Two major Relish transcripts are seen in Northern blots of adult flies. A 3.4-kb transcript is expressed constitutively and is further induced about 15-fold after infection. In contrast, a 3.1-kb transcript is undetectable in untreated animals, but is strongly induced in infected flies with induction kinetics similar to the Cecropin A1 gene. Relish is thus induced much more strongly than either of the two other Drosophila Rel protein genes, Dif and dorsal, which are only induced about 3-fold under these conditions. The dorsal gene is known for its role in early embryogenesis. In contrast, Dif is only expressed late in embryogenesis and probably serves no developmental function. To test if Relish is expressed during development, Northern blots were prepared with RNA from different staged embryos, larvae, pupae, and adults. A 2.7-kb Relish transcript is present in 0-2 h embryos and uninjected adult females. This transcript is present at low levels in 2-4 h embryos and is not detectable in embryos after 8 h, or in larvae, pupae, or adult males. This pattern of expression is similar to dorsal and other maternally transcribed genes, and suggests that Relish mRNA is provided to the egg by the mother (Dushay, 1996).

Effects of Mutation or Deletion

Mutations in the Relish gene have a striking effect on the antimicrobial immune response. No induction of Cecropin A1 expression could be detected in mutants E20 and E38 after a bacterial injection, while the induction of this gene is normal in the other lines. Since different peptides are believed to depend on different signal transduction pathways, the same filter was reprobed with probes for several other genes. The result with Diptericin is very similar to that of Cecropin, and there is essentially no induction of this gene in Relish mutants, although a faint signal could occasionally be seen in E38. Relish is required for full induction of Drosomycin, Metchnikowin, and Attacin, although for these genes some residual inducibility remains in the mutants. Quantification of Northern blots shows that on average about 10% of the induction of Attacin and Metchnikowin remains in E20, and about 20% of Drosomycin. In E38, the corresponding values are at least twice as high. In addition, there is a significant background of expression of Attacin and Drosomycin, even in uninduced flies (Hedengren, 1999).

The drastic effects on the immune response seen in E20 and E38 are likely to be due to the Relish mutation, not to any effect on expression of the closely linked Nmdmc in these strains since the immune response is normal in both E21 and E14, both of which also lack Nmdmc transcript B, and in R6, which is an Nmdmc null mutant. Furthermore, the inducibility of Cecropin and Diptericin is rescued in flies overexpressing Relish in an E20 background. The suppressed immune response in the E20 mutants is probably due to a direct effect of Relish and not indirectly via Dif since the expression of Dif in the E20 mutant is not seriously affected (Hedengren, 1999).

Since the inducible antimicrobial peptides are believed to be important for the immune defense in Drosophila, it was expected that the Relish mutants would be sensitive to infection. Indeed, under standard conditions for the induction of the immune response (injecting flies with a 10-fold dilution of an overnight culture of Enterobacter cloacae ß12) all flies from mutant lines E20 and E38 succumb to the bacteria within 17 and 27 hr, respectively. Wild-type flies, such as Canton-S and the perfect excision line E23, generally survive this treatment. Lower doses of bacteria also killed the Relish mutants, but after a time delay. Even at an average estimated dose of 0.2 bacteria per fly, a corresponding fraction of the mutant flies is killed, indicating that a single injected cell of E. cloacae is sufficient to kill Relish mutants. Again, this is due to the Relish mutation, not to Nmdmc, since E21, E14, and R6 flies show normal resistance to infection and since the resistance is rescued in the E20 flies after overexpression of Relish (Hedengren, 1999).

Since the induction of antifungal peptides, such as drosomycin, cecropin, and metchnikowin is also impaired in the Relish mutants, tests were performed to see if the resistance against fungal infections is affected. Four different fungal species were tested. Beauveria bassiana and Metarhizium anisopliae are well-known insect pathogenic fungi, and Geotrichum candidum and Dipodascopsis uninucleata are yeast-like fungi isolated from the normal environment of Drosophila. The Relish null mutant E20 is considerably more sensitive than wild-type flies to injected Geotrichum, Dipodascopsis, and Metarhizium, although killing is slower than after a bacterial infection. The majority of the mutant flies are killed by the fungi within a week, whereas there is good survival in the perfect excision control, E23. For the E38 mutant, there is an intermediate effect. The effect of the Relish mutations is less striking in the case of Beauveria bassiana, since this insect pathogen kills even the control flies (Hedengren, 1999).

It is concluded that the Relish mutants have a profound effect on the antibacterial defence and that the resistance against fungi is also strongly affected These results show that the Relish gene is of central importance for the humoral immune response. The possible role of this gene in cellular immunity was examined. In preliminary experiments, total hemocyte cell number and morphology in E20, E38, E23, and Canton-S larvae were examined. No significant difference was found between the strains, and the lymph glands are also not visibly affected. The phagocytic activity of hemocytes was examined after an injection of fluorescently labeled Gram-positive or Gram-negative bacteria. However, the phagocytic activity of larval plasmatocytes from the Relish mutants E20 or E38 is indistinguishable from that in wild-type controls. Similar experiments were carried out with larval E23 and R6 and with adult E20, E38, E23, and Canton-S flies. Samples were taken at time intervals up to 5 hr, but no difference could be detected between the strains in the number of phagocytic cells (Hedengren, 1999).

The capacity of mutant larvae to encapsulate parasites was examined. The encapsulation response is not affected in the Relish mutants. When larvae of E20 or E38 are infested with the parasitoid wasp, Leptopilina boulardi, a majority of them show an efficient encapsulation response, and very few adult wasps emerge from the infested animals. There is no significant difference in this respect between the Relish mutants and the perfect excision control, E23, and the success of the parasitic infestation also falls within the range seen in wild-type strains such as Canton-S and Oregon-R. For comparison, two strains, S strain 22 and R strain 940, which have been selected for susceptibility and resistance to Leptopilina, respectively, were examined. In contrast to the Relish mutants, the S strain 22 has a much reduced response against the parasite. It should also be noted that the activation of the phenoloxidase system is normal in the Relish mutants. Encapsulated Leptopilina eggs become melanized, and injection wounds also show normal melanization in the fly (Hedengren, 1999).

Adaptive evolution of Relish, a Drosophila NF-kappaB/IkappaB protein

NF-kappaB and IkappaB proteins have central roles in regulation of inflammation and innate immunity in mammals. Homologs of these proteins also play an important role in regulation of the Drosophila immune response. A molecular population genetic analysis is presented for Relish, a Drosophila NF-kappaB/IkappaB protein, in Drosophila simulans and D. melanogaster. Strong evidence is found for adaptive protein evolution in D. simulans, but not in D. melanogaster. The adaptive evolution appears to be restricted to the IkappaB domain. A possible explanation for these results is that Relish is a site of evolutionary conflict between flies and their microbial pathogens (Begun, 2000).

A history of directional selection on amino acid variation in D. simulans has been convincingly established. How might this analysis impinge on broader issues of the evolution of fly immunity and the biological role of Relish? One potentially relevant finding is that there is strong evidence for adaptive evolution in the IkappaB domain, yet no evidence for adaptive protein evolution in the NF-kappaB domain. Models of IkappaB function posit that such proteins are modulated primarily through kinase-dependent phosphorylation and subsequent ubiquitin-dependent targeting to proteolytic degradation pathways. An interesting issue is whether adaptive amino acid evolution at large numbers of residues throughout the IkappaB region of Relish is likely to be caused strictly by selection resulting from interactions of this protein with internal signaling components. If this is thought to be unlikely, an alternative possibility is that selection pressures acting on the IkappaB domain of Relish arise from direct interactions with other molecules; those deriving directly from pathogens are obvious candidates (Begun, 2000).

