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The Drosophila Imd Signaling Pathway Henna Myllymäki, Susanna Valanne and Mika Rämet J Immunol 2014; 192:3455-3462; ; This information is current as doi: 10.4049/jimmunol.1303309 of September 26, 2021. http://www.jimmunol.org/content/192/8/3455 Downloaded from References This article cites 125 articles, 49 of which you can access for free at: http://www.jimmunol.org/content/192/8/3455.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2014 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Th eJournal of Brief Reviews Immunology

The Drosophila Imd Signaling Pathway x Henna Myllyma¨ki,* Susanna Valanne,* and Mika Ra¨met*,†,‡, The fruit fly, Drosophila melanogaster, has helped us to controls the expression of a limited set of immune response genes understand how innate immunity is activated. In ad- in Drosophila. The immune deficiency (Imd) pathway, which dition to the Toll receptor and the Toll signaling path- regulates the activity of a third Drosophila NF-kBproteincalled way, the Drosophila immune response is regulated by Relish (19), controls the expression of most of the Drosophila another evolutionarily conserved signaling cascade, the AMPs and, thus, is indispensable for normal immunity (20). immune deficiency (Imd) pathway, which activates NF- kB. In fact, the Imd pathway controls the expression of Peptidoglycan-recognition proteins and activation of the Imd pathway most of the antimicrobial peptides in Drosophila;thus, Microbial recognition is the first step in initiating the immune response via the Imd signaling pathway. For pathogen de- it is indispensable for normal immunity in flies. In this Downloaded from article, we review the current literature on the Drosophila tection, the innate immune system uses specific, genome- Imd pathway, with special emphasis on its role in the encoded pattern recognition molecules that bind conserved (patho)physiology of differentorgans.Wediscussthesys- structures, which are found on pathogens but are absent in the temic response, as well as local responses, in the epithelial host (21). Peptidoglycan (PGN) is a good example of such a structure: it is present on most bacteria and is composed of and mucosal surfaces and the nervous system. The b Journal of Immunology, 2014, 192: 3455–3462. conserved polymers of -1,4-linked N-acetylglucosamine and http://www.jimmunol.org/ N-acetylmuramic acid cross-linked by short stem peptides, which vary between different types of bacteria. PGN is rec- ulticellular organisms constantly encounter po- ognized and bound by conserved host proteins, called PGN- tentially harmful microorganisms. Although insects recognition proteins (PGRPs), which share a 160-aa PGRP M lack an adaptive immune system in the classical domain. Mammals have a family of four PGRPs, whereas sense, it has long been recognized that they too possess powerful insects have more (e.g., 13 genes coding for 19 proteins in means of fighting infections, such as the ability to phagocytose Drosophila) (22, 23). The mammalian PGRPs are secreted bacteria and encapsulate parasites. In addition, insects can proteins that bind bacterial muramyl peptides. Some mam- mount a humoral response against intruders. This is charac- malian PGRPs have an amidase activity, probably to eliminate by guest on September 26, 2021 terized by the secretion of antimicrobial peptides (AMPs) into the proinflammatory PGN, whereas others are more diverged the hemolymph. This aspect was analyzed carefully first by from the insect genes and function directly as bactericidal Boman et al. (1), who eventually isolated the first AMPs from proteins (24–26). It appears that mammalian PGRPs do not the silkmoth Hyalophora cecropia (2–4). The cloning of more possess signaling activity. AMPs from Drosophila and other insect models revealed con- Insect PGRPs can act both as enzyme-active amidases that served binding sites for NF-kB proteins in their promoter degrade PGN and activate signal-transduction pathways and regions. This suggested that the mechanisms that induce AMP proteolytic cascades (Fig. 1). Insect PGRPs are classified as production in response to infection might be evolutionarily short (S) or long (L), according to their transcript size: short conserved (5–7). In addition to its function in development, PGRPs have signal peptides and can be extracellular proteins, the dorsal gene, coding for an NF-kB transcription factor, was whereas long PGRPs can be intracellular, extracellular, and discovered to mediate immune responses in adult flies, together transmembrane proteins. Of the Drosophila PGRPs, PGRP- with another NF-kB protein-coding gene, DIF, and an addi- SA and PGRP-SD are needed for activating the Toll pathway tional set of genes, including Toll, tube, and pelle, belonging to (14, 27, 28). No immunological in vivo phenotype has been theTollsignalingpathway(8–13,reviewedinRef.14).Subse- demonstrated for the amidases PGRP-SB1 and PGRP-SB2, quently, genes coding for TLRs also were found in the human even though PGRP-SB1 is strongly induced postinfection, genome. The discovery of these pattern recognition receptors and its bactericidal activity is shown in vitro (29–31). The highlighted similarities between the human and Drosophila in- function of PGRP-LD is unclear. The other PGRPs either nate immune systems (15–18). However, the Toll pathway only positively or negatively regulate the Imd pathway.

