Avaliação da expressão de receptores celulares e da produção de citocinas e eicosanoides por neutrófilos humanos em resposta a glicoproteína gp43 do Paracoccidioides brasiliensis
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Data
2019-07-12
Autores
Gardizani, Taiane Priscila [UNESP]
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Universidade Estadual Paulista (Unesp)
Resumo
A paracoccidioidomicose (PCM) é uma doença granulomatosa sistêmica, prevalente na América Latina, que tem por agentes etiológicos diferentes espécies de Paracoccidioides. Entre eles encontra-se o Paracoccidioides brasiliensis (P. brasiliensis), espécie composta por um complexo de agrupamentos geneticamente isolados, classificados como espécies filogenéticas: S1, PS2, PS3 e PS4. O P. brasiliensis possui distribuído pela parede fúngica e dentro de vesículas citoplasmáticas uma glicoproteína de 43KDa, a gp43. Esta molécula é considerada como o principal componente antigênico produzido e secretado pelo fungo, a qual é encontrada no soro de pacientes acometidos pela PCM e, está associada aos fatores de virulência e escape do patógeno. Sabendo disso, tornou-se extremamente importante a avaliação da interação dessa proteína com as células do sistema imune inato e sua consequente atuação sobre os mecanismos moduladores celulares. Assim, nosso estudo avaliou os neutrófilos polimorfonucleares (PMNs) que estão presentes em abundância desde o início e durante a infecção pelo Paracoccidioides, e que apresentam além de suas ações efetoras diretas contra o fungo, uma importante ação moduladora da resposta imune. Estas ações envolvem o reconhecimento do fungo por Receptores de Reconhecimento de Padrão (PRRs) que culminam em ativação celular com consequente produção e liberação de diferentes mediadores inflamatórios. Dessa maneira, o presente trabalho avaliou o envolvimento dos receptores celulares TLR2 e TLR4 no reconhecimento e na produção de citocinas e eicosanoides por PMNs humanos de indivíduos saudáveis estimulados com a gp43. As células foram inicialmente incubadas na presença ou ausência de anticorpos monoclonais anti-TLR2 e anti-TLR4, individualmente ou em conjunto, por 2h, a 37°C, em uma atmosfera de 5% CO2, para o bloqueio dos receptores. Em seguida, os PMNs foram estimulados ou não com a gp43 (20ng/ml) por 4h, nas mesmas condições anteriormente citadas. A expressão de TLR2 e TLR4 bem como a detecção intracitoplasmática de IL-17A e IL-4 foram feitas por citometria de fluxo. Já a concentração de PGE2, LTB4, IL-6, IL-10, IL-12, IFN-γ e TNF-α foi avaliada em sobrenadante de cultura por ELISA. Nossos resultados mostraram que a gp43 interage com TLR2 e TLR4, induzindo a liberação de PGE2, mediada por TLR2, e IL-17A, via TLR4. Tais dados corroboram a ação da gp43 como um importante fator de virulência do P. brasiliensis, uma vez que estimulou os PMNs a produzirem PGE2 e IL-17A, mediadores envolvidos na susceptibilidade do hospedeiro à infecção fúngica na PCM.