One can speculate that microbial pathogens could benefit by interfering with activation of the Drosophila immune response. Pathogenic bacteria possessing type III secretion systems are able to carry out contact-mediated transport of proteins directly into the cytoplasm of host cells. These bacterial proteins can specifically interfere with host-cell signal transduction or other processes. Thus, there is a well-established mechanistic basis for specific manipulation of animal cytoplasmic proteins by microbial pathogens, though there has been no exploration of the phenomenon in Drosophila. Manipulation of IkappaB proteins such that nuclear translocation of NF-kappaB proteins (which regulate transcription of other immune system proteins) is inhibited would be a potential mechanism whereby microbial pathogens could suppress the Drosophila immune response. Drosophila populations would experience strong natural selection to evade such strategies. In this scenario, a putative arms race is manifested in an evolutionary conflict (mediated through interactions with IkappaB proteins) between fly and pathogen over control of subcellular localization of NF-kappaB proteins. These hypotheses must be considered to be very speculative. The ability to formulate evolutionary hypotheses about Relish is limited by a poor understanding of the biology of this protein and its precise role in the Drosophila immune response (Begun, 2000).

Nevertheless, the data provide at least one potential experimental foothold into the evolutionary or ecological genetics of Drosophila-microbe interactions. For example, analysis of phenotypic consequences of standing variation at Relish could prove interesting from both a mechanistic and evolutionary/ecological perspective. Experiments to elucidate functional consequences of interspecific differences in Relish in the context of natural pathogens might also be interesting. The recent discovery of numbers of Drosophila mutants affecting nuclear localization of Rel proteins suggests that there could be numerous arenas for conflict between flies and their microbial pathogens (Begun, 2000).

The Toll and Imd pathways are the major regulators of the immune response in Drosophila

Microarray studies have shown recently that microbial infection leads to extensive changes in the Drosophila gene expression program. However, little is known about the control of most of the fly immune-responsive genes, except for the antimicrobial peptide (AMP)-encoding genes, which are regulated by the Toll and Imd pathways. Oligonucleotide microarrays have been used to monitor the effect of mutations affecting the Toll and Imd pathways on the expression program induced by septic injury in Drosophila adults. Toll and Imd cascades were found to control the majority of the genes regulated by microbial infection in addition to AMP genes and are involved in nearly all known Drosophila innate immune reactions. However, some genes controlled by septic injury were identified that are not affected in double mutant flies where both Toll and Imd pathways are defective, suggesting that other unidentified signaling cascades are activated by infection. Interestingly, it was observed that some Drosophila immune-responsive genes are located in gene clusters, which often are transcriptionally co-regulated (De Gregorio, 2002).

To identify the target genes of the Toll and Imd pathways in response to microbial infection, the gene expression programs induced by septic injury have been compared in wild-type and mutant adult male flies using oligonucleotide microarrays. In parallel, the survival rate and the expression level of various AMP genes have been monitored after infection by various microorganisms. For the Toll pathway, a strong homozygous viable allele of spz (rm7) was selected. The spz, Tl and pll mutations, alone or in combination with rel, have similar effects on both the survival rate and pattern of AMP gene expression after microbial infection. These findings suggest that the effects of spz mutation on the transcription program induced by infection reflect the role of the entire Toll pathway in the immune response. For the Imd pathway, a null viable allele of relish (E20) was selected. Similarly to the Toll pathway, previous comparative studies did not reveal any striking difference between mutations in relish and null mutations in the genes encoding the other members of the Imd pathway such as kenny, ird5 and dredd, with the sole exception of mutations in dTAK1, which have a slightly weaker phenotype. Again, these data suggest that the effects of rel mutation on the immune response reflect the role of the whole Imd pathway. However, other pathways, including Toll, cannot be excluded from having a minor role in Relish activation (De Gregorio, 2002).

The septic injury experiments were performed using a mixture of Gram-positive and Gram-negative bacteria. This type of infection activates a wide immune response and allows the simultaneous analysis of several categories of immune-responsive genes. However, it has been shown that Toll and Imd pathways are activated selectively by different classes of microorganisms; thus, the use of a bacterial mixture might increase the redundancy of the two pathways in the control of common target genes (De Gregorio, 2002).

The microarray analysis demonstrates that the functions of Toll and Imd pathways in Drosophila immunity can be extended beyond the regulation of AMP genes. The majority of the Drosophila immune-regulated genes (DIRGs) are affected by the mutations in the Toll or Imd pathways. Many of these genes are unknown (see for a complete list); others can be assigned to several immune functions. The susceptibility of the Imd and Toll pathway mutants to different types of microbial infection suggested a dual aspect to the control of the antifungal response by the Toll pathway: a major role for the Toll pathway for the response to Gram-positive bacteria with a minor contribution of Imd, and a predominant role of Imd with a minor contribution of Toll to the resistance against Gram-negative bacteria. In agreement, microarray analysis shows that the Toll pathway controls most of the late genes induced by fungal infection and cooperates with the Imd pathway for the control of genes implicated in several immune reactions such as coagulation, AMP production, opsonization, iron sequestration and wound healing. Interestingly, defensin, which encodes the most effective antimicrobial peptide directed against Gram-positive bacteria, is co-regulated by both the Imd and Toll pathways. The hierarchical cluster analysis of the expression profiles combining the effect of the mutations after septic injury with the response to fungal infection provides a wealth of information that may help to elucidate the function of some of the uncharacterized DIRGs. Until now, the increased susceptibility to infection of Imd- or Toll-deficient flies has been attributed to the lack of expression of AMP genes, and it has been shown recently that the constitutive expression of single AMP genes in imd;spz double mutant flies can increase the survival rate of some types of bacterial infection. The finding that the Toll and Imd pathways are the major regulators of the Drosophila immune response now suggests that other immune defence mechanisms might contribute to the increased susceptibility to infection displayed by mutant flies (De Gregorio, 2002).

The interactions between the Toll and Imd pathways are more complex than merely regulating the same target genes. In agreement with Northern blot analysis, it has been shown that the transcriptional control of relish in response to infection receives a modest input from the Toll pathway, revealing an additional level of interaction between the two cascades. The activation of Toll may increase the level of Relish to allow a more efficient response to bacterial infection. This finding is in agreement with previous observations showing that in mutants where the Toll pathway is constitutively active (Tl10b), all the antibacterial peptides genes, including diptericin, are induced with more rapid kinetics than in wild-type flies. Furthermore, the higher susceptibility to E.coli infection of the rel,spz double mutant compared with the rel single mutants flies indicates that Toll also has a direct, Relish-independent effect on the resistance to infection by Gram-negative bacteria. Northern blot analysis shows that relish induction in response to infection is significantly reduced in dTAK1 and dredd mutants, indicating that the Imd pathway undergoes autoregulation. Interestingly, the Imd pathway can influence the Toll pathway through the control of PGRP-SA, which encodes a recognition protein essential for the activation of the Toll pathway by Gram-positive bacteria. Again, it is interesting to notice that this interaction between the Toll and Imd pathways correlates with the contribution of both pathways to fight infection with Gram-positive bacteria. Interestingly, all the genes encoding components of the Toll pathway required for both antibacterial and antifungal responses (necrotic, spaetzle, Toll, pelle, cactus and Dif) are not controlled by the Imd pathway and are subjected to autoregulation (De Gregorio, 2002).

