Figure 6. NOX-dependent ROS production triggers mitochondrial-dependent apoptosis in DENV-infected cells and activates bystander cells.

(A) The percentage of apoptotic cells was assessed by Annexin-V staining at 6–48 h post-infection. Data are the means ± SEM from independent experiments performed in triplicate on five individual donors. P values were determined based on the comparison with uninfected cells at the appropriate time (B) mRNA relative expression of apoptosis-associated genes was detected by qPCR at various times post DENV2 infection. Values are representative of one donor. Experiment has been repeated on three individual donors. (C) Levels of mitoSOX red, DiOC₆(3), cleaved caspase 3 (_CLcaspase3), Annexin-V and DENV E protein expression were evaluated by flow cytometry 48 h post-DENV infection. Values represent the means ± SEM from at least three individual donors. (D) The correlation between (i) the percentage of DENV2+ cells at 24 h and the percentage of apoptotic cells at 48 h; (ii) the percentage of apoptotic cells and the percentage of mitoSOX+ cells at 48 h, and (iii) the percentage of apoptotic cells and the percentage of DiOC₆(3)^low cells at 48 h was calculated in Mo-DC using a Spearman test. (E) Percentage of apoptotic cells in DENV-infected Mo-DC was detected 48 h post-infection in the presence or absence of DPI (0.3 µM). Histograms represent the means ± SEM of three experiments performed in duplicate on three independent donors. (F) Percentage of apoptotic cells in DENV-infected Mo-DC was detected 48 h post-infection in the presence or absence of the p53 inhibitor pifithrin-α (10 µM) or the pan-caspase inhibitor Z-VAD-fmk (20 µM). Histograms represent the means ± SEM of one representative experiment performed in triplicate. P values were determined based on the comparison with DENV2-infected cells. (G) CD83 expression level was evaluated on Annexin-V⁺ (red) and Annexin-V⁻ (blue) DENV-infected cell population (MOI 1) (H) Mo-DC were pre-treated with increasing concentrations of DPI (0.3–3 µM) before DENV challenge (MOI 0.5). Percent of CD83⁺ Annexin-V⁻ cells was detected by flow cytometry 48 h after infection. P values were determined based on the comparison with DENV2-infected cells. Data are the means ± SEM of two experiments performed in triplicate. (I) Mo-DC pre-treated or not with increasing concentrations of DPI were cultured in the presence of DENV2 (MOI 20). After 24 h of infection, supernatants were collected and transferred for 8 h on naïve Mo-DC and cells were subsequently infected by DENV2 (MOI 20). DENV infection was assessed 24 h later by flow cytometry. The values are means ± SEM from one experiment performed in triplicate. P values were determined based on the comparison with DENV2 infected cells.