RESUMO
Autophagy degrades cytoplasmic components and is important for development and human health. Although autophagy is known to be influenced by systemic intercellular signals, the proteins that control autophagy are largely thought to function within individual cells. Here, we report that Drosophila macroglobulin complement-related (Mcr), a complement ortholog, plays an essential role during developmental cell death and inflammation by influencing autophagy in neighboring cells. This function of Mcr involves the immune receptor Draper, suggesting a relationship between autophagy and the control of inflammation. Interestingly, Mcr function in epithelial cells is required for macrophage autophagy and migration to epithelial wounds, a Draper-dependent process. This study reveals, unexpectedly, that complement-related from one cell regulates autophagy in neighboring cells via an ancient immune signaling program.
Assuntos
Autofagia , Proteínas do Sistema Complemento/imunologia , Drosophila melanogaster/crescimento & desenvolvimento , Animais , Citocinas , Proteínas de Drosophila , Drosophila melanogaster/citologia , Drosophila melanogaster/imunologia , Inflamação/imunologia , Larva/crescimento & desenvolvimento , Larva/imunologia , Macrófagos/imunologia , Glândulas Salivares/citologia , Glândulas Salivares/crescimento & desenvolvimento , Glândulas Salivares/metabolismo , SerpinasRESUMO
Fine control of macrophage activation is needed to prevent inflammatory disease, particularly at barrier sites such as the lungs. However, the dominant mechanisms that regulate the activation of pulmonary macrophages during inflammation are poorly understood. We found that alveolar macrophages (AlvMs) were much less able to respond to the canonical type 2 cytokine IL-4, which underpins allergic disease and parasitic worm infections, than macrophages from lung tissue or the peritoneal cavity. We found that the hyporesponsiveness of AlvMs to IL-4 depended upon the lung environment but was independent of the host microbiota or the lung extracellular matrix components surfactant protein D (SP-D) and mucin 5b (Muc5b). AlvMs showed severely dysregulated metabolism relative to that of cavity macrophages. After removal from the lungs, AlvMs regained responsiveness to IL-4 in a glycolysis-dependent manner. Thus, impaired glycolysis in the pulmonary niche regulates AlvM responsiveness during type 2 inflammation.
Assuntos
Inflamação/imunologia , Pulmão/imunologia , Ativação de Macrófagos/imunologia , Macrófagos Alveolares/imunologia , Animais , Inflamação/genética , Inflamação/metabolismo , Interleucina-4/genética , Interleucina-4/imunologia , Interleucina-4/metabolismo , Larva/imunologia , Larva/fisiologia , Pulmão/metabolismo , Pulmão/patologia , Ativação de Macrófagos/genética , Macrófagos Alveolares/metabolismo , Macrófagos Alveolares/parasitologia , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Mucina-5B/genética , Mucina-5B/imunologia , Mucina-5B/metabolismo , Nippostrongylus/imunologia , Nippostrongylus/fisiologia , Proteína D Associada a Surfactante Pulmonar/genética , Proteína D Associada a Surfactante Pulmonar/imunologia , Proteína D Associada a Surfactante Pulmonar/metabolismo , Infecções por Strongylida/genética , Infecções por Strongylida/imunologia , Infecções por Strongylida/parasitologiaRESUMO
Insect respiration has long been thought to be solely dependent on an elaborate tracheal system without assistance from the circulatory system or immune cells1,2. Here we describe that Drosophila crystal cells-myeloid-like immune cells called haemocytes-control respiration by oxygenating Prophenoloxidase 2 (PPO2) proteins. Crystal cells direct the movement of haemocytes between the trachea of the larval body wall and the circulation to collect oxygen. Aided by copper and a neutral pH, oxygen is trapped in the crystalline structures of PPO2 in crystal cells. Conversely, PPO2 crystals can be dissolved when carbonic anhydrase lowers the intracellular pH and then reassembled into crystals in cellulo by adhering to the trachea. Physiologically, larvae lacking crystal cells or PPO2, or those expressing a copper-binding mutant of PPO2, display hypoxic responses under normoxic conditions and are susceptible to hypoxia. These hypoxic phenotypes can be rescued by hyperoxia, expression of arthropod haemocyanin or prevention of larval burrowing activity to expose their respiratory organs. Thus, we propose that insect immune cells collaborate with the tracheal system to reserve and transport oxygen through the phase transition of PPO2 crystals, facilitating internal oxygen homeostasis in a process that is comparable to vertebrate respiration.