The Rel/NF-kappaB proteins Dif, Dorsal and Relish, which are the transactivators induced by the Toll and Imd pathways, bind to the kappaB sites present in the promoters of target genes, such as AMP genes, regulating their expression. Therefore, the analysis of the promoters of the DIRGs controlled by Toll or Imd pathways could help to identify all the direct NF-kappaB targets during infection. However, some of the effects of mutations affecting the Toll or Imd pathways that were monitored by microarray analysis might be mediated by the regulation of other transcription factors or signaling cascades. It has been shown recently in larvae that the Tep1 gene is regulated by the JAK-STAT pathway and can be activated by the Toll pathway, suggesting that Toll can control, at least partially, the JAK-STAT cascade. Two genes encoding components of the JNK pathway (puc and d-Jun) are partially regulated by Toll and Imd in response to septic injury (De Gregorio, 2002).

The presence of DIRGs independent of or only partially dependent on both the Imd and Toll pathways suggests the presence of other signaling cascades activated after septic injury. Potential candidates are MAPK and JAK-STAT pathways. Beside their developmental functions, the MAPK pathways have been implicated in wound healing (JNK) and the stress response (MEKK). The JAK-STAT pathway controls the Drosophila complement-like gene TepI. The stimuli that trigger these cascades are not known and it is not clear if these cascades are activated by exogenous or host factors. Interestingly, in vertebrates, the JAK-STAT pathway is activated by cytokines during the immune response. The microarray analysis of mutants in these pathways might help to reveal their exact contribution to the Drosophila immune response. The observation that Toll and Imd pathways control most of the DIRGs raises the question of whether these two pathways are the sole signaling cascades directly activated by microbial elictors, while the other signaling pathways are triggered by other stimuli associated with infection such as wound, stress, cytokine-like factors and Toll and Imd activities (De Gregorio, 2002).

In vertebrates, many genes involved in the immune response are grouped in large chromosomal complexes. The recent completion of the Drosophila genome did not reveal any striking chromosomal organization beside clustering of genes belonging to the same family, probably reflecting recent duplication events. In this study, it was observed that some of the genes responding to microbial infection are located in the same cytological region or are associated in transcriptionally co-regulated genomic clusters. Interestingly, microarray analysis of circadian gene expression in Drosophila has led to the identification of similar clusters of genes. Other microarray analyses might reveal the importance of the genome organization in the definition of adequate transcription programs in response to a variety of stimuli (De Gregorio, 2002).

Sequential activation of signaling pathways during innate immune responses in Drosophila

Innate immunity is essential for metazoans to fight microbial infections. Genome-wide expression profiling was used to analyze the outcome of impairing specific signaling pathways after microbial challenge. These transcriptional patterns can be dissected into distinct groups. In addition to signaling through the Toll/NFkappaB or Imd/Relish pathways, signaling through the JNK and JAK/STAT pathways controls distinct subsets of targets induced by microbial agents. Each pathway shows a specific temporal pattern of activation and targets different functional groups, suggesting that innate immune responses are modular and recruit distinct physiological programs. In particular, the results may imply a close link between the control of tissue repair and antimicrobial processes (Boutros, 2002).

Lipopolysaccharides (LPS) are the principal cell wall components of gram-negative bacteria. In mammals, exposure to LPS causes septic shock through a Toll-like receptor TLR4-dependent signaling pathway. LPS treatment of Drosophila SL2 cells leads to rapid expression of antimicrobial peptides, such as Cecropins (Cec). SL2 cells resemble embryonic hemocytes and have also been used as a model system to study JNK and other signaling pathways. LPS-responsive induction of the antimicrobial peptides AttacinA (AttA), Diptericin (Dipt), and Cec relies on IKK and Relish. In order to obtain a broad overview on the transcriptional response to LPS in Drosophila, genome-wide expression profiles of SL2 cells were generated at different time points following LPS treatment. Altered expression of 238 genes was detected (Boutros, 2002).

In time-course experiments, a complex pattern of gene expression was observed that can be separated into different temporal clusters. A first group, with peak expression at 60 min after LPS, primarily consists of cytoskeletal regulators, signaling, and proapoptotic factors. This group includes cytoskeletal and cell adhesion modulators such as Matrix metalloprotease-1, WASp, Myosin, and Ninjurin, proapoptotic factors such as Reaper, and signaling proteins such as Puckered and VEGF-2. A second group, with peak expression at 120 min, includes many known defense and immunity genes, such as Cec, Mtk, and AttA, but not the gram-positive-induced peptide Drs. Interestingly, this cluster also includes PGRP-SA, which is a gram-positive pattern recognition receptor in vivo, suggesting possible crossregulation between gram-positive- and gram-negative-induced factors. A third group is transiently downregulated upon LPS stimulation. This cluster includes genes that play a role in cell cycle control, such as String and Rca1. Altogether, these results show that, in response to LPS, a defined gram-negative stimulus, cells elicit a complex transcriptional response (Boutros, 2002).

In adult Drosophila, gram-negative bacteria elicit an antimicrobial response mediated by a signaling pathway that involves the intracellular factors Imd, Tak1, IkappaB kinase Kenny (Key), and Rel. On the basis the expression profiling results, it was reasoned that the temporal waves of transcriptional activity in SL2 cells might reflect different signaling pathway contributions. It was therefore asked whether selectively removing signaling components by RNA interference (RNAi) would block induction of all, or only parts, of the transcriptional response to LPS (Boutros, 2002).

The effect of removing key or rel by RNAi was investigated. The expression profiles demonstrate that removing key or rel diminishes the induction of antimicrobial peptides. However, the induction of cytoskeletal and proapoptotic factors was not affected. In contrast, removing tak1 reduces the level of induction or repression for all identified genes, indicating that LPS-induced signaling is transmitted through Tak1 and that specific pathways branch downstream of Tak1 (Boutros, 2002).

In the Rel-independent group, several transcripts were identified that are indicative of other signaling events. For example, puc is transcriptionally regulated by JNK signaling during embryonic development. Therefore, the effect of removing SAPK/JNK activity was tested on LPS-induced transcripts. mkk4/hep dsRNA-treated cells lose the ability to induce the Rel-independent cluster, indicating that LPS signaling branches downstream of Tak1 into separate Rel- and JNK-dependent branches. To validate the results obtained from the microarray experiments, quantitative PCR (qPCR) was performed using puc and cec mRNA levels as indicators for Imd/Rel- or Mkk4/Hep-dependent pathways. Additionally, the effect of removing imd, which, in vivo, acts upstream of Tak1, was tested to clarify whether, in addition to Tak1, other known upstream components of a gram-negative signaling pathway are required for both Rel- and Mkk4/Hep-dependent pathways. These qPCR experiments confirm that cec is dependent for its expression on Imd, Tak1, Rel, and Key, whereas LPS-induced puc expression is dependent on Imd, Tak1, and Mkk4/Hep. Hence, the immunity signaling pathway in response to LPS bifurcates downstream of Imd and Tak1 into Rel- and SAPK/JNK-dependent branches. Both the Rel and SAPK/JNK pathways regulate different functional groups of downstream target genes (Boutros, 2002).

While both Rel and Mkk4/Hep pathways are downstream of Imd and Tak1 in response to LPS, the two downstream branches elicit different temporal expression patterns. It was then asked whether the first transcriptional response is controlled by downstream targets that might negatively feed back into the signaling circuit. puc was a candidate for such a transcriptionally induced negative regulator. Expression profiles of cells depleted for puc were tested before and after a 60 min LPS treatment. These experiments showed that transcripts dependent on the Mkk4/Hep branch of LPS signaling are upregulated, even without further LPS stimulus. In contrast, Rel branch targets are not influenced. puc dsRNA-treated cells show loss of the typical round cell shape. These cells appear flat and have a delocalized Actin staining, consistent with a deregulation of cytoskeletal modulators in puc-deficient cells (Boutros, 2002).

