BIOLOGICAL OVERVIEW

Relish was identified in a screen for genes involved in the Drosophila immune system by using PCR differential display to identify genes induced in infected flies. Unlike the single domain proteins Dorsal and Cactus (respectively the classic NF-kappaB- and IkappaB-related proteins of Drosophila), Relish contains both a Rel homology domain (hence the name Relish) and an IkappaB-like domain with six ankyrin repeats. Thus Relish is a dual domain protein. In this respect Relish is similar to the compound mammalian NF-kappaB precursors p100 and p105, although no obvious similarity is seen outside the two conserved domains (Dushay, 1996).

Interesting features have been noted in the regions outside the conserved domains. Like RelB, Relish has an unusually long region N-terminal of the Rel homology domain. Furthermore, just downstream of the Rel homology domain there is a serine-rich stretch, corresponding to the position where p100 and p105 have a glycine-rich region that serves as a processing signal for the generation Rel domain containing p50. The serine-rich sequence in Relish may serve a similar function. Another serine-rich region is found in the N-terminal region of Relish. Finally, there are several potential target sites for phosphorylation by casein kinase II, including four in the spacer between the Rel and ankyrin domains, and five either in or near the PEST region. Casein kinase II has been implicated in the constitutive phosphorylation of the PEST region in IkappaB, and the signal-induced phosphorylation of the same protein is mediated by a kinase with similar target sites (Dushay, 1996).

There is experimental evidence for at least three independent signal transduction pathways in the activation of the immune response in Drosophila. One pathway, defined by the Toll gene, is involved in the defense against fungi. A constitutively active form of Toll can mediate superinduction of the Cecropin gene promoter in the transfected hemocyte line mbn-2. Toll is preferentially involved in the induction of antifungal components such as Drosomycin and metchnikowin. The Toll pathway also requires other dorsal group genes such as tube, pelle, cactus, and spatzle (spz), but not dorsal itself. A second pathway directs an antibacterial response. It is defined by the immune deficiency (imd) mutation, which affects the expression of diptericin and other bactericidal factors, but not of drosomycin. In agreement with this model, factors that have both antibacterial and antifungal activity, such as the cecropins and metchnikowin, appear to respond to both pathways. In addition to these two pathways, recent experiments with the Toll-like gene, 18-wheeler, indicate that a third pathway may exist. Mutations in 18-wheeler affect the induction of Attacin, but not Diptericin or Drosomycin (Hedengren, 1999 and references therein).

Although one or more Rel factors are likely to mediate signals from these pathways in the immune response, the specific role of the three known Rel factors in Drosophila is unclear. Dorsal is not required, because dorsal mutants have no phenotypic effect on the induction of the antimicrobial genes. However, recent data show that Dif is specifically involved in the induction of drosomycin and that Dorsal may play a redundant role in the same pathway. Using P element-mediated mutagenesis, a series of deletions in the Relish gene have been created. These mutations have a profound effect on the induction of the entire set of antimicrobial genes, and it is concluded that Relish is a key factor that controls the antibacterial as well as the antifungal defense in Drosophila. The most straightforward interpretation of the broad effect of the Relish mutants on the antibacterial and antifungal responses is that Relish is involved in all three immunity pathways of Drosophila (Hedengren, 1999)

Relish is a key factor in the induction of the humoral immune response in Drosophila, including antibacterial as well as antifungal factors. In striking contrast, there is a complete lack of effect of Relish on cellular immune reactions and hematopoiesis. These observations indicate that cellular and humoral immune responses are controlled by distinct and independent systems in Drosophila. Hematopoiesis, and perhaps the activation of cellular reactions, may instead be controlled by another Rel factor, Dorsal. Overexpression of this factor has been shown to induce lamellocyte differentiation and cause the formation of melanotic capsules, and similar effects have been observed with mutations that lead to the activation of the Toll pathway (Hedengren, 1999 and references therein).

It is interesting to compare the roles of different Rel factors in the cellular and humoral immune responses of Drosophila with the situation in mammals. A parallel can be drawn between the phenotype of the Relish mutants and the effects seen in knockout mice that carry a disruption of the p105 gene. Such mice have a normal complement of lymphocytes, but they are deficient in both specific and nonspecific responses to infection (Sha, 1995). In contrast, disruption of the RelA and RelB genes in mice gives more complex developmental phenotypes, including effects on hematopoiesis. Thus, although the picture is less clear in mice than in Drosophila, it appears to be a conserved feature that different Rel factors regulate immune responses and hematopoiesis (Hedengren, 1999 and references therein).

The relative importance of the humoral factors in Drosophila immunity is dramatically illustrated by the phenotype of the Relish mutants. A single bacterial cell is sufficient to kill these mutants and the resistance to fungal infections is also impaired, even though the cellular immune defense appears to be intact and parasites are efficiently eliminated. This extreme sensitivity to a bacterial infection is surprising, since phagocytosis of bacteria appears to be normal. One possible explanation is that the antibacterial effector functions of the phagocytic cells are impaired in the Relish mutants. Alternatively, and perhaps more likely, enterobacteria such as Enterobacter cloacae may be relatively resistant to the killing mechanisms of the hemocytes. Indeed, injected cells of Enterobacter cloacae or Escherichia coli can survive for a long time in wild-type Drosophila, even though bacteria injected later are eliminated efficiently by the induced immune response. The surviving bacteria probably reside within phagocytic cells, where they are protected from the humoral factors. Thus, the humoral immune response may be the only efficient defense against bacteria such as Enterobacter cloacae or Escherichia coli (Hedengren, 1999 and references therein).

In spite of the dramatic effects on the immune response, homozygous Relish mutants are fertile and give rise to normal offspring. This lack of Relish developmental effects is surprising, considering the fact that early embryos express a specific, maternally contributed form of Relish that lacks the N-terminal domain (Dushay, 1996). The existence of a specific embryonic form suggests that Relish, like dorsal, could play a role in embryogenesis. However, the function of Relish in the early embryo, if any, must be redundant or else it is involved in a more subtle process that is not required for survival (Hedengren, 1999).

Two alternative models are suggested for the role of Relish in the three genetically defined induction pathways of immune responses in Drosophila. The most straightforward interpretation of the broad effect of the Relish mutants on the antibacterial and antifungal responses is that Relish is involved in all three pathways. The case is most clear for the imd pathway, which is of particular importance for the induction of Diptericin and other genes of the antibacterial defense. Since Diptericin induction has an absolute requirement for a functional Relish gene, it has been concluded that Relish is likely to be part of this pathway. Furthermore, Relish could also be involved in the Toll and 18-wheeler pathways, since the inducibility of both Drosomycin and Attacin is significantly reduced in Relish mutants. However, additional factors have to be invoked in order to explain how different pathways can specifically induce different genes. For the Toll pathway, this specificity could be provided by Dif and/or dorsal since these factors have been shown to participate in the Toll-dependent induction of Drosomycin, perhaps in the form of a Relish-Dif (or Relish-Dorsal) heterodimer. In this case, a homo- or a hetero-dimer of Dif and/or Dorsal could partially substitute for loss or Relish, explaining the residual expression of Drosomycin in the Relish mutants. Data supporting this model were very recently obtained by Han (1999). A synergistic effect on Drosomycin expression was found when Relish was overexpressed together with Dif. This indicates that the two factors may indeed cooperate, perhaps as a heterodimer, in the Toll pathway. For the other pathways, no candidates for such specificity factors have been found. The induction of Drosomycin and Attacin is not completely abolished in the Relish mutants, and for this reason at least one Relish-independent pathway must be included in the model (Hedengren, 1999 and references therein).

A second model, which is also consistent with these findings, is suggested by the fact that there is a residual inducibility of Drosomycin in Toll mutants and of Attacin in 18-wheeler mutants. According to this model, Relish and Toll (the latter probably via Dif) act independently to induce Drosomycin, and similarly Relish and 18-wheeler form independent pathways for the induction of Attacin (Hedengren, 1999 and references therein).

It is interesting to observe the complexity of the different signal transduction pathways that converge to activate the immune responses in Drosophila. At least two of these pathways depend on receptors of the Toll family. Several other members of this family can be identified in the database of the Drosophila genome project, and it is possible that yet other signal pathways will be identified as the role of these receptors becomes clear. The availability of Relish and other mutants will help in understanding these different pathways and their role in immunity, not only in Drosophila but probably also in humans (Hedengren, 1999).

Two roles for the Drosophila IKK complex in the activation of Relish and the induction of antimicrobial peptide genes

The Drosophila NF-kappaB transcription factor Relish is an essential regulator of antimicrobial peptide gene induction after gram-negative bacterial infection. Relish is a bipartite NF-kappaB precursor protein, with an N-terminal Rel homology domain and a C-terminal IkappaB-like domain, similar to mammalian p100 and p105. Unlike these mammalian homologs, Relish is endoproteolytically cleaved after infection, allowing the N-terminal NF-kappaB module to translocate to the nucleus. Signal-dependent activation of Relish, including cleavage, requires both the Drosophila IkappaB kinase [consisting of 2 subunits: a catalytic kinase subunit encoded by ird5 (IKKβ) and a regulatory subunit encoded by kenny (IKKγ)] and death-related ced-3/Nedd2-like protein (DREDD), the Drosophila caspase-8 like protease. This report shows that the IKK complex controls Relish by direct phosphorylation on serines 528 and 529. Surprisingly, these phosphorylation sites are not required for Relish cleavage, nuclear translocation, or DNA binding. Instead they are critical for recruitment of RNA polymerase II and antimicrobial peptide gene induction, whereas IKK functions noncatalytically to support Dredd-mediated cleavage of Relish (Ertürk-Hasdemir, 2009).

These data suggest a new model for the regulation of Relish activity. In this model, Relish is controlled by 2 distinct mechanisms, both of which signal downstream of the receptor Peptidoglycan recognition protein LC (PGRP-LC). One arm controls the cleavage of Relish and requires IMD, FADD, DREDD, and the IKK complex. The other arm controls Relish phosphorylation through TAK1 and the IKK complex. Robust induction of antimicrobial peptide expression requires that both mechanisms of control are fully active; Relish must be cleaved and phosphorylated (Ertürk-Hasdemir, 2009).

