TLR2 on blood monocytes senses dengue virus infection and its expression correlates with disease pathogenesis

Abstract

Vascular permeability and plasma leakage are immune-pathologies of severe dengue virus (DENV) infection, but the mechanisms underlying the exacerbated inflammation during DENV pathogenesis are unclear. Here, we demonstrate that TLR2, together with its co-receptors CD14 and TLR6, is an innate sensor of DENV particles inducing inflammatory cytokine expression and impairing vascular integrity in vitro. Blocking TLR2 prior to DENV infection in vitro abrogates NF-κB activation while CD14 and TLR6 block has a moderate effect. Moreover, TLR2 block prior to DENV infection of peripheral blood mononuclear cells prevents activation of human vascular endothelium, suggesting a potential role of the TLR2-responses in vascular integrity. TLR2 expression on CD14 + + classical monocytes isolated in an acute phase from DENV-infected pediatric patients correlates with severe disease development. Altogether, these data identify a role for TLR2 in DENV infection and provide insights into the complex interaction between the virus and innate receptors that may underlie disease pathogenesis.

Fig. 1. Sustained high expression of TLR2 and increased frequency of monocytes correlates with DENV disease severity.

a–e PBMCs were isolated from 15 age-matched healthy donors (HD) and 54 patients undergoing acute DENV infection (DENV+) who developed relatively mild (DF, n = 32) or severe (DHF/DSS, n = 22) disease. a Monocyte subsets distribution in healthy and DENV+ patients (two-tailed Mann–Whitney test, ****P < 0.0001). b Percentages of cells expressing TLR2 were determined for each monocyte subset (two-tailed Mann–Whitney test, ****P < 0.0001) and c stratified by disease severity (two-tailed Mann–Whitney test, *P < 0.05; **P < 0.01). d Percentages of NS3+ infected cells in DENV-positive patients (n = 15) stained intracellularly for DENV NS3 (two-tailed Mann–Whitney test, ***P < 0.001; ****P < 0.0001). e Monocyte subsets distribution in patients, stratified by disease severity (two-tailed Mann–Whitney test, *P < 0.05). CM classical monocytes, IM intermediate monocytes, NM non-classical monocytes. Bars represent median with interquartile range (IQR). Source data are provided as a Source data file.

Fig. 2. DENV engages TLR2/6 and CD14 to activate NF-κB and to establish infection.

a NF-κB activation in HEK-Blue™ hTLR2 cells (mock-) treated with PAM3CSK4 (PAM3, 25 ng/mL), DENV2 (MOG1000), UV-I DENV2 (MOG1000) or purified DENV2 (pDENV2) (MOG1000) for 24 h (n = 3 one-way ANOVA, Dunnett post hoc test, ***P < 0.001). OD630 values represent induction of NF-κB. b, c NF-κB activation in HEK-Blue™ hTLR2 cells pretreated for 2 h with b αTLR2 (n = 3, one-tailed paired t test, ***P < 0.001; ****P < 0.0001) or c αTLR1, αTLR6 and αCD14 (15 µg/mL) before exposure to PAM3 (25 ng/mL), PAM2CSK4 (PAM2, 1 ng/mL) or DENV2 (MOG1000) for 24 h (n = 3, one-way ANOVA, Dunnett post hoc test, *P < 0.05; **P < 0.01; ***P < 0.001). d NF-κB activation in HEK-Blue™ hTLR2 pretreated for 1 h with endocytosis inhibitors pitstop (PS, 60 µM), ammonium chloride (NH4Cl, 50 mM) and wortmannin (W, 2 µM) prior to exposure to PAM3 (25 ng/mL), PAM2 (1 ng/mL) or DENV2 (MOG1000) for 24 h (n = 3, one-way ANOVA, Dunnett post hoc test, *P < 0.05). Percentages of DENV (E)—positive cells were determined by flow cytometry in e HEK-Blue™ hTLR2 cells (n = 5, one-way ANOVA, Dunnett post hoc test, *P < 0.05; ****P < 0.0001) or f monocytes in the context of PBMCs, in the presence or absence of TLR2 axis blockade after 24 and 48 h, respectively (n = 3, three donors and three different DENV2 preparations, one-way ANOVA, Dunnett post hoc test, ****P < 0.0001). Bars represent the mean ± SEM. CC (cellular control), PAM2, PAM3, and DENV2 as controls of their respective blocking/treatment conditions. N refers to the number of independent biological experiments unless otherwise specified. g Correlation of mean fluorescence intensity (MFI) of CD14 expression on different monocyte subsets and DENV infection (NS3) in our patient’s cohort. Statistical differences were determined by Spearman correlation analysis. Source data are provided as a Source data file.

Fig. 3. Active DENV infection upregulates TLR2 and increases CD16 expression in a TLR2/TLR6 dependent manner.

PBMCs from healthy donors were (mock-) treated with αTLR2, αTLR1 and αTLR6 (5 µg/mL) for 2 h prior to infection with DENV2 at MOI of 10 or its UV-inactivated equivalent (UV-DENV2) for 48 h. a MFI of TLR2 expression (n = 5, five different donors and up to 4 different DENV2 preparations, paired one-tailed t test, *P < 0.05; **P < 0.01, ***P < 0.001) and b Percenatge of TLR2-positive cells within each of monocytes subset (n = 5, five different donors and up to four different DENV2 preparations, paired one-tailed t test, *P < 0.05; **P < 0.01, ***P < 0.001, ****P < 0.0001). c CD14 and CD16 expression were detected for total monocytes in blocking and non-blocking condition (n = 2, two different donors and three different viral preparations, paired one-tailed t test, *P < 0.05; **P < 0.01) and d per monocyte subset (n = 2, two different donors and three different viral preparations, paired one-tailed t test, *P < 0.05; **P < 0.01). CM classical monocytes, IM intermediate monocytes and NM: non-classical monocytes. Data represented as fold-changes in MFI relative to the respective mock or as a percentage of positive cells of the respective marker. Bars represent mean ± SEM. Source data are provided as a Source data file.

Fig. 4. Active DENV infection induces inflammatory mediators in a TLR2-dependent manner.

a HUVEC were incubated for 6 h with cell-free supernatants from PBMCs infected in the presence or absence of TLR2 blockade. b Boxplots show the fold-changes in surface expression of E-selectin, VCAM-1 and ICAM-1 compared to the respective mock (n = 4, paired one-tailed t test, *P < 0.05; **P < 0.01). The horizontal line represents the median and the whiskers the minimum and maximum values. c Fold-changes in gene expression levels of E-selectin, VCAM-1, and ICAM-1 relative to the respective mock (n = 2). d Fold-changes in gene expression levels of IL-6, IL-8, MCP-1, and CXCL6 relative to the respective mock (n = 2). N refers to the number of independent biological experiments in HUVEC. Source data are provided as a Source data file.

Fig. 5. TLR2/CD14-dependent cytokines induced by DENV2 infection.

PBMCs from healthy donors were (mock)—treated with αTLR2, αTLR1, αTLR6 (5 µg/mL) and αCD14 (3 µg/mL) for 2 h prior to infection with DENV2 at MOI of 20 for 48 h (n = 2, two different donors and 3 different DENV2 preparations, one-way ANOVA, Dunnett post hoc test, **P < 0.01). Cytokine production was measured by flow cytometry using LegendPlex. Each boxplot in the graphs shows the production in picograms per milliliter (pg/mL) of the respective cytokine. The horizontal line represents the mean and the bottom and top of the box show the minimum and the maximum values. Source data are provided as a Source data file.