Dynamic Regulation of NF-κB Response in Innate Immunity: The Case of the IMD Pathway in Drosophila.

Metazoans have developed strategies to protect themselves from pathogenic attack. These preserved mechanisms constitute the immune system, composed of innate and adaptive responses. Among the two kinds, the innate immune system involves the activation of a fast response. NF-κB signaling pathways are activated during infections and lead to the expression of timely-controlled immune response genes. However, activation of NF-κB pathways can be deleterious when uncontrolled. Their regulation is necessary to prevent the development of inflammatory diseases or cancers. The similarity of the NF-κB pathways mediating immune mechanisms in insects and mammals makes Drosophila melanogaster a suitable model for studying the innate immune response and learning general mechanisms that are also relevant for humans. In this review, we summarize what is known about the dynamic regulation of the central NF-κB-pathways and go into detail on the molecular level of the IMD pathway. We report on the role of the nuclear protein Akirin in the regulation of the NF-κB Relish immune response. The use of the Drosophila model allows the understanding of the fine-tuned regulation of this central NF-κB pathway.

Protein Phosphatase 4 Negatively Regulates the Immune Deficiency-NF-κB Pathway during the Drosophila Immune Response.

The evolutionarily conserved immune deficiency (IMD) signaling pathway shields Drosophila against bacterial infections. It regulates the expression of antimicrobial peptides encoding genes through the activation of the NF-κB transcription factor Relish. Tight regulation of the signaling cascade ensures a balanced immune response, which is otherwise highly harmful. Several phosphorylation events mediate intracellular progression of the IMD pathway. However, signal termination by dephosphorylation remains largely elusive. Here, we identify the highly conserved protein phosphatase 4 (PP4) complex as a bona fide negative regulator of the IMD pathway. RNA interference-mediated gene silencing of PP4-19c, PP4R2, and Falafel, which encode the catalytic and regulatory subunits of the phosphatase complex, respectively, caused a marked upregulation of bacterial-induced antimicrobial peptide gene expression in both Drosophila melanogaster S2 cells and adult flies. Deregulated IMD signaling is associated with reduced lifespan of PP4-deficient flies in the absence of any infection. In contrast, flies overexpressing this phosphatase are highly sensitive to bacterial infections. Altogether, our results highlight an evolutionarily conserved function of PP4c in the regulation of NF-κB signaling from Drosophila to mammals.

La Société de Biologie de Strasbourg : 100 ans au service de la science et de la société.

Founded in 1919, the Society of Biology of Strasbourg (SBS) is a learned society whose purpose is the dissemination and promotion of scientific knowledge in biology. Subsidiary of the Society of Biology, the SBS celebrated its Centenary on Wednesday, the 16th of October 2019 on the Strasbourg University campus and at the Strasbourg City Hall. This day allowed retracing the various milestones of the SBS, through its main strengths, its difficulties and its permanent goal to meet scientific and societal challenges. The common thread of this day was the transmission of knowledge related to the past, the present, but also the future. At the start of the 21st century, the SBS must continue to reinvent itself to pursue its objective of transmitting scientific knowledge in biology and beyond. Scientific talks performed by senior scientists and former SBS thesis prizes awardees, a round table, and informal discussions reflected the history and the dynamism of the SBS association. All SBS Centennial participants have set the first milestone for the SBS Bicentennial.

TITLE: La Société de Biologie de Strasbourg : 100 ans au service de la science et de la société. ABSTRACT: Filiale de la Société de Biologie, la Société de Biologie de Strasbourg (SBS) est une société savante qui a pour objet la diffusion et la promotion du savoir scientifique en biologie et en médecine. Fondée en 1919, La SBS a célébré son Centenaire le mercredi 16 octobre 2019. Cette journée a permis de retracer les différents jalons de la SBS, à travers ses lignes de forces, ses difficultés et sa volonté permanente de mettre en exergue les défis scientifiques et sociétaux auxquels participent les recherches strasbourgeoises. Le fil rouge de cette journée a été la transmission d'un savoir en lien avec le passé, le présent, mais également le futur. En ce début du 21e siècle, la SBS se doit de continuer de se réinventer pour poursuivre son objectif de transmission des connaissances scientifiques en biologie et au-delà. L'ensemble des participants du Centenaire de la SBS a ainsi posé la première pierre du Bicentenaire de la SBS.

Hyd ubiquitinates the NF-κB co-factor Akirin to operate an effective immune response in Drosophila.