Phosphorylation of Relish is critical for signal-dependent transcriptional activation of target genes. By using mass spectrometry and in vitro kinase assays, serines 528 and 529 were identified as targets of IKKβ phosphorylation. These serines are phosphorylated rapidly, in cell lines and flies, after immune challenge. Mutation of these residues to alanine resulted in a protein that acted as dominant negative in cell culture, inhibiting the PGN-induced expression of antimicrobial peptide genes Diptericin and Attacin. A recent study reported that ectopic expression of REL-68 (the N-terminal portion of Relish) was not sufficient to drive the expression of the antimicrobial peptide genes Attacin and Cecropin (Wiklund, 2009). Because REL-68 is not expected to be phosphorylated, these results support the conclusion that phosphorylation is critical for Relish-mediated transcriptional activation of AMP genes. However, this article also reported that REL-68 was able to induce Diptericin, which appears to contradict the current findings. In these experiments, transgenic REL-68 is overexpressed, which may contribute to these confusing results (Ertürk-Hasdemir, 2009).

Further supporting the conclusion that Relish phosphorylation is critical, kinase-dead IKKβ transgenic rescue supported only very weak induction of AMP genes in flies. In these experiments, Relish is expressed at normal levels. Surprisingly, serines 528 and 529, and IKKβ catalytic activity itself, were not required for signal-dependent Relish cleavage. Serines 528 and 529 were also not essential for nuclear translocation or DNA binding. Instead, ChIP experiments show that these serines are required for the efficient recruitment of RNA Pol II to the Diptericin locus (Ertürk-Hasdemir, 2009).

These ChIP assays used the 8WG16 mAb, which preferentially recognizes unphosphorylated CTD repeats of the largest subunit of RNA Pol II. The unphosphorylated CTD is associated with the preinitiating RNA Pol II complex recruited to promoters. Thus, these results argue that phosphorylation of Relish on serines 528 and 529 is required for efficient recruitment of RNA Pol II to the Diptericin and Diptericin-B promoters. An alternate possibility, suggested by recent findings on gene regulation in Drosophila, is that phosphorylated Relish could stimulate elongation from paused RNA Pol II. However, a genome-wide analysis of promoters containing stalled RNA Pol II has found that many Drosophila antimicrobial peptide genes are not sites of paused RNA Pol II. The use of 8WG16 in the experiments presented in this study, further argues that phosphorylation of serines 528 and 529 does not modulate RNA Pol II pausing but instead regulates polymerase recruitment to the preinitiation complex at the Diptericin locus. The exact mechanism by which phosphorylation of serines 528 and 529 affect RNA Pol II recruitment remains to be elucidated. It may involve interaction with coactivators, such as components of the mediator complex, or it may involve the recently discovered IMD component Akirin (Goto, 2008), which is argued to function in the nucleus, downstream of Relish (Ertürk-Hasdemir, 2009).

This report also provides further supporting evidence that DREDD may be the caspase that directly cleaves Relish. This study shows that overexpression of DREDD is sufficient to cause Relish cleavage. Relish cleavage required catalytically active DREDD and expression of another apical caspase, the caspase-9 like DRONC, did not generate cleaved Relish. Interestingly, DREDD-mediated Relish cleavage did not lead to Relish phosphorylation and was not sufficient to drive Diptericin expression. Furthermore, immunopurified DREDD, but not drICE, cleaved Relish in vitro, albeit not very efficiently. The poor efficiency of Relish cleavage, in vitro, may be due to the highly oligomeric state of purified Relish and/or the low activity of DREDD, which has proven to be very difficult to produce in an active form. It was also found that a biotinylated peptide with the Relish cleavage site bound active DREDD; although strong evidence for a direct interaction, this assay is not particularly specific. Together, these data strongly suggest that DREDD directly cleaves Relish, but it cannot yet be concluded with certainty that other proteases, such as an effector caspase, are not involved (Ertürk-Hasdemir, 2009).

In addition to DREDD, Relish cleavage also requires both IKK subunits. However, Relish cleavage does not require catalytically active IKKβ. Delaney (2006) showed that TAK1 is not required for Relish cleavage. Because TAK1 is required for the immune-induced activation of the IKK kinase, this result is consistent with the data indicating that IKK catalytic activity is not involved in Relish cleavage. Instead, IKK complex may function as a scaffold or adaptor, but not as a kinase, in controlling the cleavage of Relish (Ertürk-Hasdemir, 2009).

Taken together, these data demonstrate that Relish is regulated by 2 distinct mechanisms. Relish is probably cleaved by DREDD and phosphorylated by the IKK complex. These 2 regulatory mechanisms appear to be independent, because phosphorylation can occur without cleavage, and vice versa, although they are both triggered by PGN stimulation of the receptor PGRP-LC. Surprisingly, the IKK complex also plays a role in the cleavage of Relish, but not through its kinase activity. Instead, IKK-mediated phosphorylation of Relish on serines 528 and 529, within its N-terminal transcription factor module, is necessary for transcriptional activation of target genes (Ertürk-Hasdemir, 2009).

Caspase-Mediated Cleavage, IAP binding, and ubiquitination: linking three mechanisms crucial for Drosophila NF-kappaB signaling

Innate immune responses are critical for the immediate protection against microbial infection. In Drosophila, infection leads to the rapid and robust production of antimicrobial peptides through two NF-kappaB signaling pathways - IMD and Toll. The IMD pathway is triggered by diaminopimelic (DAP)-type peptidoglycan, common to most Gram-negative bacteria. Signaling downstream from the peptidoglycan (PGN) receptors is thought to involve K63 ubiquitination and caspase-mediated cleavage, but the molecular mechanisms remain obscure. This study shows that PGN stimulation causes caspase-mediated cleavage of the Imd protein, exposing a highly conserved IAP-binding motif (IBM) at its neo-N terminus. A functional IBM is required for the association of cleaved IMD with the ubiquitin E3-ligase DIAP2. Through its association with DIAP2, IMD is rapidly conjugated with K63-linked polyubiquitin chains. These results mechanistically connect caspase-mediated cleavage and K63 ubiquitination in immune-induced NF-kappaB signaling (Paquette, 2010).

Activation of the Drosophila IMD pathway by DAP-type peptidoglycan (PGN) leads to the robust and rapid production of a battery of antimicrobial peptides (AMPs) and other immune-responsive genes. Two peptidoglycan recognition protein (PGRP) receptors are responsible for the recognition of DAP-type PGN, the cell surface receptor PGRP-LC and the cytosolic receptor PGRP-LE. DAP-type PGN binding causes these receptors to multimerize or cluster, triggering signal transduction. IMD signaling culminates in activation of the NF-κB precursor Relish and transcriptional induction of AMP genes (Paquette, 2010 and references therein).

Currently, the molecular mechanisms linking these PGN-binding receptors and activation of Relish remain unclear. Genetic experiments suggest that the most receptor-proximal component of the pathway is the imd protein, while the MAP3 kinase TAK1 appears to function downstream. In turn, TAK1 is required for activation of the Drosophila IKK complex, which is essential for the immune-induced cleavage and activation of the NF-κB precursor Relish, the key transcription factor required for immune-responsive AMP gene expression. In addition to NF-κB signaling, TAK1 also mediates immune-induced JNK signaling (Paquette, 2010 and references therein).

Other major components in the IMD pathway include the caspase-8-like DREDD and its adaptor FADD. RNAi-based studies suggest that these proteins have two distinct roles in IMD pathway signaling, one relatively early in the cascade and the second further downstream. Using RNAi, DREDD and FADD have been shown to be required for immune-induced activation of the IKK complex. These data suggested that DREDD and FADD function downstream of IMD but upstream of TAK1; however, it was not established if this upstream role for DREDD involves its protease activity. In its second role, DREDD is thought to proteolytically cleave Relish (Paquette, 2010).

In addition to the components outlined above, several studies have suggested that ubiquitination plays a critical role in the IMD signaling cascade. Recently, Drosophila inhibitor of apoptosis 2 (DIAP2) was shown to be a crucial component of the IMD pathway. Typical of IAP proteins, DIAP2 has three N-terminal BIR domains, which are involved in interactions with proteins carrying conserved IAP-binding motifs (IBMs). In addition, some IAPs, including DIAP2, carry a C-terminal RING finger domain that provides these proteins with ubiquitin E3-ligase activity. Although it is unclear where in the pathway DIAP2 functions, one study showed that the RING finger is indispensable for its role in the immune response, suggesting it operates as an E3-ubiquitin ligase. Also it has been shown, using RNAi-based approaches, that the E2-ubiquitin-conjugating enzymes Uev1a and Ubc13 (bendless) are critical components of the IMD pathway. Notably, Ubc13 and Uev1a function together in a complex to generate K63-linked polyubiquitin chains. K63-polyubiquitin chains are not linked to proteasomal degradation but instead are thought to play regulatory roles. However, no K63-ubiquitinated target protein(s) has been identified in the IMD pathway. Although no connection between DIAP2 and the Bend/Uev1a E2 complex has been established, one attractive scenario is that DIAP2 functions as an E3 together with the Bend-Uev1a E2 complex (Paquette, 2010 and references therein).

The imd¹ allele is a strong hypomorphic mutation that impairs innate immune responses. Surprisingly, this allele encodes a conservative amino acid substitution, alanine (A) to valine (V) at position 31, and is positioned in a region with no obvious structural motifs. The reason for the strong hypomorphic phenotype associated with the A31V substitution remains unclear. This work, demonstrates that imd protein is rapidly cleaved following PGN stimulation. Cleavage requires the caspase DREDD and occurs at caspase recognition motif ₂₇LEKD/A₃₁, creating a neo-N terminus at A31 that is critical for the immune-induced association of IMD with DIAP2. Substitution of the neo-N terminus with valine, as in imd¹, disrupts the IMD-DIAP2 interaction. Moreover, once associated with DIAP2, cleaved IMD is rapidly K63-polyubiquitinated. Together, these data resolve a number of outstanding questions in IMD signal transduction and present a clear molecular mechanism linking caspase-mediated cleavage to NF-κB activation (Paquette, 2010).