The analysis of expression profiles shows that, while SAPK/JNK and Rel signaling are controlled by the same Imd/Tak1 cascade, they appear to have different feedback loops. Whereas Rel signaling induces Rel expression and thereby generates a self-sustaining loop, possibly leading to the maintenance of target gene expression, the SAPK/JNK branch induces an inhibitor and thereby establishes a self-correcting feedback loop. These results may explain how a single upstream cascade can lead to different dynamic patterns (Boutros, 2002).

Septic injury of adult Drosophila is a widely used model system to study innate immune responses in vivo. To explore the signaling pathways that control induced genes in vivo, genome-wide expression profiles were generated of adult Drosophila infected by septic injury. Equal numbers of male and female adult Oregon R flies were infected with a mixture of E. coli (gram negative) and M. luteus (gram positive). Subsequently, flies were collected at 1, 3, 6, 24, 48, and 72 hr time points post-septic injury to measure temporal changes in gene expression levels. Computational analysis identified a list of 223 genes that were differentially regulated and matched the filtering criteria for at least two time points after microbial infection. This set includes 197 genes that are transiently upregulated and 26 that are transiently downregulated upon immune challenge. Different temporal profiles of gene expression can be detected in this analysis; clusters of genes differed significantly in the timing and persistence of induction. For example, whereas many genes are expressed transiently shortly after infection, others are induced late and are still upregulated at a 72 hr time point. A significant number of genes of both early and late clusters are differentially expressed at a 6 hr time point after infection, which was chosen for further analysis (Boutros, 2002).

Interestingly, some genes do not fit either pattern, suggesting that they are regulated by other pathways. One group of genes, including cytoskeletal factors such as actin88F, flightin, and tpnC41C, is induced in Tl10b, but not in cact, mutants. In contrast, totM and CG11501 are expressed at high levels in cact mutant flies but are not expressed in Tl10b mutant flies. In addition, these transcripts are highly inducible in a tube genetic background, but they are not inducible in key or rel. This may suggest that Toll, Tube, and Cact do not act in a linear pathway under all circumstances. Moreover, rel shows an expression pattern suggesting that it is regulated by both the Imd/Rel and Toll pathways. Thus, these results indicate that, in addition to the canonical Toll and Imd pathways, other signaling events and possibly signaling pathway branching contribute to the complex expression patterns after septic injury. Finally, there is a strong correlation between pathway requirement and temporal expression pattern. Whereas Toll targets are exclusively found in the sustained cluster, Imd/Rel targets are expressed early and transiently after septic injury. The two additional clusters with noncanonical patterns show temporal patterns distinct from either Toll or Imd pathways (Boutros, 2002).

NFkappaB pathways play a central role for innate and adaptive immune response in mammals. In innate immune responses, TLRs on dendritic cells recognize microbial agents and activate NFkappaB, leading to the expression of proinflammatory cytokines and other costimulatory factors required to initiate an adaptive immune response. Additionally, other signaling pathways have been implicated at later stages during immune responses in mammals, but their physiological role in innate immunity remains rather poorly understood. For example, several cytokines, such as IL-6 and IL-11, signal through a JAK/STAT pathway to induce the expression of acute phase proteins. Similarly, JNK pathways are activated in response to TNF and IL-1, may lead to the expression of immune modulators, and are required for T cell differentiation. In Drosophila, studies have investigated two distinct NFkappaB-pathways --Toll and Imd/Rel -- that have been shown to mediate gram-positive/fungal and gram-negative responses. Both pathways induce specific antimicrobial peptides and thereby focus the response on the invading microbial agent. Genetic analysis has shown that functions of the NFkappaB-pathways are separable; flies that are mutant for only one of these pathways are susceptible to subgroups of pathogens. Could the contribution of NFkappaB-dependent and, possibly, other signaling pathways be identified by examining global expression profiles? The obtained data set demonstrates that NFkappaB-independent signaling pathways contribute to the transcriptional patterns observed after microbial infection. Both in cells and in vivo, JNK-dependent targets precede the peak expression of antimicrobial peptides that require NFkappaB. JAK/STAT targets are induced with a distinct temporal pattern that shows late, but only transient, expression characteristics. The stereotyped pathway patterns after microbial challenge suggest that the correct temporal execution of signaling events, similar to signaling during development, may play an important role in the regulation of homeostasis (Boutros, 2002).

In conclusion, genome-wide expression profiling was employed to examine the contribution of different signaling pathways in complex tissues and to assign targets to candidate pathways. Both a cell culture model system and an in vivo analysis were used to show the temporal order of NFkappaB-dependent and -independent pathways after septic injury. An interesting question that remains is, how do the extracellular events leading to pathway activation reflect the nature of the pathogen? Clean injury experiments induce a largely overlapping set of induced genes, but to a lower extent than septic injury. This is consistent with experiments showing that septic injury with only gram-negative E. coli induces both anti-gram-negative and anti-gram-positive responses. These results can be interpreted to suggest that wounding, in itself, might be sufficient to induce a transient (and unspecific) innate immune response. However, further studies are needed to understand the nature of the inducing agent (Boutros, 2002).

Signaling role of hemocytes in Drosophila JAK/STAT-dependent response to septic injury

To characterize the features of JAK/STAT signaling in Drosophila immune response, totA was identified as a gene that is regulated by the JAK/STAT pathway in response to septic injury. Septic injury triggers the hemocyte-specific expression of upd3, a gene encoding a novel Upd-like cytokine that is necessary for the JAK/STAT-dependent activation of totA in the Drosophila counterpart of the mammalian liver, the fat body. In addition, totA activation is shown to require the NF-KB-like Relish pathway, indicating that fat body cells integrate the activity of NF-KB and JAK/STAT signaling pathways upon immune response. This study reveals that, in addition to the pattern recognition receptor-mediated NF-kappaB-dependent immune response, Drosophila undergoes a complex systemic response that is mediated by the production of cytokines in blood cells, a process that is similar to the acute phase response in mammals (Agaisse, 2003).

In order to identify genes that are regulated by the JAK/STAT pathway in response to septic injury in adult flies, a screen was performed for candidates that display an inducible expression upon immune challenge and that are constitutively expressed in flies carrying a gain-of-function mutation in the JAK/STAT pathway. To this end, custom-made cDNA microarrays were used to compare gene expression profiles of nonchallenged wild-type flies to gene expression profiles of challenged wild-type flies and to gene expression profiles of nonchallenged TumL flies displaying a gain-of-function mutation in the Drosophila JAK kinase Hopscotch. MP1 was identified as a gene that fulfilled both criteria for induction upon challenge and constitutive expression in a JAK/STAT gain-of-function mutation. MP1 expression was not induced in challenged flies displaying loss-of-function mutation in hop (hopM38/hopmsv1), confirming the involvement of Drosophila JAK in MP1 expression (Agaisse, 2003).

Sequence analysis of MP1 cDNA reveals that MP1 codes for Turandot A (TotA), a polypeptide that is produced by the larval fat body and accumulates in hemolymph in response to various stress conditions in flies. totA expression is mainly fat body specific in adult flies. totA was weakly expressed in the fat body of unchallenged flies and strongly induced after septic injury (Agaisse, 2003).