Assuntos
Catecol Oxidase , Proteínas de Drosophila , Drosophila melanogaster , Precursores Enzimáticos , Hemócitos , Oxigênio , Transição de Fase , Respiração , Animais , Feminino , Masculino , Transporte Biológico , Anidrases Carbônicas/metabolismo , Catecol Oxidase/metabolismo , Cobre/metabolismo , Cristalização , Drosophila melanogaster/anatomia & histologia , Drosophila melanogaster/citologia , Drosophila melanogaster/enzimologia , Drosophila melanogaster/imunologia , Drosophila melanogaster/metabolismo , Proteínas de Drosophila/metabolismo , Precursores Enzimáticos/metabolismo , Hemocianinas/metabolismo , Hemócitos/imunologia , Hemócitos/metabolismo , Homeostase , Concentração de Íons de Hidrogênio , Hiperóxia/metabolismo , Hipóxia/metabolismo , Larva/anatomia & histologia , Larva/citologia , Larva/imunologia , Larva/metabolismo , Oxigênio/metabolismoRESUMO
Group 2 innate lymphoid cells (ILC2s) regulate immunity, inflammation, and tissue homeostasis. Two distinct subsets of ILC2s have been described: steady-state natural ILC2s and inflammatory ILC2s, which are elicited following helminth infection. However, how tissue-specific cues regulate these two subsets of ILC2s and their effector functions remains elusive. Here, we report that interleukin-33 (IL-33) promotes the generation of inflammatory ILC2s (ILC2INFLAM) via induction of the enzyme tryptophan hydroxylase 1 (Tph1). Tph1 expression was upregulated in ILC2s upon activation with IL-33 or following helminth infection in an IL-33-dependent manner. Conditional deletion of Tph1 in lymphocytes resulted in selective impairment of ILC2INFLAM responses and increased susceptibility to helminth infection. Further, RNA sequencing analysis revealed altered gene expression in Tph1 deficient ILC2s including inducible T cell co-stimulator (Icos). Collectively, these data reveal a previously unrecognized function for IL-33, Tph1, and ICOS in promoting inflammatory ILC2 responses and type 2 immunity at mucosal barriers.
Assuntos
Imunidade Celular , Proteína Coestimuladora de Linfócitos T Induzíveis/imunologia , Interleucina-33/imunologia , Nippostrongylus/imunologia , Infecções por Strongylida/imunologia , Subpopulações de Linfócitos T/imunologia , Triptofano Hidroxilase/imunologia , Animais , Linhagem da Célula/genética , Linhagem da Célula/imunologia , Suscetibilidade a Doenças , Regulação da Expressão Gênica/imunologia , Imunidade Inata , Imunidade nas Mucosas , Proteína Coestimuladora de Linfócitos T Induzíveis/genética , Interleucina-33/genética , Larva/crescimento & desenvolvimento , Larva/imunologia , Larva/patogenicidade , Linfonodos/imunologia , Linfonodos/parasitologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Nippostrongylus/crescimento & desenvolvimento , Nippostrongylus/patogenicidade , Cultura Primária de Células , Transdução de Sinais , Infecções por Strongylida/genética , Infecções por Strongylida/parasitologia , Infecções por Strongylida/patologia , Subpopulações de Linfócitos T/classificação , Subpopulações de Linfócitos T/parasitologia , Triptofano Hidroxilase/genéticaRESUMO
Tissue damage and infection are deemed likewise triggers of innate immune responses. But whereas neutrophil responses to microbes are generally protective, neutrophil recruitment into damaged tissues without infection is deleterious. Why neutrophils respond to tissue damage and not just to microbes is unknown. Is it a flaw of the innate immune system that persists because evolution did not select against it, or does it provide a selective advantage? Here we dissect the contribution of tissue damage signaling to antimicrobial immune responses in a live vertebrate. By intravital imaging of zebrafish larvae, a powerful model for innate immunity, we show that prevention of tissue damage signaling upon microbial ear infection abrogates leukocyte chemotaxis and reduces animal survival, at least in part, through suppression of cytosolic phospholipase A2 (cPla2), which integrates tissue damage- and microbe-derived cues. Thus, microbial cues are insufficient, and damage signaling is essential for antimicrobial neutrophil responses in zebrafish.
Assuntos
Doenças dos Peixes/imunologia , Infiltração de Neutrófilos/imunologia , Transdução de Sinais/imunologia , Peixe-Zebra/imunologia , Animais , Animais Geneticamente Modificados , Doenças dos Peixes/microbiologia , Imunidade Inata/imunologia , Larva/imunologia , Larva/microbiologia , Neutrófilos/imunologia , Neutrófilos/metabolismo , Fosfolipases A2 Citosólicas/imunologia , Fosfolipases A2 Citosólicas/metabolismo , Peixe-Zebra/genética , Peixe-Zebra/microbiologia , Proteínas de Peixe-Zebra/imunologia , Proteínas de Peixe-Zebra/metabolismoRESUMO
We examined the role of innate cells in acquired resistance to the natural murine parasitic nematode, Nippostrongylus brasiliensis. Macrophages obtained from lungs as late as 45 d after N. brasiliensis inoculation were able to transfer accelerated parasite clearance to naive recipients. Primed macrophages adhered to larvae in vitro and triggered increased mortality of parasites. Neutrophil depletion in primed mice abrogated the protective effects of transferred macrophages and inhibited their in vitro binding to larvae. Neutrophils in parasite-infected mice showed a distinct transcriptional profile and promoted alternatively activated M2 macrophage polarization through secretory factors including IL-13. Differentially activated neutrophils in the context of a type 2 immune response therefore prime a long-lived effector macrophage phenotype that directly mediates rapid nematode damage and clearance.
