Imd Pathway Components Research Articles

Immune signaling pathways in Rhodnius prolixus in the context of Trypanosoma rangeli infection: cellular and humoral immune responses and microbiota modulation.

Rhodnius prolixus is a hematophagous insect and one of the main vectors for Trypanosoma cruzi and Trypanosoma rangeli parasites in Latin America. Gut microbiota and insect immune responses affect T. cruzi and T. rangeli infection within triatomines. Particularly the Toll and IMD signaling pathways activations and how they orchestrate the antimicrobial peptides (AMPs) expressions in R. prolixus, especially when infected by T. rangeli. Examine how T. rangeli infection modulates R. prolixus cellular and humoral immunity and its impacts on insect microbiota. R. prolixus was fed on blood containing epimastigotes of T. rangeli, and infection was quantified in insect tissues. The gene expression of dorsal, cactus, relish, PGRP, and AMPs was examined in the midgut, fat body, and salivary glands by quantitative real-time PCR. Microbiota composition was analyzed using RT-q PCR targeting specific bacterial species. Hemocyte numbers and phenoloxidase activity were quantified to assess cellular immune responses. T. rangeli infection modulated triatomine immunity in midgut and hemocoel, activating the expression of the NF-kB gene dorsal, associated with the Toll pathway; increasing expression of the gene encoding PGRP receptor, a component involved in the IMD pathway, both in the intestine and fat body; repressing the expression of the relish transcription factor, mainly in salivary glands. Among the R. prolixus AMPs studied, T. rangeli infection repressed all AMP gene expression, other than defensin C which increased mRNA levels. The PO activity was enhanced in the hemolymph of infected insects. T. rangeli infection did not induce hemocyte number alterations compared to control insects. However, an increase in hemocyte microaggregation was detected in infected insects. R. prolixus recognizes T. rangeli infection and triggers humoral and cellular immune responses involving Toll pathway activation, defensin C synthesis, increased phenoloxidase activity, and enhanced hemocyte aggregation. On the other hand, T. rangeli infection suppressed some IMD pathway components, suggesting that, in R. prolixus, this pathway is involved in defensins A and B gene regulation. Importantly, these immune responses altered the bacterial microbiota composition, potentially favoring T. rangeli establishment in the insect vector.

Effects of TmTak1 silencing on AMP production as an Imd pathway component in Tenebrio molitor

Mealworms beetles, Tenebrio molitor, are the limelight next-generation food for humans due to their high nutrient contents. Since Tenebrio molitor is used as feed for pets and livestock in addition to their ability to decompose polystyrene and plastic waste, it is recognized as an insect with an industrial core value. Therefore, it is important to study the immune mechanism related to the development and infection of mealworms for mass breeding purposes. The immune deficiency (Imd) signaling is one of the main pathways with pivotal roles in the production of antimicrobial peptides (AMPs). Transforming growth factor-β activated kinase (TAK1) is one of the Imd pathway components, forms a complex with TAK1 binding protein 2 (TAB2) to ultimately help activate the transcription factor Relish and eventually induce host to produce AMPs. Relatively, little has been revealed about TAK1 in insect models, especially in the T. molitor. Therefore, this study was conducted to elucidate the function of TmTak1 in T. molitor. Our results showed that the highest and lowest mRNA expression of TmTak1 were found in egg and young larvae respectively. The tissue-specific expression patterns were reported in the gut of T. molitor larvae and the fat bodies of adults. Systemic microbial challenge illustrated TmTak1 high expression following the fungal infection in all dissected tissues except for the whole body. However, silencing TmTak1 experiments showed that the survivability of T. molitor larvae affected significantly following Escherichia coli infection. Accordingly, AMP induction after TmTak1 knock down was mainly reported in the integument and the fat bodies.

JNK pathway-a key mediator of antiviral immunity in mosquito salivary glands

Background: Mosquito salivary glands play crucial role in transmission of arboviral diseases like Dengue (DENV), Zika (ZIKV) and Chikungunya (CHIKV). We aimed to characterize virus responsive gene expression in Aedes aegypti salivary glands against these pathogenic arboviruses. Methods: We performed high throughput RNA-sequencing on uninfected and virus-infected (DENV, ZIKV, CHIKV) female Ae. aegypti salivary glands to elucidate differential expression of genes at the transcript level. We validated the transcriptomic analysis by qPCR and performed RNA-i based functional characterization of virus-induced immune genes. Results: DENV, ZIKV or CHIKV infected salivary gland transcriptome revealed regulations of genes related to blood feeding, metabolism, apoptosis, and immunity; the latter including Toll, IMD, and JNK pathway components. Silencing of Toll and IMD pathway components did not increase replication of all three viruses. However, depletion of infection induced JNK pathway activator and repressor showed conserved antiviral response of this pathway against the viral infections. We further showed that JNK activation by arboviruses is mediated by antiviral complement and apoptosis activation. Conclusion: This study determined the previously unknown antiviral mechanism of JNK pathway in mosquito salivary glands against important pathogenic arboviruses. This pathway shows potential to be utilized for developing effective transmission blocking tools.

