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

Abstract

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.

Keywords: Trypanosoma cruzi; miRNAs; placenta; trophoblast differentiation.

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.

Figure 2

HPE can be effectively transfected with miR-512-3p and miR-515-5p mimics and antagomirs. HPE was transfected with 100 nM of miR-512-3p (A) or miR-515-5p (B) antagomirs (A-512 or A-515), mimics (M-512 or M-515), and their respective scrambles (AS or MS) for 24 h. The miRNA expression was determined by means of real-time PCR. All values are given as the means ± SD; data were normalized to control values and analyzed via one-way ANOVA and Dunnett’s post-test. ** p ≤ 0.01; *** p ≤ 0.001.

Figure 3

HPE transfects are localized in the trophoblast. HPE was transfected with 100 nM of AntagomiR-Cy3 for 24 h. Samples were processed using routine fluorescence methods; nuclei were stained with DAPI. White arrows indicate the fluorescence label in the trophoblast. Scale bar: 20 μm.

Figure 4

T. cruzi increases and decreases miR-512-3p and miR-515-5p levels, respectively, in HPE. HPE was transfected with 100 nM of miR-512-3p (A) or miR-515-5p (B) antagomirs (A-512 or A-515), mimics (M-512 or M-515), and their respective scrambles (AS or MS) for 24 h and then challenged with 10⁵ T. cruzi trypomastigotes for 2 h. The miRNA expression was determined by means of real-time PCR. All values are given as the means ± SD; data were normalized to control values and analyzed via one-way ANOVA and Dunnett’s post-test. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001.

Figure 5

T. cruzi does not affect c-FLIP expression. HPE was transfected with 100 nM of miR-512-3p (A,B) antagomirs (A-512), mimics (M-512), and their respective scrambles (AS or MS) for 24 h and then challenged with 10⁵ T. cruzi trypomastigotes for 2 h. The c-FLIP mRNA expression was determined by means of real-time PCR. All values are given as the means ± SD; data were normalized to control values and analyzed via one-way ANOVA and Dunnett’s post-test. * p ≤ 0.05.

Figure 6

MiR-515-5p and miR-512-3p regulate T. cruzi-induced increase in hGCM-1. HPE was transfected with 100 nM of miR-515-5p (A,B) or miR-512-3p (C,D) antagomirs (A-512 or A-515), mimics (M-512 or M-515), and their respective scrambles (AS or MS) for 24 h and then challenged with 10⁵ T. cruzi trypomastigotes for 2 h. The hGCM-1 mRNA expression was determined by means of real-time PCR. All values are given as the means ± SD; data were normalized to control values and analyzed via one-way ANOVA and Dunnett’s post-test. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001.

Figure 7

MiR-512-3p and miR-515-5p regulate T. cruzi-induced increase in hCG. HPE were transfected with 100 nM of miR-512-3p (A,B) or miR-515-5p (C,D) antagomirs (A-512 or A-515), mimics (M-512 or M-515), and their respective scrambles (AS or MS) for 24 h and then challenged with 10⁵ T. cruzi trypomastigotes for 2 h. The hCG mRNA expression was determined by means of real-time PCR. All values are given as the means ± SD; data were normalized to control values and analyzed via one-way ANOVA and Dunnett’s post-test. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001.

Figure 8

MiR-512-3p and miR-515-5p levels determine HPE susceptibility to T. cruzi infection. HPE was transfected with 100 nM of miR-512-3p (A) or miR-515-5p (B) antagomirs (A-512 or A-515), mimics (M-512 or M-515), and their respective scrambles (AS or MS) for 24 h and then challenged with 10⁵ T. cruzi trypomastigotes for 2 h. Parasite DNA was detected using real-time qPCR. Data analysis was performed using the ΔΔCt method. All values are presented as the mean ± SD (of normalized values) and analyzed via one-way ANOVA and Dunnett’st’s post-test. ** p ≤ 0.01; **** p ≤ 0.0001.