To analyze the regulation of totA expression in fat body cells, totA expression was monitored in response to clean injury, septic injury with gram-negative bacteria (E. coli), or septic injury with gram-positive bacteria (M. luteus). Clean injury and septic injury with M. luteus resulted in a modest but significant induction of totA expression: 4-fold induction 6 hr after challenge and 7-fold induction 18 hr after challenge. In sharp contrast, septic injury with E. coli resulted in a robust induction of totA expression: 25-fold induction at 6 hr and 35-fold induction at 18 hr. Gram-negative bacteria therefore constitute the best inducer for totA expression. It is well established in flies that immune response to gram-negative bacteria is mediated by the Imd pathway through activation of TAK1 and the NF-KB-like transcription factor Relish. Therefore totA expression was analyzed in TAK1 and in relish mutant flies. totA activation after challenge was totally abolished in these mutants, indicating that, in addition to being JAK/STAT dependent, totA expression also requires the activity of the Relish pathway. Whether the activity of the Relish pathway is specifically required in the fat body was analyzed. To this end, relish dsRNA was overexpressed in fat body using the UAS-irel construct and the yolk-GAL4 driver. dsRNA-mediated silencing of relish expression leads to a failure in totA activation, indicating that Relish activity is specifically required in the fat body. Finally, whether Relish activation in the fat body is sufficient to activate totA expression was analyzed. Overexpression of Imd in fat body cells has been shown to lead to activation of Relish and therefore constitutive expression of the antimicrobial peptide genes, such as diptericin, in the absence of immune challenge. totA is not constitutively expressed in the corresponding flies, indicating that Relish activation is required in fat body but is not sufficient to activate totA expression (Agaisse, 2003).

Upd was first identified as a secreted molecule that activates the JAK/STAT pathway during Drosophila embryogenesis. Evidence is provided for the existence of a component of the JAK/STAT pathway: Upd3 that is produced in hemocytes in response to immune challenge. Although cytokine-like activities, such as IL1 and TNFα, have been previously reported as being produced by hemocytes from Lepidopteran larvae in response to LPS stimulation, none of these activities have been shown to have a physiological function in vivo. upd3 is thus the first example of a gene coding for a cytokine that is expressed in hemocytes and is required for signaling in fat body. This study therefore constitutes the first demonstration that sentinel cells, such as hemocytes, play a signaling role in the Drosophila immune response. The nature of the signals that are detected by hemocytes and the signaling pathway(s) that trigger upd3 activation in response to septic injury remain to be determined. Preliminary experiments indicate that upd3 expression is severely impaired in TAK1 flies after septic injury, suggesting that components of the Relish pathway (as defined in fat body cells) might be involved in upd3 activation in hemocytes in response to bacterial infection. However, further analysis in PGRP-LC and relish mutant backgrounds was not consistent with this hypothesis. Clearly, the mechanisms involved in upd3 regulation potentially constitute a new paradigm for studying the signals and the transduction machinery involved in the control of gene expression in activated hemocytes (Agaisse, 2003).

Inhibitor of apoptosis 2 and TAK1-binding protein are components of the Drosophila Imd pathway

The Imd signaling cascade, similar to the mammalian TNF-receptor pathway, controls antimicrobial peptide expression in Drosophila. A large-scale RNAi screen was performed to identify novel components of the Imd pathway in Drosophila S2 cells. In all, 6713 dsRNAs from an S2 cell-derived cDNA library were analyzed for their effect on Attacin promoter activity in response to Escherichia coli. Seven gene products required for the Attacin response in vitro were identified, including two novel Imd pathway components: inhibitor of apoptosis 2 (Iap2) and transforming growth factor-activated kinase 1 (TAK1)-binding protein (TAB). Iap2 is required for antimicrobial peptide response also by the fat body in vivo. Both these factors function downstream of Imd. Neither TAB nor Iap2 is required for Relish cleavage, but may be involved in Relish nuclear localization in vitro, suggesting a novel mode of regulation of the Imd pathway. These results show that an RNAi-based approach is suitable to identify genes in conserved signaling cascades (Kleino, 2005).

Drosophila has developed a highly sophisticated immune defense, which is required for living in a natural environment that is rich in bacteria and fungi. In contrast to mammals, Drosophila has no adaptive, that is, antibody-mediated immunity, which makes it a good model for studying the pattern recognition receptors and signaling pathways of innate immunity. In Drosophila, there are two major pathways that respond to microbes: the Imd and the Toll pathways. Both of them are strikingly well conserved throughout evolution. Thus, novel findings from work on Drosophila immune response can fuel discoveries in the mammalian systems (Kleino, 2005).

In Drosophila, evolutionarily conserved peptidoglycan recognition proteins (PGRPs) are of paramount importance for microbial recognition. Several Drosophila PGRPs are necessary for normal resistance to bacteria . Secreted PGRP-SA is essential for induction of immune response genes via the Toll pathway in response to certain Gram-positive bacteria in vivo. In contrast, PGRP-LC is the first component of the Imd pathway (Choe, 2002; Gottar, 2002; Rämet, 2002). It is located on the cell membrane where it appears to act as a pattern recognition receptor for bacteria either alone or together with other PGRPs (Takehana, 2004). Recently, intracellular domain of PGRP-LC was shown to bind directly to the Imd, which is the next known component downstream of PGRP-LC (Choe, 2005). Imd contains a death domain with homology to the mammalian receptor-interacting protein 1. The signal is propagated via transforming growth factor-activated kinase 1 (TAK1) to Drosophila homologs for IKKgamma and IKKalpha/ß (Key and Ird5, respectively) (Rutschmann, 2000; Lu, 2001). Whether TAK1 phosphorylates the Drosophila IKKs directly is uncertain and the mechanism of TAK1 activation is elusive. TAK1 has also been shown to play a role in the regulation of the c-Jun N-terminal kinase (JNK) pathway (Park, 2004). Finally, the signal leads to the activation of the Drosophila NF-kappaB homolog Relish, involving its phosphorylation by the IKK complex (Silverman, 2000) and cleavage by a caspase currently believed to be Dredd (Leulier, 2000: Stöven, 2000; Stöven, 2003), which forms a complex with BG4, a homolog to mammalian Fas-associated death domain protein (FADD; Leulier, 2002). The phosphorylated and cleaved Relish is then translocated to the nucleus, where it binds to DNA leading to synthesis of antimicrobial peptides (Kleino, 2005 and references therein).

Normal response to most Gram-negative bacteria in Drosophila depends on the Imd pathway, which is very similar to the TNF receptor signaling pathway in mammals. In order to determine whether there are still unknown components in the Imd signaling pathway, a large-scale RNAi-based screen was carried out in Drosophila S2 cells using a luciferase-reporter-based quantitative assay. The activity of the pathway was assayed using Attacin-luciferase (Att-luc) reporter. Transfection efficiency and cell viability were monitored using Act5C-ß-gal reporter. The Imd signaling pathway was activated with heat-killed Escherichia coli. At first, tests were carried out to see if dsRNA targeting a known component of the pathway caused a decrease in Att-luc activity. Relish (Rel) RNAi blocked the Imd pathway activity in a dose-dependent manner. 10 ng of Rel dsRNA per 5.0 x 105 S2 cells in 500 microl of medium reduced the luciferase activity by >50% and more than 0.1 microg of Rel dsRNA blocked the luciferase activity almost completely. Therefore, RNAi very effectively silences the expression of the targeted gene in this assay, which thus can be used to identify essential components of the Imd pathway (Kleino, 2005).

6713 dsRNAs from an S2 cell-derived cDNA library were assayed for their effect on the Imd signaling pathway in S2 cells using Att-luc reporter as a read-out. Most dsRNA treatments had little or no effect. Seven genes decreased Att-luc activity by >80% without decreasing Act5C-ß-gal activity by more than 40%, indicating that viability and the translation machinery were unaffected. These genes included three (PGRP-LC, imd and Rel) out of eight known components of the Imd pathway. Rel was identified three times. Novel genes identified were kayak, longitudinals lacking (lola), inhibitor of apoptosis 2 (Iap2) and CG7417. The CG7417 protein is a homolog to the mammalian TAK1-binding proteins 2 and 3 (TAB2 and TAB3), hereafter called TAB. Interestingly, a dsRNA treatment silencing Rel, TAB, PGRP-LC, imd or lola also strongly decreased the Drosomycin reporter (Drs-luc) activity induced by the constitutively active form of Toll (Toll10b). Therefore, it appears that a low level of Rel activity is required also for normal Drs response via the Toll pathway in S2 cells (Kleino, 2005).

