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. 2023 Jun 17;9(6):682.
doi: 10.3390/jof9060682.

Characterization of Lung Inflammatory Response to Aspergillus fumigatus Spores

Alexandra Bouyssi  1 , Tanguy Déméautis  1 , Alexis Trecourt  1   2 , Marie Delles  1 , Fany Agostini  1 , Guillaume Monneret  3 , Olivier Glehen  4 , Martine Wallon  5 , Florence Persat  1   5 , Gilles Devouassoux  1   6 , Abderrazzak Bentaher  1 , Jean Menotti  1   5
Affiliations

Characterization of Lung Inflammatory Response to Aspergillus fumigatus Spores

Alexandra Bouyssi et al. J Fungi (Basel). .

Abstract

The airway exposure to Aspergillus fumigatus spores (AFsp) is associated with an inflammatory response, potentially leading to allergic and/or chronic pulmonary aspergillosis. The aim of our study is to better understand the host response, first in vitro, then in vivo, following the chronic exposure of mice to AFsp. We investigated the inflammatory response to AFsp in cell mono- and co-culture systems with murine macrophages and alveolar epithelial cells. The mice were subjected to two intranasal instillations using 105 AFsp. Their lungs were processed for inflammatory and histopathological analyses. In cell culture, the gene expressions significantly increased for TNF-α, CXCL-1, CXCL-2, IL-1β, IL-1α and GM-CSF in macrophages, with these increases being limited for TNF-α, CXCL-1 and IL-1α in epithelial cells. In co-culture, increases in the TNF-α, CXCL-2 and CXCL-1 gene expressions were observed to be associated with increased protein levels. The in vivo lung histological analyses of mice challenged by AFsp showed cellular infiltrates in the peribronchial and/or alveolar spaces. A Bio-Plex approach on the bronchoalveolar lavage revealed significant increases in the protein secretion of selected mediators of the challenged mice compared to the unchallenged mice. In conclusion, the exposure to AFsp resulted in a marked inflammatory response of macrophages and epithelial cells. These inflammatory findings were confirmed in mouse models associated with lung histologic changes.

Keywords: Aspergillus fumigatus; epithelial cell; immune response; lung; macrophages; spores.

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Conflict of interest statement

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

Figures

Figure 1
Figure 1
Gene expression of defined inflammatory mediators after a 4 h exposure of RAW 264.7 and MLE-15 cells to A. fumigatus spores. (A) Quantification of the gene expression of inflammatory markers in RAW 264.7 cells via RT-qPCR. (B) Quantification of the gene expression of inflammatory markers in MLE-15 cells via RT-qPCR. (*** p < 0.001; ** p < 0.01; * p < 0.05 versus untreated cells, n = 3; ns, not significant.) Data are represented as mean with SD.
Figure 2
Figure 2
Immunofluorescence micrograph (×250 magnification) of the co-culture system (A); the white arrow points to E-cadherin staining for epithelial cells in red, and the arrowhead depicts CD-68 staining for macrophages in green-yellow (ratio: 2 MLE-15/1 RAW264.7). Gene expression of inflammatory mediators (B) and cytokine secretion in the supernatants after a 4 h exposure of the co-culture model to A. fumigatus spores (C) (**** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.05 versus untreated cells, n = 4). Data are represented as mean with SD.
Figure 3
Figure 3
Histopathological features of inflammatory reaction (location and intensity) in mice challenged with A. fumigatus spores (A–F) and control mice (G–I). (A) (Hematoxylin–eosin–saffron (HES), ×200 magnification). Mild peribronchiolar inflammation (red arrows): inflammatory cells easy to count around the bronchioles (green arrow). (B) (HES, ×200 magnification). Moderate perivascular inflammation (red arrow): inflammatory cells difficult to count around the vascular wall (green arrow). (C) (HES, ×200 magnification). Severe perivascular inflammation (red arrow): inflammatory cells impossible to count around the vascular wall (green arrow). (D) (HES, ×400 magnification). Mild alveolar inflammation with lymphocytes, plasma cells and few neutrophils and eosinophils (red arrow). (E) (HES, ×190 magnification). Moderate alveolar inflammation (red arrow) with lymphocytes, plasma cells and macrophages with thickening of the alveolar walls (green arrow). (F) (HES, ×100 magnification). Perivascular edema: edema (red asterisks) surrounding the vessel wall (red arrow). (G–I) (HES, ×200 magnification). No alveolar or peribronchiolar inflammation (except in 1 mouse out of 7), no fibrosis and no edema were observed in the control group.
Figure 4
Figure 4
Total BAL cell counts (A) and cell types in BAL (B) from control mice or mice exposed to spores (** p < 0.01; * p < 0.05). Total cell counts in the BAL was measured using an Automatic Cell Counter with trypan blue. The cellularity was estimated on cytospin slides. Data are represented as mean with SD.
Figure 5
Figure 5
Fungal burden in mouse lung after exposure to two instillations of 105 A. fumigatus spores (sAF) 5 weeks apart (**** p < 0.0001). Data are represented as Min to Max in box and whiskers form.
Figure 6
Figure 6
Gene expression of defined inflammatory mediators after exposure of mice to two instillations of 105 A. fumigatus spores (sAF) 5 weeks apart (*** p < 0.001, ** p < 0.01, * p < 0.05 versus control mice, n = 7). Data are represented as mean with SD.
Figure 7
Figure 7
Quantification of defined secreted cytokines in bronchoalveolar lavage (BAL) of mice exposed to A. fumigatus spores (sAF) (**** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.05 versus control mice, n = 7). Data are represented as mean with SD.
Figure 8
Figure 8
Quantification of defined secreted cytokines in sera of mice exposed to A. fumigatus spores (sAF) (*** p < 0.001, ** p < 0.01, * p < 0.05 versus control mice, n = 7). Data are represented as mean with SD.
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