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FOXO mediated PeroxiredoxinV expression regulates redox homeostasis during Drosophila melanogaster gut infection
Developmental and Comparative Immunology 38 (2012) 466–473
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Developmental and Comparative Immunology journal homepage: www.elsevier.com/locate/dci
JNK/FOXO mediated PeroxiredoxinV expression regulates redox homeostasis during Drosophila melanogaster gut infection Hye-Mi Ahn a,b,1, Kyu-Sun Lee a,c,1, Dong-Seok Lee b,⇑, Kweon Yu a,c,⇑ a
Aging Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon 305-806, Republic of Korea College of Natural Sciences, Kyungpook National University, Daegu 702-701, Republic of Korea c Functional Genomics Program, University of Science and Technology (UST), Daejeon 305-333, Republic of Korea b
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Article history: Received 30 April 2012 Revised 4 July 2012 Accepted 5 July 2012 Available online 31 July 2012 Keywords: Drosophila melanogaster Gut infection ROS Duox Peroxiredoxin V JNK FOXO
a b s t r a c t Innate immunity plays an important role in combating microbial infection in animals. During bacterial infection in Drosophila melanogaster gut, Dual oxidase (Duox) generates reactive oxygen species (ROS) to fight against the infected microbes. Concurrently, antioxidant systems eliminate residual ROS and protect the hosts. Here we found that Drosophila melanogaster Peroxiredoxin V (dPrxV) is an immune-related antioxidant enzyme which maintains intestinal redox homeostasis. dPrxV was highly expressed in gut and induced by the oral infection of Erwinia carotovora carotovora. dPrxV expression was increased by the gut-specific Duox overexpression but decreased by Duox inhibition. Moreover, dPrxV expression was mediated by the JNK/FOXO signaling and dPrxV mutant reduced survival after gut infection. These results suggest that JNK/FOXO mediated dPrxV expression plays a critical role in Drosophila melanogaster gut during bacterial infection in protecting the host gut epithelial cells from oxidative damage. Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction Animal guts are constantly exposed to various ingested microbes. Pathogenic microbes trigger immune response and prolonged infection causes gastrointestinal diseases (Macdonald and Monteleone, 2005). Understanding the immune defense mechanism is important for alleviating gastrointestinal diseases. Drosophila melanogaster gut immune response consists of two types of molecular defense mechanisms which act synergistically to restrict the proliferation of infected bacteria. One type is the production of bacteria killing reactive oxygen species (ROS) by Duox, which is the first line of defense (Ha et al., 2005a). The other defense mechanism is the generation of antimicrobial peptides (AMP) through the IMD-Relish pathway, which is the second line of defense (Buchon et al., 2009b). Since the residual ROS can damage gut epithelia, antioxidant enzymes should work to maintain redox homeostasis in the fly Abbreviations: ROS, Reactive Oxygen Species; Ecc15, Erwinia carotovora carotovora; Ecc15-GFP, Green fluorescence protein-tagged Ecc15; dPrxV, Drosophila melanogaster Peroxiredoxin V; Duox, Dual oxidase; JNK, c-Jun N-terminal kinase. ⇑ Corresponding authors. Address: Kyungpook National Univ., Sangyeok 3-dong, Bung-gu, Daegu, Republic of Korea. Tel.: 82 53 950 7366 (D.S. Lee), Tel.: 82 42 860 4642 (K. Yu). E-mail addresses: [email protected] (D.-S. Lee), [email protected] (K. Yu). 1 These authors contributed equally to this work. 0145-305X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.dci.2012.07.002
gut. In a microarray analysis, the infection of Erwinia carotovora carotovora (Ecc15) triggers an immediate stress response which includes the production of antioxidant enzymes (Buchon et al., 2009b). Antioxidant systems involve SODs, catalases, glutathione peroxidases (Gpxs), and Peroxiredoxins (Prxs) (Foyer and Noctor, 2009). A secreted form of catalase immune-regulated catalase (IRC) is required in the fly gut because IRC knock-down flies show high mortality after gut infection by non-lethal bacteria (Ha et al., 2005b). Prxs catalyze the reduction of hydrogen peroxide, organic hydroperoxides, and peroxynitrite (Radyuk et al., 2009). Prx family consists of six subtypes (PrxI to VI) distinguished by their structure, subcellular location, and enzyme activities. PrxV is found in the cytosol, nucleus, mitochondria, and peroxisomes (Radyuk et al., 2001). Mammalian Prxs are necessary for removing residual ROS and maintaining redox balance during bacterial infection or inflammation by lipopolysaccharide (LPS) via activated macrophages (Abbas et al., 2009; Bast et al., 2010; Li et al., 2009). Like mammalian Prxs, Drosophila melanogaster Prxs are also involved in the modulation of immune responses and protecting hosts against oxidative damage (Ha et al., 2005b; Lee et al., 2009; Radyuk et al., 2010, 2009). JNK/FOXO signaling pathway is a well-defined form of signaling against oxidative stress (Kops et al., 2002; Rubin and Spradling, 1982; Wang et al., 2003, 2005). The activation of JNK/FOXO signaling in Drosophila melanogaster neurons can tolerate oxidative
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stress-induced damage and extend the Drosophila melanogaster lifespan by inducing Jafrac1 (dPrxII) (Lee et al., 2009). Jafrac1 is also involved in the tissue reconstruction during aging in Drosophila melanogaster gut (Biteau et al., 2010). Mutation of Jafrac1 induces the unbalance of redox homeostasis, which accelerates age-related degeneration in the fly gut epithelia (Hochmuth et al., 2011). These results suggest that dPrxs have important roles in maintaining redox homeostasis in Drosophila melanogaster gut during aging and in immune response. In this report, we demonstrate that dPrxV expression in Drosophila melanogaster gut was regulated by the JNK/FOXO signaling and it protected host gut epithelia against oxidative stress during bacterial infection. 