Kayak is a known component of the JNK signaling pathway; RNAi targeting kayak caused an 88 +/- 7% decrease in Att-luc activity. This is in accordance with recent results, which indicate that JNK is essential for normal antimicrobial peptide release in S2 cells (Kallio, 2005). RNAi targeting lola caused 87 +/- 5% decrease in Att-luc activity. Lola is a nuclear factor that is required for axon growth in the Drosophila embryo and normal phagocytosis of bacteria in S2 cells (Rämet, 2002). Lola has not been indicated to play a role in the synthesis of antimicrobial peptides. In the reporter assay, lola RNAi decreased Att-luc activity slightly less than known components of the Imd pathway. Of note, RNAi silencing of lola also decreased Drs-luc activity induced by Toll10b in S2 cells (Kleino, 2005).

In all, 35 dsRNA treatments representing 22 genes caused a greater than three-fold increase in the Att-luc activity in response to heat-killed E. coli after ecdysone treatment in S2 cells. These genes could be divided into the following categories based on the putative function of their encoding protein: (1) genes involved in microtubule organization or actin cytoskeleton regulation (par-1, Rab-protein 11, multiple ankyrin repeats single KH domain [mask], alpha-Tubulin at 84B, CG6509 and PDGF- and VEGF receptor related [Pvr]); (2) helicases and other genes involved in DNA replication (Helicase 89B, Rm62, kismet, mutagen-sensitive 209 and double parked); (3) signaling molecules (daughter of sevenless, CG32782 and Ecdysone-induced protein 75B); (4) transcription factors (E2F transcription factor and Zn-finger homeodomain 1) and (5) uncharacterized genes. Of note, kismet was identified eight times, Pvr six times and E2F transcription factor twice in this screen. The mechanisms for how these genes affect signaling through the Imd pathway remain to be studied. Of note, none of these dsRNA treatments notably induced the Imd pathway without E. coli (Kleino, 2005).

Two novel components of the Imd pathway, Iap2 and TAB, were identified that appear to be absolutely necessary for induction of Att-luc activity in S2 cells in response to heat-killed E. coli. dsRNA targeting either Iap2 or TAB causes a drastic, 98 +/- 1% decrease in Att-luc activity. Iap2 or TAB RNAi has no effect on cell growth as determined by cell counts, indicating that the result is not due to increased cell death. To verify that the observed phenotypes were caused by decreased expression of Iap2 and TAB, targeted RNAi with gene-specific primers was carried out. Specific dsRNA treatments targeting either Iap2 or TAB drastically decreases the Att-luc activity. TAB RNAi also decreases the Drs reporter activity via the Toll10b-induced Toll pathway. Whether the effect of Iap2 or TAB RNAi was ecdysone dependent was examined. If ecdysone was not used, Att-luc induction was clearly (35 +/- 2%, N=3) weaker, but also this induction was blocked by RNAi targeting either Iap2 or TAB, indicating an ecdysone-independent mechanism (Kleino, 2005).

To ascertain that the results were not due to an artifact related to the use of a reporter construct, the expression level of Cecropin A1 (CecA1), another well-characterized antimicrobial gene regulated by the Imd pathway, was analyzed by semiquantitative RT-PCR. A 6-h exposure to heat-killed E. coli increased the mRNA level of CecA1. This increase could be blocked entirely by RNAi targeting either Rel or TAB. In addition, induction was reduced by RNAi targeting Iap2. Corresponding results were obtained also for Att D and Diptericin (Dpt). dsRNA treatments targeting either Rel or TAB totally blocked the induction, while the effect of Iap2 RNAi was somewhat more moderate (Kleino, 2005).

To investigate whether these in vitro findings are of in vivo relevance, the inducible expression of Iap2 dsRNA was used in Drosophila in vivo. The UAS/GAL4 binary system to drive expression of dsRNA in a defined tissue has been previously used to block the expression of defined genes. To this end, transgenic flies were generated carrying the UAS-Iap2-IR. This construct has two 500 bp long inverted repeats (IR) of the gene, separated by an unrelated DNA sequence that acts as a spacer, to give a hairpin-loop-shaped RNA. These transgenic flies were crossed to flies carrying various GAL4 drivers in order to activate transcription of the hairpin-encoding transgene in the progeny. Iap2 has been shown to be required for the regulation of apoptosis in Drosophila (F. Leulier, personal communication to Kleino, 2005), and overexpression of UAS-Iap2-IR with the ubiquitous and strong daughterless-GAL4 (da-GAL4) driver leads to lethality at the pupal stage. To address the role of Iap2 in antimicrobial gene expression, the UAS-Iap2-IR transgene was expressed using the C564-GAL4 driver that expresses GAL4 in the adult fat body. Flies were kept at 25°C to avoid the induction of apoptosis in the fat body. Flies that express Iap2-IR ubiquitously through C564 showed no detectable defects. However, the expression of the antibacterial peptide gene Dpt was strongly reduced after infection with the Gram-negative bacteria Erwinia carotovora. This phenotype was similar, although weaker, than those generated by BG4-IR RNAi. Importantly, the expression of Drs remained inducible in Iap2-IR; C564 flies, indicating that Iap2 did not block the Toll pathway and that the fat body remained functional (Kleino, 2005).

To map the locations of Iap2 and TAB in the Imd signaling cascade, known components of the cascade were overexpressed including a constitutively active form of Relish (Rel DeltaS29-S45), wild-type Relish or wild-type Imd. All these caused an activation of Att expression in S2 cells. Att induction caused by expression of either Relish construct could not be blocked by RNAi targeting either imd, TAB or Iap2, indicating that both TAB and Iap2 are located upstream of Relish. In contrast, Att induction caused by overexpression of Imd is blocked by RNAi targeting either Rel, imd, TAB or Iap2, indicating that both TAB and Iap2 lie downstream of Imd in the hemocyte-like S2 cells (Kleino, 2005).

To assess whether Iap2 is located downstream of Imd in the fat body in vivo, the UAS-imd construct with a heat-shock-GAL4 (hs-GAL4) driver was overexpressed; this induced expression of the Dpt gene in the absence of infection. Although there is some constitutive Dpt expression in these flies, the level of Dpt increases after heat shock. Using these flies, Dpt expression was reduced by coexpression of UAS-Iap2-IR by 44 +/- 8% (N=2) in these flies. Total RNA was extracted from unchallenged adult flies, collected 6 or 16 h after a heat shock (37°C, 1 h) and RT-PCR analysis was used to monitor the expression level of Dpt. This indicates that Iap2 functions, genetically, downstream of Imd in the fat body in vivo (Kleino, 2005).

To map the exact location of Iap2 in the Imd signaling cascade, Iap2 was overexpressed in S2 cells, which resulted in a minimal but reproducible induction of Att expression. This induction was completely blocked by dsRNAs targeting the known components of the Imd pathway, except dsRNAs targeting either imd or TAK1. These results indicate that Iap2 lies downstream of TAK1 in the Imd signaling pathway. To ascertain efficacies of the dsRNA treatments used, effect of the dsRNA treatments on E. coli-induced Att response was simultaneously measured. All of the dsRNA treatments strongly decreased the Att response, suggesting that the expression of targeted genes was effectively silenced. Of note, it was not possible to stimulate the Imd pathway with the expression vector containing the full-length cDNA of TAB (Kleino, 2005).