Paracoccidioidomycosis (PCM) is a systemic granulomatous disease, prevalent in Latin America and has as etiological agents Paracoccidioides spp. One of them is Paracoccidioides brasiliensis (P. brasiliensis) composed by a complex of genetically isolated clusters, classified as phylogenetic species: S1, PS2, PS3 and PS4. P. brasiliensis present along the cell wall and inside cytoplasmic vesicles a 43 KDa glycoprotein, known as gp43. This molecule is recognized as the main antigenic component produced and secreted by the fungus into sera of PCM patients and is associated with the escape and virulence factors of the pathogen. Thereby, becomes extremely important to evaluate the interaction mechanisms of this protein with cells from the innate immune system and their consequent action on cellular functions. Polymorphonuclear neutrophils (PMNs) has prominent participation in PCM, once these cells are present in a great amount at the beginning and during Paracoccidioides infection. PMNs perform effector actions directly against the fungi as well as an important immune modulatory function. Such actions involve the recognition of fungal components by pattern recognition receptors (PRRs) that leads to cell activation and consequent production of inflammatory mediators. So, the present study evaluated the involvement of TLR2 and TLR4 on cytokines and eicosanoids release by PMNs from healthy human stimulated with gp43. Cells were initially incubated in the presence or absence of monoclonal antibodies anti-TLR2 and anti-TLR4, individually or in combination, for 2h, at 37°C, in a 5% CO2 atmosphere, for receptor blockage. PMNs were then stimulated or not with gp43 (20 ng/ml) during 4h, at the same conditions. The expression of TLR2 and TLR4 as well the intracytoplasmic detection of IL-17A and IL-4 were done by flow cytometry. The concentration of PGE2, LTB4, IL-6, IL-10, IL-12, IFN-γ and TNF-α was measured in cultures supernatants by ELISA. Our results showed that gp43 interacts with TLR2 and TLR4 as also induced the release of PGE2 via TLR2 and the production of IL-17A through TLR4. Therefore, our data corroborate the gp43 action as the main virulence factor of P. brasiliensis, once it stimulated PMNs to release PGE2 and IL-17A, which are inflammatory mediators responsible to enhance the host susceptibility to the fungal infection in PCM.
Paracoccidioidomycosis (PCM) is a systemic granulomatous disease, prevalent in Latin America and has as etiological agents Paracoccidioides spp. One of them is Paracoccidioides brasiliensis (P. brasiliensis) composed by a complex of genetically isolated clusters, classified as phylogenetic species: S1, PS2, PS3 and PS4. P. brasiliensis present along the cell wall and inside cytoplasmic vesicles a 43 KDa glycoprotein, known as gp43. This molecule is recognized as the main antigenic component produced and secreted by the fungus into sera of PCM patients and is associated with the escape and virulence factors of the pathogen. Thereby, becomes extremely important to evaluate the interaction mechanisms of this protein with cells from the innate immune system and their consequent action on cellular functions. Polymorphonuclear neutrophils (PMNs) has prominent participation in PCM, once these cells are present in a great amount at the beginning and during Paracoccidioides infection. PMNs perform effector actions directly against the fungi as well as an important immune modulatory function. Such actions involve the recognition of fungal components by pattern recognition receptors (PRRs) that leads to cell activation and consequent production of inflammatory mediators. So, the present study evaluated the involvement of TLR2 and TLR4 on cytokines and eicosanoids release by PMNs from healthy human stimulated with gp43. Cells were initially incubated in the presence or absence of monoclonal antibodies anti-TLR2 and anti-TLR4, individually or in combination, for 2h, at 37°C, in a 5% CO2 atmosphere, for receptor blockage. PMNs were then stimulated or not with gp43 (20 ng/ml) during 4h, at the same conditions. The expression of TLR2 and TLR4 as well the intracytoplasmic detection of IL-17A and IL-4 were done by flow cytometry. The concentration of PGE2, LTB4, IL-6, IL-10, IL-12, IFN-γ and TNF-α was measured in cultures supernatants by ELISA. Our results showed that gp43 interacts with TLR2 and TLR4 as also induced the release of PGE2 via TLR2 and the production of IL-17A through TLR4. Therefore, our data corroborate the gp43 action as the main virulence factor of P. brasiliensis, once it stimulated PMNs to release PGE2 and IL-17A, which are inflammatory mediators responsible to enhance the host susceptibility to the fungal infection in PCM.
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Palavras-chave
gp43, neutrófilos polimorfonucleares, TLR2, TLR4, citocinas, eicosanoides, polymorphonuclear neutrophils, cytokines, eicosanoids