Extracellular Vesicles from Candida albicans and Aspergillus fumigatus Differentially Integrate PRR and cGAS-STING Signaling to Shape Macrophage Responses.

Fungal infections remain a major global health challenge, underscoring the need to better understand host-pathogen interactions. Extracellular vesicles (EVs) released by fungal pathogens are emerging as key modulators of immune responses. Here, we investigated how EVs from Candida albicans and Aspergillus fumigatus influence macrophage activation, focusing on the integration of pattern recognition receptors (PRRs) and cytosolic sensing pathways. EVs were characterized by nanoparticle tracking analysis, zeta potential, and cryo-electron microscopy. Human THP-1-derived macrophages were stimulated with fungal EVs, and cytokine production, gene expression, and signaling pathway activation were assessed by ELISA, RT-qPCR, and Western blotting. C. albicans EVs induced a strong pro-inflammatory response, with increased production of IL-1β, IL-6, TNF, IL-8, and IFN-β after 24 h. In contrast, A. fumigatus EVs elicited a more limited response, characterized by IL-4 production and minimal pro-inflammatory cytokine induction. Both EV populations modulated PRR expression and activated the cGAS-STING-IRF3 axis; however, C. albicans EVs promoted coordinated activation of TLR4, TLR9, and Dectin-1-CARD9 signaling, whereas A. fumigatus EVs downregulated CARD9 and dampened inflammation. These findings demonstrate that fungal EVs differentially shape macrophage responses through species-specific integration of immune sensing pathways.