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1.
J Virol ; 96(11): e0063422, 2022 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-35575553

RESUMO

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/virologia
2.
PLoS One ; 17(2): e0263620, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35134064

RESUMO

Insect immune responses to multiple pathogen groups including viruses, bacteria, fungi, and entomopathogenic nematodes have traditionally been documented in model insects such as Drosophila melanogaster, or medically important insects such as Aedes aegypti. Despite their potential importance in understanding the efficacy of pathogens as biological control agents, these responses are infrequently studied in agriculturally important pests. Additionally, studies that investigate responses of a host species to different pathogen groups are uncommon, and typically focus on only a single time point during infection. As such, a robust understanding of immune system responses over the time of infection is often lacking in many pest species. This study was conducted to understand how 3rd instar larvae of the major insect pest Helicoverpa zea responded through the course of an infection by four different pathogenic groups: viruses, bacteria, fungi, and entomopathogenic nematodes; by sampling at three different times post-inoculation. Physiological immune responses were assessed at 4-, 24-, and 48-hours post-infection by measuring hemolymph phenoloxidase concentrations, hemolymph prophenoloxidase concentrations, hemocyte counts, and encapsulation ability. Transcriptional immune responses were measured at 24-, 48-, and 72-hours post-infection by quantifying the expression of PPO2, Argonaute-2, JNK, Dorsal, and Relish. This gene set covers the major known immune pathways: phenoloxidase cascade, siRNA, JNK pathway, Toll pathway, and IMD pathway. Our results indicate H. zea has an extreme immune response to Bacillus thuringiensis bacteria, a mild response to Helicoverpa armigera nucleopolyhedrovirus, and little-to-no detectable response to either the fungus Beauveria bassiana or Steinernema carpocapsae nematodes.


Assuntos
Mariposas/genética , Mariposas/microbiologia , Controle Biológico de Vetores/métodos , Animais , Bacillus thuringiensis/patogenicidade , Agentes de Controle Biológico , Hemócitos/metabolismo , Hemolinfa/metabolismo , Imunidade , Proteínas de Insetos/genética , Larva/imunologia , Larva/metabolismo , Lepidópteros/genética , Lepidópteros/imunologia , Mariposas/imunologia , Nucleopoliedrovírus/patogenicidade , Controle de Pragas/métodos
3.
PLoS One ; 17(1): e0259386, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-34982789

RESUMO

Bacillus cereus is a spore forming bacteria recognized among the leading agents responsible for foodborne outbreaks in Europe. B. cereus is also gaining notoriety as an opportunistic human pathogen inducing local and systemic infections. The real incidence of such infection is likely underestimated and information on genetic and phenotypic characteristics of the incriminated strains is generally scarce. We have recently analyzed a large strain collection of varying pathogenic potential. Screening for biomarkers to differentiate among clinical and non-clinical strains, a gene encoding an alcohol dehydrogenase-like protein was identified among the leading candidates. This family of proteins has been demonstrated to be involved in the virulence of several bacterial species. The relevant gene was knocked out to elucidate its function with regards to resistance to host innate immune response, both in vitro and in vivo. Our results demonstrate that the adhB gene plays a significant role in resistance to nitric oxide and oxidative stress in vitro, as well as its pathogenic ability with regards to in vivo toxicity. These properties may explain the pathogenic potential of strains carrying this newly identified virulence factor.


Assuntos
Álcool Desidrogenase/metabolismo , Bacillus cereus/patogenicidade , Proteínas de Bactérias/metabolismo , Biomarcadores/metabolismo , Imunidade Inata/fisiologia , Virulência/genética , Álcool Desidrogenase/genética , Animais , Bacillus cereus/crescimento & desenvolvimento , Proteínas de Bactérias/genética , Peróxido de Hidrogênio/farmacologia , Insetos/crescimento & desenvolvimento , Insetos/microbiologia , Larva/imunologia , Larva/microbiologia , Mutação , Óxido Nítrico/farmacologia , Estresse Oxidativo/efeitos dos fármacos
4.
Dev Biol ; 483: 107-111, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-35007518

RESUMO

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/metabolismo
5.
Front Immunol ; 12: 745160, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34867970