Previous work has demonstrated that the caspase-8-like protease DREDD and its binding partner FADD are required upstream in the IMD pathway, at a position similar to Ubc13 and Uev1a (Zhou, 2005). However, it was not clear from these studies if the protease activity of DREDD is also required in this role upstream in the IMD pathway. This study shows that upon immune stimulation the imd protein is rapidly cleaved in a DREDD- and FADD-dependent manner. In fact, expression of DREDD, without immune stimulation, is sufficient to cause IMD cleavage. A caspase recognition site was identified in IMD, with cleavage predicted to occur after aspartate 30. Substitution of this residue with alanine prevents signal-induced cleavage and creates a dominant-negative allele of imd. This putative cleavage site in IMD (₂₇LEKD/A₃₁) is similar to the Relish cleavage site (₅₄₂LQHD/G₅₄₆), consistent with the notion that both proteins are cleaved by the same protease. Likewise, when IMD cleavage was blocked by caspase inhibitors, IMD was no longer ubiquitinated. Alignment of imd protein sequences from 12 Drosophila species and the Anopheles mosquito showed that the cleavage site is highly conserved (LEKD or LETD in all cases). These findings strongly argue that IMD cleavage after position 30 is mediated by DREDD and that this cleavage is critical for further downstream signaling events (Paquette, 2010).

Cleavage of IMD exposes a highly conserved IBM, which then binds the BIR2/3 domains of DIAP2. In the context of programmed cell-death regulation, these IBM motifs are best defined by their neo-N terminal alanine as well as the proline at position 3, both of which are also present in cleaved IMD, supporting the notion that IMD includes an IBM starting at position 31. The notion that IMD carries an IBM also provides a molecular explanation for the hypomorphic phenotype observed in the imd¹ mutant, which carries a valine substitution for this alanine at position 1 of cleaved IMD. Although several IAP proteins have been implicated in mammalian innate immune/NF-κB signaling, the significance of their associated BIR domains, as well as their possible binding to proteins with exposed IBMs, has remained largely unexplored. This study shows that the BIR/IBM association plays a crucial role in innate immune NF-κB signaling in Drosophila. These findings present a unique role for the BIR-IBM interaction module outside of the cell-death arena (Paquette, 2010).

Furthermore, characterization of signaling in the imd¹, diap2, dredd, and PGRP-LC/LE mutant flies provides critical in vivo verification of the cell-culture data and leads to a proposed model. In particular, the molecular mechanism suggests that immune stimulation leads to the DREDD-dependent cleavage of IMD, perhaps by recruiting IMD, FADD, and DREDD to a receptor complex. Consistent with this aspect of the model, dredd mutants and receptor mutants failed to cleave (or ubiquitinate IMD) following infection. Once cleaved, the exposed IBM of IMD interacts with BIR2 and BIR3 of DIAP2. Currently, it is not known precisely where in the cell the IMD/DIAP2 association occurs. Once associated with DIAP2, cleaved IMD is rapidly K63 ubiquitinated. As the RING-mutated version of diap2 failed to support IMD ubiquitination in flies, DIAP2 likely functions as the E3 for this reaction. Furthermore, the imd¹ allele, which fails to interact with DIAP2 because of a mutation in the IBM, demonstrates the critical nature of the IMD-DIAP2 interaction for innate immune signaling. Consistent with the notion that cleavage precedes ubiquitination, mutants that fail to generate ubiquitinated IMD (i.e., diap2 and imd¹) actually accumulate more cleaved IMD than is observed in wild-type flies. Presumably, in wild-type animals, cleaved IMD is efficiently ubiquitinated and thus is difficult to detect in assays. In contrast, dredd mutants or mutants lacking the key immunoreceptors (PGRP-LC/LE) failed to cleave and ubiquitinate IMD, consistent the cell-culture data (Paquette, 2010).

Previous work has suggested that ubiquitination plays a critical role in IMD signaling in the Drosophila immune response. However, the molecular target(s) of ubiquitination and the mechanisms of its activation have remained elusive. The data presented in this study indicate that DIAP2 functions as the E3-ligase in the IMD pathway, a function usually attributed to the TRAF or, more recently, cIAP proteins in mammalian NF-κB signaling pathways (Bertrand, 2009). The E2 complex of Bend and Uev1a also appears to be involved in IMD ubiquitination. RNAi targeting of these K63-ubiquitinating enzymes reproducibly decreases IMD ubiquitination and the induction of target genes; however, the degree of inhibition is variable and never complete (Zhou, 2005). This study show that a third E2 enzyme, Effete, the Drosophila Ubc5 homolog, also plays a vital role in ubiquitination of IMD. RNAi treatment targeting Effete, in concert with Uev1a and/or Bendless reproducibly eliminated IMD ubiquitination and the induction of Diptericin (Paquette, 2010).

Several lines of evidence argue that IMD is the critical target for K63 ubiqutination in this pathway. First, IMD is by far the most robustly modified component that identified, and the only one in which modifications can be detected in whole animals. Second, the protein produced as a result of the imd¹ mutation, which does not signal, is also not ubiquitinated. Third, a deletion mutant, IMDΔ5, is present that is not ubiquitinated and fails to signal. Finally, Thevenon (2009) recently identified the Drosophila ubiquitin-specific protease, USP36, as a negative regulator of IMD ubiquitination. Functionally, USP36 is able to remove K63-polyubiquitin chains from IMD, promoting K48-mediated polyubiquitination and degradation of IMD. Consistent with the current model, animals which overexpress USP36 show decreased levels of IMD ubiquitination and reduced IMD pathway activation as monitored by Diptericin RNA expression, and are susceptible to bacterial infection. Together, these data strongly argue that IMD is the critical substrate for K63-polyubiqutination in IMD pathway signaling, although other proteins may also be conjugated to lesser degree (as shown in this study for DIAP2) and could potentially substitute for IMD as the platform for ubiquitin conjugation. Interestingly, Xia (2009) recently showed that unanchored K63-polyubiquitin chains (i.e., ubiquitin chains that are not conjugated to a target substrate) are sufficient to activate the mammalian TAK1 and IKK kinase complexes. Furthermore, unanchored polyubiquitin chains are produced after stimulation of HEK cells with IL-1β (Xia, 2009). Thus, the presence (or absence) of K63-polyubiquitin chains may be more important than their conjugation substrate (Paquette, 2010).

K63-polyubiquitin chains are likely to serve as scaffolds to recruit the key kinases TAK1 and IKK in the IMD pathway. Both of these kinases include regulatory subunits with highly conserved K63-polyubiquitin binding domains. Drosophila TAB2, which complexes with TAK1, and the IKKγ subunit are predicted to contain conserved K63-polyubiquitin-binding domains. Thus, it is hypothesized that K63-polyubiquitin chains will recruit both the TAB2/TAK1 complex and the IKK complex, creating a local environment for optimal kinase activation and signal transduction; however, this aspect of the model is still speculative (Paquette, 2010).

Although mammalian caspase-8 and FADD are best known for their role in apoptosis, a growing body of literature indicates that these factors, along with RIP1 (which has some homology to IMD), also function in RIG-I signaling to NF-κB. In addition, caspase-8 has been implicated in NF-κB signaling in B cell, T cell, and LPS signaling. Cells, from mice or humans, lacking caspase-8 have defects in immune activation, cytokine production, and nuclear translocation of NF-κB p50/p65. Furthermore, recent evidence also shows that during mammalian NOD signaling the RIP2 protein is ubiquitinated in a cIAP1/2-dependent manner. Given that Drosophila homologs of RIP1, FADD cIAP1/2, and caspase-8 also function in the IMD pathway, the results presented in this study may help elucidate the mechanism by which these factors function in these mammalian immune signaling pathways (Paquette, 2010).

Analysis of Drosophila STING Reveals an Evolutionarily Conserved Antimicrobial Function

The vertebrate protein STING, an intracellular sensor of cyclic dinucleotides, is critical to the innate immune response and the induction of type I interferon during pathogenic infection. This study showed that a STING ortholog (dmSTING) exists in Drosophila, which, similar to vertebrate STING, associates with cyclic dinucleotides to initiate an innate immune response. Following infection with Listeria monocytogenes, dmSTING activates an innate immune response via activation of the NF-kappaB transcription factor Relish, part of the immune deficiency (IMD) pathway. DmSTING-mediated activation of the immune response reduces the levels of Listeria-induced lethality and bacterial load in the host. Of significance, dmSTING triggers an innate immune response in the absence of a known functional cyclic guanosine monophosphate (GMP)-AMP synthase (cGAS) ortholog in the fly. Together, these results demonstrate that STING is an evolutionarily conserved antimicrobial effector between flies and mammals, and it comprises a key component of host defense against pathogenic infection in Drosophila (Martin, 2018).

Pathogenic infection of Drosophila induces the secretion of antimicrobial peptides by the fat body, an organ analogous to the mammalian liver, which accumulate in the hemolymph. Antimicrobial peptides are small, cationic molecules that are capable of killing bacteria and fungi. Like mammals, flies encode a number of pattern recognition receptors (PRRs) that recognize conserved pathogen motifs called pathogen-associated molecular patterns (PAMPs). The recognition of pathogens in Drosophila initiates a signaling cascade where one of the termination points is the induction of antimicrobial peptides (Martin, 2018).

