Cryptococcus neoformans (C. neoformans), an opportunistic fungal pathogen with a worldwide distribution, is responsible for fatal meningitis in immunocompromised and immunocompetent populations. Extracellular vesicles (EVs) derived from C. neoformans embody bioactivities that contribute to fungal interaction and survival. In the present study, we demonstrated that EVs from the encapsulated strain H99 increased CD44 expression while decreasing occludin expression. In contrast, EVs from acapsular strains were ineffective in CD44 and occludin expression. Furthermore, we discovered that H99 EVs could induce the formation of neutrophil extracellular traps (NETs). Mechanistically, the inhibition of PAD4 and p65 and the use of an NADPH inhibitor reduced the formation of NETs induced by H99 EVs. However, Δcap59 EV s and Δcap67 EV s failed to induce NETs. Moreover, NETs induced by H99 EVs disrupted tight junctions (TJs) in brain endothelial cells (bEnd.3), leading to a reduction in the expression of claudin-5 and occludin. This disruption was accompanied by the activation of STAT3, NF-κB, and MyD88-MAPK (p38/JNK) signaling pathways. Consequently, using an in vitro blood‒brain-barrier (BBB) model, we demonstrated that H99-derived EVs and H99 EVs-induced NETs contributed to the adhesion to and penetration of brain endothelial cells by C. neoformans. In summary, we have revealed that EVs derived from C. neoformans induce the formation of NETs, potentially facilitating fungal infection by disrupting tight junctions in brain endothelial cells.
