Elimination of Aspergillus fumigatus conidia from the airways of mice with allergic airway inflammation

Abstract

Background: Aspergillus fumigatus conidia can exacerbate asthma symptoms. Phagocytosis of conidia is a principal component of the host antifungal defense. We investigated whether allergic airway inflammation (AAI) affects the ability of phagocytic cells in the airways to internalize the resting fungal spores.

Methods: Using BALB/c mice with experimentally induced AAI, we tested the ability of neutrophils, macrophages, and dendritic cells to internalize A. fumigatus conidia at various anatomical locations. We used light microscopy and differential cell and conidium counts to determine the ingestion potential of neutrophils and macrophages present in bronchoalveolar lavage (BAL). To identify phagocyte-conidia interactions in conducting airways, conidia labeled with tetramethylrhodamine-(5-(and-6))-isothiocyanate were administered to the oropharyngeal cavity of mice. Confocal microscopy was used to quantify the ingestion potential of Ly-6G+ neutrophils and MHC II+ antigen-presenting cells located in the intraepithelial and subepithelial areas of conducting airways.

Results: Allergen challenge induced transient neutrophil recruitment to the airways. Application of A. fumigatus conidia at the acute phase of AAI provoked recurrent neutrophil infiltration, and consequently increased the number and the ingestion potential of the airway neutrophils. In the absence of recurrent allergen or conidia provocation, both the ingestion potential and the number of BAL neutrophils decreased. As a result, conidia were primarily internalized by alveolar macrophages in both AAI and control mice at 24 hours post-inhalation. Transient influx of neutrophils to conducting airways shortly after conidial application was observed in mice with AAI. In addition, the ingestion potential of conducting airway neutrophils in mice with induced asthma exceeded that of control mice. Although the number of neutrophils subsequently decreased, the ingestion capacity remained elevated in AAI mice, even at 24 hours post-conidia application.

Conclusions: Aspiration of allergen to sensitized mice enhanced the ingestion potential of conducting airway neutrophils. Such activation primes neutrophils so that they are sufficient to control dissemination of non-germinating A. fumigatus conidia. At the same time, it can be a reason for the development of sensitivity to fungi and subsequent asthma exacerbation.

Figure 1

Internalization of A. fumigatus conidia by BAL cells during AAI. (A) Mice received three i.p. injections of OVA (on days 0, 7, and 21; 10 μg/mouse/injection), and were challenged on day 27 with OVA (1% in PBS) (OVA/OVA group) or PBS (OVA/PBS group). Mice were inoculated with A. fumigatus conidia via inhalation at 24 hours post-allergen challenge. BAL samples were analyzed at 0, 2, 4, and 24 hours post-conidial administration. (B) Total and differential cell numbers immediately before conidia application were evaluated. NM – non-treated mice. (C) The neutrophil to macrophage ratio in BALs of OVA/PBS (open bars) or OVA/OVA (black bars) mice at the indicated time points following conidial application. (D) The ratio of internalized by neutrophils to internalized by macrophages conidia numbers at the different time points following conidial application to OVA/PBS (open bars) and OVA/OVA (black bars) mice. Data are shown as means ± SEM for two representative experiments, with three and five mice per group. OVA/OVA versus OVA/PBS group or OVA/PBS versus NM: * (p < 0.05), *** (p < 0.001), and ns: not significant. Neutrophil to macrophage ratio at the indicated time point versus the time point immediately before conidial application: § (p < 0.01).

Figure 2

Distribution of A. fumigatus conidia and Ly-6G⁺ neutrophils along the mouse conducting airway. The conducting airway was dissected from the right inferior lung lobe of intact mice that received 5 × 10⁶A. fumigatus conidia 4 hours prior to dissection. (A) Microdissected mouse airway represented as montage of the number of confocal images scanned with low magnification from the luminal side of the specimen was arbitrarily separated (dashed lines) into proximal, distal, dorsal, and ventral regions. (B) Representative three-dimensional image showing the distribution of Ly-6G⁺ neutrophils (green) and TRITC-labeled A. fumigatus conidia (red) in the conducting airway mucosa. (C) The single conidium and (D) neutrophil indicated in (B) are represented as the three-dimensional objects. (E) The conidium located on the luminal side of the conducting airway epithelium, (F) in the subepithelial area, and (G) parenchymal tissue, is indicated by arrows on Z-projections (top view: E, F, G upper panels) and Y-projections (front view: E, F, G lower panels). Epithelial and subepithelial compartments were separated based on epithelium auto-fluorescence (dashed line on E, F, G lower panels). Scale bar = 20 μm (B, E, F, G); scale bar and greed spacing = 1 μm (C), and 5 μm (D).

