The Imd pathway-mediated immune response in Drosophila

Kohtaamme elinympäristössämme päivittäin monenlaisia mikrobeja. Vaikka useimmat niistä ovat meille haitattomia tai jopa hyödyllisiä, osa voi kuitenkin aiheuttaa sairauksia. Nisäkkäillä, joihin ihminenkin kuuluu, kyky puolustautua mikrobeja vastaan perustuu sekä synnynnäiseen että hankittuun immuniteettiin. Synnynnäinen immuniteetti on välttämätön niin infektioiden ehkäisyssä kuin hankitun immuunivasteen kehityksessä ja säätelyssä. Se perustuu perimän koodaamien proteiinien kykyyn tunnistaa erilaisia mikrobien pintarakenteita ja viestiä tästä soluille signalointireittien välityksellä. Eräs näistä signalointireiteistä, TNFR -signalointireitti, ja sen käynnistämä sytokiinieritys, ovat tarkkaan säädeltyjä ja välttämättömiä normaalille immuunivasteelle. Signaloinnin mekanismeja ja niiden säätelyä ei kuitenkaan vielä täysin tunneta. Synnynnäisen immuniteetin signalointireitit ovat säilyneet hyvin evoluutiossa hyönteisistä ihmisiin. Siksi niiden toimintaa ja säätelyä tutkittaessa voidaan käyttää mallina kodeissakin biojäteastian liepeillä usein tavattavaa banaanikärpästä (Drosophila melanogaster). Banaanikärpäsen Imd-signalointireitti muistuttaa nisäkkäiden TNFR-signalointireittiä. Tässä tutkimusprojektissa pyrimme RNA-häirintää (RNAi) ja muita molekyylibiologian menetelmiä hyödyntäen tunnistamaan Imd-signalointireittiin kuuluvia ja sen säätelyyn osallistuvia proteiineja. Tutkimuksessa löydettiin kolme uutta säätelijää, Tab2, Iap2 ja Pirk. Näiden proteiinien toimintaa ja merkitystä banaanikärpäsen immuunivasteelle selvitettiin tarkemmin. Osoitimme kärpässolumallia apuna käyttäen, että Tab2 ja Iap2 ovat välttämättömiä Imd-signaloinnille. Lisäksi havaitsimme, että banaanikärpäset, joilta Iap2 on poistettu, ovat herkkiä Gram-negatiivisten bakteerien aiheuttamille infektioille. Pirk puolestaan on aiemmin tuntematon proteiini, jonka osoitimme hillitsevän Imd signalointia sekä solumallissa että elävissä kärpäsissä. Pirkin vaikutus oli niin tehokas, että geenin yli-ilmentäminen kärpäsissä riitti herkistämään ne Gram-negatiivisten bakteerien aiheuttamille infektioille. Tehty tutkimus osoittaa, että Imd signalointi on tarkkaan säädeltyä ja aiempaa luultua monimutkaisempaa. Banaanikärpänen mallieläimenä tarjoaa mahdollisuuden tutkia tehokkaasti synnynnäisen immuniteetin säätelyä. Tab2:lla ja Iap2:lla on vastineensa myös nisäkkäissä, joten on mahdollista, että tutkimuksesta saadut tulokset tuovat uusia näkökulmia myös nisäkkäiden immuunisignaloinnin toiminnan selvittämiseen.Innate immunity is the first line of defense against microbes and it is indispensable in preventing infections as well as in the development and regulation of the adaptive immune system. Innate immunity is based on the ability of genome-encoded proteins to recognize and bind microbial surface structures, which is followed by the activation of the innate immune response via various cell signaling pathways. Tumor necrosis factor receptor (TNFR) signaling and cytokine release are strictly regulated and essential for a normal immune response. However, in certain diseases, like infections and autoimmune diseases, cytokines are produced in excess, which prolongs the inflammation and causes tissue damage. Clinical medicine is trying to prevent cytokine overproduction by suppressing TNFR signaling. However, this is challenging, since despite the extensive research carried out in this field in recent years, the mechanisms and regulation of TNFR signaling are not thoroughly understood. The pathways of innate immunity signaling are evolutionarily conserved from insects to humans. Unlike mammals, insects have no adaptive immunity, and hence they are completely dependent on their innate immune response. For these reasons the fruit fly (Drosophila melanogaster) is a suitable model organism for studying innate immunity. In Drosophila, the immune response to Gram-negative bacteria is mediated mainly via the Imd (immune deficiency) signaling pathway, whose intracellular parts resemble the mammalian TNFR signaling pathway. The aim of this research project was to identify and molecularly characterize the components of the Imd pathway and regulatory proteins using a large-scale RNA interference (RNAi) screen. The function and importance of three of the identified regulators, namely Tak1-associated binding protein 2 (Tab2), Inhibitor of apoptosis 2 (Iap2), and Poor Imd response upon knock-in (Pirk), were then further assessed. Using Drosophila S2 cells we showed that Tab2 is essential for both the early and sustained immune responses, while Iap2 mainly regulates the sustained immune response. In addition, we discovered that when Iap2 was removed from fruit flies by in vivo RNAi the flies became susceptible to Gram-negative bacterial infections. Pirk was a previously unknown protein that we demonstrated could suppress the Imd pathway activity both in S2 cells and in flies. The inhibitory action of Pirk was shown to be efficient enough to sensitize the flies to Gram-negative bacterial infections. We found that Pirk interacts with the receptor of the Imd pathway, PGRP-LC (Peptidoglycan recognition protein LC), and the downstream component IMD. However, the elucidation of the exact mechanism of Pirk action requires further studies. The present study demonstrates that Imd signaling is strictly regulated and more complex than was previously thought. Drosophila as a model organism provides tools to efficiently study innate immunity. In addition, the results gained from this research in flies can provide new perspectives and may help understand also the mechanisms of signaling in the mammalian innate immune system

