Cyclooxygenase (COX)-2 Inhibitors Reduce Toxoplasma gondii Infection and Upregulate the Pro-inflammatory Immune Response in Calomys callosus Rodents and Human Monocyte Cell Line

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

Functional evaluation of Toll-Like Receptor 4 (TLR4) in human villous/extravillous trophoblastic cells and gestational third-trimester human placental villi infected by Toxoplasma gondii

Toll-like receptors (TLRs) are receptors that are located on the cell surface or in cell compartments. The objective of this work was to evaluate the influence of the Toll-like receptor 4 (TLR4) pathway on trophoblastic cells, BeWo, JEG-3 and HTR-8/SVneo and on explants of chorionic human villi infected or not with Toxoplasma gondii. BeWo cells infected and treated with LPS, regardless of treatment time, showed a decrease in total parasitism. However, in JEG-3 and HTR8/SVneo cells, there was increased proliferation of the parasite or no significant change in parasitic intracellular growth. In explants of human chorionic villi there was a reduction of total parasitism under the conditions in which the TLR4 pathway was activated for 48 hours by LPS. Starting from cytokine profile analysis, it was observed that, in infected and untreated BeWo cells, there was an increase in IL-10 and a decrease in IFN-γ. However, when treated with LPS, there was an increase in the IFN-γ and reduction of TGF-β1, IL-10 and TNF. In JEG-3 cells, there was an increase in the production of TGF-β1, TNF and IFN-γ, but increased IL-10 after 24 hours and a decrease after 48 hours of LPS treatment. In HTR-8/SVneo, the LPS treatment promoted an increase in the production of IFN-γ, and a decrease in the production of TGF-β1, MIF and TNF. In human chorionic villi, LPS induced IFN-γ and TGF-β1, but reduced the production of TNF, IL-10, MIF and IL-8. It was observed that the inhibition of MyD88 further reduced parasitism in BeWo, however the inhibition of TRIF increased the growth of T. gondii in BeWo, while no significant effect was observed in the other cells. Finally, the inhibition of MyD88 and TRIF increased T. gondii infection in the chorionic villi. It can be concluded that the TLR4 pathway is important for the control of T. gondii replication in the villi and BeWo cells, since inhibition of MyD88 and/or TRIF reversed the activation of TLR4 by LPS, which was not observed in JEG-3 and HTR8/SVneo. This may mean that chorionic villi and transformed cells behave differently in their immune response to Toxoplasma infection.CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível SuperiorDissertação (Mestrado)Toll-like receptors (TLRs) são receptores localizados na superfície celular ou nos compartimentos celulares. O objetivo deste trabalho foi avaliar a influência da via TLR4 em células trofoblásticas, BeWo, JEG-3 e HTR-8/SVneo e em explantes de vilosidades coriônicas humanas infectadas ou não com Toxoplasma gondii. Células BeWo infectadas e tratadas com LPS, independentemente do tempo de tratamento, mostraram uma diminuição no parasitismo total. No entanto, nas células JEG-3 e HTR8/SVneo, houve aumento da proliferação do parasita ou nenhuma mudança significativa no crescimento intracelular parasitário. Nos explantes de vilosidades coriônicas humanas houve redução do parasitismo total nas condições em que a via do TLR4 foi ativada por 48 horas pelo LPS. A partir da análise do perfil de citocinas, observou-se que, nas células BeWo infectadas e não tratadas, houve aumento da IL-10 e diminuição do IFN-γ. No entanto, quando tratados com LPS, houve aumento do IFN-γ e redução de TGF-β1, IL-10 e TNF. Nas células JEG-3, houve um aumento na produção de TGF-β1, TNF e IFN-γ, mas aumentou a IL-10 após 24 horas e uma diminuição após 48 horas de tratamento com LPS. Em HTR-8/SVneo, o tratamento com LPS promoveu aumento na produção de IFN-γ e diminuição na produção de TGF-β1, MIF e TNF. Em vilosidades coriônicas humanas, o LPS induziu IFN-γ e TGF-β1, mas reduziu a produção de TNF, IL-10, MIF e IL-8. Observou-se que a inibição do MyD88 reduziu ainda mais o parasitismo no BeWo, porém a inibição do TRIF aumentou o crescimento do T. gondii na BeWo, enquanto nenhum efeito significativo foi observado nas demais células. Finalmente, a inibição de MyD88 e TRIF aumentou a infecção por T. gondii nas vilosidades coriônicas. Pode-se concluir que a via TLR4 é importante para o controle da replicação de T. gondii nas vilosidades e células BeWo, pois a inibição de MyD88 e/ou TRIF reverteu a ativação de TLR4 por LPS, o que não foi observado em JEG-3 e HTR8/SVneo. Isso pode significar que vilosidades coriônicas e células transformadas se comportam diferentemente em sua resposta imune à infecção por Toxoplasma