As innate immunity is an ancient, evolutionarily conserved form of host defense, there is a high degree of similarity in the innate immune responses between flies and mammals. Mammalian PRRs consist of Toll-like receptors (TLR) and RIG-I-like receptors (RLR), among other families of PRRs. Activation of PRRs with their respective PAMPs leads to an innate immune response via the nuclear translocation of NF-κB or interferon (IFN) regulatory factor 3 (IRF3). This cascade culminates with the induction of hundreds of IRF-responsive genes, including IFN-β, a cytokine produced during the early stages of infection and the host defense response, which binds to the IFN-α/β receptor and induces IFN-stimulated gene expression. In Drosophila, two classical innate immune pathways function through either Toll or IMD (immune deficient). During Gram-positive bacteria infection, activation of peptidoglycan recognition protein SA (PGRP-SA), the serine protease Persephone, and Gram-negative binding protein 1 (GNBP1) lead to proteolytically processing of Spatzle and stimulation of the Toll receptor, which activates dMyD88, Tube, Pelle, and the NF-κB homolog DIF (dorsal-related immunity factor). The IMD pathway is stimulated by PGRP-LE and PGRP-LC, which recognize diaminopimelic acid (DAP) type peptidoglycan (PGN) on the surface of bacteria and activate autophagy or the IMD pathway through the NF-κB molecule Relish. Together, the Toll and IMD pathways make up two NF-κB pathways in Drosophila that function in the humoral response to pathogenic infection. Signaling through Drosophila NF-κB pathways is similar to the mammalian TLR pathways, both in pathway structure and the proteins involved in signaling. Antimicrobial peptide genes induced as part of the Toll and IMD pathways include Drosomycin (Drs), AttacinA (AttA), and CecropinA2 (CecA2), among others. Each pathway preferentially induces its own set of antimicrobial peptides, and mutations in the Toll-mediated NF-κB molecule DIF render flies susceptible and unable to induce Toll-mediated antimicrobial peptides during Gram-positive bacteria or fungal infections, while mutations in IMD or Relish render flies susceptible and unable to induce IMD-mediated antimicrobial peptides during Gram-negative bacteria infections. However, there is crosstalk between the Toll and IMD pathways, resulting in the sets of peptides being stimulated together. Ultimately, the fruit fly innate immune response must be fully functional for the proper secretion of antimicrobial peptides into the hemolymph to neutralize the pathogenic infection and curb mortality (Martin, 2018).

Another class of PAMPs in mammals that has not been extensively studied in Drosophila is one that recognizes cytosolic DNA or cyclic dinucleotides (CDNs). In mammals, these molecules trigger signaling pathways controlled by STING (stimulator of interferon genes) and lead to NF-κB and IRF3 activation and ultimately the induction of IFN-β. STING is a transmembrane protein that activates an innate immune response during viral or bacterial infection. STING activation in response to Listeria monocytogenes functions through CDNs, that are byproducts of Listeria infection known to induce IFN-β. Recent studies have also identified cyclic di-guanosine monophosphate (di-GMP) as a major signaling molecule in the Listeria life cycle that is able to activate STING. Indeed, during infection with Chlamydia trachomatis, bacterial CDNs directly activate STING to activate a type I IFN response. Cyclic GMP-AMP synthase (cGAS) signals upstream of STING by binding to cytosolic DNA, triggering cGAS to metabolize ATP and GTP into non-canonical cyclic-GMP-AMP (cGAMP) containing 2'-5' and 3'-5' mixed phosphodiester linkages, which are then able to activate STING. While the roles of STING and cGAS in sensing cytosolic nucleic acids have been comprehensively studied in mammalian immunity, less is known about their role in invertebrate immunity (Martin, 2018).

To date, the major nucleic acid sensors that have been identified in Drosophila are the Dicer proteins involved in the RNAi pathway. Dicer-2 is a pathogen-recognition receptor that senses viral nucleic acids and initiates the RNAi pathway to mount innate and antiviral responses to DNA viruses such as invertebrate iridescent virus 6 (IIV6) (Bronkhorst, 2012). Furthermore, Dicer-2 and the mammalian proteins MDA5 and RIG-I share sequence similarity at their RNA-binding helicase domains . While vertebrates sense cytosolic DNA with a variety of proteins, including IFI16, AIM2, and cGAS, only cGAS has an ortholog in Drosophila, namely CG7194. However, CG7194 lacks the zinc-ribbon domain and a positively charged N terminus, which are functionally important for DNA binding (Martin, 2018).

This study sought to identify a CDN-binding protein in Drosophila, and it was found that the Drosophila protein CG1667, henceforth referred to as dmSTING, is orthologous to the vertebrate STING protein. DmSTING retains its ability to bind cyclic di-GMP, leading to the induction of innate immune response genes. Knockdown of dmSTING during Listeria infection led to a loss of innate immune gene induction, increased bacterial burden, and consequent animal mortality. Conversely, overexpression of dmSTING led to increased antimicrobial peptide induction and activation of the Drosophila NF-κB homolog Relish. Interestingly, dmSTING was functional in mammalian cells, and it was able to induce mammalian NF-κB. Finally, epistasis analysis in flies indicated that dmSTING functioned predominantly through the IMD pathway and Relish to achieve antimicrobial peptide induction. Taken together, these results indicate that STING functions through an evolutionarily conserved host defense pathway, whose antimicrobial function, along with the RNAi, Toll, and JAK-STAT pathways, protects the invertebrate host against microbial infection (Martin, 2018).

In Drosophila, dmSTING is conserved at the amino acid level (22% identity and 57% similarity) with hsSTING, especially at regions that are crucial for binding to CDNs. Recent studies have performed evolutionary analyses to confirm that functional cGAS orthologs do not exist in insects (Martin, 2018).

Interestingly, a functional cGAS-STING pathway exists in the sea anemone Nematostella vectensis. However, purified sea anemone cGAS is not active in vitro but will function in human cells, suggesting that there are additional co-factors that are required for cGAS activity. Experiments using dsDNA virus infection in flies containing P elements in CG7194, a putative cGAS ortholog in Drosophila, further suggest the lack of a cGAS-STING axis in Drosophila since the induction of defense response genes or the dependence of dmSTING on survival to IIV6 infection was not observed. Rather, Drosophila Dicer-2, that contains a RIG-I-like helicase domain, activates an antiviral response to IIV6 through the RNAi pathway, and Dicer-2 plays a role in the defense response to both RNA and DNA virus infection in Drosophila through viral RNA sensing and subsequent degradation to protect the host. Taken together, the current results suggest that in Drosophila, STING senses CDNs, and in the absence of a functional cGAS molecule, bacterial CDNs directly lead to STING activation and a subsequent innate immune response. It is contended that dmSTING signals through the IMD pathway; however, gene expression analyses did show that some Toll-specific genes, including Relish, were less induced during Listeria infection when dmSTING was knocked down, likely due to synergism between the two pathways (Martin, 2018).

From an evolutionary standpoint, invertebrates utilize the RNAi pathway as a defense response to exogenous DNA and RNA encountered during viral infections, whereas RNAi plays less of a role, if any, in the defense response to viral infection in vertebrates. Rather, in vertebrates, antiviral immunity is mediated primarily through the RLR-MAVS axis for RNA viruses. Regarding DNA virus infection in vertebrate hosts, cGAS gained functionality in its ability to bind cytosolic DNA and metabolize CDNs as second messengers to activate STING and thus amplify antiviral immunity. As elegantly described by Kranzusch, 2015, while Nematostella vectensis contains a cGAS homolog (nv-cGAS) that stimulates STING signaling in human cells, nv-cGAS does not respond to dsDNA in vitro, also likely due to the absence of the zinc-ribbon domain and a positively charged N terminus, which are also lacking in CG7194. Like the STING homolog in N. vectensis, the role of dmSTING in innate immunity is to sense CDNs in the absence of amplification via cGAS. It should be noted that Kranzusch showed that insect STING orthologs, including dmSTING, did not associate with CDNs in their assay. However, in these experiments, a full-length dmSTING construct was used, containing the hydrophobic N-terminal transmembrane domains, which may inhibit CDN binding when the protein is not in its natural in vivo state. Crystal structures of dmSTING may be needed to uncover its precise interactions with CDNs (Martin, 2018).

During infection in mammals, bacteria such as Chlamydia generate CDNs that activate the innate immune response in a STING-dependent and cGAS-independent manner. Listeria secretes c-di-AMPs to induce an IFN response that stimulates the STING pathway. Additionally, Listeria generates c-di-GMP during its life cycle , which may be secreted or released intracellularly upon bacterial lysis to directly activate STING. However, the activation of STING in mice and the subsequent induction of IFN did not have an effect on Listeria load in the animals. In fact, the production of IFN during Listeria infection in mice is deleterious to survival, as type I IFN and IRF3 knockout mice are resistant to Listeria infection, since IFN promotes lymphocyte apoptosis. Conversely, an innate immune response in Drosophila to Listeria infection that induces antimicrobial peptides reduces Listeria-induced mortality and bacterial replication. Protection is mediated in part by the induction of IMD-mediated antimicrobial peptides such as Attacin, Cecropin, and Listericin. In the current experiments, STING-mediated induction of Attacin, Cecropin, and Listericin was observed during Listeria infection that was associated with decreased mortality and bacterial replication. Additionally, increased dmSTING-mediated Relish activation was observed, which in addition to inducing the antimicrobial peptides Attacin, Cecropin, and Listericin also positively regulates Zip3 and spirit (Martin, 2018).

A proposed mechanism by which dmSTING leads to the induction of antimicrobial peptides through the NF-κB homolog, Relish, is bolstered by the fact that dmSTING is able to induce mammalian NF-κ. The results indicate that dmSTING functions upstream of the Drosophila NF-κB ortholog Relish and likely also upstream of IMD, since knockdown of Relish and IMD in dmSTING-overexpressing flies resulted in decreased antimicrobial peptide induction during Listeria infection. Functional genomics and epistasis analysis indicates that the loss of dmSTING results in the loss of IMD signaling suggesting that dmSTING aids in inducing a defense response through the IMD signaling pathway. As compared to hsSTING, dmSTING is lacking 31 amino acids from its C terminus. The CTT in mammalian STING, which contain multiple phosphorylation sites, may have evolved to control the IRF family of transcription factors, since mammalian STING variants lacking these regions are unable to activate IRF3 but retain an ability to activate NF-κB. Additionally, the mammalian STING CTT may repress NF-κB activity, since significantly reduced mammalian NF-κB activity was observed when the CTT was appended onto dmSTING. Future experiments to assess the ability of dmSTING to activate NF-κB in other non-mammalian and invertebrate species would provide insight into how the STING-NF-κB signaling axis evolved (Martin, 2018).