Assuntos
Imunidade Adaptativa/imunologia , Macrófagos/imunologia , Neutrófilos/imunologia , Nippostrongylus/imunologia , Infecções por Strongylida/imunologia , Animais , Adesão Celular/imunologia , Adesão Celular/fisiologia , Células Cultivadas , Citocinas/genética , Citocinas/imunologia , Citocinas/metabolismo , Resistência à Doença/imunologia , Feminino , Citometria de Fluxo , Interações Hospedeiro-Parasita/imunologia , Interleucina-13/genética , Interleucina-13/imunologia , Interleucina-13/metabolismo , Subunidade alfa de Receptor de Interleucina-4/genética , Subunidade alfa de Receptor de Interleucina-4/imunologia , Subunidade alfa de Receptor de Interleucina-4/metabolismo , Larva/imunologia , Larva/fisiologia , Pulmão/imunologia , Pulmão/metabolismo , Pulmão/parasitologia , Macrófagos/metabolismo , Camundongos Endogâmicos BALB C , Camundongos Knockout , Neutrófilos/metabolismo , Nippostrongylus/fisiologia , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Infecções por Strongylida/genética , Infecções por Strongylida/parasitologia , Transcriptoma/imunologiaRESUMO
Neutrophils accumulate early in tissue injury. However, the cellular and functional heterogeneity of neutrophils during homeostasis and in response to tissue damage remains unclear. In this study, we use larval zebrafish to understand neutrophil responses to thermal injury. Single-cell transcriptional mapping of myeloid cells during a 3-d time course in burn and control larvae revealed distinct neutrophil subsets and their cell-cell interactions with macrophages across time and conditions. The trajectory formed by three zebrafish neutrophil subsets resembles human neutrophil maturation, with varying transition patterns between conditions. Through ligand-receptor cell-cell interaction analysis, we found that neutrophils communicate more in burns in a pathway and temporal manner. Finally, we identified the correlation between zebrafish myeloid signatures and human burn severity, establishing GPR84+ neutrophils as a potential marker of early innate immune response in burns. This work builds a comparative single-cell transcriptomic framework to identify neutrophil markers of tissue damage using model organisms.
Assuntos
Queimaduras , Larva , Neutrófilos , Análise de Célula Única , Peixe-Zebra , Animais , Peixe-Zebra/imunologia , Neutrófilos/imunologia , Queimaduras/imunologia , Larva/imunologia , Larva/genética , Transcriptoma , Humanos , Imunidade Inata , Modelos Animais de Doenças , Macrófagos/imunologia , Comunicação Celular/imunologiaRESUMO
BACKGROUND: Innate immune responses can be activated by pathogen-associated molecular patterns (PAMPs), danger signals released by damaged tissues, or the absence of self-molecules that inhibit immunity. As PAMPs are typically conserved across broad groups of pathogens but absent from the host, it is unclear whether they allow hosts to recognize parasites that are phylogenetically similar to themselves, such as parasitoid wasps infecting insects. RESULTS: Parasitoids must penetrate the cuticle of Drosophila larvae to inject their eggs. In line with previous results, we found that the danger signal of wounding triggers the differentiation of specialized immune cells called lamellocytes. However, using oil droplets to mimic infection by a parasitoid wasp egg, we found that this does not activate the melanization response. This aspect of the immune response also requires exposure to parasite molecules. The unidentified factor enhances the transcriptional response in hemocytes and induces a specific response in the fat body. CONCLUSIONS: We conclude that a combination of danger signals and the recognition of nonself molecules is required to activate Drosophila's immune response against parasitic insects.