Cyclooxygenase (COX)-2 modulates Toxoplasma gondii infection, immune response and lipid droplets formation in human trophoblast cells and villous explants

Abstract Congenital toxoplasmosis is represented by the transplacental passage of Toxoplasma gondii from the mother to the fetus. Our studies demonstrated that T. gondii developed mechanisms to evade of the host immune response, such as cyclooxygenase (COX)-2 and prostaglandin E2 (PGE2) induction, and these mediators can be produced/stored in lipid droplets (LDs). The aim of this study was to evaluate the role of COX-2 and LDs during T. gondii infection in human trophoblast cells and villous explants. Our data demonstrated that COX-2 inhibitors decreased T. gondii replication in trophoblast cells and villous. In BeWo cells, the COX-2 inhibitors induced an increase of pro-inflammatory cytokines (IL-6 and MIF), and a decrease in anti-inflammatory cytokines (IL-4 and IL-10). In HTR-8/SVneo cells, the COX-2 inhibitors induced an increase of IL-6 and nitrite and decreased IL-4 and TGF-β1. In villous explants, the COX-2 inhibitors increased MIF and decreased TNF-α and IL-10. Furthermore, T. gondii induced an increase in LDs in BeWo and HTR-8/SVneo, but COX-2 inhibitors reduced LDs in both cells type. We highlighted that COX-2 is a key factor to T. gondii proliferation in human trophoblast cells, since its inhibition induced a pro-inflammatory response capable of controlling parasitism and leading to a decrease in the availability of LDs, which are essentials for parasite growth

Rottlerin impairs early and late steps of Toxoplasma gondii infection in human trophoblast cells and villous explants

Congenital toxoplasmosis, caused by the opportunistic protozoan parasite T. gondii, can cause stillbirths, miscarriages and fetal abnormalities, as well as encephalitis and chorioretinitis in newborns. Available treatment options rely on antiparasitic drugs that have been linked to serious side effects, high toxicity and the development of drug-resistant parasites. The search for alternative therapeutics to treat this disease without acute toxicity for the mother and child is essential for the advancement of current therapeutic procedures. The present study aimed to unravel the mode of the anti-T. gondii action of Rottlerin, a natural polyphenol with multiple pharmacological properties described. Herein, we further assessed the antiparasitic activity of Rottlerin against T. gondii infection on the human trophoblastic cells (BeWo cells) and, for the first time, on human villous explants. We found that non-cytotoxic doses of Rottlerin impaired early and late steps of parasite infection with an irreversible manner in BeWo cells. Rottlerin caused parasite cell cycle arrest in G1 phase and compromised the ability of tachyzoites to infect new cells, thus highlighting the possible direct action on parasites. An additional and non-exclusive mechanism of action of Rottlerin involves the modulation of host cell components, by affecting lipid droplet formation, mitochondrial function and upregulation of the IL-6 and MIF levels in BeWo cells. Supporting our findings, Rottlerin also controlled T. gondii proliferation in villous explants with low toxicity and reduced the IL-10 levels, a cytokine associated with parasite susceptibility. Collectively, our results highlighted the potential use of Rottlerin as a promising tool to prevent and/or treat congenital toxoplasmosis