Aspergillus fumigatus, a saprophytic fungus, causes clinical syndromes ranging from colonization to deep-seated infection in lung tissue of individuals with a challenged immune system. Current studies primarily involve the use of animal models and cell lines to understand the fungal invasion process which have many limitations. Thus, we set up a perfused 3-dimensional in vitro cell culture model containing primary, differentiated human bronchial epithelial cells and immune cells important to sense fungal pathogens, i.e. alveolar macrophages or dendritic cells. This model will help us better understand fungal-epithelial-immune interactions in more detail at the entry sites of the pathogen.
Development, differentiation and interactions with A. fumigatus of epithelial - immune cell co-cultures under static or perfused conditions were studied using confocal, scanning electron (SEM) and live cell microscopy. Additionally, cytokine analyses from Aspergillus-exposed tissues and TEER measurements were performed. Analysis over time by confocal microscopy showed that the respiratory cells differentiated in an air-liquid interface culture to form tight junctions, produced mucus and developed cilia. This development was significantly accelerated under perfused compared to static conditions. SEM and confocal studies of the respiratory cells infected with Aspergillus demonstrated the progression of infection with time. In epithelial-immune cell co-cultures, migration of the macrophages from the basolateral to the apical site was detected upon fungal infection, where macrophages co-localized with fungal hyphae.
Our model will provide novel immunologic and mechanistic insights into Aspergillus-infection processes within 3D space. Once optimized, this model will support better understanding of pathogenesis, detection, and treatment of polymicrobial infections. Additionally, animal experimentation can be significantly reduced by use of this highly developed human system, thereby contributing to ethical considerations and higher biological relevance in terms of avoiding interspecies differences.
