Bothrops jararaca Snake Venom Inflammation Induced in Human Whole Blood: Role of the Complement System.

The clinical manifestations of envenomation by Bothrops species are complex and characterized by prominent local effects that can progress to tissue loss, physical disability, or amputation. Systemic signs can also occur, such as hemorrhage, coagulopathy, shock, and acute kidney failure. The rapid development of local clinical manifestations is accompanied by the presence of mediators of the inflammatory process originating from tissues damaged by the bothropic venom. Considering the important role that the complement system plays in the inflammatory response, in this study, we analyzed the action of Bothrops jararaca snake venom on the complement system and cell surface receptors involved in innate immunity using an ex vivo human whole blood model. B. jararaca venom was able to induce activation of the complement system in the human whole blood model and promoted a significant increase in the production of anaphylatoxins C3a/C3a-desArg, C4a/C4a-desArg, C5a/C5a-desArg and sTCC. In leukocytes, the venom of B. jararaca reduced the expression of CD11b, CD14 and C5aR1. Inhibition of the C3 component by Cp40, an inhibitor of C3, resulted in a reduction of C3a/C3a-desArg, C5a/C5a-desArg and sTCC to basal levels in samples stimulated with the venom. Exposure to B. jararaca venom induced the production of inflammatory cytokines and chemokines such as TNF-α, IL-8/CXCL8, MCP-1/CCL2 and MIG/CXCL9 in the human whole blood model. Treatment with Cp40 promoted a significant reduction in the production of TNF-α, IL-8/CXCL8 and MCP-1/CCL2. C5aR1 inhibition with PMX205 also promoted a reduction of TNF-α and IL-8/CXCL8 to basal levels in the samples stimulated with venom. In conclusion, the data presented here suggest that the activation of the complement system promoted by the venom of the snake B. jararaca in the human whole blood model significantly contributes to the inflammatory process. The control of several inflammatory parameters using Cp40, an inhibitor of the C3 component, and PMX205, a C5aR1 antagonist, indicates that complement inhibition may represent a potential therapeutic tool in B. jararaca envenoming.

Papel do sistema complemento na inflamação induzida pelo veneno da serpente Bothrops jararaca

The genus Bothrops, family Viperidae, integrates a group of snakes with more than 30 species and subspecies, widely distributed in the neotropical region, occurring from southern Mexico to northern Argentina and some islands of the Caribbean. In Brazil, in 2021, they were responsible for around 70% of the more than 29,000 snakebites registered. The snake venom of this genus has a wide repertoire of molecules in its composition, such as metalloproteases, serineproteases, type C lectin, bradykininenhancing peptides (BPPs), among others. The clinical manifestations of the envenomation are complex and characterized by prominent local effects, including pain, edema, ecchymosis, blisters, abscesses and necrosis, which can progress to tissue loss, physical disability or amputation. Systemic signs can also occur, such as hemorrhage, coagulopathy, shock, and acute kidney failure. Parenteral administration of commercial polyvalent antivenom is the only scientifically validated therapeutic tool for the treatment of snakebites. However, despite the effective neutralization of the systemic effects, this treatment is still ineffective in reversing local clinical manifestations. The rapid development of local clinical manifestations is accompanied by the presence of mediators of the inflammatory process, originated from tissues damaged by the bothropic venom. Studies from our group have shown that the Bothrops jararaca venom is able to activate the complement system, suggesting that this activation may contribute to the symptoms observed in these envenomations. Considering the important role that the complement system plays in the inflammatory response, this study aimed to analyze the action of B. jararaca snake venom on the complement system and cell surface receptors involved in innate immunity. For this, we used the ex-vivo human whole blood model proposed by Mollnes et al. (2002) and recently adapted by Johnson et al. (2018). With this model, by means of immunoassays, it was evaluated the complement system activation: from the generation of anaphylatoxins and soluble terminal complement complex (sTCC /SC5b-9); the production of cytokines (IL-1β, IL-6, IL-10, IL-12p70 e TNF-α) and chemokines (IL-8, IP-10, MCP-1, MIG, RANTES); the expression of leukocyte receptors such as TLRs 2 and 4; CD14; CD11b; C3aR and C5aR; the modulation of these parameters by the use of specific inhibitors of the complement system (Cp40, PMX205). The B. jararaca venom was able to induce activation of the complement system in the human whole blood model, generating a significant increase in the production of anaphylatoxins C3a/C3a-desArg, C4a/C4a-desArg, C5a/C5a-desArg and sTCC. In leukocytes, the venom of B. jararaca induced increased expression of TLR2 and reduced expression of C5aR. The expression of CD11b, CD14, C3aR and TLR4 was not altered by the action of the venom. Inhibition of the C3 component by Cp40 resulted in a reduction to basal levels of C3a/C3a-desArg and sTCC in samples stimulated with the venom. Cp40 also caused a significant reduction in the levels of C5a/C5a-desArg, but not of C4a/C4a-desArg. Exposure to B. jararaca venom induced the production of inflammatory cytokines and chemokines such as TNF-α, IL-8, MCP1 and MIG in the human whole blood model. Treatment with Cp40 promoted a significant reduction in the production of TNF-α, IL-8 and MCP-1. The use of the C5a receptor 1 antagonist, PMX205, promoted a reduction to basal levels of TNF-α and IL8 in samples stimulated with venom. In conclusion, the data presented here suggest that the activation of the complement system, promoted by the venom of the snake B. jararaca in the human whole blood model, significantly contributes to the inflammatory process. The control of several inflammatory parameters by Cp40, an inhibitor of the C3 component, and of PMX205, a C5a receptor 1 antagonist, indicate that complement inhibition may be a possible therapeutic tool in B. jararaca envenomation.

