Análise e identificação de vias de sinalização moduladas por mediadores produzidos pela ação da fosfolipase-d de Loxosceles intermedia

Abstract

Resumo: As aranhas do gênero Loxosceles, popularmente conhecidas como aranhas-marrom estão associadas a numerosos acidentes com humanos, que resultam em um quadro clínico de envenenamento denominado loxoscelismo. O loxoscelismo pode manifestar-se de forma cutânea ou sistêmica. A variante cutânea apresenta-se com sintomas como edema, eritema, equimose, isquemia tecidual e lesões dermonecróticas de progressão gravitacional. Já o loxoscelismo sistêmico pode incluir hemólise intravascular e insuficiência renal aguda. As toxinas presentes no veneno, com destaque para as fosfolipases-D (FLDs), são responsáveis pelos sintomas característicos do loxoscelismo. As FLDs clivam diferentes fosfolipídios, desencadeando processos de inflamação, necrose tecidual e outras respostas. Afim de entender essas respostas, este estudo utilizou e uma abordagem de biologia de sistemas para investigar esses mecanismos, modelando redes de sinalização celular estimuladas pelas FLDs e identificando possíveis interações proteína-proteína e vias gênicas associadas às respostas desencadeadas por estas toxinas. Foram incluídos alvos com alta intermediação em processos celulares desencadeados pelas FLDs, com ligação a respostas inflamatórias, estresse oxidativo, sobrevivência e senescência celular, resultando na seleção dos genes IL-1??, IL6 e TNF-??, src, c-jun, AKT-1, relA, Stat-3, TP53 como alvos deste estudo. Com base nesses alvos resultantes, culturas de células presentes na derme e epiderme, incluindo queratinócitos, fibroblastos e células endoteliais, foram utilizadas para validar esses achados. Essas células foram expostas à LiRecDT1, uma isoforma recombinante ativa altamente expressa de PLD do veneno de L. intermedia. Após extração do mRNA dessas células tratadas, as amostras foram submetidas a ensaios de RT-qPCR com oligonucleotídeos específicos para os alvos mencionados acima. Os dados obtidos permitiram observar que os três tipos celulares envolvidos possuem os genes selecionados moduladas pela ação da LiRecDT1, sendo estimulados após 4 horas e com a persistência de níveis de expressão de alguns desses genes aumentados após 24 horas. Com esses dados, foi feita uma análise de vias gênicas, que sugeriu respostas de regulação positiva relacionadas às vias de IL-6 e IL-8, além de resposta inflamatória aguda, sobrevivência celular e estímulo de vias adesão de leucócitos ao endotélio para todos os tipos celulares. Além disso, foi avaliada a capacidade de LiRecDT1 estimular diretamente a adesão de leucócitos à membrana celular de células endoteliais em cultura, já que esse processo é relevante para reação inflamatória na derme observada no loxoscelismo. Meios condicionados de queratinócitos e fibroblastos tratadas com LiRecDT1 também foram incubados com células endoteliais em cultura para avaliar se aumentava a adesão de leucócitos sobre essas células.. Foi possível observar estímulo à adesão de polimorfonucleares à membrana das células endoteliais, tanto estimuladas diretamente pela LiRecDT1, quanto por meio condicionado das células testadas. Esse achado indica que, in vivo, a FLD pode atuar na membrana dessas células sinalizando e estimulando o processo de diapedese. Juntos, os resultados mostram que a FLD interfere na expressão dos alvos envolvidos em processos celulares em especial em vias relacionadas a inflamação nas células presentes na pele. Ademais, os dados revelam que essa toxina é capaz de estimular a adesão de leucócitos em células endoteliais, e que sua ação sobre queratinócitos e fibroblastos leva a esse mesmo estímulo por meio de sinalização parácrina sobre células endoteliais, indicando um papel chave desses processos sobre a ocorrência de diapedese in vivo. Estes achados destacam o papel desses tipos celulares presentes na pele, que é o local de inoculação do veneno, no desenvolvimento do loxoscelismo cutâneo

