Biogenesis and function of extracellular vesicles of neutrophilic Granulocytes

The opportunistic human fungal pathogen A. fumigatus can cause a wide variety of diseases, including potentially fatal invasive aspergillosis in immunocompromised hosts. The innate immune response, especially polymorphonuclear neutrophils (PMNs), are important for antifungal defence, and recently, extracellular vesicles (EVs) of PMNs were shown to exhibit antifungal capacity. This thesis aims to extend our knowledge of host cell-derived EVs during fungal infection. By using lipophilic dyes and a genetic system fusing EV-associated proteins with fluorescent proteins, we were able to visualise host EVs by confocal laser scanning microscopy, revealing their association with fungal hyphae in vitro. While staining allowed for the visualisation of PMN EVs, the genetic system cannot be translated to primary PMNs as they are not amendable to genetic manipulation. To overcome complications of working with PMNs, we established dimethylformamide-differentiated PLB-985 cells to serve as a neutrophil-like cell model that were able to phagocytose A. fumigatus conidia, produce neutrophil extracellular traps, and secrete EVs exhibiting antimicrobial proteins and antifungal activity in vitro. In addition, optimisation and extension of the EV isolation technique identified a previously overlooked subset of EVs that was antifungal against A. fumigatus conidia in vitro when isolated from PMNs that were infected with melanised and non-melanised conidia prior to EV isolation. These EVs carried antimicrobial proteins, including the iron-chelating protein lactoferrin as well as chitinases that were hypothesised to play an additional role in antifungal defence. Future studies should focus on elucidating the difference between antifungal and non-antifungal EVs, their mode of action, and their applicability in in vivo model systems. In the future, host cell-derived EVs could even serve as a platform for targeted antifungal therapy against human fungal pathogens.