*Laboratory of Experimental Immunology, BioMediTech, University of Tampere, FIN- Abbreviations used in this article: AMP, antimicrobial peptide; Iap2, inhibitor of apo- 33014 Tampere, Finland; †Department of Pediatrics, Tampere University Hospital, ptosis 2; IKK, IkB kinase; Imd, immune deﬁciency; PGN, peptidoglycan; PGRP, FIN-33521 Tampere, Finland; ‡Department of Pediatrics, Medical Research Center peptidoglycan-recognition protein, TCT, tracheal cytotoxin. x Oulu, University of Oulu, FIN-90014 Oulu, Finland; and Department of Children and Adolescents, Oulu University Hospital, FIN-90029 Oulu, Finland Copyright Ó 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00 Received for publication December 10, 2013. Accepted for publication February 11, 2014. Address correspondence and reprint requests to Prof. Mika Ra¨met,UniversityofTampere, FIN-33014 Tampere, Finland. E-mail address: mika.ramet@uta.ﬁ

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1303309 3456 BRIEF REVIEWS: THE DROSOPHILA Imd SIGNALING PATHWAY

is sufficient to activate AMP expression, in a cell-autonomous fashion, in the absence of infection. It also was shown that cytoplasmic PGRP-LE can activate the Imd pathway, independently of PGRP-LC, by interacting with Imd (40–43). PGRP- LE is the only intracellular pathogen receptor identified in Dro- sophila. The intracellular form is able to activate autophagy, whereas the transmembrane form can activate a prophenoloxidase cascade together with PGRP-LC (42, 44, 45). PGRP-LF is a transmembrane protein that resembles PGRP- LC but lacks the intracellular signaling domain and does not bind PGN. PGRP-LF acts as an inhibitor of Imd signaling by binding PGRP-LC and preventing its dimerization (46–48). PGRP-LA is also predicted not to bind PGN and recently was shown to be dispensable for systemic infections. However, consistent with its expression profile, PGRP-LA appears to positively regulate the Imd pathway in barrier epithelia, such as the trachea and the gut (49). PGRP-LB, PGRP-SC1A,

PGRP-SC1B, and PGRP-SC2 have an amidase activity and Downloaded from are shown to play somewhat redundant roles in down- regulating the Imd pathway during a systemic response. PGRP-LB is the major regulator in the gut. The amidase PGRPs digest PGN into short, nonimmunogenic or less im- munogenic fragments and, therefore, prevent or reduce the

activation of defense mechanisms (30, 31). http://www.jimmunol.org/

Regulation of the Imd signaling pathway The Drosophila Imd pathway is often compared with TNFR signaling in mammals, although the Imd pathway also shares similarities with the TLR signaling pathways. The Imd and TNFR signaling pathways are shown schematically in Fig. 2, with the conserved signaling molecules indicated by similar shapes and colors. In essence, activation of the Imd pathway by guest on September 26, 2021 and TNF-a target genes require activation of the transcription factor Relish and NF-kB, respectively (19). In mammalian NF-kB signaling, activation of the transcription factor is achieved by phosphorylation of the inhibitor protein IkBby FIGURE 1. The Drosophila PGRPs and their roles in immunity. References the IkB kinase (IKK) complex. The phosphorylated IkBis are shown in parentheses. Lys-type PGN, lysine-type PGN. degraded, and NF-kB is released.

