Crotoxina modula macrófagos infectados pelo Encephalitozoon cuniculi para perfil microbicida M1
Documento
Informações
Título
Crotoxina modula macrófagos infectados pelo Encephalitozoon cuniculi para perfil microbicida M1
Autor(es)
Cristina Gabriela Nascimento de Oliveira
Orientador(es)
Maria Anete Lallo
Data de Defesa
08/03/2023
Resumo
A crotoxina (CTX), uma fração bioativa isolada do veneno da serpente Crotalus durissus terrificus, possui propriedades antibacteriana, antitumorais e antiinflamatória identificadas em estudos in vitro e in vivo. O microsporídio Encephalitozoon cuniculi pertence a um grupo amplo de fungos oportunistas, intracelulares obrigatórios que infectam vertebrados e invertebrados e apresentam grande resistência aos fármacos convencionais. Assim, o objetivo desse estudo foi avaliar os efeitos fungicida e/ou fungistático da CTX sobre os esporos do microsporídio Encephalitozoon cuniculi, bem como avaliar sua capacidade de modular a atividade microbicida de macrófagos in vitro. Esporos de E. cuniculi foram tratados com duas concentrações de CTX (2,4 e 4,8 μg/mL) e, posteriormente, cultivados em células RK-13 para análise de sua viabilidade. Adicionalmente, macrófagos foram obtidos a partir de células peritoneais aderentes (APerC) após lavados peritoneais de camundongos BALB/c. APerCs foram infectadas com esporos de E. cuniculi e após uma hora tratadas com CTX por 3h, para posterior análise do perfil dos macrófagos, da produção de citocinas, da viabilidade dos macrófagos e dos esporos. Não foi demonstrado efeito fungicida, contudo com o tratamento dos esporos de E. cuniculi com 2,4 μg/mL de CTX teve efeito fungistático, com a redução da proliferação e viabilidade. Os esporos tratados com CTX determinaram maior porcentagem de necrose das células RK infectadas. Nessa mesma concentração de CTX (2,4 μg/mL), macrófagos desafiados com E. cuniculi e tratados apresentaram predomínio de macrófagos com perfil M1, o que foi confirmado pela razão entre CD40/CD206 com predomínio de M1 em relação ao M2, com produção de aumentada de citocina pró-inflamatórias TNF- e IL-6 e com atividade microbicida confirmada pela menor viabilidade dos esporos demonstrada pela menor razão de proliferação. A CTX aumentou a morte por necrose de macrófagos infectados ou não com E. cuniculi. Os resultados permitem concluir que a CTX modulou macrófagos infectados pelo E. cuniculi perfil M1 com maior produção de citocinas proinflamatórias e com maior atividade microbicida, assim como teve efeito fungistático sobre os esporos.
Resumo (EN)
Crotoxin (CTX), a bioactive fraction isolated from the venom of the snake Crotalus durissus terrificus, has antibacterial, antitumor and anti-inflammatory properties identified in in vitro and in vivo studies. Microsporidia Encephalitozoon cuniculi belongs to a broad group of opportunistic, obligate intracellular fungi that infect vertebrates and invertebrates and are highly resistant to conventional drugs. Thus, the aim of this study was to evaluate the fungicidal and/or fungistatic effects of CTX on spores of the microsporidian E. cuniculi, as well as to evaluate its ability to modulate the microbicidal activity of macrophages in vitro. E. cuniculi spores were treated with two concentrations of CTX (2.4 and 4.8 μg/mL) and subsequently cultivated in RK-13 cells for viability analysis. Additionally, macrophages were obtained from adherent peritoneal cells (APerC) after peritoneal washings from BALB/c mice. APerCs were infected with spores of E. cuniculi and after one hour treated with CTX for 3h, for further analysis of the profile of macrophages, cytokine production, viability of macrophages and spores. No fungicidal effect was demonstrated, however, treatment of E. cuniculi spores with 2.4 μg/mL of CTX had a fungistatic effect, with a reduction in proliferation and viability. Spores treated with CTX determined a higher percentage of necrosis in infected RK cells. At the same concentration of CTX (2.4 μg/mL), macrophages challenged with E. cuniculi and treated showed a predominance of the M1 profile, confirmed by the CD40/CD206 ratio, with increased production of the pro-inflammatory cytokine TNF- and IL-6 and higher microbicidal activity confirmed by reduced spore viability demonstrated by lower proliferation ratio. CTX increased death by necrosis of macrophages infected or not with E. cuniculi. The results allow us to conclude that CTX modulated macrophages infected by E. cuniculi profile M1 with greater production of proinflammatory cytokines and with greater microbicidal activity, as well as having a fungistatic effect on spores.
Tipo
Tese
Palavras-chave
Crotoxin (CTX), a bioactive fraction isolated from the venom of the snake Crotalus durissus terrificus, has antibacterial, antitumor and anti-inflammatory properties identified in in vitro and in vivo studies. Microsporidia Encephalitozoon cuniculi belongs to a broad group of opportunistic, obligate intracellular fungi that infect vertebrates and invertebrates and are highly resistant to conventional drugs. Thus, the aim of this study was to evaluate the fungicidal and/or fungistatic effects of CTX on spores of the microsporidian E. cuniculi, as well as to evaluate its ability to modulate the microbicidal activity of macrophages in vitro. E. cuniculi spores were treated with two concentrations of CTX (2.4 and 4.8 μg/mL) and subsequently cultivated in RK-13 cells for viability analysis. Additionally, macrophages were obtained from adherent peritoneal cells (APerC) after peritoneal washings from BALB/c mice. APerCs were infected with spores of E. cuniculi and after one hour treated with CTX for 3h, for further analysis of the profile of macrophages, cytokine production, viability of macrophages and spores. No fungicidal effect was demonstrated, however, treatment of E. cuniculi spores with 2.4 μg/mL of CTX had a fungistatic effect, with a reduction in proliferation and viability. Spores treated with CTX determined a higher percentage of necrosis in infected RK cells. At the same concentration of CTX (2.4 μg/mL), macrophages challenged with E. cuniculi and treated showed a predominance of the M1 profile, confirmed by the CD40/CD206 ratio, with increased production of the pro-inflammatory cytokine TNF- and IL-6 and higher microbicidal activity confirmed by reduced spore viability demonstrated by lower proliferation ratio. CTX increased death by necrosis of macrophages infected or not with E. cuniculi. The results allow us to conclude that CTX modulated macrophages infected by E. cuniculi profile M1 with greater production of proinflammatory cytokines and with greater microbicidal activity, as well as having a fungistatic effect on spores.
Área de Concentração
Patologia Ambiental e Experimental
Linha de Pesquisa
Patologia Integrada e Translacional
Instituição
Universidade Paulista
Direito de Acesso
Acesso Aberto