O gênero Bothrops, família Viperidae, integra um grupo de serpentes com mais de 30 espécies e subespécies, amplamente distribuídas na região neotropical, ocorrendo desde o Sul do México até o norte da Argentina e em algumas ilhas do Caribe. No Brasil, em 2021, foram responsáveis por cerca de 70% dos mais de 29.000 acidentes ofídicos registrados. O veneno das serpentes deste gênero possui amplo repertório de moléculas em sua composição, como metaloproteases, serinoproteases, lectina do tipo C, peptídeos potenciadores de bradicinina (BPPs), entre outras. As manifestações clínicas do envenenamento são complexas e caracterizadas por efeitos locais proeminentes, incluindo dor, edema, equimose, bolhas, abcessos e necrose, que podem evoluir para perda tecidual, incapacidade física ou amputação. Sinais sistêmicos também podem ocorrer, tais como hemorragia, coagulopatia, choque e insuficiência renal aguda. A administração parenteral do antiveneno polivalente comercial constitui o único recurso terapêutico cientificamente validado para o tratamento dos acidentes ofídicos. Contudo, apesar da efetiva neutralização dos efeitos sistêmicos, esse tratamento ainda se mostra ineficiente na reversão das manifestações clínicas locais. O rápido desenvolvimento das manifestações clínicas locais é acompanhado pela presença de mediadores do processo inflamatório, originados a partir de tecidos lesados pelo veneno botrópico. Estudos do nosso grupo mostraram que o veneno da Bothrops jararaca é capaz de ativar o sistema complemento, sugerindo que essa ativação possa contribuir para os sintomas observados nesses envenenamentos. Considerando o importante papel que o sistema complemento desempenha na reposta inflamatória, o presente estudo teve como objetivo analisar a ação do veneno da serpente B. jararaca sobre o sistema complemento e receptores de superfície celular envolvidos na imunidade inata. Para isto, utilizamos o modelo ex-vivo de sangue total humano, proposto por Mollnes e colaboradores (2002) e, recentemente, adaptado por Johnson e colaboradores (2018). Com este modelo, por meio de imunoensaios, foram avaliadas a ativação do sistema complemento: a partir da geração de anafilatoxinas e complexo terminal do complemento solúvel (sTCC /SC5b-9); produção de citocinas (IL-1β, IL-6, IL-10, IL12p70 e TNF-α) e quimiocinas (IL-8, IP-10, MCP-1, MIG, RANTES); expressão de receptores de leucócitos, como TLRs 2 e 4; CD14; CD11b; C3aR e C5aR; modulação destes parâmetros pelo uso de inibidores específicos do sistema complemento (Cp40, PMX205). O veneno da B. jararaca foi capaz de induzir ativação do sistema complemento no modelo de sangue total humano, gerando aumento significativo na produção das anafilatoxinas C3a/C3a-desArg, C4a/C4a-desArg, C5a/C5a-desArg e sTCC. Nos leucócitos, o veneno da B. jararaca induziu aumento da expressão de TLR2 e redução da expressão de C5aR. A expressão de CD11b, CD14, C3aR e TLR4 não foi alterada pela ação do veneno. A inibição do componente C3 pelo Cp40 resultou na redução para níveis basais de C3a/C3a-desArg e do sTCC nas amostras estimuladas com o veneno. O Cp40 também causou uma redução significativa nos níveis de C5a/C5a-desArg, mas não de C4a/C4a-desArg. A exposição ao veneno da B. jararaca induziu a produção de citocinas e quimiocinas inflamatórias como TNF-α, IL-8, MCP-1 e MIG no modelo de sangue total humano. O tratamento com Cp40 promoveu uma redução significativa na produção de TNF-α, IL- 8 e MCP-1. O uso do antagonista do receptor 1 de C5a, PMX205, promoveu redução para níveis basais de TNF-α e IL-8 em amostras estimuladas com veneno. Em conclusão, os dados aqui apresentados sugerem que a ativação do sistema complemento, promovida pelo veneno da serpente B. jararaca no modelo de sangue total humano, contribui significativamente para o processo inflamatório. O controle de vários parâmetros inflamatórios pelo uso do Cp40, inibidor do componente C3, e do PMX205, antagonista do receptor 1 de C5a, indicam que a inibição do complemento possa ser uma possível ferramenta terapêutica no envenenamento por B. jararaca.

