RESUMO
Crotoxin (CTX), the predominant toxin in Crotalus durissus terrificus snake venom (CdtV), has anti-inflammatory and immunomodulatory effects. Despite its inhibitory action on neutrophil migration and phagocytosis, CTX does not directly affect the production of reactive oxygen species (ROS) by the neutrophils. In contrast, it enhances the generation of reactive oxygen and nitrogen intermediates by macrophages. Given the importance of macrophage-neutrophil interactions in innate antimicrobial defense, the aim of this study was to investigate the effect of CTX on neutrophil ROS production and killing activity, either through CTX-treated macrophage co-culture or conditioned medium of CTX-treated macrophages. The results showed an important modulatory action of CTX on the neutrophil function as well as neutrophil-macrophage interactions, as demonstrated by the increased production of hydrogen peroxide, hypochlorous acid, nitric oxide and TNF- a , along with the increased fungicidal activity of neutrophils.
RESUMO
Crotoxin (CTX), the predominant toxin in Crotalus durissus terrificus snake venom (CdtV), has anti-inflammatory and immunomodulatory effects. Despite its inhibitory action on neutrophil migration and phagocytosis, CTX does not directly affect the production of reactive oxygen species (ROS) by the neutrophils. In contrast, it enhances the generation of reactive oxygen and nitrogen intermediates by macrophages. Given the importance of macrophage-neutrophil interactions in innate antimicrobial defense, the aim of this study was to investigate the effect of CTX on neutrophil ROS production and killing activity, either through CTX-treated macrophage co-culture or conditioned medium of CTX-treated macrophages. The results showed an important modulatory action of CTX on the neutrophil function as well as neutrophil-macrophage interactions, as demonstrated by the increased production of hydrogen peroxide, hypochlorous acid, nitric oxide and TNF- a , along with the increased fungicidal activity of neutrophils.
RESUMO
Crotoxin (CTX), the major toxin of Crotalus durissus terrificus snake venom, induces immunomodulatory action, particularly on the macrophages function, crucial cells to innate defense mechanisms. In these cells, dual action of this toxin is observed, since both inhibition of some functional parameters such as spreading and phagocytosis, as well as respiratory burst (hydrogen peroxide-H2O2 generation and nitric oxide-NO generation), Lipoxin A4 (LXA4) and its stable analog (15-Epi-LXA4) secretion and glucose and glutamine metabolism increased. Recent studies have demonstrated the importance of these CTX stimulatory actions on the metabolism of macrophages for the control of infectious inflammatory response and for tumor progression. Furthermore, FPRs are crucial for the different effects of CTX on macrophage function and metabolism. In spite of these evidences, the mechanisms involved with the CTX stimulatory actions on the metabolism of these cells are not known in their entirety. Among the mechanisms involved in the regulation of energetic metabolism of macrophages, purinergic signaling is essential in the stimulation of cytokine secretion and the reactive oxygen species and reactive nitrogen species generation by macrophages, besides being receptors responsive to stimuli via FPRs. Therefore, the objective of the present project was to investigate the possible participation of purinergic receptors on the H2O2 production by CTX treated THP-1 monocytic cells and whether FPRs participate in this signaling. For this purpose, THP-1 cells were used, such as monocytes or macrophage-differentiated or LPS-stimulated cells. The results show that THP-1 monocytes, such as macrophages differentiated from THP-1, by previous incubation with PMA, showed a significant increase of H2O2 production in the presence of different concentrations of CTX. CTX-induced H2O2 production increased was also observed after 24 hours by differentiated and LPS-stimulated macrophages. Different CTX concentrations lead to a significant increase in the ATP releasing by THP-1, in the absence or presence of LPS. Furthermore, different concentrations of CTX enhanced visualization of P2Y11 purinergic receptors and FPRs in LPS-stimulated THP-1. Blocking of P2X and P2Y purinergic receptors by non-selective antagonists abolished CTX-induced H2O2 production. Blocking of the FPRs by the selective antagonist partially interfered with the production of this toxin-induced reaction. The results together demonstrate, in an unprecedented way, that human monocytes are responsive to the action stimulatory CTX on ROS production and release of ATP and demonstrate for the first time that purinergic receptors are involved in this CTX-stimulatory action, with the FPRs participation.
A Crotoxina (CTX), toxina majoritária do veneno de serpente Crotalus durissus terrificus (VCdt) acarreta ação imunomoduladora, particularmente sobre a funcionalidade de macrófagos, células fundamentais para os mecanismos da defesa inata. Nestas células, é observado o dualismo na ação desta toxina, uma vez que foi observada tanto inibição de alguns parâmetros funcionais, como espraiamento e fagocitose, quanto à estimulação do “burst” respiratório (da geração de peróxido de hidrogênio-H2O2 e óxido nítrico-NO), da secreção de lipoxina A4 (LXA4) e seu análogo estável (15-Epi-LXA4) e do metabolismo de glicose e glutamina. Estudos recentes vêm demonstrando a importância dessas ações estimulatórias da CTX sobre o metabolismo de macrófagos para o controle da resposta inflamatória infecciosa e para a progressão tumoral. Ainda, receptores para peptídeo formil (Formyl Peptide Receptors-FPRs), ligantes de LXA4/15-Epi-LXA4 são cruciais para os diferentes efeitos da CTX sobre função e metabolismo de macrófagos. Apesar dessas evidencias não são conhecidos, na sua totalidade, os mecanismos envolvidos com as ações estimulatórias da CTX sobre o metabolismo destas células. Dentre os mecanismos envolvidos na regulação do metabolismo energético de macrófagos, a sinalização purinérgica é essencial na estimulação de secreção de citocinas e na geração de espécies reativas do oxigênio e do nitrogênio por macrófagos, além de serem receptores responsivos a estímulos via FPRs. Portanto, o objetivo do presente projeto foi investigar a possível participação dos receptores purinérgicos (RPs) sobre a produção de H2O2 por células monocíticas da linhagem THP-1, tratadas com CTX e se FPRs participam desta sinalização. Para tanto, foram utilizadas as células THP-1, como monócitos ou diferenciados em macrófagos ou estimulados por LPS. Os resultados mostram que os monócitos THP-1, como macrófagos diferenciados a partir de THP-1, por meio da incubação prévia com PMA, apresentaram aumento significativo da produção de H2O2 na presença das diferentes concentrações de CTX. O aumento da produção de H2O2 induzido pela CTX também foi observado após 24 horas, por macrófagos diferenciados e estimulados com LPS. Diferentes concentrações de CTX acarretam importante aumento de liberação de ATP por THP-1, na ausência ou presença de LPS. Ainda, as diferentes concentrações de CTX induziram aumento da visualização de receptores purinérgicos P2Y11 e FPRs em THP-1 estimuladas com LPS. O bloqueio dos receptores purinérgicos P2X e P2Y por antagonistas não seletivos aboliu a produção de H2O2 induzida pela CTX. O bloqueio dos FPRs pelo antagonista seletivo interferiu parcialmente com a produção deste reativo induzido pela toxina. Os resultados em conjunto demonstram, de maneira inédita, que monócitos humanos são responsivos à ação estimulatória da CTX sobre a produção de ROS e liberação de ATP e demonstram, pela primeira vez, que receptores purinérgicos estão envolvidos nesta ação estimulatória da CTX, com a participação dos FPRs.