Upon Imd pathway activation, the NF-kappaB homolog Relish becomes phosphorylated by the IKK complex and thereafter cleaved by a caspase putatively thought to be Dredd. Finally, Relish is translocated to the nucleus. To study the role of TAB and Iap2 on Relish cleavage, Drosophila hemocyte-like mbn-2 and S2 cells were stimulated with commercial lipopolysaccharide (LPS) known to contain a bacterial component that activates the Imd pathway, followed by Western blotting with Relish antibody (alpha-C; Stöven, 2000). In unstimulated, GFP dsRNA-treated mbn-2 cells, most of Relish is uncleaved (Relish-110), whereas upon LPS stimulus, Relish cleavage is induced. As expected, in Dredd and key dsRNA-treated cells Relish cleavage was blocked. Interestingly, TAB, Iap2 or TAK1 dsRNA did not affect Relish cleavage. Similar results were obtained also in S2 cells. This points to a novel mechanism of regulation of Relish activity. There was no Relish detected in Rel dsRNA-treated cells, indicating that the half-life of REL-49 (C-terminal Relish cleavage product) is less than the duration of the dsRNA treatment. Of note, REL-49 was observed also in all Dredd dsRNA-treated cells. It is possible that after RNAi knockdown, there is a small amount of Dredd left, sufficient to cleave REL-110 in unstimulated cells. Alternatively, there is some constitutively cleaved Relish in cell lines and this cleavage is Dredd independent (Kleino, 2005).

To investigate whether Iap2 or TAB play a role in the nuclear localization of the activated Relish protein, dsRNA-treated S2 cells were stained with alpha-RHD antibody (Stöven, 2000). In GFP dsRNA-treated cells, Relish is translocated into the nucleus upon LPS stimulus. As expected, there is no nuclear staining of Relish in Dredd or key dsRNA-treated, LPS-stimulated S2 cells. Importantly, the nuclear translocation of Relish appears to be affected in both Iap2 and TAB dsRNA-treated cells compared to GFP dsRNA-treated controls. This suggests that cleavage of Relish is not sufficient for translocation of Relish to the nucleus but another, yet to be characterized signal that is propagated via Iap2 and TAB is required. Alternatively, a different staining pattern could be due to decreased stability of nuclear Relish or slower kinetics. Of note, compared to key and Dredd dsRNA-treated cells, very faint nuclear staining can be seen in Iap2 dsRNA-treated cells. Surprisingly, Relish nuclear localization was normal in TAK1 dsRNA-treated cells. This implies a possibility that the role of TAK1 in the Imd pathway signaling is downstream of translocation of Relish into the nucleus. Altogether, these results show that the regulation of Relish activity is more complex than previously thought. The involvement of TAB and Iap2 in nuclear localization but not cleavage of Relish indicates a novel mode of regulation in the Imd pathway (Kleino, 2005).

Iap2 codes for a 498 amino-acid (aa) protein that has three N-terminal BIR (baculovirus IAP repeat) domains and a C-terminal RING-finger (Really Interesting New Gene) domain. Drosophila Iap2 is well conserved throughout phylogeny and has high sequence similarity with many mammalian Iap2s, such as human, rat and mouse (E values 9 x 10-66, 2 x 10-66 and 3 x 10-66, respectively). Interestingly, the CARD (caspase recruitment domain), identified in apoptotic signaling proteins, is present in the mammalian homologs but missing from Drosophila. It has been shown that RING domain containing proteins, including IAPs, bind E2 ubiquitin-conjugating enzymes catalyzing the transfer of ubiquitin from E2 to a substrate, therefore acting as E3 ligases. Ubiquitination can lead to either proteasomal degradation, or, in the case of non-K48-linked polyubiquitination, to multiple outcomes such as activation or relocalization of the substrate protein. Human c-Iap2 is expressed most strongly in immune tissues including spleen and thymus and has been proposed to associate with TRAFs through its BIR domains (Rothe, 1995). However, in a luciferase assay, neither TRAF1 nor TRAF2 dsRNA treatment reduced the Imd pathway activity, indicating that TRAFs are not essential for Imd pathway activity in Drosophila S2 cells (Kleino, 2005).

The Drosophila TAB codes for an 831 aa protein that has an N-terminal CUE domain (97-139 aa) and a C-terminal zinc-finger (ZnF) domain (765-789 aa). Only two other Drosophila genes code for a CUE domain: CG2701, and CG12024. Their function is unknown. TAB is the only Drosophila protein with both CUE and ZnF domains. These domains are homologous to the respective domains in mammalian TABs. The CUE domain carries a ubiquitin-binding motif, whereas the ZnF domain has an alpha-helical coiled-coil region. It has been shown that Drosophila TAK1 binds TAB (CG7417) in a two-hybrid protein interaction system (Giot, 2003). In humans, the C-terminal coiled-coil domain of TAB3 mediates the association with TAK1, and it is also required for stimulation of TAB3 ubiquitination by TRAF6 (Ishitani, 2003). Apart from these two domains, there is very little sequence similarity, suggesting that these domains are functionally important. Indeed, it has been shown that the ZnF domain and the CUE domain, to a lesser extent, of human TAB2 and TAB3 are important to NF-kappaB activation (Kanayama, 2004). The exact molecular mechanism by which Iap2 and TAB modulate signaling via the Imd pathway in Drosophila remains to be studied (Kleino, 2005).

This study identified two novel components of the Imd signaling cascade: Iap2 and TAB. Both of these have mammalian homologs, further indicating high conservation of this signaling cascade. TAB is an 831 aa protein that has conserved CUE and ZnF domains. As in mammals, it is plausible that TAB regulates TAK1 activity also in Drosophila, since TAB was the only protein to bind TAK1 in a two-hybrid protein interaction system (Giot, 2003). Both Iap2 and TAB are located downstream of Imd. Interestingly, Relish is cleaved appropriately without Iap2 or TAB, but there appears to be an effect to the transportation of Relish to the nucleus. Therefore, it is speculated that there is another, previously unidentified level of regulation needed for Relish activation. Since the RING domain-containing Iap2 is a putative E3 ligase, it is hypothesized that this regulation could involve ubiquitination of Relish -- or another protein regulating the activity of Relish -- by Iap2. Possible interaction of Iap2 with the other Imd pathway components remains to be studied (Kleino, 2005).

Surprisingly, Relish nuclear localization is normal in TAK1 dsRNA-treated cells. This implies a possibility that the role of TAK1 in the Imd pathway signaling is downstream of translocation of Relish into the nucleus. These results are in line with recent results from Delaney (in preparation, reported by Kleino, 2005), which indicate that Relish activation is intact in TAK1 mutant flies. Therefore, TAK1 may control the activity of another transcription factor -- possibly via the JNK pathway -- required for normal antimicrobial peptide response in Drosophila. This is in line with identification of Kayak as an important factor for Att response in this study and with earlier results indicating that the JNK pathway is required for normal Att response in S2 cells (Kallio, 2005). Of note, the effect of dsRNA treatments targeting JNK pathway components is more modest compared to TAK1 RNAi in this experimental setting. This is in line with the earlier results of Silverman (2003), who showed that in S2* cells TAK1 RNAi totally blocks Dpt, Cecropin and Att response to LPS, whereas RNAi targeting JNK pathway components hemipterous and basket have a more moderate effect. Nevertheless, the regulatory interplay that has been detected between the Imd and the JNK pathway in the Drosophila innate immune response (Boutros, 2002: Park, 2004) is likely to attract more attention in the future (Kleino, 2005).

This present study underlines the convenience of RNAi-based screening in S2 cells. Importantly, two novel components of the Imd pathway have been identified. The exact roles Iap2 and TAB play in the activation of Relish remain to be solved. In addition, these findings will likely focus attention to investigate the importance of Iap2 in mammalian TNF receptor signaling. This methodology can be readily applied to study other conserved signaling cascades (Kleino, 2005).

