Ex Vivo Infection of Human Placental Explants by Trypanosoma cruzi Reveals a microRNA Profile Similar to That Seen in Trophoblast Differentiation

Congenital Chagas disease, caused by the protozoan parasite Trypanosoma cruzi, is responsible for 22.5% of new cases each year. However, placental transmission occurs in only 5% of infected mothers and it has been proposed that the epithelial turnover of the trophoblast can be considered a local placental defense against the parasite. Thus, Trypanosoma cruzi induces cellular proliferation, differentiation, and apoptotic cell death in the trophoblast, which are regulated, among other mechanisms, by small non-coding RNAs such as microRNAs. On the other hand, ex vivo infection of human placental explants induces a specific microRNA profile that includes microRNAs related to trophoblast differentiation such as miR-512-3p miR-515-5p, codified at the chromosome 19 microRNA cluster. Here we determined the expression validated target genes of miR-512-3p and miR-515-5p, specifically human glial cells missing 1 transcription factor and cellular FLICE-like inhibitory protein, as well as the expression of the main trophoblast differentiation marker human chorionic gonadotrophin during ex vivo infection of human placental explants, and examined how the inhibition or overexpression of both microRNAs affects parasite infection. We conclude that Trypanosoma cruzi-induced trophoblast epithelial turnover, particularly trophoblast differentiation, is at least partially mediated by placenta-specific miR-512-3p and miR-515-5p and that both miRNAs mediate placental susceptibility to ex vivo infection of human placental explants. Knowledge about the role of parasite-modulated microRNAs in the placenta might enable their use as biomarkers, as prognostic and therapeutic tools for congenital Chagas disease in the future.


Introduction
Chagas disease, also known as American trypanosomiasis, is caused by the protozoan parasite Trypanosoma cruzi (T. cruzi), and is one of the 13 most neglected tropical diseases [1][2][3]. The parasite can be transmitted from mother to child, causing congenital CD with potential long-term consequences. The World Health Organization estimates that 1,125,000 women of fertile age are infected with T. cruzi, with an incidence of congenital infection of 8668 cases/year in the 21 Latin American countries where Chagas Disease is currently endemic [4]. Furthermore, congenital transmission is responsible for 22.5% of new annual infections in countries with controlled domestic vector infestations [4,5]. In Figure 1. MiR-512-3p-and miR-515-5p = mediated trophoblast differentiation. MiR-512-3p promotes trophoblast differentiation by repressing the caspase 8 inhibitor c-FLIP and, consequently, increases caspase 8 expression, promoting cellular fusion in the trophoblast. Conversely, miR-515-5p inhibits trophoblast differentiation by repressing the transcription factor hGCM-1, which mediates hCG expression.
In this study, we aimed to determine the expression of miR-512-3p-and miR-515-5pregulated genes, specifically hGCM-1 and c-FLIP, as well as the expression of the main trophoblast differentiation marker hCG during ex vivo infection of HPE, and examined how inhibition or overexpression of both miRNAs affects parasite infection.

Discussion
MiRNAs regulate the expression of more than 30% of fundamental genes that, in turn, are involved in critical biological processes, including cellular differentiation and immune responses, determining the success or failure of infection [15,24,25].