2. Materials and methods 2.1. Drosophila melanogaster culture and stocks Flies were kept at 25 °C and cultured using the standard methods. Wild-type (Oregon R), w-, UAS-FOXO.p, and dPrxVEY02106 were obtained from Bloomington stock Center (Bloomington, USA). dPrxVEY02106 carries a p-element insertion in the first exon of the coding region (Radyuk et al., 2009). Homozygous dPrxVEY02106 flies have no detectable dPrxV mRNA (Supplementary Fig. 1). NP3084Gal4 (gut driver), UAS-dPrxV-RNAi, and UAS-bsk-RNAi were obtained from Kyoto stock Center (Kyoto, Japan). UAS-dPrxV transgenic flies were generated by the p element-mediated germ-line transformation with the UAS-dPrxV construct containing the full length dPrxV cDNA in the pUAST vector (Rubin and Spradling, 1982). UAS-Duox, UAS-Duox-RNAi (Ha et al., 2005a), UAS-dFOXO21 and UAS-dFOXO25 were gifts from WJ Lee, JK Jung, E. Hafen, and kJ Min. 2.2. Gut infection 3–5 day old adult flies were starved for 2 h and then transferred to a vial containing the filter paper soaked with 5% sucrose solution including cultured bacteria (Ha et al., 2005a). Bacteria used in this study were Erwinia carotovora carotovora15 (Ecc15) and Green Fluorescence Protein-tagged Erwinia carotovora carotovora15 (Ecc15-GFP). Ecc15 was cultured in LB medium while Ecc15-GFP was cultured in LB medium with spectinomycin (100 lg/ml) at 30 °C. Bacterial culture at OD600 = 2.0 was used for experiments. Ecc15 strains can colonize in Drosophila melanogaster gut and activate the immune system (Basset et al., 2003). Ecc15 and Ecc15GFP were gifts from WJ Lee. 2.3. Measuring ROS levels in Drosophila melanogaster gut For measuring intracellular ROS in Drosophila melanogaster gut epithelia, the method described by Strayer was used (Strayer et al., 2003). The infected fly gut was dissected and washed out with PBS to remove materials in the lumen. Then, the washed gut was homogenized in 200 ll of PBST (PBS containing 0.1% Tween 20) and 90 ll of the supernatant was transferred to a 96-well plate. After adding 50 ll of 50 lM DCF-DA to the samples, the fluorescence intensity (excitation 485 nm and emission 640 nm) was measured every 5 min for 25 min using the fluorescence microplate FLUOstar Optima reader (BMG Laboratory). Non-fluorescent 2,7-dichlorofluorescein di-acetate (Molecular Probes, DCF-DA) cell permeable dye is converted to 2,7-dichlorofluoroscein (DCF) in the presence of ROS (Royall and Ischiropoulos, 1993). For measuring the lumen ROS in the gut, FOX (Ferric-Xylenol Orange) assay was used (Ha et al., 2005a). Infected gut was dissected, cut into small pieces, and dipped into 50 ll of H2O containing aminotriazol (2 mg/ml). After the sample was centrifuged for 5 min, the supernatant was used for
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measuring diffused ROS by the FOX assay. Three independent experiments with 10 flies in each experiment were performed. 2.4. RT-PCR and quantitative RT-PCR analysis The semi-quantitative RT-PCR analysis with an adult fly gut was performed as previously described (Lee et al., 2004). Total RNA was isolated from infected adult gut using Easy-Blue RNA extraction kit (iNtRON Biotechnology, Korea). The first stranded cDNA was synthesized by using Superscript III Reverse Transcription system (Invitrogen, CA). PCR reactions were performed using AccuPower PCR premix (Bioneer, Korea). Various primer sets used in the experiments are in Supplementary Table 1. For quantitative RT– PCR analysis, ABI Prism 7900 Sequence Detector (Applied Biosystems, CA) and SYBR Green PCR Core reagents (Applied Biosystems, CA) were used. mRNA expression levels were presented as the relative fold change against the equalized rp49 mRNA expression level. The relative cycle threshold (Ct) (User Bulletin 2, Applied Biosystems, CA) was used to evaluate the data. All experiments were repeated at least three times. The data were shown as the mean and the error bar (±S.E.M.). 2.5. Generation of dPrxV antiserum and Western blot analysis dPrxV polyclonal antibody was generated by the immunization of rabbits with the synthetic peptides (85PGYVSSADEKSKQG99 and 153 RSKRYSLVVENGKVTE169) matching to the amino acid residues of the dPrxV protein. For the Western blot, adult fly guts were dissected in PBS and total proteins were isolated using the PROPREP protein extraction buffer (iNtRON Biotechnology, Korea). Immunoblotting was performed as the previously described method (Lee et al., 2004). Anti-b-actin antibody was used as the loading control. The phospho-JNK antibody was used to detect the activated form of JNK (Lee et al., 2009). The anti-rabbit IgG and anti-mouse IgG were used as secondary antibodies. 2.6. Immunohistochemistry To detect dFOXO localization in the fly gut, the infected fly gut was dissected and immunostained with the FOXO antibody, a procedure that was performed as the previously described method (Lee et al., 2008). The anti-rabbit IgG Alexa 594 was used as the secondary antibody. The imaging was visualized by the FluoView FV1000 Confocal Microscope (Olympus, Japan). 2.7. Lethality test For measuring a survival rate, 3–5 day old adult flies were raised in the vials containing the filter paper soaked with cultured bacterial solution. Filter papers were replaced twice a day and dead flies were counted. The test was repeated at least three times for each genotype. 2.8. Statistical analysis Data are presented as means ±S.E.M. Comparisons were performed using the One-Way ANOVA analysis. Statistical analyses for lethality tests were carried out using the survival curve analysis in the Prism software (GraphPad, CA). ⁄P < 0.05 is considered as statistically significant and ⁄⁄P < 0.001 is statistically more significant. 3. Results 3.1. dPrxV has a high homology with mammalian PrxVs and is highly expressed in Drosophila melanogaster gut The putative Drosophila melanogaster PeroxiredoxinV gene (CG7217, FlyBase) was identified based on the amino acid sequence
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similarity with mammalian PeroxiredoxinV genes. It belongs to the subtype of 2-cysteine Peroxiredoxins (Michalak et al., 2008) (Fig. 1A, boxes). The amino acid sequence of the dPrxV shows significant homology with the human, rat, and mouse PrxV (58%) (Fig. 1A, black background). During development, dPrxV was expressed in all developmental stages (Fig. 1B). While dPrxV was expressed in the central nervous system (CNS), fat body (FB), gut, and Malpighian tubules (MT) at the larval stage, the expression level of dPrxV in the gut was relatively higher than other tissues (Fig. 1C). In this study, we focused on the function of dPrxV in the gut during immune response by bacterial oral infection.
