The identification of the Drosophila melanogaster Toll pathway cascade and the subsequent characterization of TLRs have reshaped our understanding of the immune system. Ever since, Drosophila NF-κB signaling has been actively studied. In flies, the Toll receptors are essential for embryonic development and immunity. In total, nine Toll receptors are encoded in the Drosophila genome, including the Toll pathway receptor Toll. The induction of the Toll pathway by Gram-positive bacteria or fungi leads to the activation of cellular immunity as well as the systemic production of certain antimicrobial peptides. The Toll receptor is activated when the proteolytically cleaved ligand Spatzle binds to the receptor, eventually leading to the activation of the NF-κB factors Dorsal-related immunity factor or Dorsal. In this study, we review the current literature on the Toll pathway and compare the Drosophila and mammalian NF-κB pathways.

The Toll pathway was initially identified in a series of genetic screens for genes involved in early Drosophila embryonic development. These screens were based on the revolutionary saturation mutagenesis screen developed by C. Nüsslein-Volhard and E.F. Wieschaus, who identified 15 genes that control embryonic segmentation (1). This approach earned them, together with E.B. Lewis, the Nobel Prize in Medicine in 1995 (http://nobelprize.org/nobel_prizes/medicine/laureates/1995/). Subsequent genetic screens led to the discovery of genes important in the dorsal-ventral (DV) patterning of the embryo (i.e., the dorsal group of genes, including Toll, tube, pelle, cactus, the NF-κB homolog dorsal, and seven genes upstream of Toll) (2).

Because NF-κB was implied to be involved in mammalian immunity, and because the moth Hyalophora cecropia expresses an NF-κB–like immunoresponsive factor (3), it gradually became evident that parallels between the signaling pathways in Drosophila embryonic development and activation of the immune system exist (4). Hultmark and colleagues (5) first identified Toll (Toll-1) as an activator of the immune response in a Drosophila cell line in 1995. Around the same time, a human homolog of Toll was identified and mapped to chromosome 4p14 (6). Soon after this, a compelling in vivo study in Drosophila demonstrated that the DV regulatory gene cassette signaling from the Toll ligand spatzle to cactus is involved in the antifungal response in Drosophila (7). The first mammalian TLR was described 1 y later in 1997 (8). This was shortly followed by the characterization of five human TLRs (9) establishing the role of the Drosophila Toll pathway as an evolutionarily conserved signaling cascade. However, mammalian TLRs are believed to have no role in development (10), whereas the Drosophila Toll pathway is involved both in immunity (7) and developmental processes (2, 11, 12).

The Drosophila immune system is composed of humoral and cellular components. A Gram-positive or fungal infection triggers the activation of the Toll pathway, which leads to the systemic production of antimicrobial peptides (AMPs) (13, 14). The antifungal peptide Drosomycin appears to be the principal target of the Toll humoral response. The Toll pathway also plays a role in the cellular immune response, which includes the phagocytosis of microbes, and the encapsulation and killing of parasites (15). Infecting Drosophila with the parasitic wasp Leptopilina boulardi activates a cellular immune response (16), which is manifested by increased production of circulating plasmatocytes, and the differentiation of a group of plasmatocytes into another specialized class of hemocyte, the lamellocyte. Lamellocytes participate in the encapsulation and killing of the parasite. Mutations in the gene cactus, a gain-of-function mutation in the Toll receptor gene, or the constitutive expression of dorsal can induce lamellocyte differentiation and cause the formation of melanotic tumor phenotypes (12, 17). Moreover, the Toll signaling pathway together with other pathways has been found to control hemocyte proliferation and hemocyte density (16, 18). In Drosophila larvae, Toll signaling is required for melanization (19). In gain-of-function Toll mutant flies, or cactus mutant flies that exhibit melanotic tumor phenotypes, the Toll-responsive NF-κB factor Dorsal is constitutively nuclear (17). However, this melanotic tumor phenotype is independent of Dorsal, suggesting a redundant role for Dorsal and the Dorsal-related immunity factor (Dif) in this context (17).

The cellular response can also affect the activation of the Toll pathway. Under normal conditions, Spn77Ba, a protease inhibitor of the serpin family, inhibits a phenol oxidase protease cascade. It was reported that tracheal melanization resulting from Spn77Ba disruption induces the systemic expression of the antifungal Drosomycin via the Toll pathway (20). Such signaling between local and systemic immune responses may be an alarm mechanism that prepares the host in case a pathogen breaches the epithelial barrier (20).

To date, nine genes encoding Toll-related receptors have been identified in the Drosophila genome. Toll, or Toll-1, was the first Toll identified and is responsible for AMP induction via the Toll pathway. All Drosophila Toll receptors share a similar molecular structure, with an ectodomain mainly composed of leucine-rich repeat and cystein-rich flanking motifs. Phylogenetically, Toll-5 is the closest relative to Toll (21). In contrast to other Tolls, Toll-9 has only one cystein-rich motif between the transmembrane domain and leucine-rich repeats, a structure very similar to mammalian TLRs (22). Drosophila Tolls and the IL-1Rs in mammals share a cytosolic homology domain called Toll/IL-1R (TIR) domain, which interacts with adaptor molecules, thereby activating downstream events (22).

As all mammalian TLRs are involved in the immune response, it is tempting to speculate the involvement of other Drosophila Tolls in immunity. Some Tolls could well play roles in immunological events; for example, Toll-5 may induce Drosomycin and Metchnikowin expression (21, 23, 24). In addition, Toll-5 has been shown to interact with the intracytoplasmic domains of Toll and Pelle, leading to the activation of Dorsal-dependent transcription in a synergistic manner with Toll (24). Also, Toll-9 has been reported to activate the constitutive expression of Drosomycin (25), and for this, Toll-9 may take advantage of the Toll signaling pathway components (26).

To activate the Drosophila Toll pathway either in development or in immunity, extracellular recognition factors initiate protease cascades leading to the activation of the Toll receptor ligand Spatzle ( or Spaetzle, Spätzle [Spz]) (27, 28). In nonsignaling conditions, the prodomain of Spz masks a predominantly hydrophobic C-terminal Spz region. Activation induces proteolysis, which causes a conformational change exposing determinants that are critical for binding of the Toll receptor (29). Interestingly, the prodomain remains associated with the C terminus and is only released when the Toll extracellular domain binds to the complex (30). Two models for the binding of Spz to Toll have been suggested, the first of which implies that one Spz dimer binds to two Toll receptors (31). In a newer model, two Spz dimers, each binding to the N terminus of one of the two Toll receptors, trigger a conformational change in the Tolls to activate downstream signaling (32) (Fig. 1).

FIGURE 1.

Extracellular cleavage of Spz leading to Toll pathway activation. In early embryogenesis, the protease cascade Gastrulation Defective-Snake activates the protease Easter, which cleaves full-length Spz. In the immune response, three protease cascades lead to the activation of SPE to cleave full-length Spz; the Persephone (PSH) cascade senses virulence factors and is activated by live Gram-positive bacteria and fungi. The other two cascades are activated by pattern recognition receptors binding cell wall components from Gram-positive bacteria and fungi, respectively. All cascades converge at ModSP-Grass for downstream activation of SPE. Upon proteolytical processing, the Spz prodomain is cleaved, exposing the C-terminal Spz parts critical for binding of Toll. Spz binding to the Toll receptor initiates intracellular signaling.

FIGURE 1.