RESUMO

In insects, a complex and effective immune system that can be rapidly activated by a plethora of stimuli has evolved. Although the main cellular and humoral mechanisms and their activation pathways are highly conserved across insects, the timing and the efficacy of triggered immune responses can differ among different species. In this scenario, an insect deserving particular attention is the black soldier fly (BSF), Hermetia illucens (Diptera: Stratiomyidae). Indeed, BSF larvae can be reared on a wide range of decaying organic substrates and, thanks to their high protein and lipid content, they represent a valuable source of macromolecules useful for different applications (e.g., production of feedstuff, bioplastics, and biodiesel), thus contributing to the development of circular economy supply chains for waste valorization. However, decaying substrates bring the larvae into contact with different potential pathogens that can challenge their health status and growth. Although these life strategies have presumably contributed to shape the evolution of a sophisticated and efficient immune system in this dipteran, knowledge about its functional features is still fragmentary. In the present study, we investigated the processes underpinning the immune response to bacteria in H. illucens larvae and characterized their reaction times. Our data demonstrate that the cellular and humoral responses in this insect show different kinetics: phagocytosis and encapsulation are rapidly triggered after the immune challenge, while the humoral components intervene later. Moreover, although both Gram-positive and Gram-negative bacteria are completely removed from the insect body within a few hours after injection, Gram-positive bacteria persist in the hemolymph longer than do Gram-negative bacteria. Finally, the activity of two key actors of the humoral response, i.e., lysozyme and phenoloxidase, show unusual dynamics as compared to other insects. This study represents the first detailed characterization of the immune response to bacteria of H. illucens larvae, expanding knowledge on the defense mechanisms of this insect among Diptera. This information is a prerequisite to manipulating the larval immune response by nutritional and environmental factors to increase resistance to pathogens and optimize health status during mass rearing.


Assuntos
Imunidade/imunologia , Larva/imunologia , Larva/microbiologia , Simuliidae/imunologia , Simuliidae/microbiologia , Animais , Bactérias/imunologia
6.
Front Immunol ; 12: 771277, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34868031

RESUMO

Histone H2A is a nuclear molecule tightly associated in the form of the nucleosome. Our previous studies have demonstrated the antibacterial property of piscine H2A variants against gram-negative bacteria Edwardsiella piscicida and Gram-positive bacteria Streptococcus agalactiae. In this study, we show the function and mechanism of piscine H2A in the negative regulation of RLR signaling pathway and host innate immune response against spring viremia of carp virus (SVCV) infection. SVCV infection significantly inhibits the expression of histone H2A during an early stage of infection, but induces the expression of histone H2A during the late stage of infection such as at 48 and 72 hpi. Under normal physiological conditions, histone H2A is nuclear-localized. However, SVCV infection promotes the migration of histone H2A from the nucleus to the cytoplasm. The in vivo studies revealed that histone H2A overexpression led to the increased expression of SVCV gene and decreased survival rate. The overexpression of histone H2A also significantly impaired the expression levels of those genes involved in RLR antiviral signaling pathway. Furthermore, histone H2A targeted TBK1 and IRF3 to promote their protein degradation via the lysosomal pathway and impair the formation of TBK1-IRF3 functional complex. Importantly, histone H2A completely abolished TBK1-mediated antiviral activity and enormously impaired the protein expression of IRF3, especially nuclear IRF3. Further analysis demonstrated that the inhibition of histone H2A nuclear/cytoplasmic trafficking could relieve the protein degradation of TBK1 and IRF3, and blocked the negative regulation of histone H2A on the SVCV infection. Collectively, our results suggest that histone H2A nuclear/cytoplasmic trafficking is essential for negative regulation of RLR signaling pathway and antiviral immune response in response to SVCV infection.


Assuntos
Histonas/imunologia , Imunidade Inata/imunologia , Fator Regulador 3 de Interferon/imunologia , Lisossomos/imunologia , Rhabdoviridae/imunologia , Proteínas de Peixe-Zebra/imunologia , Peixe-Zebra/imunologia , Animais , Linhagem Celular , Núcleo Celular/imunologia , Núcleo Celular/metabolismo , Citoplasma/imunologia , Citoplasma/metabolismo , Regulação da Expressão Gênica/imunologia , Histonas/genética , Histonas/metabolismo , Interações Hospedeiro-Patógeno/imunologia , Fator Regulador 3 de Interferon/genética , Fator Regulador 3 de Interferon/metabolismo , Larva/imunologia , Larva/metabolismo , Larva/virologia , Lisossomos/metabolismo , /metabolismo , Transporte Proteico/imunologia , Proteólise , Rhabdoviridae/fisiologia , Peixe-Zebra/metabolismo , Peixe-Zebra/virologia , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
7.
J Insect Sci ; 21(6)2021 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-34865031

RESUMO

The innate immunity of insects has been widely studied. Although the effect of sex on insect immunity has been extensively discussed, differences in immunity between the sexes of larvae insects remain largely unstudied. Studying larval sex differences in immunity may provide valuable information about the mechanisms underlying the insect immune system, which, in turn, can be valuable for the development and improvement of pest management. Here we compared the antibacterial activity in both the midgut tissue and cell-free hemolymph of Lymantria dispar L. (Lepidoptera: Erebidae) females and males at the larval stage without and after a challenge by entomopathogenic bacterium Bacillus thuringiensis Berliner. We also evaluated the sex-specific mortality of L. dispar induced by B. thuringiensis infection. We find that antibacterial activity in the midgut is activated by infection, but only in females. Thus, sex differences in immunity can have important effects even before sexual differentiation at adulthood.


