work has shown that the intensity and duration of the immune responses initiating with these receptors is tightly regulated at multiple levels, by a series of negative regulators. Through two-hybrid screening with PGRP-LC, we identified Rudra, a new regulator of the IMD pathway, and demonstrate that it is a critical feedback inhibitor of peptidoglycan receptor signaling. Following stimulation of the IMD pathway, rudra expression was rapidly induced. In cells, RNAi targeting of rudra caused a marked up-regulation of antimicrobial peptide gene expression. rudra mutant flies also hyper-activated antimicrobial peptide genes and were more resistant to infection with the insect pathogen Erwinia carotovora carotovora. Molecularly, Rudra was found to bind and interfere with both PGRP-LC and PGRP-LE, disrupting their signaling complex. These results show that Rudra is a critical component in a negative feedback loop, whereby immune-induced gene expression rapidly produces a potent inhibitor that binds and inhibits pattern recognition receptors.

Serine/threonine acetylation of TGFβ-activated kinase (TAK1) by Yersinia pestis YopJ inhibits innate immune signaling

The Gram-negative bacteria Yersinia pestis, causative agent of plague, is extremely virulent. One mechanism contributing to Y. pestis virulence is the presence of a type-three secretion system, which injects effector proteins, Yops, directly into immune cells of the infected host. One of these Yop proteins, YopJ, is proapoptotic and inhibits mammalian NF-κB and MAP-kinase signal transduction pathways. Although the molecular mechanism remained elusive for some time, recent work has shown that YopJ acts as a serine/threonine acetyl-transferase targeting MAP2 kinases. Using Drosophila as a model system, we find that YopJ inhibits one innate immune NF-κB signaling pathway (IMD) but not the other (Toll). In fact, we show YopJ mediated serine/threonine acetylation and inhibition of dTAK1, the critical MAP3 kinase in the IMD pathway. Acetylation of critical serine/threonine residues in the activation loop of Drosophila TAK1 blocks phosphorylation of the protein and subsequent kinase activation. In addition, studies in mammalian cells show similar modification and inhibition of hTAK1. These data present evidence that TAK1 is a target for YopJ-mediated inhibition.

Ecdysone triggered PGRP‐LC expression controls Drosophila innate immunity

Throughout the animal kingdom, steroid hormones have been implicated in the defense against microbial infection, but how these systemic signals control immunity is unclear. Here, we show that the steroid hormone ecdysone controls the expression of the pattern recognition receptor PGRP‐LC in Drosophila, thereby tightly regulating innate immune recognition and defense against bacterial infection. We identify a group of steroid‐regulated transcription factors as well as two GATA transcription factors that act as repressors and activators of the immune response and are required for the proper hormonal control of PGRP‐LC expression. Together, our results demonstrate that Drosophila use complex mechanisms to modulate innate immune responses, and identify a transcriptional hierarchy that integrates steroid signalling and immunity in animals.

The Caspase-8 Homolog Dredd Cleaves Imd and Relish but Is Not Inhibited by p35

Background: During the Drosophila immune response, both Imd and Relish are cleaved in a manner dependent on the caspase-8 homolog Dredd. Results: Dredd cleaves Imd and Relish, but not the caspase inhibitor p35, without interdomain autoprocessing. Conclusion: Imd and Relish are direct substrates for full-length Dredd. Significance: Dredd is similar to some mammalian initiator caspases, which can function without interdomain cleavage. In Drosophila, the Imd pathway is activated by diaminopimelic acid-type peptidoglycan and triggers the humoral innate immune response, including the robust induction of antimicrobial peptide gene expression. Imd and Relish, two essential components of this pathway, are both endoproteolytically cleaved upon immune stimulation. Genetic analyses have shown that these cleavage events are dependent on the caspase-8 like Dredd, suggesting that Imd and Relish are direct substrates of Dredd. Among the seven Drosophila caspases, we find that Dredd uniquely promotes Imd and Relish processing, and purified recombinant Dredd cleaves Imd and Relish in vitro. In addition, interdomain cleavage of Dredd is not required for Imd or Relish processing and is not observed during immune stimulation. Baculovirus p35, a suicide substrate of executioner caspases, is not cleaved by purified Dredd in vitro. Consistent with this biochemistry but contrary to earlier reports, p35 does not interfere with Imd signaling in S2* cells or in vivo.

miR-718 represses proinflammatory cytokine production through targeting phosphatase and tensin homolog (PTEN).

