• Characterisation of Aspergillus fumigatus Endocytic Trafficking within Airway Epithelial Cells Using High-Resolution Automated Quantitative Confocal Microscopy

      Ben-Ghazzi, Nagwa; email: nagwaben@gmail.com; Moreno-Velásquez, Sergio; email: s.moreno.velasquez@gmail.com; Seidel, Constanze; email: dr.constanze.seidel@gmail.com; Thomson, Darren; orcid: 0000-0002-4800-7717; email: darren.thomson@manchester.ac.uk; Denning, David W.; email: david.denning@manchester.ac.uk; Read, Nick D.; email: nick.read@manchester.ac.uk; Bowyer, Paul; email: paul.bowyer@manchester.ac.uk; Gago, Sara; orcid: 0000-0002-7027-4598; email: sara.gago-2@manchester.ac.uk (MDPI, 2021-06-07)
      The precise characterization of the mechanisms modulating Aspergillus fumigatus survival within airway epithelial cells has been impaired by the lack of live-cell imaging technologies and user-friendly quantification approaches. Here we described the use of an automated image analysis pipeline to estimate the proportion of A. fumigatus spores taken up by airway epithelial cells, those contained within phagolysosomes or acidified phagosomes, along with the fungal factors contributing to these processes. Coupling the use of fluorescent A. fumigatus strains and fluorescent epithelial probes targeting lysosomes, acidified compartments and cell membrane, we found that both the efficacy of lysosome recruitment to phagosomes and phagosome acidification determines the capacity of airway epithelial cells to contain A. fumigatus growth. Overall, the capability of the airway epithelium to prevent A. fumigatus survival was higher in bronchial epithelial than alveolar epithelial cells. Certain A. fumigatus cell wall mutants influenced phagosome maturation in airway epithelial cells. Taken together, this live-cell 4D imaging approach allows observation and measurement of the very early processes of A. fumigatus interaction within live airway epithelial monolayers.
    • Differential Proinflammatory Responses to Aspergillus fumigatus by Airway Epithelial Cells In Vitro Are Protease Dependent

      Rowley, Jessica; email: j.rowley@imperial.ac.uk; Namvar, Sara; orcid: 0000-0001-5571-368X; email: s.namvar@salford.ac.uk; Gago, Sara; orcid: 0000-0002-7027-4598; email: sara.gago-2@manchester.ac.uk; Labram, Briony; email: briony.labram@nc3rs.org.uk; Bowyer, Paul; email: paul.bowyer@manchester.ac.uk; Richardson, Malcolm D.; orcid: 0000-0001-5672-9552; email: Malcolm.Richardson@manchester.ac.uk; Herrick, Sarah E.; orcid: 0000-0002-9085-5664; email: Sarah.Herrick@manchester.ac.uk (MDPI, 2021-06-10)
      Aspergillus fumigatus is an important human respiratory mould pathogen. In addition to a barrier function, airway epithelium elicits a robust defence against inhaled A. fumigatus by initiating an immune response. The manner by which A. fumigatus initiates this response and the reasons for the immunological heterogeneity with different isolates are unclear. Both direct fungal cell wall–epithelial cell interaction and secretion of soluble proteases have been proposed as possible mechanisms. Our aim was to determine the contribution of fungal proteases to the induction of epithelial IL-6 and IL-8 in response to different A. fumigatus isolates. Airway epithelial cells were exposed to conidia from a low or high protease-producing strain of A. fumigatus, and IL-6 and IL-8 gene expression and protein production were quantified. The role of proteases in cytokine production was further determined using specific protease inhibitors. The proinflammatory cytokine response correlated with conidia germination and hyphal extension. IL-8 induction was significantly reduced in the presence of matrix metalloprotease or cysteine protease inhibitors. With a high protease-producing strain of A. fumigatus, IL-6 release was metalloprotease dependent. Dectin-1 antagonism also inhibited the production of both cytokines. In conclusion, A. fumigatus-secreted proteases mediate a proinflammatory response by airway epithelial cells in a strain-dependent manner.