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

Banaanikärpäsen immuunivasteen toiminnallinen tutkimus

Ihmisen immuunijärjestelmä muodostuu synnynnäisestä ja hankitusta immuniteetistä. Synnynnäinen immuniteetti on tärkeä osa meidän immuunivastettamme, sillä se toimii ensilinjan puolustusjärjestelmänä taudinaiheuttajia vastaan. Synnynnäisen immuniteetin mekanismit perustuvat genomiin koodattujen reseptorimolekyylien kykyyn tunnistaa taudinaiheuttajien pinnalla olevia yleisiä rakenteita. Taudinaiheuttajien tunnistus aktivoi nopeasti synnynnäisen immuniteetin mekanismeja, joihin kuuluvat muun muassa immuunisolujen harjoittama mikro-organismien fagosytoosi, signalointireittien aktivoitumien sekä tulehdusvälittäjäaineiden ja muiden immuunivasteeseen osallistuvien molekyylien tuottaminen ja erittäminen. Toisin kuin synnynnäinen immuniteetti hankittu immuniteetti, johon kuuluvat esimerkiksi vasta-aineiden tuotto ja immunologinen muisti, käynnistyy vasta myöhemmin kehityksen aikana ja on synnynnäistä immuniteettia hitaampi reagoimaan taudinaiheuttajiin. Koska synnynnäinen immuniteetti on erityisen tärkeä yksilön elinkaaren alkuvaiheessa sekä myös infektion aikaisessa vaiheessa, synnynnäisen immuniteetin mekanismien tutkiminen on hyvin perusteltua. Banaanikärpänen, latinankieliseltä nimeltään Drosophila melanogaster, on osoittautunut erinomaiseksi mallieläimeksi synnynnäisen immuniteetin tutkimiseen, koska siltä puuttuu täysin hankittu immuniteetti, ja koska synnynnäisen immuniteetin mekanismit ovat hyvin säilyneet evoluutiossa. Tämän tutkimuksen tarkoituksena oli määrittää valikoitujen, aikaisemmin laajamittaisisten geneettisten seulontojen avulla tunnistettujen geenien toimintaa banaanikärpäsen immuunivasteessa. Erityisesti keskityttiin tutkimaan fagosytoosiin liittyvän geenin 14-3-3ζ:n (14-3-3 zeeta) ja infektiossa indusoituvan geenin edinin (elevated during infection) merkitystä banaanikärpäsen immuunivasteelle. Käyttämällä RNA-häirintään perustuvaa kudosspesifistä geeninhiljennystä pystyimme osoittamaan, että kofiliinin säätelijänä toimiva 14 3-3ζ on tärkeä tekijä bakteereiden fagosytoosissa, ja että sen toiminta on evoluutiossa hyvin säilynyt. Tutkimuksemme osoitti myös, että fagosytoosi on olennainen osa immuunipuolustusta bakteeri-infektion yhteydessä, koska 14-3-3ζ:n hiljentäminen herkisti banaanikärpäset bakteeri-infektiolle. Osoitimme lisäksi, että sekä bakteeri-infektio että Leptopilina boulardi -pistiäisen aiheuttama loisinfektio sai aikaan edinin ilmentymisen banaanikärpäsessä. Laajamittaisten in vitro ja in vivo kokeiden perusteella pystyimme kuitenkin osoittamaan Edinillä olevan vain vähäinen tehtävä bakteeri-infektion yhteydessä. Sen sijaan tutkimuksemme perusteella Edin säätelee banaanikärpäsen verisoluja pistiäisinfektion yhteydessä. Tämä tutkimus osoitti 14-3-3ζ:n ja Edinin olevan uusia banaanikärpäsen immuunivasteeseen osallistuvia tekijöitä, jotka toimivat erityisesti soluvälitteisen immuniteetin säätelijöinä. Tutkimustuloksemme tuovat lisätietoa synnynnäisen immuunivasteen toiminnasta sekä soluvälitteisen ja humoraalisen immuunivasteen vuorovaikutuksesta. Tästä tutkimuksesta saatu uusi tieto voi tulevaisuudessa auttaa ymmärtämään paremmin myös ihmisen synnynnäistä immuunivastetta johtuen synnynnäisen immuniteetin mekanismien samankaltaisuudesta banaanikärpäsen ja ihmisen välillä.The human immune system constitutes of the innate and the adaptive immunity. Innate immune responses are an important part of our immune defense as they provide the first line of defense against pathogens. Innate immunity is based on the ability of genome-encoded receptors to recognize common features on the surface of pathogens. This leads to rapid responses in the host, including the phagocytosis of microorganisms by immune cells, the activation of signaling cascades and the production and secretion of cytokines and other effector molecules. In contrast, the mechanisms of the adaptive immunity, which include, for example the production of antibodies and immunological memory, arise later on in development and are slower to react to an immune challenge. Because of the crucial importance of the innate immunity in the early stages of an individual’s life cycle, and also in the early stages of an infection, a profound understanding of the regulators and mediators involved in the innate immune responses is needed. The fruit fly, Drosophila melanogaster, is an excellent model organism for studying the mechanisms of innate immunity, because it lacks adaptive immune responses and because the mechanisms of innate immunity are evolutionarily conserved. This study focused on the functional characterization of selected Drosophila genes that had been previously identified as novel immunity-related genes in large-scale in vitro screens. Especially, the mechanisms of function of the phagocytosis-related gene 14-3-3ζ (14-3-3 zeta) and the infection-inducible gene edin (elevated during infection) were studied. Using tissue-specific RNA-interference (RNAi) mediated gene silencing we were able to show that the cofilin regulator 14-3-3ζ is an evolutionarily conserved protein required for the phagocytosis of bacteria both in Drosophila larvae and in adult flies. Our study also showed that phagocytosis is required for an efficient immune response against bacteria, because silencing 14-3-3ζ with RNAi sensitized the flies to bacterial infections. Additionally, we showed that the expression of edin is upregulated in response to both a bacterial infection and wasp parasitism by Leptopilina boulardi, although after both in vitro and in vivo analyses Edin proved to have only a minor role in the host defense against bacteria. Instead, we were able to demonstrate that Edin is an important determinant in the defense against wasp parasitism, where it acts as a regulator of Drosophila blood cells. The present study identified 14-3-3ζ and Edin as novel mediators of the Drosophila host defense, where they were found to take part especially in the cellular immune response. Our findings add more clarity to the mechanisms of innate immunity and provide more evidence of an active interaction between the humoral and cellular arms of the immune defense. In the future, the knowledge obtained from this study may serve as a novel starting point for human research due to the ancient origin of the mechanisms of innate immunity and their evolutionary conservation from fly to man

