Mechanistic insights into Nicotine-derived nitrosamine ketone (NNK) in multiple sclerosis via integrated systems analyses.

Multiple sclerosis (MS) is characterized by recurrent neuroinflammatory episodes that drive progressive neurodegeneration, with cigarette smoking recognized as a major environmental risk factor. Nicotine-derived nitrosamine ketone (NNK), a potent tobacco-specific nitrosamine, has been implicated in MS; however, its mechanistic contribution to disease pathogenesis remains poorly understood. Here, we applied an integrative multi-level approach to investigate the potential role of NNK in MS. Mendelian randomization (MR) analysis identified genetically predicted dipeptidyl peptidase-4 (DPP4) activity as being associated with MS susceptibility. Molecular docking further demonstrated feasible interactions between NNK and candidate proteins, including DPP4, providing theoretical plausibility for chemical-protein interactions. In experimental autoimmune encephalomyelitis (EAE) mice, systemic NNK exposure accelerated disease onset, aggravated neurological deficits, and enhanced immune cell infiltration and demyelination within the central nervous system (CNS), accompanied by increased DPP4 expression in inflamed regions. Consistent with these observations, in vitro NNK treatment impaired endothelial barrier integrity in bEnd.3 cells by reducing tight junction proteins (ZO-1, Claudin-5, and Occludin) and upregulating adhesion molecules (VCAM-1 and ICAM-1). NNK exposure also triggered inflammatory activation in BV2 microglia, resulting in elevated expression of TNF-α, IL-1β, and IL-6. Importantly, DPP4 silencing partially restored endothelial junctional integrity and attenuated pro-adhesive signaling in endothelial cells while reducing inflammatory cytokine expression in microglia, suggesting that DPP4 is functionally involved in NNK-induced neurovascular inflammation. Together, these findings suggest that NNK exposure may modulate neuroinflammatory processes relevant to MS through blood-brain barrier (BBB) disruption and microglial activation, with DPP4 potentially being involved in this process. This study provides mechanistic insights into how smoking-associated toxins may influence MS progression and highlights DPP4-related pathways as potential targets for therapeutic investigation.