Assuntos
Bacillus thuringiensis , Imunidade Inata , Larva/imunologia , Mariposas , Caracteres Sexuais , Animais , Bacillus thuringiensis/patogenicidade , Infecções Bacterianas/imunologia , Infecções Bacterianas/veterinária , Feminino , Masculino , Mariposas/imunologia
8.
J Therm Biol ; 101: 103082, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34879910

RESUMO

Changes in temperature resulting from climate change can impact the distribution and survival of species, including bees, where temperature may also affect their immune system. Evaluation of immune system activity is often performed by the total count of circulating hemocytes in the hemolymph. However, there are few studies on bees examining the relationship between the amount of circulating hemocytes and temperature. This study evaluated changes of circulating hemocytes in Apis mellifera hemolymph at different temperatures and development stages. Total hemocytes of bees were determined at - 8, 16, 24, and 32 °C - and at different development stages - in vivo larvae, in vitro larvae, newly emerged, and forager bees. A. mellifera larvae had a greater number of circulating hemocytes compared to the other development stages (newly emerged and foragers). Additionally, temperature was an important factor explaining variation of circulating hemocytes in the hemolymph, according to principal component analyses (PCA), as the number of circulating hemocytes was greater at higher temperatures. Therefore, extreme events arising from climate change, such as variation in temperature, can directly impact the immune system of bees, both individually and at the colony level, threatening the distribution and survival of several species.


Assuntos
Abelhas/imunologia , Temperatura , Animais , Hemócitos/imunologia , Hemolinfa/imunologia , Larva/imunologia
9.
Int J Mol Sci ; 22(19)2021 Oct 08.
Artigo em Inglês | MEDLINE | ID: mdl-34639230

RESUMO

The cystine knot protein Spätzle is a Toll receptor ligand that modulates the intracellular signaling cascade involved in the nuclear factor kappa B (NF-κB)-mediated regulation of antimicrobial peptide (AMP)-encoding genes. Spätzle-mediated activation of the Toll pathway is critical for the innate immune responses of insects against Gram-positive bacteria and fungi. In this study, the open reading frame (ORF) sequence of Spätzle-like from T. molitor (TmSpz-like) identified from the RNA sequencing dataset was cloned and sequenced. The 885-bp TmSpz-like ORF encoded a polypeptide of 294 amino acid residues. TmSpz-like comprised a cystine knot domain with six conserved cysteine residues that formed three disulfide bonds. Additionally, TmSpz-like exhibited the highest amino acid sequence similarity with T. castaneum Spätzle (TcSpz). In the phylogenetic tree, TmSpz-like and TcSpz were located within a single cluster. The expression of TmSpz-like was upregulated in the Malpighian tubules and gut tissues of T. molitor. Additionally, the expression of TmSpz-like in the whole body and gut of the larvae was upregulated at 24 h post-E. coli infection. The results of RNA interference experiments revealed that TmSpz-like is critical for the viability of E. coli-infected T. molitor larvae. Eleven AMP-encoding genes were downregulated in the E. coli-infected TmSpz-like knockdown larvae, which suggested that TmSpz-like positively regulated these genes. Additionally, the NF-κB-encoding genes (TmDorX1, TmDorX2, and TmRelish) were downregulated in the E. coli-infected TmSpz-like knockdown larvae. Thus, TmSpz-like plays a critical role in the regulation of AMP production in T. molitor in response to E. coli infection.


