Fig 1.
In Trypanosoma cruzi-infected endothelial cells, cell adhesion molecule surface expression is increased.
Endothelial—like (EA.hy926) (A, B) and HUVECs (C, D) were infected with T. cruzi (Dm28c) trypomastigotes at a MOI of 10. E-Selectin, ICAM-1 and VCAM-1 expression was determined at the times indicated in the figures. Flow cytometry was performed using mouse IgG antibodies conjugated with PE, FITC-A, and APC against human E-Selectin, ICAM-1, and VCAM-1, respectively. Panels A and C represent mean fluorescence intensity (MFI) calculations, and panels B and D demonstrate representative histograms at 48 and 72 hours post-infection. Each group was compared with the respective IgG1 and IgG2A isotype match and normalized. The data represent the mean ± SD of MFI values from three independent experiments. *** p<0.0001; **p<0.01; *p<0.5 vs. control using two-way ANOVA and Bonferroni post-test analysis.
Fig 2.
Simvastatin and benznidazole prevent cell adhesion molecule expression on endothelial cells during Trypanosoma cruzi infection.
Endothelial-like (EA.hy926) and HUVEC cells were incubated with 5 μM simvastatin or 20 μM benznidazole dissolved in DMSO 0.025% v/v final concentration. After 24 hours, the medium was replaced, and the cells were infected for 16 hours with a 1:10 ratio of T. cruzi trypomastigotes. E-Selectin, ICAM-1, and VCAM-1 expression in EA.hy926 cells (A, C) and HUVECs (B, D) was determined at the end of the infection time. Flow cytometry was performed using human antibodies conjugated with PE, FITC, and APC for E-Selectin, ICAM-1, and VCAM-1, respectively. Panels A and B represent mean fluorescence intensity (MFI) calculations and panels C and D demonstrate representative histograms. Each group was compared with the respective IgG1 and IgG2A isotype match and normalized. In E and F, EA.hy926 cells were washed with PBS and incubated with IgG anti human E-selectin, ICAM-1, and VCAM-1 antibodies. Subsequently, the cells were incubated with anti-IgG mouse FITC-conjugated secondary antibodies. E. Representative immunofluorescence images (40X) and F. Quantitative analysis of relative fluorescence for each adhesion molecule. All controls were incubated with DMSO vehicle alone. The data are expressed as the mean ± SD from three independent experiments. The data represent the mean ± SD of MFI values (A and B) from four independent experiments. Comparisons were made by two-way ANOVA and Tukey’s post-test analysis. *** p<0.001; ** p<0.01; ‡ p< 0.001 (F).
Table 1.
Effect of simvastatin or benznidazol on cellular viability*.
Fig 3.
Simvastatin and benznidazole decreased cell adhesion molecule amount during Trypanosoma cruzi infection of endothelial cells.
Endothelial-like (EA.hy926) and HUVEC cells were incubated with 5 μM simvastatin or 20 μM benznidazole 0.025% v/v final concentration. After 24 hours, the medium was replaced, and the cells were infected at a MOI of 10. After 16 hours of infection, cells were harvested, and total protein expression was determined by Western blot. The data are presented as total of E-Selectin (A), VCAM-1 (B) and ICAM-1 (C) content. Upper panels correspond to representative blot images. Bottom panels correspond to relative blot quantification using β-tubulin as a control. Controls were incubated with DMSO vehicle alone. The data are expressed as the mean ± SD from three independent experiments. One-way ANOVA and Tukey’s post-test analysis were used to assess significant differences. ‡ p<0.001 vs. control; * p<0.05; *** p< 0.001 vs. T. cruzi.
Fig 4.
Intracellular T. cruzi load is not affected by simvastatin or benznidazole treatment before cell infection.
Endothelial-like (EA.hy926) and HUVEC cells were incubated with 5 μM simvastatin or 20 μM benznidazole 0.025% v/v final concentration. After 24 hours, the medium was replaced, and the cells were infected at a MOI of 10. After 16 hours of infection, cells were washed twice with fresh medium and the drug-free medium was changed daily. Then, after 72 hours of culture, the cells were fixed in cold methanol, and nuclei were stained with DAPI. Each panel is a representative micrograph (40X) of T. cruzi-infected, DAPI stained cells. Each experimental condition was performed in duplicate and, for each duplicate, 10 photographs were taken. In average, there were counted 20.12±3.8 cells per photograph. Infected cells averaged 5.57±1.16 and healthy cells were 14.92±3.1. Infected to uninfected cells ration was 1:5. Controls were incubated with DMSO vehicle alone. The data are expressed as the mean ± SD from three independent experiments. NS: not significant after one-way ANOVA analysis.
Fig 5.
Cell adhesion was decreased by simvastatin and benznidazole during Trypanosoma cruzi infection.