The Immune Deficiency (IMD) pathway in Drosophila melanogaster is activated upon microbial challenge with Gram-negative bacteria to trigger the innate immune response. In order to decipher this nuclear factor κB (NF-κB) signaling pathway, we undertook an in vitro RNAi screen targeting E3 ubiquitin ligases specifically and identified the HECT-type E3 ubiquitin ligase Hyperplastic discs (Hyd) as a new actor in the IMD pathway. Hyd mediated Lys63 (K63)-linked polyubiquitination of the NF-κB cofactor Akirin was required for efficient binding of Akirin to the NF-κB transcription factor Relish. We showed that this Hyd-dependent interaction was required for the transcription of immunity-related genes that are activated by both Relish and Akirin but was dispensable for the transcription of genes that depend solely on Relish. Therefore Hyd is key in NF-κB transcriptional selectivity downstream of the IMD pathway. Drosophila depleted of Akirin or Hyd failed to express the full set of genes encoding immune-induced anti-microbial peptides and succumbed to immune challenges. We showed further that UBR5, the mammalian homolog of Hyd, was also required downstream of the NF-κB pathway for the activation of Interleukin 6 (IL6) transcription by LPS or IL-1ß in cultured human cells. Our findings link the action of an E3 ubiquitin ligase to the activation of immune effector genes, deepening our understanding of the involvement of ubiquitination in inflammation and identifying a potential target for the control of inflammatory diseases.

The Circulating Protease Persephone Is an Immune Sensor for Microbial Proteolytic Activities Upstream of the Drosophila Toll Pathway.

Microbial or endogenous molecular patterns as well as pathogen functional features can activate innate immune systems. Whereas detection of infection by pattern recognition receptors has been investigated in details, sensing of virulence factors activities remains less characterized. In Drosophila, genetic evidences indicate that the serine protease Persephone belongs to a danger pathway activated by abnormal proteolytic activities to induce Toll signaling. However, neither the activation mechanism of this pathway nor its specificity has been determined. Here, we identify a unique region in the pro-domain of Persephone that functions as bait for exogenous proteases independently of their origin, type, or specificity. Cleavage in this bait region constitutes the first step of a sequential activation and licenses the subsequent maturation of Persephone to the endogenous cysteine cathepsin 26-29-p. Our results establish Persephone itself as an immune receptor able to sense a broad range of microbes through virulence factor activities rather than molecular patterns.

The SUMO-targeted ubiquitin ligase, Dgrn, is essential for Drosophila innate immunity.

The ability of metazoans to combat pathogenic infection involves both systemic and local responses to the invading pathogens. Ubiquitin and SUMO pathways molecularly regulate the response to infection, immune signaling and gene expression. Here, we report that Degringolade (Dgrn, CG10981), a SUMO-targeted ubiquitin ligase connecting the two pathways, is essential for the innate immunity response in Drosophila. dgrnDK null and heterozygous mutant adult flies are severely immune-compromised and succumb rapidly to both pathogenic bacteria and fungi infections. The sensitivity to infection stems from the inability to produce multiple anti-microbial peptides, and transcriptional analyses suggest that the overexpression of Dgrn enhances the transcriptional output of the NF-ĸB related Toll and immune deficiency (IMD)-pathways. Moreover, expression of Dgrn alleviated the inhibitory impact of the cytoplasmic NF-ĸB inhibitor Cactus and the nuclear co-repressor Groucho/TLE (Gro). Additionally, we found that Dgrn is required for the local regenerative response of the mid-gut following infection. Upon oral infection, dgrn mutant flies fail to activate the Delta-Notch pathway in stem cells and enteroblasts, and are unable to regenerate and replace the damaged and dying enterocytes. Interestingly, the ubiquitin-specific protease CG8334 (dUSP32/dUSP11) antagonizes Dgrn activity in the gut, and halving the dose of CG8334 restores Delta-Notch signaling and rescues the lethality observed in dgrn mutants. Collectively, our data suggest that Dgrn is essential for both systemic and local tissue response to infection.

Advances in Myeloid-Like Cell Origins and Functions in the Model Organism Drosophila melanogaster.

Drosophila has long served as a valuable model for deciphering many biological processes, including immune responses. Indeed, the genetic tractability of this organism is particularly suited for large-scale analyses. Studies performed during the last 3 decades have proven that the signaling pathways that regulate the innate immune response are conserved between Drosophila and mammals. This review summarizes the recent advances on Drosophila hematopoiesis and immune cellular responses, with a particular emphasis on phagocytosis.

