Hormonal regulation of Drosophila microRNA let-7 and miR-125 that target innate immunity

The steroid 20-hydroxy-ecdysone (20-HE) and the sesquiterpenoid Juvenile Hormone (JH) coordinate insect life stage transitions. 20-HE exerts these effects by the sequential induction of response genes. In the nematode Caenorhabditis elegans hormones also play a role in such transitions, but notably, microRNA such as let-7 and lin-4 have likewise been found to help order developmental steps. Little is known about the corresponding function of homologous microRNA in Drosophila melanogaster, and the way microRNA might be regulated by 20-HE in the fly is ambiguous. Here we used Drosophila S2 cells to analyze the effects of 20-HE on D. melanogaster microRNA let-7 and miR-125, the homolog of lin-4. The induction by 20-HE of let-7 and miR-125 in S2 cells is inhibited by RNAi knockdown of the ecdysone receptor and, as previously shown, by knockdown of its cofactor broad-complex C. To help resolve the currently ambiguous role of 20-HE in the control of microRNA, we show that nanomolar concentrations of 20-HE primes cells to subsequently express microRNA when exposed to micromolar levels of 20-HE. We then explore the role microRNA plays in the established relationship between 20-HE and the induction of innate immunity. We show that the 3'UTR of the antimicrobial peptide diptericin has a let-7 binding site and that let-7 represses translation from this site. We conclude that 20-HE facilitates the initial expression of innate immunity while it simultaneously induces negative regulation via microRNA control of antimicrobial peptide translation.     

Insect immunity: a genetic factor (hrtp) is essential for antibacterial peptide expression in Drosophila after infection by parasitoid wasps

We have used a parasitoid wasp Drosophila melanogaster system to investigate the relationship between the humoral and cellular immune responses in insects. Expression of the gene encoding diptericin, an antibacterial peptide in various D. melanogaster strains parasitized by several species of parasitoid wasps, was studied by Northern blot. These strains have the capacity to encapsulate parasitoid eggs. Two strains appeared to produce diptericin mRNA after parasitoid challenge, regardless of their cellular immune reaction to the wasp species. This suggests that a specific genetic factor, or factors, here designated humoral response to parasitoid (hrtp), is present in these two strains of D. melanogaster and is implicated in the expression of the antibacterial gene after parasite infection. This hrtp genetic factor is recessively expressed and located on the second chromosome, suggesting that it is monofactorial. The transgenic strain Dipt.2.2-lacZ:1, in which the transgene is present on the first chromosome, is normally susceptible to the parasitoid wasp. The chromosome bearing the hrtp factor was transferred to this transgenic strain, which then became reactive when triggered by wasp infection. The hrtp factor appears necessary for the activation of diptericin by the parasitoid wasp. No correlation between the cellular immune capacity and the humoral response was observed, suggesting that the two components of insect immunity are regulated independently. Arch.     

unpaired (upd)-3 expression and other immune-related functions are stimulated by interleukin-8 in Drosophila melanogaster SL2 cell line

Invertebrate innate immunity relies on both cellular and humoral components. Among humoral factors, there is less information on soluble molecules able to act as signals during the immune response (i.e. cytokines). In Drosophila melanogaster, the cytokine Unpaired (Upd)-3, is known to activate the JAK/STAT pathway, but it is still not clear which molecules and pathways are responsible for its induction and secretion. It has been proposed that highly chemotactic factors may increase the expression of upd-3. In this respect, we have studied the effects of the chemotactic human recombinant (hr) interleukin (IL)-8 on the immune functions of Drosophila SL2 macrophage-like cells. The hrIL-8 increases the percentage of phagocytic cells with a specific timing and enhances the expression of the cytokine, upd-3, as well as that of the putative cytokine Drosophila helical factor (dhf). The antimicrobial peptides defensin, cecropin A1 and diptericin, are all influenced in their expression by the human chemokine, while hrIL-8 leaves unaffected the expression of drosomycin, i.e. the antimicrobial peptide more strictly connected with the Toll pathway. Similar effects to those registered for hrIL-8 are also provoked by a specific activator of the Imd pathway, i.e. the Escherichia coli peptidoglycan. RNAi experiments demonstrated that the silencing of the Imd pathway-associated kinase dTAK1, leaves unaffected the induction of upd-3, while it completely abolishes the effects of hrIL-8-on the expression of dhf. Our data suggest that the Imd pathway is not fundamental in regulating the levels of upd-3, whereas it controls the expression of the putative cytokine dhf.     

