RESUMO
Since the first record of the five founder members of the group of Natterin proteins in the venom of the medically significant fish Thalassophryne nattereri, new sequences have been identified in other species. In this work, we performed a detailed screening using available genome databases across a wide range of species to identify sequence members of the Natterin group, sequence similarities, conserved domains, and evolutionary relationships. The high-throughput tools have enabled us to dramatically expand the number of members within this group of proteins, which has a remote origin (around 400 million years ago) and is spread across Eukarya organisms, even in plants and primitive Agnathans jawless fish. Overall, the survey resulted in 331 species presenting Natterin-like proteins, mainly fish, and 859 putative genes. Besides fish, the groups with more species included in our analysis were insects and birds. The number and variety of annotations increased the knowledge of the obtained sequences in detail, such as the conserved motif AGIP in the pore-forming loop involved in the transmembrane barrel insertion, allowing us to classify them as important constituents of the innate immune defense system as effector molecules activating immune cells by interacting with conserved intracellular signaling mechanisms in the hosts.
Assuntos
Venenos de Peixe , Proteínas Citotóxicas Formadoras de Poros , Animais , Venenos de Peixe/química , Venenos de Peixe/genética , Venenos de Peixe/imunologia , Estrutura Molecular , Filogenia , Proteínas Citotóxicas Formadoras de Poros/química , Proteínas Citotóxicas Formadoras de Poros/genética , Proteínas Citotóxicas Formadoras de Poros/imunologiaRESUMO
We hypothesized that beyond the Thalassophryne nattereri venoms ability to induce in mice a strong specific-Th2 response with high levels of specific IgE/IgG1, it would be able to trigger anaphylaxis in sensitized individuals. To investigate whether the venom is capable of inducing an allergic reaction in mice and characterize soluble and cellular mediators involved in this process, BALB/c female mice were sensitized intraperitoneally with decreasing-dose of venom at weekly intervals for 4 weeks and challenged by intraperitoneal, oral or epicutaneous routes with venom 2 weeks later. Our data show that sensitized-mice challenged by all routes showed intense symptoms of anaphylaxis, dependent on the anaphylactic IgG1 and IgE antibodies and mast cells. The late-phase reaction developed after initial symptoms was characterized by the influx of eosinophils, dependent on IL-5, IL-17A and eotaxin produced by Th2 cells in inflamed lungs and skin draining lymph-nodes. Using C57BL/6 deficient mice we demonstrated that IL-4 KO mice failed to develop anaphylactic symptoms or local Th2 inflammation, producing low levels of IgG1 and increased levels of IgG2a. Together our results demonstrated that the venom of T. nattereri has allergenic proteins that can trigger an allergic process, a phenomenon IgE-IgG1 dependent, IL-4-mediated and negatively regulated by IFN-γ.
Assuntos
Anafilaxia/imunologia , Batracoidiformes/metabolismo , Venenos de Peixe/efeitos adversos , Interleucina-4/genética , Interleucina-4/metabolismo , Administração Cutânea , Administração Oral , Anafilaxia/induzido quimicamente , Animais , Modelos Animais de Doenças , Feminino , Venenos de Peixe/imunologia , Técnicas de Inativação de Genes , Humanos , Imunoglobulina E/imunologia , Imunoglobulina G/imunologia , Injeções Intraperitoneais , Interferon gama/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , RatosRESUMO
Interleukin (IL) 17A in chronic inflammation is also produced by innate immune cells as neutrophils. Mice with chronic humoral response induced by venom of Thalassophryne nattereri (VTn) proved to be a good tool for evaluating the impact of IL-17A on the development of long-lived plasma cells in the inflamed peritoneal cavity. Here, we report that VTn induces IL-17A production by neutrophils accumulating in the peritoneal cavity and triggers the extrusion of IL-17A along with neutrophil extracellular traps (NETs). Neutrophil depletion reduced the number of IL17A-producing cells in VTn-immunized mice and blocked the differentiation of long-lived plasma cells. Specific antibody production and survival of long-lived plasma cells was ablated in VTn-immunized mice deficient in CD4, while CD28 signaling had the opposite effect on differentiation of long-lived plasma cells. Further, maturation of long-lived plasma cells in inflamed peritoneal cavity was IL-1R1 and COX-2 dependent. Finally, when both the Raf-MEK-ERK pathway and the IL-17A or IL-1R1 activities were blocked, neutrophils were unable to promote the differentiation of memory B cells into long-lived plasma cells, confirming the essential role of neutrophils and IL-17A along with NETs in an IL-1/IL-1R-dependent manner as the novel helping partner for plasma cell differentiation in chronically inflamed tissues.