The multilayered innate immune defense of the gut.

In the wild, the fruit fly Drosophila melanogaster thrives on rotten fruit. The digestive tract maintains a powerful gut immune barrier to regulate the ingested microbiota, including entomopathogenic bacteria. This gut immune barrier includes a chitinous peritrophic matrix that isolates the gut contents from the epithelial cells. In addition, the epithelial cells are tightly sealed by septate junctions and can mount an inducible immune response. This local response can be activated by invasive bacteria, or triggered by commensal bacteria in the gut lumen. As with chronic inflammation in mammals, constitutive activation of the gut innate immune response is detrimental to the health of flies. Accordingly, the Drosophila gut innate immune response is tightly regulated to maintain the endogenous microbiota, while preventing infections by pathogenic microorganisms.

Akirin specifies NF-κB selectivity of Drosophila innate immune response via chromatin remodeling.

The network of NF-κB-dependent transcription that activates both pro- and anti-inflammatory genes in mammals is still unclear. As NF-κB factors are evolutionarily conserved, we used Drosophila to understand this network. The NF-κB transcription factor Relish activates effector gene expression following Gram-negative bacterial immune challenge. Here, we show, using a genome-wide approach, that the conserved nuclear protein Akirin is a NF-κB co-factor required for the activation of a subset of Relish-dependent genes correlating with the presence of H3K4ac epigenetic marks. A large-scale unbiased proteomic analysis revealed that Akirin orchestrates NF-κB transcriptional selectivity through the recruitment of the Osa-containing-SWI/SNF-like Brahma complex (BAP). Immune challenge in Drosophila shows that Akirin is required for the transcription of a subset of effector genes, but dispensable for the transcription of genes that are negative regulators of the innate immune response. Therefore, Akirins act as molecular selectors specifying the choice between subsets of NF-κB target genes. The discovery of this mechanism, conserved in mammals, paves the way for the establishment of more specific and less toxic anti-inflammatory drugs targeting pro-inflammatory genes.

big bang gene modulates gut immune tolerance in Drosophila.

Chronic inflammation of the intestine is detrimental to mammals. Similarly, constant activation of the immune response in the gut by the endogenous flora is suspected to be harmful to Drosophila. Therefore, the innate immune response in the gut of Drosophila melanogaster is tightly balanced to simultaneously prevent infections by pathogenic microorganisms and tolerate the endogenous flora. Here we describe the role of the big bang (bbg) gene, encoding multiple membrane-associated PDZ (PSD-95, Discs-large, ZO-1) domain-containing protein isoforms, in the modulation of the gut immune response. We show that in the adult Drosophila midgut, BBG is present at the level of the septate junctions, on the apical side of the enterocytes. In the absence of BBG, these junctions become loose, enabling the intestinal flora to trigger a constitutive activation of the anterior midgut immune response. This chronic epithelial inflammation leads to a reduced lifespan of bbg mutant flies. Clearing the commensal flora by antibiotics prevents the abnormal activation of the gut immune response and restores a normal lifespan. We now provide genetic evidence that Drosophila septate junctions are part of the gut immune barrier, a function that is evolutionarily conserved in mammals. Collectively, our data suggest that septate junctions are required to maintain the subtle balance between immune tolerance and immune response in the Drosophila gut, which represents a powerful model to study inflammatory bowel diseases.

Conserved cis-regulatory regions in a large genomic landscape control SHH and BMP-regulated Gremlin1 expression in mouse limb buds.

