Figure 5. Cellular oxidative stress response is required to mediate DENV-induced innate immune responses.

Mo-DC were pre-treated with DPI (0.3 or 3 µM) for 1 h, and subsequently infected with DENV2 (MOI 20) for 24 h. (A) Antiviral and inflammatory responses were monitored by immunoblotting. Data are representative of at least two independent experiments on separate donors. (B) Intracellular levels of phosphorylated STAT1 were detected by PhosFlow in Mo-DC infected for 24 h by DENV2 (MOI 20) or stimulated for 30 min with IFN-β (1000 IU/mL) and pre-treated or not with DPI (1 µM). Data represent the means ± SEM from two experiments performed on separate donors. (C) High throughput analysis of gene expression evaluated by qPCR BioMark analysis. Gene expression levels were calculated using the ΔΔCt method and gene-wise standardized expression (z-score) were generated for each gene. The scale represents z-score values where red shows an up-regulation and blue a down-regulation in gene expression. Data are representative of one experiment performed on three individual donors. Each box of the heatmap represents one donor. (D) Cytokine release was evaluated by cytokine bead array (CBA) on the supernatants of DENV infected cells pre-treated or not with DPI (0.3–3 µM). Data represent the means ± SEM from three individual donors. P values were determined based on the comparison with DENV2-infected cells. (E) Mo-DC were pre-treated with DPI (3 µM), Apocynin (3 mM), TEMPOL (3 mM), Ebselen (10 µM), PDTC (40 µM) and Trolox (5 µM) for 1 h, and subsequently infected with DENV2 (MOI 20) for 24 h. Antiviral and inflammatory gene expression was determined by qPCR. Data represent the means ± SEM from one experiment performed on three individual donors. P values were determined based on the comparison with DENV2-infected cells. (F) Mo-DC were transfected with control or gp91 phox siRNA and 48 h later were infected with DENV2 (MOI 20). IFIT1 and gp91 phox protein expression levels were measured by immunoblot analysis. Result is representative of one experiment. (G) Mo-DC were pre-treated with DPI (0.1–1 µM) for 1 h, and subsequently infected with DENV2 (MOI 1) for 48 h. Percentage of infected cells was determined by intracellular staining of DENV E protein (i–ii). DENV titers were determined by transferring supernatants from Mo-DC-infected cells on A549 cells and staining for DENV E protein (iii). DENV titers were expressed as the number of infectious units/mL. Data represent the means ± SEM of experiments performed on four (i–ii) and two different donors (iii).