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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior The interaction between human extravillous trophoblast cells and macrophages has an important role in the implantation and placentation during a successful pregnancy. However any dysfunction in this communication is associated with pregnancy complications. The aim of this study was investigate the influence of Toxoplasma gondii in the occurrence of apoptosis triggered by macrophages in HTR-8/SVneo cells. For this purpose, HTR-8/SVneo cells were treated or not with the supernatant from uninfected or infected macrophages and then infected or not by T. gondii. Cytokine secretion and soluble FasL were analyzed by ELISA, apoptosis index and expression of Fas/CD95 were determined by flow cytometry and intracellular parasite proliferation was analyzed by enzymatic assay. IL-6 secretion by HTR-8/SVneo cells was synergistically increased with both stimulus, supernatant from macrophages and T. gondii infection. The apoptosis index of infected HTR-8/SVneo cells was decreased in the presence or absence of supernatant from infected macrophage. On the other hand, the apoptosis of infected HTR-8/SVneo cells increased when these cells were treated with supernatant from uninfected macrophages. Also, a low expression of Fas/CD95 and a high soluble FasL release were observed in these conditions. Finally, we did not verify any change in the proliferation of T. gondii. However, there is downmodulation of apoptosis in HTR-8/SVneo cells by the parasite which probably relates to favoring its establishment in the host cell, while the functional activities of macrophages toward restoring these death rates cell, to keep gestational events of invasion and placentation appropriate. All together, these results contribute to better understanding of the mechanisms of cellular communication existing between the extravillous trophoblastic cells and macrophages, and demonstrate that infection with T. gondii can interfere with this interaction through modulation of cell death by apoptosis. A interação entre células trofoblásticas extravilosas humanas e macrófagos tem importante papel tanto na implantação quanto na placentação no curso de uma gestação de sucesso. No entanto, alterações nesta comunicação, causada ou não por agentes patogênicos, podem causar problemas gestacionais. Assim, o objetivo desse estudo foi investigar a influência de Toxoplasma gondii na ocorrência da apoptose induzida por macrófagos em células trofoblásticas extravilosas humanas. Para este propósito, células trofoblásticas extravilosas da linhagem HTR-8/SVneo foram tratadas ou não com sobrenadante de macrófagos e/ou posteriormente infectadas por T. gondii. A secreção de citocinas e a liberação de FasL solúvel foram verificadas pelo ELISA, o índice de apoptose e a expressão de Fas/CD95 foram determinadas por meio do citômetro de fluxo, e a proliferação intracelular do parasito foi analisada pelo ensaio enzimático. Nossos resultados mostraram que a produção de IL-6 por células HTR-8/SVneo foi sinergicamente aumentada na presença de ambos estímulos: sobrenadante de macrófagos e infecção por T. gondii. O índice de apoptose de células HTR-8/SVneo infectadas diminuiu na presença ou ausência do sobrenadante de macrófagos infectados. Por outro lado, a apoptose de células HTR-8/SVneo infectadas aumentou quando essas células foram tratadas com o sobrenadante de macrófagos não infectados. Além disso, a baixa expressão de Fas/CD95 e a alta liberação de FasL solúvel foram observadas nessas condições. Finalmente, não foi verificada nenhuma mudança na proliferação de T. gondii nas diferentes situações experimentais. No entanto, observa-se uma regulação negativa da apoptose em células HTR-8/SVneo pelo parasito que provavelmente relaciona-se com o favorecimento de seu estabelecimento na célula hospedeira, enquanto as atividades funcionais dos macrófagos foram no sentido de restaurar esses índices de morte celular, para manter os eventos gestacionais de invasão e placentação adequados. Assim, nossos resultados contribuíram para melhor entendimento dos mecanismos de comunicação celular existente entre as células trofoblásticas extravilosas e macrófagos, além de demonstrar que a infecção por T. gondii pode interferir nessa interação por meio da modulação da morte celular por apoptose. Mestre em Imunologia e Parasitologia Aplicadas

