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Aspergillus fumigatus Cell Wall Promotes Apical Airway Epithelial Recruitment of Human Neutrophils.

ABSTRACT: Aspergillus fumigatus is a ubiquitous fungal pathogen capable of causing multiple pulmonary diseases, including invasive aspergillosis, chronic necrotizing aspergillosis, fungal colonization, and allergic bronchopulmonary aspergillosis. Intact mucociliary barrier function and early airway neutrophil responses are critical for clearing fungal conidia from the host airways prior to establishing disease. Following inhalation, Aspergillus conidia deposit in the small airways, where they are likely to make their initial host encounter with epithelial cells. Challenges in airway infection models have limited the ability to explore early steps in the interactions between A. fumigatus and the human airway epithelium. Here, we use inverted air-liquid interface cultures to demonstrate that the human airway epithelium responds to apical stimulation by A. fumigatus to promote the transepithelial migration of neutrophils from the basolateral membrane surface to the apical airway surface. Promoting epithelial transmigration with Aspergillus required prolonged exposure with live resting conidia. Swollen conidia did not expedite epithelial transmigration. Using A. fumigatus strains containing deletions of genes for cell wall components, we identified that deletion of the hydrophobic rodlet layer or dihydroxynaphthalene-melanin in the conidial cell wall amplified the epithelial transmigration of neutrophils, using primary human airway epithelium. Ultimately, we show that an as-yet-unidentified nonsecreted cell wall protein is required to promote the early epithelial transmigration of human neutrophils into the airspace in response to A. fumigatus Together, these data provide critical insight into the initial epithelial host response to Aspergillus.

SUBMITTER: Feldman MB

PROVIDER: S-EPMC6977131 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description: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. fumigatus IMPORTANCE 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.

Project description:During the initiation of invasive aspergillosis, inhaled Aspergillus fumigatus conidia are deposited on the epithelial cells lining the bronchi, terminal bronchioles, and alveoli. While the interactions of A. fumigatus with bronchial and type II alveolar cell lines have been investigated in vitro, little is known about the interactions of this fungus with terminal bronchiolar epithelial cells. Using the HSAEC1-KT human small airway epithelial (HSAE) cell line, we developed an in vitro model to study the interaction of two strains of A. fumigatus with these cells. We then compared the interactions of A. fumigatus with the A549 type II alveolar epithelial cell line and the HSAE cell line. We found that A. fumigatus conidia were poorly endocytosed by A549 cells, but avidly endocytosed by HSAE cells. A. fumigatus germlings invaded both cell types by induced endocytosis, but not by active penetration. A549 cell endocytosis of A. fumigatus was independent of fungal viability, more dependent on host microfilaments than microtubules, and induced by A. fumigatus CalA interacting with host cell integrin α5β1. By contrast, HSAE cell endocytosis required fungal viability, was more dependent on microtubules than microfilaments, and did not require CalA or integrin α5β1. HSAE cells were more susceptible than A549 cells to damage caused by direct contact with killed A. fumigatus germlings and by secreted fungal products. In response to A. fumigatus infection, A549 cells secreted a broader profile of cytokines and chemokines than HSAE cells. Taken together, these results demonstrate that studies of HSAE cells provide complementary data to A549 cells and thus represent a useful model for probing the interactions of A. fumigatus with bronchiolar epithelial cells in vitro. Importance During the initiation of invasive aspergillosis, Aspergillus fumigatus interacts with the epithelial cells that line the airways and alveoli. Previous studies of A. fumigatus-epithelial cell interactions in vitro used either large airway epithelial cell lines or the A549 type II alveolar epithelial cell line; the interactions of fungi with terminal bronchiolar epithelial cells were not investigated. Using the TERT-immortalized human small airway epithelial HSAEC1-KT (HSAE) cell line, we developed an in vitro model of the interactions of A. fumigatus with bronchiolar epithelial cells. We discovered that A. fumigatus invades and damages A549 and HSAE cell lines by distinct mechanisms. Also, the proinflammatory responses of the cell lines to A. fumigatus are different. These results provide insight into how A. fumigatus interacts with different types of epithelial cells during invasive aspergillosis and demonstrate that HSAE cells are useful in vitro model for investigating the interactions of this fungus with bronchiolar epithelial cells.

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Aspergillus fumigatus Cell Wall Promotes Apical Airway Epithelial Recruitment of Human Neutrophils.

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