Dengue virus (DV) infection is the most prevalent mosquito-borne viral disease and its manifestation has been shown to be contributed in part by the host immune responses. In this study, pathogen recognition receptors, Toll-like receptor (TLR) 2 and TLR6 were found to be up-regulated in DV-infected human PBMC using immunofluorescence staining, flow cytometry and Western blot analyses. Using ELISA, IL-6 and TNF-α, cytokines downstream of TLR2 and TLR6 signaling pathways were also found to be up-regulated in DV-infected PBMC. IL-6 and TNF-α production by PBMC were reduced when TLR2 and TLR6 were blocked using TLR2 and TLR6 neutralizing antibodies during DV infection. These results suggested that signaling pathways of TLR2 and TLR6 were activated during DV infection and its activation contributed to IL-6 and TNF-α production. DV NS1 protein was found to significantly increase the production of IL-6 and TNF-α when added to PBMC. The amount of IL-6 and TNF-α stimulated by DV NS1 protein was reduced when TLR2 and TLR6 were blocked, suggesting that DV NS1 protein is the viral protein responsible for the activation of TLR2 and TLR6 during DV infection. Secreted alkaline phosphatase (SEAP) reporter assay was used to further confirm activation of TLR2 and TLR6 by DV NS1 protein. In addition, DV-infected and DV NS1 protein-treated TLR6-/- mice have higher survivability compared to DV-infected and DV NS1 protein-treated wild-type mice. Hence, activation of TLR6 via DV NS1 protein could potentially play an important role in the immunopathogenesis of DV infection.
Despite the prevalence of dengue virus infection and the heavy economic burden it puts on the endemic countries, the immunopathogenesis of dengue virus infection remains unclear. Plasma leakage in dengue hemorrhagic fever (DHF) develops not when the viremia is at its peak in infected patients but when viremia has been significantly reduced or cleared. This suggests that host immune response is responsible for the development DHF. The interactions of the viral factors with host factors which trigger the host immune responses are likely to play a significant role in the development of dengue diseases, thus are of great interests. In this study, we found that dengue NS1 protein activates TLR2 and TLR6, leading to increase proinflammatory cytokine production. In addition, the interaction of viral factor with TLR6 was found to play an important role in the manifestation of dengue virus infection. Our study provides new insights into the involvement of TLR6 in dengue virus infection and the potential of using TLR6 anatagonist in therapeutic treatment for DV infection.