BACKGROUND: Mouse limb bud is a prime model to study the regulatory interactions that control vertebrate organogenesis. Major aspects of limb bud development are controlled by feedback loops that define a self-regulatory signalling system. The SHH/GREM1/AER-FGF feedback loop forms the core of this signalling system that operates between the posterior mesenchymal organiser and the ectodermal signalling centre. The BMP antagonist Gremlin1 (GREM1) is a critical node in this system, whose dynamic expression is controlled by BMP, SHH, and FGF signalling and key to normal progression of limb bud development. Previous analysis identified a distant cis-regulatory landscape within the neighbouring Formin1 (Fmn1) locus that is required for Grem1 expression, reminiscent of the genomic landscapes controlling HoxD and Shh expression in limb buds. RESULTS: Three highly conserved regions (HMCO1-3) were identified within the previously defined critical genomic region and tested for their ability to regulate Grem1 expression in mouse limb buds. Using a combination of BAC and conventional transgenic approaches, a 9 kb region located ~70 kb downstream of the Grem1 transcription unit was identified. This region, termed Grem1 Regulatory Sequence 1 (GRS1), is able to recapitulate major aspects of Grem1 expression, as it drives expression of a LacZ reporter into the posterior and, to a lesser extent, in the distal-anterior mesenchyme. Crossing the GRS1 transgene into embryos with alterations in the SHH and BMP pathways established that GRS1 depends on SHH and is modulated by BMP signalling, i.e. integrates inputs from these pathways. Chromatin immunoprecipitation revealed interaction of endogenous GLI3 proteins with the core cis-regulatory elements in the GRS1 region. As GLI3 is a mediator of SHH signal transduction, these results indicated that SHH directly controls Grem1 expression through the GRS1 region. Finally, all cis-regulatory regions within the Grem1 genomic landscape locate to the DNAse I hypersensitive sites identified in this genomic region by the ENCODE consortium. CONCLUSIONS: This study establishes that distant cis-regulatory regions scattered through a larger genomic landscape control the highly dynamic expression of Grem1, which is key to normal progression of mouse limb bud development.

Analysis of thioester-containing proteins during the innate immune response of Drosophila melanogaster.

Thioester-containing proteins (TEPs) are conserved proteins among insects that are thought to be involved in innate immunity. In Drosophila, the Tep family is composed of 6 genes named Tep1-Tep6. In this study, we investigated the phylogeny, expression pattern and roles of these genes in the host defense of Drosophila. Protostomian Tep genes are clustered in 3 distinct branches, 1 of which is specific to mosquitoes. Most D. melanogaster Tep genes are expressed in hemocytes, can be induced in the fat body, and are expressed in specific regions of the hypodermis. This expression pattern is consistent with a role in innate immunity. However, we find that TEP1, TEP2, and TEP4 are not strictly required in the body cavity to fight several bacterial and fungal infections. One possibility is that Drosophila TEPs act redundantly or that their absence can be compensated by other components of the immune response. TEPs may thus provide a subtle selective advantage during evolution. Alternatively, they may be required in host defense against specific as yet unidentified natural pathogens of Drosophila.

The DExD/H-box helicase Dicer-2 mediates the induction of antiviral activity in drosophila.

Drosophila, like other invertebrates and plants, relies mainly on RNA interference for its defense against viruses. In flies, viral infection also triggers the expression of many genes. One of the genes induced, Vago, encodes a 18-kilodalton cysteine-rich polypeptide. Here we provide genetic evidence that the Vago gene product controlled viral load in the fat body after infection with drosophila C virus. Induction of Vago was dependent on the helicase Dicer-2. Dicer-2 belongs to the same DExD/H-box helicase family as do the RIG-I-like receptors, which sense viral infection and mediate interferon induction in mammals. We propose that this family represents an evolutionary conserved set of sensors that detect viral nucleic acids and direct antiviral responses.

Impairing retinoic acid signalling in the neural crest cells is sufficient to alter entire eye morphogenesis.

Retinoic acid (RA) is known to be required at various levels of eye patterning via Retinoic Acid Receptors (RAR); however the molecular and cellular mechanisms triggered by these nuclear receptors are still obscure. The genetic studies performed here enable us to present a new model to study RA action during eye development. By inactivating the three RARs, specifically in the periocular mesenchyme, we discriminate the individual contribution of each RAR during eye development and describe a new function for RARs during the formation of the optic nerve. We demonstrate that RARalpha is the only receptor that mediates RA signalling in the neurectoderm during ocular development. Surprisingly, and despite a sophisticated pattern of RA-activity in the developing retina, we observed that RA signalling is not autonomously required in this tissue for eye formation. We show that the action of RA during eye morphogenesis is occurring specifically in neural crest-derived periocular mesenchyme and is mediated by all three RARs. Furthermore, we point out that Pitx2, which encodes a homeodomain transcription factor, is a key RA-responsive gene in neural crest cells during eye development. Interestingly, we observed that RA is required in the neural crest cells for normal position of the extraocular muscle.

Retinoic acid-dependent eye morphogenesis is orchestrated by neural crest cells.