Discussion
MiRNAs regulate the expression of more than 30% of fundamental genes that, in turn, are involved in critical biological processes, including cellular differentiation and immune responses, determining the success or failure of infection [15,24,25].
During host-pathogen interactions, different pathogens, including protozoan parasites, modulate the host s gene expression to avoid its clearance and to establish infection [15,26].
In HPE, T. cruzi not only induces a specific mRNA profile [27], it also induces a specific miRNA profile that is different from that of another protozoan parasite, Toxoplasma gondii (T. gondii), which might explain the low congenital transmission rates observed for T. cruzi and the high transmission rates for T. gondii [15]. In that previous study, miR-512-3p and miR-515-5p were validated as miRNAs modulated by T. cruzi and this finding was concordantly confirmed in the present work ( Figure 4). Both miRNAs are interesting molecules since they are encoded in chromosome 19 (C19MC) (19q13.41), which is the largest miRNA cluster in humans and is almost exclusively expressed in the placenta, undifferentiated embryonic stem cells, and germ cells [16,17]. Furthermore, C19MC-derived miRNAs have been associated with placental development [13], pregnancy-related pathologies, and infections [13,16,28,29]. This organ-specific expression of the studied miRNAs makes them attractive molecules for further studies as diagnostic, prognostic, or therapeutic tools for infections. Notably, C19MC miRNAs are among the most abundant miRNAs expressed in human trophoblastic cells, at least in term placenta [17].
Both miRNAs regulate trophoblast differentiation [20,22], a cellular process related to trophoblast epithelial turnover, considered a local placental defense mechanism against T. cruzi [2,8,9,21,30]. The epithelial turnover is considered part of the innate immune system since pathogens, before cell invasion, must attach to the surface of cells. As these cells are continuously eliminated, the attached pathogens are removed [8,31]. Moreover, infected epithelial cells have an alarm system to alert uninfected neighboring cells by transferring danger signals via the gap junction, enabling the epithelium to dispose of infected host cells [31,32]. Danger signals are propagated via nuclear factor kappa B (NFkB) and mitogen-activating protein kinase (MAPK) signaling, pathways that T. cruzi activates in HPE [33,34].
MiR-512-3p regulates trophoblast differentiation by repressing the caspase 8 inhibitor c-FLIP, leading to increased caspase 8 activity [20]. Caspase 8 regulates trophoblast differentiation and apoptotic cell death and is activated by T. cruzi. Moreover, caspase 8 inhibition promotes parasite infection in BeWo cells, evidenced by an increase in the parasite DNA load and the number of parasites per cell [21]. Here, we confirmed that, in HPE, c-FLIP expression is regulated by miR-512-3p, but its expression is not altered by T. cruzi ( Figure 5). In addition, we also confirmed that miR-515-5p regulates its validated target hGCM-1 in HPE and that the parasite modulates its expression ( Figure 6A,B). However, transfection with the miR-515-5p mimic could not prevent the increase in parasite-induced hGCM-1 expression, nor did the inhibition with the antagomir augment its expression. Interestingly, the miR-512-3p antagomir was able to decrease hGCM-1 in spite of the fact that hGCM-1 is not its direct target. However, similarly to the results observed for miR-515-5p, the antagomir could not prevent the parasite-induced increase in hGCM-1 and the miR-512-3p mimics did not alter the T. cruzi-induced increase in hGCM-1 expression ( Figure 6C,D). On the other hand, miR-512-3p mimic ( Figure 7A) and miR-515-5p antagomir ( Figure 7C) transfection increased hCG expression, but only the miR-512-3p antagomir ( Figure 7B) could completely prevent the parasite-induced increase in the main trophoblast differentiation marker. Interestingly, both miR-515-p mimics and antagomirs partially prevented the T. cruzi-induced increase in hCG. It is probable that, in order to prevent the parasiteinduced increase in hGCM-1 and hCG, other miRNAs that share the same seed sequence or target gene are needed, as has been reported for C19MC cluster miRNAs [13,35]. For instance, eight miRNAs belonging to the C19MC cluster are upregulated (miR-521, -520h, -517c, -519d, -517d, -542-3p, -518e, and -519a) in women with preeclampsia [33,36], and hGCM-1 is targeted by at least two other miRNAs (miR-106a and miR-19b) [37], providing evidence for the complex regulation of mRNA expression by miRNAs. In addition, miR-515-5p also represses aromatase P450 (hCYP19A1) [37] and frizzled 5 (Fzd5) [38] genes. In the placenta, aromatase P450 catalyzes the synthesis of estrogens from C 19 steroids that promote trophoblast differentiation in an autocrine manner [37], and Fzd5 is part of the hetero-dimeric receptor family that binds secreted Wingless (Wnt) proteins and promote cell cycle progression and differentiation through the canonical Wnt signaling pathway [35].
Regarding the susceptibility to parasite infection, as expected, the inhibition of miR-512-3p and increased miR-515-5p levels augmented the T. cruzi DNA load in the HPE (Figure 8). Thus, impairing trophoblast differentiation through both miRNAs (Figure 1), the parasite can more easily overcome the placental barrier [8]. Interestingly, the inhibition of miR-515-5p also increased parasite infection ( Figure 8B). MiR-515-5p regulates cellular proliferation through the Wnt pathway [38] as well as Notch1 (neurogenic locus notch homolog protein 1), and its deregulation is related to tumorigenesis [39], the promotion of bacterial growth, and disbalance of the gut microbiota [40]. Importantly, cellular proliferation is also a fundamental part of the trophoblast epithelial turnover process [2,8,10], and its de-regulation alters the normal physiology of the placental barrier. Therefore, any change in the anatomical placental barrier might lead to an increase in parasite infection.
Importantly, knowledge about the role of parasite-modulated microRNAs in the placenta might allow their use as biomarkers in the future, providing prognostic and therapeutic tools for congenital Chagas disease.

