confocal-laser-scanning-microscopy-based-quantitative-analysis

Confocal Laser Scanning Microscopy-Based Quantitative Analysis of <em>Aspergillus fumigatus</em> Conidia Distribution in Whole-Mount Optically Cleared Mouse Lung

Aspergillus fumigatus conidia are airborne pathogens that can penetrate human airways. Immunocompetent people without allergies exhibit resistance and ...

Russian Academy of Sciences

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.

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

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  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  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  infection. Conducting airway wall neutrophils are affected by both neutropenia and immunosuppression.

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

People are constantly exposed to airborne fungal spores, including  conidia that can cause life-threatening conditions in immunocompromised patients or acute exacerbations in allergics. However, immunocompetent hosts do not exhibit mycoses or systemic inflammation, due to the sufficient but not excessive antifungal immune response that prevent fungal invasion. Intraepithelial dendritic cells (IE-DCs) of the conducting airway mucosa are located in the primary site of the inhalant pathogen entry; these cells can sense  conidia and maintain homeostasis. The mechanisms by which IE-DCs contribute to regulating the antifungal immune response and controlling conidia dissemination are not understood. To clarify the role of IE-DCs in the balance between pathogen sensing and immune tolerance we investigated the  conidia distribution in optically cleared mouse lungs and estimated the kinetics of the local phagocytic response during the course of inflammation. MHCII antigen-presenting cells, including IE-DCs, and CD11b phagocytes were identified by immunohistochemistry and three-dimensional fluorescence confocal laser-scanning microscopy of conducting airway whole-mounts. Application of  conidia increased the number of CD11b phagocytes in the conducting airway mucosa and induced the trafficking of these cells through the conducting airway wall to the luminal side of the epithelium. Some CD11b phagocytes internalized conidia in the conducting airway lumen. During the migration through the airway wall, CD11b phagocytes formed clusters. Permanently located in the airway wall IE-DCs contacted both single CD11b phagocytes and clusters. Based on the spatiotemporal characteristics of the interactions between IE-DCs and CD11b phagocytes, we provide a novel anatomical rationale for the contribution of IE-DCs to controlling the excessive phagocyte-mediated immune response rather than participating in pathogen uptake.

Marina A. Shevchenko

Department of Immunology,
Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry,
Department of Immunology, Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry

Russian Academy of Sciences

Confocal Laser Scanning Microscopy-Based Quantitative Analysis of Aspergillus fumigatus Conidia Distribution in Whole-Mount Optically Cleared Mouse Lung

Marina A. Shevchenko

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Immunology,Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry,Department of Immunology, Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry

Russian Academy of Sciences

Confocal Laser Scanning Microscopy-Based Quantitative Analysis of Aspergillus fumigatus Conidia Distribution in Whole-Mount Optically Cleared Mouse Lung

Department of Immunology,
Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry,
Department of Immunology, Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry