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Master's Dissertation
DOI
Document
Author
Full name
Anna Cláudia Calvielli Castelo Branco
Institute/School/College
Knowledge Area
Date of Defense
Published
São Paulo, 2019
Supervisor
Committee
Sato, Maria Notomi (President)
Campa, Ana
Hinds, Luciana Barros de Arruda
Martins, Joilson de Oliveira
Title in Portuguese
Ativação de vilos placentários humanos com agonista de receptor toll-like 4 na infecção in vitro por Zika vírus
Keywords in Portuguese
células de Hofbauer
imunomodulação
inflamação
receptor Toll-like
Zika vírus
Abstract in Portuguese
O Zika vírus (ZIKV) pertence à família flaviviridae que inclui espécies transmitidas principalmente por mosquitos Aedes sp. No Brasil, casos de microcefalia fetal foram associados à infecção congênita por ZIKV. Vários mecanismos de imunorregulação operantes durante a gestação como a inflamação gerada e resposta antiviral são vitais para o entendimento da infecção congênita do ZIKV. Desta forma, é proposto avaliar se a inflamação via ativação do receptor Toll-like 4 (TLR4-LPS) em vilos placentários humanos e em modelo murino interfere na infecção por ZIKV. Para isso, explantes de vilos placentários humanos de 3o trimestre foram ativados com LPS e posteriormente infectados com ZIKV. Nossos achados mostram que a ativação com LPS é capaz de aumentar a replicação de ZIKV em vilos placentários e de elevar a atividade enzimática da lactato desidrogenase, indicativo de ativação celular. A replicação viral foi inibida com o tratamento dos explantes com um antioxidante, Naringenina, mostrando potencial terapêutico. A ativação placentária com LPS inibiu a via IRF-3, diminuindo a atividade antiviral e aumentando a clivagem proteica do componente da via de autofagia LC3, sendo que estes mecanismos podem estar relacionados com o aumento da replicação viral. Em paralelo, foi detectado aumento da produção de citocinas inflamatórias e hiperplasia das células de Hofbauer nos explantes inflamados e infectados, indicando que estes macrófagos fetais podem ser um nicho de replicação viral. Em modelo murino, evidenciamos que a inoculação intravaginal de LPS em fêmeas prenhas previamente à infecção com ZIKV é capaz de promover o aumento de carga viral no cérebro das mães e da prole, com agravamento do quadro de microcefalia nos fetos. Em conjunto, os dados mostram que em modelo de explante placentário humano e, in vivo , em camundongos, a inflamação é fator predisponente da replicação do ZIKV. As estratégias imunomoduladoras mostram potencial terapêutico, atenuando a resposta inflamatória e a diminuição da replicação viral.
Title in English
Activation of human placental villi with toll-like receptor 4 agonist during in vitro infection by Zika vírus
Keywords in English
Zika virus; inflammation; Toll-like receptor; Hofbauer cells; immunomodulation
Abstract in English
The Zika virus (ZIKV) belongs to the flaviviridae family that includes species transmitted mainly by mosquitoes Aedes sp. In Brazil, cases of fetal microcephaly were associated with ZIKV congenital infection. Several mechanisms of immunoregulation operative during gestation as the inflammation generated and antiviral response are vital for the understanding of congenital ZIKV infection. Thus, it is proposed to evaluate whether inflammation via Toll-like receptor 4 (TLR4-LPS) activation in human and murine placental villi interferes with ZIKV infection. For this, human placental explants of 3rd trimester were activated with LPS and later infected with ZIKV. Our findings show that LPS activation is capable of increasing ZIKV replication in placental villi and elevating the enzymatic activity of lactate dehydrogenase, indicative of cellular activation. Viral replication was inhibited with the treatment of explants with an antioxidant, Naringenin, showing therapeutic potential. LPS placental activation inhibited the IRF-3 pathway, decreasing antiviral activity and increasing protein cleavage of the autophagy pathway component LC3, and these mechanisms may be related to increased viral replication. In parallel, increased production of inflammatory cytokines and Hofbauer cell hyperplasia were detected in inflamed and infected explants, indicating that these fetal macrophages may be a niche for viral replication. In the murine model, we demonstrated that the intravaginal inoculation of LPS in pregnant females prior to infection with ZIKV is able to promote the increase of viral load in the brains of mothers and offspring, with worsening of the microcephaly in fetuses. Together, the data show that in the model of human placental explant and, in vivo, in mice, inflammation is a predisposing factor of ZIKV replication. Immunomodulatory strategies show therapeutic potential, attenuating the inflammatory response and decreasing viral replication.
 
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Release Date
2021-04-28
Publishing Date
2019-05-07
 
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