Extracellular cleavage of Spz leading to Toll pathway activation. In early embryogenesis, the protease cascade Gastrulation Defective-Snake activates the protease Easter, which cleaves full-length Spz. In the immune response, three protease cascades lead to the activation of SPE to cleave full-length Spz; the Persephone (PSH) cascade senses virulence factors and is activated by live Gram-positive bacteria and fungi. The other two cascades are activated by pattern recognition receptors binding cell wall components from Gram-positive bacteria and fungi, respectively. All cascades converge at ModSP-Grass for downstream activation of SPE. Upon proteolytical processing, the Spz prodomain is cleaved, exposing the C-terminal Spz parts critical for binding of Toll. Spz binding to the Toll receptor initiates intracellular signaling.

Close modal

Spz is synthesized and secreted as an inactive precursor consisting of a prodomain and a C-terminal region (C-106) (33). In DV patterning, Spz is processed into its active C-106 form by a serine protease cascade including Nudel, Gastrulation Defective, Snake, and Easter (34, 35). In addition, sulfotransferase Pipe is required independently of the protease cascade to activate Easter (36). In microbe recognition, Spz-processing enzyme (SPE) is responsible for Spz cleavage (37). The current model for activation of SPE contains three upstream cascades depending on the activating microorganism (Fig. 1). Two protease cascades leading to the activation of Gram-positive–specific serine protease (Grass) are initiated by cell wall components of both fungi (β-glucan) and Gram-positive bacteria (Lysine-type peptidoglycan) (38). Grass was originally identified to be specifically involved in the recognition of Gram-positive bacteria (39), but was later shown to be important also for the recognition of fungal components (38). In addition, four other serine proteases, namely spirit, spheroide, and sphinx1/2, were identified in response to both fungi and Gram-positive bacteria (39). Upstream of Grass, a modular serine protease (ModSP), conserved in insect immune reactions, plays an essential role in integrating signals from the recognition molecules Gram-negative binding protein (GNBP) 3 and PGN recognition protein (PGRP)-SA to the Grass-SPE-Spatzle cascade (40). A third protease cascade leading to the activation of SPE is mediated by the protease Persephone, which is proteolytically matured by the secreted fungal virulence factor PR1 (41) and Gram-positive bacterial virulence factors (38). Similar detection mechanisms have been suggested to occur in mammals, in which TLRs or Nod-like receptors directly detect virulence factors or endogenous proteins released by damaged cells (42, 43).

The recognition of the Gram-positive bacterial lysine-type peptidoglycan and/or the β-glucan from fungal cell walls is mediated by extracellular recognition factors. GNBP3 is responsible for yeast recognition (41). The other identified factors, namely GNBP1, PGRP-SA, and PGRP-SD, appear to mainly recognize Gram-positive bacteria. Upon Gram-positive bacterial recognition, PGRP-SA and GNBP1 physically interact and form a complex (4446). Thereafter, activated GNBP1 hydrolyzes the Lys-type PGN and produces new glycan reducing ends, which are presented to PGRP-SA (47). In contrast, Buchon et al. (40) showed that full-length GNBP1 had no enzymatic activity. They suggested a role for GNBP1 as a linker between PGRP-SA and ModSP. PGRP-SD functions as a receptor for Gram-positive bacteria with partial redundancy to the PGRP-SA–GNBP1 complex (48). It appears that PGRP-SD can also recognize diaminopimelic acid (DAP)-type PGNs from Gram-negative bacteria, thereby activating the Toll pathway (49).

After binding the processed Spz, the activated Toll receptor binds to the adaptor protein MyD88 via intracellular TIR domains (5052). Upon this interaction, MyD88, an adaptor protein, Tube, and the kinase Pelle are recruited to form a MyD88-Tube-Pelle heterotrimeric complex through death domain (DD)-mediated interactions (5254). MyD88 and Pelle do not come into contact with each other; instead, two distinct DD surfaces in the adaptor protein Tube separately bind MyD88 and Pelle (52). Recently, a highly conserved Pelle/IL-1R–associated kinase (IRAK) interacting protein Pellino was shown to act as a positive regulator of Toll signaling (55). Drosophila Pellino mutants have impaired Drosomycin expression and reduced survival against Gram-positive bacteria (55). As all Pellinos contain a RING domain, it is tempting to speculate that Drosophila Pellino may ubiquitinate Pelle in a similar fashion as mammalian Pellinos polyubiquitinate IRAK1 (56).

From the oligomeric MyD88-Tube-Pelle complex, the signal proceeds to the phosphorylation and degradation of the Drosophila IκB factor Cactus. In nonsignaling conditions, Cactus is bound to the NF-κB transcription factor(s) Dorsal and/or Dif in a context-dependent manner, inhibiting their activity and nuclear localization. So, the nuclear translocation of both Dorsal and Dif requires Cactus degradation (57). To be degraded, Cactus needs to be phosphorylated, and although it has not been directly shown, it is possible that this is achieved by Pelle, because its kinase activity is required for Cactus phosphorylation (58). Also, in a recent screening (59) in which 476 dsRNA were targeted against all the known and predicted Drosophila kinases, Pelle was found to be the only kinase implicated in Cactus phosphorylation. Cactus needs to be phosphorylated in two distinct N-terminal motifs (60) that resemble IκB kinase (IKK) targets, yet the Drosophila IKK-β (Ird5) or IKK-γ (Kenny) are not involved in the Toll/Cactus pathway (61, 62). After phosphorylation, nuclear translocation of Dorsal/Dif leads to activation of the transcription of several sets of target genes. The Drosophila core Toll signaling pathway is shown in Fig. 2.

FIGURE 2.

Comparison of Drosophila Imd, Toll, and mammalian TLR signaling pathways. Homologies between signaling components are depicted by similar shape. The Imd pathway is activated by DAP-type PGN binding of the PGRP-LC dimer. Other PGRP family members play either negative or positive roles. IMD is connected to the caspase DREDD via the adaptor protein Fas-associated DD protein (FADD). DREDD proteolytically cleaves IMD and Relish. Cleaved IMD associates with the E3-ligase IAP2, E2-ubiquitin-conjugating enzymes UEV1a, Bendless (Ubc13), and Effete (Ubc5) and is K63 polyubiquitinated. This activates the downstream kinase cascade leading to the phosphorylation and activation of Relish and AP-1, which activate the transcription of AMP and stress genes, respectively. Akirin is required for Imd pathway function at the level of Relish (105). Pirk (106), Caspar (107), and Dnr1 (108) are negative regulators of the Imd pathway. The Toll pathway is activated by Spz. One Spz dimer is depicted to bind the N terminus of Toll and to induce a conformational change leading to the formation of a 4Spz:2Toll complex. Intracellular signaling leads to the phosphorylation and degradation of Cactus, which releases Dif and/or Dorsal to translocate to the nucleus and activate transcription. Gprk2 associates with Cactus in a kinase domain (KD)-dependent manner. DEAF-1 is required to induce Toll pathway target genes at or downstream of Dif/Dorsal. Mammalian TLRs are activated by bacterial-, viral-, and self-derived products. Depicted are MyD88-dependent signal transduction events. TLR1, -2, -4, -5, and -6 signal through the plasma membrane, whereas TLR7, -8, and -9 function in the endosome. TLR1, -2, -4, and -6 use the adaptors TIR domain-containing adaptor protein (TIRAP)/MyD88 adaptor-like (Mal) and MyD88, whereas TLR5, -7, -8, and -9 use MyD88 only. MyD88 recruits IRAKs and TRAF6, which activates the TAK1/TAB complex via K63-linked ubiquitination. The activated TAK1 complex stimulates the IKK complex and the MAPK pathway, thereby activating NF-κB and AP-1, respectively. Activated NF-κB translocates to the nucleus to activate transcription. The signal from the endosome activates a complex containing TRAF3 in addition to MyD88, TRAF6, IRAKs, and IKK-α. The activated complex phosphorylates and activates IFN regulatory factor 7 (IRF7) for its nuclear translocation and subsequent transcriptional activation of target genes.

