PLB-985 Neutrophil-Like Cells as a Model To Study Aspergillus fumigatus Pathogenesis.

Fungal infections remain a major global concern. Emerging fungal pathogens and increasing rates of resistance mean that additional research efforts and resources must be allocated to advancing our understanding of fungal pathogenesis and developing new therapeutic interventions. Neutrophilic granulocytes are a major cell type involved in protection against the important fungal pathogen Aspergillus fumigatus, where they employ numerous defense mechanisms, including production of antimicrobial extracellular vesicles. A major drawback to work with neutrophils is the lack of a suitable cell line system for the study of fungal pathogenesis. To address this problem, we assessed the feasibility of using differentiated PLB-985 neutrophil-like cells as an in vitro model to study A. fumigatus infection. We find that dimethylformamide-differentiated PLB-985 cells provide a useful recapitulation of many aspects of A. fumigatus interactions with primary human polymorphonuclear leukocytes. We show that differentiated PLB-985 cells phagocytose fungal conidia and acidify conidia-containing phagolysosomes similar to primary neutrophils, release neutrophil extracellular traps, and also produce antifungal extracellular vesicles in response to infection. In addition, we provide an improved method for the isolation of extracellular vesicles produced during infection by employing a size exclusion chromatography-based approach. Advanced liquid chromatography-tandem mass spectrometry (LC-MS/MS) proteomics revealed an enrichment of extracellular vesicle marker proteins and a decrease of cytoplasmic proteins in extracellular vesicles isolated using this improved method. Ultimately, we find that differentiated PLB-985 cells can serve as a genetically tractable model to study many aspects of A. fumigatus pathogenesis. IMPORTANCE Polymorphonuclear leukocytes are an important defense against human fungal pathogens, yet our model systems to study this group of cells remain very limited in scope. In this study, we established that differentiated PLB-985 cells can serve as a model to recapitulate several important aspects of human polymorphonuclear leukocyte interactions with the important human fungal pathogen Aspergillus fumigatus. The proposed addition of a cultured neutrophil-like cell line to the experimental toolbox to study fungal pathogenesis will allow for a more mechanistic description of neutrophil antifungal biology. In addition, the easier handling of the cell line compared to primary human neutrophils allowed us to use PLB-985 cells to provide an improved method for isolation of neutrophil-derived extracellular vesicles using size exclusion chromatography. Together, these results provide significant tools and a baseline knowledge for the future study of neutrophil-derived extracellular vesicles in the laboratory.

Host-derived extracellular vesicles for antimicrobial defense.

Extracellular vesicles are of increasing importance in the clinic, as diagnostics for complex diseases and as potential delivery systems for therapeutics. Over the past several decades, extracellular vesicles have emerged as a widespread, conserved mechanism of intercellular and interkingdom communication. The ubiquitous distribution of extracellular vesicles across life offers at least two compelling opportunities: first a path forward in the design of targeted antimicrobial delivery systems; and second, a new way to view host pathogenesis during infection. Both avenues of research are well underway. In particular, preliminary studies showing that plant and human host-derived extracellular vesicles can deliver natural antimicrobial cargos to invading fungal and bacterial pathogens are captivating. Further, modification of host extracellular vesicle populations may ultimately lead to enhanced killing and serve as a starting point for the development of more advanced therapeutic options, especially against difficult to treat pathogens. Despite the rapid pace of growth surrounding extracellular vesicle biology, many questions remain unanswered. For example, the heterogeneity of vesicle populations continues to be a confounding factor in ascribing clear functions to a vesicular subset, and the molecular cargos responsible for specific antimicrobial actions of extracellular vesicles during infection remain especially poorly described. In this short review, we will summarize the current state of affairs surrounding the antimicrobial function, and potential, of host-derived extracellular vesicles.

Human Neutrophils Produce Antifungal Extracellular Vesicles against Aspergillus fumigatus.

Polymorphonuclear granulocytes (PMNs) are indispensable for controlling life-threatening fungal infections. In addition to various effector mechanisms, PMNs also produce extracellular vesicles (EVs). Their contribution to antifungal defense has remained unexplored. We reveal that the clinically important human-pathogenic fungus Aspergillus fumigatus triggers PMNs to release a distinct set of antifungal EVs (afEVs). Proteome analyses indicated that afEVs are enriched in antimicrobial proteins. The cargo and the release kinetics of EVs are modulated by the fungal strain confronted. Tracking of afEVs indicated that they associated with fungal cells and even entered fungal hyphae, resulting in alterations in the morphology of the fungal cell wall and dose-dependent antifungal effects. To assess as a proof of concept whether the antimicrobial proteins found in afEVs might contribute to growth inhibition of hyphae when present in the fungal cytoplasm, two human proteins enriched in afEVs, cathepsin G and azurocidin, were heterologously expressed in fungal hyphae. This led to reduced fungal growth relative to that of a control strain producing the human retinol binding protein 7. In conclusion, extracellular vesicles produced by neutrophils in response to A. fumigatus infection are able to associate with the fungus, limit growth, and elicit cell damage by delivering antifungal cargo. This finding offers an intriguing, previously overlooked mechanism of antifungal defense against A. fumigatusIMPORTANCE Invasive fungal infections caused by the mold Aspergillus fumigatus are a growing concern in the clinic due to the increasing use of immunosuppressive therapies and increasing antifungal drug resistance. These infections result in high rates of mortality, as treatment and diagnostic options remain limited. In healthy individuals, neutrophilic granulocytes are critical for elimination of A. fumigatus from the host; however, the exact extracellular mechanism of neutrophil-mediated antifungal activity remains unresolved. Here, we present a mode of antifungal defense employed by human neutrophils against A. fumigatus not previously described. We found that extracellular vesicles produced by neutrophils in response to A. fumigatus infection are able to associate with the fungus, limit growth, and elicit cell damage by delivering antifungal cargo. In the end, antifungal extracellular vesicle biology provides a significant step forward in our understanding of A. fumigatus host pathogenesis and opens up novel diagnostic and therapeutic possibilities.