Fig 1.
prM-DENV2 induces an early onset of soluble inflammatory responses.
(A) Experimental scheme: HUVEC were incubated with cell-free supernatants harvested at indicated time points from hPBMCs (n = 3, three different donors) exposed to prM-DENV2 (MOG 300), std DENV2 (MOG300) or mock treatment. Surface expression of E-selectin, VCAM-1 and ICAM-1 on HUVEC was determined by flow cytometry and represented as (B) percentage of positive cells and (C) MFI normalized to relative mock values. Data represents the mean ± SEM. P values were obtained by unpaired one-tailed t test (*/#/@P<0.05; **/##/@@P<0.01). (A) Figure created with BioRender.com.
Fig 2.
Immature DENV2 virions engage TLR2/6/CD14 to activate NF-κB.
(A) NF-κB activation in HEK-Blue hTLR2 cells (mock)-treated with PAM3CSK4 (PAM3, 50 ng/mL, n = 3) and prM-DENV2 (MOG 250, n = 5; MOG 1000, n = 6) for 24h. (B) NF-κB activation in HEK-Blue hTLR2 cells pretreated for 2h with αTLR2, αTLR1, αTLR6 and αCD14 (15 μg/mL) before exposure to prM-DENV2 (MOG1000) for 24h, n = 3. (C) NF-κB activation in HEK-Blue hTLR2 cells pretreated for 1h with endocytosis inhibitors pitstop (PS, 60μM), ammonium chloride (NH4Cl, 50mM) and wortmannin (WN, 2 μM), before exposure to prM-DENV2 (MOG1000) for 24h, n = 3. NF-κB stimulation was assessed by QUANTI-Blue, OD values show the induction of NF-κB. Data represents the mean ± SEM. P values were obtained by one-way ANOVA, Dunnett post hoc test (**P<0.01; ***P<0.001; ****P<0.0001). n = independent biological experiments. CC: cellular control. PAM3 and prM-DENV2 as a control of their respective blocking/treatment conditions.
Fig 3.
Schematic representation of the signaling pathways and molecules subjected to pharmacological targeting downstream TLR2 axis.
C29 (C16H15NO4) is a TLR2 inhibitor that blocks the interaction of the receptor TIR domain with MyD88; Dexamethasone (DEX) blocks NF-κB and MAPK activation during TLR engagement; Bay-11inhibits the phosphorylation of IkB which results in the inactivation of NF-kB; MG-132 inhibits the degradation of IκB by blocking the proteolytic activity of the 26S proteasome. Figure was created with BioRender.com.
Fig 4.
TNF-α- and IL-1β intracellular accumulation induced by prM-DENV2 is TLR2/6/CD14 and NF-κB mediated.
(A, B and D) PBMCs from healthy donors (n = 3–4, three to four different donors) were (mock) treated with αTLR2 (5 μg/mL), αTLR1 (5 μg/mL), TLR6 (5 μg/mL), αCD14 (3 μg/mL), C29 (100 μM) or its solvent control (DMSO) for 2 hours prior exposure with prM-DENV2 at MOG 300 for 6h. Cytokine production (in picograms per milliliter (pg/mL)) in the PBMCs’ supernatants was measured by flow cytometry using LegendPlex. (C, E and F) PBMCs from healthy donors (n = 3–5, three to five different donors) were (mock) treated with αTLR2 (5 μg/mL), αTLR1 (5 μg/mL), TLR6 (5 μg/mL), αCD14 (3 μg/mL), DEX, (10μM), Bay-11 (5μM) and MG-132 (9.5 μg/mL) for 2 hours prior exposure with prM-DENV2 at MOG 300 or PAM3CSK4 (PAM3, 600 ng/mL) for 12h in the presence of Brefeldin-A. (C, E and F) Percentage of monocytes (in PBMCs) with intracellular expression of TNF-α and IL-1β was measured by flow cytometry. P values were obtained by unpaired one-tailed t test. (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001).
Fig 5.
TLR2/TLR6/CD14 axis controls the early, but not the late onset of soluble inflammatory responses induced by prM-DENV2.
(A) HUVEC were incubated for 6h with cell-free supernatants from PBMCs (n = 2–5, two to five different donors) (mock)-exposed for (B) 6h and (C) 48h to prM-DENV2 at a MOG of 300 in the presence or absence of TLR2/TLR1/TLR6/CD14 blocking antibodies. Surface expression of E-selectin, VCAM-1 and ICAM-1was determined by flow cytometry and represented as percentage of positive cells. P values were determined by unpaired one-tailed t-test. (*P<0.05, **P<0.01, ***P<0.001). (A) Figure created with BioRender.com.
Fig 6.
prM-DENV2 downregulates TLR2 surface expression on monocytes within the PBMCs.
PBMCs from healthy donors (n = 3, three different donors) were exposed to prM-DENV2 at MOG 300 for 6h, 12h, 24h, 30h and 48h. (A) Percentages of TLR2 positive monocytes were determined by flow cytometry. (B) Fold changes in surface expression of TLR2 relative to the mock. (C) Concentration of sTLR2 in the supernatants exposed to prM-DENV2 determined by ELISA. Error bars represent mean± SEM. P values were obtained by one-tailed t test. (*P<0.05; ***P<0.001).
Fig 7.
Prolonged TLR2 block induces the release of TNF-α by monocytes exposed to prM-DENV2 and drives EC activation.
(A) Experimental scheme: hPBMCs (n = 3, three different donors) were treated with anti-TLR2 or TLR2 isotype control antibody (5 μg/mL) for 2 h prior exposure to prM-DENV2 (MOG 300) for 6h, 24h, and 48hpi in the presence of Brefeldin-A as indicated in the scheme. (B) Percentage of monocytes (in PBMCs) with intracellular expression of TNF-α and IL-1β was measured by flow cytometry. (C) Experimental scheme: supernatants from PBMCs (n = 3, three different donors) exposed to prM-DENV2 for 6h and 48 hpi, were incubated for 1h in the presence or absence of anti-TNF-α antibody (4 μg/ml). PBMCs exposed to prM-DENV2 for 48h were treated with anti-TLR2 2h prior prM-DENV2 stimulation. TNF-α-depleted PBMCs supernatants were used to stimulate HUVEC for (D) 6 hpi and (E) 48hpi. Surface expression of E-selectin, VCAM-1 and ICAM-1was determined by flow cytometry and represented as MFI. (B) Bars represents mean± SEM. P values were obtained by two-way ANOVA with Tukey’s post-test or unpaired one-tailed t-test (NS: not significant; *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001). (A and C) Figures created with BioRender.com.
Fig 8.
Proposed model of immature DENV sensing by TLR2.
Sensing of prM-DENV2 by monocytes (within PBMCs) induce an early onset (6h) of soluble inflammatory responses leading to the activation of endothelial cells, as evidence by the upregulation of adhesion molecules E-selectin, VCAM-1 and ICAM-1 on endothelial cells (ECs). The production of inflammatory mediators including TNF-α, IL-1β and IL-6 was in control of TLR2 and its co-receptors TLR6 and CD14 and relies on the activation of the transcription factor NF-κB. Importantly, at later time points (48hpi) TLR2 axis functions to dampen the inflammatory responses as evidenced by downregulation of adhesion molecules on ECs. Figure created with BioRender.com.