PGRP-LC is a transmembrane receptor that preferably binds a meso-diaminopimelic acid–type PGN found on Gram- negative bacteria and certain Gram-positive bacteria, such as Bacillus spp. (32, 33). It functions as the principal receptor for mediating the activation of the Imd pathway in a systemic infection and locally in the most anterior part of the midgut (34–37). PGRP-LC is spliced into several isoforms, three of which have been characterized. PGRP-LCx recognizes polymeric PGN; PGRP-LCa does not directly bind PGN, but it acts as a coreceptor with PGRP-LCx to bind monomeric PGN fragments called tracheal cytotoxin (TCT) (32, 38, 39). PGRP-LC mutant ﬂies are still responsive toward TCT. This is due to PGRP-LE, which is found in two forms (40, 41). The short form is secreted and binds PGN in the hemolymph (42) and is thought to assist Imd signaling by presenting PGN to PGRP-LC, although the export mechanism of PGRP-LE has not been characterized. The full-length PGRP-LE remains in FIGURE 2. the cytoplasm, where it is thought to recognize TCT frag- Schematic representation of the Drosophila Imd pathway and human TNFR signaling. Conserved components are indicated by similar ments that gain access to the cell. Binding of TCT leads to the shapes and colors. bend, bendless; eff, effete; Key, Kenny; K63 Ub, K63 oligomerization of cytoplasmic PGRP-LE in a head-to-tail polyubiquitination; NEMO, NF-kB essential modulator; TAB, TAK1-binding fashion (39). Ectopic expression of PGRP-LE in the fat body protein; TRADD, TNFR1-associated death domain. The Journal of Immunology 3457

However, like in the mammalian Rel proteins p100 and proteins, Ird5 and Kenny, are associated with SUMO pro- p105, IkB is contained in the C terminus of Relish. This is teins, but only Ird5 is sumoylated on a conserved site, K152, thought to mask the nuclear localization signal in the N ter- which is needed for the activation of the Imd pathway (82). A minus and inhibit Relish dimerization by the Rel homology proteomics analysis described in the same study identified 102 domain. Therefore, in addition to phosphorylation, the acti- gene products that interacted with the core Imd pathway vation of Relish requires cleavage of the inhibitory C-terminal components and affected the activity of several reporter genes part. This is likely accomplished by the caspase Dredd, be- in response to a Gram-negative bacterial challenge in RNA cause it was shown to cleave Relish in vitro (50). interference assays. These gene products included a number Binding of PGN to the receptor results in recruitment of of histone acetyltransferases and components of chromatin- a signaling complex consisting of Imd, a death domain pro- remodeling complexes, such as the SWI/SNF complex. These tein homologous to mammalian RIP1, the adaptor protein discoveries indicate that the regulation of the Imd pathway dFadd, and the caspase-8 homolog Dredd (20, 51–53). Dredd involves DNA modifications (82). The Imd pathway is also becomes activated by ubiquitination by the E3-ligase inhibi- positively regulated by a nuclear protein called Akirin (83). A tor of apoptosis 2 (Iap2) (54), which associates with the E2- recent study showed that, during myogenesis, Akirin coloc- ubiquitin–conjugating enzymes UEV1a, Bendless (Ubc13), alizes and genetically interacts with subunits of the SWI/SNF- and Effete (Ubc5) (55). Once activated, Dredd cleaves Imd, class chromatin-remodeling complex to optimize gene ex- removing a 30-aa N-terminal fragment, and creates a novel pression by the transcription factor Twist. It remains to be binding site for Iap2, which can then K63-ubiquitinate Imd studied whether Akirin provides a similar link between tran- Downloaded from (54, 56). This leads to the recruitment and activation of the scription factors and the chromatin-remodeling machinery in Tab2/Tak1 complex, which is responsible for the phosphor- Imd signaling (84). Akirin shows high conservation in both ylation and activation of the Drosophila IKK complex (57– sequence and function, and in addition to Drosophila, it was 60). Relish is activated by the phosphorylation of multiple found to positively regulate NF-kB signaling in other insects, sites at its N terminus by the IKK complex (59). It was shown mammals, and reptiles (83). Akirin may even provide a target that phosphorylation of serine residues S528 and S529 is Ag for the development of a vaccine against insect vector- http://www.jimmunol.org/ required for efficient recruitment of RNA polymerase II to borne diseases (e.g., Ref. 85). the promoters of Relish target genes (59, 61). The C-terminal Moreover, the activity of the Imd pathway is negatively reg- part (Rel-49) remains in the cytoplasm, and the active ulated at the transcriptional level by the transcription factors N-terminal part (Rel-68) translocates into the nucleus to acti- zfh1 and the AP-1/Stat92E complex. Zfh1 strongly down- vate the transcription of genes coding for AMPs, such as regulates the Imd pathway activity in vitro, but its role in vivo Diptericin and Cecropin (50, 62). Like in mammalian TNFR is more difficult to study because of developmental effects signaling, the Drosophila Imd pathway bifurcates into the JNK (83, 86–88). The JNK pathway transcription factor AP-1,