The Aedes aegypti IMD pathway is a critical component of the mosquito antifungal immune response

Successful infection of the insect body by entomopathogenic fungi is the result of complex molecular interactions between the host and the invading pathogenic fungi. The mosquito antifungal response is multifaceted and is regulated in part by the Toll and Jak-STAT pathways. Here, we assessed the role of the IMD pathway in the mosquito Ae. aegypti antifungal immune response when challenged with one of two entomopathogenic fungi, Beauveria bassiana and Isaria javanica. IMD pathway components of the mosquito immune system were elicited in response to infection with both entomopathogenic fungi, primarily in the fat body of mosquitoes. Furthermore, we observed induction of antimicrobial peptides that in turn appear to be tissue and fungal strain-specific. IMD pathway impairment by RNAi gene silencing resulted in higher fungal proliferation and reduction in survival of fungi-infected mosquitoes. Collectively, these data indicates that the IMD pathway plays a more significant role in the antifungal immune response than previously recognized.

The NF-κB Factor Relish Regulates Atg1 Expression and Controls Autophagy

SUMMARYMacroautophagy and cell death both contribute to innate immunity, but little is known about how these processes integrate. Drosophila larval salivary glands require autophagy for developmentally programmed cell death, and innate immune signaling factors increase in these dying cells. Here, we show that the nuclear factor κB (NF-κB) factor Relish, a component of the immune deficiency (Imd) pathway, is required for salivary gland degradation. Surprisingly, of the classic Imd pathway components, only Relish and the PGRP receptors were involved in salivary gland degradation. Significantly, Relish controls salivary gland degradation by regulating autophagy but not caspases. In addition, expression of either Relish or PGRP-LC causes premature autophagy induction and subsequent gland degradation. Relish controls autophagy by regulating the expression of Atg1, a core component and activator of the autophagy pathway. Together these findings demonstrate that a NF-κB pathway regulates autophagy during developmentally programmed cell death.

Signaling pathways regulating innate immune responses in shrimp

The first line of defense against microbial infections in animals is innate immune response which triggers diverse humoral and cellular activities via signal transduction pathways. Toll, IMD and JAK/STAT pathways are regarded as the main pathways regulating the immune response of invertebrates. This paper reviews the main progress of the investigation on the immune response to pathogen's infection in shrimp and supposes that these three signal pathways exist in shrimp. Most of the components (proteins or genes) involved in Toll pathway of Drosophila have been cloned also in shrimp which suggested the existence of Toll pathway in shrimp. The data update shows that the Toll pathway of shrimp is responsive not only to Gram-positive bacteria, Gram-negative bacteria, but also to WSSV. Challenge of WSSV can lead to the variation of transcription level of all identified components in shrimp Toll pathway, which supported that Toll pathway in shrimp played important roles during WSSV infection. Two major homologs to the components of IMD pathway of Drosophila, IMD and Relish, have been identified in shrimp, which indicated that IMD pathway should be existed in shrimp and might play important roles in regulating the immune response of shrimp to bacteria and virus infection. Relish in IMD pathway and dorsal in Toll pathway of shrimp were both involved in the immune response of shrimp to bacteria and virus infection, which implied that these two pathways are not completely separated during the immune response of shrimp. The transcription of STAT in shrimp was modulated after WSSV infection, which suggested that a putative JAK/STAT pathway might exist in shrimp and be very important to virus infection. Study on the signaling pathway regulating the immune response in shrimp could help us to understand the innate immune system, and would provide instructions to shrimp disease control. Obviously, to get more clear ideas about the innate immunological pathways in shrimp, more solid functional studies should be done in the future.

