Innate immunity pathways activate cell proliferation after penetrating traumatic brain injury in adult Drosophila

In Drosophila, the synthesis of antimicrobial peptides in response to microbial infections is under the control of the Toll and immune deficiency (Imd) signaling pathway. The Toll signaling pathway responds mainly to the lysine-type peptidoglycan of Gram-positive bacteria and fungal β-1,3-glucan, whereas the Imd pathway responds to the meso-diaminopimelic acid (DAP)-type peptidoglycan of Gram-negative bacteria and certain Gram-positive bacilli. Recently we determined the activation mechanism of a Toll signaling pathway biochemically using a large beetle, Tenebrio molitor. However, DAP-type peptidoglycan recognition mechanism and its signaling pathway are still unclear in the fly and beetle. Here, we show that polymeric DAP-type peptidoglycan, but not its monomeric form, formed a complex with Tenebrio peptidoglycan recognition protein-SA, and this complex activated the three-step proteolytic cascade to produce processed Spätzle, a Toll receptor ligand, and induced Drosophila defensin-like antimicrobial peptide in Tenebrio larvae similarly to polymeric lysine-type peptidoglycan. Monomeric DAP-type peptidoglycan induced Drosophila diptericin-like antimicrobial peptide in Tenebrio hemocytes. In addition, both polymeric and monomeric DAP-type peptidoglycans induced expression of Tenebrio peptidoglycan recognition protein-SC2, which is DAP-type peptidoglycan-selective N-acetylmuramyl-l-alanine amidase that functions as a DAP-type peptidoglycan scavenger, appearing to function as a negative regulator of the DAP-type peptidoglycan signaling by cleaving DAP-type peptidoglycan in Tenebrio larvae. Taken together, these results demonstrate that molecular recognition mechanism for polymeric DAP-type peptidoglycan is different between Tenebrio larvae and Drosophila adults, providing biochemical evidences of biological diversity of innate immune responses in insects.

Destruxins are a class of insecticidal, anti-viral, and phytotoxic cyclic depsipeptides that are also studied for their toxicity to cancer cells. They are produced by various fungi, and a direct relationship has been established between Destruxin production and the virulence of the entomopathogen Metarhizium anisopliae. Aside from opening calcium channels, their in vivo mode of action during pathogenesis remains largely uncharacterized. To better understand the effects of a Destruxin, we looked at changes in gene expression following injection of Destruxin A into the fruit fly Drosophila melanogaster. Microarray results revealed reduced expression of various antimicrobial peptides that play a major role in the humoral immune response of the fly. Flies co-injected with a non-lethal dose of Destruxin A and the normally innocuous Gram-negative bacteria Escherichia coli, showed increased mortality and an accompanying increase in bacterial titers. Mortality due to sepsis was rescued through ectopic activation of components in the IMD pathway, one of two signal transduction pathways that are responsible for antimicrobial peptide induction. These results demonstrate a novel role for Destruxin A in specific suppression of the humoral immune response in insects.

Insect Immunity: The Post-Genomic Era

Neurodegenerative diseases such as Alzheimer's and Parkinson's currently affect ∼25 million people worldwide. The global incidence of traumatic brain injury (TBI) is estimated at ∼70 million/year. Both neurodegenerative diseases and TBI remain without effective treatments. We are utilizing adult Drosophila melanogaster to investigate the mechanisms of brain regeneration with the long-term goal of identifying targets for neural regenerative therapies. We specifically focused on neurogenesis, i.e., the generation of new cells, as opposed to the regrowth of specific subcellular structures such as axons. Like mammals, Drosophila have few proliferating cells in the adult brain. Nonetheless, within 24 hours of a penetrating traumatic brain injury (PTBI) to the central brain, there is a significant increase in the number of proliferating cells. We subsequently detect both new glia and new neurons and the formation of new axon tracts that target appropriate brain regions. Glial cells divide rapidly upon injury to give rise to new glial cells. Other cells near the injury site upregulate neural progenitor genes including asense and deadpan and later give rise to the new neurons. Locomotor abnormalities observed after PTBI are reversed within 2 weeks of injury, supporting the idea that there is functional recovery. Together, these data indicate that adult Drosophila brains are capable of neuronal repair. We anticipate that this paradigm will facilitate the dissection of the mechanisms of neural regeneration and that these processes will be relevant to human brain repair.