3.2. Ecc15-GFP oral infection induces Duox and dPrxV expression in the Drosophila melanogaster gut To induce immune response in the fly gut, wild-type flies were fed with the Green Fluorescence Protein-tagged Ecc15 (Ecc15-GFP). After the oral infection, GFP was detected in the fly abdomen (Fig. 2A) and ROS was induced in the fly gut. The ROS level in the gut peaked at 1 h, reduced until 3 h, and increased again after 3 h of oral infection (Fig. 2B). Duox mRNA was increased at 4 h after Ecc15-GFP infection (Fig. 2C). On the contrary, dPrxV mRNA (Fig. 2D) and protein (Fig. 2E) were gradually increased and peaked
Fig. 1. The comparison of Drosophila melanogaster Peroxiredoxin V (dPrxV) amino acid sequences with mammalian PrxVs and its developmental and tissue expression. (A) The amino acid sequence alignments among hPrxV, rPrxV, mPrxV, and dPrxV show high identities (58%, black background) and similarities (gray background). Conserved 2Cysteine residues were boxed. (B) dPrxV was expressed throughout the developmental stages. (C) In the larval stage, dPrxV was expressed in central nervous system (CNS), fat body (FB), gut, and malphigian tubules (MT). Data are presented as mean ± S.E.M. ⁄P < 0.05 and ⁄⁄P < 0.001.
H.-M. Ahn et al. / Developmental and Comparative Immunology 38 (2012) 466–473
around 3–4 h after Ecc15-GFP infection. These results indicate that Ecc15-GFP induces ROS, Duox mRNA, and PrxV mRNA and protein in the fly gut during the early stage of infection. 3.3. Duox regulates dPrxV expression, which may control residual ROS in the fly gut To find out whether Duox regulates dPrxV expression, we measured dPrxV levels in the fly gut at the different Duox genetic backgrounds. When Duox was overexpressed in the gut epithelia by the NP3084-Gal4 driver (NP3084 > Duox), dPrxV expression was increased compared with the NP3084-Gal4 control. On the contrary, when Duox was inhibited in the gut epithelia (NP3084 > Duox-Ri), dPrxV expression was reduced Fig. 3(A) and (B). These results indicate that Duox regulates dPrxV expression in the fly gut. Since Duox generates ROS in the fly gut to combat infected microbes and residual ROS should be eliminated to pro-
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tect the host gut (Ha et al., 2009), we checked whether dPrxV removes residual ROS in the gut. dPrxV overexpression (DA > dPrxV) in the Ecc15-GFP infected fly gut reduced intracellular ROS levels compared with the DA-Gal4 control while dPrxV inhibition (DA > dPrxV-Ri) increased intracellular ROS levels at 2 h and 4 h after infection (Fig. 3(C) and 3D). In contrast, dPrxV overexpression and inhibition did not change diffused ROS levels in gut lumen (Supplementary Fig. 1B). These results indicate that dPrxV can protect bacterial infection induced ROS in the fly gut epithelia. 3.4. JNK/FOXO signaling induces dPrxV expression in fly gut by the Ecc15-GFP oral infection In Drosophila melanogaster, oxidative stress activates JNK/FOXO signaling to induce target genes which protect flies (Wang et al., 2003, 2005). When wild-type flies were infected with Ecc15-GFP,
Fig. 2. Ecc15-GFP oral infection induces Duox and dPrxV expression in the Drosophila melanogaster gut. (A) In the Ecc15-GFP oral infection, GFP was detected in the fly abdomen. (B) The intracellular ROS level in the infected gut epithelia. (C, D) RT-qPCR analysis of Duox and dPrxV expression levels after Ecc15-GFP infection. (E) dPrxV protein expression and quantitation after Ecc15-GFP infection. b-actin was the loading control. Data is presented as mean ± S.E.M. ⁄P < 0.05 and ⁄⁄P < 0.001.
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Fig. 3. Duox regulates dPrxV expression, which may control residual ROS in the fly gut. (A) dPrxV expression was increased in the Duox gut overexpression (NP3084 > Duox) while it was suppressed in the Duox gut inhibition (NP3084 > Duox-Ri) compared to the NP3084-Gal4 control. (B) Duox expression levels in NP3084-Gal4/+, NP3084 > Duox, and NP3084 > Duox-Ri fly gut. (C) dPrxV overexpression (DA > dPrxV) in the Ecc15-GFP infected fly gut reduced intracellular ROS levels compared with the DA-Gal4/+ control while dPrxV inhibition (DA > dPrxV-Ri) increased ROS levels. (D) dPrxV expression levels in DA-Gal4/+, DA > dPrxV, and DA > dPrxV-Ri fly gut. Data are presented as mean ± S.E.M. ⁄ P < 0.05 and ⁄⁄P < 0.001.