Banaanikärpäsen immuunivasteen toiminnallinen tutkimus

Ihmisen immuunijärjestelmä muodostuu synnynnäisestä ja hankitusta immuniteetistä. Synnynnäinen immuniteetti on tärkeä osa meidän immuunivastettamme, sillä se toimii ensilinjan puolustusjärjestelmänä taudinaiheuttajia vastaan. Synnynnäisen immuniteetin mekanismit perustuvat genomiin koodattujen reseptorimolekyylien kykyyn tunnistaa taudinaiheuttajien pinnalla olevia yleisiä rakenteita. Taudinaiheuttajien tunnistus aktivoi nopeasti synnynnäisen immuniteetin mekanismeja, joihin kuuluvat muun muassa immuunisolujen harjoittama mikro-organismien fagosytoosi, signalointireittien aktivoitumien sekä tulehdusvälittäjäaineiden ja muiden immuunivasteeseen osallistuvien molekyylien tuottaminen ja erittäminen. Toisin kuin synnynnäinen immuniteetti hankittu immuniteetti, johon kuuluvat esimerkiksi vasta-aineiden tuotto ja immunologinen muisti, käynnistyy vasta myöhemmin kehityksen aikana ja on synnynnäistä immuniteettia hitaampi reagoimaan taudinaiheuttajiin. Koska synnynnäinen immuniteetti on erityisen tärkeä yksilön elinkaaren alkuvaiheessa sekä myös infektion aikaisessa vaiheessa, synnynnäisen immuniteetin mekanismien tutkiminen on hyvin perusteltua. Banaanikärpänen, latinankieliseltä nimeltään Drosophila melanogaster, on osoittautunut erinomaiseksi mallieläimeksi synnynnäisen immuniteetin tutkimiseen, koska siltä puuttuu täysin hankittu immuniteetti, ja koska synnynnäisen immuniteetin mekanismit ovat hyvin säilyneet evoluutiossa. Tämän tutkimuksen tarkoituksena oli määrittää valikoitujen, aikaisemmin laajamittaisisten geneettisten seulontojen avulla tunnistettujen geenien toimintaa banaanikärpäsen immuunivasteessa. Erityisesti keskityttiin tutkimaan fagosytoosiin liittyvän geenin 14-3-3ζ:n (14-3-3 zeeta) ja infektiossa indusoituvan geenin edinin (elevated during infection) merkitystä banaanikärpäsen immuunivasteelle. Käyttämällä RNA-häirintään perustuvaa kudosspesifistä geeninhiljennystä pystyimme osoittamaan, että kofiliinin säätelijänä toimiva 14 3-3ζ on tärkeä tekijä bakteereiden fagosytoosissa, ja että sen toiminta on evoluutiossa hyvin säilynyt. Tutkimuksemme osoitti myös, että fagosytoosi on olennainen osa immuunipuolustusta bakteeri-infektion yhteydessä, koska 14-3-3ζ:n hiljentäminen herkisti banaanikärpäset bakteeri-infektiolle. Osoitimme lisäksi, että sekä bakteeri-infektio että Leptopilina boulardi -pistiäisen aiheuttama loisinfektio sai aikaan edinin ilmentymisen banaanikärpäsessä. Laajamittaisten in vitro ja in vivo kokeiden perusteella pystyimme kuitenkin osoittamaan Edinillä olevan vain vähäinen tehtävä bakteeri-infektion yhteydessä. Sen sijaan tutkimuksemme perusteella Edin säätelee banaanikärpäsen verisoluja pistiäisinfektion yhteydessä. Tämä tutkimus osoitti 14-3-3ζ:n ja Edinin olevan uusia banaanikärpäsen immuunivasteeseen osallistuvia tekijöitä, jotka toimivat erityisesti soluvälitteisen immuniteetin säätelijöinä. Tutkimustuloksemme tuovat lisätietoa synnynnäisen immuunivasteen toiminnasta sekä soluvälitteisen ja humoraalisen immuunivasteen vuorovaikutuksesta. Tästä tutkimuksesta saatu uusi tieto voi tulevaisuudessa auttaa ymmärtämään paremmin myös ihmisen synnynnäistä immuunivastetta johtuen synnynnäisen immuniteetin mekanismien