果蝇先天性免疫研究进展

果蝇是生命科学与人类疾病研究的重要模式生物,虽然不具有人类高度专一的获得性免疫,但也有对病原微生物感染作出快速有效反应的先天性免疫应答系统,主要包括体液免疫,细胞免疫和黑化反应。文章结合国外最新研究,详细介绍果蝇体液免疫中控制抗菌肽合成的Toll信号通路和Imd信号通路中涉及的蛋白及其相互作用,并对果蝇细胞免疫中的吞噬、包埋功能和黑化反应作简要阐述。研究表明,果蝇的Toll和Imd信号通路分别与人类的TLR4和TNRF-1信号通路存在着惊人的相似之处,说明果蝇与人类在免疫调控通路方面可能存在着共同的进化起源。     

Functional analysis of immune response genes in Drosophila identifies JNK pathway as a regulator of antimicrobial peptide gene expression in S2 cells

The templates of innate immunity have ancient origins. Thus, such model animals as the fruit fly, Drosophila melanogaster, can be used to identify gene products that also play a key role in the innate immunity in mammals. We have used oligonucleotide microarrays to identify genes that are responsive to gram-negative bacteria in Drosophila macrophage-like S2 cells. In total, 53 genes were induced by greater than threefold in response to Escherichia coli. The induction of all these genes was peptidoglycan recognition protein LC (PGRP-LC) dependent. Twenty-two genes including 10 of the most strongly induced genes are also known to be up-regulated by septic injury in vivo. Importantly, we identified 31 genes that are not known to respond to bacterial challenge. We carried out targeted dsRNA treatments to assess the functional importance of these gene products for microbial recognition, phagocytosis and antimicrobial peptide release in Drosophila S2 cells in vitro. RNAi targeting three of these genes, CG7097, CG15678 and beta-Tubulin 60D, caused altered antimicrobial peptide release in vitro. Our results indicate that the JNK pathway is essential for normal antimicrobial peptide release in Drosophila in vitro.     

A putative helical cytokine functioning in innate immune signalling in Drosophila melanogaster

In invertebrates and vertebrates, innate immunity is considered the first line of defense mechanism against non-self material. In vertebrates, cytokines play a critical role in innate immune signalling. To date, however, the existence of genes encoding for invertebrate helical cytokines has been anticipated, but never demonstrated. Here, we report the first structural and functional evidence of a gene encoding for a putative helical cytokine in Drosophila melanogaster. Functional experiments demonstrate that its expression, as well as that of the antimicrobial factors defensin and cecropin A1, is significantly increased after immune stimulation. These observations suggest the involvement of helical cytokines in the innate immune response of invertebrates.     

Diptericin-like protein: an immune response gene regulated by the anti-bacterial gene induction pathway in Drosophila.

Insects produce various anti-microbial peptides in response to injury and infection. In Drosophila, diptericin has previously been studied as an anti-bacterial immune response gene. Here, we report the cloning of the diptericin-like protein (dptlp) gene as a paralog of Drosophila diptericin. By comparison of their sequences, we found that the dptlp gene has all of the functional domains conserved in the diptericin gene and other anti-bacterial proteins. The dptlp gene was rapidly induced by bacterial infections and showed different time-dependent gene expression patterns from those of diptericin. Like diptericin, dptlp was specifically produced from the fat body, and its expression was strictly dependent on bacterial infections. In addition, the dptlp gene expression was almost completely abolished in the imd mutant, which implicates that its expression is regulated by the anti-bacterial arm of the Drosophila innate immune regulatory pathways. In support of this, we found GATA, interferon consensus responding element, and kappa B binding sites, which is known to be important for the proper expression of anti-bacterial genes, in the proximal promoter region of the dptlp gene. Taken together, our findings support that dptlp is a novel anti-bacterial peptide whose expression is regulated by the anti-bacterial immune response mechanism.     