active phospho-JNK was detected immediately after infection and reduced from 3 h in the fly gut (Fig. 4A). JNK inhibition in the bacteria infected fly gut (NP3084 > bsk-Ri) did not induce dPrxV expression and the NP3084-Gal4 control induced dPrxV expression in the early stage of infection (Fig. 4B). NP3084 > bsk-Ri did not activate JNK in the fly gut (Supplementary Fig. 1C). These results suggest that residual ROS can activate JNK and induce dPrxV expression. Next, to find out whether FOXO is a transcriptional factor for the dPrxV induction, we examined the localization FOXO during the gut infection. FOXO, a forkhead domain-containing transcription factor, is a key regulator of growth, tolerance to stress, metabolism, and life span in various organisms including Drosophila melanogaster (Accili and Arden, 2004; Hwangbo et al., 2004). FOXO was localized mainly in cytoplasm in the non-infected control while FOXO was localized in nuclei in the infected gut (Fig. 4C). dPrxV expression was almost absent in the infected gut of the FOXO null mutant (FOXO21/25) while dPrxV was expressed in the w- control fly gut (Fig. 4D). Neuronal expression of FOXO also increased dPrxV expression (Supplementary Fig. 1D). These findings indicate that JNK/FOXO signaling induces dPrxV expression in the fly gut after Ecc15-GFP infection. 3.5. The lethality of dPrxV mutants is increased during bacterial infection To find the role of dPrxV, we measured the lethality. In both male and female dPrxV mutant flies, Ecc15 infection increased lethality compared to the w- control flies (Fig. 5A, B). dPrxV inhibition
(DA > dPrxV-Ri) also increased lethality compared to the DA-Gal4/+ control (Supplementary Fig. 2). These results demonstrate that dPrxV is critical for survival by protecting the host gut against bacterial infection induced ROS.
4. Discussion Among antioxidant enzymes, an immune responsive catalase (IRC) is a key enzyme to remove the infection induced ROS in Drosophila melanogaster. IRC knock-down flies show high mortality after gut infection by non-lethal bacteria (Ha et al., 2005b). IRC is a secretory antioxidant enzyme which removes the total in vivo ROS including gut lumen (Ha et al., 2005b). In this study, we detected that infection-induced intracellular ROS was reduced by dPrxV, whereas the level of ROS in gut lumen did not change by PrxV (Fig. 3C, Supplementary Fig. 1B). In addition, total IRC mRNA is dramatically increased by septic injury (Ha et al., 2005b). These suggest that IRC removes total in vivo ROS induced by septic injury or gut infection while dPrxV has a limited role which controls intracellular ROS during gut infection. Duox activity and expression are required to produce ROS which kills infected bacteria in the fly gut. When gut cells come in contact with bacteria, active Duox immediately produces ROS to kill the bacteria. Then, when the gut needs more ROS, Duox expression is increased (Ha et al., 2009). The first peak of ROS in Fig. 2B seems to be produced by the active Duox and the second peak of ROS may be a result of increased Duox expression.
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Fig. 4. JNK/FOXO signaling induces dPrxV expression in the fly gut by Ecc15-GFP oral infection. (A) Activated JNK was detected after Ecc15-GFP oral infection. b-actin was a loading control. (B) JNK inhibition in the fly gut (NP3084 > bsk-Ri) did not induce dPrxV expression while the NP3084-Gal4/+ control induced dPrxV expression in the early stage of infection. (C) FOXO was localized mainly in the cytoplasm of the non-infected control fly gut epithelia while FOXO was localized in the nuclei (arrows) of the infected gut. (D) dPrxV expression was almost absent in the infected gut of the FOXO null mutant (FOXO21/25) while dPrxV was expressed in the w- control fly gut. Data are presented as mean ± S.E.M. ⁄P < 0.05 and ⁄⁄P < 0.001.
Oxidative stress modulates the activation of JNK pathway by Ecc15 infection within 30 min (Buchon et al., 2009a). JNK signaling mediates the nuclear localization of FOXO and the expression of antioxidant enzymes (Biteau et al., 2011). In Drosophila melanogaster, the activation of JNK signaling induces resistance to oxidative stress by changing the cellular transcriptome (Wang et al., 2003). The transcription of Jafrac1 regulated by JNK/FOXO signaling promotes resistance to oxidative stress and extends life span (Lee et al., 2009). Recent studies show that dPrxV is associated with longevity and immune response. dPrxV deficient flies are vulnerable
to oxidative stress and apoptosis, and reduced life span (Radyuk et al., 2009). Contrary to our data, dPrxV deficient flies are more resistant to the septic infection and dPrxV is considered to be the negative regulator of JNK during septic infection induced-immune response (Radyuk et al., 2010). Unexpectedly, ubiquitous overexpression of dPrxV (DA > dPrxV) increased the lethality of the gut infection compared to the DA-Gal4/+ control (Supplementary Fig. 2). The lethality is worse than that of the dPrxV inhibition (DA > dPrxV-Ri). Recently, Wu et al.,(2012) demonstrates that local production of ROS by gut
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Fig. 5. dPrxV mutants increase lethality during bacterial infection. In both male (A) and female dPrxV mutant flies (B), non-lethal pathogen Ecc15 infection increased lethality compared with the w-control.
infection triggers systemic AMP production in fat body. Therefore, overexpression of dPrxV (DA > dPrxV) eliminates residual ROS and may not trigger enough AMP production in fat body, which results in increased lethality. In this study, we show that dPrxV is a novel target for JNK/ FOXO signaling during bacterial infection in gut. Active JNK level peaked within 1 h and then gradually reduced until 5 h by gut infection (Fig. 4A) but dPrxV expression gradually increased and peaked around 4 h after gut infection (Fig. 2D). This data suggests that ROS induced JNK activation may lead to dPrxV expression and then clearance of ROS by dPrxV results in decreased JNK activity. We can speculate that dPrxV mutants die early because a certain level of ROS can activate JNK continuously and induce JNK mediated apoptosis in the fly gut. Our findings provide important information of how host animals protect their gut against microbial infection. Acknowledgements This work was supported by the grants from the Research Foundation of Korea (2009-0080870 and 2011-0030134) and KRIBB Research Initiative Program. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.dci.2012.07.002.