Assuntos
Peptídeos Catiônicos Antimicrobianos/metabolismo , Infecções por Escherichia coli/microbiologia , Escherichia coli/imunologia , Imunidade Inata/imunologia , Proteínas de Insetos/metabolismo , Staphylococcus aureus/imunologia , Tenebrio/imunologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Infecções por Escherichia coli/imunologia , Infecções por Escherichia coli/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Insetos/genética , Larva/genética , Larva/imunologia , Larva/metabolismo , Larva/microbiologia , Filogenia , Homologia de Sequência de Aminoácidos , Infecções Estafilocócicas , Tenebrio/genética , Tenebrio/metabolismo , Tenebrio/microbiologia
10.
Front Immunol ; 12: 715766, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34475874

RESUMO

Parasitic nematodes such as hookworms actively penetrate the skin of their hosts, encountering skin-resident innate immune cells that represent the host´s first line of defense. Here we use Strongyloides ratti as a model for an intestinal helminth parasite with tissue migrating stages. We show that interception and killing of migrating larvae in mice during a 1st infection occurred predominantly in skin and muscle tissue before larvae migrated via lung and head tissue to the intestine. Inhibition of larval migration was even more efficient in immune mice during a 2nd infection where larvae barely left the site of entry i.e. the foot. Using cell-deficient mice we show that interception in the tissue was predominantly mediated by neutrophils and eosinophils while basophils and mast cells were dispensable in vivo. Likewise, neutrophils and eosinophils inhibited S. ratti L3 motility in vitro in the context of ETosis. Thereby eosinophils were strictly dependent on the presence of anti-S. ratti antibodies while neutrophils inhibited L3 motility as such. Also, MPO and MMP-9 were released by neutrophils in response to L3 alone, but immune plasma further stimulated MPO release in an antibody-dependent manner. In summary, our findings highlight the central role of the skin as first line of defense against helminth parasites in both, innate and adaptive immunity.


Assuntos
Eosinófilos/imunologia , Armadilhas Extracelulares/imunologia , Interações Hospedeiro-Parasita/imunologia , Neutrófilos/imunologia , Strongyloides ratti/imunologia , Estrongiloidíase/imunologia , Estrongiloidíase/parasitologia , Animais , Degranulação Celular/imunologia , Citotoxicidade Imunológica , Modelos Animais de Doenças , Suscetibilidade a Doenças , Armadilhas Extracelulares/parasitologia , Imunidade Inata , Larva/imunologia , Camundongos , Estrongiloidíase/metabolismo
11.
Front Immunol ; 12: 737403, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34489981

RESUMO

The global amphibian declines are compounded by ranavirus infections such as Frog Virus 3 (FV3), and amphibian tadpoles more frequently succumb to these pathogens than adult animals. Amphibian gastrointestinal tracts represent a major route of ranavirus entry, and viral pathogenesis often leads to hemorrhaging and necrosis within this tissue. Alas, the differences between tadpole and adult amphibian immune responses to intestinal ranavirus infections remain poorly defined. As interferon (IFN) cytokine responses represent a cornerstone of vertebrate antiviral immunity, it is pertinent that the tadpoles and adults of the anuran Xenopus laevis frog mount disparate IFN responses to FV3 infections. Presently, we compared the tadpole and adult X. laevis responses to intestinal FV3 infections. Our results indicate that FV3-challenged tadpoles mount more robust intestinal type I and III IFN responses than adult frogs. These tadpole antiviral responses appear to be mediated by myeloid cells, which are recruited into tadpole intestines in response to FV3 infections. Conversely, myeloid cells bearing similar cytology already reside within the intestines of healthy (uninfected) adult frogs, possibly accounting for some of the anti-FV3 resistance of these animals. Further insight into the differences between tadpole and adult frog responses to ranaviral infections is critical to understanding the facets of susceptibility and resistance to these pathogens.


Assuntos
Proteínas de Anfíbios/metabolismo , Infecções por Vírus de DNA/virologia , Interferons/metabolismo , Intestinos/virologia , Células Mieloides/virologia , Ranavirus/patogenicidade , Xenopus laevis/virologia , Fatores Etários , Animais , Infecções por Vírus de DNA/imunologia , Infecções por Vírus de DNA/metabolismo , Suscetibilidade a Doenças , Feminino , Interações Hospedeiro-Patógeno , Intestinos/embriologia , Intestinos/imunologia , Larva/imunologia , Larva/metabolismo , Larva/virologia , Masculino , Células Mieloides/imunologia , Células Mieloides/metabolismo , Ranavirus/imunologia , Carga Viral , Xenopus laevis/embriologia , Xenopus laevis/imunologia , Xenopus laevis/metabolismo
12.
Trop Med Int Health ; 26(12): 1634-1644, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34510662