FIGURE 2.

Comparison of Drosophila Imd, Toll, and mammalian TLR signaling pathways. Homologies between signaling components are depicted by similar shape. The Imd pathway is activated by DAP-type PGN binding of the PGRP-LC dimer. Other PGRP family members play either negative or positive roles. IMD is connected to the caspase DREDD via the adaptor protein Fas-associated DD protein (FADD). DREDD proteolytically cleaves IMD and Relish. Cleaved IMD associates with the E3-ligase IAP2, E2-ubiquitin-conjugating enzymes UEV1a, Bendless (Ubc13), and Effete (Ubc5) and is K63 polyubiquitinated. This activates the downstream kinase cascade leading to the phosphorylation and activation of Relish and AP-1, which activate the transcription of AMP and stress genes, respectively. Akirin is required for Imd pathway function at the level of Relish (105). Pirk (106), Caspar (107), and Dnr1 (108) are negative regulators of the Imd pathway. The Toll pathway is activated by Spz. One Spz dimer is depicted to bind the N terminus of Toll and to induce a conformational change leading to the formation of a 4Spz:2Toll complex. Intracellular signaling leads to the phosphorylation and degradation of Cactus, which releases Dif and/or Dorsal to translocate to the nucleus and activate transcription. Gprk2 associates with Cactus in a kinase domain (KD)-dependent manner. DEAF-1 is required to induce Toll pathway target genes at or downstream of Dif/Dorsal. Mammalian TLRs are activated by bacterial-, viral-, and self-derived products. Depicted are MyD88-dependent signal transduction events. TLR1, -2, -4, -5, and -6 signal through the plasma membrane, whereas TLR7, -8, and -9 function in the endosome. TLR1, -2, -4, and -6 use the adaptors TIR domain-containing adaptor protein (TIRAP)/MyD88 adaptor-like (Mal) and MyD88, whereas TLR5, -7, -8, and -9 use MyD88 only. MyD88 recruits IRAKs and TRAF6, which activates the TAK1/TAB complex via K63-linked ubiquitination. The activated TAK1 complex stimulates the IKK complex and the MAPK pathway, thereby activating NF-κB and AP-1, respectively. Activated NF-κB translocates to the nucleus to activate transcription. The signal from the endosome activates a complex containing TRAF3 in addition to MyD88, TRAF6, IRAKs, and IKK-α. The activated complex phosphorylates and activates IFN regulatory factor 7 (IRF7) for its nuclear translocation and subsequent transcriptional activation of target genes.

Close modal

The Drosophila Dorsal is a Rel protein originally identified as an important morphogen in DV polarization. In larvae and adult Drosophila, Dorsal is expressed in the fatbody, and both its expression level (63) and nuclear localization (17) are enhanced upon microbial challenge. Dorsal interacts with Pelle, Tube, and Cactus (6466), and, upon pathway activation, Dorsal translocates to the nucleus and binds to the κB-related sequence of AMP genes (63). Dorsal can activate the diptericin promoter in vitro (67), and, moreover, bacterial culture supernatants can stimulate nuclear translocation of Dorsal in vivo in dissected fatbodies in a hemolymph-dependent manner (19). Also, Dorsal activity is required to restrict the infectivity of Pseudomonas aeruginosa in adult Drosophila, providing evidence for Dorsal function in resistance against microorganisms (68).

Dif was identified in Drosophila as a dorsal-related immune responsive gene that does not participate in DV patterning. Instead, it mediates an immune response in Drosophila larvae (69) and interacts with Cactus in vitro (70). Dif (71), but not Dorsal (7), mediates Toll-dependent induction of the antifungal peptide gene Drosomycin in Drosophila adults, whereas Dorsal and Dif seem to be redundant in larvae (71, 72). Furthermore, Dif and Dorsal can form heterodimers in vitro (67), and in a Drosophila macrophage-like S2 cell line, Dorsal seems to play a more important role in Drosomycin promoter activation than does Dif (73).

Drosophila cells are ideal for large-scale in vitro RNA interference (RNAi) screens (74, 75). Long dsRNA fragments up to several kilobases are readily internalized and processed by Drosophila S2 cells, which makes RNAi in S2 cells a very feasible tool for identifying genes involved in various processes (76, 77). In general, robust degradation of target RNA is obtained without a need for any transfection reagents. RNAi screening strategies have revealed several new important findings related to Drosophila Toll signaling. Recently, deformed epidermal autoregulatory factor-1 (DEAF-1), which was first identified as a transcription factor that binds to Metchnikowin and Drosomycin promoters (78), was confirmed to be required for full Drosomycin expression as well as for defending against fungal infections (79). Moreover, endocytic machinery components, including Myopic, were indicated to play a role in the endocytosis of the Toll receptor upon pathway activation (59).

In a recent genome-wide RNAi screen in S2 cells, G Protein-coupled receptor kinase 2 (Gprk2) was identified as a regulator of the Toll pathway (73). Gprk2 was found to be important against a Gram-positive bacterial infection as well as in Toll pathway-mediated hemocyte activation in Drosophila in vivo. Gprk2 interacts with Cactus in S2 cells, but is not involved in Cactus degradation, adding a new level of complexity to Drosophila Toll/Cactus regulation (73). Other genes identified in the screen include a Friend of GATA factor U-shaped and Toll activation mediating protein (TAMP; CG15737), with a previously unknown function. RNAi knockdown of ush or TAMP was shown to reduce the activity of the Drosomycin reporter in S2 cells in vitro as well as Drosomycin expression in vivo in infected flies. However, the molecular mechanisms of the effect of these components on the Toll pathway remain to be investigated (73).

It is clear that the Drosophila Toll pathway plays a key role in Gram-positive bacterial and fungal infections (80). In turn, Imd signaling is initiated by the PGRP-LC–mediated recognition of mainly a DAP-type PGN from Gram-negative bacteria (74, 81). Imd pathway activation ultimately leads to the activation of the NF-κB factor Relish (8284), its translocation to the nucleus, and the transcriptional activation of a group of target genes including AMPs (13, 14) (Fig. 2). Although the Drosophila immunity pathways get selectively activated to a certain degree (85), synergistic interactions between the Toll and the Imd pathways have gradually become evident (73, 8688). For example, although the bacterial branch of the Toll pathway is mainly activated by a Lys-type PGN, the crystal structure of the Toll pathway mediator PGRP-SD suggests binding to a DAP-type PGN rather than a Lys-type one (49). Moreover, in a Drosophila cell line, Relish RNAi reduces the expression of the Toll10b-induced Drosomycin reporter gene (86), and the Drosomycin reporter can be synergistically activated by Toll10b and Gram-negative bacteria (73). Furthermore, the expression of Drosomycin and Defensin are best induced by the Relish/Dif and the Relish/Dorsal heterodimers, respectively (89). In vivo, postinfection with Escherichia coli, the double mutants for Dif and the Imd pathway component kenny die earlier than kenny mutants (90). The same holds for the Relish,spz, and Relish,Toll double mutants compared with Relish mutants (88).

