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Components of microbial cell walls are potent activators of innate immune responses in animals. For example, the mammalian TLR4 signaling pathway is activated by bacterial lipopolysaccharide and is required for resistance to infection by Gram-negative bacteria. Other components of microbial surfaces, such as peptidoglycan, are also potent activators of innate immune responses, but less is known about how those components activate host defense. Here we show that a peptidoglycan recognition protein, PGRP-LC, is absolutely required for the induction of antibacterial peptide genes in response to infection in Drosophila and acts by controlling activation of the NF-κB family transcription factor Relish.

Rel/NF- Kappa B transcription factors include a collection of proteins, conserved from Drosophila to humans. Among model organisms, these transcription factors are notably absent in yeast and C. elegans; in part, this may be because one of the primary roles of these factors is to control a variety of physiological aspects of immune and inflammatory responses. A pathway similar to the Rel/NF- Kappa B signaling pathway may also control certain defense responses in plants. The Rel/NF- Kappa B proteins are related through a highly conserved DNA-binding/dimerization domain called the Rel homology (RH) domain. However, they can be divided into two classes based on sequences C-terminal to the RH domain. Members of one class (p105, p100, and Drosophila Relish) have long C-terminal domains that contain multiple copies of ankyrin repeats, which act to inhibit these molecules. Members of this class become active, shorter DNA-binding proteins (p105 to p50, p100 to p52) by either limited proteolysis or arrested translation. As such, members of this first class are generally not activators of transcription, except when they form dimers with members of the second class of Rel/NF- Kappa B transcription factors. The second class includes c-Rel (and its homologue v-Rel), RelB, RelA (p65), Dorsal, and Dif. These Rel proteins contain C-terminal activation domains, which are often not conserved at the sequence level across species, even though they can activate transcription in a variety of species.

NF-kappaB/Rel transcription factors are central regulators of mammalian immunity and are also implicated in the induction of cecropins and other antibacterial peptides in insects. We identified the gene for Relish, a compound Drosophila protein that, like mammalian p105 and p100, contains both a Rel homology domain and an IkappaB-like domain. Relish is strongly induced in infected flies, and it can activate transcription from the Cecropin A1 promoter. A Relish transcript is also detected in early embryos, suggesting that it acts in both immunity and embryogenesis. The presence of a compound Rel protein in Drosophila indicates that similar proteins were likely present in primordial immune systems and may serve unique signaling functions.

The Drosophila Rel/NF-kappaB transcription factors - Dorsal, Dif, and Relish - control several biological processes, including embryonic pattern formation, muscle development, immunity, and hematopoiesis. Molecular-genetic analysis of 12 mutations that cause embryonic dorsal/ventral patterning defects has defined the steps that control the formation of this axis. Regulated activation of the Toll receptor leads to the establishment of a gradient of nuclear Dorsal protein, which in turn governs the subdivision of the axis and specification of ventral, lateral and dorsal fates. Phenotypic analysis of dorsal-ventral embryonic mutants and the characterization of the two other fly Rel proteins, Dif and Relish, have shown that the intracellular portion of the Toll to Cactus pathway also controls the innate immune response in Drosophila. Innate immunity and hematopoiesis are regulated by analogous Rel/NF-kappaB-family pathways in mammals. The elucidation of the complex regulation and diverse functions of Drosophila Rel proteins underscores the relevance of basic studies in Drosophila.

Khush et al comment on studies by Choe et al and Michel et al that identify two peptidoglycan recognition proteins (PGRPs) in the fruit fly that are probable pattern recognition receptors for the insect innate immune response. The growing appreciation of the conservation of some immune responses in insects and mammals has produced an exchange of ideas and results that has invigorated the field of innate immunity.

The NF-κB family of transcription factors are central to signaling from Toll family receptors. The IKKγ protein of Drosophila has been found to regulate NF-κB family–member Relish, but not the similar protein DIF, in the fly innate immune response.

Circadian clocks orchestrate temporal regulation of diverse physiological processes, including innate immunity and oxidative stress responses. However, the molecular mechanisms by which core clock components modulate immune tone and redox homeostasis remain elusive. Here, the circadian transcription factor CLOCK (CLK) is identified as a key regulator of oxidative stress resistance in Drosophila melanogaster. Loss of clk significantly enhances survival under oxidative stress, accompanied by constitutive activation of innate immune pathways. Mechanistically, the RNA‐binding protein Achilles (ACHL) is identified as a critical downstream effector of CLK. Indeed, CLK drives rhythmic transcription of Achl, and ACHL post‐transcriptionally represses the NF‐κB homolog Relish by promoting its mRNA degradation, thereby limiting immune overactivation. Disruption of this regulatory cascade, through loss of either clk or Achl, leads to increased Relish abundance, excessive immune gene expression, and enhanced oxidative stress resistance. Genetic suppression of Relish reverses these phenotypes, establishing a functional CLK–ACHL–Relish axis that links circadian output to immune restraint. Importantly, this regulatory mechanism is evolutionarily conserved, as Clock‐deficient mammalian cells exhibit increased resistance to oxidative injury. Together, the findings uncover a post‐transcriptional immune checkpoint controlled by circadian networks, linking immune quiescence with redox adaptation. This study identifies the circadian transcription factor CLOCK (CLK) as a key regulator of oxidative stress resistance in fruit flies. CLK controls immune responses by driving rhythmic transcription of an RNA‐binding protein‐Achilles (ACHL) in the fly brain, which post‐transcriptionally represses the NF‐κB homolog Relish, limiting immune activation and promoting redox homeostasis.