Innate immunity is the first line of defense against invading pathogens. It functions to eliminate pathogens and also to control infections. The innate immune response is also important for the development of pathogen-specific adaptive immune responses. As a result, the study of innate immune signaling pathways is crucial for understanding the interactions between host and pathogen. Unlike mammals, insects lack a classical adaptive immune response and rely mostly on innate immune responses. Innate immune mechanisms have been widely studied in the fruit fly, Drosophila melanogaster . The genetic and molecular tools available in the Drosophila system make it an excellent model system for studying immunity. Furthermore, the innate immune signaling pathways used by Drosophila show strong homology to those of vertebrates making them ideal for studying these pathways. Drosophila immunity relies on cellular and humoral innate immune responses to fight pathogens. The hallmark of the Drosophila humoral immune response is the rapid induction of antimicrobial peptide genes in the fat body. The production of these antimicrobial peptides is regulated by two immune signaling pathways-Toll and Immune Deficency (IMD) pathways. The Toll pathway responds to many Gram-positive bacterial and fungal infections , while the IMD pathway is potently activated by DAP-type peptidoglycan (PGN) from Gram-negative bacteria and certain Gram-positive bacteria. Two receptors, PGRP-LC and PGRP-LE, are able to recognize DAP-type PGN at the cell surface or in the cytosol, respectively, and trigger the IMD pathway. Upon binding DAP-type PGN, both PGRP-LC and PGRP-LE dimerize/ multimerize and signal to the downstream components of IMD pathway. It is unclear how the receptor activates its downstream components. My work has focused on understanding the molecular events that take place at the receptors following there activation. In these studies I have identified a common motif in the N-terminal domains of both the receptors, known as the RHIM-like domain. The RHIM-like domain is critical for signaling by either receptor, but the mechanism(s) involved remain unclear. IMD, a downstream component of the pathway, associates with both PGRP-LC and -LE but the interaction of PGRP-LC with IMD is not mediated through its RHIM-like domain. Also, mutations affecting the PGRP-LC RHIM-like motif are defective in all known downstream signaling events. However, the RHIM-like mutant receptors are capable of serving as a platform for the assembly of all known components of a receptor proximal signaling complex. These results suggest that another, unidentified component of the IMD signaling pathway may function to mediate interaction with the RHIM-like motif. I performed a yeast two-hybrid screen to identify proteins that might interact with the receptor PGRP-LC through its RHIM- like domain. With this approach, two new components of the IMD pathway were identified. The first component I characterized is…

Innate immunity is the first line of defense against invading pathogens. Vertebrate innate immunity provides both initial protection, and activates adaptive immune responses, including memory. As a result, the study of innate immune signaling is crucial for understanding the interactions between host and pathogen. Unlike mammals, the insect Drosophila melanogaster lack classical adaptive immunity, relying on innate immune signaling via the Toll and IMD pathways to detect and respond to invading pathogens. Once activated these pathways lead to the rapid and robust production of a variety of antimicrobial peptides. These peptides are secreted directly into the hemolymph and assist in clearance of the infection. The genetic and molecular tools available in the Drosophila system make it an excellent model system for studying immunity. Furthermore, the innate immune signaling pathways used by Drosophila show strong homology to those of vertebrates making them ideal for the study of activation, regulation and mechanism. Currently a number of questions remain regarding the activation and regulation of both vertebrate and insect innate immune signaling. Over the past years many proteins have been implicated in mammalian and insect innate immune signaling pathways, however the mechanisms by which these proteins function remain largely undetermined. My work has focused on understanding the molecular mechanisms of innate immune activation in Drosophila. In these studies I have identified a xii