In addition to κB binding sites for Rel proteins, the transcriptional regulation of many Drosophila AMP genes depends on GATA binding sites in their promoter proximal regions (91). Drosophila has five GATA factors, namely Pannier (dGATAa), Serpent (dGATAb), Grain (dGATAc), dGATAd, and dGATAe. Pannier and a Friend of GATA factor U-shaped were recently identified as regulators of the Toll pathway in S2 cells (73). Serpent is the major GATA transcription factor in the larval fat body, and synergy between Relish and Serpent in the activation of the full immune response in larvae has been shown (92). Moreover, evidence is presented for dGATAe-mediated immune responses in the gut (93). It appears that, in most cases, Rel proteins and GATA factors act in concert to activate immune responses. Also, at least full Metchnikowin expression requires DEAF-1 (78).

To date, 10 TLRs have been identified in humans and 12 in mice. The significance of TLRs was unknown until the mouse Tlr4 gene was identified as essential for LPS signaling (94). TLRs have since been shown to act as pattern recognition receptors for bacterial-, viral-, and self-derived products (reviewed in Ref. 95). When the signal is transduced, Tolls and TLRs associate with MyD88 via their intracytoplasmic TIR domains, activating the homologous protein kinases Pelle (in Drosophila) and IRAK (in mammals) (22). A recent study provides evidence for orthology between Tube and IRAK4 as well as Pelle and IRAK1 (96). In contrast, it has also been suggested that Drosophila Tube is at least functionally equivalent, and maybe distantly related in sequence, to the human TLR pathway adaptor protein MyD88 adaptor-like (97). In mammals, six MyD88, four IRAK4, and four IRAK2 DDs form a helical oligomer complex called Myddosome for downstream signaling (98). A similar three-component system, albeit with a different stoichiometry, is used in Drosophila: dimers of MyD88, Tube, and Pelle are needed for complex formation (54). In mammals, signal transmission downstream of MyD88 triggers the cooperation of several IRAKs, after which the IRAK complex interacts with TNFR-associated factor (TRAF) 6, which mediates the signal forward, via ubiquitination events, to the TGF-β–activated kinase 1 (TAK1) and TAK1 binding protein (TAB) complexes. TRAF homologs have been identified in the Drosophila genome, but they do not appear to participate in immune signaling (52, 86).

It appears that downstream from TAK1/TAB, the mammalian TLR pathway and the Drosophila Imd pathway, rather than the Toll pathway, share homologous components (95, 99). In mammals, the signal bifurcates at the level of a complex containing TAK1 and TABs, where one signal leads to the phosphorylation of the IKK complex and another via MAPKs to the activation of the JNK pathway and the eventual nuclear translocation of AP-1. The IKK complex phosphorylates IκB, leading to its ubiquitination and degradation. This results in the nuclear translocation of NF-κB factor(s) and the activation of transcription (95). Similarly, in the Drosophila Imd pathway, two signals from a complex containing Tak1, Tab2, and inhibitor of apoptosis 2 (86) are transmitted, one to the JNK pathway and one to the IKK complex, which phosphorylates the Rel protein Relish. After this, the caspase death-related Ced-3/Nedd2-like protein (DREDD) cleaves the C-terminal inhibitory domain of Relish (100). As was recently reported, DREDD is also involved in the cleavage of the Imd protein (101). The Drosophila Toll and Imd pathways are compared with related mammalian TLR pathways in Fig. 2.

Events downstream of MyD88 in the Drosophila Toll pathway appear somewhat different from the mammalian MyD88-dependent TLR pathways. The IKK complex is not involved in the phosphorylation and degradation of the IκB protein Cactus. However, conserved mechanisms in downstream parts of the Toll pathway and mammalian NF-κB signaling are evident. The Drosophila Gprk2 protein, which was shown to be involved in Toll pathway regulation and to interact with Cactus (73), is homologous to the murine GRK5, which was recently implicated in TNF-α–induced NF-κB signaling via direct interaction with IκB (102). Furthermore, the GRK5 knockout mice have attenuated LPS response, suggesting an evolutionarily conserved role for Gprk2/GRK5 (103).

In mammals, TLR7/TLR7, TLR9/TLR9, and TLR7/TLR8 act on the endosomal membrane also in a MyD88-dependent way to recognize nucleic acids from, for example, viruses (95, 104). Upon activation, the signal is propagated via several cytoplasmic IRAK proteins leading to the phosphorylation and nuclear translocation of IFN regulatory factor 7 (95). Interestingly, it was recently reported (59) that the Drosophila Mop (myopic) and Hrs (hepatocyte growth factor-regulated tyrosine kinase substrate), which are critical components of the endocytosis complex, colocalize with the Toll receptor in endosomes. Also, the Bro1 domain in Mop, which points to endosomal localization, is required for Toll signaling. So, it is plausible that endocytosis has an evolutionarily conserved role in Drosophila Toll and mammalian TLR signaling (59).

Since the initial discovery of the Toll pathway in fruit fly development 25 y ago, research in the field has firmly established the role of Toll signaling in immunity as well. In recent years, studies on microbe recognition and events upstream of Spz activation have revealed new components of the pathway. In addition, large-scale RNAi screens on the core intracellular pathway have revealed new essential components, putative conserved mechanisms, and cooperation of the fly immune pathways.

Mammalian TLR signaling mechanisms share similarities with the Drosophila Toll pathway, but also important differences exist; for example, the Toll receptor is a cytokine receptor, whereas TLRs are pattern recognition receptors. Also, among the nine Drosophila Tolls, a clear immunological role has only been assigned to Toll, whereas the others have putative roles in development. In contrast, all mammalian TLRs appear to have roles in immunity. Future work on the Drosophila Toll and other immune response pathways will undoubtedly continue to increase our understanding of these conserved NF-κB mechanisms in mammals.

We thank Dr. Helen Cooper for revising the language of the manuscript.

Disclosures The authors have no financial conflicts of interest.

This work was supported by grants from the Academy of Finland, the Foundation for Pediatric Research, the Sigrid Juselius Foundation, and the Emil Aaltonen Foundation (to M.R.), the Foundation of the Finnish Anti-Tuberculosis Association (to S.V.), the Tampere Tuberculosis Foundation, Competitive Research Funding of the Pirkanmaa Hospital District, and Biocenter Finland (to M.R. and S.V.).

Abbreviations used in this article:

AMP

antimicrobial peptide

DAP

diaminopimelic acid

DD

death domain

DEAF-1

deformed epidermal autoregulatory factor-1

Dif

dorsal-related immunity factor

DREDD

death-related Ced-3/Nedd2-like protein

DV

dorsal-ventral

GNBP

Gram-negative binding protein

Gprk2

G protein-coupled receptor kinase 2

Grass

Gram-positive–specific serine protease

IKK

IκB kinase

IRAK

IL-1R–associated kinase

ModSP

modular serine protease

PGN

peptidoglycan

PGRP

peptidoglycan recognition protein

RNAi

RNA interference

SPE

Spatzle-processing enzyme

Spz

Spa(e)tzle

TAB

TGF-β–activated kinase 1 binding protein

TAK1

TGF-β–activated kinase 1

TIR

Toll/IL-1R

TRAF

TNFR-associated factor.

