Transcriptome analysis of the host response to DENV2 infection in Mo-DC.

<p>Mo-DC were infected with DENV2 (MOI 20) for designated periods of time. Samples were analyzed by Illumina gene expression array and differentially expressed genes (DEGs) that satisfied a p value (<0.05) with ≥1.3 fold change (up or down) were selected. (A) Waterfall plot representing the total number of up-regulated and down-regulated genes at each time point. (B) Heat map shows statistically significant canonical pathways (Ingenuity Pathway Analysis Software) commonly regulated at 6 h, 12 h, 18 h and 24 h when compared to baseline. Genes that had adjusted p-value <0.05 at each time point and fold change >1.3 or <−1.3 and associated with a canonical pathway in Ingenuity's Knowledge Base were used for pathway analysis. Heat map colors represent the ratio of regulated genes/pathway genes after dengue infection (red and blue correspond to over- and under-represented, respectively). The over-representation test was performed using Fisher Exact Test. Statistical significance achieved at p<0.05. The data are representative of one experiment performed on three different donors. (C) Gene expression heatmap of the top 50 differentially expressed genes induced by dengue infection in Mo-DC at various times when compared to baseline. Genes are selected as differentially expressed in at least one comparison following ANOVA F test as implemented in the LIMMA package. The scale shows the level of gene expression where red and blue correspond to up- and down-regulation respectively. A panel of antiviral (black) and antioxidant (red) DEG is represented on the right hand side of the heatmap. (D) Word clouds representing potentially activated (red)/inhibited (green) transcription factors at 6 h and 24 h after DENV2 challenge. IPA Upstream Regulator Analysis was used to identify molecules upstream of the genes in the data set that could explain the detected expression changes. The p-value of overlap, which measures the enrichment of network-regulated genes in the data set, is represented by the size of the word. The activation z-score which predicts likely regulating molecules was used to color the predicted activation state.</p

Functional characterization of genes differentially expressed by DENV2 infection.

<p>Differentially expressed genes were subjected to Ingenuity Pathway Analysis for both 6 h (A) and 24 h (B) time points based on <i>p</i>-values (<0.001) and fold change ≥±1 (log2). Red represent genes induced by DENV, and green indicated genes downregulated by the virus; the intensity of the color is representative of the fold change. Larger circles indicate transcription factors. Genes enriched for a pathway are represented as a cloud.</p

DENV-infected cells accumulate intracellular NOX-derived ROS which potentiate the immune response.

<p>(A) ROS generation in DENV-infected Mo-DC (MOI 20) was monitored by FACS using CM-H2DCFDA (1 µM) 18 h after infection. Data represent the mean ± SEM from experiments performed on four different donors. (B) Accumulation of ROS was measured by flow cytometry using CM-H2DCFDA (1 µM) 24 h after infection with increasing amount of virus. Data represent the mean ± SEM from one experiments performed on three different donors. P values were determined based on the comparison with uninfected cells (C) The correlation between the percentage of infected cells determined by intracellular staining of DENV E protein expression and the intracellular accumulation of ROS measured by DCFDA staining was calculated (n = 15; Spearman test). (D) Oxidative stress generation was detected by flow cytometry using the CM-H2DCFDA (1 µM) fluorescent probe on Mo-DC pretreated with DPI (3 µM) and infected with DENV2 (MOI 20) for 3 h or treated with the stress-inducer Pyocyanin (100 µM). Data represent the geometric mean fluorescence ± SEM from an experiment performed on two different donors. (E) Expression level of the phosphorylated NADPH-oxidase p47 subunit was assessed by immunoblotting 3 h after DENV2 infection. Histograms represent the fold change ratio of phosphorylated-p47 over total p47. Data are the means ± SEM of independent experiments performed on three different donors. P value was determined based on the comparison with uninfected cells. (F) Mo-DC were transfected with control or gp91 phox siRNA and 48 h later were infected with DENV2 (MOI 20) for 18 h. ROS accumulation was measured by FACS using the CM-H2DCFDA fluorescent probe. Data are the means ± SEM of three independent experiments performed on different donors. P value was determined based on the comparison with cells infected with DENV and transfected with the control siRNA sequences (G) Mo-DC were treated with increasing concentrations of hydrogen peroxide (10–50 µM) in the presence or absence of DENV2 (MOI 20). CCL5 and CXCL10 mRNA expression levels were monitored by qPCR 24 h following treatment and infection. Data represent the means ± SEM from one experiment performed on three individual donors. Experiment has been repeated twice. P values were determined based on the comparison with DENV2-infected cells.</p

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

<p>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).</p

Mo-DC are highly susceptible to DENV infection and generate a broad antiviral and inflammatory response.

