Extracellular vesicles of macrophages and neutrophils in response to Aspergillus fumigatus

The global burden of fungal infections has increased over the past century. Among pathogenic fungi, Aspergillus fumigatus is especially concerning and classified by the World Health Organization as causing a critical health threat. This thesis explores immune mechanisms against A. fumigatus, with a focus on the antifungal properties of neutrophil- and macrophage-derived extracellular vesicles (EVs). Neutrophils were shown to produce antifungal EVs (afEVs) in response to A. fumigatus exposure. These EVs were enriched with antimicrobial peptides, including neutrophil elastase, myeloperoxidase, and cathepsin G. The afEVs inhibited hyphal growth, a key pathogenic feature of A. fumigatus, and led to fragmentation of the mitochondrial network within hyphae, a strong hint for a detrimental effect of afEVs. Furthermore, we observed that afEVs released after contact with A. fumigatus conidia are highly specific against dihydroxynaphthalene (DHN) melanin-producing Aspergillus spp. but not against some of the tested bacteria. We were able to uncover DHN melanin on the surface of conidia as a possible trigger for the formation of afEVs in human neutrophils. This thesis also established a model for in vitro studies of neutrophil function in A. fumigatus infection, using DMF-differentiated PLB 985 cells. By using this cell line we were able to display critical properties of neutrophil defense mechanisms against A. fumigatus, e.g., phagocytosis, phagolysosome maturation, NET formation, and afEV release. Additionally, we developed a streamlined protocol for the isolation and characterization of macrophage-derived EVs. Our investigations showed that after contact with A. fumigatus conidia macrophages release a distinct population of EVs that transport molecules apparently promoting pro-inflammatory cytokine production in macrophages. Together, this underscores the role of innate immune EVs in antifungal defense and their effect on neighboring cells.