Trypanosoma cruzi-derived exovesicles contribute to parasite infection, tissue damage, and apoptotic cell death during ex vivo infection of human placental explants

Figures

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  Figure 1. TcEVs increase the parasite load and cause tissue damage in HPEs. HPEs were co-cultured for 24 hours in the presence and absence of 0.2 ug/ml of TcEVs or iTcEVs and in presence and absence of 105 parasites/ml. Additionally, HPE were also pre-incubated for 2 hours with TcEVs or iTcEVs and then challenged with T. cruzi. (A) In addition, HPEs were co-incubated with 0.2 ug/ml or 0.5 ug/mg of TcEVs (B). The graphs present parasite DNA load data from real-time quantification using the ΔΔCt method. Co-incubated HPEs were further analyzed for histopathological damage (C, D). The graph (D) quantifies the histopathological damage according to the parameters detailed in Table 2 . All values are presented as mean ± S.D. from at least 3 independent experiments performed in triplicate. ***###p < 0.001; ****####p < 0.0001. ns: not significative. Bars marked with asterisks refer to conditions compared to control conditions; hashtags refer to comparisons between different experimental conditions.

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  Figure 2. TcEVs cause damage to the basal membrane that separates the trophoblast from the villous stroma in HPEs HPEs were co-incubated for 24 hours in the presence or absence of TcEVs and iTcEVs, with or without T. cruzi trypomastigotes. Samples underwent routine histological analysis and were stained with PAS (A) and for collagen IV immunohistochemistry (D). Graphs quantify PAS staining intensity (B), scores basal membrane continuity (C) ( Table 2 ), and collagen IV immunohistochemistry (E). The data presented in panels (B, D) was analyzed using Image J software. All values are presented as mean ± S.D. from at least 3 independent experiments performed in triplicate. *#p < 0.05; ##p < 0.01; ***p < 0.001; ****p < 0.0001. Bars marked with asterisks refer to conditions compared to control conditions; hashtags refer to comparisons between different experimental conditions.

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  Figure 3. TcEVs damage the villous stroma in HPEs and contribute to the destruction of the placental barrier caused by T. cruzi. HPEs were co-incubated for 24 hours in the presence or absence of TcEVs and iTcEVs, with or without T. cruzi trypomastigotes. Samples underwent routine histological analysis and were stained with Masson’s Trichrome (A), Picro-Sirius Red (C), and vimentin immunohistochemistry (F). Graphs show the quantification of collagen trichrome histochemistry (B), collagen I (D), and III (E) histochemistry, as well as vimentin immunohistochemistry (G) using the Image J software. All values are presented as mean ± S.D. from at least 3 independent experiments performed in triplicate. *#p < 0.05; **##p < 0.01; ***p < 0.001; ****####p < 0.0001. Bars marked with asterisks refer to conditions compared to control conditions; hashtags refer to comparisons between different experimental conditions.

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  Figure 4. TcEVs induce apoptotic cell death in HPEs. HPEs were co-incubated for 24 hours in the presence or absence of TcEVs and iTcEVs, with or without T. cruzi trypomastigotes (A). Staurosporine was used as a positive control for apoptotic cell death. DNA fragmentation was determined by the TUNEL method (A, B). Graphs (C, D) display the percentage of TUNEL-positive cells. Caspase 8 (C) and caspase 3 (D) enzymatic activities were measured using commercial kits. The p18 active fragment of caspase 8 was detected by routine immunohistochemistry (E, F), and the graph (F) quantifies the immunoreactivity and is analyzed with the Image J software. All values are presented as mean ± S.D. from at least 3 independent experiments performed in triplicate. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.