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Developmental and Comparative Immunology journal homepage: www.elsevier.com/locate/dci
JNK/FOXO mediated PeroxiredoxinV expression regulates redox homeostasis during Drosophila melanogaster gut infection Hye-Mi Ahn a,b,1, Kyu-Sun Lee a,c,1, Dong-Seok Lee b,⇑, Kweon Yu a,c,⇑ a
Aging Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon 305-806, Republic of Korea College of Natural Sciences, Kyungpook National University, Daegu 702-701, Republic of Korea c Functional Genomics Program, University of Science and Technology (UST), Daejeon 305-333, Republic of Korea b
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Article history: Received 30 April 2012 Revised 4 July 2012 Accepted 5 July 2012 Available online 31 July 2012 Keywords: Drosophila melanogaster Gut infection ROS Duox Peroxiredoxin V JNK FOXO
a b s t r a c t Innate immunity plays an important role in combating microbial infection in animals. During bacterial infection in Drosophila melanogaster gut, Dual oxidase (Duox) generates reactive oxygen species (ROS) to fight against the infected microbes. Concurrently, antioxidant systems eliminate residual ROS and protect the hosts. Here we found that Drosophila melanogaster Peroxiredoxin V (dPrxV) is an immune-related antioxidant enzyme which maintains intestinal redox homeostasis. dPrxV was highly expressed in gut and induced by the oral infection of Erwinia carotovora carotovora. dPrxV expression was increased by the gut-specific Duox overexpression but decreased by Duox inhibition. Moreover, dPrxV expression was mediated by the JNK/FOXO signaling and dPrxV mutant reduced survival after gut infection. These results suggest that JNK/FOXO mediated dPrxV expression plays a critical role in Drosophila melanogaster gut during bacterial infection in protecting the host gut epithelial cells from oxidative damage. Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction Animal guts are constantly exposed to various ingested microbes. Pathogenic microbes trigger immune response and prolonged infection causes gastrointestinal diseases (Macdonald and Monteleone, 2005). Understanding the immune defense mechanism is important for alleviating gastrointestinal diseases. Drosophila melanogaster gut immune response consists of two types of molecular defense mechanisms which act synergistically to restrict the proliferation of infected bacteria. One type is the production of bacteria killing reactive oxygen species (ROS) by Duox, which is the first line of defense (Ha et al., 2005a). The other defense mechanism is the generation of antimicrobial peptides (AMP) through the IMD-Relish pathway, which is the second line of defense (Buchon et al., 2009b). Since the residual ROS can damage gut epithelia, antioxidant enzymes should work to maintain redox homeostasis in the fly Abbreviations: ROS, Reactive Oxygen Species; Ecc15, Erwinia carotovora carotovora; Ecc15-GFP, Green fluorescence protein-tagged Ecc15; dPrxV, Drosophila melanogaster Peroxiredoxin V; Duox, Dual oxidase; JNK, c-Jun N-terminal kinase. ⇑ Corresponding authors. Address: Kyungpook National Univ., Sangyeok 3-dong, Bung-gu, Daegu, Republic of Korea. Tel.: 82 53 950 7366 (D.S. Lee), Tel.: 82 42 860 4642 (K. Yu). E-mail addresses: [email protected] (D.-S. Lee), [email protected] (K. Yu). 1 These authors contributed equally to this work. 0145-305X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.dci.2012.07.002
gut. In a microarray analysis, the infection of Erwinia carotovora carotovora (Ecc15) triggers an immediate stress response which includes the production of antioxidant enzymes (Buchon et al., 2009b). Antioxidant systems involve SODs, catalases, glutathione peroxidases (Gpxs), and Peroxiredoxins (Prxs) (Foyer and Noctor, 2009). A secreted form of catalase immune-regulated catalase (IRC) is required in the fly gut because IRC knock-down flies show high mortality after gut infection by non-lethal bacteria (Ha et al., 2005b). Prxs catalyze the reduction of hydrogen peroxide, organic hydroperoxides, and peroxynitrite (Radyuk et al., 2009). Prx family consists of six subtypes (PrxI to VI) distinguished by their structure, subcellular location, and enzyme activities. PrxV is found in the cytosol, nucleus, mitochondria, and peroxisomes (Radyuk et al., 2001). Mammalian Prxs are necessary for removing residual ROS and maintaining redox balance during bacterial infection or inflammation by lipopolysaccharide (LPS) via activated macrophages (Abbas et al., 2009; Bast et al., 2010; Li et al., 2009). Like mammalian Prxs, Drosophila melanogaster Prxs are also involved in the modulation of immune responses and protecting hosts against oxidative damage (Ha et al., 2005b; Lee et al., 2009; Radyuk et al., 2010, 2009). JNK/FOXO signaling pathway is a well-defined form of signaling against oxidative stress (Kops et al., 2002; Rubin and Spradling, 1982; Wang et al., 2003, 2005). The activation of JNK/FOXO signaling in Drosophila melanogaster neurons can tolerate oxidative
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stress-induced damage and extend the Drosophila melanogaster lifespan by inducing Jafrac1 (dPrxII) (Lee et al., 2009). Jafrac1 is also involved in the tissue reconstruction during aging in Drosophila melanogaster gut (Biteau et al., 2010). Mutation of Jafrac1 induces the unbalance of redox homeostasis, which accelerates age-related degeneration in the fly gut epithelia (Hochmuth et al., 2011). These results suggest that dPrxs have important roles in maintaining redox homeostasis in Drosophila melanogaster gut during aging and in immune response. In this report, we demonstrate that dPrxV expression in Drosophila melanogaster gut was regulated by the JNK/FOXO signaling and it protected host gut epithelia against oxidative stress during bacterial infection. 