1
Nüsslein-Volhard
C.
,
Wieschaus
E.
.
1980
.
Mutations affecting segment number and polarity in Drosophila.
Nature
287
:
795
801
.
2
Belvin
M. P.
,
Anderson
K. V.
.
1996
.
A conserved signaling pathway: the Drosophila toll-dorsal pathway.
Annu. Rev. Cell Dev. Biol.
12
:
393
416
.
3
Sun
S. C.
,
Faye
I.
.
1992
.
Cecropia immunoresponsive factor, an insect immunoresponsive factor with DNA-binding properties similar to nuclear-factor κ B.
Eur. J. Biochem.
204
:
885
892
.
4
Wasserman
S. A.
1993
.
A conserved signal transduction pathway regulating the activity of the rel-like proteins dorsal and NF-κ B.
Mol. Biol. Cell
4
:
767
771
.
5
Rosetto
M.
,
Engström
Y.
,
Baldari
C. T.
,
Telford
J. L.
,
Hultmark
D.
.
1995
.
Signals from the IL-1 receptor homolog, Toll, can activate an immune response in a Drosophila hemocyte cell line.
Biochem. Biophys. Res. Commun.
209
:
111
116
.
6
Taguchi
T.
,
Mitcham
J. L.
,
Dower
S. K.
,
Sims
J. E.
,
Testa
J. R.
.
1996
.
Chromosomal localization of TIL, a gene encoding a protein related to the Drosophila transmembrane receptor Toll, to human chromosome 4p14.
Genomics
32
:
486
488
.
7
Lemaitre
B.
,
Nicolas
E.
,
Michaut
L.
,
Reichhart
J. M.
,
Hoffmann
J. A.
.
1996
.
The dorsoventral regulatory gene cassette spätzle/Toll/cactus controls the potent antifungal response in Drosophila adults.
Cell
86
:
973
983
.
8
Medzhitov
R.
,
Preston-Hurlburt
P.
,
Janeway
C. A.
 Jr
.
1997
.
A human homologue of the Drosophila Toll protein signals activation of adaptive immunity.
Nature
388
:
394
397
.
9
Rock
F. L.
,
Hardiman
G.
,
Timans
J. C.
,
Kastelein
R. A.
,
Bazan
J. F.
.
1998
.
A family of human receptors structurally related to Drosophila Toll.
Proc. Natl. Acad. Sci. USA
95
:
588
593
.
10
Kimbrell
D. A.
,
Beutler
B.
.
2001
.
The evolution and genetics of innate immunity.
Nat. Rev. Genet.
2
:
256
267
.
11
Halfon
M. S.
,
Hashimoto
C.
,
Keshishian
H.
.
1995
.
The Drosophila toll gene functions zygotically and is necessary for proper motoneuron and muscle development.
Dev. Biol.
169
:
151
167
.
12
Qiu
P.
,
Pan
P. C.
,
Govind
S.
.
1998
.
A role for the Drosophila Toll/Cactus pathway in larval hematopoiesis.
Development
125
:
1909
1920
.
13
Hetru
C.
,
Hoffmann
J. A.
.
2009
.
NF-kappaB in the immune response of Drosophila.
Cold Spring Harb. Perspect. Biol.
1
:
a000232
.
14
Aggarwal
K.
,
Silverman
N.
.
2008
.
Positive and negative regulation of the Drosophila immune response.
BMB Rep.
41
:
267
277
.
15
Hultmark
D.
2003
.
Drosophila immunity: paths and patterns.
Curr. Opin. Immunol.
15
:
12
19
.
16
Zettervall
C. J.
,
Anderl
I.
,
Williams
M. J.
,
Palmer
R.
,
Kurucz
E.
,
Ando
I.
,
Hultmark
D.
.
2004
.
A directed screen for genes involved in Drosophila blood cell activation.
Proc. Natl. Acad. Sci. USA
101
:
14192
14197
.
17
Lemaitre
B.
,
Meister
M.
,
Govind
S.
,
Georgel
P.
,
Steward
R.
,
Reichhart
J. M.
,
Hoffmann
J. A.
.
1995
.
Functional analysis and regulation of nuclear import of dorsal during the immune response in Drosophila.
EMBO J.
14
:
536
545
.
18
Sorrentino
R. P.
,
Melk
J. P.
,
Govind
S.
.
2004
.
Genetic analysis of contributions of dorsal group and JAK-Stat92E pathway genes to larval hemocyte concentration and the egg encapsulation response in Drosophila.
Genetics
166
:
1343
1356
.
19
Bettencourt
R.
,
Asha
H.
,
Dearolf
C.
,
Ip
Y. T.
.
2004
.
Hemolymph-dependent and -independent responses in Drosophila immune tissue.
J. Cell. Biochem.
92
:
849
863
.
20
Tang
H.
,
Kambris
Z.
,
Lemaitre
B.
,
Hashimoto
C.
.
2008
.
A serpin that regulates immune melanization in the respiratory system of Drosophila.
Dev. Cell
15
:
617
626
.
21
Tauszig
S.
,
Jouanguy
E.
,
Hoffmann
J. A.
,
Imler
J. L.
.
2000
.
Toll-related receptors and the control of antimicrobial peptide expression in Drosophila.
Proc. Natl. Acad. Sci. USA
97
:
10520
10525
.
22
Imler
J. L.
,
Hoffmann
J. A.
.
2001
.
Toll receptors in innate immunity.
Trends Cell Biol.
11
:
304
311
.
23
Imler
J. L.
,
Tauszig
S.
,
Jouanguy
E.
,
Forestier
C.
,
Hoffmann
J. A.
.
2000
.
LPS-induced immune response in Drosophila.
J. Endotoxin Res.
6
:
459
462
.
24
Luo
C.
,
Shen
B.
,
Manley
J. L.
,
Zheng
L.
.
2001
.
Tehao functions in the Toll pathway in Drosophila melanogaster: possible roles in development and innate immunity.
Insect Mol. Biol.
10
:
457
464
.
25
Ooi
J. Y.
,
Yagi
Y.
,
Hu
X.
,
Ip
Y. T.
.
2002
.
The Drosophila Toll-9 activates a constitutive antimicrobial defense.
EMBO Rep.
3
:
82
87
.
26
Bettencourt
R.
,
Tanji
T.
,
Yagi
Y.
,
Ip
Y. T.
.
2004
.
Toll and Toll-9 in Drosophila innate immune response.
J. Endotoxin Res.
10
:
261
268
.
27
Morisato
D.
,
Anderson
K. V.
.
1994
.
The spätzle gene encodes a component of the extracellular signaling pathway establishing the dorsal-ventral pattern of the Drosophila embryo.
Cell
76
:
677
688
.
28
Schneider
D. S.
,
Jin
Y.
,
Morisato
D.
,
Anderson
K. V.
.
1994
.
A processed form of the Spätzle protein defines dorsal-ventral polarity in the Drosophila embryo.
Development
120
:
1243
1250
.
29
Arnot
C. J.
,
Gay
N. J.
,
Gangloff
M.
.
2010
.
Molecular mechanism that induces activation of Spätzle, the ligand for the Drosophila Toll receptor.
J. Biol. Chem.
285
:
19502
19509
.
30
Weber
A. N.
,
Gangloff
M.
,
Moncrieffe
M. C.
,
Hyvert
Y.
,
Imler
J. L.
,
Gay
N. J.
.
2007
.
Role of the Spatzle Pro-domain in the generation of an active toll receptor ligand.
J. Biol. Chem.
282
:
13522
13531
.
31
Weber
A. N.
,
Moncrieffe
M. C.
,
