Ana Liempi

Trypanosoma cruzi induces trophoblast differentiation: A potential local antiparasitic mechanism of the human placenta?

INTRODUCTION The congenital transmission of Trypanosoma cruzi (T. cruzi) is responsible for one-third of new Chagas disease cases each year. During congenital transmission, the parasite breaks down the placental barrier formed by the trophoblast, basal laminae and villous stroma. The observation that only 5% of infected mothers transmit the parasite to the fetus implies that the placenta may impair parasite transmission. The trophoblast undergoes continuous epithelial turnover, which is considered part of innate immunity. Therefore, we propose that T. cruzi induces differentiation in the trophoblast as part of a local antiparasitic mechanism of the placenta. METHODS We analyzed β-human chorionic gonadotropin (β-hCG) and syncytin protein expression in HPCVE and BeWo cells using immunofluorescence and western blotting. Additionally, β-hCG secretion into the culture medium was measured by ELISA. We assessed the differentiation of trophoblastic cells in BeWo cells using the two-color fusion assay and by determining desmoplakin re-distribution. RESULTS T. cruzi trypomastigotes induce β-hCG secretion and protein expression as well as syncytin protein expression in HPCVE and BeWo cells. Additionally, the parasite induces the trophoblast fusion of BeWo cells. DISCUSSION T. cruzi induces differentiation of the trophoblast, which may contribute to increase the trophoblast turnover. The turnover could be a component of local antiparasitic mechanisms in the human placenta.

A local innate immune response against Trypanosoma cruzi in the human placenta: The epithelial turnover of the trophoblast

Congenital Chagas disease, caused by Trypanosoma cruzi, is partially responsible for the progressive globalization of Chagas disease despite of its low transmission rate. The probability of congenital transmission depends on complex interactions between the parasite, the maternal and fetus/newborn immune responses and placental factors, being the latter the least studied one. During transplacental transmission, the parasite must cross the placental barrier where the trophoblast, a continuous renewing epithelium, is the first tissue to have contact with the parasite. Importantly, the epithelial turnover is considered part of the innate immune system since pathogens, prior to cell invasion, must attach to the surface of cells. The trophoblast turnover involves cellular processes such as proliferation, differentiation and apoptotic cell death, all of them are induced by the parasite. In the present review, we analyze the current evidence about the trophoblast epithelial turnover as a local placental innate immune response.

Toxic and therapeutic effects of Nifurtimox and Benznidazol on Trypanosoma cruzi ex vivo infection of human placental chorionic villi explants

Nifurtimox (Nfx) and Benznidazole (Bnz) are the only available drugs in use for the treatment of Chagas disease. These drugs are recommended but not fully validated in evidence-based medicine and reports about the differential toxicity of both drugs are controversial. Here, we evaluated the toxic and therapeutic effects of Nfx and Bnz on human placental chorionic villi explants (HPCVE) during ex vivo infection of Trypanosoma cruzi, performing histopathological, histochemical, immunohistochemical as well as immunofluorescence analysis of the tissue. Additionally, we determined the effect of both drugs on parasite load by real time PCR. Bnz prevents the parasite induced tissue damage in ex vivo infected HPCVE compared to Nfx, which is toxic per se. The presence of T. cruzi antigens and DNA in infected explants suggests that these drugs do not impair parasite invasion into the HPCVE. Additionally, our results confirm reports suggesting that Bnz is less toxic than Nfx and support the need for the development of more effective and better-tolerated drugs.

Simvastatin and Benznidazole-Mediated Prevention of Trypanosoma cruzi-Induced Endothelial Activation: Role of 15-epi-lipoxin A4 in the Action of Simvastatin.