In addition to the classical Toll, IMD, JAK-STAT, and RNAi pathways of innate immunity in Drosophila, autophagy and apoptosis play major roles in the defense response to infection. However, previous reports suggest that the pathway through which each function to induce a host defense response differs. For example, when chromosomal DNA escapes apoptotic degradation, there is induction of the IMD pathway, but not the Toll pathway. However, in response to viral infection, an antiviral state is induced via hemocyte-mediated phagocytosis of virions and, to a minor extent, autophagy through Atg7, independent of the canonical Toll, IMD, and JAK-STAT pathways. With regards to Listeria infection, both the Toll and IMD pathways are important to combat infection, as well as autophagy. While both PGRP-LC and -LE induce antimicrobial peptides in response to monomeric PGN stimulation , only PGRP-LE controls autophagy during Listeria infection. Since mammalian STING contributes to autophagy, it would be prudent to test the role of dmSTING in autophagy (Martin, 2018).

Further understanding of STING function in Drosophila, and more importantly, how it functions during pathogenic infection, will have an important impact on how methods are developed to target STING for therapeutic intervention, particularly with regards to insect vector-borne diseases. Additionally, studies of dmSTING may help in uncovering evolutionarily conserved mechanisms of autoimmunity, since STING activity must be kept under tight control to prevent autoimmune disease in humans (Martin, 2018).

NF-kappaB shapes metabolic adaptation by attenuating Foxo-mediated lipolysis in Drosophila

Metabolic and innate immune signaling pathways have co-evolved to elicit coordinated responses. However, dissecting the integration of these ancient signaling mechanisms remains a challenge. Using Drosophila, this study uncovered a role for the innate immune transcription factor nuclear factor kappaB (NF-kappaB)/Relish in governing lipid metabolism during metabolic adaptation to fasting. Relish in fat bodies was found to be required to restrain fasting-induced lipolysis, and thus conserve cellular triglyceride levels during metabolic adaptation, through specific repression of ATGL/Brummer lipase gene expression in adipose tissue (fat body). Fasting-induced changes in Brummer expression and, consequently, triglyceride metabolism are adjusted by Relish-dependent attenuation of Foxo transcriptional activation function, a critical metabolic transcription factor. Relish limits Foxo function by influencing fasting-dependent histone deacetylation and subsequent chromatin modifications within the Bmm locus. These results highlight that the antagonism of Relish and Foxo functions are crucial in the regulation of lipid metabolism during metabolic adaptation, which may further influence the coordination of innate immune-metabolic responses (Molaei, 2019).

In order to explore mechanistic connections between NF-κB and various metabolic control networks, lipid homeostasis was assessed in Drosophila lacking functional Relish (utilizing the rel^E20 allele) independent of pathogenic infection. Relish is similar to mammalian p100/p105 NF-κB proteins and contains a Rel-homology domain, as well as ankyrin repeats (found in mammalian inhibitory IκBs). During ad libitum feeding, NF-κB/Rel mutant adult female flies (rel^E20/rel^E20) have significantly less organismal TAGs than genetically matched controls (either OreR or rel^E20 / + heterozygote flies, 7 days old post eclosion). However, these changes in TAG correlated with decreases in acute and chronic feeding and can be rescued by high-calorie (sugar) diets, suggesting that steady-state differences in lipid homeostasis are potentially driven by changes in feeding behavior. Assaying the major fat storage tissues, this study also found that TAG-level reduction in mutant animals correlates with strong, but variable, decreases in neutral lipid content in fat body and/or adipose but not in the intestine (Molaei, 2019).

Since ad libitum effects on lipid homeostasis appear to correspond with feeding deficits (i.e., they are potentially indirect), changes in fat metabolism were assayed in Relish mutant animals during metabolic adaptation to fasting. rel^E20/rel^E20 mutant flies were sensitive to starvation. Furthermore, Relish-deficient animals displayed accelerated decreases in organismal TAG levels during acute fasting (at time points before significant death occurred), while controls flies showed little to no change at the same time points (always comparing within sibling genotypes). These changes in TAG levels correlate with a strong reduction of stored neutral lipids and/or lipid droplets in carcass fat body, suggesting that there is enhanced or accelerated lipid breakdown during metabolic adaptation in these animals (Molaei, 2019).

The insect fat body acts as a key sensor to link nutrient status and energy expenditure and, as such, is the major lipid depository (mainly TAGs) that combines energy storage, de novo synthesis, and breakdown functions of vertebrate adipose and hepatic tissues. This tissue is also essential for Toll- and Relish-mediated innate immune responses to bacterial infection. Critically, the fat body is integral to properly balance lipid catabolism and anabolism in order to modulate organismal energy homeostasis (through lipid supply to other tissues) in response to metabolic or dietary adaptation. Expression of full-length Relish in the fat body (CGGal4>UAS-Rel) can rescue reduced starvation survival rates and the accelerated loss of lipid storage in rel^E20/rel^E20 mutant flies during fasting. These data suggest that Relish function in the fat body is required to acutely maintain lipid homeostasis throughout the course of metabolic adaptation (Molaei, 2019).

To further confirm an autonomous and potentially direct role for Relish in the regulation of fasting-mediated changes in lipid metabolism, Relish was specifically in the fat body (using multiple, independent RNAi lines: named UAS-RelRNAi KK and GD). Attenuating Relish in the fat body of female flies (CGGal4>UAS-RelRNAi) leads to starvation sensitivity, as well as accelerated loss of organismal TAG levels and fat body lipid storage in response to fasting; additional RNAi control experiments can be found in Figures S1F-S1J. As expected, fasting-induced changes in fat body lipid storage occur before significant decreases in total TAG levels of whole animals are observed. Phenotypes were confirmed with an independent fat body driver (PplGal4; Figures S2A-S2C), and similar results were found utilizing males but not when utilizing another immune cell (hemocyte) driver (HmlGal4). Conversely, over-expressing full-length Relish (CGGal4>UAS-Rel) or a constitutively active N-terminal fragment (CGGal4>UAS-Rel.68) in the fat body significantly limits fasting-mediated decreases in lipids compared to controls (Molaei, 2019).

Furthermore, attenuation of upstream components of the Relish signaling pathway phenocopies these Relish loss-of-function effects on lipid metabolism during metabolic adaption. Relish is governed by conserved regulators TAK1 and the IKK (IκB Kinase) signalosome (which consists of homologs of both IKKβ [Drosophila Ird5] and IKKgamma [Drosophila Kenny (key)]), while the apical caspase DREDD is required for the proteolytic cleavage of the IkB domain, allowing for nuclear translocation. Inhibiting Kenny or DREDD in the fat body of female flies (CGGal4>UAS-DREDDRNAior KeyRNAi) leads to starvation sensitivity, as well as accelerated loss of organismal TAG levels and fat body lipid storage in response to fasting (compared to control flies (CGGal4>w1118). Similarly, attenuating upstream receptors usually required for NF-κB/Relish activation (PGRP family members PGRP-LC [trans-membrane] or PGRP-LE ) also leads to decreased lipid storage in the fat body after starvation, suggesting that at least part of the canonical innate immune pathway is required for these metabolic phenotypes (Molaei, 2019).

Taken together, these data show that Relish can autonomously regulate lipid metabolism in the fat body during metabolic adaptation and suggest that Relish may direct specific metabolic responses to control the breakdown of TAGs (Molaei, 2019).

Properly balancing energy homeostasis in response to metabolic adaptation depends on the ability to coordinate storage, breakdown, and mobilization of lipids, primarily TAG. This coordination requires precise control of metabolic response networks, including changes in metabolic gene expression. To determine potential mechanisms by which the Relish transcription factor could direct cellular TAG metabolism during fasting, transcriptional changes of various metabolic genes related to lipid catabolism or anabolism were measured in Relish-deficient animals. Specifically, the lipase Brummer (Bmm) was identified as being regulated by Relish. Bmm is the Drosophila homolog of mammalian adipose TAG lipase (ATGL), an enzyme that is critical for lipolysis. Bmm plays an essential and conserved role in TAG breakdown and, subsequently, fatty acid mobilization, from lipid droplets in fat storage tissues during metabolic adaptation. In control flies, bmm transcription is mildly induced during acute fasting, but in rel^E20/rel^E20 mutant flies, bmm expression is strongly up-regulated (from whole flies). These Relish-dependent changes in bmm transcription appear unique, as Relish deficiency does not impact fasting-induced changes in other lipases such as Drosophila hormone-sensitive lipase (dHSL), Drosophila lipase 4 (dlip4), or CG5966. Similar results were found in dissected fat body with specific attenuation of NF-κB/Relish in the same tissue (CGGal4>UAS-RelRNAi KK). These results suggest that Relish function is required to repress or limit Bmm expression in response to metabolic adaptation and subsequently restrain TAG breakdown (Molaei, 2019).

To correlate this difference in gene expression to differences in lipolysis, an assay was employed to measure dynamic changes in lipid content based on the incorporation of radiolabeled glucose (14C-glucose) into lipids during fatty acid synthesis in vivo. After acute feeding (1.5 h) of a diet containing 14C-glucose, Relish-mutant flies show drastic changes in glucose incorporation (synthesis) that is likely due to changes in feeding behavior. 16 h of feeding minimized these differences in synthesis, and subsequent analysis of newly synthesized 14C-labeled lipids during fasting showed an increased rate of breakdown in rel^E20/rel^E20mutant flies (47% in mutants compared to 20% in controls). This change in the rate of breakdown correlated with increases in free fatty acids. Finally, genetically attenuating Bmm lipase in the fat body (CGGal4>UAS-BmmRNAi) can rescue the accelerated loss of TAGs in rel^E20/rel^E20 mutant flies during fasting (Molaei, 2019).

These data collectively reveal that Relish function is required to limit fasting-induced Bmm gene expression and subsequently restrain TAG lipolysis during metabolic adaptation (Molaei, 2019).

Following these results, the mechanism by which the Relish transcription factor can context-dependently attenuate Bmm expression was explored. Utilizing Cis-element OVERrepresentation (Clover) software, this study identified conserved NF-κB DNA binding motifs (κB sequence sites identified as GGG R N YYYYY) throughout the first intron of the Bmm locus. To assess binding, a previously characterized Relish antibody was used to perform chromatin immunoprecipitation (ChIP)-qPCR experiments. Relish binding in fed or fasted wild-type flies is significantly enriched (compared to immunoprecipitations using serum controls) at binding motif(s) approximately 1 kb downstream from the transcriptional start site. This putative Bmm regulatory region was cloned upstream of RFP (red fluorescent protein) in order to generate in vivo expression reporters. While the unaltered region only slightly influenced RFP reporter activity in fed or fasted conditions, eliminating the Relish binding site leads to minimal enhanced reporter activity under fed conditions and strong increases in RFP activity during fasting (primarily in the fat body of the carcass and head). Thus, this Relish-binding site within the first Bmm intron acts as an important regulatory region to limit induced gene expression (Molaei, 2019).