Gangloff
M.
,
Imler
J. L.
,
Gay
N. J.
.
2005
.
Ligand-receptor and receptor-receptor interactions act in concert to activate signaling in the Drosophila toll pathway.
J. Biol. Chem.
280
:
22793
22799
.
32
Gangloff
M.
,
Murali
A.
,
Xiong
J.
,
Arnot
C. J.
,
Weber
A. N.
,
Sandercock
A. M.
,
Robinson
C. V.
,
Sarisky
R.
,
Holzenburg
A.
,
Kao
C.
,
Gay
N. J.
.
2008
.
Structural insight into the mechanism of activation of the Toll receptor by the dimeric ligand Spätzle.
J. Biol. Chem.
283
:
14629
14635
.
33
DeLotto
Y.
,
DeLotto
R.
.
1998
.
Proteolytic processing of the Drosophila Spätzle protein by easter generates a dimeric NGF-like molecule with ventralising activity.
Mech. Dev.
72
:
141
148
.
34
Chasan
R.
,
Jin
Y.
,
Anderson
K. V.
.
1992
.
Activation of the easter zymogen is regulated by five other genes to define dorsal-ventral polarity in the Drosophila embryo.
Development
115
:
607
616
.
35
Hong
C. C.
,
Hashimoto
C.
.
1995
.
An unusual mosaic protein with a protease domain, encoded by the nudel gene, is involved in defining embryonic dorsoventral polarity in Drosophila.
Cell
82
:
785
794
.
36
Cho
Y. S.
,
Stevens
L. M.
,
Stein
D.
.
2010
.
Pipe-dependent ventral processing of Easter by Snake is the defining step in Drosophila embryo DV axis formation.
Curr. Biol.
20
:
1133
1137
.
37
Jang
I. H.
,
Chosa
N.
,
Kim
S. H.
,
Nam
H. J.
,
Lemaitre
B.
,
Ochiai
M.
,
Kambris
Z.
,
Brun
S.
,
Hashimoto
C.
,
Ashida
M.
, et al
.
2006
.
A Spätzle-processing enzyme required for toll signaling activation in Drosophila innate immunity.
Dev. Cell
10
:
45
55
.
38
El Chamy
L.
,
Leclerc
V.
,
Caldelari
I.
,
Reichhart
J. M.
.
2008
.
Sensing of ‘danger signals’ and pathogen-associated molecular patterns defines binary signaling pathways ‘upstream’ of Toll.
Nat. Immunol.
9
:
1165
1170
.
39
Kambris
Z.
,
Brun
S.
,
Jang
I. H.
,
Nam
H. J.
,
Romeo
Y.
,
Takahashi
K.
,
Lee
W. J.
,
Ueda
R.
,
Lemaitre
B.
.
2006
.
Drosophila immunity: a large-scale in vivo RNAi screen identifies five serine proteases required for Toll activation.
Curr. Biol.
16
:
808
813
.
40
Buchon
N.
,
Poidevin
M.
,
Kwon
H. M.
,
Guillou
A.
,
Sottas
V.
,
Lee
B. L.
,
Lemaitre
B.
.
2009
.
A single modular serine protease integrates signals from pattern-recognition receptors upstream of the Drosophila Toll pathway.
Proc. Natl. Acad. Sci. USA
106
:
12442
12447
.
41
Gottar
M.
,
Gobert
V.
,
Matskevich
A. A.
,
Reichhart
J. M.
,
Wang
C.
,
Butt
T. M.
,
Belvin
M.
,
Hoffmann
J. A.
,
Ferrandon
D.
.
2006
.
Dual detection of fungal infections in Drosophila via recognition of glucans and sensing of virulence factors.
Cell
127
:
1425
1437
.
42
Sansonetti
P. J.
2006
.
The innate signaling of dangers and the dangers of innate signaling.
Nat. Immunol.
7
:
1237
1242
.
43
Matzinger
P.
2002
.
The danger model: a renewed sense of self.
Science
296
:
301
305
.
44
Michel
T.
,
Reichhart
J. M.
,
Hoffmann
J. A.
,
Royet
J.
.
2001
.
Drosophila Toll is activated by Gram-positive bacteria through a circulating peptidoglycan recognition protein.
Nature
414
:
756
759
.
45
Gobert
V.
,
Gottar
M.
,
Matskevich
A. A.
,
Rutschmann
S.
,
Royet
J.
,
Belvin
M.
,
Hoffmann
J. A.
,
Ferrandon
D.
.
2003
.
Dual activation of the Drosophila toll pathway by two pattern recognition receptors.
Science
302
:
2126
2130
.
46
Pili-Floury
S.
,
Leulier
F.
,
Takahashi
K.
,
Saigo
K.
,
Samain
E.
,
Ueda
R.
,
Lemaitre
B.
.
2004
.
In vivo RNA interference analysis reveals an unexpected role for GNBP1 in the defense against Gram-positive bacterial infection in Drosophila adults.
J. Biol. Chem.
279
:
12848
12853
.
47
Wang
L.
,
Weber
A. N.
,
Atilano
M. L.
,
Filipe
S. R.
,
Gay
N. J.
,
Ligoxygakis
P.
.
2006
.
Sensing of Gram-positive bacteria in Drosophila: GNBP1 is needed to process and present peptidoglycan to PGRP-SA.
EMBO J.
25
:
5005
5014
.
48
Bischoff
V.
,
Vignal
C.
,
Boneca
I. G.
,
Michel
T.
,
Hoffmann
J. A.
,
Royet
J.
.
2004
.
Function of the drosophila pattern-recognition receptor PGRP-SD in the detection of Gram-positive bacteria.
Nat. Immunol.
5
:
1175
1180
.
49
Leone
P.
,
Bischoff
V.
,
Kellenberger
C.
,
Hetru
C.
,
Royet
J.
,
Roussel
A.
.
2008
.
Crystal structure of Drosophila PGRP-SD suggests binding to DAP-type but not lysine-type peptidoglycan.
Mol. Immunol.
45
:
2521
2530
.
50
Horng
T.
,
Medzhitov
R.
.
2001
.
Drosophila MyD88 is an adapter in the Toll signaling pathway.
Proc. Natl. Acad. Sci. USA
98
:
12654
12658
.
51
Tauszig-Delamasure
S.
,
Bilak
H.
,
Capovilla
M.
,
Hoffmann
J. A.
,
Imler
J. L.
.
2002
.
Drosophila MyD88 is required for the response to fungal and Gram-positive bacterial infections.
Nat. Immunol.
3
:
91
97
.
52
Sun
H.
,
Bristow
B. N.
,
Qu
G.
,
Wasserman
S. A.
.
2002
.
A heterotrimeric death domain complex in Toll signaling.
Proc. Natl. Acad. Sci. USA
99
:
12871
12876
.
53
Xiao
T.
,
Towb
P.
,
Wasserman
S. A.
,
Sprang
S. R.
.
1999
.
Three-dimensional structure of a complex between the death domains of Pelle and Tube.
Cell
99
:
545
555
.
54
Moncrieffe
M. C.
,
Grossmann
J. G.
,
Gay
N. J.
.
2008
.
Assembly of oligomeric death domain complexes during Toll receptor signaling.
J. Biol. Chem.
283
:
33447
33454
.
55
Haghayeghi
A.
,
Sarac
A.
,
Czerniecki
S.
,
Grosshans
J.
,
Schöck
F.
.
2010
.
Pellino enhances innate immunity in Drosophila.
Mech. Dev.
127
:
301
307
.
56
Moynagh
P. N.
2009
.
The Pellino family: IRAK E3 ligases with emerging roles in innate immune signalling.
Trends Immunol.
30
:
33
42
.
57
Wu
L. P.
,
Anderson