Innate immunity is the first line of defense against invading microorganisms and provides clues to adaptive immunity for the development of memory for subsequent infections. Insects, similar to other invertebrates, do not have adaptive immunity and thus rely on their innate immune system to combat infections. We have analyzed the role of the peptidoglycan (PGN) receptor protein (PGRP) family and other components of innate immune signaling pathways in the immune defense of the mosquito Anopheles gambiae, the main vector of human malaria in sub-Saharan Africa. The PGRP gene family consists of seven genes with ten PGRP domains. We have shown that from all PGRP genes only PGRPLC has a role in the resistance to bacterial infections of both Gram types. In our experiments we have used the Gram-positive bacterium Staphylococcus aureus and the Gram-negative bacterium Escherichia coli. The PGRPLC gene encodes at least three isoforms that derive from infection-driven alternative splicing of a pool of immature transcripts. Each isoform contains a different PGRP domain, encoded by three exons that all contribute equally to the PGN binding pocket. Structural modeling of the PGRPLC isoforms revealed a potential for all isoforms to bind both types of PGN, the Lys-type, which is mostly found in Gram-positive bacteria and the DAP-type, mostly found in Gram-negative bacteria. The isoform PGRPLC3 seems to be the most important in the defense against both bacteria species, although PGRPLC1 also has a crucial role in the defense against S. aureus. Bacterial defense is mediated by the NF-κB transcription factor REL2, which is the ortholog of the Drosophila Relish. The REL2 gene also encodes two protein isoforms: REL2-S, which only has the NF-κB domain, and REL2-F, which carries an I-κB inhibitory domain and a death domain in addition. REL2-F functions together with the receptor adaptor protein IMD to deal with S. aureus infections, whereas REL2-S has a role in the defense against E. coli. The PGRPLC/IMD/REL2-F pathway (IMD) is also partly responsible for the losses of Plasmodium berghei, which can be observed during the first stages of malaria infection of A. gambiae. P. berghei is a rodent malaria parasite, which has been used as a model in our studies. Whether the pathway is able to recognize the malaria parasite through PGRPLC or another associated receptor is still unclear. Another possibility is that the pathway is activated by the proliferation of commensal bacteria in the mosquito gut following a blood meal. However, we have shown that more than one of the three main isoforms of PGRPLC are required for the reaction to P. berghei. Other PGRP genes, which have been proven to play a role during infection with P. berghei, are PGRPLA2, which also mediates parasite killing, and the almost identical and thus hardly indistinguishable PGRPS2 and S3, which appear to inhibit parasite killing. This is possibly achieved by negative regulation of the IMD pathway through sequestration of PGN, which derives from the commensal bacteria and constitutively activates the pathway. We have shown that REL1, the second NF-κB transcription factor of A. gambiae, which is orthologous to the Drosophila Dorsal (Dif does not exist in Anopheles), is not involved in the mosquito resistance to bacterial infections. This fact provides additional evidence that the REL2-associated pathways are of utmost importance in the A. gambiae defense to bacteria. In addition, REL1 has no role in the documented P. berghei killing. However, silencing of the REL1 inhibitor CACT (the ortholog of the Drosophila Cactus) during a parasite infection leads to a very strong refractoriness phenotype: most of the midgut-invading ookinetes are eliminated (presumably by lysis) and the remaining of the ookinetes are melanized. We thus assume that under wild-type infection conditions the parasite is either evading recognition by the REL1-associated receptors or actively modulating activation of REL1. In conclusion, the data reported in this PhD thesis suggest significant divergence of immune signaling between the mosquito A. gambiae and the fruit fly D. melanogaster. The observed differences most likely reflect their different lifestyles and, consequently, different infectious agents, which the two insects encountered during their evolutionary lifetimes. In mosquitoes one of these agents is the malaria parasite.