Assuntos
Hemócitos , Interações Hospedeiro-Parasita , Imunidade Inata , Vespas , Animais , Vespas/fisiologia , Interações Hospedeiro-Parasita/imunologia , Hemócitos/imunologia , Drosophila melanogaster/parasitologia , Drosophila melanogaster/imunologia , Drosophila melanogaster/fisiologia , Larva/imunologia , Larva/parasitologia , Drosophila/parasitologia , Drosophila/imunologiaRESUMO
BACKGROUND: Trichinella spiralis (T. spiralis) is a parasitic helminth that causes a globally prevalent neglected zoonotic disease, and worms at different developmental stages (muscle larvae, adult worms, newborn larvae) induce immune attack at different infection sites, causing serious harm to host health. Several innate immune cells release extracellular traps (ETs) to entrap and kill most pathogens that invade the body. In response, some unicellular pathogens have evolved a strategy to escape capture by ETs through the secretion of nucleases, but few related studies have investigated multicellular helminths. RESULTS: In the present study, we observed that ETs from neutrophils capture adult worms of T. spiralis, while ETs from macrophages trap muscle larvae and newborn larvae, and ETs had a killing effect on parasites in vitro. To defend against this immune attack, T. spiralis secretes plancitoxin-1, a DNase II-like protein, to degrade ETs and escape capture, which is essential for the survival of T. spiralis in the host. CONCLUSIONS: In summary, these findings demonstrate that T. spiralis escapes ET-mediated capture by secreting deoxyribonuclease as a potential conserved immune evasion mechanism, and plancitoxin-1 could be used as a potential vaccine candidate.
Assuntos
Armadilhas Extracelulares , Evasão da Resposta Imune , Trichinella spiralis , Animais , Trichinella spiralis/fisiologia , Trichinella spiralis/imunologia , Armadilhas Extracelulares/imunologia , Armadilhas Extracelulares/metabolismo , Camundongos , Proteínas de Helminto/metabolismo , Larva/imunologia , Larva/parasitologiaRESUMO
Immune cells provide defense against non-self and have recently been shown to also play key roles in diverse processes such as development, metabolism, and tumor progression. The heterogeneity of Drosophila immune cells (hemocytes) remains an open question. Using bulk RNA sequencing, we find that the hemocytes display distinct features in the embryo, a closed and rapidly developing system, compared to the larva, which is exposed to environmental and metabolic challenges. Through single-cell RNA sequencing, we identify fourteen hemocyte clusters present in unchallenged larvae and associated with distinct processes, e.g., proliferation, phagocytosis, metabolic homeostasis, and humoral response. Finally, we characterize the changes occurring in the hemocyte clusters upon wasp infestation, which triggers the differentiation of a novel hemocyte type, the lamellocyte. This first molecular atlas of hemocytes provides insights and paves the way to study the biology of the Drosophila immune cells in physiological and pathological conditions.
Assuntos
Proteínas de Drosophila/imunologia , Hemócitos/imunologia , Animais , Proteínas de Drosophila/genética , Drosophila melanogaster , Larva/genética , Larva/imunologiaRESUMO
Insects rely on their innate immune system to eliminate pathogenic microbes. As a system component, cytokines transmit intercellular signals to control immune responses. Growth-blocking peptide (GBP) is a member of the stress-responsive peptide family of cytokines found in several orders of insects, including Drosophila. However, the physiological role of GBP in defence against pathogens is not thoroughly understood. In this study, we explored the functions of GBP in a lepidopteran pest, Ostrinia furnacalis. Injection of recombinant O. furnacalis GBP (OfGBP) precursor (proGBP) and chemically synthesised GBP significantly induced the transcription of antimicrobial peptides (AMPs) and other immunity-related genes including immune deficiency (IMD) and Dorsal. The level of OfGBP mRNA was upregulated after bacterial infection. Knockdown of OfGBP expression led to a decrease in IMD, Relish, MyD88 and Dorsal mRNA levels. OfGBP induced phenoloxidase activity and affected hemocyte behaviours in O. furnacalis larvae. In summary, GBP is a potent cytokine, effectively regulating AMP synthesis, melanization response and cellular immunity to eliminate invading pathogens.
Assuntos
Proteínas de Insetos , Larva , Mariposas , Animais , Mariposas/imunologia , Mariposas/genética , Mariposas/crescimento & desenvolvimento , Proteínas de Insetos/metabolismo , Proteínas de Insetos/genética , Larva/crescimento & desenvolvimento , Larva/imunologia , Peptídeos Antimicrobianos/farmacologia , Peptídeos Antimicrobianos/genética , Peptídeos Antimicrobianos/metabolismo , Hemócitos/metabolismo , Imunidade InataRESUMO
REPAT (response to pathogen) is an immune-associated gene family that plays important roles in insect immune response to pathogens. Although nine REPAT genes have been identified in Spodoptera frugiperda (Lepidoptera: Noctuidae) currently, their functions and mechanisms in the immune response to pathogens still remain unclear. Therefore, SfREPAT38, a pathogen response gene (REPAT) of S. frugiperda, was characterised and its function was analysed. The results showed that SfREPAT38 contains a signal peptide and a transcription activator MBF2 (multi-protein bridging factor 2) domain. Quantitative real-time polymerase chain reaction analysis showed that SfREPAT38 was highly expressed in the sixth-instar larvae (L6) and was the highest in expression in the midgut of L6. We found that the expression of SfREPAT38 could be activated by challenge with four microbial pathogens (Bacillus thuringiensis, Metarhizium anisopliae, Spodoptera exigua nuclearpolyhedrosis and Escherichia coli), except 12 h after E. coli infection. Furthermore, the SfREPAT38 expression levels significantly decreased at 24, 48 and 72 h after SfREPAT38 dsRNA injection or feeding. Feeding with SfREPAT38 dsRNA significantly decreased the weight gain of S. frugiperda, and continuous feeding led to the death of S. frugiperda larvae from the fourth day. Moreover, SfREPAT38 dsRNA injection resulted in a significant decrease of weight gain on the fifth day. Silencing SfREPAT38 gene down-regulated the expression levels of immune genes belonging to the Toll pathway, including SPZ, Myd88, DIF, Cactus, Pell and Toll18W. After treatment with SfREPAT38 dsRNA, S. frugiperda became extremely sensitive to the B. thuringiensis infection, and the survival rate dramatically increased, with 100% mortality by the eighth day. The weight of S. frugiperda larvae was also significantly lower than that of the control groups from the second day onwards. In addition, the genes involved in the Toll signalling pathway and a few antibacterial peptide related genes were down-regulated after treatment. These results showed that SfREPAT38 is involved in the immune response of S. frugiperda larvae through mediating Toll signalling pathway.