Trypanosoma cruzi is the causal agent of Chagas Disease that is endemic in Latin American, afflicting more than ten million people approximately. This disease has two phases, acute and chronic. The acute phase is often asymptomatic, but with time it progresses to the chronic phase, affecting the heart and gastrointestinal tract and can be lethal. Chronic Chagas cardiomyopathy involves an inflammatory vasculopathy. Endothelial activation during Chagas disease entails the expression of cell adhesion molecules such as E-selectin, vascular cell adhesion molecule-1 (VCAM-1) and intercellular cell adhesion molecule-1 (ICAM-1) through a mechanism involving NF-κB activation. Currently, specific trypanocidal therapy remains on benznidazole, although new triazole derivatives are promising. A novel strategy is proposed that aims at some pathophysiological processes to facilitate current antiparasitic therapy, decreasing treatment length or doses and slowing disease progress. Simvastatin has anti-inflammatory actions, including improvement of endothelial function, by inducing a novel pro-resolving lipid, the 5-lypoxygenase derivative 15-epi-lipoxin A4 (15-epi-LXA4), which belongs to aspirin-triggered lipoxins. Herein, we propose modifying endothelial activation with simvastatin or benznidazole and evaluate the pathways involved, including induction of 15-epi-LXA4. The effect of 5 μM simvastatin or 20 μM benznidazole upon endothelial activation was assessed in EA.hy926 or HUVEC cells, by E-selectin, ICAM-1 and VCAM-1 expression. 15-epi-LXA4 production and the relationship of both drugs with the NFκB pathway, as measured by IKK-IKB phosphorylation and nuclear migration of p65 protein was also assayed. Both drugs were administered to cell cultures 16 hours before the infection with T. cruzi parasites. Indeed, 5 μM simvastatin as well as 20 μM benznidazole prevented the increase in E-selectin, ICAM-1 and VCAM-1 expression in T. cruzi-infected endothelial cells by decreasing the NF-κB pathway. In conclusion, Simvastatin and benznidazole prevent endothelial activation induced by T. cruzi infection, and the effect of simvastatin is mediated by the inhibition of the NFκB pathway by inducing 15-epi-LXA4 production.

Simvastatin Attenuates Endothelial Activation through 15-Epi-Lipoxin A4 Production in Murine Chronic Chagas Cardiomyopathy.

ABSTRACT Current treatments for chronic Chagas cardiomyopathy, a disease with high mortality rates and caused by the protozoan Trypanosoma cruzi, are unsatisfactory. Myocardial inflammation, including endothelial activation, is responsible for the structural and functional damage seen in the chronic phase. The clinical efficacy of benznidazole could be improved by decreasing chronic inflammation. Statins, which have anti-inflammatory properties, may improve the action of benznidazole. Here, the action of simvastatin in a murine model of chronic Chagas cardiomyopathy and the link with the production of the proresolving eicosanoid 15-epi-lipoxin A4, produced by 5-lipoxygenase, are evaluated. Simvastatin decreased the expression of the adhesion molecules E-selectin, intracellular adhesion molecule type 1 (ICAM-1), and vascular cell adhesion molecule type 1 (VCAM-1) in T. cruzi-infected mice. However, when this drug was administered to 5-lipoxygenase-deficient mice, the anti-inflammatory effect was not observed unless exogenous 15-epi-lipoxin A4 was administered. Thus, in chronic Chagas disease, 5-epi-lipoxin A4 induced by simvastatin treatment could improve the pathophysiological condition of patients by increasing the trypanocidal action of benznidazole.

Ex vivo infection of human placental chorionic villi explants with Trypanosoma cruzi and Toxoplasma gondii induces different Toll-like receptor expression and cytokine/chemokine profiles

Trypanosoma cruzi and Toxoplasma gondii present, respectively, low and high congenital transmission rates. The placenta as an immune regulatory organ expresses TLRs, leading to the secretion of cytokines. Both parasites are recognized by TLR‐2, TLR‐4, and TLR‐9. Here, we studied if the parasites induce differences in TLR protein expression, cytokine profiles, and whether receptor inhibition is related to parasite infection.

Trypanosoma cruzi infectivity assessment in "in vitro" culture systems by automated cell counting.