K. V.
.
1998
.
Regulated nuclear import of Rel proteins in the Drosophila immune response.
Nature
392
:
93
97
.
58
Towb
P.
,
Bergmann
A.
,
Wasserman
S. A.
.
2001
.
The protein kinase Pelle mediates feedback regulation in the Drosophila Toll signaling pathway.
Development
128
:
4729
4736
.
59
Huang
H. R.
,
Chen
Z. J.
,
Kunes
S.
,
Chang
G. D.
,
Maniatis
T.
.
2010
.
Endocytic pathway is required for Drosophila Toll innate immune signaling.
Proc. Natl. Acad. Sci. USA
107
:
8322
8327
.
60
Fernandez
N. Q.
,
Grosshans
J.
,
Goltz
J. S.
,
Stein
D.
.
2001
.
Separable and redundant regulatory determinants in Cactus mediate its dorsal group dependent degradation.
Development
128
:
2963
2974
.
61
Lu
Y.
,
Wu
L. P.
,
Anderson
K. V.
.
2001
.
The antibacterial arm of the drosophila innate immune response requires an IkappaB kinase.
Genes Dev.
15
:
104
110
.
62
Rutschmann
S.
,
Jung
A. C.
,
Zhou
R.
,
Silverman
N.
,
Hoffmann
J. A.
,
Ferrandon
D.
.
2000
.
Role of Drosophila IKK γ in a toll-independent antibacterial immune response.
Nat. Immunol.
1
:
342
347
.
63
Reichhart
J. M.
,
Georgel
P.
,
Meister
M.
,
Lemaitre
B.
,
Kappler
C.
,
Hoffmann
J. A.
.
1993
.
Expression and nuclear translocation of the rel/NF-κ B-related morphogen dorsal during the immune response of Drosophila.
C. R. Acad. Sci. III
316
:
1218
1224
.
64
Kidd
S.
1992
.
Characterization of the Drosophila cactus locus and analysis of interactions between cactus and dorsal proteins.
Cell
71
:
623
635
.
65
Yang
J.
,
Steward
R.
.
1997
.
A multimeric complex and the nuclear targeting of the Drosophila Rel protein Dorsal.
Proc. Natl. Acad. Sci. USA
94
:
14524
14529
.
66
Edwards
D. N.
,
Towb
P.
,
Wasserman
S. A.
.
1997
.
An activity-dependent network of interactions links the Rel protein Dorsal with its cytoplasmic regulators.
Development
124
:
3855
3864
.
67
Gross
I.
,
Georgel
P.
,
Kappler
C.
,
Reichhart
J. M.
,
Hoffmann
J. A.
.
1996
.
Drosophila immunity: a comparative analysis of the Rel proteins dorsal and Dif in the induction of the genes encoding diptericin and cecropin.
Nucleic Acids Res.
24
:
1238
1245
.
68
Lau
G. W.
,
Goumnerov
B. C.
,
Walendziewicz
C. L.
,
Hewitson
J.
,
Xiao
W.
,
Mahajan-Miklos
S.
,
Tompkins
R. G.
,
Perkins
L. A.
,
Rahme
L. G.
.
2003
.
The Drosophila melanogaster toll pathway participates in resistance to infection by the gram-negative human pathogen Pseudomonas aeruginosa.
Infect. Immun.
71
:
4059
4066
.
69
Ip
Y. T.
,
Reach
M.
,
Engstrom
Y.
,
Kadalayil
L.
,
Cai
H.
,
González-Crespo
S.
,
Tatei
K.
,
Levine
M.
.
1993
.
Dif, a dorsal-related gene that mediates an immune response in Drosophila.
Cell
75
:
753
763
.
70
Tatei
K.
,
Levine
M.
.
1995
.
Specificity of Rel-inhibitor interactions in Drosophila embryos.
Mol. Cell. Biol.
15
:
3627
3634
.
71
Rutschmann
S.
,
Jung
A. C.
,
Hetru
C.
,
Reichhart
J. M.
,
Hoffmann
J. A.
,
Ferrandon
D.
.
2000
.
The Rel protein DIF mediates the antifungal but not the antibacterial host defense in Drosophila.
Immunity
12
:
569
580
.
72
Manfruelli
P.
,
Reichhart
J. M.
,
Steward
R.
,
Hoffmann
J. A.
,
Lemaitre
B.
.
1999
.
A mosaic analysis in Drosophila fat body cells of the control of antimicrobial peptide genes by the Rel proteins Dorsal and DIF.
EMBO J.
18
:
3380
3391
.
73
Valanne
S.
,
Myllymäki
H.
,
Kallio
J.
,
Schmid
M. R.
,
Kleino
A.
,
Murumägi
A.
,
Airaksinen
L.
,
Kotipelto
T.
,
Kaustio
M.
,
Ulvila
J.
, et al
.
2010
.
Genome-wide RNA interference in Drosophila cells identifies G protein-coupled receptor kinase 2 as a conserved regulator of NF-kappaB signaling.
J. Immunol.
184
:
6188
6198
.
74
Rämet
M.
,
Manfruelli
P.
,
Pearson
A.
,
Mathey-Prevot
B.
,
Ezekowitz
R. A.
.
2002
.
Functional genomic analysis of phagocytosis and identification of a Drosophila receptor for E. coli.
Nature
416
:
644
648
.
75
Boutros
M.
,
Kiger
A. A.
,
Armknecht
S.
,
Kerr
K.
,
Hild
M.
,
Koch
B.
,
Haas
S. A.
,
Paro
R.
,
Perrimon
N.
Heidelberg Fly Array Consortium
.
2004
.
Genome-wide RNAi analysis of growth and viability in Drosophila cells.
Science
303
:
832
835
.
76
Ulvila
J.
,
Parikka
M.
,
Kleino
A.
,
Sormunen
R.
,
Ezekowitz
R. A.
,
Kocks
C.
,
Rämet
M.
.
2006
.
Double-stranded RNA is internalized by scavenger receptor-mediated endocytosis in Drosophila S2 cells.
J. Biol. Chem.
281
:
14370
14375
.
77
Saleh
M. C.
,
van Rij
R. P.
,
Hekele
A.
,
Gillis
A.
,
Foley
E.
,
O’Farrell
P. H.
,
Andino
R.
.
2006
.
The endocytic pathway mediates cell entry of dsRNA to induce RNAi silencing.
Nat. Cell Biol.
8
:
793
802
.
78
Reed
D. E.
,
Huang
X. M.
,
Wohlschlegel
J. A.
,
Levine
M. S.
,
Senger
K.
.
2008
.
DEAF-1 regulates immunity gene expression in Drosophila.
Proc. Natl. Acad. Sci. USA
105
:
8351
8356
.
79
Kuttenkeuler
D.
,
Pelte
N.
,
Ragab
A.
,
Gesellchen
V.
,
Schneider
L.
,
Blass
C.
,
Axelsson
E.
,
Huber
W.
,
Boutros
M.
.
2010
.
A large-scale RNAi screen identifies Deaf1 as a regulator of innate immune responses in Drosophila.
J. Innate Immun.
2
:
181
194
.
80
Lemaitre
B.
,
Hoffmann
J.
.
2007
.
The host defense of Drosophila melanogaster.
Annu. Rev. Immunol.
25
:
697
743
.
81
Choe
K. M.
,
Werner
T.
,
Stöven
S.
,
Hultmark
D.
,
Anderson
K. V.
.
2002
.
Requirement for a peptidoglycan recognition protein (PGRP) in Relish activation and antibacterial immune responses in Drosophila.
Science
296
:
359
362
.
82
Hedengren
M.
,
Asling
B.
,
Dushay
M. S.
,
Ando
I.
,
Ekengren
S.
,
Wihlborg
M.
,
Hultmark
D.
.
1999
.
