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

Abstract

Aspergillus fumigatus is an opportunistic fungal pathogen that can cause life-threatening invasive lung infections in immunodeficient patients. The cellular and molecular processes of infection during onset, establishment and progression are highly complex and depend on both fungal attributes and the immune status of the host. Therefore, preclinical animal models are paramount to investigate and gain better insight into the infection process. Yet, despite their extensive use, commonly employed murine models of invasive pulmonary aspergillosis are not well understood due to analytical limitations. Here we present quantitative light sheet fluorescence microscopy (LSFM) to describe fungal growth and the local immune response in whole lungs at cellular resolution within its anatomical context. We analyzed three very common murine models of pulmonary aspergillosis based on immunosuppression with corticosteroids, chemotherapy-induced leukopenia or myeloablative irradiation. LSFM uncovered distinct architectures of fungal growth and degrees of tissue invasion in each model. Furthermore, LSFM revealed the spatial distribution, interaction and activation of two key immune cell populations in antifungal defense: alveolar macrophages and polymorphonuclear neutrophils. Interestingly, the patterns of fungal growth correlated with the detected effects of the immunosuppressive regimens on the local immune cell populations. Moreover, LSFM demonstrates that the commonly used intranasal route of spore administration did not result in a complete intra-alveolar deposition, as more than 60% of fungal growth occurred outside of the alveolar space. Hence, LSFM allows for more rigorous characterization than previously used methods of murine models of invasive pulmonary aspergillosis and pinpointing their strengths and limitations.