Using genetic approaches in the mouse, we show that the primary target tissue of retinoic acid (RA) action during eye morphogenesis is not the retina nor the corneal ectoderm, which both express RA-synthesizing retinaldehyde dehydrogenases (RALDH1 and RALDH3), but the neural crest cell-derived periocular mesenchyme (POM), which is devoid of RALDH. In POM, the effects of the paracrine RA signal are mediated by the nuclear RA receptors heterodimers RXRalpha/RARbeta and RXRalpha/RARgamma. These heterodimers appear to control: (1) the remodeling of the POM through activation of Eya2-related apoptosis; (2) the expression of Foxc1 and Pitx2, which play crucial roles in anterior eye segment development; and (3) the growth of the ventral retina. We additionally show that RALDH1 and RALDH3 are the only enzymes that are required for RA synthesis in the eye region from E10.5 to E13.5, and that patterning of the dorsoventral axis of the retina does not require RA.

Contribution of cellular retinol-binding protein type 1 to retinol metabolism during mouse development.

Within cells, retinol (ROL) is bound to cytoplasmic proteins (cellular retinol-binding proteins [CRBPs]), whose proposed function is to protect it from unspecific enzymes through channeling to retinoid-metabolizing pathways. We show that, during development, ROL and retinyl ester levels are decreased in CRBP type 1 (CRBP1) -deficient embryos and fetuses by 50% and 80%, respectively. The steady state level of retinoic acid (RA) is also decreased but to a lesser extent. However, CRBP1-null fetuses do not exhibit the abnormalities characteristic of a vitamin A-deficiency syndrome. Neither CRBP1 deficiency alters the expression patterns of RA-responding genes during development, nor does CRBP1 availability modify the expression of an RA-dependent gene in primary embryonic fibroblasts treated with ROL. Therefore, CRBP1 is required in prenatal life to maintain normal amounts of ROL and to ensure its efficient storage but seems of secondary importance for RA synthesis, at least under conditions of maternal vitamin A sufficiency.

A newborn lethal defect due to inactivation of retinaldehyde dehydrogenase type 3 is prevented by maternal retinoic acid treatment.

The retinoic acid (RA) signal, produced locally from vitamin A by retinaldehyde dehydrogenase (Raldh) and transduced by the nuclear receptors for retinoids (RA receptor and 9-cis-RA receptor), is indispensable for ontogenesis and homeostasis of numerous tissues. We demonstrate that Raldh3 knockout in mouse suppresses RA synthesis and causes malformations restricted to ocular and nasal regions, which are similar to those observed in vitamin A-deficient fetuses and/or in retinoid receptor mutants. Raldh3 knockout notably causes choanal atresia (CA), which is responsible for respiratory distress and death of Raldh3-null mutants at birth. CA is due to persistence of nasal fins, whose rupture normally allows the communication between nasal and oral cavities. This malformation, which is similar to isolated congenital CA in humans and may result from impaired RA-controlled down-regulation of Fgf8 expression in nasal fins, can be prevented by a simple maternal treatment with RA.

Retinoic acid-induced developmental defects are mediated by RARbeta/RXR heterodimers in the pharyngeal endoderm.

Fusion and hypoplasia of the first two branchial arches, a defect typically observed in retinoic acid (RA) embryopathy, is generated in cultured mouse embryos upon treatment with BMS453, a synthetic compound that exhibits retinoic acid receptor beta (RARbeta) agonistic properties in transfected cells. By contrast, no branchial arch defects are observed following treatment with synthetic retinoids that exhibit RARalpha or RARgamma agonistic properties. The BMS453-induced branchial arch defects are mediated through RAR activation, as they are similar to those generated by a selective pan-RAR agonist, are prevented by a selective pan-RAR antagonist and cannot be mimicked by exposure to a pan-RXR agonist alone. They are enhanced in the presence of a pan-RXR agonist, and cannot be generated in Rarb-null embryos. Furthermore, they are accompanied, in the morphologically altered region, by ectopic expression of Rarb and of several other direct RA target genes. Therefore, craniofacial abnormalities characteristic of the RA embryopathy are mediated through ectopic activation of RARbeta/RXR heterodimers, in which the ligand-dependent activity of RXR is subordinated to that of RARbeta. Endodermal cells lining the first two branchial arches respond to treatment with the RARbeta agonist, in contrast to neural crest cells and ectoderm, which suggests that a faulty endodermal regionalization is directly responsible for RA-induced branchial arch dysmorphologies. Additionally, we provide the first in vivo evidence that the synthetic RARbeta agonist BMS453 exhibits an antagonistic activity on the two other RAR isotypes.