Relish binding at this region is similar in fed and fasted states. No evidence was found of classical Relish transcriptional activation function during acute fasting. First, innate immune target gene expression (Drosomycin and Diptericin) and Relish DNA binding to innate immune gene promoters (Diptericin) were not changed during fasting. Second, metabolic adaptation did not significantly alter nuclear localization of Relish in the fat body. Thus, in order to explore how Relish limits or represses fasting-induced Bmm expression, despite its constitutive binding to DNA and distinct from its transcriptional activation function, histone and/or chromatin changes were assessed in Relish-deficient flies. Histone deacetylases (HDACs) have been shown to accumulate in the nucleus during metabolic adaptation, influencing gene expression in a fasting-dependent manner through chromatin regulation and transcription factor deacetylation. Furthermore, previous studies have linked interactions of NF-κB transcription factors and HDACs with NF-κB-dependent transcriptional repression. It was thus hypothesized that Relish might repress Bmm gene expression through influencing histone modifications during fasting, when histone modifiers (such as HDACs) in the nucleus are elevated. Using ChIP-qPCR, histone 3 lysine 9 acetylation (H3K9ac, a post-translational modification generally associated with transcriptional activation) was monitored at this Bmm regulatory region in Relish-deficient animals and controls. During feeding, there is no change in H3K9ac enrichment at this locus between genotypes. However, during fasting rel^E20/rel^E20 mutant flies display a significant enrichment (compared to controls) of H3K9ac at the site of Relish binding, indicative of promoter or enhancer activation. Analysis of modEncode ChIP sequencing databases associated with histone modifications (in adult female flies) also revealed that this site is generally enriched for other modifications linked to gene expression regulation (such as H3K27ac, H3K4me3, and H3K4me1), further indicating that this locus is an important regulatory region. Additionally, inhibiting a single HDAC in the fat body (Rpd3 [Drosophila HDAC1]; CGGal4>UAS-Rpd3RNAi) can drive small, but significant, increases in fasting-induced Bmm transcription (from whole flies; Figure 3D) and accelerate fat body lipid usage (Molaei, 2019).

Taken together, these data show that Relish can bind to a putative regulatory region within the Bmm locus during both feeding and fasting. In response to fasting, the presence of Relish can influence fasting-dependent histone acetylation and chromatin changes that are consistent with transcriptional repression (Molaei, 2019).

The unique ability of Relish to limit or repress fasting-induced Bmm transcription correlates with attenuation of H3K9ac at Bmm regulatory regions. It was thus hypothesized that Relish binding to the Bmm locus leads to fasting-dependent chromatin changes, which subsequently limit transcription activation function of other factors that are induced during metabolic adaptation. Various metabolic transcription factors were assessed and Foxo, a critical regulator of lipolysis and catabolism in general, was shown to be required for Relish-dependent changes in ATGL/Bmm expression during metabolic adaptation. Firstly, Foxo (of which there is a single ortholog in Drosophila) is activated during metabolic adaptation and required for fasting-induced ATGL/Bmm expression across taxa, including in the fly fat body. Full Relish/Foxo double mutant animals (using the foxo²⁴ allele) are synthetic lethal during metamorphosis (rel^E20, foxo24/rel^E20). However, simply reducing Foxo gene dose in NF-κB/Relish mutant flies (rel^E20, foxo²⁴/rel^E20) completely rescues fasting-dependent increases in Bmm expression, starvation survival rates, and increases in lipolysis (accelerated loss of lipid storage) in Relish-deficient flies during metabolic adaptation. Molecular analysis of Foxo transcription activation function also showed that Foxo binding to the Bmm promoter is slightly, but significantly, elevated in Relish-deficient flies only during fasting. Furthermore, attenuating Foxo specifically in the fat body (CGGal4>UAS-FoxoRNAi) rescues the enhanced depletion of lipid storage and starvation sensitivity associated with rel^E20/rel^E20 mutant flies during fasting. Foxo transcription activation function is thus required for Relish-dependent changes in lipid metabolism, highlighting that Relish/Foxo integration and antagonism is critical to maintain TAG metabolism throughout the course of metabolic adaptation (Molaei, 2019).

In summary, this study has uncovered a role for the innate immune transcription factor Relish in governing lipid metabolism during metabolic adaptation to fasting utilizing Drosophila. Relish is required to restrain fasting-induced lipolysis and thus conserve cellular TAG levels and promote survival during metabolic adaptation, through specific repression of Bmm lipase gene induction in the fat body/adipose. Fasting-induced changes in Bmm expression and TAG metabolism are adjusted by Relish-dependent attenuation of Foxo transcriptional activation function, likely through regulation of histone acetylation (Molaei, 2019).

These findings thus suggest that association of Relish with histone modifiers (and subsequent changes in chromatin accessibility at gene regulatory regions) functions to control or limit the induced level of certain metabolic genes. Indeed, a few previous studies have highlighted that mammalian p65/RelA or Drosophila Relish can negatively regulate gene expression, including innate immune targets, through changes in chromatin. This modification of chromatin and repression of gene expression likely occurs through recruitment of HDACs (HDAC 1/2) to promoter or enhancer regions of target genes, thus influencing histone acetylation, chromatin remodeling, and function of NF-κB itself or other transcriptional activators. This study shows that Relish can bind a metabolic target gene during both feeding and fasting but that uniquely during fasting, it can influence H3K9 acetylation levels at this target gene and subsequently limit transcriptional activation function of positive regulators (such as Foxo). This allows NF-κB transcription factors precise control of specific metabolic gene expression, potentially through metabolic changes in HDAC nuclear localization, in a context-dependent manner. To this end, this study has uncovered other metabolic target genes of Relish (in unique tissues with unique metabolic functions) regulated through similar mechanisms (Molaei, 2019).

It remains unclear whether Relish binding to metabolic target genes in fed states, independent of metabolic stress, can influence gene expression and physiology. However, this may represent a constitutive or steady-state function of NF-κB (described in mammals) and thus could act as a priming mechanism that promotes the maintenance of metabolic homeostasis during acute stress (such as fasting) (Molaei, 2019).

While these data show that NF-κB can direct lipid metabolism independent of infection, Foxo (as well as HDACs) is integrated with innate immune responses through a variety of mechanisms. Additional experimental evidence suggests that Relish and, perhaps, Relish-Foxo antagonism and Bmm gene regulation manipulate TAG metabolism after a chronic systemic bacterial infection. It is thus possible that NF-κB-dependent regulation of TAG catabolism, by limiting Foxo-mediated lipolysis, also plays a role in governing innate immune homeostasis in response to pathogenic infections (Molaei, 2019).

Microenvironmental innate immune signaling and cell mechanical responses promote tumor growth

Tissue homeostasis is achieved by balancing stem cell maintenance, cell proliferation and differentiation, as well as the purging of damaged cells. Elimination of unfit cells maintains tissue health: however, the underlying mechanisms driving competitive growth when homeostasis fails, for example, during tumorigenesis, remain largely unresolved. Using a Drosophila intestinal model, this study found that tumor cells outcompete nearby enterocytes (ECs) by influencing cell adhesion and contractility. This process relies on activating the immune-responsive Relish/NF-κB pathway to induce EC delamination and requires a JNK-dependent transcriptional upregulation of the peptidoglycan recognition protein PGRP-LA. Consequently, in organisms with impaired PGRP-LA function, tumor growth is delayed and lifespan extended. This study identifies a non-cell-autonomous role for a JNK/PGRP-LA/Relish signaling axis in mediating death of neighboring normal cells to facilitate tumor growth. It is proposed that intestinal tumors 'hijack' innate immune signaling to eliminate enterocytes in order to support their own growth (Zhou, 2021).

The intestinal epithelium separates the organism from the environment and plays essential roles in nutrient uptake and immune and regenerative processes. Intestinal renewal requires dynamic regulation of cell-cell contacts between enterocytes, and this is achieved by highly proliferative stem cells, proper differentiation, and cell loss by cell extrusion and apoptosis. Dysregulation of cell death in the intestinal epithelium can lead to pathologies such as intestinal bowel diseases and cancer (Zhou, 2021).

Studies in Drosophila have made important contributions toward an understanding of intestinal homeostasis, innate immunity, and aging. In adult flies, intestinal stem cells (ISCs) self-renew and produce progenitor cells called enteroblasts (EBs). These EBs can differentiate into either enteroendocrine cells (EEs) or enterocytes (ECs). The intestinal epithelium undergoes rapid stem cell division and differentiation to continuously replace damaged ECs and ensure tissue integrity and homeostasis, similar to mammalian intestines. Previous studies have shown that bacterial infection induces ISC proliferation and elimination of damaged ECs, thereby leading to remodeling of the intestinal epithelium. Enteric infection also triggers the evolutionarily conserved NF-ΚB pathway through the recognition of pathogen-associated molecular patterns (PAMPs) by pattern recognition receptors (PRRs), leading to the production of antimicrobial peptides (AMPs) for host immune defense (Zhou, 2021).

In addition to the role of the NF-κB pathway in AMP production in different cell types, several studies have identified non-immune functions. For example, constitutive activation of NF-κB reduces animal lifespan, NF-κB activity has been implicated in age-related neurodegenerative diseases, and NF-κB regulates Mef2 to coordinate its immune functions with metabolism. Further evidence links NFκB to Ras/MAPK and JAK/STAT signaling pathways. This allows for the proper balance of immune responses with cell growth and proliferation. Moreover, it has been recently reported that the NF-κB pathway in Drosophila is involved in infection-induced EC shedding, which facilitates maintenance of barrier function during intestinal regeneration (Zhou, 2021).