samankaltaisuudesta banaanikärpäsen ja ihmisen välillä.The human immune system constitutes of the innate and the adaptive immunity. Innate immune responses are an important part of our immune defense as they provide the first line of defense against pathogens. Innate immunity is based on the ability of genome-encoded receptors to recognize common features on the surface of pathogens. This leads to rapid responses in the host, including the phagocytosis of microorganisms by immune cells, the activation of signaling cascades and the production and secretion of cytokines and other effector molecules. In contrast, the mechanisms of the adaptive immunity, which include, for example the production of antibodies and immunological memory, arise later on in development and are slower to react to an immune challenge. Because of the crucial importance of the innate immunity in the early stages of an individual’s life cycle, and also in the early stages of an infection, a profound understanding of the regulators and mediators involved in the innate immune responses is needed. The fruit fly, Drosophila melanogaster, is an excellent model organism for studying the mechanisms of innate immunity, because it lacks adaptive immune responses and because the mechanisms of innate immunity are evolutionarily conserved. This study focused on the functional characterization of selected Drosophila genes that had been previously identified as novel immunity-related genes in large-scale in vitro screens. Especially, the mechanisms of function of the phagocytosis-related gene 14-3-3ζ (14-3-3 zeta) and the infection-inducible gene edin (elevated during infection) were studied. Using tissue-specific RNA-interference (RNAi) mediated gene silencing we were able to show that the cofilin regulator 14-3-3ζ is an evolutionarily conserved protein required for the phagocytosis of bacteria both in Drosophila larvae and in adult flies. Our study also showed that phagocytosis is required for an efficient immune response against bacteria, because silencing 14-3-3ζ with RNAi sensitized the flies to bacterial infections. Additionally, we showed that the expression of edin is upregulated in response to both a bacterial infection and wasp parasitism by Leptopilina boulardi, although after both in vitro and in vivo analyses Edin proved to have only a minor role in the host defense against bacteria. Instead, we were able to demonstrate that Edin is an important determinant in the defense against wasp parasitism, where it acts as a regulator of Drosophila blood cells. The present study identified 14-3-3ζ and Edin as novel mediators of the Drosophila host defense, where they were found to take part especially in the cellular immune response. Our findings add more clarity to the mechanisms of innate immunity and provide more evidence of an active interaction between the humoral and cellular arms of the immune defense. In the future, the knowledge obtained from this study may serve as a novel starting point for human research due to the ancient origin of the mechanisms of innate immunity and their evolutionary conservation from fly to man

Edin expression in the fat body is required in the defense against parasitic wasps in Drosophila melanogaster