Assuntos
Diferenciação Celular/imunologia , Armadilhas Extracelulares/metabolismo , Interleucina-17/metabolismo , Neutrófilos/imunologia , Neutrófilos/metabolismo , Plasmócitos/imunologia , Plasmócitos/metabolismo , Receptores Tipo I de Interleucina-1/metabolismo , Animais , Ciclo-Oxigenase 2/metabolismo , Ensaio de Imunoadsorção Enzimática , Armadilhas Extracelulares/imunologia , Venenos de Peixe/imunologia , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Memória Imunológica , Ativação Linfocitária , Masculino , Camundongos , Camundongos Knockout , Anafilaxia Cutânea Passiva/imunologia , Plasmócitos/citologiaRESUMO
Switched CD19-positive memory B cells purified from mice with chronic immune response against Thalassophrynenattereri venom proteins were cultured with venom or cytokines. Our results confirm the existence of a hierarchic process of differentiation: activated memory B cells progressively acquire increasing levels of CD138 and decreasing levels of CD45R/B220 to finally arrive at ASC with B220(neg) phenotype, which are IgG1-secreting cells. Only Bmem from peritoneal cavity or bone marrow of VTn immunized mice presented the capacity to generate ASC functionally active. IL-17A or IL-21/IL-23/IL-33 improves the ability of venom to induce intracellular IgG of peritoneal derived-ASC. Cognate stimulation with venom and IL-17A is sufficient to down-regulate the expression of CD45R/B220. BAFF-R is up-regulated in splenic or medullar derived-ASC stimulated by venom, CpG or cytokines. Only splenic derived-ASC up-regulate Bcl-2 expression after CpG or the combination of IL-21/IL-23/IL-33 stimulation. Finally, the activation of ASC for IgG1 secretion is triggered by venom proteins in peritoneal cavity and by IL-17A in medullar niche. These results show the importance of the integration of signals downstream of BCR and IL17-A receptors in modulating ASC differentiation, focusing in the microenvironment niche of their generation.
Assuntos
Células Produtoras de Anticorpos/citologia , Antígenos/imunologia , Diferenciação Celular/imunologia , Interleucina-17/metabolismo , Transdução de Sinais , Animais , Células Produtoras de Anticorpos/efeitos dos fármacos , Células Produtoras de Anticorpos/metabolismo , Antígenos CD19/metabolismo , Apoptose/efeitos dos fármacos , Apoptose/imunologia , Receptor do Fator Ativador de Células B/metabolismo , Linfócitos B/imunologia , Diferenciação Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Venenos de Peixe/imunologia , Imunoglobulina G/biossíntese , Memória Imunológica/efeitos dos fármacos , Interleucina-23/farmacologia , Interleucinas/farmacologia , Antígenos Comuns de Leucócito/metabolismo , Contagem de Linfócitos , Masculino , Camundongos , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Recombinantes/farmacologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia , Baço/citologia , Receptor Toll-Like 9/metabolismoRESUMO
Thalassophryne nattereri envenoming represents a great cost to North and Northeast Brazilian communities in terms of public healths, leisure and tourism. Victims rapidally develop symptoms as pain, local swelling, erythema followed by intense necrosis that persist for long days. The aim of this work was tested the immune competence of neutralizing antibodies in pre-immunized mice against principal toxic activities induced by venom. During the primary antibody response in mice, an elevation of IgG antibody levels was only observed on day 28. After boosting, high antibody levels were detected between days 49 and 70, with a 12-fold