Regulation of the Drosophila Imd pathway by signaling amyloids

Fruit flies elicit effective defense responses against numerous microbes. The responses against Gram-negative bacteria are mediated by the Imd pathway, an evolutionarily conserved NF-kappaB pathway recognizing meso-diaminopimelic acid (DAP)-type peptidoglycan from bacterial cell walls. Several reviews already provide a detailed view of ligand recognition and signal transduction during Imd signaling, but the formation and regulation of the signaling complex immediately downstream of the peptidoglycan-sensing receptors is still elusive. In this review, we focus on the formation of the Imd amyloidal signaling center and post-translational modifications in the assembly and disassembly of the Imd signaling complex

IIV-6 Inhibits NF-kappaB Responses in Drosophila

The host immune response and virus-encoded immune evasion proteins pose constant, mutual selective pressure on each other. Virally encoded immune evasion proteins also indicate which host pathways must be inhibited to allow for viral replication. Here, we show that IIV-6 is capable of inhibiting the two Drosophila NF-kappaB signaling pathways, Imd and Toll. Antimicrobial peptide (AMP) gene induction downstream of either pathway is suppressed when cells infected with IIV-6 are also stimulated with Toll or Imd ligands. We find that cleavage of both Imd and Relish, as well as Relish nuclear translocation, three key points in Imd signal transduction, occur in IIV-6 infected cells, indicating that the mechanism of viral inhibition is farther downstream, at the level of Relish promoter binding or transcriptional activation. Additionally, flies co-infected with both IIV-6 and the Gram-negative bacterium, Erwinia carotovora carotovora, succumb to infection more rapidly than flies singly infected with either the virus or the bacterium. These findings demonstrate how pre-existing infections can have a dramatic and negative effect on secondary infections, and establish a Drosophila model to study confection susceptibility

Functional Characterization of the Infection-Inducible Peptide Edin in Drosophila melanogaster

Drosophila is a well-established model organism for studying innate immunity because of its high resistance against microbial infections and lack of adaptive immunity. In addition, the immune signaling cascades found in Drosophila are evolutionarily conserved. Upon infection, activation of the immune signaling pathways, Toll and Imd, leads to the expression of multiple immune response genes, such as the antimicrobial peptides (AMPs). Previously, we identified an uncharacterized gene edin among the genes, which were strongly induced upon stimulation with Escherichia coli in Drosophila S2 cells. Edin has been associated with resistance against Listeria monocytogenes, but its role in Drosophila immunity remains elusive. In this study, we examined the role of Edin in the immune response of Drosophila both in vitro and in vivo. We report that edin expression is dependent on the Imd-pathway NF-κB transcription factor Relish and that it is expressed upon infection both in vitro and in vivo. Edin encodes a pro-protein, which is further processed in S2 cells. In our experiments, Edin did not bind microbes, nor did it possess antimicrobial activity to tested microbial strains in vitro or in vivo. Furthermore, edin RNAi did not significantly affect the expression of AMPs in vitro or in vivo. However, edin RNAi flies showed modestly impaired resistance to E. faecalis infection. We conclude that Edin has no potent antimicrobial properties but it appears to be important for E. faecalis infection via an uncharacterized mechanism. Further studies are still required to elucidate the exact role of Edin in the Drosophila immune response

IIV-6 Inhibits NF-κB Responses in Drosophila.

