Valproic Acid Downregulates Cytokine Expression in Human Macrophages Infected with Dengue Virus

Abstract

Natural infection with dengue virus (DENV) induces an increase in the production of cytokines that play an important role in disease pathogenesis. Despite numerous scientific studies, there are still no commercially available disease-specific therapeutics. Previous evidence shows that inhibiting histone deacetylase enzymes (HDACs) regulates the immune response in several inflammatory disease models. The aim of the current study was to evaluate the effect of HDAC inhibition in the production of inflammatory cytokines in human monocyte-derived macrophages infected with DENV serotype 2 (DENV-2). To this end, human monocyte-derived macrophages (MDMs) were treated with valproic acid (VPA) before or after infection and the inflammatory cytokine concentration was quantified by flow cytometry. We found that infected MDMs secreted IL-8, IL-1b, IL-6, TNF-alpha, and IL-10, but not IL-12. Strikingly, treatment of infected cells with VPA had a differential and concentration-dependent effect on the production of specific cytokines without eliciting significant changes in cell viability. Using the highest concentration of VPA, a significant reduction in the production of all cytokines was observed. These results suggest that HDAC inhibition during DENV-2 infection could exert an important regulatory effect in the production of inflammatory cytokines, representing a significant advance in the design of novel therapeutic dengue treatments.

Keywords: HDAC inhibitors; cytokines; dengue virus; macrophage; valproic acid.

Figure 1

Trichostatin A (TSA) and valproic acid (VPA) pretreatment of peripheral blood mononuclear cells (PBMCs) infected with dengue virus (DENV-2) exhibit a decrease TNF-alpha and IL-6 production. PBMCs were treated with TSA (A) or VPA (B) for 3 h and then were infected with DENV-2. At 24 h post-infection, the supernatants were used to quantify cytokine production. Data is expressed as relative cytokine production compared with infected non-treated control cells. Error bars represent the mean ± SEM of three independent experiments. * p values were calculated using the non-parametric two-tailed Mann-Whitney test, comparing each experimental condition with the infected non-treated control. A p value < 0.05 was considered significant. PBMCs were treated with TSA (C) or VPA (D) for three hours and were then infected with DENV-2. At 3 h post-infection, total RNA was collected to quantify cytokine mRNA. Gene expression was normalized to human β-actin expression. The figure shown is representative data from one of three independent experiments.

Figure 2

Cell viability of PBMCs pre-treated with TSA and VPA. (A) PBMCs were treated with different concentrations of TSA (left panel) or VPA (right panel) and their metabolic activity was quantified after 3, 6 and 24 h using resazurin. The percentage viability was calculated as described in the methods section. Error bars represent the mean ± SEM of three independent experiments; (B) PBMCs were treated with different concentrations of VPA, and after 24 h, CD14+ cells were analyzed for apoptosis using Annexin V.

Figure 3

VPA treatment of monocyte-derived macrophages (MDMs) infected with DENV2 induced a significant decrease in inflammatory cytokine production. MDMs were infected with DENV-2 at a multiplicity of infection (MOI) of 1 and treated with VPA (1, 2, or 4 mM) three hours before or after infection. At 24 h post-infection, the supernatants were used to quantify the production of TNF-alpha (A); IL-6 (B); IL-8 (C); IL-10 (D) and IL-1beta (E). Error bars represent the mean ± SEM of three independent experiments. * p values were calculated using the non-parametric two-tailed Mann-Whitney test. A p value < 0.05 was considered significant.