DENV Induces Platelet Activation and Platelet-Monocyte Signaling though TREM-1 and TLR3

Dengue is a tropical disease caused by DENV that can progress to severe disease with vascular leakage, hemorrhage, and thrombocytopenia. Beyond coagulation, platelets participate in immunity and recognize and internalize DENV through CLEC-2, DC-SIGN, and heparan sulfate. However, mechanisms of platelet activation remain unclear. Our molecular docking data indicates DENV E protein binds to TREM-1. We hypothesized that TREM-1 and viral genome recognition by endosomal TLRs contribute to platelet activation and platelet-mediated monocyte activation in vitro. Platelets from healthy volunteers (n=7) were isolated and infected with DENV-2 for 6 h (IRB: 85978925.4.1001.5147). TREM-1 and endosomal TLRs were inhibited using VJDT and TAC-5, respectively, 30-min before infection. Platelet activation was assessed by CD62P, CD63, and PAC-1 expression through flow cytometry and quantification of secreted mediators by ELISA. DENV-2 increased CD62P and CD63 expression and the secretion of PF4, PDGF, MIF, sCD62P, VEGF, sCD40L, sTLT-1, and TGF-β. TREM-1 inhibition significantly reduced VEGF, sTLT-1, and PF4, whereas endosomal TLR inhibition reduced CD63 and VEGF. We then investigated which endosomal TLR might be involved in DENV-induced platelet activation. TLR3 inhibition reduced CD62P and CD63 expression and VEGF, PF4, and PDGF secretion, supporting a role for TLR3 in DENV-induced platelet activation. To investigate whether these receptors play a role in platelet-dependent  monocyte modulation, we performed  24-hour cocultures with DENV-infected platelets under TREM-1 and TLR3 inhibition, and measured cytokine levels by ELISA. DENV-infected platelets increased monocyte TNF-α and sTREM-1 secretion and reduced IL-10 release. TREM-1 inhibition in platelets reduced monocyte sTREM-1, suggesting a role for platelet TREM-1 in platelet–monocyte crosstalk. Overall, our findings indicate that DENV activates platelets via TREM-1 and TLR3, modulating monocyte inflammatory responses.