The Drosophila BMP2/4 homolog Decapentaplegic (Dpp) is involved in multiple developmental processes. The Dpp signal is transduced by the type I receptor Thickveins (Tkv) and type II receptor Punt that phosphorylate Drosophila Smad transcriptional factors such as Mothers against Dpp (Mad), Medea (Med), and the coregulator Schnurri (Shn) to regulate gene expression. Inactivation of BMP signaling components in the Drosophila intestine leads to intestinal tumor formation resembling juvenile polyposis syndrome (JPS). In humans, loss of BMP signaling leads to JPS, which has been associated with increased risks of developing gastrointestinal cancer (Zhou, 2021).

Cell replenishment and rearrangement are common mechanisms to sustain tissue homeostasis, which is also essential for development. Tissue growth requires dynamic cell rearrangements including cell elimination by mechanical competition. For example, epithelial cells can be eliminated by cell extrusion to maintain tissue homeostasis. Recent studies also suggested that tumor cells outcompete and eliminate their neighboring cells to clear space for their expansion. However, the underlying mechanisms and how tumor cells eliminate normal cells in the tumor microenvironment remain largely unknown (Zhou, 2021).

This study demonstrates a critical role for mechanical competition in the tumor microenvironment to promote tumorigenesis. Mechanistically, it was shown that tumor induces DE-cadherin- and myosin-dysregulation-associated mechanical stresses to nearby ECs. These processes trigger the ROCK-associated JNK signaling and subsequent activation of PGRP-LA/NF-κrB/ Relish in surrounding ECs to regulate the expression of pro- apoptotic genes and thereby promote cell delamination and apoptosis. The dying ECs then induce paracrine JAK/STAT signaling to trigger regeneration and further promote tumorigenesis. Importantly, tumors with associated activation of JNK/ PGPR-LA/Relish cascades can be inhibited by preventing apoptosis or by administering ROCK inhibitors. These results thus establish a tumor-cell-driven inflammatory feedback mechanism for competitive growth (Zhou, 2021).

This study demonstrates a non-cell-autonomous feedback mechanism that facilitates tumor development. First, tumor growth outcompetes its microenvironment by inducing mechanical forces. This triggers stress related ROCK/JNK signaling and induces EC elimination through activation of PGRP-LA/Relish signaling and downstream pro-apoptotic genes. Subsequently, dying ECs produce cytokines that activate JAK/STAT signaling in tumor cells to further promote tumor growth, thereby establishing a positive amplification loop between the tumor and its microenvironment (Zhou, 2021).

Cells undergoing rapid proliferation will push on their neighbors, which leads to local increase in mechanical pressures and triggers cell delamination. Previous studies have shown that the expansion of tumor cells triggers cell competition, which involves mechanical interactions. For instance, ectopic expression of Ras oncogene drives mechanical competitive growth and induces delamination of nearby wild-type cells in Drosophila. Hence, these data are in line with the current interpretation that mechanical competition also drives tumor competitive growth in the Drosophila intestine. In the epithelium, the mechanical modulation of surface tension is regulated by the actomyosin complex, counterbalanced by the Cadherin-dependent cell-cell adhesions. This study shows that tumors interact with their microenvironment and trigger ROCK/ JNK related cell death. A similar mechanism was observed in mammalian Madin-Darbin canine kidney (MDCK) cells, which, when deficient in the polarity gene scribble, are eliminated by mechanical cell competition, a process that requires the activation of the ROCK-p38-p53 pathway (Zhou, 2021).

The intestinal epithelium requires homeostatic mechanisms to counterbalance stem cell division and elimination of damaged or unfit cells. Dysregulation of either of these homeostatic programs can lead to tumor development. This study found that tumor cells induce immune-responsive PGRP- LA/Relish signaling for cell delamination and apoptosis. The role of NF-κB in the regulation of apoptosis has been discussed before for Drosophila Imd and mammalian TNFR1 pathways, which share key components to regulate NF-κB-related immune response and caspase-dependent apoptosis. Several studies have shown that tumor cells displace the nearby ECs through activation of Hippo and JNK signaling for tumor progression. In addition, JNK acts in parallel with NF-κB to control EC shedding during intestinal regeneration. In mammals, intestinal TNFR1 signaling is also required for EC detachment and apoptosis (Zhou, 2021).

Previous studies revealed a role for NF-κB signaling in cell death of outcompeted cells during development. In Drosophila wing discs, Myc-induced cell competition triggers Imd/Relish-related activation of the pro-apoptotic gene Hid for cell death. The Toll-signaling transcription factors Dorsal and Dif have been suggested to be required for Minute-induced cell competition by inducing Reaper-dependent apoptosis of outcompeted cells. However, further evidence showed axenic conditions abolished Toll-inhibition-induced competitive growth in the outcompeted cells. This suggested that infection contributes to Toll pathway inhibition induced cell competition. The current experiments indicate that axenic conditions failed to abolish the Imd activation in the tumor-surrounding ECs, suggesting a tumor-associated role of Imd/Relish induced EC cell death. Furthermore, a recent study discovered that cells with growth advantages, such as high protein synthesis, induce NF-κB-dependent autophagy to eliminate neighboring unfit cells in developing tissues. NF-κB and its upstream activating receptor are also required for salivary gland degradation through autophagy. Eye disc tumors also trigger a cell-autonomous feedback loop to promote proliferation by activation of JNK, Yki, and JAK/STAT signaling. In a distinct organ with a high rate of turnover, the data suggest that the NF-κB/Rel-dependent EC cell death cooperates with compensatory stem cell proliferation through the non-cell-autonomous activation of JNK and JAK/ STAT signaling for tumor progression (Zhou, 2021).

PGRPs are known as immune modulators of NF-κB signaling through binding and recognizing bacterial peptidoglycans. Several PGRPs have been implicated in other important biological processes beyond immunity such as host-microbe homeostasis, systemic inflammatory response, tissue integrity, and aging. PGRPs contain a RIP RHIM domain which has been proposed to activate NF-κB signaling. However, unlike in mammals, the Drosophila RHIMs may not be required for cell death. Consistently, it was found that the PGRP-LAD containing the RHIM domain does not induce cell death. However, PGRP-LAF lacking RHIM drives EC delamination and apoptosis. Previous studies suggested a regulatory role of PGRP-LA in controlling NF-κB activity rather than binding to peptidoglycan. In this study, PGRP-LA depletion extends the lifespan of tumor-bearing flies. However, the expression of PGRP-LA is low in the intestine during normal homeostasis, suggesting PGRP-LA may not be involved in normal aging and intestinal homeostasis. Whether the programed cell death pathways impact metazoan lifespan remains unknown, and this requires further research (Zhou, 2021).

This study revealed that tumor induces cytoplasmic enrichment of DE-cadherin::GFP and activates p-Myosin signal in nearby ECs with elongated cell morphology. The DE-cadherin reporter and p-Myosin staining have been previously used as mechano-transduction sensors in Drosophila. The results therefore suggest that the tumor induces DE-cadherin- and myosin-dysregulation-associated mechanical stresses. However, changes in mechanical tension in cells adjacent to tumor cells is a rapid process and more evidence will be required to illustrate mechanical competition, e.g., by monitoring mechanosensors in live tissue. Unfortunately, this remains technically difficult in the Drosophila intestine because of constraints on live imaging and a lack of molecular markers. Moreover, how cells sense and respond to mechanical stress in the context of tumor growth requires further investigations (Zhou, 2021).

Glial immune-related pathways mediate effects of closed head traumatic brain injury on behavior and lethality in Drosophila

In traumatic brain injury (TBI), the initial injury phase is followed by a secondary phase that contributes to neurodegeneration, yet the mechanisms leading to neuropathology in vivo remain to be elucidated. To address this question, this study developed a Drosophila head-specific model for TBI termed Drosophila Closed Head Injury (dCHI), where well-controlled, nonpenetrating strikes are delivered to the head of unanesthetized flies. This assay recapitulates many TBI phenotypes, including increased mortality, impaired motor control, fragmented sleep, and increased neuronal cell death. TBI results in significant changes in the transcriptome, including up-regulation of genes encoding antimicrobial peptides (AMPs). To test the in vivo functional role of these changes, TBI-dependent behavior and lethality were examined in mutants of the master immune regulator NF-κB (Relish), important for AMP induction: while sleep and motor function effects were reduced, lethality effects were enhanced. Similarly, loss of most AMP classes also renders flies susceptible to lethal TBI effects. These studies validate a new Drosophila TBI model and identify immune pathways as in vivo mediators of TBI effects (van Alphen, 2022).

This study has developed a straightforward and reproducible Drosophila model for closed head TBI where precisely controlled strikes are delivered to the head of individually restrained, unanesthetized flies. This TBI paradigm is validated by recapitulating many of the phenotypes observed in mammalian TBI models, including increased mortality, increased neuronal cell death, impaired motor control, decreased/fragmented sleep, and hundreds of TBI-induced changes to the transcriptome, including the activation of many AMPs, indicating a strong activation of the immune response. These results set the stage to leverage Drosophila genetic tools to investigate the role of the immune response as well as novel pathways in TBI pathology (van Alphen, 2022).

The single fly paradigm is a more valid Drosophila model for TBI that circumvents the lack of specificity of currently available models or the use of anesthesia. Both previous assays induce TBI by subjecting the whole fly to trauma, which makes it hard to distinguish whether observed phenotypes are a due to TBI or a consequence of internal injuries. A recently published method uses a pneumatic device to strike an anesthetized fly's head. This method is an improvement of earlier assays and results in increased mortality in a stimulus strength-dependent manner. However, it only shows a modest reduction in locomotor activity, without demonstrating any other TBI-related phenotypes such as neuronal cell death or immune activation. The dependence on CO2 anesthesia further impairs the usefulness of this assay, as prolonged behavioral impairments in Drosophila occur even after brief exposure to CO2 anesthesia. Additionally, anesthetics that are administered either during or shortly after TBI induction can offer neuroprotective effects and alter cognitive, motor, and histological outcomes in mammalian models of TBI as well as affecting mortality in a whole body injury model in flies. The Drosophila model allows study of how TBI affects behavior and gene expression without the confounding effects of anesthesia, making it a more valid model for TBI that occurs under natural conditions (van Alphen, 2022).

