Aspergillus fumigatusInfection-Induced Neutrophil Recruitment and Location in the Conducting Airway of Immunocompetent, Neutropenic, and Immunosuppressed Mice

Abstract

Susceptibility to fungal infection is commonly associated with impaired neutrophil responses. To study the mechanisms underlying this association, we investigated neutrophil recruitment to the conducting airway wall after Aspergillus fumigatus conidium inhalation in mouse models of drug-induced immunosuppression and antibody-mediated neutrophil depletion (neutropenia) by performing three-dimensional confocal laser-scanning microscopy of whole-mount primary bronchus specimens. Actin staining enabled visualization of the epithelial and smooth muscle layers that mark the airway wall. Gr-1+ or Ly6G+ neutrophils located between the epithelium and smooth muscles were considered airway wall neutrophils. The number of airway wall neutrophils for immunocompetent, immunosuppressed, and neutropenic mice before and 6 h after A. fumigatus infection were analyzed and compared. Our results show that the number of conducting airway wall neutrophils in immunocompetent mice significantly increased upon inflammation, while a dramatic reduction in this number was observed following immunosuppression and neutropenia. Interestingly, a slight increase in the infiltration of neutrophils into the airway wall was detected as a result of infection, even in immunosuppressed and neutropenic mice. Taken together, these data indicate that neutrophils are present in intact conducting airway walls and the number elevates upon A. fumigatus infection. Conducting airway wall neutrophils are affected by both neutropenia and immunosuppression.

Three-Dimensional Light Sheet Fluorescence Microscopy of Lungs To Dissect Local Host Immune-Aspergillus fumigatus Interactions

“…Therefore, we employed LSFM to reveal spatial information with respect to the distribution of A. fumigatus in the context of the pulmonary microanatomy to address the issue of whether or not the localization of the infective conidia is within the alveoli. To this end, we stained lung epithelial cells with an antipodoplanin antibody, visualizing the epithelial surface to measure the distance of fungal burden to bronchioles by excluding surfaces for the alveoli with diameters less than 50 m, which is a typical diameter of an alveolus in mice (34,35) (Fig. 4A).…”

“…Thus, histology sections serve as a reference for fungal germination/hyphal enlargement or presence/absence of immune cells but provide qualitative rather than quantitative data. Recently, Shevchenko and colleagues utilized confocal microscopy to investigate neutrophil recruitment and location in the conducting airway upon A. fumigatus infection (35). In a further development, Kowalski and colleagues utilized the passive clarity technique (PACT) to investigate the architecture of in vivo fungal growth in cleared lungs using confocal microscopy (36).…”

3D light sheet fluorescence microscopy of lungs to dissect local host immune -Aspergillus fumigatusinteractions

“…Thus, histology sections serve as a reference of fungal germination/hyphal enlargement or presence/absence of immune cells, but provide qualitative rather than quantitative data. Recently, Shevchenko and colleagues utilized confocal microscopy to investigate neutrophil recruitment and location in the conducting airway upon A. fumigatus infection [37]. Although this is a significant improvement, this technique is still limited by the small fraction of tissue imaged, which makes any quantification and anatomical information relative.…”

Murine Intraepithelial Dendritic Cells Interact With Phagocytic Cells During Aspergillus fumigatus-Induced Inflammation

“…Neutropenia was mimicked by injecting neutrophil-depleting antibodies rat anti-mouse Ly6G (BioLegend), clone 1A8, 170 µg per mouse. Antibody dosage was chosen in accordance with our previous findings (22). Control groups received rat IgG2a (BioLegend), 170 µg per mouse.…”

“…Image stacks were analyzed using Imaris version 7.6.5 software (Bitplane Scientific Software, Zurich, Switzerland). MHCII + APCs, CD11c + cells, Ly6G + neutrophils, CD11b + phagocytes and A. fumigatus conidia, along with the epithelial and smooth muscle layers, were identified and processed via "threedimensional surface rendering" of the appropriate channel, as previously described (22). The threshold and filter settings were optimized by visually comparing the result with the maximum intensity projection.…”