PeptidoGlycan Recognition Proteins (PGRPs) are key regulators of the insect innate antibacterial response. Even if they have been intensively studied, some of them have yet unknown functions. Here, we present a functional analysis of PGRP-LA, an as yet uncharacterized Drosophila PGRP. The PGRP-LA gene is located in cluster with PGRP-LC and PGRP-LF, which encode a receptor and a negative regulator of the Imd pathway, respectively. Structure predictions indicate that PGRP-LA would not bind to peptidoglycan, pointing to a regulatory role of this PGRP. PGRP-LA expression was enriched in barrier epithelia, but low in the fat body. Use of a newly generated PGRP-LA deficient mutant indicates that PGRP-LA is not required for the production of antimicrobial peptides by the fat body in response to a systemic infection. Focusing on the respiratory tract, where PGRP-LA is strongly expressed, we conducted a genome-wide microarray analysis of the tracheal immune response of wild-type, Relish, and PGRP-LA mutant larvae. Comparing our data to previous microarray studies, we report that a majority of genes regulated in the trachea upon infection differ from those induced in the gut or the fat body. Importantly, antimicrobial peptide gene expression was reduced in the tracheae of larvae and in the adult gut of PGRP-LA-deficient Drosophila upon oral bacterial infection. Together, our results suggest that PGRP-LA positively regulates the Imd pathway in barrier epithelia.

Abstract The nuclear factor binding the κ light chain in B-cells (NFκB) is involved in a wide range of cellular processes including development, growth, innate immunity, and sleep. However, genetic studies of the role of specific NFκB transcription factors in sleep have been limited. Drosophila fruit flies carry three genes encoding NFκB transcription factors, Dorsal, Dorsal Immunity Factor (Dif), and Relish. We previously found that loss of the Relish gene from fat body suppressed daily nighttime sleep, and abolished infection-induced sleep. Here we show that Dif regulates daily sleep and recovery sleep following prolonged wakefulness. Mutants of Dif showed reduced daily sleep and suppressed recovery in response to sleep deprivation. Pan-neuronal knockdown of Dif strongly suppressed daily sleep, indicating that in contrast to Relish, Dif functions from the central nervous system to regulate sleep. Based on the unique expression pattern of a Dif- GAL4 driver, we hypothesized that its effects on sleep were mediated by the pars intercerebralis (PI). While RNAi knock-down of Dif in the PI reduced daily sleep, it had no effect on the recovery response to sleep deprivation. However, recovery sleep was suppressed when RNAi knock-down of Dif was distributed across a wider range of neurons. Induction of the nemuri (nur) antimicrobial peptide by sleep deprivation was reduced in Dif mutants and pan-neuronal overexpression of nur also suppressed the Dif mutant phenotype by significantly increasing sleep and reducing nighttime arousability. Together, these findings indicate that Dif functions from brain to target nemuri and to promote deep sleep. Graphical Abstract Graphical Abstract

Stimulator of interferon genes (STING) is crucial for the innate immune to defend against pathogenic infections. Our previous study showed that a STING homolog from Litopenaeus vannamei (LvSTING) was involved in antibacterial response via regulating antimicrobial peptides (AMPs). Nevertheless, how LvSTING induces AMPs expression to inhibit bacterial infection remains unknown. Herein, we revealed that the existence of a STING–IKKβ–Relish–AMPs axis in shrimp that was essential for opposing to Vibrio parahaemolyticus invasion. We observed that LvRelish was essential for host defense against V. parahaemolyticus infection via inducing several AMPs, such as LvALF1, LvCRU1, LvLYZ1 and LvPEN4. Knockdown of LvSTING or LvIKKβ in vivo led to the attenuated phosphorylation and diminished nuclear translocation of LvRelish, as well as the impaired expression levels of LvRelish-regulated AMPs. Accordingly, shrimps with knockdown of LvSTING or LvIKKβ or both were vulnerable to V. parahaemolyticus infection. Finally, LvSTING could recruit LvRelish and LvIKKβ to form a complex, which synergistically induced the promoter activity of several AMPs in vitro . Taken together, our results demonstrated that the shrimp STING–IKKβ–Relish–AMPs axis played a critical role in the defense against bacterial infection, and provided some insights into the development of disease prevention strategies in shrimp culture.