increase in IgG level over control values at day 49. We confirmed the in vitro neutralizing capacity of T. nattereri anti-venom against toxic effects and thereafter we show that neutralizing antibodies obtained in a persistent immune response are more effective, inclusive against edematous reaction. After boosting during the secondary response mice with high antibody levels do not present any alterations in venule or arteriole after topical application of venom on cremaster muscle. In addition, CK activity diminished in these mice with high neutralizing antibody levels corroborating the attenuation of the myonecrotic effect by venom. In addition, we determined the presence of high IgG antibodies levels in patients 6 months after injury by T. nattereri. In conclusion, the presence of neutralizing antibodies against to T. nattereri venom in the serum of pre-immunized mice could change the outcome of lesion at site of posterior envenoming. Antigen-specific antibodies of high affinity in consequence to specific immune response, dependent of T lymphocyte activation, could minimize the symptoms of intense and immediate inflammatory reaction caused by T. nattereri venom. These finding prompt us to the possibility of development of immune therapeutic strategies using specific anti-venom as an efficient intervention for protecting human victims.
Assuntos
Antivenenos/farmacologia , Batracoidiformes , Venenos de Peixe/antagonistas & inibidores , Animais , Antivenenos/sangue , Linfócitos B/imunologia , Venenos de Peixe/imunologia , Venenos de Peixe/toxicidade , Humanos , Imunização , Imunoglobulina G/sangue , Memória Imunológica/efeitos dos fármacos , Masculino , CamundongosRESUMO
Cathorops spixii is one of the most abundant venomous fish of the southeastern coast of the State of São Paulo, and consequently causes a great part of the accidents seen there. The accidents affect mainly fishermen, swimmers and tourists and are characterized by punctiform or wide wounds, erythema, edema, pain, sudoresis, indisposition, fever, nausea, vomiting and secondary infection. The objective of this work was to characterize the inflammatory response induced in mice by both venoms (mucus and sting) of the catfish C. spixii. Our results demonstrated that both venoms induced a great number of rolling and adherent leukocytes in the post-capillary venules of cremaster muscle of mice, and an increase in the vascular permeability in peritoneal cavity. Mucus induced the recruitment of neutrophils immediately after injection followed later by macrophage infiltration. In contrast, the cellular infiltration elicited by sting venom was rapidly resolved. The peritonitis reaction provoked by venoms was characterized by cytokine (IL-6), chemokines (MCP-1 and KC) or lipid mediator (LTB4) production in the peritoneal cavity. The macrophages from 7-day mucus venom-induced exudates upon in vitro mucus venom stimulation, expressed CD11c x MHC class II and release bioactive IL-12p70. On the other hand, sting venom-elicited peritoneal macrophages lost the ability to differentiate into dendritic cells, following re-stimulation in vitro with sting venom, they do not express CD11c, nor do they exhibit sufficient levels of MHC class II. In conclusion, both types of venoms (mucus or sting) promote inflammatory reaction with different profiles, and the inflammatory reaction induced by the first was characterized by antigen persistence in peritoneal cavity that allowed the activation of phagocytic cells with capacity of antigenic presentation.