Assuntos
Proteínas de Insetos , Larva , Transdução de Sinais , Spodoptera , Animais , Spodoptera/imunologia , Spodoptera/genética , Spodoptera/crescimento & desenvolvimento , Larva/crescimento & desenvolvimento , Larva/imunologia , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Imunidade Inata , Receptores Toll-Like/genética , Receptores Toll-Like/metabolismoRESUMO
Cystic echinococcosis is caused by the tissue-dwelling larva (hydatid) of Echinococcus granulosus sensu lato. A salient feature is that this larva is protected by the acellular laminated layer (LL). As the parasite grows, the LL sheds abundant particles that can accumulate in the parasite's vicinity. The potential of LL particles to induce inflammation in vivo has not been specifically analysed. It is not known how each of its two major components, namely highly glycosylated mucins and calcium inositol hexakisphosphate (InsP6) deposits, impacts inflammation induced by the LL as a whole. In this work, we show that LL particles injected intraperitoneally cause infiltration of eosinophils, neutrophils and monocytes/macrophages as well as the disappearance of resident (large peritoneal) macrophages. Strikingly, the absence of calcium InsP6 enhanced the recruitment of all the inflammatory cell types analysed. In contrast, oxidation of the mucin carbohydrates caused decreased recruitment of neutrophils. The carbohydrate-oxidised particles caused cell influx nonetheless, which may be explained by possible receptor-independent effects of LL particles on innate immune cells, as suggested by previous works from our group. In summary, LL particles can induce acute inflammatory cell recruitment partly dependent on its mucin glycans, and this recruitment is attenuated by the calcium InsP6 component.
Assuntos
Echinococcus granulosus , Ácido Fítico , Animais , Echinococcus granulosus/imunologia , Ácido Fítico/farmacologia , Ácido Fítico/metabolismo , Equinococose/imunologia , Equinococose/parasitologia , Inflamação , Neutrófilos/imunologia , Mucinas/metabolismo , Camundongos , Macrófagos/imunologia , Macrófagos/metabolismo , Eosinófilos/imunologia , Feminino , Larva/imunologiaRESUMO
Toll receptors are important regulators of insects' innate immune system which, upon binding of pathogen molecules, activate a conserved signal transduction cascade known as the Toll pathway. RNA interference (RNAi) is a powerful tool to study the function of genes via reverse genetics. However, due to the reported refractory of RNAi efficiency in lepidopteran insects, successful reports of silencing of Toll receptors in the silkworm Bombyx mori have not been reported yet. In this study, a Toll receptor of the silkworm Bombyx Toll9-2 (BmToll9-2) was cloned and its expression and function were analyzed. The results showed that BmToll9-2 contains an ectodomain (ECD) with a signal peptide and nine leucine-rich repeats, a transmembrane helix, and a cytoplasmic region with a Toll/interleukin-1 domain. Phylogenetic analysis indicates that BmToll9-2 clusters with other insect Toll9 receptors and mammalian Toll-like receptor 4. Oral infection of exogenous pathogens showed that the Gram-negative bacterium Escherichia coli and its main cell wall component lipopolysaccharide (LPS), as well as the Gram-positive bacterium Staphylococcus aureus and its main cell wall component peptidoglycan, significantly induce BmToll9-2 expression in vivo. LPS also induced the expression of BmToll9-2 in BmN4 cells in vitro. These observations indicate its role as a sensor in the innate immunity to exogenous pathogens and as a pathogen-associated receptor that is responsive to LPS. RNAi of BmToll9-2 was effective in the midgut and epidermis. RNAi-mediated knock-down of BmToll9-2 reduced the weight and growth of the silkworm. Bacterial challenge following RNAi upregulated the expression of BmToll9-2 and rescued the weight differences of the silkworm, which may be related to its participation in the immune response and the regulation of the microbiota in the midgut lumen of the silkworm larvae.
