Activation of TLR2 and TLR6 by Dengue NS1 Protein and Its Implications in the Immunopathogenesis of Dengue Virus Infection

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.

登革病毒（Dengue virus, DV）感染是全球范围内最流行的虫媒病毒性疾病，现有研究证实宿主免疫应答在其发病进程中发挥了部分调控作用。本研究中，研究人员通过免疫荧光染色、流式细胞术及蛋白质印迹（Western blot）分析，发现登革病毒感染的人外周血单个核细胞（peripheral blood mononuclear cell, PBMC）内，模式识别受体Toll样受体（Toll-like receptor, TLR）2与TLR6的表达水平显著上调。采用酶联免疫吸附测定（Enzyme-Linked Immunosorbent Assay, ELISA）检测发现，作为TLR2与TLR6信号通路下游的细胞因子白细胞介素-6（Interleukin-6, IL-6）与肿瘤坏死因子-α（Tumor Necrosis Factor-α, TNF-α），在登革病毒感染的PBMC中同样呈现上调表达。当在登革病毒感染过程中使用TLR2与TLR6中和抗体阻断这两个受体后，PBMC分泌的IL-6与TNF-α水平显著降低。上述结果表明，登革病毒感染期间TLR2与TLR6的信号通路被激活，且该激活过程可促进IL-6与TNF-α的产生。研究进一步发现，将登革病毒NS1蛋白（DV NS1 protein）加入PBMC培养体系后，可显著诱导IL-6与TNF-α的分泌；而预先阻断TLR2与TLR6时，登革病毒NS1蛋白诱导的IL-6与TNF-α分泌量明显下降，提示登革病毒NS1蛋白是介导登革病毒感染过程中TLR2与TLR6激活的关键病毒蛋白。研究人员还采用分泌型碱性磷酸酶（secreted alkaline phosphatase, SEAP）报告基因实验，进一步验证了登革病毒NS1蛋白对TLR2与TLR6的激活作用。此外，与野生型小鼠相比，经登革病毒感染或登革病毒NS1蛋白处理的TLR6基因敲除（TLR6-/-）小鼠存活率更高。综上，通过登革病毒NS1蛋白激活TLR6，可能在登革病毒感染的免疫发病机制中扮演重要角色。