The host immune response and virus-encoded immune evasion proteins pose constant, mutual selective pressure on each other. Virally encoded immune evasion proteins also indicate which host pathways must be inhibited to allow for viral replication. Here, we show that IIV-6 is capable of inhibiting the two Drosophila NF-κB signaling pathways, Imd and Toll. Antimicrobial peptide (AMP) gene induction downstream of either pathway is suppressed when cells infected with IIV-6 are also stimulated with Toll or Imd ligands. We find that cleavage of both Imd and Relish, as well as Relish nuclear translocation, three key points in Imd signal transduction, occur in IIV-6 infected cells, indicating that the mechanism of viral inhibition is farther downstream, at the level of Relish promoter binding or transcriptional activation. Additionally, flies co-infected with both IIV-6 and the Gram-negative bacterium, Erwinia carotovora carotovora, succumb to infection more rapidly than flies singly infected with either the virus or the bacterium. These findings demonstrate how pre-existing infections can have a dramatic and negative effect on secondary infections, and establish a Drosophila model to study confection susceptibility

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

Inhibitor of apoptosis 2 and TAK1-binding protein are components of the Drosophila Imd pathway

The Imd signaling cascade, similar to the mammalian TNF-receptor pathway, controls antimicrobial peptide expression in Drosophila. We performed a large-scale RNAi screen to identify novel components of the Imd pathway in Drosophila S2 cells. In all, 6713 dsRNAs from an S2 cell-derived cDNA library were analyzed for their effect on Attacin promoter activity in response to Escherichia coli. We identified seven gene products required for the Attacin response in vitro, including two novel Imd pathway components: inhibitor of apoptosis 2 (Iap2) and transforming growth factor-activated kinase 1 (TAK1)-binding protein (TAB). Iap2 is required for antimicrobial peptide response also by the fat body in vivo. Both these factors function downstream of Imd. Neither TAB nor Iap2 is required for Relish cleavage, but may be involved in Relish nuclear localization in vitro, suggesting a novel mode of regulation of the Imd pathway. Our results show that an RNAi-based approach is suitable to identify genes in conserved signaling cascades