pathway at the level of Tak1 and Tab2 (63–65). the Stat92E transcription factor, and Dsp1 can form a complex by guest on September 26, 2021 The activity of Imd signaling is fine-tuned at multiple levels that binds to the promoters of Relish target genes. Complex by several molecules and mechanisms (66–68). For example, formation leads to recruitment of the histone deacetylase the ubiquitination state of various pathway components is dHDAC1, the replacement of Relish, and target gene down- delicately regulated. The E3-ligase Iap2 has been identified in regulation, indicating that cross-talk between the Drosophila many large-scale screens for Imd pathway regulators (60, 66, immune signaling pathways is needed for proper regulation of 69, 70), and it also was shown to be essential for Imd path- these genes (86, 87). way–mediated AMP expression and bacterial resistance in One of the target genes of the mammalian NF-kB protein is vivo (60, 65, 71). In addition, a number of ubiquitinating and its own inhibitor, IkB. This creates a negative feedback loop, deubiquitinating enzymes have been implicated in the nega- which is a major regulatory mechanism for the signaling tive regulation of the signaling pathway. The ubiquitin-specific pathways (89). Because Relish itself contains IkB, this method protease dUSP36/Scny prevents the accumulation of the ac- of regulation is not feasible in Drosophila. Instead, the Imd tivated, K63-ubiquitinated Imd and promotes its K48-linked pathway is subject to other negative feedback mechanisms, ubiquitination and subsequent degradation (72). faf is a deu- such as the rapid induction of a gene called pirk (90, 91). Pirk biquitinating enzyme that also was proposed to regulate the expression appears to be induced by the Ras/MAPK pathway ubiquitination state of Imd (73). Tak1 is targeted for ubiq- as well, suggesting that Ras/MAPK signaling functions in uitination and degradation by the RING-finger protein immunity by negatively regulating the Imd pathway (92). POSH (74). CYLD is a deubiquitinating enzyme that down- Pirk interacts with the receptors PGRP-LC and PCRP-LE regulates mammalian TNFR signaling by inactivating TRAF2. and the adaptor protein Imd, thus likely interrupting signal Drosophila CYLD interacts with the IKK protein Kenny and transduction at the receptor complex (91, 93, 94). also negatively regulates Imd signaling (75, 76). Activated 20-Hydroxyecdysterone (ecdysone) is a steroid hormone Relish is targeted for ubiquitination and proteasomal degra- known to coordinate tissue remodeling in flies undergoing dation by the SCF complex (77), possibly promoted by the metamorphosis, including the phagocytosis of dead cells. In ubiquitin-binding protein dRYBP (78). Dredd is inhibited by addition, it is needed for the optimal phagocytic activity of the RING-domain containing protein Dnr1 (79, 80), whereas hemocytes in response to bacteria (95). Ecdysone also was Caspar, a homolog of the mammalian FAF1, inhibits the reported to regulate the activity of the Imd pathway in adult Dredd-mediated cleavage of Relish (81). flies via its target genes, whose products are needed for In addition to ubiquitination, another posttranscriptional PGRP-LC expression. In addition, some of these proteins modification that regulates signaling molecules is the addition appear to directly regulate the expression of a subset of the of SUMO. Recently, it was shown that both Drosophila IKK Imd pathway target genes (96). 3458 BRIEF REVIEWS: THE DROSOPHILA Imd SIGNALING PATHWAY

