Dengue protease-mediated activation and polarization of macrophages.

Structural proteins of viruses are well recognized as major determinants of host immune responses. However, increasing evidence indicates that viral non-structural proteins can also be released from infected cells under inflammatory conditions, including via extracellular vesicles and inflammasome-associated cell death. The immunological consequences of such extracellular exposure of viral non-structural proteins remain poorly understood. Here, we investigated a non-canonical extracellular function of the dengue virus NS2B/NS3 protease and examined its ability to modulate macrophage activation and functional programming in human and murine systems using complementary in vitro and ex vivo models. Extracellular stimulation of murine macrophage cell lines with purified NS2B/NS3 induced robust classical macrophage activation and polarization, characterized by increased activation of mitogen-activated protein kinases (MAPK) and c-Jun N-terminal kinase (JNK) pathways, enhanced intracellular accumulation of pro-inflammatory cytokines, elevated reactive oxygen species (ROS) production, and pronounced nuclear translocation of NF-κB p65. Importantly, macrophage activation was markedly attenuated when cells were exposed to heat-denatured or chemically inactivated NS2B/NS3, indicating that both the native structural integrity of the protease and associated enzymatic activity contribute to its immunostimulatory capacity. Across the working concentration range used in this study, NS2B/NS3 did not exert detectable cytotoxic effects on macrophages. These findings have been further validated in human peripheral blood-derived monocyte-macrophages. Collectively, we report an unrecognized extracellular immunomodulatory role of the dengue virus NS2B/NS3 protease and demonstrate that exposure to this viral non-structural protein is sufficient to drive pro-inflammatory activation and polarization of macrophages.