Epithelial uptake ofAspergillus fumigatusdrives efficient fungal clearancein vivoand is aberrant in Chronic Obstructive Pulmonary Disease (COPD)

Abstract

Hundreds of spores of the common mould Aspergillus fumigatus (Af) are inhaled daily by human beings, representing a constant, often fatal, threat to our respiratory health. The small size of Af spores suggest that interactions with Airway Epithelial Cells (AECs) are frequent and we and others have previously demonstrated that AECs are able to internalise Af spores. We thus hypothesised that Af spore uptake and killing by AECs is important for driving efficient fungal clearance in vivo and that defective spore uptake and killing would represent major risk factors for Aspergillus-related diseases. In order to test this, we utilised single-cell approaches based on Imaging Flow Cytometry (IFC) and live-cell microfluidic imaging to measure spore uptake and outcomes in vitro, in vivo and using primary human AECs. In vitro, viability of immortalised AECs was largely unaffected by Af uptake and AECs were able to significantly curtail the growth of internalised spores. Applying our approach directly to infected mouse lungs we demonstrated, for the first time, that Af spores are internalised and killed by AECs during whole animal infection, whereby only ~3% of internalised spores remained viable after 8 hours of co-incubation with murine AECs. Finally, in vitro analysis of primary human AECs from healthy and at-risk donors revealed significant alterations in the uptake and consequent outcomes in Chronic Obstructive Pulmonary Disease (COPD), whereby gorging COPD-derived AECs were unable to quell intracellular Af as efficiently as healthy primary AECs. We have thus demonstrated that AECs efficiently kill Af spores upon uptake in vivo and that this process is altered in COPD, a well-known risk factor for debilitating fungal lung disease, thereby suggesting that AECs critically contribute to the efficient clearance of inhaled Af spores and that dysregulation of curative AEC responses represents a potent driver of Aspergillus-related diseases.

Novel Insights into Aspergillus fumigatus Pathogenesis and Host Response from State-of-the-Art Imaging of Host–Pathogen Interactions during Infection

“…Numerous studies have demonstrated that A. fumigatus conidia are readily internalised by AECs in vitro, with both alveolar and bronchial epithelial cells internalising 30–50% of the conidia they encounter [ 17 , 31 , 120 , 121 , 122 , 123 , 124 , 125 , 126 , 127 , 128 , 129 ]. In vitro A. fumigatus uptake by AECs is a time-dependent process, which increases proportionally with longer co-incubations or a higher multiplicity of infections [ 17 , 126 , 128 ]. AECs exert antifungal activity, with intracellular conidia showing impaired germination relative to extracellular conidia [ 17 , 126 ].…”

“…In vitro A. fumigatus uptake by AECs is a time-dependent process, which increases proportionally with longer co-incubations or a higher multiplicity of infections [ 17 , 126 , 128 ]. AECs exert antifungal activity, with intracellular conidia showing impaired germination relative to extracellular conidia [ 17 , 126 ]. This antifungal activity has been largely attributed to phagosome acidification [ 122 , 126 ], and, indeed, population- and single-cell studies have demonstrated that the vast majority of internalised conidia (90–97%) are killed by AECs within 12–24 h of infection; however, a small subpopulation is able to avoid clearance and to germinate intracellularly [ 17 , 122 ].…”

“…AECs exert antifungal activity, with intracellular conidia showing impaired germination relative to extracellular conidia [ 17 , 126 ]. This antifungal activity has been largely attributed to phagosome acidification [ 122 , 126 ], and, indeed, population- and single-cell studies have demonstrated that the vast majority of internalised conidia (90–97%) are killed by AECs within 12–24 h of infection; however, a small subpopulation is able to avoid clearance and to germinate intracellularly [ 17 , 122 ]. In response to challenging intracellular conditions, this subpopulation shows unique morphological changes and surviving intracellular conidia frequently form secondary germ tubes and contain an increased number of vacuoles in their hyphae [ 126 ].…”

“…In response to challenging intracellular conditions, this subpopulation shows unique morphological changes and surviving intracellular conidia frequently form secondary germ tubes and contain an increased number of vacuoles in their hyphae [ 126 ]. Interestingly, AECs that internalise conidia do not undergo increased cell death (relative to uninfected cells), suggesting that, while A. fumigatus clearly leads to damage to AEC monolayers, this damage is not primarily associated with uptake [ 17 ]. Indeed, a small proportion of germinated intracellular conidia are able to escape AECs while avoiding the rupturing of host cells, and, following non-lytic escape, hyphal extensions can invade the adjacent AECs without further perpetuating host cell damage [ 126 , 130 ].…”

“…We aim to review the advanced imaging techniques applied to invertebrate and murine models of aspergillosis, which have recently provided important visually-quantitative insights into the host–pathogen interactions within the infected host. Previous reviews have already expertly addressed the current knowledge on, and dynamic intricacy of, the A. fumigatus host–pathogen interaction (some examples are [ 10 , 11 , 12 , 13 , 14 ]); therefore, in this review, we will discuss recent contributions and future potential applications of state-of-the-art imaging techniques by answering four outstanding questions regarding A. fumigatus pathogenesis and antifungal response during A. fumigatus infection ( Figure 1 ) [ 15 , 16 , 17 , 18 ].…”