Fluconazole tolerance is associated with altered extracellular vesicle-mediated epithelial immune responses in...

Candida albicans is a major opportunistic fungal pathogen and a leading cause of oropharyngeal candidiasis, particularly in immunocompromised individuals. Extracellular vesicles (EVs) have emerged as important mediators of fungal-host communication; however, whether antifungal tolerance is associated with EV-mediated epithelial immune responses remains poorly understood. Here, we investigated the immunomodulatory effects of EVs released by fluconazole-tolerant and fluconazole-susceptible C. albicans clinical isolates on human oral epithelial cells (FaDu). EVs from tolerant and susceptible isolates exhibited comparable size distributions, protein content, cytotoxicity, uptake efficiency, and effects on epithelial barrier integrity. Despite these similarities, EVs from fluconazole-tolerant isolates consistently induced stronger epithelial immune activation, including significantly higher expression of proinflammatory cytokines (IL-1β, IL-6, TNF-α), antimicrobial peptides (hBD-2 and hBD-3), and increased nitric oxide production compared with EVs from susceptible isolates. These differences were reproducible across independent biological replicates and were independent of EV association, cytotoxicity, or barrier disruption. Collectively, these findings indicate that fluconazole tolerance is associated with altered EV-mediated immunomodulatory capacity of C. albicans EVs at the epithelial interface within this experimental system. This work provides a functional framework for understanding how tolerant isolates may modulate mucosal immunity and could contribute to mucosal persistence through immune conditioning rather than increased invasive capacity.