2. Materials and methods 2.1. Drosophila melanogaster culture and stocks Flies were kept at 25 °C and cultured using the standard methods. Wild-type (Oregon R), w-, UAS-FOXO.p, and dPrxVEY02106 were obtained from Bloomington stock Center (Bloomington, USA). dPrxVEY02106 carries a p-element insertion in the first exon of the coding region (Radyuk et al., 2009). Homozygous dPrxVEY02106 flies have no detectable dPrxV mRNA (Supplementary Fig. 1). NP3084Gal4 (gut driver), UAS-dPrxV-RNAi, and UAS-bsk-RNAi were obtained from Kyoto stock Center (Kyoto, Japan). UAS-dPrxV transgenic flies were generated by the p element-mediated germ-line transformation with the UAS-dPrxV construct containing the full length dPrxV cDNA in the pUAST vector (Rubin and Spradling, 1982). UAS-Duox, UAS-Duox-RNAi (Ha et al., 2005a), UAS-dFOXO21 and UAS-dFOXO25 were gifts from WJ Lee, JK Jung, E. Hafen, and kJ Min. 2.2. Gut infection 3–5 day old adult flies were starved for 2 h and then transferred to a vial containing the filter paper soaked with 5% sucrose solution including cultured bacteria (Ha et al., 2005a). Bacteria used in this study were Erwinia carotovora carotovora15 (Ecc15) and Green Fluorescence Protein-tagged Erwinia carotovora carotovora15 (Ecc15-GFP). Ecc15 was cultured in LB medium while Ecc15-GFP was cultured in LB medium with spectinomycin (100 lg/ml) at 30 °C. Bacterial culture at OD600 = 2.0 was used for experiments. Ecc15 strains can colonize in Drosophila melanogaster gut and activate the immune system (Basset et al., 2003). Ecc15 and Ecc15GFP were gifts from WJ Lee. 2.3. Measuring ROS levels in Drosophila melanogaster gut For measuring intracellular ROS in Drosophila melanogaster gut epithelia, the method described by Strayer was used (Strayer et al., 2003). The infected fly gut was dissected and washed out with PBS to remove materials in the lumen. Then, the washed gut was homogenized in 200 ll of PBST (PBS containing 0.1% Tween 20) and 90 ll of the supernatant was transferred to a 96-well plate. After adding 50 ll of 50 lM DCF-DA to the samples, the fluorescence intensity (excitation 485 nm and emission 640 nm) was measured every 5 min for 25 min using the fluorescence microplate FLUOstar Optima reader (BMG Laboratory). Non-fluorescent 2,7-dichlorofluorescein di-acetate (Molecular Probes, DCF-DA) cell permeable dye is converted to 2,7-dichlorofluoroscein (DCF) in the presence of ROS (Royall and Ischiropoulos, 1993). For measuring the lumen ROS in the gut, FOX (Ferric-Xylenol Orange) assay was used (Ha et al., 2005a). Infected gut was dissected, cut into small pieces, and dipped into 50 ll of H2O containing aminotriazol (2 mg/ml). After the sample was centrifuged for 5 min, the supernatant was used for
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measuring diffused ROS by the FOX assay. Three independent experiments with 10 flies in each experiment were performed. 2.4. RT-PCR and quantitative RT-PCR analysis The semi-quantitative RT-PCR analysis with an adult fly gut was performed as previously described (Lee et al., 2004). Total RNA was isolated from infected adult gut using Easy-Blue RNA extraction kit (iNtRON Biotechnology, Korea). The first stranded cDNA was synthesized by using Superscript III Reverse Transcription system (Invitrogen, CA). PCR reactions were performed using AccuPower PCR premix (Bioneer, Korea). Various primer sets used in the experiments are in Supplementary Table 1. For quantitative RT– PCR analysis, ABI Prism 7900 Sequence Detector (Applied Biosystems, CA) and SYBR Green PCR Core reagents (Applied Biosystems, CA) were used. mRNA expression levels were presented as the relative fold change against the equalized rp49 mRNA expression level. The relative cycle threshold (Ct) (User Bulletin 2, Applied Biosystems, CA) was used to evaluate the data. All experiments were repeated at least three times. The data were shown as the mean and the error bar (±S.E.M.). 2.5. Generation of dPrxV antiserum and Western blot analysis dPrxV polyclonal antibody was generated by the immunization of rabbits with the synthetic peptides (85PGYVSSADEKSKQG99 and 153 RSKRYSLVVENGKVTE169) matching to the amino acid residues of the dPrxV protein. For the Western blot, adult fly guts were dissected in PBS and total proteins were isolated using the PROPREP protein extraction buffer (iNtRON Biotechnology, Korea). Immunoblotting was performed as the previously described method (Lee et al., 2004). Anti-b-actin antibody was used as the loading control. The phospho-JNK antibody was used to detect the activated form of JNK (Lee et al., 2009). The anti-rabbit IgG and anti-mouse IgG were used as secondary antibodies. 2.6. Immunohistochemistry To detect dFOXO localization in the fly gut, the infected fly gut was dissected and immunostained with the FOXO antibody, a procedure that was performed as the previously described method (Lee et al., 2008). The anti-rabbit IgG Alexa 594 was used as the secondary antibody. The imaging was visualized by the FluoView FV1000 Confocal Microscope (Olympus, Japan). 2.7. Lethality test For measuring a survival rate, 3–5 day old adult flies were raised in the vials containing the filter paper soaked with cultured bacterial solution. Filter papers were replaced twice a day and dead flies were counted. The test was repeated at least three times for each genotype. 2.8. Statistical analysis Data are presented as means ±S.E.M. Comparisons were performed using the One-Way ANOVA analysis. Statistical analyses for lethality tests were carried out using the survival curve analysis in the Prism software (GraphPad, CA). ⁄P < 0.05 is considered as statistically significant and ⁄⁄P < 0.001 is statistically more significant. 3. Results 3.1. dPrxV has a high homology with mammalian PrxVs and is highly expressed in Drosophila melanogaster gut The putative Drosophila melanogaster PeroxiredoxinV gene (CG7217, FlyBase) was identified based on the amino acid sequence
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similarity with mammalian PeroxiredoxinV genes. It belongs to the subtype of 2-cysteine Peroxiredoxins (Michalak et al., 2008) (Fig. 1A, boxes). The amino acid sequence of the dPrxV shows significant homology with the human, rat, and mouse PrxV (58%) (Fig. 1A, black background). During development, dPrxV was expressed in all developmental stages (Fig. 1B). While dPrxV was expressed in the central nervous system (CNS), fat body (FB), gut, and Malpighian tubules (MT) at the larval stage, the expression level of dPrxV in the gut was relatively higher than other tissues (Fig. 1C). In this study, we focused on the function of dPrxV in the gut during immune response by bacterial oral infection.