果蝇抗微生物肽基因的免疫诱导模式

果蝇作为一种模式昆虫,为研究昆虫和人类的先天免疫发挥了重要作用。目前对果蝇体内免疫诱导产生的抗微生物肽多基因家族在分子进化、抗菌功能的分子特征和免疫诱导表达的信号传递机制等方面的研究进展,进一步加深了人们对昆虫乃至其他动物和人类先天免疫模式的认识,为研究其他昆虫特别是作为主要农林害虫的鳞翅目昆虫的先天免疫机制发挥了重要作用。本文集中对黑腹果蝇Drosophila melanogaster抗微生物肽及其免疫模式的研究结果和最新进展进行了介绍,其中包括作者近几年的研究结果。     

Immune response of Drosophila melanogaster to infection with the flagellate parasite Crithidia spp

Insects are able to recognize invading microorganisms and to mount an immune response to bacterial and fungal infections. Recently, the fruitfly Drosophila melanogaster has emerged as a promising invertebrate model to investigate innate immunity because of its well-characterized genetics. Insects are also vectors of numerous parasites which can trigger an immune response. We have investigated the interaction of Drosophila melanogaster with the flagellate protozoan Crithidia spp. We show that a per os parasitic infection triggers the synthesis of several antimicrobial peptides. By reverse phase HPLC and mass spectrometry, peptides were shown to be present in the hemolymph and not in the gut tissue, suggesting the presence of immune messengers between the site of the infection, namely the gut, and the fat body, the main site of synthesis for antimicrobial peptides. Interestingly, we have identified one molecule which is specifically induced in the hemolymph after infection with Crithidia, but not with bacteria, suggesting that Drosophila can discriminate between pathogens. When flagellates were injected into the hemolymph, a low synthesis of antimicrobial peptides was observed together with phagocytosis of parasites by circulating hemocytes. The data presented here suggest that Drosophila-Crithidia spp. represents an interesting model to study host defense against protozoan parasites.     

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

Innate immunity is an evolutionarily conserved self-defense mechanism against microbial infections. In Drosophila, induction of antimicrobial peptides is a major immune response that is regulated by two distinct signaling pathways called the IMD pathway and the Toll pathway, similar to the tumor necrosis factor-alpha signaling and Toll-like receptor/interleukin-1 signaling pathways, respectively, in mammals. In mammals, innate immunity interacts with adaptive immunity and has a key role in the regulated immune response. Therefore, innate immunity is a pharmaceutical target for the development of immune regulators. Previously, based on the striking conservation between the mechanisms that regulate Drosophila immunity and human innate immunity, we established an ex vivo culture in which compounds acting on innate immunity can be evaluated using a reporter gene that reflects activation of the IMD pathway [Yajima et al. [Yajima, M., Takada, M., Takahashi, N., Kikuchi, H., Natori, S., Oshima, Y., Kurata, S., 2003. A newly established in vitro culture using transgenic Drosophila reveals functional coupling between the phospholipase A2-generated fatty acid cascade and lipopolysaccharide-dependent activation of the immune deficiency (imd) pathway in insect immunity. The Biochemical Journal 371(Pt 1), 205-210] Biochem J 371, 205-210]. Here, we combined the ex vivo culture with a reporter gene that reflects the heat shock response and demonstrated that the resulting systems are useful for screening compounds that act specifically on innate immunity, including mammalian innate immune responses. Identification of target molecules is essential for the development of more potent medicines with fewer side effects. In this study, we also established ex vivo systems capable of identifying target molecules of the identified compounds using targeted activation of the IMD pathway.     