Chagas disease is an endemic, neglected tropical disease in Latin America that is caused by the protozoan parasite Trypanosoma cruzi. In vitro models constitute the first experimental approach to study the physiopathology of the disease and to assay potential new trypanocidal agents. Here, we report and describe clearly the use of commercial software (MATLAB(®)) to quantify T. cruzi amastigotes and infected mammalian cells (BeWo) and compared this analysis with the manual one. There was no statistically significant difference between the manual and the automatic quantification of the parasite; the two methods showed a correlation analysis r(2) value of 0.9159. The most significant advantage of the automatic quantification was the efficiency of the analysis. The drawback of this automated cell counting method was that some parasites were assigned to the wrong BeWo cell, however this data did not exceed 5% when adequate experimental conditions were chosen. We conclude that this quantification method constitutes an excellent tool for evaluating the parasite load in cells and therefore constitutes an easy and reliable ways to study parasite infectivity.

Old yellow enzyme from Trypanosoma cruzi exhibits in vivo prostaglandin F-2 alpha synthase activity and has a key role in parasite infection and drug susceptibility

The discovery that trypanosomatids, unicellular organisms of the order Kinetoplastida, are capable of synthesizing prostaglandins raised questions about the role of these molecules during parasitic infections. Multiple studies indicate that prostaglandins could be related to the infection processes and pathogenesis in trypanosomatids. This work aimed to unveil the role of the prostaglandin F2α synthase TcOYE in the establishment of Trypanosoma cruzi infection, the causative agent of Chagas disease. This chronic disease affects several million people in Latin America causing high morbidity and mortality. Here, we propose a prokaryotic evolutionary origin for TcOYE, and then we used in vitro and in vivo experiments to show that T. cruzi prostaglandin F2α synthase plays an important role in modulating the infection process. TcOYE overexpressing parasites were less able to complete the infective cycle in cell culture infections and increased cardiac tissue parasitic load in infected mice. Additionally, parasites overexpressing the enzyme increased PGF2α synthesis from arachidonic acid. Finally, an increase in benznidazole and nifurtimox susceptibility in TcOYE overexpressing parasites showed its participation in activating the currently anti-chagasic drugs, which added to its observed ability to confer resistance to hydrogen peroxide, highlights the relevance of this enzyme in multiple events including host–parasite interaction.

Differential infectivity of two Trypanosoma cruzi strains in placental cells and tissue

Congenital Chagas disease, caused by Trypanosoma cruzi (T. cruzi), has become epidemiologically relevant. The probability of congenital transmission depends on the maternal and developing fetal/newborn immune responses, placental factors and importantly, the virulence of the parasite. It has been proposed, that different genotypes of T. cruzi and their associated pathogenicity, virulence and tissue tropism may play an important role in congenital infection. Since there is no laboratory or animal model that recapitulates the complexities of vertical transmission in humans, here we studied parasite infectivity in human placental explants (HPE) as well as in the human trophoblast-derived cell line BeWo of the Y(DTU II) and the VD (TcVI) T. cruzi strains; the latter was isolated from a human case of congenital infection. Our results show that the VD strain is more infective and pathogenic than the Y strain, as demonstrated by qPCR and cell counting as well as by histopathological analysis. The present study constitutes the first approach to study the relationship between parasite two parasite strains from different genotypes and the infection efficiency in human placenta.

The immune response against Trypanosoma cruzi in the human placenta

Congenital Chagas disease, caused by Trypanosoma cruzi ( T. cruzi ), is partially responsible for the increasing globalization of Chagas disease despite its low transmission. During congenital transmission, the parasite reaches the fetus by crossing the placental barrier. However, the success or impairment of congenital transmission of the parasite is the product of a complex interaction between the parasite, the maternal and fetus/newborn immune responses and placental factors. There is other evidence apart from the low congenital transmission rates, which suggests the presence of defense mechanisms against T. cruzi . Thus, the typical amastigote nests (intracellular parasites) cannot be observed in placentas from mothers with chronic Chagas disease nor in human placental chorionic villi explants infected in vitro with the parasite. In the latter, only a few parasite antigens and DNA are identified. Accordingly, other infections of the placenta are not commonly observed. All these evidences suggest that the placenta can mount defense mechanisms against T. cruzi .