3.2. Ecc15-GFP oral infection induces Duox and dPrxV expression in the Drosophila melanogaster gut To induce immune response in the fly gut, wild-type flies were fed with the Green Fluorescence Protein-tagged Ecc15 (Ecc15-GFP). After the oral infection, GFP was detected in the fly abdomen (Fig. 2A) and ROS was induced in the fly gut. The ROS level in the gut peaked at 1 h, reduced until 3 h, and increased again after 3 h of oral infection (Fig. 2B). Duox mRNA was increased at 4 h after Ecc15-GFP infection (Fig. 2C). On the contrary, dPrxV mRNA (Fig. 2D) and protein (Fig. 2E) were gradually increased and peaked
Fig. 1. The comparison of Drosophila melanogaster Peroxiredoxin V (dPrxV) amino acid sequences with mammalian PrxVs and its developmental and tissue expression. (A) The amino acid sequence alignments among hPrxV, rPrxV, mPrxV, and dPrxV show high identities (58%, black background) and similarities (gray background). Conserved 2Cysteine residues were boxed. (B) dPrxV was expressed throughout the developmental stages. (C) In the larval stage, dPrxV was expressed in central nervous system (CNS), fat body (FB), gut, and malphigian tubules (MT). Data are presented as mean ± S.E.M. ⁄P < 0.05 and ⁄⁄P < 0.001.
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around 3–4 h after Ecc15-GFP infection. These results indicate that Ecc15-GFP induces ROS, Duox mRNA, and PrxV mRNA and protein in the fly gut during the early stage of infection. 3.3. Duox regulates dPrxV expression, which may control residual ROS in the fly gut To find out whether Duox regulates dPrxV expression, we measured dPrxV levels in the fly gut at the different Duox genetic backgrounds. When Duox was overexpressed in the gut epithelia by the NP3084-Gal4 driver (NP3084 > Duox), dPrxV expression was increased compared with the NP3084-Gal4 control. On the contrary, when Duox was inhibited in the gut epithelia (NP3084 > Duox-Ri), dPrxV expression was reduced Fig. 3(A) and (B). These results indicate that Duox regulates dPrxV expression in the fly gut. Since Duox generates ROS in the fly gut to combat infected microbes and residual ROS should be eliminated to pro-
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tect the host gut (Ha et al., 2009), we checked whether dPrxV removes residual ROS in the gut. dPrxV overexpression (DA > dPrxV) in the Ecc15-GFP infected fly gut reduced intracellular ROS levels compared with the DA-Gal4 control while dPrxV inhibition (DA > dPrxV-Ri) increased intracellular ROS levels at 2 h and 4 h after infection (Fig. 3(C) and 3D). In contrast, dPrxV overexpression and inhibition did not change diffused ROS levels in gut lumen (Supplementary Fig. 1B). These results indicate that dPrxV can protect bacterial infection induced ROS in the fly gut epithelia. 3.4. JNK/FOXO signaling induces dPrxV expression in fly gut by the Ecc15-GFP oral infection In Drosophila melanogaster, oxidative stress activates JNK/FOXO signaling to induce target genes which protect flies (Wang et al., 2003, 2005). When wild-type flies were infected with Ecc15-GFP,
Fig. 2. Ecc15-GFP oral infection induces Duox and dPrxV expression in the Drosophila melanogaster gut. (A) In the Ecc15-GFP oral infection, GFP was detected in the fly abdomen. (B) The intracellular ROS level in the infected gut epithelia. (C, D) RT-qPCR analysis of Duox and dPrxV expression levels after Ecc15-GFP infection. (E) dPrxV protein expression and quantitation after Ecc15-GFP infection. b-actin was the loading control. Data is presented as mean ± S.E.M. ⁄P < 0.05 and ⁄⁄P < 0.001.
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Fig. 3. Duox regulates dPrxV expression, which may control residual ROS in the fly gut. (A) dPrxV expression was increased in the Duox gut overexpression (NP3084 > Duox) while it was suppressed in the Duox gut inhibition (NP3084 > Duox-Ri) compared to the NP3084-Gal4 control. (B) Duox expression levels in NP3084-Gal4/+, NP3084 > Duox, and NP3084 > Duox-Ri fly gut. (C) dPrxV overexpression (DA > dPrxV) in the Ecc15-GFP infected fly gut reduced intracellular ROS levels compared with the DA-Gal4/+ control while dPrxV inhibition (DA > dPrxV-Ri) increased ROS levels. (D) dPrxV expression levels in DA-Gal4/+, DA > dPrxV, and DA > dPrxV-Ri fly gut. Data are presented as mean ± S.E.M. ⁄ P < 0.05 and ⁄⁄P < 0.001.