Abstract: Spiders of the Loxosceles genus, commonly known as brown recluse spiders, are associated with numerous human envenomation cases, resulting in a clinical condition known as loxoscelism. Loxoscelism can manifest in either cutaneous or systemic forms. The cutaneous variant presents symptoms such as edema, erythema, ecchymosis, tissue ischemia, and dermonecrotic lesions with progressive worsening. Meanwhile, systemic loxoscelism can include intravascular hemolysis and acute renal failure. The toxins present in the venom, particularly phospholipases-D (PLDs), are responsible for the characteristic symptoms of loxoscelism. PLDs cleave different phospholipids, triggering inflammatory processes, tissue necrosis, and other responses. To better understand these responses, this study employed a systems biology approach to investigate these mechanisms, modeling cellular signaling networks stimulated by PLDs and identifying potential protein-protein interactions and gene pathways associated with the responses triggered by these toxins. Targets with high centrality in cellular processes activated by PLDs were included, with links to inflammatory responses, oxidative stress, cell survival, and senescence. This selection resulted in the identification of IL-1ß, IL-6, TNF-a, src, c-jun, AKT-1, relA, Stat-3, and TP53 as key targets in this study. Based on these targets, cell cultures derived from the dermis and epidermis, including keratinocytes, fibroblasts, and endothelial cells, were used to validate these findings. These cells were exposed to LiRecDT1, a highly expressed recombinant active isoform of PLD from L. intermedia venom. After extracting mRNA from the treated cells, the samples were subjected to RT-qPCR assays using specific oligonucleotides for the argets. The data obtained revealed that all three cell types showed modulation of the selected genes by LiRecDT1, with stimulation occurring after 4 hours and increased expression levels of some of these genes persisting after 24 hours. Based on these results, a gene pathway analysis suggested positive regulation responses related to IL-6 and IL-8 pathways, as well as acute inflammatory response, cell survival, and stimulation of leukocyte adhesion pathways to the endothelium in all cell types. Additionally, the ability of LiRecDT1 to directly stimulate leukocyte adhesion to the cell membrane of endothelial cells in culture was assessed, as this process is relevant to the inflammatory reaction in the dermis observed in loxoscelism. Conditioned media from keratinocytes and fibroblasts treated with LiRecDT1 were also incubated with endothelial cells in culture to evaluate whether leukocyte adhesion was increased. It was observed that polymorphonuclear adhesion to endothelial cell membranes was stimulated both directly by LiRecDT1 and indirectly through conditioned media from the tested cells. This finding indicates that, in vivo, PLD may act on these cell membranes, signaling and stimulating the diapedesis process. TogetheSpiders of the Loxosceles genus, commonly known as brown recluse spiders, are associated with numerous human envenomation cases, resulting in a clinical condition known as loxoscelism. Loxoscelism can manifest in either cutaneous or systemic forms. The cutaneous variant presents symptoms such as edema, erythema, ecchymosis, tissue ischemia, and dermonecrotic lesions with progressive worsening. Meanwhile, systemic loxoscelism can include intravascular hemolysis and acute renal failure. The toxins present in the venom, particularly phospholipases-D (PLDs), are responsible for the characteristic symptoms of loxoscelism. PLDs cleave different phospholipids, triggering inflammatory processes, tissue necrosis, and other responses. To better understand these responses, this study employed a systems biology approach to investigate these mechanisms, modeling cellular signaling networks stimulated by PLDs and identifying potential protein-protein interactions and gene pathways associated with the responses triggered by these toxins. Targets with high centrality in cellular processes activated by PLDs were included, with links to inflammatory responses, oxidative stress, cell survival, and senescence. This selection resulted in the identification of IL-1ß, IL-6, TNF-a, src, c-jun, AKT-1, relA, Stat-3, and TP53 as key targets in this study. Based on these targets, cell cultures derived from the dermis and epidermis, including keratinocytes, fibroblasts, and endothelial cells, were used to validate these findings. These cells were exposed to LiRecDT1, a highly expressed recombinant active isoform of PLD from L. intermedia venom. After extracting mRNA from the treated cells, the samples were subjected to RT-qPCR assays using specific oligonucleotides for the argets. The data obtained revealed that all three cell types showed modulation of the selected genes by LiRecDT1, with stimulation occurring after 4 hours and increased expression levels of some of these genes persisting after 24 hours. Based on these results, a gene pathway analysis suggested positive regulation responses related to IL-6 and IL-8 pathways, as well as acute inflammatory response, cell survival, and stimulation of leukocyte adhesion pathways to the endothelium in all cell types. Additionally, the ability of LiRecDT1 to directly stimulate leukocyte adhesion to the cell membrane of endothelial cells in culture was assessed, as this process is relevant to the inflammatory reaction in the dermis observed in loxoscelism. Conditioned media from keratinocytes and fibroblasts treated with LiRecDT1 were also incubated with endothelial cells in culture to evaluate whether leukocyte adhesion was increased. It was observed that polymorphonuclear adhesion to endothelial cell membranes was stimulated both directly by LiRecDT1 and indirectly through conditioned media from the tested cells. This finding indicates that, in vivo, PLD may act on these cell membranes, signaling and stimulating the diapedesis process. Together, the results show that PLD interferes with the expression of targets involved in cellular processes, particularly in inflammation-related pathways in skin cells. Moreover, the data reveal that this toxin can stimulate leukocyte adhesion in endothelial cells and that its action on keratinocytes and fibroblasts leads to the same stimulation via paracrine signaling on endothelial cells, highlighting a key role of these processes in the occurrence of in vivo diapedesis. These findings emphasize the role of these skin-resident cell types—being the site of venom inoculation—in the development of cutaneous loxoscelismr, the results show that PLD interferes with the expression of targets involved in cellular processes, particularly in inflammation-related pathways in skin cells. Moreover, the data reveal that this toxin can stimulate leukocyte adhesion in endothelial cells and that its action on keratinocytes and fibroblasts leads to the same stimulation via paracrine signaling on endothelial cells, highlighting a key role of these processes in the occurrence of in vivo diapedesis. These findings emphasize the role of these skin-resident cell types—being the site of venom inoculation—in the development of cutaneous loxoscelism