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.