Three-Dimensional 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 of A. fumigatus infections are highly complex and depend on both fungal attributes and the immune status of the host. Therefore, preclinical animal models are of paramount importance 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 complete intra-alveolar deposition, as about 80% of fungal growth occurred outside the alveolar space. Hence, characterization by LSFM is more rigorous than by previously used methods employing murine models of invasive pulmonary aspergillosis and pinpoints their strengths and limitations. IMPORTANCE The use of animal models of infection is essential to advance our understanding of the complex host-pathogen interactions that take place during Aspergillus fumigatus lung infections. As in the case of humans, mice need to suffer an immune imbalance in order to become susceptible to invasive pulmonary aspergillosis (IPA), the most serious infection caused by A. fumigatus. There are several immunosuppressive regimens that are routinely used to investigate fungal growth and/or immune responses in murine models of invasive pulmonary aspergillosis. However, the precise consequences of the use of each immunosuppressive model for the local immune populations and for fungal growth are not completely understood. Here, to pin down the scenarios involving commonly used IPA models, we employed light sheet fluorescence microscopy (LSFM) to analyze whole lungs at cellular resolution. Our results will be valuable to optimize and refine animal models to maximize their use in future research.

From 2D to 3D: Promising Advances in Imaging Lung Structure

“…Light sheet microscopy has been employed to investigate inflammation, cancer, hemopoiesis and infection in lung and other tissues ( 18 – 21 ). It has also has been used to investigate the distribution of Aspergillosis fumigatus and identify the local inflammatory response secondary to pulmonary infections ( 22 ). However, this technique is limited by optical aberrations and a point-by-point scanning technique ( 17 ).…”

3D reconstruction of SARS-CoV-2 infection in ferrets emphasizes focal infection pattern in the upper respiratory tract

“…The samples were dehydrated in a graded ethanol series (30% [v/v], 50%, 70%, and twice in 100%; each for ≥ 8 h at 4 °C, diluted in aqua ad iniectabilia, and pH-adjusted to 9). Following a two-hour wash with n -hexane at room temperature [60], n -hexane was gradually replaced with the clearing agent ECi and samples were incubated until optically transparent.…”

Biosensors and Diagnostics for Fungal Detection

“…Antigen testing will be more helpful in patients with neutropenia or other conditions affecting humoral or cellular immunity [ 53 ]. For example, testing for the fungal antigens galactomannan, mannan and β-D-glucan in serum samples and testing for galactomannan in BAL samples are among those routinely performed serological tests for the detection of infections due to Aspergillus, Candida and some other mould infections in immunocompromised patients [ 18 , 54 , 55 , 56 , 57 ]. Common diagnostic tests for antibody detection such as, CF and ID are useful markers of infection with histoplasmosis and other endemic dimorphic pathogens, chronic aspergillosis and candidosis.…”