The combination of sulfadiazine and pyrimethamine plus folinic acid is the conventional treatment for congenital toxoplasmosis. However, this classical treatment presents teratogenic effects and bone marrow suppression. In this sense, new therapeutic strategies are necessary to reduce these effects and improve the control of infection. In this context, biogenic silver nanoparticles (AgNp-Bio) appear as a promising alternative since they have antimicrobial, antiviral, and antiparasitic activity. The purpose of this study to investigate the action of AgNp-Bio in BeWo cells, HTR-8/SVneo cells and villous explants and its effects against Toxoplasma gondii infection. Both cells and villous explants were treated with different concentrations of AgNp-Bio or combination of sulfadiazine + pyrimethamine (SDZ + PYZ) in order to verify the viability. After, cells and villi were infected and treated with AgNp-Bio or SDZ + PYZ in different concentrations to ascertain the parasite proliferation and cytokine production profile. AgNp-Bio treatment did not reduce the cell viability and villous explants. Significant reduction was observed in parasite replication in both cells and villous explants treated with silver nanoparticles and classical treatment. The AgNp-Bio treatment increased of IL-4 and IL-10 by BeWo cells, while HTR8/SVneo cells produced macrophage migration inhibitory factor (MIF) and IL-4. In the presence of T. gondii, the treatment induced high levels of MIF production by BeWo cells and IL-6 by HTR8SV/neo. In villous explants, the AgNp-Bio treatment downregulated production of IL-4, IL-6, and IL-8 after infection. In conclusion, AgNp-Bio can decrease T. gondii infection in trophoblast cells and villous explants. Therefore, this treatment demonstrated the ability to reduce the T. gondii proliferation with induction of inflammatory mediators in the cells and independent of mediators in chorionic villus which we consider the use of AgNp-Bio promising in the treatment of toxoplasmosis in BeWo and HTR8/SVneo cell models and in chorionic villi.

A interação entre os trofoblastos extravilosos humanos e as células endoteliais desempenha um importante papel nos processos de implantação embrionária e placentação. Alterações nos padrões de comunicação entre estas duas populações celulares estão associadas com complicações gestacionais, neste contexto, a infecção por Toxoplasma gondii pode ser um problema em potencial para essa relação. Assim, o objetivo desse estudo foi avaliar os fenômenos de invasão, migração e morte celular de células trofoblásticas extravilosas (linhagem HTR-8/SVneo) sob a influência de células endoteliais venosas (linhagem HUVEC) infectadas por T. gondii. As células HTR-8/SVneo foram co-cultivadas com as células HUVEC infectadas ou não por T. gondii, no sentido de avaliar a produção de citocinas detectadas pelo método de ELISA, bem como funções biológicas das células trofoblásticas extravilosas. A capacidade invasiva das células trofoblásticas foi analisada diante a utilização de insertos de transwell previamente cobertos com Matrigel, a capacidade migratória dessas células foi verificado pelo ensaio de scratch e o índice de morte celular foi mensurado através do método de citometria de fluxo. A interação entre as células HTR-8/SVneo e HUVEC induziram a secreção de IL-6 e IL-8 pelas células trofoblásticas. Já a secreção de MIF foi diminuída pelas células HTR-8/SVneo após a co-cultura com as células HUVEC não infectadas, e maiores níveis dessa citocina foram observados após a co-cultura com células HUVEC infectadas. O estímulo da co-cultura com células HUVEC não infectadas reduziu a capacidade invasiva e aumentou a migração de células HTR-8/SVneo. De forma contrária, as condições de células HUVEC infectadas aumentaram a capacidade invasiva, reduziram a migração e induziram o índice de morte celular das células trofoblásticas. O tratamento com ISO-1, inibidor altamente específico de MIF, alterou os eventos relacionados com a capacidade invasiva e morte celular das células HTR-8/SVneo quando comparados com cada condição respectiva sem o tratamento com inibidor de MIF. A comunicação entre as células trofoblásticas extravilosas e células endoteliais venosas foi modificada pela infecção por T. gondii e a indução da produção de MIF pelas células HTR-8/SVneo juntamente com a alteração dos índices de morte celular, podem ter sido alguns dos fatores responsáveis pela variação das funções trofoblásticas relacionadas à capacidade invasiva e migratória das células trofoblásticas