Hormonal regulation of Drosophila microRNA let-7 and miR-125 that target innate immunity

The steroid 20-hydroxy-ecdysone (20-HE) and the sesquiterpenoid Juvenile Hormone (JH) coordinate insect life stage transitions. 20-HE exerts these effects by the sequential induction of response genes. In the nematode Caenorhabditis elegans hormones also play a role in such transitions, but notably, microRNA such as let-7 and lin-4 have likewise been found to help order developmental steps. Little is known about the corresponding function of homologous microRNA in Drosophila melanogaster, and the way microRNA might be regulated by 20-HE in the fly is ambiguous. Here we used Drosophila S2 cells to analyze the effects of 20-HE on D. melanogaster microRNA let-7 and miR-125, the homolog of lin-4. The induction by 20-HE of let-7 and miR-125 in S2 cells is inhibited by RNai knockdown of the ecdysone receptor and, as previously shown, by knockdown of its cofactor broad-complex C. To help resolve the currently ambiguous role of 20-HE in the control of microRNa, we show that nanomolar concentrations of 20-HE primes cells to subsequently express microRNa when exposed to micromolar levels of 20-HE. We then explore the role microRNa plays in the established relationship between 20-HE and the induction of innate immunity. We show that the 3′UTR of the antimicrobial peptide diptericin has a let-7 binding site and that let-7 represses translation from this site. We conclude that 20-HE facilitates the initial expression of innate immunity while it simultaneously induces negative regulation via microRNa control of antimicrobial peptide translation.Key words: microRNA, ecdysone, innate immunity, let-7, diptericin, inflammation     

The Drosophila systemic immune response: sensing and signalling during bacterial and fungal infections

A hallmark of the potent, multifaceted antimicrobial defence of Drosophila melanogaster is the challenge-induced synthesis of several families of antimicrobial peptides by cells in the fat body. The basic mechanisms of recognition of various types of microbial infections by the adult fly are now understood, often in great detail. We have further gained valuable insight into the infection-induced gene reprogramming by nuclear factor-kappaB (NF-kappaB) family members under the dependence of complex intracellular signalling cascades. The striking parallels between the adult fly response and mammalian innate immune defences described below point to a common ancestry and validate the relevance of the fly defence as a paradigm for innate immunity.     

The Drosophila Toll signaling pathway

The identification of the Drosophila melanogaster Toll pathway cascade and the subsequent characterization of TLRs have reshaped our understanding of the immune system. Ever since, Drosophila NF-κB signaling has been actively studied. In flies, the Toll receptors are essential for embryonic development and immunity. In total, nine Toll receptors are encoded in the Drosophila genome, including the Toll pathway receptor Toll. The induction of the Toll pathway by gram-positive bacteria or fungi leads to the activation of cellular immunity as well as the systemic production of certain antimicrobial peptides. The Toll receptor is activated when the proteolytically cleaved ligand Spatzle binds to the receptor, eventually leading to the activation of the NF-κB factors Dorsal-related immunity factor or Dorsal. In this study, we review the current literature on the Toll pathway and compare the Drosophila and mammalian NF-κB pathways.     

Specificity and complexity of the Caenorhabditis elegans innate immune response

In response to infection, Caenorhabditis elegans produces an array of antimicrobial proteins. To understand the C. elegans immune response, we have investigated the regulation of a large, representative sample of candidate antimicrobial genes. We found that all these putative antimicrobial genes are expressed in tissues exposed to the environment, a position from which they can ward off infection. Using RNA interference to inhibit the function of immune signaling pathways in C. elegans, we found that different immune response pathways regulate expression of distinct but overlapping sets of antimicrobial genes. We also show that different bacterial pathogens regulate distinct but overlapping sets of antimicrobial genes. The patterns of genes induced by pathogens do not coincide with any single immune signaling pathway. Thus, even in this simple model system for innate immunity, striking specificity and complexity exist in the immune response. The unique patterns of antimicrobial gene expression observed when C. elegans is exposed to different pathogens or when different immune signaling pathways are perturbed suggest that a large set of yet to be identified pathogen recognition receptors (PRRs) exist in the nematode. These PRRs must interact in a complicated fashion to induce a unique set of antimicrobial genes. We also propose the existence of an "antimicrobial fingerprint," which will aid in assigning newly identified C. elegans innate immunity genes to known immune signaling pathways.