Bothrops jararaca snake venom inflammation induced in human whole blood: role of the complement system

The clinical manifestations of envenomation by Bothrops species are complex and characterized by prominent local effects that can progress to tissue loss, physical disability, or amputation. Systemic signs can also occur, such as hemorrhage, coagulopathy, shock, and acute kidney failure. The rapid development of local clinical manifestations is accompanied by the presence of mediators of the inflammatory process originating from tissues damaged by the bothropic venom. Considering the important role that the complement system plays in the inflammatory response, in this study, we analyzed the action of Bothrops jararaca snake venom on the complement system and cell surface receptors involved in innate immunity using an ex vivo human whole blood model. B. jararaca venom was able to induce activation of the complement system in the human whole blood model and promoted a significant increase in the production of anaphylatoxins C3a/C3a-desArg, C4a/C4a-desArg, C5a/C5a-desArg and sTCC. In leukocytes, the venom of B. jararaca reduced the expression of CD11b, CD14 and C5aR1. Inhibition of the C3 component by Cp40, an inhibitor of C3, resulted in a reduction of C3a/C3a-desArg, C5a/C5a-desArg and sTCC to basal levels in samples stimulated with the venom. Exposure to B. jararaca venom induced the production of inflammatory cytokines and chemokines such as TNF-α, IL-8/CXCL8, MCP-1/CCL2 and MIG/CXCL9 in the human whole blood model. Treatment with Cp40 promoted a significant reduction in the production of TNF-α, IL-8/CXCL8 and MCP-1/CCL2. C5aR1 inhibition with PMX205 also promoted a reduction of TNF-α and IL-8/CXCL8 to basal levels in the samples stimulated with venom. In conclusion, the data presented here suggest that the activation of the complement system promoted by the venom of the snake B. jararaca in the human whole blood model significantly contributes to the inflammatory process. The control of several inflammatory parameters using Cp40, an inhibitor of the C3 component, and PMX205, a C5aR1 antagonist, indicates that complement inhibition may represent a potential therapeutic tool in B. jararaca envenoming.