The Imd pathway-mediated immune response in Drosophila

Kohtaamme elinympäristössämme päivittäin monenlaisia mikrobeja. Vaikka useimmat niistä ovat meille haitattomia tai jopa hyödyllisiä, osa voi kuitenkin aiheuttaa sairauksia. Nisäkkäillä, joihin ihminenkin kuuluu, kyky puolustautua mikrobeja vastaan perustuu sekä synnynnäiseen että hankittuun immuniteettiin. Synnynnäinen immuniteetti on välttämätön niin infektioiden ehkäisyssä kuin hankitun immuunivasteen kehityksessä ja säätelyssä. Se perustuu perimän koodaamien proteiinien kykyyn tunnistaa erilaisia mikrobien pintarakenteita ja viestiä tästä soluille signalointireittien välityksellä. Eräs näistä signalointireiteistä, TNFR -signalointireitti, ja sen käynnistämä sytokiinieritys, ovat tarkkaan säädeltyjä ja välttämättömiä normaalille immuunivasteelle. Signaloinnin mekanismeja ja niiden säätelyä ei kuitenkaan vielä täysin tunneta. Synnynnäisen immuniteetin signalointireitit ovat säilyneet hyvin evoluutiossa hyönteisistä ihmisiin. Siksi niiden toimintaa ja säätelyä tutkittaessa voidaan käyttää mallina kodeissakin biojäteastian liepeillä usein tavattavaa banaanikärpästä (Drosophila melanogaster). Banaanikärpäsen Imd-signalointireitti muistuttaa nisäkkäiden TNFR-signalointireittiä. Tässä tutkimusprojektissa pyrimme RNA-häirintää (RNAi) ja muita molekyylibiologian menetelmiä hyödyntäen tunnistamaan Imd-signalointireittiin kuuluvia ja sen säätelyyn osallistuvia proteiineja. Tutkimuksessa löydettiin kolme uutta säätelijää, Tab2, Iap2 ja Pirk. Näiden proteiinien toimintaa ja merkitystä banaanikärpäsen immuunivasteelle selvitettiin tarkemmin. Osoitimme kärpässolumallia apuna käyttäen, että Tab2 ja Iap2 ovat välttämättömiä Imd-signaloinnille. Lisäksi havaitsimme, että banaanikärpäset, joilta Iap2 on poistettu, ovat herkkiä Gram-negatiivisten bakteerien aiheuttamille infektioille. Pirk puolestaan on aiemmin tuntematon proteiini, jonka osoitimme hillitsevän Imd signalointia sekä solumallissa että elävissä kärpäsissä. Pirkin vaikutus oli niin tehokas, että geenin yli-ilmentäminen kärpäsissä riitti herkistämään ne Gram-negatiivisten bakteerien aiheuttamille infektioille. Tehty tutkimus osoittaa, että Imd signalointi on tarkkaan säädeltyä ja aiempaa luultua monimutkaisempaa. Banaanikärpänen mallieläimenä tarjoaa mahdollisuuden tutkia tehokkaasti synnynnäisen immuniteetin säätelyä. Tab2:lla ja Iap2:lla on vastineensa myös nisäkkäissä, joten on mahdollista, että tutkimuksesta saadut tulokset tuovat uusia näkökulmia myös nisäkkäiden immuunisignaloinnin toiminnan selvittämiseen.Innate immunity is the first line of defense against microbes and it is indispensable in preventing infections as well as in the development and regulation of the adaptive immune system. Innate immunity is based on the ability of genome-encoded proteins to recognize and bind microbial surface structures, which is followed by the activation of the innate immune response via various cell signaling pathways. Tumor necrosis factor receptor (TNFR) signaling and cytokine release are strictly regulated and essential for a normal immune response. However, in certain diseases, like infections and autoimmune diseases, cytokines are produced in excess, which prolongs the inflammation and causes tissue damage. Clinical medicine is trying to prevent cytokine overproduction by suppressing TNFR signaling. However, this is challenging, since despite the extensive research carried out in this field in recent years, the mechanisms and regulation of TNFR signaling are not thoroughly understood. The pathways of innate immunity signaling are evolutionarily conserved from insects to humans. Unlike mammals, insects have no adaptive immunity, and hence they are completely dependent on their innate immune response. For these reasons the fruit fly (Drosophila melanogaster) is a suitable model organism for studying innate immunity. In Drosophila, the immune response to Gram-negative bacteria is mediated mainly via the Imd (immune deficiency) signaling pathway, whose intracellular parts resemble the mammalian TNFR signaling pathway. The aim of this research project was to identify and molecularly characterize the components of the Imd pathway and regulatory proteins using a large-scale RNA interference (RNAi) screen. The function and importance of three of the identified regulators, namely Tak1-associated binding protein 2 (Tab2), Inhibitor of apoptosis 2 (Iap2), and Poor Imd response upon knock-in (Pirk), were then further assessed. Using Drosophila S2 cells we showed that Tab2 is essential for both the early and sustained immune responses, while Iap2 mainly regulates the sustained immune response. In addition, we discovered that when Iap2 was removed from fruit flies by in vivo RNAi the flies became susceptible to Gram-negative bacterial infections. Pirk was a previously unknown protein that we demonstrated could suppress the Imd pathway activity both in S2 cells and in flies. The inhibitory action of Pirk was shown to be efficient enough to sensitize the flies to Gram-negative bacterial infections. We found that Pirk interacts with the receptor of the Imd pathway, PGRP-LC (Peptidoglycan recognition protein LC), and the downstream component IMD. However, the elucidation of the exact mechanism of Pirk action requires further studies. The present study demonstrates that Imd signaling is strictly regulated and more complex than was previously thought. Drosophila as a model organism provides tools to efficiently study innate immunity. In addition, the results gained from this research in flies can provide new perspectives and may help understand also the mechanisms of signaling in the mammalian innate immune system

UnZIPping mechanisms of effector-triggered immunity in animals

The mechanisms by which epithelial cells distinguish pathogens from commensal microbes have long puzzled us. Now, McEwan et al. (2012) and Dunbar et al. (2012), in this issue of Cell Host and Microbe, demonstrate that in C. elegans, microbial toxin-induced inhibition of host cellular functions, especially blockade of protein translation, activates the effector-triggered immune response dependent on the transcription factor ZIP-2