Relish, a central factor in the control of humoral but not cellular immunity in Drosophila.
Mol. Cell
4
:
827
837
.
83
Dushay
M. S.
,
Asling
B.
,
Hultmark
D.
.
1996
.
Origins of immunity: Relish, a compound Rel-like gene in the antibacterial defense of Drosophila.
Proc. Natl. Acad. Sci. USA
93
:
10343
10347
.
84
Silverman
N.
,
Zhou
R.
,
Stöven
S.
,
Pandey
N.
,
Hultmark
D.
,
Maniatis
T.
.
2000
.
A Drosophila IkappaB kinase complex required for Relish cleavage and antibacterial immunity.
Genes Dev.
14
:
2461
2471
.
85
Lemaitre
B.
,
Reichhart
J. M.
,
Hoffmann
J. A.
.
1997
.
Drosophila host defense: differential induction of antimicrobial peptide genes after infection by various classes of microorganisms.
Proc. Natl. Acad. Sci. USA
94
:
14614
14619
.
86
Kleino
A.
,
Valanne
S.
,
Ulvila
J.
,
Kallio
J.
,
Myllymäki
H.
,
Enwald
H.
,
Stöven
S.
,
Poidevin
M.
,
Ueda
R.
,
Hultmark
D.
, et al
.
2005
.
Inhibitor of apoptosis 2 and TAK1-binding protein are components of the Drosophila Imd pathway.
EMBO J.
24
:
3423
3434
.
87
Tanji
T.
,
Hu
X.
,
Weber
A. N.
,
Ip
Y. T.
.
2007
.
Toll and IMD pathways synergistically activate an innate immune response in Drosophila melanogaster.
Mol. Cell. Biol.
27
:
4578
4588
.
88
De Gregorio
E.
,
Spellman
P. T.
,
Tzou
P.
,
Rubin
G. M.
,
Lemaitre
B.
.
2002
.
The Toll and Imd pathways are the major regulators of the immune response in Drosophila.
EMBO J.
21
:
2568
2579
.
89
Han
Z. S.
,
Ip
Y. T.
.
1999
.
Interaction and specificity of Rel-related proteins in regulating Drosophila immunity gene expression.
J. Biol. Chem.
274
:
21355
21361
.
90
Rutschmann
S.
,
Kilinc
A.
,
Ferrandon
D.
.
2002
.
Cutting edge: the toll pathway is required for resistance to gram-positive bacterial infections in Drosophila.
J. Immunol.
168
:
1542
1546
.
91
Engström
Y.
,
Kadalayil
L.
,
Sun
S. C.
,
Samakovlis
C.
,
Hultmark
D.
,
Faye
I.
.
1993
.
kappaB-like motifs regulate the induction of immune genes in Drosophila.
J. Mol. Biol.
232
:
327
333
.
92
Petersen
U. M.
,
Kadalayil
L.
,
Rehorn
K. P.
,
Hoshizaki
D. K.
,
Reuter
R.
,
Engström
Y.
.
1999
.
Serpent regulates Drosophila immunity genes in the larval fat body through an essential GATA motif.
EMBO J.
18
:
4013
4022
.
93
Senger
K.
,
Harris
K.
,
Levine
M.
.
2006
.
GATA factors participate in tissue-specific immune responses in Drosophila larvae.
Proc. Natl. Acad. Sci. USA
103
:
15957
15962
.
94
Poltorak
A.
,
Smirnova
I.
,
He
X.
,
Liu
M. Y.
,
Van Huffel
C.
,
McNally
O.
,
Birdwell
D.
,
Alejos
E.
,
Silva
M.
,
Du
X.
, et al
.
1998
.
Genetic and physical mapping of the Lps locus: identification of the toll-4 receptor as a candidate gene in the critical region.
Blood Cells Mol. Dis.
24
:
340
355
.
95
Takeuchi
O.
,
Akira
S.
.
2010
.
Pattern recognition receptors and inflammation.
Cell
140
:
805
820
.
96
Towb
P.
,
Sun
H.
,
Wasserman
S. A.
.
2009
.
Tube Is an IRAK-4 homolog in a Toll pathway adapted for development and immunity.
J. Innate Immun.
1
:
309
321
.
97
Dunne
A.
,
Ejdeback
M.
,
Ludidi
P. L.
,
O’Neill
L. A.
,
Gay
N. J.
.
2003
.
Structural complementarity of Toll/interleukin-1 receptor domains in Toll-like receptors and the adaptors Mal and MyD88.
J. Biol. Chem.
278
:
41443
41451
.
98
Lin
S. C.
,
Lo
Y. C.
,
Wu
H.
.
2010
.
Helical assembly in the MyD88-IRAK4-IRAK2 complex in TLR/IL-1R signalling.
Nature
465
:
885
890
.
99
Silverman
N.
,
Maniatis
T.
.
2001
.
NF-kappaB signaling pathways in mammalian and insect innate immunity.
Genes Dev.
15
:
2321
2342
.
100
Leulier
F.
,
Rodriguez
A.
,
Khush
R. S.
,
Abrams
J. M.
,
Lemaitre
B.
.
2000
.
The Drosophila caspase Dredd is required to resist gram-negative bacterial infection.
EMBO Rep.
1
:
353
358
.
101
Paquette
N.
,
Broemer
M.
,
Aggarwal
K.
,
Chen
L.
,
Husson
M.
,
Ertürk-Hasdemir
D.
,
Reichhart
J. M.
,
Meier
P.
,
Silverman
N.
.
2010
.
Caspase-mediated cleavage, IAP binding, and ubiquitination: linking three mechanisms crucial for Drosophila NF-kappaB signaling.
Mol. Cell
37
:
172
182
.
102
Patial
S.
,
Luo
J.
,
Porter
K. J.
,
Benovic
J. L.
,
Parameswaran
N.
.
2010
.
G-protein-coupled-receptor kinases mediate TNFα-induced NF-κB signalling via direct interaction with and phosphorylation of IkBα.
Biochem. J.
425
:
169
178
.
103
Patial
S.
,
Shahi
S.
,
Saini
Y.
,
Lee
T.
,
Packiriswamy
N.
,
Appledorn
D. M.
,
Lapres
J. J.
,
Amalfitano
A.
,
Parameswaran
N.
.
2010
.
G-protein coupled receptor kinase 5 mediates lipopolysaccharide-induced NFκB activation in primary macrophages and modulates inflammation in vivo in mice.
J. Cell. Physiol.
DOI:10.1002/jcp.22460
.
104
Kawai
T.
,
Akira
S.
.
2006
.
TLR signaling.
Cell Death Differ.
13
:
816
825
.
105
Goto
A.
,
Matsushita
K.
,
Gesellchen
V.
,
El Chamy
L.
,
Kuttenkeuler
D.
,
Takeuchi
O.
,
Hoffmann
J. A.
,
Akira
S.
,
Boutros
M.
,
Reichhart
J. M.
.
2008
.
Akirins are highly conserved nuclear proteins required for NF-kappaB-dependent gene expression in drosophila and mice.
Nat. Immunol.
9
:
97
104
.
106
Kleino
A.
,
Myllymäki
H.
,
Kallio
J.
,
Vanha-aho
L. M.
,
Oksanen
K.
,
Ulvila
J.
,
Hultmark
D.
,
Valanne
S.
,
Rämet
M.
.
2008
.
Pirk is a negative regulator of the Drosophila Imd pathway.
J. Immunol.
180
:
5413
5422
.
107
Kim
M.
,
Lee
J. H.
,
Lee
S. Y.
,
Kim
E.
,
Chung
J.
.
2006
.
Caspar, a suppressor of antibacterial immunity in Drosophila.
Proc. Natl. Acad. Sci. USA
103
:
16358
16363
.
108
Foley
E.
,
O’Farrell
P. H.
.
2004
.
Functional dissection of an innate immune response by a genome-wide RNAi screen.
PLoS Biol.
2
:
E203
.