Extracellular vesicles of Anisakis simplex s. s. modulate inflammatory signaling and oxidative homeostasis in human intestinal epithelial cells

Extracellular vesicles (EVs) released by parasitic helminths are increasingly recognized as key mediators of host-parasite communication; however, their functional impact on human intestinal epithelial cells remains poorly characterized. Here, we investigated the immunomodulatory and redox-related effects of EVs secreted by third-stage larvae of the zoonotic nematode Anisakis simplex s. s. on human Caco-2 intestinal epithelial cells. EVs after isolation were rigorously characterized by nanoparticle tracking analysis, transmission electron microscopy, and particle-to-protein ratio assessment. Exposure of Caco-2 cells to A. simplex EVs (Anis-EVs) resulted in a concentration-dependent reshaping of the epithelial immune profile, characterized by suppression of the pro-inflammatory chemokine IL-8 and enhanced secretion of the anti-inflammatory cytokines IL-10 and IL-13, while IL-4, IL-6, and IFN-γ levels remained largely unaffected. Anis-EVs also suppressed the secretion of the epithelial alarmins IL-33 and TSLP in a dose-dependent manner, representing the first evidence for Anis-EVs-mediated downregulation of these innate danger signals in an intestinal epithelial cell model. In parallel, Anis-EVs treatment significantly modulated cellular redox homeostasis. Anis-EVs-exposed cells displayed altered total antioxidant capacity and glutathione levels, increased superoxide dismutase activity, and reduced lipid peroxidation, indicating a finely tuned antioxidant response rather than overt oxidative damage. Together, these findings demonstrate that Anis-EVs act as multifunctional effectors that simultaneously attenuate inflammatory signaling, suppress epithelial alarmin release, and modulate oxidative stress pathways in human intestinal epithelial cells.