<p>(A) The correlation between the percentage of infected cells determined by intracellular staining of DENV E protein expression and the percentage of CD14⁻ CD1a⁺ Mo-DC was calculated during the course of Mo-DC differentiation (n = 15; Spearman test). The FACS panels on the left hand side represent the progression of Mo-DC differentiation at days 0, 3 and 7. (B) DENV RNA levels as well as <i>IFN-β</i>, <i>IFIT1</i>, and <i>CCL5</i> gene expression levels were determined by qPCR at various times of DENV2 infection (MOI 10). P values were determined based on the comparison with cells at time 3 h after infection. Data are from one experiment performed on three individual donors. (C) Mo-DC were infected with increasing amounts of virus (MOI 0.04-MOI 20) for 24 h. Percentage of DENV2-infected cells was determined by intracellular staining (ICS) of DENV E protein expression using flow cytometry. Data are the means ± SEM from one experiment performed on three different donors. (D) Mo-DC were challenged with DENV2 (MOI 20) and antiviral and inflammatory responses were examined by immunoblotting. Data are from one representative experiment. (E) Cytokine release was evaluated by cytokine bead array (CBA) 24 h after DENV2 challenge (MOI 10) in the supernatants of infected cells. P values were determined based on the comparison with uninfected cells. Data are the means ± SEM from one experiment performed on three different donors.</p

Nrf2 transcription factor limits DENV infection and modulates the innate immune and apoptotic responses.

<p>(A) Gene expression heatmap of antioxidant expressed genes modulated by dengue infection in Mo-DC at various times of infection (6–24 h) when compared to baseline. The scale shows the level of gene expression where red and blue correspond to up- and down-regulation respectively. Each box of the heatmap represents one donor. (B) The expression level of Nrf2-regulated genes was determined by qPCR at various times after DENV challenge. The data are representative of three donors. (C) Mo-DC were pre-treated with DPI (3 µM) and subsequently challenged with increasing amounts of DENV (MOI 0.1–10). The gene and protein expression levels of HMOX-1 and SOD2 were evaluated by qPCR and immunoblot, respectively. Data are the means ± SEM of one experiment performed on three individual donors. Immunoblots are representative of the results from one donor. (<b>D–E</b>) Mo-DC were transfected with control or Nrf2 siRNA and 48 h later were infected with DENV (MOI 1). Nrf2 mRNA expression level (D) and DENV viral RNA (E) were assessed by qPCR. (<b>F–G</b>) Mo-DC were transfected with Control or Nrf2 siRNA and 48 h later were infected with DENV (MOI 20). Percentage of DENV-infected cells and ROS accumulation was determined in the same samples at 18 h after infection. (H–I) mRNA expression levels of antiviral (H) and apoptotic genes (I) were measured by qPCR in Nrf2-depleted cells that were infected with DENV (MOI 1). Data are the mean ± SEM of two independent experiments performed in duplicate on two donors. P values were determined based on the comparison with DENV2-infected si-control-transfected cells.</p

Schematic of DENV induces NOX-dependent ROS production required for antiviral and apoptotic responses.

<p>DENV2 infection generates intracellular NOX-derived ROS accumulation in Mo-DC (A). DENV2-induced ROS formation is essential for the activation of the NF-κB inflammatory response (B) and for the IRF3-mediated antiviral response (C). ROS accumulation leads to p53 stimulation which generates a mitochondrial and caspase-dependent apoptosis (D). Finally, oxidative stress generation also stimulates the cytoprotective transcription factor Nrf2 (E) which tightly regulates ROS levels (f) as well as innate immune and apoptotic responses to DENV infection.</p

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

<p>(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.</p