RESUMO

OBJECTIVES: The aims of the study were two-fold: (1) antigen (Ag) preparation and evaluation of three antigens of Gnathostoma spinigerum infective larvae (GsL3), crude somatic antigen (CSAg), excretory-secretory antigen (ESAg) and partially purified antigens (namely P1Ag, P2Ag and P3Ag) to differentiate IgE, IgG, IgG1-4 and IgM for human gnathostomiasis diagnosis; and (2) application of the selected ELISA for following up stored sera of patients treated with ivermectin (IVM) and albendazole (ABZ). METHODS: Different antigens were analysed by antibodies of gnathostomiasis cases, other parasite infections and healthy controls using indirect ELISA to differentiate IgE, IgG, IgG1-4 and IgM. Then, prominent antigen and immunoglobulin were used in antibody predictions of gnathostomiasis cases treated with albendazole or ivermectin. RESULTS: Sensitivity of all evaluated ELISAs: IgM-, IgG-, IgG1- and IgG4-ELISA, was 100%. IgM-ELISA with CSAg and P3Ag exhibited the highest specificity of 99%. IgG-ELISA with P2Ag resulted in the highest specificity of 92.3%. IgG1-ELISA with P2Ag and P3Ag showed excellent results with 100% specificity. Finally, P2Ag evaluated IgG1 of the followed-up cases with ABZ and IVM. Decreasing antibody IgG1 levels were mostly found in both treatments at Month 9 and long follow-up was over 12 months. A Gnathostoma worm was extracted from each two treated patients. CONCLUSIONS: Using IgG1-ELISA against P2Ag and P3Ag gave excellent results with 100% sensitivity and specificity. These tests can be an alternative to immunoblotting for gnathostomiasis. IgG1 decreased at least 9 months in most cases, so long-term treatment should be performed over 1 year.


Assuntos
Antígenos de Helmintos/imunologia , Gnathostoma/imunologia , Gnatostomíase/sangue , Gnatostomíase/diagnóstico , Testes Imunológicos/métodos , Albendazol/uso terapêutico , Animais , Anticorpos Anti-Helmínticos/sangue , Anticorpos Anti-Helmínticos/imunologia , Antiparasitários/uso terapêutico , Gnatostomíase/tratamento farmacológico , Gnatostomíase/imunologia , Humanos , Imunoglobulina E/sangue , Imunoglobulina E/imunologia , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Imunoglobulina M/sangue , Imunoglobulina M/imunologia , Ivermectina/uso terapêutico , Larva/imunologia , Sensibilidade e Especificidade
13.
Int Immunopharmacol ; 100: 108120, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34537480

RESUMO

AIMS: The objective of this investigation is to evaluate the mechanisms Anisakis simplex employs to modify its host immune system, regarding the larval antigens interactions with Toll-Like-Receptors (TLRs). METHODS AND RESULTS: In a previous study, we described that the stimulation of bone marrow derived dendritic cells (BMDCs) with A. simplex larval antigens drive an acute inflammatory response in BALB/c mice, but a more discrete and longer response in C57BL/6J. Moreover, when A. simplex larval antigens were combined with TLR agonists (TLR 1/2-9), they modified mainly TLR2, TLR4 and TLR9 agonists responses in both mice strains, and also TLR3, TLR5 and TLR7 in BALB/c. Antigen-presenting ability was analyzed by the detection of CD11c + cells expressing surface markers (CD80-86, MHC I-II), intracellular cytokines (IL-10, IL-12, TNF-α) and intracellular proteins (Myd88, NF-κß) by Flow Cytometry. Secreted IL-10 was measured by ELISA. CONCLUSION: Our findings confirm not only that the host genetic basis plays a role in the development of a Th2/Th1/Treg response, but also it states A. simplex larval antigens present specific mechanisms to modify the innate response of the host. As allergies share common pathways with the immune response against this particular helminth, our results provide a better understanding into the specific mechanisms of A. simplex allergy related diseases.


Assuntos
Anisakis/imunologia , Antígenos/imunologia , Imunomodulação , Larva/imunologia , Receptores Toll-Like/imunologia , Animais , Feminino , Antígenos H-2 , Interleucina-10/metabolismo , Interleucina-12/metabolismo , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Receptor 2 Toll-Like , Receptores Toll-Like/agonistas
14.
Front Immunol ; 12: 677994, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34557186