Assuntos
Peixes-Gato , Venenos de Peixe/toxicidade , Inflamação/induzido quimicamente , Animais , Biomarcadores/análise , Permeabilidade Capilar/efeitos dos fármacos , Venenos de Peixe/química , Venenos de Peixe/imunologia , Imunidade Celular/efeitos dos fármacos , Inflamação/imunologia , Masculino , Camundongos , Cavidade Peritoneal/irrigação sanguínea , Cavidade Peritoneal/citologia , Testes de ToxicidadeRESUMO
Murine experimental model have been useful to understanding the toxic as well as the pharmacological properties of the Thalassophryne nattereri venom. However, the specific immune response to T. nattereri venom in mice is yet unclear. Our results showed that the venom elicited in BALB/c mice high levels of specific IgG1 and total IgE isotype with high affinity, accompanied by a striking IL-5 production, what point out to a Th2-like response. Meanwhile, the production of IFN-gamma by lymphocytes pool expanded upon mitogen stimulus, suggests that the venom was also able to activate Th1 clones. Elevated number of antigen-presenting cells expressing CD11c or CD11b from day 4 to 6 supported ongoing antigen presentation process in the primary response and efficient T-cell expansion (increase of CD4(+) cells). In contrast, decreased B220 expression was observed, suggesting that the formation of memory long lived cell compartment. In conclusion, T. natterri venom stimulates an association of cytokine of both Th1 and Th2 profile, with a notable IL-5 production and specific IgG1 and total IgE isotypes secretion. Furthermore, our finding showed that T. natterri venom can affect the B cell fate and induce a memory antibody response through the secretion of protective IgG subclasses. Further studies with the venom protein toxins may provide clues to molecular mechanism regulating proliferation and differentiation of antibody-secreting cells in our model. A better understating of how T. natterri venom can modulate immune response could be useful in therapeutic strategies.
Assuntos
Batracoidiformes , Venenos de Peixe/imunologia , Interleucina-5/biossíntese , Subpopulações de Linfócitos T/imunologia , Animais , Células Cultivadas , Concanavalina A/farmacologia , Venenos de Peixe/farmacologia , Imunoglobulina E/imunologia , Imunoglobulina G/imunologia , Interferon gama/biossíntese , Antígenos Comuns de Leucócito/análise , Antígenos Comuns de Leucócito/metabolismo , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/imunologia , Linfócitos/efeitos dos fármacos , Linfócitos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Mitógenos/farmacologia , Modelos Animais , Baço/citologia , Baço/efeitos dos fármacos , Baço/imunologia , Subpopulações de Linfócitos T/efeitos dos fármacos , Subpopulações de Linfócitos T/metabolismo , VacinaçãoRESUMO
A produção de toxinas por animais aquáticos é uma estratégia importante que garante sua sobrevivência em um ecossistema altamente competitivo. Constituem-se de uma rica fonte de agentes bioquímicos altamente ativos o que aumenta ainda mais a relevância das pesquisas nessa área. Nossos estudos objetivaram caracterizar as respostas imune inata e específica induzidas pelas peçonhas do muco e do ferrão do bagre Cathorops agassizii. A coleta dos espécimes foi realizada no complexo Baía-Estuário de Santos e São Vicente, localizado no litoral sul do Estado de São Paulo. As peçonhas (do Muco e do Ferrão) apresentaram perfil eletroforético similar entre si. Induzida a inflamação aguda em um modelo experimental murino, as peçonhas apresentaram igualmente a capacidade de induzir aumento da permeabilidade vascular e também edema de pata. A detecção de Leucotrieno B4 e Prostaglandina E2 no lavado da cavidade peritoneal dos camundongos injetados, com ambas as peçonhas, corroboram esta hipótese. Nossos resultados através da mícroscopia intravital mostraram que as peçonhas induzem um grande número de leucócitos rolantes nas vênulas pós-capilares com focos de extravasamento leucocitárío, principalmente de neutrófilos seguido pelo influxo de macrófagos. Além disso, a peçonha do Ferrão induziu uma resolução mais rápida do influxo leucocitário ao contrário da peçonha do Muco que manteve o infiltrado macrofágico por até 7 dias. De maneira interessante, somente a citocina IL -6 foi detectada no lavado peritoneal induzida principalmente pela peçonha do Muco e as quimiocinas KC e MCP-l, por ambas as peçonhas, expressando naquele momento, a participação destes mediadores no recrutamento de neutrófilos e macrófagos para o sítio da lesão. As peçonhas foram eficazes ao induzir uma produção primária e secundária de anticorpos das classes IgM e IgG anti-venenos...