EA.hy926 cells (A) or HUVECs (B) were incubated with 5 μM simvastatin and 20 μM benznidazole 0.025% v/v final concentration. After 24 hours, the medium was replaced, and the cells were infected at a MOI of 10. After 16 hours of infection, HL-60 leukocytes that were loaded with Leuko Tracker were added to the EA.hy926 monolayer and incubated for 1.5 hours. After washing with PBS, cells were lysed, and fluorescence was measured at 480/520 nm. Controls were incubated with DMSO vehicle alone. Graphs represent the mean ± SD fold change of the relative fluorescence from three independent experiments. One-way ANOVA and Tukey’s post-test analysis were used to assess significant differences. * p<0.05; *** p<0.001.
Fig 6.
Simvastatin and benznidazole block NF-κB activation in Trypanosoma cruzi infection of EA.hy926 endothelial-like cells.
Endothelial-like (EA.hy926) and HUVEC cells were incubated with 5 μM simvastatin or 20 μM benznidazole 0.025% v/v final concentration. After 24 hours, the medium was replaced with drug-free medium, and the cells were at a MOI of 10 for 1 hour. Then, the cells were harvested at the time points indicated in the figure, and total protein expression was determined by Western blot using rabbit monoclonal antibodies against human IKK, p-IKK, IκB, and p-IκB. Representative blots are shown for p-IKK and IKK in untreated (A) and treated (C) cells and for p-IkB and IkB in untreated (B) and treated (D) cells. Bottom panels in A-D correspond to relative blot quantification using α-tubulin as a control. All controls were incubated with DMSO vehicle alone. The data are expressed as the mean ± SD from three independent experiments. One-way ANOVA and Tukey’s post-test analysis were used to find significant differences. ‡ p<0.001 vs. control; * p<0.05; *** p< 0.001 vs. T. cruzi. E. Representative photographs of the p65 localization by confocal microscopy after contact of EA.hy926 cells with T. cruzi for 1 hour with or without previous 5 μM simvastatin and 20 μM benznidazole treatment.
Fig 7.
Simvastatin-induced 15-epi-lipoxin A4 mediates cell adhesion molecule expression after endothelial activation by Trypanosoma cruzi infection.
A) EA.hy926 cells were infected with T. cruzi trypomastigotes (Dm28c) at a MOI of 10 for 16 hours. After washing with fresh culture medium, 5 μM simvastatin was added to the cells and harvested at the time point indicated. 15-epi-lipoxin A4 content was determined in the supernatants by competitive ELISA. The data are expressed as the mean ± SD from three independent experiments. Statistical analysis was performed using ANOVA compared with zero time. * P<0.05; ***p<0.0001. B) EA.hy926 cells were incubated with 5 μM simvastatin and 25 and 50 μM AA-861 (a competitive 5-lipoxygenase inhibitor) for 24 hours. After washing the cells, they were infected with T. cruzi trypomastigotes (Dm28c) at a 1:10 ratio for 16 hours. ECAM expression was determined on the EA.hy926 cell surface by flow cytometry using human antibodies conjugated with PE, FITC, and APC for E-Selectin, ICAM-1, and VCAM-1, respectively. Controls were incubated with DMSO vehicle alone. Results represent the normalized mean fluorescent intensity (MFI) ± SD from three independent experiments. One-way ANOVA and Tukey’s post-test analysis were used to assess significant differences *p<0.01; *** p<0.001 vs. T. cruzi.
Table 2.
Mass spectra data and quantification of 15-epi-lipoxin A4 determined by DSA-TOF.
Fig 8.
15-epi-lipoxinA4 prevents ECAMs expression on EA.hy926 during Trypanosoma cruzi infection and blocks NF-κB activation.
A. EA.hy926 cells were incubated with 15-epi-LXA4 at the concentrations indicated in the figure. After 24 hours, the medium was replaced, and the cells were infected with a 1:10 ratio of T. cruzi trypomastigotes. After 1 hour, the cells were harvested, and total protein expression was determined by Western blot using rabbit monoclonal antibodies against human IKK, p-IKK, IκB, and p-IκB. B-C correspond to the relative blot quantification using α-tubulin as a control. The data are expressed as the mean ± SD from three independent experiments. One-way ANOVA and Tukey’s post-test analysis were used to find significant differences. ‡ p<0.001 vs. control; * p<0.05; *** p<0.001 vs. T. cruzi. D. Representative photographs of p65 localization by confocal microscopy after EA.hy926 cell contact with T. cruzi for 1 hour with or without previous 15-epi-LXA4 treatment. E. EA.hy926 cells were incubated with 1 and 10 nM 15-epi-LXA4. After 24 hours, cells were infected with T. cruzi trypomastigotes at a 1:10 ratio for 16 hours. ECAM expression was determined on the EA.hy926 cell surface by flow cytometry using human antibodies conjugated with PE, FITC, and APC for E-Selectin, ICAM-1, and VCAM-1, respectively. All controls were incubated with DMSO vehicle alone. Results represent the normalized mean fluorescent intensity (MFI) ± SD from three independent experiments. One way ANOVA and Tukey’s post-test analysis were used to assess significant differences *p<0.05; **p<0.01; ***p<0.001 vs. T. cruzi.