RESUMO

Neutrophils are key players in innate immunity and originate from the bone marrow of the adult mammalian organism. In mammals, mature neutrophils are released from the bone marrow into the peripheral blood where they circulate until their recruitment to sites of inflammation in a multistep adhesion cascade. Here, adhesion molecules of the ß2 integrin family (CD11/CD18) are critically required for the initial neutrophil adhesion to the inflamed endothelium and several post-adhesion steps allowing their extravasation into the inflamed tissue. Within the mammalian tissue, interstitial neutrophil migration can occur widely independent of ß2 integrins. This is in sharp contrast to neutrophil recruitment in zebrafish larvae (Danio rerio) where neutrophils originate from the caudal hematopoietic tissue and mainly migrate interstitially to sites of lesion upon the early onset of inflammation. However, neutrophils extravasate from the circulation to the inflamed tissue in zebrafish larvae at later-time points. Although zebrafish larvae are a widely accepted model system to analyze neutrophil trafficking in vivo, the functional impact of ß2 integrins for neutrophil trafficking during acute inflammation is completely unknown in this model. In this study, we generated zebrafish with a genetic deletion of CD18, the ß subunit of ß2 integrins, using CRISPR/Cas9 technology. Sequence alignments demonstrated a high similarity of the amino acid sequences between zebrafish and human CD18 especially in the functionally relevant I-like domain. In addition, the cytoplasmic domain of CD18 harbors two highly conserved NXXF motifs suggesting that zebrafish CD18 may share functional properties of human CD18. Accordingly, CD18 knock-out (KO) zebrafish larvae displayed the key symptoms of patients suffering from leukocyte adhesion deficiency (LAD) type I due to defects in ITGB2, the gene for CD18. Importantly, CD18 KO zebrafish larvae showed reduced neutrophil trafficking to sites of sterile inflammation despite the fact that an increased number of neutrophils was detectable in the circulation. By demonstrating the functional importance of CD18 for neutrophil trafficking in zebrafish larvae, our findings shed new light on neutrophil biology in vertebrates and introduce a new model organism for studying LAD type I.


Assuntos
Antígenos CD18/metabolismo , Adesão Celular/genética , Movimento Celular/genética , Infiltração de Neutrófilos/genética , Neutrófilos/imunologia , Peixe-Zebra/genética , Peixe-Zebra/imunologia , Sequência de Aminoácidos , Animais , Animais Geneticamente Modificados , Antígenos CD11/química , Antígenos CD11/genética , Antígenos CD11/metabolismo , Antígenos CD18/química , Antígenos CD18/genética , Adesão Celular/imunologia , Movimento Celular/imunologia , Modelos Animais de Doenças , Deleção de Genes , Técnicas de Inativação de Genes , Inflamação/genética , Inflamação/imunologia , Integrinas/metabolismo , Larva/genética , Larva/imunologia , Síndrome da Aderência Leucocítica Deficitária/imunologia , Infiltração de Neutrófilos/imunologia
15.
PLoS One ; 16(9): e0257792, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34559852

RESUMO

Spray-dried animal plasma (SDP) in feed for several animal species provides health benefits, but research about use of SDP in shrimp feed is very limited. The objectives of the present study were to investigate the effects of dietary SDP on growth performance, feed utilization, immune responses, and prevention of Vibrio parahaemolyticus infection in Pacific white shrimp (Litopenaeus vannamei). In Experiment 1, the post-larvae were divided into five groups (four tank/group and 80 shrimp/tank) and fed four times daily diets with porcine SDP at 0, 1.5, 3, 4.5, and 6% of the diet for 45 days. In Experiment 2, the surviving shrimp from Experiment 1 were redistributed into six groups: four SDP groups as in Experiment 1 plus the positive and negative controls (four tank/group and 30 shrimp/tank). They were then challenged with V. parahaemolyticus by immersion at 105 colony-forming units (CFU)/mL and were fed with the same diets for another 4 days. In Experiment 1, shrimp fed 4.5% or 6% SDP diets had significantly higher body weight, survival rate, and improved feed conversion ratio. The immune parameters (total hemocyte count and phagocytic, phenoloxidase, and superoxide dismutase activities) of the shrimp fed 3-6% SDP diets also showed significant enhancement compared to the control. In Experiment 2, the survival rates of the 3-6% SDP groups were significantly higher than the positive control at day 4 after the immersion challenge. Likewise, the histopathological study revealed milder signs of bacterial infection in the hepatopancreas of the 3-6% SDP groups compared to the challenged positive control and 1.5% SDP groups. In conclusion, shrimp fed diets with SDP, especially at 4.5-6% of the diet, showed significant improvement in overall health conditions and better resistance to V. parahaemolyticus infection.