Dual Detection of Fungal Infections in Drosophila via Recognition of Glucans and Sensing of Virulence Factors

Antimicrobial peptide gene induction, involvement of Toll and IMD pathways and defense against bacteria in the red flour beetle, Tribolium castaneum

Drosophila Sex-Peptide Stimulates Female Innate Immune System after Mating via the Toll and Imd Pathways

BackgroundThe mosquito Aedes aegypti is a major vector for the arthropod-borne viruses (arboviruses) chikungunya, dengue, yellow fever and Zika viruses. Vector immune responses pose a major barrier to arboviral transmission, and transgenic insects with altered immunity have been proposed as tools for reducing the global public health impact of arboviral diseases. However, a better understanding of virus-immune interactions is needed to progress the development of such transgenic insects. Although the NF-κB-regulated Toll and ‘immunodeficiency’ (Imd) pathways are increasingly thought to be antiviral, relevant pattern recognition receptors (PRRs) and pathogen-associated molecular patterns (PAMPs) remain poorly characterised in A. aegypti.Methodology/Principle findingsWe developed novel RT-qPCR and luciferase reporter assays to measure induction of the Toll and Imd pathways in the commonly used A. aegypti-derived Aag2 cell line. We thus determined that the Toll pathway is not inducible by exogenous stimulation with bacterial, viral or fungal stimuli in Aag2 cells under our experimental conditions. We used our Imd pathway-specific assays to demonstrate that the viral dsRNA mimic poly(I:C) is sensed by the Imd pathway, likely through intracellular and extracellular PRRs. The Imd pathway was also induced during infection with the model insect-specific virus cricket paralysis virus (CrPV).Conclusions/SignificanceOur demonstration that a general PAMP shared by many arboviruses is sensed by the Imd pathway paves the way for future studies to determine how viral RNA is sensed by mosquito PRRs at a molecular level. Our data also suggest that studies measuring inducible immune pathway activation through antimicrobial peptide (AMP) expression in Aag2 cells should be interpreted cautiously given that the Toll pathway is not responsive under all experimental conditions. With no antiviral therapies and few effective vaccines available to treat arboviral diseases, our findings provide new insights relevant to the development of transgenic mosquitoes as a means of reducing arbovirus transmission.

Establishment of ex vivo systems to identify compounds acting on innate immune responses and to determine their target molecules using transgenic Drosophila

Enhancing the efficacy of innate immune agonists: could nanolipoprotein particles hold the key?

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

The gut microbiome plays a fundamental role in host health and homeostasis, yet immune mechanisms regulating this relationship remain poorly understood in commercially important invertebrate such as the black tiger shrimp (Penaeus monodon). We employed a multiomics approach, combining RNA interference (RNAi) with transcriptomic, metabolomic, and 16S rRNA gene profiling, to investigate how the innate immune Toll and IMD pathways regulate gut health. We systematically suppressed key signaling components, MyD88 (Toll) and Relish (IMD), under non-pathogenic conditions. Knockdown of the IMD pathway transcription factor, Relish, triggered a profound and selective response across all measured biological layers. We observed a disproportionately large transcriptomic change, with 1,362 differentially expressed genes (DEGs) in the Relish knockdown group compared to only 333 DEGs in the MyD88 knockdown group. This was accompanied by a targeted alteration in immune effectors, including the upregulation of lysozyme C-like (log2 fold change = 2.44) and a strong suppression of penaeidin 5 (log2 fold change = −3.62). At the microbial level, while overall community structure remained stable, a selective shift was observed, the abundance of specific Gram-negative genera, particularly Photobacterium and Shewanella, was significantly reduced, yet Pseudoalteromonas were enriched in the Relish knockdown group. Metabolomic analysis further revealed that the Relish-suppressed shrimp had a distinct metabolic signature, marked by a decrease in bacterial-associated metabolites like D-alanyl-D-alanine and an increase in pro-inflammatory markers such as succinic acid and 8-HETE. Our findings showed that in P. monodon, the IMD pathway is the primary and central regulator of gut microbiome and metabolic homeostasis. This study provides novel insights into the dynamic interplay between innate immunity and the gut microbiome in a crustacean, identifying the IMD pathway as a promising target for developing strategies to enhance shrimp health and the sustainability of the global aquaculture industry.