Assuntos
Bombyx , Escherichia coli , Proteínas de Insetos , Larva , Lipopolissacarídeos , Filogenia , Animais , Bombyx/imunologia , Bombyx/genética , Bombyx/crescimento & desenvolvimento , Bombyx/microbiologia , Bombyx/metabolismo , Larva/imunologia , Larva/crescimento & desenvolvimento , Larva/microbiologia , Larva/genética , Larva/metabolismo , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Lipopolissacarídeos/farmacologia , Receptores Toll-Like/metabolismo , Receptores Toll-Like/genética , Imunidade Inata , Staphylococcus aureus , Trato Gastrointestinal/imunologia , Trato Gastrointestinal/microbiologia , Trato Gastrointestinal/metabolismo , Sequência de Aminoácidos , Interferência de RNARESUMO
Epoxyoctadecamonoenoic acids (EpOMEs) are produced from linoleic acid by a cytochrome P450 monooxygenase (CYP) and play a crucial role in terminating excessive and unnecessary immune responses during the late infection stage in insects. This suggests that an increase in the EpOME level may enhance the virulence of insect pathogens against pests. This study tested this hypothesis using a specific inhibitor against soluble epoxide hydrolase (sEH) to degrade EpOMEs, which leads to elevated endogenous EpOME levels. A baculovirus, Autographa californica multiple nucleopolyhedrovirus (AcMNPV), was used to infect three different lepidopteran insects (Spodoptera exigua, Maruca vitrata, and Plutella xylostella) by oral feeding or hemocoelic injection treatments. Within one hour, the viral infection induced the expression of three different phospholipase A2 (PLA2) genes and, after 12 h, up-regulated the expressions of CYP and sEH genes in Spodopera exigua. As expected, AcMNPV virulence was suppressed by the addition of arachidonic acid (a catalytic product of PLA2) but was enhanced by the addition of either of the EpOME regioisomers. In addition, treatment with a specific sEH inhibitor (AUDA) increased AcMNPV virulence against three different lepidopteran insects, presumably by increasing endogenous EpOME levels. This enhanced effect of EpOMEs on virulence was further supported by specific RNA interference (RNAi), in which RNAi specific to CYP expression decreased AcMNPV virulence while a specific RNAi against sEH expression significantly enhanced virulence. In response to AcMNPV infection, TUNEL assay results showed that S. exigua larvae exhibited apoptosis in the midgut, fat body, and epidermis. Inhibition of apoptosis by a pan-caspase inhibitor, Z-VAD-FMK, significantly increased virulence. Similarly, the addition of AUDA to the viral treatment suppressed the gene expression of five inducible caspases and cytochrome C to suppress apoptosis, which led to a significant increase in the tissue viral titers. These results indicate that EpOMEs play a role in terminating excessive and unnecessary immune responses against viral infection during the late stage by down-regulating antiviral apoptosis in lepidopteran insects.
Assuntos
Mariposas , Nucleopoliedrovírus , Animais , Mariposas/virologia , Mariposas/imunologia , Virulência , Nucleopoliedrovírus/patogenicidade , Spodoptera/virologia , Spodoptera/imunologia , Larva/virologia , Larva/imunologiaRESUMO
C-type lectins (CTLs) play essential roles in humoral and cellular immune responses of invertebrates. Previous studies have demonstrated the involvement of CTLs in the humoral immunity of Tribolium castaneum, a worldwide pest in stored products. However, the function of CTLs in cellular immunity remains unclear. Here, we identified a CTL gene located on chromosome X and designated it as CTL2 (TcCTL2) from T. castaneum. It encodes a protein of 305 amino acids with a secretion signal peptide and a carbohydrate-recognition domain. TcCTL2 was mainly expressed in the early pupae and primarily distributed in the hemocytes in the late larvae. It was significantly upregulated after larvae were infected with Escherichia coli or Staphylococcus aureus, while knockdown of TcCTL2 exacerbates larval mortality and bacterial colonization after infection. The purified recombinant TcCTL2 (rTcCTL2) can bind to pathogen-associated molecular patterns and microbes and promote hemocyte-mediated encapsulation, melanization and phagocytosis in vitro. rTcCTL2 also induced bacterial agglutination in a Ca2+-dependent manner. Knockdown of TcCTL2 drastically suppressed encapsulation, melanization, and phagocytosis. Furthermore, silencing of TcCTL2 followed by bacterial infection significantly decreased the expression of transcription factors in Toll and IMD pathways, antimicrobial peptides, and prophenoloxidases and phenoloxidase activity. These results unveiled that TcCTL2 mediates both humoral and cellular immunity to promote bacterial clearance and protect T. castaneum from infectious microbes, which will deepen the understanding of the interaction between CTLs and innate immunity in T. castaneum and permit the optimization of pest control strategies by a combination of RNAi technology and bacterial infection.