Parasite Culture and Harvesting
Semiconfluent VERO cells were incubated with a culture of Y strain epimastigotes (a non-infective cellular form of the parasite) in the late stationary phase, containing about 5% of infective trypomastigotes. Trypomastigotes invade fibroblasts and replicate intracellularly as amastigotes. After 72 h, amastigotes transform into trypomastigotes, which lyse the host cells. The parasites were recovered via low-speed centrifugation (500× g), producing trypomastigotes in the supernatant and amastigotes in the sediment [41].

Human Placental Explant (HPE) Culture and Parasite Infection
Ten human-term placentas were obtained from uncomplicated pregnancies from vaginal or cesarean deliveries. Informed consent for the experimental use of the placenta was provided by each patient as stipulated by the Code of Ethics of the Faculty of Medicine of the University of Chile and Servicio de Salud Metroplitano Norte (Approval number AE 010/2019). The exclusion criteria for the patients were the following: major fetal abnormalities, placental tumor, intrauterine infection, obstetric pathology, or any other maternal disease. The organs were collected in a cold, sterile saline-buffered solution (PBS) and processed no more than 30 min after delivery. The maternal and fetal surfaces were discarded, and villous tissue was obtained from the central part of the cotyledons. The isolated chorionic villi were washed with PBS to remove blood, dissected into approximately 0.5 cm 3 fragments, and co-cultured with T. cruzi trypomastigotes (1 × 10 5 /mL) for 2 h or forskolin (100 uM; as a positive control for trophoblast differentiation) in 1 mL of RPMI culture medium supplemented with complement-inactivated FBS and antibiotics [9,42].

HPE Transfection with miRNAs Mimics and Antagomirs
HPE samples were transfected with 100 nM of mimics or antagomirs of miR-512-3p or miR-515-5p and their respective negative controls by incubating them in 1 mL of RPMI culture medium (without phenol red, supplemented with complement-inactivated FBS and antibiotics) for 24 h at 37 • C [43]. Following transfection, HPE was co-cultivated with T. cruzi trypomastigotes as described above. HPE was collected in RNAlater solution (ThermoFisher Scientific ® , Burlington, ONT, Canada), stored at 4 • C for 24 h and later at −80 • C for posterior miRNA and mRNA isolation. In addition, explants were held at 4 • C in 75% ethanol for DNA extraction. Tissue localization and effective transfection were determined by detecting fluorescently-labeled molecules. Thus, HPE was transfected as described above with 100 nM of a Cy3-conjugated off-target antagomir (Antagomir-Cy3) (abm ® Richmond, BC, Canada) [43], and transfection efficiency was determined by means of fluorescence microscopy [44]. The supernatant in which the different experimental conditions were incubated was kept at −20 • C until processing to evaluate lactate dehydrogenase activity.