Drosophila Sex-peptide stimulates female innate immune system after mating via the Toll and Imd pathways

Insect immune defense is mainly based on humoral factors like antimicrobial peptides (AMPs) that kill the pathogens directly or is based on cellular processes involving phagocytosis and encapsulation by hemocytes. In Drosophila, the Toll pathway (activated by fungi and gram-positive bacteria) and the Imd pathway (activated by gram-negative bacteria) leads to the synthesis of AMPs. But AMP genes are also regulated without pathogenic challenge, e.g., by aging, circadian rhythms, and mating. This study shows that AMP genes are differentially expressed in mated females. Metchnikowin (Mtk) expression is strongly stimulated in the first 6 hr after mating. Sex-peptide (SP), a male seminal peptide transferred during copulation, is the major agent eliciting transcription of Mtk and of other AMP genes. Both pathways are needed for Mtk induction by SP. Furthermore, SP induces additional AMP genes via the Toll (Drosomycin) and the Imd (Diptericin) pathways. SP affects the Toll pathway at or upstream of the gene spätzle, and the Imd pathway at or upstream of the gene imd. Mating may physically damage females and pathogens may be transferred. Thus, endogenous stimulation of AMP transcription by SP at mating might be considered as a preventive step to encounter putative immunogenic attacks (Peng, 2005).

The Toll and Imd signaling cascades are the major and best-characterized pathways involved in the activation of AMPs after pathogenic challenges. The effect of SP on AMP expression was studied by comparing the expression of Mtk, Drs, and Dipt in wt females or in females mutant in the Toll and Imd pathways, respectively, before and after mating with wt males. RNA was extracted from virgin and mated females and analyzed by quantitative PCR (Peng, 2005).

With the exception of dorsal (dl), all loss-of-function mutants of the Toll and Imd pathways abolish or strongly reduce Mtk expression after mating. Thus, Mtk expression induced by SP is dependent on both pathways. Furthermore, since spz and imd females fail to induce Mtk transcription after mating, SP must act on or upstream of spz and imd. dl and its functional homolog dif have been reported to be involved in AMP gene transcription under pathogenic challenge in the larval stage, but not functional in the adult immune defense. A partial response is observed in dl females, indicating that dl may be partially involved in the innate immune response elicited by SP in adult females (Peng, 2005).

Drs expression, controlled by the Toll pathway, is completely abolished in spz and Tl mutants. Correspondingly, Dipt expression, which is controlled by the Imd pathway, is completely abolished in the Imd pathway loss-of-function mutants imd, Tak1, and rel. It is concluded that SP can activate the Toll and the Imd pathways. The Toll pathway is essential for Drs expression, whereas the Imd pathway is essential for Dipt expression (Peng, 2005).

The SP-induced immune response activates the transcription of all three AMP genes studied. After pathogenic infections, Drs is induced by the Toll pathway and Dipt by the Imd pathway, whereas both pathways induce Mtk expression. The results obtained with the loss-of-function mutants follow this scheme. Whereas loss-of-function mutants of both pathways reduce or abolish Mtk expression after mating, induction of Drs expression is only abolished by loss-of-function mutants of the Toll pathway, whereas induction of Dipt expression is only lost in mutants of the Imd pathway. In sum, the classical pathways are activated to induce the transcription of AMP genes after mating as after microbial or fungal infections (Peng, 2005).

Detection of microorganisms and triggering the appropriate pathway is achieved by pattern recognition receptors (PRRs), immune proteins recognizing general microbial components. Two families of PRRs have been identified in Drosophila: the peptidoglycan recognition proteins (PGRPs) and the gram-negative binding proteins (GNBPs). Some of the 13 PGRPs encoded in the D. melanogaster genome have been implicated in the activation of specific immune responses. However, the signaling cascades between the PRRs and the Toll and the Imd pathways are not well characterized. Since in spz and imd null mutants AMP induction by SP is specifically abolished, the inducing signals must affect the signaling cascades at or upstream of those genes. At this stage, it cannot be determined whether SP enters the pathways at the PRR level or at an intermediate level between the PRRs and spz or imd, respectively. Furthermore, the induction of AMPs may occur systemically (e.g., in the fat body) or locally in the reproductive tract. Microarray analysis of AMP expression after mating of wt females with either wt or SP0 males, respectively, suggests that AMPs are mainly induced in the abdomen, but it does not discriminate between a systematic response in the abdomen and a specific response in the genital tract (Peng, 2005).

Drosophila females undergo dramatic physiological changes after mating, predominantly induced by SP. Mating may also physically damage females and may expose the female to pathogens transferred by the male as shown for the milkweed leaf beetle. Thus, the activation of the innate immune system to encounter putative immunogenic attacks during this sensitive phase of the life history of females makes biological sense. The signal is plausibly coupled to copulation in the form of SP transferred in the seminal fluid. Such a mechanism might allow the female to respond preventively to potential threats. In sum, these findings may describe the result of an optimal economical balance between spending costly energy for the innate immune response and preventive measures to fight a putative pathogenic attack (Peng, 2005).

p120 catenin is required for the stress response in Drosophila

p120ctn is a ubiquitously expressed core component of cadherin junctions and essential for vertebrate development. Surprisingly, Drosophila p120ctn (dp120ctn) is dispensable for adherens junctions and development, which has discouraged Drosophila researchers from further pursuing the biological role of dp120ctn. This study demonstrate that dp120ctn loss results in increased heat shock sensitivity and reduced animal lifespan, which are completely rescued by ectopic expression of a dp120ctn-GFP transgene. Transcriptomic analysis revealed multiple relish/NF-kappaB target genes differentially expressed upon loss of dp120ctn. Importantly, this aberrant gene expression was rescued by overexpression of dp120ctn-GFP or heterozygosity for relish. These results uncover a novel role for dp120ctn in the regulation of animal stress response and immune signalling. This may represent an ancient role of p120ctn and can influence further studies in Drosophila and mammals (Stefanatos, 2013).

Inhibition of NF-kappaB in astrocytes is sufficient to delay neurodegeneration induced by proteotoxicity in neurons

This study examined responses in astrocytes induced by expression of disease-associated, aggregation-prone proteins in other cells. A role was examined for intracellular astrocytic responses in a Drosophila model for Spinocerebellar ataxia type 3 (SCA3, also known as Machado-Joseph disease), a disease caused by expansion of the polyglutamine (polyQ) stretch in the ATXN3 gene. In this Drosophila SCA3 model, eye-specific expression of a biologically relevant portion of the ATXN3 gene, containing expanded polyQ repeats (SCA3(polyQ78)) was expressed. Eye-specific expression of SCA3(polyQ78) resulted in the presence of astrocytes in the eye, suggesting putative involvement of astrocytes in SCA3. In a candidate RNAi screen, genes in astrocytes were identified that can enhance or suppress SCA3(polyQ78)-induced eye degeneration. Relish, a conserved NF-kappaB transcription factor, was identified as an enhancer of degeneration. Activity of Relish was upregulated in the SCA3 model. Relish can exert its effect via Relish-specific AMPs, since downregulation of these AMPs attenuated degeneration. Relish signaling was examined in astrocytes on neurodegeneration. Selective inhibition of Relish expression specifically in astrocytes extended lifespan of flies that expressed SCA3(polyQ78) exclusively in neurons. Inhibition of Relish signaling in astrocytes also extended lifespan in a Drosophila model for Alzheimer's disease. These data demonstrate that astrocytes respond to proteotoxic stress in neurons, and that these astrocytic responses are important contributors to neurodegeneration. The data provide direct evidence for cell-non-autonomous contributions of astrocytes to neurodegeneration, with possible implications for therapeutic interventions in multiple neurodegenerative diseases (Li, 2018).


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Relish : Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation

date revised: 23 December 2021

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