Assuntos
Imunidade Celular , Imunidade Humoral , Proteínas de Insetos , Lectinas Tipo C , Staphylococcus aureus , Tribolium , Animais , Lectinas Tipo C/metabolismo , Lectinas Tipo C/genética , Staphylococcus aureus/imunologia , Tribolium/imunologia , Tribolium/genética , Proteínas de Insetos/metabolismo , Proteínas de Insetos/genética , Hemócitos/imunologia , Hemócitos/metabolismo , Escherichia coli , Fagocitose , Larva/imunologia , Larva/microbiologiaRESUMO
The improvement of the novel foods' safety assessment algorithms is currently one of the food hygiene significant areas. Within the studying of Hermetia illucens insects' effect, the standard in vivo allergological research integrated in the protocol of medical and biological evaluation of genetically modified food has been used. The protocol was supplemented with cytokine profile indicators and pathomorphologic characteristics of immunocompetent organs' lymphoid tissue. The purpose of the research was to study the effect of black soldier fly (Hermetia illucens) larvae biomass on the rats' immune status in the experiment on the induced anaphylactic shock model. Material and methods. The effect of black soldier fly (Hermetia illucens) larvae biomass was studied in a 29-day experiment on growing (43-72 days of life) male Wistar rats fed with Hermetia illucens biomass - main group (n=29) and semi-synthetic casein diet - control group (n=29). The complex assessment of allergenic potential of Hermetia illucens biomass was carried out in the experiment on the induced anaphylactic shock model in Wistar rats. An expanded pool of immune status indicators was studied including active anaphylactic shock severity (lethality, number of severe anaphylaxis reactions, anaphylactic index); cytokine profile (content of proinflammatory and anti-inflammatory cytokines, as well as regulators of cellular and humoral immune response); IgG1 and IgG4 level before and after administration of ovalbumin permissive dose (4 mg/kg b.w.). In addition to this pathomorphologic characteristics of lymphoid tissue of the main immunocompetent organs (thymus, spleen, Payer's patches) have been obtained. Results. The significant systemic anaphylaxis reaction decrease in the main group has been shown. Comparative assessment of the serum cytokines (GM-CSF, IFN-γ, IL-10, IL-12(p70), IL-13, IL-1α, IL-1ß, IL-2, IL-4, IL-5, IL-6, TNF-α) as well as the level of immunoglobulins of the IgG1, IgG4 class before and after administration of ovalbumin permissive dose did not reveal significant differences in rats of the control and main groups. In the main group, there was a decrease in blood serum proallergic cytokines: the level of IL-4 reduced by 1.3 fold, IL-10 - 1.1 and IL-13 - 1.2 fold (p>0.05), and in animals with mild anaphylactic reaction - by 1.8, 1.4 and 1.4 times, respectively (p>0.05). The morphologic studies of the immune system organs showed no intergroup differences. Conclusion. Thus, allergological studies of black soldier fly (Hermetia illucens) larvae in the experiment with the use of systemic anaphylaxis rat model and determination of immune status indicators (anaphylactic shock severity, cytokine profile, IgG1 and IgG4 level, morphologic structure of immunocompetent organs) did not reveal any allergenic effect of the studied product.
Assuntos
Anafilaxia , Citocinas , Larva , Ratos Wistar , Animais , Ratos , Masculino , Larva/imunologia , Anafilaxia/imunologia , Anafilaxia/induzido quimicamente , Citocinas/metabolismo , Citocinas/imunologia , Biomassa , Simuliidae/imunologia , Dípteros/imunologia , Dípteros/crescimento & desenvolvimentoRESUMO
At each molt of Manduca, the large dermal secretory cells expel the protein contents of their vacuoles into the hemocoel. The constellation of proteins expelled at the last larval-pupal molt, however, differs qualitatively from those proteins released at earlier larval-larval molts. Secretory cells at the two stages not only have different lectin staining properties but also have different proteins that separate on two-dimensional gels. Numerous physiological changes accompany the termination of the last larval instar, including increased chitin synthesis, diminished oxygen delivery, and reduced humoral immunity. Secretion of trehalase that is essential for chitin synthesis and the release of hypoxia up-regulated protein to ameliorate oxygen deprivation help ensure normal transition from larva to pupa. Proteins released by dermal secretory cells at this last molt could supplement the diminished immune defenses mediated by fat body and hemocytes at the end of larval life. Additional immune defenses provided by dermal secretory cells could help ensure a safe transition during a period of increased vulnerability for the newly molted pupa with its soft, thin cuticle and reduced mobility.