Bacterial sepsis involves a complex interaction between the host immune response and bacterial LPS. LPS binds Toll-like receptor (TLR) 4, which leads to the release of proinflammatory cytokines that are essential for a potent innate immune response against pathogens. The innate immune system is tightly regulated, as excessive inflammation can lead to organ failure and death. MicroRNAs have recently emerged as important regulators of the innate immune system. Here we determined the function of miR-718, which is conserved across mammals and overlaps with the 5′ UTR of the interleukin 1 receptor-associated kinase (IRAK1) gene. As IRAK1 is a key component of innate immune signaling pathways that are downstream of most TLRs, we hypothesized that miR-718 helps regulate the innate immune response. Activation of TLR4, but not TLR3, induced the expression of miR-718 in macrophages. miR-718 expression was also induced in the spleens of mice upon LPS injection. miR-718 modulates PI3K/Akt signaling by directly down-regulating phosphatase and tensin homolog (PTEN), thereby promoting phosphorylation of Akt, which leads to a decrease in proinflammatory cytokine production. Phosphorylated Akt induces let-7e expression, which, in turn, down-regulates TLR4 and further diminishes TLR4-mediated proinflammatory signals. Decreased miR-718 expression is associated with bacterial burden during Neisseria gonorrhoeae infection and alters the infection dynamics of N. gonorrhoeae in vitro. Furthermore, miR-718 regulates the induction of LPS tolerance in macrophages. We propose a role for miR-718 in controlling TLR4 signaling and inflammatory cytokine signaling through a negative feedback regulation loop involving down-regulation of TLR4, IRAK1, and NF-κB.

Dermatophagoides pteronyssinus Major Allergen 1 Activates the Innate Immune Response of the Fruit Fly Drosophila melanogaster

Some allergens with relevant protease activity have the potential to directly interact with host structures. It remains to be elucidated whether this activity is relevant for developing their allergenic properties. The major goal of this study was to elucidate whether allergens with a strong protease activity directly interact with modules of the innate immune system, thereby inducing an immune response. We chose Drosophila melanogaster for our experiments to prevent the results from being influenced by the adaptive immune system and used the armamentarium of methods available for the fly to study the underlying mechanisms. We show that Dermatophagoides pteronyssinus major allergen 1 (Der p 1), the major allergen of the house dust mite, efficiently activates various facets of the Drosophila innate-immune system, including both epithelial and systemic responses. These responses depend on the immune deficiency (IMD) pathway via activation of the NF-κB transcription factor Relish. In addition, the major pathogen associated molecular pattern recognizing receptor of the IMD pathway, peptidoglycan recognition protein–LC, was necessary for this response. We showed that Der p 1, which has cysteine protease activity, cleaves the ectodomain of peptidoglycan recognition protein–LC and, thus, activates the IMD pathway to induce a profound immune response. We conclude that the innate immune response to this allergen-mediated proteolytic cleavage represents an ancient type of danger signaling that may be highly relevant for the primary allergenicity of compounds such as Der p 1.

Dehydration triggers ecdysone-mediated recognition-protein priming and elevated anti-bacterial immune responses in Drosophila Malpighian tubule renal cells

BackgroundDrosophila is a powerful model for the study of factors modulating innate immunity. This study examines the effect of water-loss dehydration on innate immune responsiveness in the Drosophila renal system (Malpighian tubules; MTs), and how this leads to elevated host defense and contributes to immunosenescence.ResultsA short period of desiccation-elevated peptidoglycan recognition protein-LC (PGRP-LC) expression in MTs, increased antimicrobial peptide (AMP) gene induction, and protected animals from bacterial infection. We show that desiccation increased ecdysone synthesis in MTs, while inhibition of ecdysone synthesis or ecdysone receptor expression, specifically within MTs, prevented induction of PGRP-LC and reduced protection from bacterial infection. Additionally, aged flies are constitutively water-stressed and have elevated levels of ecdysone and PGRP-LC. Conversely, adults aged at high relative humidity show less water loss and have reduced expression of PGRP-LC and AMPs.ConclusionsThe Drosophila renal system is an important contributor to host defense and can modulate immune responses in an organ autonomous manner, responding to environmental changes such as desiccation. Desiccation primes immune responsiveness by elevating PGRP-LC expression specifically in MTs. In response to desiccation, ecdysone is produced in MTs and acts in a paracrine fashion to increase PGRP-LC expression, immune responsiveness, and improve host defense. This activity of the renal system may contribute to the immunosenescence observed in Drosophila.

Characterization of poxvirus-encoded proteins that regulate innate immune signaling pathways

Innate immune recognition of pathogens is critical to the prompt control of infections, permitting the host to survive to develop long-term immunity via an adaptive immune response. Poxviruses encode a family of proteins that inhibit signaling by Toll-like receptors to their downstream signaling components, severely limiting nuclear translocation of transcription factors such as IRF3 and NF-κB and thereby decreasing production of host interferons and cytokines. We describe bioinformatics techniques for identifying candidate poxviral inhibitors of the innate immune response based on similarity to the family of proteins that includes A52, A46, and N1. Robust luciferase assays can determine whether a given poxviral gene affects innate immune signaling, and in combination with other approaches can identify the cellular targets of poxviral innate immune evasion genes. Because apoptosis is an innate immune response of the cell to viral infection, assays for identifying poxviral genes that inhibit apoptosis can also be employed. Novel poxviral innate immune inhibitors are being identified via several approaches and these techniques promise to identify further complexities in the way that poxviruses interact with the host innate immune system.

Additional file 6: of Dehydration triggers ecdysone-mediated recognition-protein priming and elevated anti-bacterial immune responses in Drosophila Malpighian tubule renal cells

Figure S6. Relative humidity (RH) and temperature recorded from demography chambers under different humidity conditions. Spikes indicate when chambers were opened to access cages and show rapid homeostasis of the humidity control system. Blue tracings show realized RH, black tracing represents temperature. (PDF 3450 kb)