Dengue virus type 3 isolated from a fatal case with visceral complications induces enhanced proinflammatory responses and apoptosis of human dendritic cells

Abstract

A recent (2007 to 2009) dengue outbreak caused by dengue virus (DENV) in Paraguay presented unusual severe clinical outcomes associated with 50% mortality rates. Although it has been reported that inflammatory responses influence the severity of dengue virus infection (T. Pang, M. J. Cardosa, and M. G. Guzman, Immunol. Cell Biol. 85:43-45, 2007), there remains a paucity of information on virus-innate immunity interactions influencing clinical outcome. Using human dendritic cells from a major innate immune cell population as an in vitro model, we have investigated signature cytokine responses as well as infectivity-replicative profiles of DENV clinical isolates from either a nonfatal case of classical dengue fever (strain DENV3/290; isolated in Brazil in 2002) or a fatal case of dengue fever with visceral complications isolated in Paraguay in 2007 (strain DENV3/5532). Strain DENV3/5532 was found to display significantly higher replicative ability than DENV3/290 in monocyte-derived dendritic cells (mdDCs). In addition, compared to DENV3/290 results, mdDCs exposed to DENV3/5532 showed increased production of proinflammatory cytokines associated with higher rates of programmed cell death, as shown by annexin V staining. The observed phenotype was due to viral replication, and tumor necrosis factor alpha (TNF-α) appears to exert a protective effect on virus-induced mdDC apoptosis. These results suggest that the DENV3/5532 strain isolated from the fatal case replicates within human dendritic cells, modulating cell survival and synthesis of inflammatory mediators.

Fig. 1.

(A) Hierarchical clustering (cluster 3.0) of the 124 modulated genes in mdDCs infected with DENV3/5532 (upregulated genes are indicated in yellow and downregulated genes in blue). (B) The functional annotation of 124 selected genes determined using Expander software and the NCBI Entrez Gene (www.ncbi.nih.gov) and Gene Ontology (www.geneontology.org) databases. ND, not defined. (C) Quantitative PCR analyzes of OAS2, IFIT1, and EIF2AK2 genes in mdDCs after infection with DENV3/5532 and DENV3/290 and mock infection. Data were analyzed using two-way ANOVA followed by a Bonferroni test; values represent means ± SDs of the results of six different experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Fig. 2.

DENV3/5532 displays higher infectivity and increased mdDC apoptosis compared to DENV3/290. (A) Percentages of infected mdDCs exposed to DENV3/290 or DENV3/5532 or mock infected. (B) Viral progeny in mdDC culture supernatants. For viral progeny, results are expressed in log₁₀ focus-forming units (ffu) in C6/36 cells/ml. (C) Percentages of apoptotic cells. mdDCs were infected as described for panel A and assessed for annexin V-positive events by flow cytometry. Data were analyzed using two-way ANOVA followed by a Bonferroni test; values represent means ± SD of the results of four different experiments. (D) Percentages of mdDCs costained for CD11c⁺ fluorescein isothiocyanate (FITC) and 4G2⁺ anti-flavivirus antibody plus anti-mouse PE conjugated by flow cytometry after 72 hpi. Data were analyzed using one-way ANOVA followed by a Bonferroni test; values represent means ± SD of the results of three different experiments. (E) Dot plot analyzes of one representative mdDC culture costained for CD11c⁺ and 4G2⁺. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Fig. 3.

DENV3/5532 enhances TNF-α, IL-6, and IL-8 production by mdDCs. mdDCs from four healthy donors were infected with DENV3/5532 or DENV3/290 or mock infected for several time points. Inflammatory cytokines were measured in cell culture supernatants by CBA as described in Materials and Methods. Data were analyzed using two-way ANOVA followed by a Bonferroni test; values represent means ± SD of the results of four different experiments. *, P < 0.05; **, P < 0.01.

Fig. 4.

Role of TNF-α in virus fitness and mdDC apoptosis. Cells undergoing apoptosis (A), percentages of infected cells (B), and levels of TNF-α (C) after 72 hpi with DENV3/290 or DENV3/5532 or mock infection and treatment with RPMI media (nontreated group), TNF-α (10 ng/well), anti-TNF-α neutralizing antibody, and an isotype control are indicated. Data were analyzed using two-way ANOVA followed by a Bonferroni test; values represent means ± SD of the results of five independent experiments. **, P < 0.01; ***, P < 0.001.

Fig. 5.

Role of virus replication in fitness, apoptosis, and secretion of proinflammatory cytokines. Percentages of infected cells (A), viral progeny (B), and cells undergoing apoptosis (C) and levels of TNF-α (D), IL-6 (E), and IL-8 (F) after 72 hpi with DENV3/290, inactivated DENV3/290, DENV3/5532, or inactivated DENV3/5532 or mock infection are indicated. Data were analyzed using one-way ANOVA followed by a Bonferroni test; values represent means ± SD of the results of five different experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001; #, P < 0.05 (compared to the native virus strain).