The role of Imd-pathway activation in systemic and epithelial immunity saliva that rinses through the digestive system (101). Impor- In addition to initiating the systemic response in the fat body, tantly, there are marked differences in the sets of genes in- the Imd pathway is activated locally in several tissues (Fig. 3). duced in different tissues. For example, a microarray study Inducible immune responses in the epithelial and mucosal comparing the immune response in the fat body, gut, and surfaces, such as the Drosophila tracheal airway system and trachea showed that there is only a small set of “universal” the digestive system, are important, because these surfaces are Imd pathway target genes, mainly consisting of AMPs and constantly exposed to potentially pathogenic bacteria, fungi, pathway components; the rest of the infection-inducible genes and yeast (97, 98). In the trachea, an infection triggers the aremoretissuespeciﬁc(49).Also, Imd pathway–mediated Imd pathway–mediated reactivation of a set of tracheal de- AMPs were shown to activate speciﬁc responses in the nervous velopment genes, a survival reaction that remodels the epi- system (e.g., Ref. 102). thelial tissue damaged upon infection (99). Also, AMPs are The gut microbial community, or microbiome, is now rec- induced, and their expression is antagonized via a signaling ognized as an integral part of the host system with important route involving a Toll family member Toll-8/Tollo (100). metabolic activities (103). The Drosophila gut microbiome is Salivary glands have the potential to contribute to intestinal a dynamic structure: its composition varies widely within and immunity by secreting soluble proteins and peptides into the among Drosophila populations and species (104), and it is Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 3. Schematic representation of tissue-specific (dys)functions and the regulation of the Imd pathway. In Drosophila, systemic AMP expression is induced in the fatbody cells by PGRP-LC–mediated recognition of PGN fragments and monomers (TCT) and leads to the secretion of AMPs into the hemolymph. In the gut, the negative regulators Pirk, Caudal, PGRP-LB, and PGRP-LF maintain tissue homeostasis by preventing extensive Imd pathway activation by commensal microbes. In contrast, pathogenic bacteria are thought to secrete more PGN and, therefore, activate Imd pathway–mediated AMP expression. TCT molecules that cross the epithelial barrier and gain access to the hemolymph can also elicit a systemic response in the fatbody. PGRP-LA and Tollo regulate Imd pathway activity, specifically in the trachea (airway epithelium). In the brain, Dredd is activated independently of the upstream pathway components, and Relish-mediated AMP expression leads to neurodegeneration. Cad, Caudal; ECT4, ectoderm-expressed 4; Spz2, Spaetzle 2. The Journal of Immunology 3459 influenced by bacteria introduced into the gut by ingestion and Imd mutants do not show signs of reduced fecundity (105). The microbial community needs to balance a tolerance (number of eggs laid) after injection with killed bacteria toward commensal bacteria and alertness toward pathogenic (119). Thus, the Imd pathway appears to be involved in invasion, as well as the homeostasis between gut microbes and mediating chronic inflammation and immune senescence, but the host. There are two parallel systems in Drosophila that the exact mechanisms need to be studied further. control this host–microbe homeostasis: the DUOX pathway(s) Many human age-related neurodegenerative diseases also (106) and the Imd pathway. The Imd pathway is activated are characterized by the activation of inflammatory pathways upon PGN binding by PGRP-LC and, specifically in the gut, in neural cells, caused by the accumulation of aberrant pro- by PGRP-LE (41, 107). Evidently, mechanisms for control- tein aggregates, such as the b-amyloid fibrils in Alzheimer’s ling the immune response play a major role in maintaining disease. Therefore, an elevation in TNF-a levels and NF-kB homeostasis. The first checkpoint is PGN sensing: pathogens activity are often found to contribute to the disease pathology that divide more rapidly than commensals shed more PGN by inducing the expression of additional proinflammatory upon cell division and, therefore, get recognized by the Imd molecules or cell death (122). Similarly, involvement of the pathway, unlike commensals (reviewed in Ref. 108). The Toll or Imd pathways in neurodegeneration has been ob- second control mechanism is the expression of negative reg- served in Drosophila. An experimental bacterial infection in ulators that downregulate the Imd pathway at all of its levels. the brain causes progressive, age-dependent neurodegeneration Amidase PGRPs (Fig. 1), especially PGRP-LB (30, 37), de- at the injection site, together with locomotive defects. The grade and scavenge PGN in the gut lumen to prevent it from phenotype is dependent on Relish, and strikingly, overex- Downloaded from crossing the gut barrier. However, in some pathogenic infec- pression of individual AMPs alone in neurons or glial cells tions, PGN fragments may translocate across the gut epithe- is sufficient to cause these symptoms. This suggests that lium and trigger a systemic response (37, 109, 110) (Fig. 3). AMPs could be directly cytotoxic to brain cells (123). Dro- Some negative regulators directly bind components of the Imd sophila neurodegeneration disease models also have been used pathway: PGRP-LF dimerizes with a PGRP-LCx isoform to to study immune-related diseases in the nervous system. form a competitive nonsignaling complex (46, 47). Pirk, which Ataxia-telangiectasia is a rare disease caused by a recessive http://www.jimmunol.org/ interacts with the receptor and Imd, is needed for normal mutation of the ATM gene, which encodes a DNA damage- immune tolerance in the gut (94). Furthermore, it was pro- responsive protein kinase. In Drosophila, a temperature- posed recently that the transglutaminase protein inhibits Relish sensitive allele of ATM or the expression of an ATM RNA activity by catalyzing the polymerization of N-terminal Relish interference construct in glial cells leads to reduced longevity molecules (111). Another level of control in the immune sys- and mobility, increased death of neurons and glial cells, and tem includes Caudal, the intestinal homeobox gene (112), increased expression of immune-response genes (124). These which is expressed in a defined compartment in the adult fly changes are dependent on Relish but not Dif or Imd (102).

