Developmental and Comparative Immunology 33 (2009) 127–134 Contents lists available at ScienceDirect Developmental and Comparative Immunology journal homepage: www.elsevier.com/locate/dci Dnr1-dependent regulation of the Drosophila immune deficiency signaling pathway Silvia Guntermann, David A. Primrose, Edan Foley * Department of Medical Microbiology and Immunology, University of Alberta, Alberta Institute for Viral Immunology, Edmonton AB, T6G 2S2, Canada ARTICLE INFO ABSTRACT Article history: Innate immunity is a critical metazoan defense strategy that rapidly detects and neutralizes invading Received 20 June 2008 microbes. As the signaling pathways that drive innate immune responses are evolutionarily conserved, Received in revised form 25 July 2008 there is considerable interest in the characterization of innate immune signaling in genetically tractable Accepted 26 July 2008 models, such as Drosophila melanogaster. Drosophila responds to detection of diamonopimelic-type Available online 5 September 2008 microbial peptidoglycan through activation of the immune deficiency (Imd) pathway, a signaling pathway with numerous similarities to the mammalian pro-inflammatory TNF pathway. In this Keywords: manuscript, we focus on a molecular and in vivo characterization of Dnr1, a putative regulator of Imd Drosophila pathway activity. A previous cell culture RNAi screen indicated that Dnr1 may serve as a negative Imd Dredd regulator of the Imd pathway. However, there are no in vivo data to validate this hypothesis and there are Dnr1 scant molecular data to identify the mechanism by which Dnr1 may inhibit the Imd pathway. In this IAP manuscript, we present in vivo data that are consistent with a negative regulatory role for Dnr1 in the Imd pathway. Additionally, we provide molecular data to indicate that Dnr1 inhibits the Imd pathway at the level of the initiator caspase Dredd. ß 2008 Elsevier Ltd. All rights reserved. 1. Introduction numerous parallels to the mammalian tumor necrosis factor (TNF) pathway (for a review of the Imd pathway see [3]). For example, The innate immune response is a powerful, phylogenetically both Imd and TNF pathways signal through conserved signal widespread first line of defense against microbial challenges [1].In transduction molecules to engage caspase, c-Jun N-terminal kinase contrast to the elaborate adaptive immune responses of higher (JNK) and NF-kB modules that coordinate a rapid response to vertebrates, innate immune responses are extremely fast and rely ongoing infections. In contrast to the TNF pathway, the transmem- exclusively on non-rearranging, germline-encoded gene products. brane peptidoglycan recognition protein (PGRP-LC) detects dia- Due to the high degree of evolutionary conservation of key minopimelic (DAP) acid-type peptidoglycan from Gram-negative regulators of innate immune signaling, genetically accessible bacteria, some Gram-positive bacteria and fungi and initiates the organisms such as the fruitfly Drosophila melanogaster are potent Imd pathway signaling cascade in response [8–12]. Imd pathway tools for deciphering innate immune signaling pathways [2,3]. For signaling proceeds through a series of signal transduction example, identification of the Toll signaling pathway as a mediator molecules and activates the JNK-responsive dAP-1 transcription of antimicrobial defenses in Drosophila led to the characterization factor and the NF-kB family member Relish (Rel). Cell culture of the critical role of Toll signaling in the activation of innate and studies indicate that the combined activities of dAP-1 and Rel adaptive defenses in higher vertebrates [4–7]. mediate the bulk of the Imd pathway transcriptional response [13]. More recently, considerable efforts have been directed to the The dJNK/dAP-1 axis of the Imd pathway is activated in a rapid and study of the Drosophila immune deficiency (Imd) signaling transitory manner. Typically, JNK activation occurs within minutes pathway, an innate immune response pathway that displays and terminates within an hour, while AP-1 transcriptional activity terminates within 4 h [13,14]. In contrast, Rel activation occurs in a more prolonged manner. Rel is a p105 homolog with an N-terminal * Corresponding author. Tel.: +1 780 4922303; fax: +1 780 4929828. NF-kB domain and a C-terminal ankyrin domain that is presumed E-mail address: [email protected] (E. Foley). to tether Rel in the cytoplasm in the absence of PGRP-LC activation Abbreviations: Imd, immune deficiency; PGRP-LC, peptidoglycan recognition [15–17]. Infection initiates a signaling cascade that culminates in protein; TNF, tumor necrosis factor; JNK, c-Jun N-terminal kinase; PGN, peptidoglycan; Rel, relish; Dnr1, defense repressor 1; att, attacin; dipt, diptericin; the proteolytic cleavage of Rel between the NF-kB and ankyrin IAP, inhibitor of apoptosis; LPS, lipopolysaccharides. domains [18,19]. The liberated NF-kB domain translocates to the 0145-305X/$ – see front matter ß 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.dci.2008.07.021 128 S. Guntermann et al. / Developmental and Comparative Immunology 33 (2009) 127–134 