extravilosas. The interaction between human extravillous trophoblast and human endothelial cells has an important role in embryonic implantation and placentation. Changes in communication patterns between these two cell populations are associated with pregnancy complications, in this context, Toxoplasma gondii infection can be a potential problem in this crosstalk. Therefore, the aim of this study was to evaluate the occurrence of invasion, migration and cell death of extravillous trophoblast cells (HTR-8/SVneo cell line) under influence of infected human venous endothelial cell (HUVEC) by T. gondii. HTR-8/SVneo cells were co-cultured with T. gondii-infected or uninfected HUVECs in order to analyze cytokine production by ELISA as well biological functions of extravillous trophoblast cells. The invasive capacity of trophoblastic cells was analyzed using transwell inserts previously covered with Matrigel, the migratory capacity of these cells was verified by scratch method and the cell death index was measured by the flow cytometry. HTR-8/SVneo cells and HUVECs interaction induced secretion of IL-6 and IL-8 by trophoblastic cells. MIF secretion was downregulated by HTR-8/SVneo cells after co-culture with uninfected HUVECs, but upregulated MIF levels were detected after co-culture with infected HUVECs. Stimuli of uninfected HUVECs co-culture reduced invasive capacity and increased migration of HTR-8/SVneo cells. Otherwise, infected HUVECs condition enhanced invasive capability, reduced migration, induced cell death index of trophoblastic cells. The treatment with ISO-1, highly specific MIF inhibitor, altered the events related to invasive capacity and cell death of HTR-8/SVneo when compared to each conditions without treatment with MIF inhibitor. The extravillous trophoblast-venous endothelial cells crosstalk was changed by T. gondii infection and induction of MIF production by HTR-8/SVneo cells together with changes in cell death index, might be some of the factors responsible for alteration in trophoblastic functions related to migratory and invasive capacity of extravillous trophoblastic cells. Tese (Doutorado)

Toxoplasma gondii is able to infect a wide range of vertebrates, including humans. Studies show that cyclooxygenase-2 (COX-2) is a modulator of immune response in multiple types of infection, such as Trypanosoma cruzi. However, the role of COX-2 during T. gondii infection is still unclear. The aim of this study was to investigate the role of COX-2 during infection by moderately or highly virulent strains of T. gondii in Calomys callosus rodents and human THP-1 cells. C. callosus were infected with 50 cysts of T. gondii (ME49), treated with COX-2 inhibitors (meloxicam or celecoxib) and evaluated to check body weight and morbidity. After 40 days, brain and serum were collected for detection of T. gondii by real-time PCR and immunohistochemistry or cytokines by CBA. Furthermore, peritoneal macrophages or THP-1 cells, infected with RH strain or uninfected, were treated with meloxicam or celecoxib to evaluate the parasite proliferation by colorimetric assay and cytokine production by ELISA. Finally, in order to verify the role of prostaglandin E2 in COX-2 mechanism, THP-1 cells were infected, treated with meloxicam or celecoxib plus PGE2, and analyzed to parasite proliferation and cytokine production. The data showed that body weight and morbidity of the animals changed after infection by T. gondii, under both treatments. Immunohistochemistry and real-time PCR showed a reduction of T. gondii in brains of animals treated with both COX-2 inhibitors. Additionally, it was observed that both COX-2 inhibitors controlled the T. gondii proliferation in peritoneal macrophages and THP-1 cells, and the treatment with PGE2 restored the parasite growth in THP-1 cells blocked to COX-2. In the serum of Calomys, upregulation of pro-inflammatory cytokines was detected, while the supernatants of peritoneal macrophages and THP-1 cells demonstrated significant production of TNF and nitrite, or TNF, nitrite and MIF, respectively, under both COX-2 inhibitors. Finally, PGE2 treatment in THP-1 cells triggered downmodulation of pro-inflammatory mediators and upregulation of IL-8 and IL-10. Thus, COX-2 is an immune mediator involved in the susceptibility to T. gondii regardless of strain or cell types, since inhibition of this enzyme induced control of infection by upregulating important pro-inflammatory mediators against Toxoplasma.