Susceptibility and response of human blood monocyte subsets to primary dengue virus infection

Abstract

Human blood monocytes play a central role in dengue infections and form the majority of virus infected cells in the blood. Human blood monocytes are heterogeneous and divided into CD16(-) and CD16(+) subsets. Monocyte subsets play distinct roles during disease, but it is not currently known if monocyte subsets differentially contribute to dengue protection and pathogenesis. Here, we compared the susceptibility and response of the human CD16(-) and CD16(+) blood monocyte subsets to primary dengue virus in vitro. We found that both monocyte subsets were equally susceptible to dengue virus (DENV2 NGC), and capable of supporting the initial production of new infective virus particles. Both monocyte subsets produced anti-viral factors, including IFN-α, CXCL10 and TRAIL. However, CD16(+) monocytes were the major producers of inflammatory cytokines and chemokines in response to dengue virus, including IL-1β, TNF-α, IL-6, CCL2, 3 and 4. The susceptibility of both monocyte subsets to infection was increased after IL-4 treatment, but this increase was more profound for the CD16(+) monocyte subset, particularly at early time points after virus exposure. These findings reveal the differential role that monocyte subsets might play during dengue disease.

Figure 1. Susceptibility of monocyte subsets to dengue virus infection.

(A) Flow cytometric profile of CD16⁻ and CD16⁺ monocytes after isolation. (B) Isolated CD16⁻ or CD16⁺ monocyte subsets were either exposed to dengue virus (DENV2, NGC) at a MOI of 10 or medium without virus. After 2 days, monocytes were fixed, permeabilized and labeled with anti-E-protein and anti-NS1 specific antibodies. Quadrants for virus exposed monocytes (right panel) were set based on monocytes without virus exposure (left panel). Percentage positive cells in each quadrant are shown. Representative data for 6 different donors. (C and D) Percentages of CD16⁻ and CD16⁺ monocytes that are NS1⁺ or E-protein⁺ over the course of 6 days after virus exposure. Results are mean ± SE of 6 different donors. (E) Plaque assays with BHK-21 cells were performed with supernatants taken from virus exposed CD16⁻ or CD16⁺ monocytes over the course of 6 days. Results are mean ± SE from 5 different donors.

Figure 2. Production of soluble factors associated with protection against dengue by monocyte subsets.

Isolated monocyte subsets were either exposed to dengue virus (DENV2, NGC) at a MOI of 10 or medium without virus. Supernatants were harvested over the course of 6 days. (A) Levels of IFN-α were determined by a multi-subtype specific ELISA kit. (B and C) Levels of CXCL10 and TRAIL were determined using multiplex bead arrays. Results are mean ± SE for 6 different donors. There were no significant differences were found between infected CD16⁻ and CD16⁺ monocytes. (D) Supernatants from CD16⁻ and CD16⁺ monocytes exposed to dengue virus or medium without virus were harvested at day 6. These supernatants were passed through 100 kDa centrifuge filters to remove dengue virus. K562 cells were pretreated for 24 hours with either culture medium, supernatants of CD16⁻ or CD16⁺ monocytes with or without virus exposure. Pre-treated K562 cells were washed and infected with dengue virus at a MOI of 2. After 2 days, the extent of infection was determined by intracellular labeling of K562 cells with anti-NS1 antibody. The percentage of NS1⁺ K562 cells after 2 days is shown. Data are representative of 2 experiments using different donors. **, p<0.005 between respective monocyte subset with and without virus.

Figure 3. Production of inflammatory cytokines by monocyte subsets.

Monocyte subsets were exposed to dengue virus or medium without virus. Supernatants were harvested over the course of 6 days. Levels of (A) IL-1β (B) TNF-α (C) IL-6 (D) CCL2 (E) CCL3 and (F) CCL4 were measured using multiplex bead arrays. Results are mean ± SE of 5 different donors. * p<0.05, **, p<0.005 between CD16⁺ and CD16⁻ monocytes with virus.

Figure 4. Viability of monocyte subsets after dengue virus exposure.

(A) Representative forward scatter (FSC) versus side scatter (SSC) plot of monocyte subsets exposed to dengue virus or medium without virus after 2 days. Numbers indicate percentages within each region gate. (B) Annexin V and 7-AAD staining of monocyte subsets exposed to dengue virus or medium without virus after 2 days. Representative data for 5 different donors. (C) Percentage of viable monocyte subsets (Annexin V and 7-AAD double negative) over the course of 6 days after exposure to dengue virus or medium without virus. Data are expressed as mean ± SE from 5 different donors. (D) Live/Dead staining of monocyte subsets exposed to dengue virus or medium without virus after 2 days. Dead cells stain strongly with the dye (right peaks) while live cells are able to actively exclude the dye and are thus stained weakly (left peaks). Representative data for 4 different donors. (E) Percentage of live cells exposed to dengue virus or medium without virus after 2 days. Data are expressed as mean ± SE from 4 different donors. (F) MTS assay. Metabolic activity of live cells produces a substrate that absorbs at OD490 nm. Absorbance at OD490 nm is proportional to the number of live cells. Data are normalized to the respective monocyte subset without virus. Representative data for 2 different donors. *, p<0.05 between respective monocyte subset with and without virus.

Figure 5. IL-4 treatment enhances the susceptibility of the CD16⁺ monocyte subset to a greater extent.

Isolated CD16⁻ or CD16⁺ monocyte subsets were pretreated with 25 ng/ml of IL-4 for two days. Cells were subsequently washed and harvested before exposure to dengue virus (DENV2, NGC) at a MOI of 10 or medium without virus. Percentages of CD16⁻ and CD16⁺ monocytes that are (A) NS1⁺ or (B) E-protein⁺ over the course of 6 days after virus exposure. Results are mean ± SE of 5 different donors. (C) Plaque assays with BHK-21 cells were performed with supernatants taken from virus exposed IL-4 treated CD16⁻ or CD16⁺ monocytes over the course of 6 days. Results are mean ± SE from 4 different donors. * p<0.05, between IL-4 treated CD16⁺ and IL-4 treated CD16⁻ monocytes with virus.