nucleus and drives a significant portion of the transcriptional component of the Imd pathway response. The transcriptional induction of several antimicrobial peptide genes such as diptericin (dipt) and attacin (att) are widely accepted signatures of Rel activation through the Imd pathway. While the Rel protease requires definitive biochemical identi- fication, several lines of evidence indicate that Rel cleavage depends on the activity of Dredd, a Drosophila initiator caspase with modest sequence similarities to mammalian caspase-8 [20]. dredd mutants display a significantly reduced ability to activate Imd signaling upon infection with Gram-negative bacteria and dredd mutants also display greatly enhanced lethality upon infection [21]. As Dredd forms a complex with Rel in Drosophila cell culture assays and Rel cleavage occurs at a caspase consensus cleavage site, it is widely assumed that Dredd is the protease responsible for Rel cleavage [19]. More recent data indicate that Fig. 1. Dnr1 forms a complex with Dredd. (A and B) Western blot analysis of lysates from S2 cells that constitutively express HADreddC408A probed with anti-myc (A) Dredd is also required to activate the JNK component of the Imd and anti-HA (B) antibodies. Cells were transfected with myc-tagged Dnr1 expression pathway, although the mechanism of Dredd-mediated JNK plasmids as indicated and immunoprecipitated with anti-myc antibodies. Whole cell activation remains unclear [22]. input lysates are shown in lanes 1–3 and the corresponding immunoprecipitates are Numerous genetic and molecular studies led to the identification shown in lanes 5–7. Molecular mass markers are shown in lane 4. We detect a co- of essential components of the Imd signal transduction pathway. In precipitation of HADreddC408A with both Dnr1 variants (lanes 6 and 7), while we only detect a weak precipitation of HADreddC408A in the absence of Dnr1 (lane 5). contrast, there are only a limited number of reports on negative regulators of Imd pathway signaling [23–28]. A recent report identified defense repressor 1 (Dnr1) as a putative negative serum, 50 U penicillin/ml and 50 mg streptomycin/ml (all Gibco). regulator of Dredd activity in the Imd pathway [29].Dnr1isa For transient transfections, plasmid DNA (1 mg/106 cells) was conserved protein with an N-terminal FERM domain and a C- incubated with DDAB (40 ml/106 cells) and serum-free cell culture terminal RING domain. RING domains are established E3 ubiquitin medium (20 ml/106 cells) for 20 min at room temperature. The ligases that target substrates for proteasomal degradation. The RING transfection mix was then added dropwise to S2 cells (106 cells per domain of Dnr1 shares striking similarities with the C-terminal RING ml) and incubated overnight at 25 8C. Stable S2 cell lines were domains frequently found on a family of caspase inhibitors known as generated by transfecting cells with the respective plasmids and inhibitor of apoptosis (IAP) proteins [30]. Depletion of Dnr1 from the pCoHygro hygromycin B resistance selection plasmid (Invitro- Drosophila S2 cells by RNAi led to the transcriptional activation of a gen) at a ratio of 19:1. Transfections were performed with 3 Â 106 Dredd/Rel responsive reporter construct (dipt-lacZ). Peptidoglycan- S2 cells in 3 ml cell culture medium and cells were passaged mediated activation of Dredd/Rel signaling also resulted in the with cell culture medium containing hygromycin B (300 mg/ml, transient stabilization of Dnr1 in S2 cell culture assays. These data Sigma) for three weeks to select for stable transfected cell lines. led to the proposal that Dnr1 is a negative regulator of the Imd For immunoprecipitation assays, S2 cells that stably express pathway and that Rel activation results in the stabilization of its own HADreddC408A were transiently transfected with mycDnr1 or inhibitor (Dnr1), thereby contributing to termination of Imd mycDnr1C563Y expression plasmids and incubated for 24 h at pathway signaling. Consistent with a role for Dnr1 in the regulation 25 8C. Cells were then collected by centrifugation at 1000 Â g for of caspase activity, we recently demonstrated that overexpression of 3 min and lysed in 1 ml lysis buffer (50 mM HEPES (pH 7.5), 10 mM Dnr1 in S2 cells blocked pharmacological or genetic induction of EDTA (pH 8.0), 50 mM KCl, 50 mM NaCl, 1 mM MgCl2, 0.1% NP40, apoptosis, while depletion of Dnr1 from S2 cells sensitized the cells protease inhibitors (Roche inhibitor cocktail tablets), phosphatase to pro-apoptotic signals [31]. inhibitors (Sigma, phosphatase inhibitor cocktail)) for 10 min at In this study, we present a comprehensive cell culture and in vivo 4 8C. Cellular debris was cleared by centrifugation at 21,000 Â g for characterization of interactions between Dnr1 and the Imd pathway. 10 min at 4 8C and the supernatant was incubated with rabbit anti- We demonstrate that Dnr1 forms a complex with Dredd in S2 cells, myc antibodies (Sigma) overnight at 4 8C. The samples were and that overexpression of Dnr1 significantly decreases Dredd incubated with Protein G Sepharose (Amersham Biosciences) for proteins levels.