The cellular immune response against parasitoid wasps in Drosophila involves the activation, mobilization, proliferation and differentiation of different blood cell types. Here, we have assessed the role of Edin (elevated during infection) in the immune response against the parasitoid wasp Leptopilina boulardi in Drosophila melanogaster larvae. The expression of edin was induced within hours after a wasp infection in larval fat bodies. Using tissue-specific RNAi, we show that Edin is an important determinant of the encapsulation response. Although edin expression in the fat body was required for the larvae to mount a normal encapsulation response, it was dispensable in hemocytes. Edin expression in the fat body was not required for lamellocyte differentiation, but it was needed for the increase in plasmatocyte numbers and for the release of sessile hemocytes into the hemolymph. We conclude that edin expression in the fat body affects the outcome of a wasp infection by regulating the increase of plasmatocyte numbers and the mobilization of sessile hemocytes in Drosophila larvae

Transdifferentiation and Proliferation in Two Distinct Hemocyte Lineages in Drosophila melanogaster Larvae after Wasp Infection

Cellular immune responses require the generation and recruitment of diverse blood cell types that recognize and kill pathogens. In Drosophila melanogaster larvae, immune-inducible lamellocytes participate in recognizing and killing parasitoid wasp eggs. However, the sequence of events required for lamellocyte generation remains controversial. To study the cellular immune system, we developed a flow cytometry approach using in vivo reporters for lamellocytes as well as for plasmatocytes, the main hemocyte type in healthy larvae. We found that two different blood cell lineages, the plasmatocyte and lamellocyte lineages, contribute to the generation of lamellocytes in a demand-adapted hematopoietic process. Plasmatocytes transdifferentiate into lamellocyte-like cells in situ directly on the wasp egg. In parallel, a novel population of infection-induced cells, which we named lamelloblasts, appears in the circulation. Lamelloblasts proliferate vigorously and develop into the major class of circulating lamellocytes. Our data indicate that lamellocyte differentiation upon wasp parasitism is a plastic and dynamic process. Flow cytometry with in vivo hemocyte reporters can be used to study this phenomenon in detail

<i>Edin</i> expression is induced upon a wasp infection.

<p><b>(A)</b> Wasp infection causes a 6.7-fold increase in <i>edin</i> expression in 2^nd instar <i>Canton S</i> larvae. Data are pooled from two independent experiments, n = 2 for each experiment, where one sample represents 10 larvae. <b>(B)</b><i>Edin</i> expression is induced in the fat bodies of <i>Canton S</i> larvae 24 hours post infection. The data are pooled from four independent experiments, and each experiment consisted of two samples, where one sample represents 8–10 larval fat bodies.</p

Knock down of <i>edin</i> in the fat body decreases the encapsulation and killing ability of <i>Drosophila</i> larvae.

<p><b>(A)</b> The encapsulation response of two different <i>edin</i> RNAi lines (<i>edin</i>^<i>14289</i> and <i>edin</i>^{<i>109528</i>}) was analyzed 27-29h after a wasp infection. The <i>C564</i>-<i>GAL4</i> (<i>C564>)</i>, <i>Fb-GAL4</i> (<i>Fb></i>) and <i>Hml</i>^<i>Δ</i>;<i>He-GAL4</i> (<i>HH></i>) drivers were used to drive the expression of the RNAi constructs. <i>w</i>^<i>1118</i> (<i>w</i>) was used as control. Data were pooled from one to eight individual experiments, as depicted on each column, each experiment with at least 50 analyzed individual infected larvae. <b>(B)</b> The ability of <i>Drosophila</i> larvae to kill wasp eggs was assessed with two different <i>edin</i> RNAi lines (<i>edin</i>^<i>14289</i> and <i>edin</i>^{<i>109528</i>}) 48-50h after infection. The <i>C564</i>-<i>GAL4</i> (<i>C564></i>) and <i>Fb-GAL4</i> (<i>Fb></i>) drivers were used to drive the expression of the RNAi constructs. <i>w</i>^<i>1118</i> (<i>w</i>) was used as control. Data are pooled from three to sixteen independent experiments, as indicated on each column, and at least 50 infected larvae were scored per experiment. Error bars in A and B show standard deviations. Knocking down the expression of <i>edin</i> in several tissues including the fat body or in the fat body alone caused a significant decrease in the encapsulation activity and killing response of <i>Drosophila</i> larvae compared to controls, whereas knocking down <i>edin</i> in hemocytes had no effect.</p