midgut (113). Silencing of Caudal constitutively activates Furthermore, Dredd has been associated with retinal degen- by guest on September 26, 2021 AMPs and leads to intestinal disorders, compromising the fit- eration caused by a mutation in an eye-specific phospholipase ness of the animal (112, 113). Caudal, together with other gut- C(norpA). In this model, the light-dependent endocytosis of specific transcription factors, has important functions in pat- rhodopsin, its accumulation in the late endosomes, and the terning the expression of AMPs and other intestinal genes resulting death of photoreceptor cells are not dependent on (113). the developmental apoptosis machinery but require Dredd. Moreover, this retinal phenotype was rescued by mutations in The Imd pathway in immune senescence and neurodegeneration Relish and key, but not Imd or Fadd, suggesting an alternative In humans, aging is associated with changes in immunity, such means for Dredd regulation. In addition, overexpression of as upregulation of the innate immune system. This can lead to active Relish during development has detrimental effects on chronic inflammation, with immune functions actually dete- various tissues and cell types, such as neurons, photoreceptor riorating, making the elderly more susceptible to infections and eye cells, and the wing (125). Thus, several Drosophila (114). In Drosophila, aging is also characterized by the in- models demonstrate the activation of Relish and its target creased expression of immune-related genes, including several genes in neurodegeneration. The neurotoxicity is dependent antimicrobial peptides, PGRPs, and Relish (115–117), in- on Dredd but not the upstream Imd pathway components, creased bacterial loads (118), and more persistent AMP acti- and aberrant Dredd activation due to a mutation in the neg- vation upon infection (119). This suggests that flies might de- ative regulator Dnr1 is also sufficient to cause neurotoxicity velop a chronic inflammation-like condition resembling that (102, 123, 125). seen in humans. Genetic studies show that overexpression of The importance of the Toll pathway in Drosophila devel- PGRP-LE or PGRP-LC in the fat body activates the Imd opment is well established, and accumulating evidence sug- pathway, which enhances the flies’ resistance against patho- gests that the Imd pathway also has functions distinct from gens and has no acute effects on reproduction or general fit- its conventional roles. Therefore, Drosophila appears to be an ness. However, long-term activation of the Imd pathway creates advantageous model for studying the role of innate immunity an inflammation-like state and results in reduced lifespan, signaling pathways and inflammatory responses in various suggesting that maintaining an active immunity is physio- contexts, such as aging, and the function and dysfunction of logically costly in the long run (114, 120). Evidence sup- the nervous system. porting this hypothesis includes a delay in the age-related immune activation in flies reared on a calorie-restricted diet Conclusions (116). Moreover, Relish mutant flies live longer than do wild- The research on Drosophila immunity has blossomed during type flies on a calorie-restricted diet (121), and female Relish the past 20 years. The Imd signaling pathway has been at the 3460 BRIEF REVIEWS: THE DROSOPHILA Imd SIGNALING PATHWAY center of this investigation. Today, the genes that are required 18. Ra¨met, M. 2012. 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Steiner, and R. Dziarski. 2000. Mammalian pep- to increased research on the pathway in this context. For tidoglycan recognition protein binds peptidoglycan with high affinity, is expressed example, the trigger for the neurotoxic activation of Relish is in neutrophils, and inhibits bacterial growth. J. Biol. Chem. 275: 24490–24499. 25. Dziarski, R., and D. Gupta. 2006. The peptidoglycan recognition proteins yet to be found. (PGRPs). Genome Biol. 7: 232. 26. Swaminathan, C. P., P. H. Brown, A. Roychowdhury, Q. Wang, R. Guan, N. Silverman, W. E. Goldman, G. J. Boons, and R. A. Mariuzza. 2006. Dual Downloaded from Acknowledgments strategies for peptidoglycan discrimination by peptidoglycan recognition proteins We thank Dr. Helen Cooper for revising the language of the manuscript. (PGRPs). Proc. Natl. Acad. Sci. USA 103: 684–689. 27. Michel, T., J. M. Reichhart, J. A. Hoffmann, and J. Royet. 2001. 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