Anopheles Imd Pathway Factors and Effectors in Infection Intensity-Dependent Anti-Plasmodium Action

The Anopheles gambiae immune response against Plasmodium falciparum, an etiological agent of human malaria, has been identified as a source of potential anti-Plasmodium genes and mechanisms to be exploited in efforts to control the malaria transmission cycle. One such mechanism is the Imd pathway, a conserved immune signaling pathway that has potent anti-P. falciparum activity. Silencing the expression of caspar, a negative regulator of the Imd pathway, or over-expressing rel2, an Imd pathway-controlled NFkappaB transcription factor, confers a resistant phenotype on A. gambiae mosquitoes that involves an array of immune effector genes. However, unexplored features of this powerful mechanism that may be essential for the implementation of a malaria control strategy still remain. Using RNA interference to singly or dually silence caspar and other components of the Imd pathway, we have identified genes participating in the anti-Plasmodium signaling module regulated by Caspar, each of which represents a potential target to achieve over-activation of the pathway. We also determined that the Imd pathway is most potent against the parasite's ookinete stage, yet also has reasonable activity against early oocysts and lesser activity against late oocysts. We further demonstrated that caspar silencing alone is sufficient to induce a robust anti-P. falciparum response even in the relative absence of resident gut microbiota. Finally, we established the relevance of the Imd pathway components and regulated effectors TEP1, APL1, and LRIM1 in parasite infection intensity-dependent defense, thereby shedding light on the relevance of laboratory versus natural infection intensity models. Our results highlight the physiological considerations that are integral to a thoughtful implementation of Imd pathway manipulation in A. gambiae as part of an effort to limit the malaria transmission cycle, and they reveal a variety of previously unrecognized nuances in the Imd-directed immune response against P. falciparum.

Relative Roles of the Cellular and Humoral Responses in the Drosophila Host Defense against Three Gram-Positive Bacterial Infections

BackgroundTwo NF-kappaB signaling pathways, Toll and immune deficiency (imd), are required for survival to bacterial infections in Drosophila. In response to septic injury, these pathways mediate rapid transcriptional activation of distinct sets of effector molecules, including antimicrobial peptides, which are important components of a humoral defense response. However, it is less clear to what extent macrophage-like hemocytes contribute to host defense.Methodology/Principal FindingsIn order to dissect the relative importance of humoral and cellular defenses after septic injury with three different Gram-positive bacteria (Micrococcus luteus, Enterococcus faecalis, Staphylococcus aureus), we used latex bead pre-injection to ablate macrophage function in flies wildtype or mutant for various Toll and imd pathway components. We found that in all three infection models a compromised phagocytic system impaired fly survival – independently of concomitant Toll or imd pathway activation. Our data failed to confirm a role of the PGRP-SA and GNBP1 Pattern Recognition Receptors for phagocytosis of S. aureus. The Drosophila scavenger receptor Eater mediates the phagocytosis by hemocytes or S2 cells of E. faecalis and S. aureus, but not of M. luteus. In the case of M. luteus and E. faecalis, but not S. aureus, decreased survival due to defective phagocytosis could be compensated for by genetically enhancing the humoral immune response.Conclusions/SignificanceOur results underscore the fundamental importance of both cellular and humoral mechanisms in Drosophila immunity and shed light on the balance between these two arms of host defense depending on the invading pathogen.

Pirk Is a Negative Regulator of the Drosophila Imd Pathway

NF-kappaB transcription factors are involved in evolutionarily conserved signaling pathways controlling multiple cellular processes including apoptosis and immune and inflammatory responses. Immune response of the fruit fly Drosophila melanogaster to Gram-negative bacteria is primarily mediated via the Imd (immune deficiency) pathway, which closely resembles the mammalian TNFR signaling pathway. Instead of cytokines, the main outcome of Imd signaling is the production of antimicrobial peptides. The pathway activity is delicately regulated. Although many of the Imd pathway components are known, the mechanisms of negative regulation are more elusive. In this study we report that a previously uncharacterized gene, pirk, is highly induced upon Gram-negative bacterial infection in Drosophila in vitro and in vivo. pirk encodes a cytoplasmic protein that coimmunoprecipitates with Imd and the cytoplasmic tail of peptidoglycan recognition protein LC (PGRP-LC). RNA interference-mediated down-regulation of Pirk caused Imd pathway hyperactivation upon infection with Gram-negative bacteria, while overexpression of pirk reduced the Imd pathway response both in vitro and in vivo. Furthermore, pirk-overexpressing flies were more susceptible to Gram-negative bacterial infection than wild-type flies. We conclude that Pirk is a negative regulator of the Imd pathway.

Fungal Peptide Destruxin A Plays a Specific Role in Suppressing the Innate Immune Response in Drosophila melanogaster

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.

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

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