Assuntos
Suplementos Nutricionais/análise , Resistência à Doença , Penaeidae/crescimento & desenvolvimento , Plasma/química , Vibrio parahaemolyticus/imunologia , Ração Animal/análise , Animais , Peso Corporal , Hemócitos/metabolismo , Imunidade Inata , Larva/crescimento & desenvolvimento , Larva/imunologia , Larva/virologia , Penaeidae/imunologia , Penaeidae/virologia , Fagócitos/metabolismo , Secagem por Atomização , Suínos
16.
Int J Biol Macromol ; 188: 32-42, 2021 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-34352318

RESUMO

Akirins, highly conserved nuclear factors, regulate diverse physiological processes such as innate immunity. The biological functions of Akirins have extensively been studied in vertebrates and many invertebrates; however, there is no report so far on lepidopteran insects. In the present study, we identified and characterized a novel Akirin from the silkworm, Bombyx mori (designated as BmAkirin), and explored its potential roles in innate immunity. The expression analysis revealed the unequal mRNA levels of BmAkirin in all the tested tissues; however, the gene's transcription level was highest in testis, followed by ovaries and hemocytes. It also had significant expression levels at the early stages of embryonic development. Expression of BmAkirin in fat bodies and hemocytes exhibited an increase in various degrees when challenged with virus, fungus, Gram-negative bacteria, and Gram-positive bacteria. Immunofluorescence analysis showed BmAkirin protein was prominently localized in the nucleus. Knockdown of BmAkirin strongly reduced the expression of AMPs and decreased the survival ability of larva upon immune stimulation. Moreover, the bacterial clearance ability of larvae was also decreased following the depletion of BmAkirin. Collectively, our results demonstrate that BmAkirin plays an indispensable role in the innate immunity of Bombyx mori (B. mori) by positively modulating AMPs expression in vivo.


Assuntos
Bombyx/genética , Imunidade Inata/genética , Proteínas de Insetos/genética , Sequência de Aminoácidos/genética , Animais , Bombyx/imunologia , Bombyx/microbiologia , Clonagem Molecular , Ecdisterona/imunologia , Regulação da Expressão Gênica/imunologia , Hemócitos/imunologia , Hemócitos/microbiologia , Proteínas de Insetos/imunologia , Larva/genética , Larva/imunologia , Larva/microbiologia , RNA Mensageiro/genética
17.
Fish Shellfish Immunol ; 117: 113-123, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34333127

RESUMO

Protection via of the immune system is indispensable to the life of organisms. Within an immune network, problems with a given link will affect the normal life activities of the organism. Octopus ocellatus is cephalopod widely distributed throughout the world's oceans. Because of its unique nervous system and locomotive organs, research on this species has gradually increased in recent years. Many immune response mechanisms associated with behaviors of O. ocellatus are still unclear. Moreover, as a factor affecting the normal growth of O. ocellatus, egg protection has rarely been considered in previous behavioral studies. In this study, we analyzed the transcriptome profile of gene expression in O. ocellatus larvae, and identified 5936 differentially expressed genes (DEGs). GO and KEGG enrichment analyses were used to search for immune-related DEGs. Protein-protein interaction networks were constructed to examine the interactions between immune-related genes. Fifteen hub genes involved in multiple KEGG signaling pathways or with multiple protein-protein interaction relationships were obtained and verified by quantitative RT-PCR. We first studied the effects of egg protection on the immunity of O. ocellatus larvae by means of protein-protein interaction networks, and the results provide valuable genetic resources for understanding the immunity of invertebrate larvae. The data serve as a foundation for further research on the egg-protecting behavior of invertebrates.


Assuntos
Comportamento Animal , Octopodiformes/genética , Octopodiformes/imunologia , Óvulo , Animais , Feminino , Perfilação da Expressão Gênica , Larva/genética , Larva/imunologia , Mapas de Interação de Proteínas , Transcriptoma
18.
Sci Rep ; 11(1): 13881, 2021 07 06.
Artigo em Inglês | MEDLINE | ID: mdl-34230553

RESUMO

With the rapid growth in the global demand, the shrimp industry needs integrated approaches for sustainable production. A high-quality shrimp larva is one of the crucial key requirements to maximize shrimp production. Survival and growth rates during larval development are often criteria to evaluate larval quality, however many aspects of gene regulation during shrimp larval development have not yet been identified. To further our understanding of biological processes in their early life, transcriptomic analysis of larval developmental stages (nauplius, zoea, mysis, and postlarva) were determined in the black tiger shrimp, Penaeus monodon using next-generation RNA sequencing. Gene clustering and gene enrichment analyses revealed that most of the transcripts were mainly related to metabolic processes, cell and growth development, and immune system. Interestingly, Spätzle and Toll receptors were found in nauplius stage, providing evidence that Toll pathway was a baseline immune system established in early larval stages. Genes encoding pathogen pattern-recognition proteins (LGBP, PL5-2 and c-type lectin), prophenoloxidase system (PPAE2, PPAF2 and serpin), antimicrobial peptides (crustin and antiviral protein), blood clotting system (hemolymph clottable protein) and heat shock protein (HSP70) were expressed as they developed further, suggesting that these immune defense mechanisms were established in later larval stages.