active phospho-JNK was detected immediately after infection and reduced from 3 h in the fly gut (Fig. 4A). JNK inhibition in the bacteria infected fly gut (NP3084 > bsk-Ri) did not induce dPrxV expression and the NP3084-Gal4 control induced dPrxV expression in the early stage of infection (Fig. 4B). NP3084 > bsk-Ri did not activate JNK in the fly gut (Supplementary Fig. 1C). These results suggest that residual ROS can activate JNK and induce dPrxV expression. Next, to find out whether FOXO is a transcriptional factor for the dPrxV induction, we examined the localization FOXO during the gut infection. FOXO, a forkhead domain-containing transcription factor, is a key regulator of growth, tolerance to stress, metabolism, and life span in various organisms including Drosophila melanogaster (Accili and Arden, 2004; Hwangbo et al., 2004). FOXO was localized mainly in cytoplasm in the non-infected control while FOXO was localized in nuclei in the infected gut (Fig. 4C). dPrxV expression was almost absent in the infected gut of the FOXO null mutant (FOXO21/25) while dPrxV was expressed in the w- control fly gut (Fig. 4D). Neuronal expression of FOXO also increased dPrxV expression (Supplementary Fig. 1D). These findings indicate that JNK/FOXO signaling induces dPrxV expression in the fly gut after Ecc15-GFP infection. 3.5. The lethality of dPrxV mutants is increased during bacterial infection To find the role of dPrxV, we measured the lethality. In both male and female dPrxV mutant flies, Ecc15 infection increased lethality compared to the w- control flies (Fig. 5A, B). dPrxV inhibition
(DA > dPrxV-Ri) also increased lethality compared to the DA-Gal4/+ control (Supplementary Fig. 2). These results demonstrate that dPrxV is critical for survival by protecting the host gut against bacterial infection induced ROS.
4. Discussion Among antioxidant enzymes, an immune responsive catalase (IRC) is a key enzyme to remove the infection induced ROS in Drosophila melanogaster. IRC knock-down flies show high mortality after gut infection by non-lethal bacteria (Ha et al., 2005b). IRC is a secretory antioxidant enzyme which removes the total in vivo ROS including gut lumen (Ha et al., 2005b). In this study, we detected that infection-induced intracellular ROS was reduced by dPrxV, whereas the level of ROS in gut lumen did not change by PrxV (Fig. 3C, Supplementary Fig. 1B). In addition, total IRC mRNA is dramatically increased by septic injury (Ha et al., 2005b). These suggest that IRC removes total in vivo ROS induced by septic injury or gut infection while dPrxV has a limited role which controls intracellular ROS during gut infection. Duox activity and expression are required to produce ROS which kills infected bacteria in the fly gut. When gut cells come in contact with bacteria, active Duox immediately produces ROS to kill the bacteria. Then, when the gut needs more ROS, Duox expression is increased (Ha et al., 2009). The first peak of ROS in Fig. 2B seems to be produced by the active Duox and the second peak of ROS may be a result of increased Duox expression.
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Fig. 4. JNK/FOXO signaling induces dPrxV expression in the fly gut by Ecc15-GFP oral infection. (A) Activated JNK was detected after Ecc15-GFP oral infection. b-actin was a loading control. (B) JNK inhibition in the fly gut (NP3084 > bsk-Ri) did not induce dPrxV expression while the NP3084-Gal4/+ control induced dPrxV expression in the early stage of infection. (C) FOXO was localized mainly in the cytoplasm of the non-infected control fly gut epithelia while FOXO was localized in the nuclei (arrows) of the infected gut. (D) dPrxV expression was almost absent in the infected gut of the FOXO null mutant (FOXO21/25) while dPrxV was expressed in the w- control fly gut. Data are presented as mean ± S.E.M. ⁄P < 0.05 and ⁄⁄P < 0.001.
Oxidative stress modulates the activation of JNK pathway by Ecc15 infection within 30 min (Buchon et al., 2009a). JNK signaling mediates the nuclear localization of FOXO and the expression of antioxidant enzymes (Biteau et al., 2011). In Drosophila melanogaster, the activation of JNK signaling induces resistance to oxidative stress by changing the cellular transcriptome (Wang et al., 2003). The transcription of Jafrac1 regulated by JNK/FOXO signaling promotes resistance to oxidative stress and extends life span (Lee et al., 2009). Recent studies show that dPrxV is associated with longevity and immune response. dPrxV deficient flies are vulnerable
to oxidative stress and apoptosis, and reduced life span (Radyuk et al., 2009). Contrary to our data, dPrxV deficient flies are more resistant to the septic infection and dPrxV is considered to be the negative regulator of JNK during septic infection induced-immune response (Radyuk et al., 2010). Unexpectedly, ubiquitous overexpression of dPrxV (DA > dPrxV) increased the lethality of the gut infection compared to the DA-Gal4/+ control (Supplementary Fig. 2). The lethality is worse than that of the dPrxV inhibition (DA > dPrxV-Ri). Recently, Wu et al.,(2012) demonstrates that local production of ROS by gut
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Fig. 5. dPrxV mutants increase lethality during bacterial infection. In both male (A) and female dPrxV mutant flies (B), non-lethal pathogen Ecc15 infection increased lethality compared with the w-control.
infection triggers systemic AMP production in fat body. Therefore, overexpression of dPrxV (DA > dPrxV) eliminates residual ROS and may not trigger enough AMP production in fat body, which results in increased lethality. In this study, we show that dPrxV is a novel target for JNK/ FOXO signaling during bacterial infection in gut. Active JNK level peaked within 1 h and then gradually reduced until 5 h by gut infection (Fig. 4A) but dPrxV expression gradually increased and peaked around 4 h after gut infection (Fig. 2D). This data suggests that ROS induced JNK activation may lead to dPrxV expression and then clearance of ROS by dPrxV results in decreased JNK activity. We can speculate that dPrxV mutants die early because a certain level of ROS can activate JNK continuously and induce JNK mediated apoptosis in the fly gut. Our findings provide important information of how host animals protect their gut against microbial infection. Acknowledgements This work was supported by the grants from the Research Foundation of Korea (2009-0080870 and 2011-0030134) and KRIBB Research Initiative Program. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.dci.2012.07.002.
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