Assuntos
Células Epiteliais/metabolismo , Hemolinfa/metabolismo , Proteínas de Insetos/metabolismo , Larva/metabolismo , Manduca/metabolismo , Muda/imunologia , Pupa/metabolismo , Animais , Quitina/biossíntese , Epitélio/metabolismo , Hemócitos/metabolismo , Hemolinfa/imunologia , Imunidade Humoral , Larva/imunologia , Manduca/imunologia , Pupa/imunologia , Via Secretória/imunologia , Trealase/metabolismoRESUMO
The global amphibian declines are compounded by infections with members of the Ranavirus genus such as Frog Virus 3 (FV3). Premetamorphic anuran amphibians are believed to be significantly more susceptible to FV3 while this pathogen targets the kidneys of both pre- and postmetamorphic animals. Paradoxically, FV3-challenged Xenopus laevis tadpoles exhibit lower kidney viral loads than adult frogs. Presently, we demonstrate that X. laevis tadpoles are intrinsically more resistant to FV3 kidney infections than cohort-matched metamorphic and postmetamorphic froglets and that this resistance appears to be epigenetically conferred by endogenous retroviruses (ERVs). Using a X. laevis kidney-derived cell line, we show that enhancing ERV gene expression activates cellular double-stranded RNA-sensing pathways, resulting in elevated mRNA levels of antiviral interferon (IFN) cytokines and thus greater anti-FV3 protection. Finally, our results indicate that large esterase-positive myeloid-lineage cells, rather than renal cells, are responsible for the elevated ERV/IFN axis seen in the tadpole kidneys. This conclusion is supported by our observation that CRISPR-Cas9 ablation of colony-stimulating factor-3 results in abolished homing of these myeloid cells to tadpole kidneys, concurrent with significantly abolished tadpole kidney expression of both ERVs and IFNs. We believe that the manuscript marks an important step forward in understanding the mechanisms controlling amphibian antiviral defenses and thus susceptibility and resistance to pathogens like FV3. IMPORTANCE Global amphibian biodiversity is being challenged by pathogens like the Frog Virus 3 (FV3) ranavirus, underlining the need to gain a greater understanding of amphibian antiviral defenses. While it was previously believed that anuran (frog/toad) amphibian tadpoles are more susceptible to FV3, we demonstrated that tadpoles are in fact more resistant to this virus than metamorphic and postmetamorphic froglets. We showed that this resistance is conferred by large myeloid cells within the tadpole kidneys (central FV3 target), which possess an elevated expression of endogenous retroviruses (ERVs). In turn, these ERVs activate cellular double-stranded RNA-sensing pathways, resulting in a greater expression of antiviral interferon cytokines, thereby offering the observed anti-FV3 protection.
Assuntos
Infecções por Vírus de DNA , Retrovirus Endógenos , Ranavirus , Xenopus laevis , Animais , Linhagem Celular , Infecções por Vírus de DNA/imunologia , Infecções por Vírus de DNA/veterinária , Resistência à Doença , Retrovirus Endógenos/imunologia , Interferons/imunologia , Rim/virologia , Larva/imunologia , Larva/virologia , RNA de Cadeia Dupla , Ranavirus/patogenicidade , Xenopus laevis/virologiaRESUMO
During infection, cellular resources are allocated toward the metabolically-demanding processes of synthesizing and secreting effector proteins that neutralize and kill invading pathogens. In Drosophila, these effectors are antimicrobial peptides (AMPs) that are produced in the fat body, an organ that also serves as a major lipid storage depot. Here we asked how activation of Toll signaling in the larval fat body perturbs lipid homeostasis to understand how cells meet the metabolic demands of the immune response. We find that genetic or physiological activation of fat body Toll signaling leads to a tissue-autonomous reduction in triglyceride storage that is paralleled by decreased transcript levels of the DGAT homolog midway, which carries out the final step of triglyceride synthesis. In contrast, Kennedy pathway enzymes that synthesize membrane phospholipids are induced. Mass spectrometry analysis revealed elevated levels of major phosphatidylcholine and phosphatidylethanolamine species in fat bodies with active Toll signaling. The ER stress mediator Xbp1 contributed to the Toll-dependent induction of Kennedy pathway enzymes, which was blunted by deleting AMP genes, thereby reducing secretory demand elicited by Toll activation. Consistent with ER stress induction, ER volume is expanded in fat body cells with active Toll signaling, as determined by transmission electron microscopy. A major functional consequence of reduced Kennedy pathway induction is an impaired immune response to bacterial infection. Our results establish that Toll signaling induces a shift in anabolic lipid metabolism to favor phospholipid synthesis and ER expansion that may serve the immediate demand for AMP synthesis and secretion but with the long-term consequence of insufficient nutrient storage.