Assuntos
Perfilação da Expressão Gênica , Estágios do Ciclo de Vida/genética , Estágios do Ciclo de Vida/imunologia , Penaeidae/genética , Penaeidae/imunologia , Animais , Regulação da Expressão Gênica no Desenvolvimento , Larva/genética , Larva/imunologia , Redes e Vias Metabólicas/genética , Modelos Biológicos , Anotação de Sequência Molecular , Penaeidae/crescimento & desenvolvimento , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reprodutibilidade dos Testes
19.
J Insect Sci ; 21(4)2021 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-34314494

RESUMO

The use of Galleria mellonella (Linnaeus) (Lepidoptera: Pyralidae), an economical insect model, for the study of enteropathogenic Escherichia coli (Migula) (EPEC), a diarrheagenic human pathogen, has been demonstrated previously but remains poorly understood. The present study characterizes the Galleria-EPEC system extensively for future studies using this system. We found that EPEC causes disease in G. mellonella larvae when injected intrahemocoelically but not orally. Disease manifests as increased mortality, decreased survival time, delayed pupation, decreased pupal mass, increased pupal duration, and hemocytopenia. Disease symptoms are dose-dependent and can be used as metrics for measuring EPEC virulence in future studies. The type III secretion system was only partially responsible for EPEC virulence in G. mellonella while the majority of the virulence remains unknown in origin. EPEC elicits insect anti-bacterial immune responses including melanization, hemolymph coagulation, nodulation, and phagocytosis. The immune responses were unable to control EPEC replication in the early stage of infection (≤3 h post-injection). EPEC clearance from the hemocoel does not guarantee insect survival. Overall, this study provided insights into EPEC virulence and pathogenesis in G. mellonella and identified areas of future research using this system.


Assuntos
Modelos Animais de Doenças , Escherichia coli Enteropatogênica/patogenicidade , Infecções por Escherichia coli/imunologia , Interações Hospedeiro-Patógeno/imunologia , Mariposas/imunologia , Animais , Infecções por Escherichia coli/mortalidade , Feminino , Larva/imunologia , Masculino
20.
J Invertebr Pathol ; 184: 107647, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34303711

RESUMO

Insect Apolipophorin-III is a multifunctional protein and also plays an important role in insect innate immunity. Early transcriptome and proteome studies indicated that the gene expression level of Bombyx mori Apolipophorin-III (BmApoLp-III) in silkworm larvae infected with Beauveria bassiana was significantly up-regulated. In this study, BmApoLp-III gene was cloned, its expression patterns in different larval tissues investigated, the BmApoLp-III protein was successfully expressed with prokaryotic expression system and its antifungal effect was verified. The results showed that the BmApoLp-III gene was expressed in all the tested tissues of the 5th instar larvae infected by B. bassiana, with the highest expression in fat body. The fungistatic zone test showed that the recombinant BmApoLp-III has a significant antifungal effect on B. bassiana. Injecting purified BmApoLp-III to the larvae delayed the onset and death of the infected larvae. Conversely, silencing BmApoLp-III gene by RNAi resulted in early morbidity and death of the infected larvae. At the same time, injecting BmApoLp-III up-regulated the expression of genes including BmßGRP4 and BmMyd88 in the Toll signaling pathway, BmCTL5 and BmHOP in the Jak/STAT signaling pathway, serine proteinase inhibitor BmSerpin5, and antimicrobial peptide BmCecA, but down-regulated the expression of BmTak1 of Imd signaling pathway; while silencing BmApoLp-III gene down-regulated the expression of BmßGRP1 and BmSpaetzle, BmCTL5 and BmHOP, BmSerpin2 and BmSerpin5, BmBAEE and BmPPO2 of relevant pathways and BmCecA, but up-regulated the expression of BmPGRP-Lc and BmTak1 of Imd pathway. These results indicate that the BmApoLp-III could not only directly inhibit B. bassiana, but also participate in regulation of the expression of immune signaling pathway related genes, promote the expression of immune effectors, and indirectly inhibit the reproduction of B. bassiana in the silkworm.


Assuntos
Apolipoproteínas/genética , Beauveria/fisiologia , Bombyx/genética , Interações Hospedeiro-Patógeno , Imunidade Inata/genética , Proteínas de Insetos/genética , Regulação para Cima/imunologia , Animais , Apolipoproteínas/metabolismo , Bombyx/crescimento & desenvolvimento , Bombyx/imunologia , Bombyx/microbiologia , Regulação Fúngica da Expressão Gênica , Proteínas de Insetos/metabolismo , Larva/genética , Larva/crescimento & desenvolvimento , Larva/imunologia , Larva/microbiologia , Transdução de Sinais
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