The force used in this study (8.34 N) is higher than the force used in the HIT assay (2.5 N). When designing the TBI paradigm, several commercially available solenoids were tested for their ability to induce TBI, and the one that gave the best results was used. A higher force may be needed because brain damage is caused by the direct impact of the solenoid to the fly head, where the fly head moves with the solenoid rather than full body injury or compression injuries used in the other Drosophila TBI assays. Although it cannot be excluded that the neck is not damaged in this assay, cell death was observed in the central brain and significant changes in glia after TBI were observed, suggesting that TBI does occur (van Alphen, 2022).

This study also elucidate, in an unbiased manner, the genomic response to TBI. Glial cells play an important role in immune responses in both mammals and Drosophila, and changes to glial morphology and function were reported in earlier Drosophila TBI models. Until now, profiling TBI-induced changes in gene expression have either been limited to a small number of preselected genes in both mammals and Drosophila or focused on whole brain tissue rather than individual cell types. Using TRAP in combination with RNA-seq, previously reported up-regulation of Attacin-C, Diptericin-B, and Metchnikowin was validated. Additionally, an acute, broad-spectrum immune response was detected, where AMPs and stress response genes are up-regulated 24 hours after TBI. These include antibacterial, antifungal, and antiviral peptides as well as peptides from the Tot family, which are secreted as part of a stress response induced by bacteria, UV, heat, and mechanical stress. Although an increase in the heatshock protein 70 family of stress response genes was reported earlier, this study detected a significant glial up-regulation only in Hsp70BC (van Alphen, 2022).

Three days after TBI, only Attacin-C, Diptericin A, and Metchnikowin are up-regulated. Seven days after TBI, AMPs or stress response genes are not detectably up-regulated. These findings match reports in mammalian TBI models, where inflammatory gene expression spikes shortly after TBI but mostly dies down during subsequent days. Using CRISPR deletions of AMP classes, this study demonstrates that most AMPs not only protect against microbes but are also crucial in promoting survival after TBI. The exception is Defensin, as loss of this peptide increases survival, indicating that the Drosophila innate immune response to TBI can have both beneficial and detrimental effects. While loss of AMPs may render flies more susceptible to TBI, the hypothesis that AMP induction after TBI actively plays a role in mediating TBI effects is favored (van Alphen, 2022).

Besides validating the Drosophila model with the detection of a strongly up-regulated immune response, several novel genes were detected among the total of 512 different glial genes that were either up- or down-regulated after TBI. Immune and stress response only make up 157 out of 512 differentially expressed glial genes. Genes involved in proteolysis and protein folding are a prominent portion (85/512) of these differentially expressed genes, yet their role in TBI is poorly understood. These results demonstrate that there are other candidate pathways that may modulate recovery, and Drosophila can be used as a first line screen to test their in vivo function and to disentangle beneficial from detrimental responses (van Alphen, 2022).

This study has successfully applied in vivo genetics to identify in vivo pathways important for TBI. Loss of master immune regulator NF-κB results in increased mortality after TBI, yet it protects against TBI-induced impairments in sleep and motor control. These findings align with previous reports showing links between sleep and the immune response in flies where NF-κB is required to alter sleep architecture after exposure to septic or aseptic injuries. It will be of interest to determine if NF-κB is required for TBI-induced cell death. One possibility is that sleep impairments can be a side effect of melanization, an invertebrate defense mechanism that requires dopamine as melanin precursor. If dopamine is up-regulated to create more melanin, decreased sleep would be a side effect. Consistent with this hypothesis, changes were observed in fumin and pale, which likely result in increased dopamine levels (van Alphen, 2022).

However, the role of sleep after injury is complex. Two recent studies demonstrated that sleep is increased after antennal transection and facilitates Wallerian degeneration and glia-mediated clearance of axonal debris, suggesting that different types of injury have different effects on sleep. Interestingly, sleep disturbances can increase the up-regulation of immune genes. Thus, it is possible that decreased sleep after TBI contributes to survival by stimulating the immune response. Some support is found for this hypothesis in the difference in TBI-induced changes to sleep in flies that survive 7 days of TBI versus flies that die within 7 days after TBI, where the survivors sleep significantly less for 4 days post-TBI and dying flies sleep is nearly unaffected. Additionally, immune response genes are up-regulated for up to 3 days after TBI, which correlates with the observed sleep impairments. Also, the engulfment receptor Draper, which mediates Wallerian degeneration, is not up-regulated in the glial TRAP-seq data, suggesting that Wallerian degeneration, and its accompanying increase in sleep, is not part of the response to dCHI (van Alphen, 2022).

TBI results in impaired climbing behavior that persists for up to 7 days, yet impairments to sleep disappear after a few days. Recently, it was shown that TBI through head compression results in impaired memory, as quantified through courtship conditioning, indicating that TBI also results in persistent memory defects (van Alphen, 2022).

Recently, it was shown that repressing neuronal NF-κB in a mouse model of TBI increases post-TBI mortality, as in the current studies, without reducing behavioral impairments, suggesting that nonneuronal NF-κB could underlie behavioral impairments after TBI. We demonstrate that behavioral responses to TBI (for example, sleep and geotaxis) are abolished in mutants of the transcription factor NF-κB Relish, which plays a central role in regulating stress-associated and inflammatory gene expression in both mammals and flies. Nonetheless, Relish null mutants show increased mortality after TBI, but none of the behavioral impairments observed in wild-type flies, indicating that these impairments might be a side effect of immune activation rather than direct injury. The demonstration of an in vivo role for TBI-regulated genes will be important for defining their function (van Alphen, 2022).

In summary, the dCHI assay recapitulates many of the physiological symptoms observed in mammals, demonstrating that fruit flies are a valid model to study physiological responses to TBI. Both a potent induction of immune pathways and a requirement for an immune master regulator was demonstrated in mediating TBI effects on behavior. This model now provides a platform to perform unbiased genetic screens to study how gene expression changes after TBI in unanesthetized, awake animals result in the long-term sequelae of TBI. These studies raise the possibility of rapidly identifying key genes and pathways that are neuroprotective for TBI, thereby providing a high-throughput approach that could facilitate the discovery of novel genes and therapeutics that offer better outcomes after TBI (van Alphen, 2022).

GENE STRUCTURE

All three Relish transcripts contain both Rel and ankyrin domains, since hybridization of Northern blots with probes from both domains show identical results. To further characterize the maternal transcript, five cDNA clones were isolated from an ovarian library. These clones are identical to those from the adult and mbn-2 cell libraries except for various degrees of truncation at the 5' end. The longest clone has an insert of 2.7 kb and thus must be near full-length. 5'-rapid amplification of cDNA end products from 0- to 2-h-old embryos show the same sequence and terminate near the 5' end of this clone. It is concluded that the maternal transcript differs from the other two Relish transcripts at the 5' end only, and that if there is a unique 5' exon it must be very short. The maternal transcript is too short to encode the N-terminal part of the open reading frame, and has an alternative translation start site (Dushay, 1996).

Relish maps near the end of the 85C region on the right arm of the third chromosome. This is a well-characterized region of the genome, covered by contiguous genomic walks around the oskar and pumilio genes, as well as by genomic clones around neuralized. Cosmid clones were obtained from the relevant interval and they were probed with the Relish cDNA clone 5.3 (Dushay, 1996). In this way, Relish could be mapped to a cosmid, cos 1698, between pumilio and neuralized. 10.8 kb around the Relish gene were sequenced and several cDNA clones corresponding to the transcripts of this region were isolated. The genomic organization in this region turns out to be complex, with overlapping transcripts from at least four different genes. Twenty cDNA clones from the Relish gene have been described (Dushay, 1996). They are all collinear, and there is no evidence for alternative splicing of the Relish gene or for the use of alternative polyadenylation sites. Nevertheless, at least three different Relish transcripts of 2.7, 3.1, and 3.4 kb can be detected on Northern blots (Dushay, 1996). The 2.7 kb transcript is expressed in females and in early embryos, presumably as a maternally contributed transcript. This conclusion is supported by the observation that Relish is expressed in nurse cells in the ovary, as detected by in situ hybridization (Hedengren, 1999).

The 3.1 kb transcript is very strongly induced in infected flies, whereas the 3.4 kb transcript is constitutively present. 5' RACE has been used to sequence and accurately map the 5' ends of these transcripts, as well as of a fourth, 3.5 kb transcript of low abundance. The latter transcript can also occasionally be detected as a faint band on Northern blots. These data show that the different Relish transcripts originate from one minor and three major transcription start sites that are arranged in tandem. The entire Relish transcription unit is situated inside an intron of the previously described Nmdmc gene (Price, 1993), which encodes a putative NAD-dependent methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase (Hedengren, 1999).

This is a bifunctional enzyme involved in tetrahydrofolate metabolism, with no obvious relationship to immune responses. From the Nmdmc gene, four different cDNA clones have been isolated, all of them representing the same splice form (transcript A) with a first exon 2.4 kb upstream of Relish, spliced to two exons downstream of the Relish gene. The first exon of this transcript differs from that of the previously published Nmdmc transcript (transcript B; Price, 1993) for which the first exon could be mapped to a position 1.2 kb upstream of Relish. Thus, the Nmdmc gene has two alternative splice forms, both of them spanning the region that contains the four Relish transcripts. The protein products encoded by the two transcripts are essentially identical. In transcript B, the first exon is noncoding, whereas transcript A encodes an additional six N-terminal amino acid residues (Hedengren, 1999).

PROTEIN STRUCTURE

Amino Acids - 817

Structural Domains

Relish contains both a Rel homology domain and an IkappaB-like domain with six ankyrin repeats. A phylogenetic reconstruction of the possible relationship between the different Rel homology domains shows that Relish may have branched off from other Rel proteins at a very early stage. The sequences of the ankyrin repeats are also quite different from those of other IkappaB-like proteins, and they are about equally close to the ankyrin repeats in the Notch, ankyrin and IkappaB families. As in other Rel proteins, a putative nuclear localization sequence is found at the C-terminal end of the Rel homology domain, and the ankyrin repeats are followed by an acidic, PEST-like sequence. PEST sequences are rich in proline, glutamic acid, serine and threonine residues, and they have been implicated in protein turnover (Dushay, 1996).

date revised: 23 June 2023

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