Lactate Deshydrogenase Activity
Enzymatic activity was determined using a Lactate Deshydrogenase Cytotoxicity Detection Kit (Cat# MK401, Takara Bio Inc ® , Shiga, Japan) as per the manufacturer's instructions. Briefly, the plates were incubated at room temperature for 30 min in the dark. Then the absorbance was determined with a Varioskan Flash Multimode microplate reader (Thermo Scientific ® , Waltham, MA, USA) at a reference wavelength of 490 nm. Finally, the results were normalized to the values obtained in the control conditions.

Histology
Routine histological methods were used for tissue analysis of the HPE. Samples were embedded in paraffin and stained with hematoxylin and eosin [42,45].  (Table 1), 10.5 µL of nuclease-free water, and 1 µL of cDNA in a 25 µL qRT-PCR reaction. For the quantification of C-Flip, hGCM-1, and hCG mRNA, the 20 µL qRT-PCR reaction contained: 10µL SensiFAST™ SYBR®Hi-ROX Kit (Bioline ®, Heidelberg, Baden Würtemberg, Germany), 1 µL of each 10nM reverse and forward primer ( Table 2), 3 µL nuclease-free water, and 5 µL cDNA. All qRT-PCR reactions were performed in three replicates under the following cycling conditions: initial denaturation at 95 • C for 2 min, followed by 40 cycles of 95 • C for 5 s, 60 • C for 30 s, and a dissociation stage was added, ranging from 60 • C to 95 • C. Gene expressions were calculated using the ∆∆CT relative expression method and normalized to the expression levels of snRNA U6 (RNU6-1) or hGAPDH [46,47].

DNA Amplification via Real-Time PCR
Genomic DNA was extracted from the placental tissue with the Wizard Genomic DNA Purification Kit (Promega ® , Madison, WI, USA) according to the manufacturer's instructions and quantified using a µDropPlate in a Varioskan Flash Multimode Reader (Thermo Scientific ® ). For the amplification of human and parasite DNA, two specific pairs of primers were used for hGADPH and T. cruzi satellite DNA (Table 3). Each reaction mix contained 0.5 µL at 10 nM of each primer (forward and reverse), 1 ng of DNA from samples, 10 µL of SensiFAST™ SYBR ® Hi-ROX Kit (Bioline ® , Heidelberg, Baden Würtemberg, Germany), and H 2 O for a total of 20 µL. Amplification was performed in an ABI Prism 7300 sequence detector (Applied Biosystems ® , Foster City, CA, USA). The cycling programs were as follows: initial denaturation at 95 • C for 3 min, followed by 40 cycles of 95 • C for 5 s, 60 • C for 30 s, and a dissociation stage was added, ranging from 60 • C to 95 • C. Relative quantification analysis of the results was expressed as RQ values using the comparative Control (∆∆Ct) method [42].

Statistical Analysis
All experiments were performed in triplicate using at least three different placentas. Results are expressed as means ± S.D, and experimental data were normalized to control values. The significance of differences was evaluated using one-way ANOVA, followed by Dunnett's post-test.

Conclusions
Our results suggest that T. cruzi-induced trophoblast epithelial turnover, particularly trophoblast differentiation, is at least partially mediated by placenta-specific miR-512-3p and miR-515-5p. In addition, both miRNAs mediate placental susceptibility to ex vivo infection of human placental explants.

Informed Consent Statement:
The studies involving human participants were reviewed and approved by Ethical Committee of the "Servicio de Salud Metropolitano Norte" Santiago de Chile, Chile. The patients/participants provided their written informed consent to participate in this study.

Data Availability Statement:
Data is contained within the article or Supplementary Material.