Figure 3

A. fumigatus conidia-induced recruitment of neutrophils to the conducting airways of mice with AAI. Representative confocal images taken from the distal ventral segment of conducting airway at 2 hours post-conidial inhalation by (A) OVA/PBS and (B) OVA/OVA mice. Images are represented as Z- (upper panels) and Y-projection (lower panels). Ly-6G⁺ neutrophils are shown in green and epithelial auto-fluorescence in dark green. Epithelial and subepithelial compartments, separated by a dashed line, were determined based on epithelial auto-fluorescence (lower panels). Neutrophil in the epithelial compartment of the conducting airway mucosa is indicated by arrow. The images are shown as MIP and the scale bar = 50 μm.

Figure 4

Internalization of A. fumigatus conidia in the epithelial compartment of conducting airways. (A) Representative confocal image taken from the proximal ventral segment of the whole-mount airway main axial pathway showing the distribution of MHC II⁺ epithelial DCs (upper left panel: MHC II⁺ DCs, yellow; epithelium auto-fluorescence, dark yellow), Ly-6G⁺ neutrophils (upper right panel, green), A. fumigatus conidia (lower left panel, red), and a merged image (lower right panel) at 4 hours post-conidial application. Scale bar = 20 μm. (B, C) The neutrophil locating in close proximity to the luminal side of the epithelium (arrowhead) and the intraepithelial DC (arrow) are indicated on Z-projection (left panels) or on X-projection (right panels) of the area displayed in (A). (D) The same neutrophil and DC are displayed on the three-dimensional larger magnification image of the region boxed in (A). Scale bar = 20 μm (A, B, C); and 10 μm (D).

Figure 5

Capture of conidia by the conducting airway epithelial DCs. (A) Three-dimensional reconstitution of the image stacks taken from the proximal ventral region of the conducting airway showing the distribution of Ly-6G⁺ neutrophils (bright green), epithelium auto-fluorescence (dark green), MHC II⁺ APCs (yellow), and A. fumigatus conidia (red) in an OVA/PBS mouse at 8 hours post-conidial application. Scale bar = 50 μm. (B) Three-dimensional reconstitution of the area indicated on (A) representing MHC II⁺ APCs (yellow) and A. fumigatus conidia (red: arrow); scale bar = 10 μm. (C) X, Y, and Z-projections of the area framed in (A) showing the precise location of an intraepithelial (epithelium auto-fluorescence, green) and epithelial DC (yellow) that internalized A. fumigatus conidia (red: arrow). Scale bar = 20 μm. (D) An epithelial DC that internalized conidia is represented as the enlarged and arbitrary rotated three-dimensional surface object. Scale bar = 5 μm.

Figure 6

Quantitative analysis of the number and ingestion effectiveness of conducting airway neutrophils and APCs. The numbers of conducting airway Ly-6G⁺ neutrophils (A) and MHC II⁺ APCs per mm² of epithelium (B) and the percentages of conidia that were internalized by neutrophils (C) and APCs from the total number of conidia in the region under observation (D) were detected. Data were acquired for OVA/OVA (black bars) or OVA/PBS (open bars) animals at different time points following A. fumigatus conidial application. Mean and SEM are presented for two independent experiments with three and four mice per group, respectively. The difference between OVA/OVA and OVA/PBS groups: ** (p< 0.01), *** (p< 0.001), and ns: not significant. Significant difference between neutrophil numbers and conidia percentages at initial and indicated time points: § (p<0.01).