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.
Humans are continuously exposed to airborne spores of the saprophytic fungus Aspergillus fumigatus. However, in healthy individuals pulmonary host defense mechanisms efficiently eliminate the fungus. In contrast, A. fumigatus causes devastating infections in immunocompromised patients. Host immune responses against A. fumigatus lung infections in immunocompromised conditions have remained largely elusive. Given the dynamic changes in immune cell subsets within tissues upon immunosuppressive therapy, we dissected the spatiotemporal pulmonary immune response after A. fumigatus infection to reveal basic immunological events that fail to effectively control invasive fungal disease. In different immunocompromised murine models, myeloid, notably neutrophils, and macrophages, but not lymphoid cells were strongly recruited to the lungs upon infection. Other myeloid cells, particularly dendritic cells and monocytes, were only recruited to lungs of corticosteroid treated mice, which developed a strong pulmonary inflammation after infection. Lymphoid cells, particularly CD4+ or CD8+ T-cells and NK cells were highly reduced upon immunosuppression and not recruited after A. fumigatus infection. Moreover, adoptive CD11b+ myeloid cell transfer rescued cyclophosphamide immunosuppressed mice from lethal A. fumigatus infection but not cortisone and cyclophosphamide immunosuppressed mice. Our findings illustrate that CD11b+ myeloid cells are critical for anti-A. fumigatus defense under cyclophosphamide immunosuppressed conditions.
Introduction: Invasive pulmonary Aspergillus fumigatus infections cause high morbidity and mortality in neutropenic patients. Granulocyte transfusions have been tested as an alternative therapy for the management of high-risk neutropenic patients with invasive A. fumigatus infections. To increase the granulocyte yield for transfusion, donors are treated with corticosteroids. Yet, the efficacy of granulocyte transfusion and functional defense mechanisms of granulocytes collected from corticosteroid treated donors remain largely elusive. Therefore, we investigated the efficacy of granulocyte transfusion and functional defense mechanisms of corticosteroid treated granulocytes using mouse models. Methods: To determine the effects of corticosteroids on granulocytes to control A. fumigatus infections, we performed granulocyte adoptive cell transfers using in vivo mouse models, in vitro human and mouse granulocyte and A. fumigatus functional co-culture experiments in combination with flow cytometry, cytokine analysis, fluorescence and electron microscopy. Results: Transfusion of granulocytes from corticosteroid treated mice did not protect cyclophosphamide immunosuppressed mice against lethal A. fumigatus infection in contrast to granulocytes from untreated mice. Upon infection increased levels of inflammatory cytokines helped to recruit granulocytes to the lungs without any recruitment defects in corticosteroid treated and infected mice or in cyclophosphamide immunosuppressed and infected mice that had received the granulocytes from corticosteroid treated mice. However, corticosteroid treated human or mouse neutrophils failed to form neutrophil extracellular traps (NETs) under in vitro and in vivo conditions. Furthermore, corticosteroid treated granulocytes exhibited impaired ROS production against A. fumigatus. Notably, corticosteroids impaired the β-glucan receptor Dectin-1 (CLEC7A) on mouse and human granulocytes to efficiently recognize and phagocytize A. fumigatus, which markedly impaired fungal killing. Conclusions: We conclude that corticosteroid treatment of granulocyte donors for increasing neutrophil yields or patients with ongoing corticosteroid treatment could result in deleterious effects on granulocyte antifungal functions, thereby limiting the benefit of granulocyte transfusion therapies against invasive fungal infections. Disclosures Einsele: Celgene: Consultancy, Honoraria, Speakers Bureau; Janssen: Consultancy, Honoraria, Speakers Bureau; Novartis: Consultancy, Honoraria; Amgen: Consultancy, Honoraria, Speakers Bureau.
Rudolf-Virchow-Zentrum für Experimentelle Biomedizin. 35 Universität Würzburg. 36 Josef-Schneider-Straße 2, Haus D15. D -97080 Würzburg 37 38 39 40 41 42 43 44 45 VISUAL ABSTRACT 46 Quantitative light sheet fluorescence microscopy to dissect local host-pathogen interactions 47 ABSTRACT 51Aspergillus fumigatus is an opportunistic fungal pathogen that can cause life-threatening 52 invasive lung infections in immunodeficient patients. The cellular and molecular processes of 53 infection during onset, establishment and progression are highly complex and depend on 54 both fungal attributes and the immune status of the host. Therefore, preclinical animal 55 models are paramount to investigate and gain better insight into the infection process. Yet, 56 despite their extensive use, commonly employed murine models of invasive pulmonary 57 aspergillosis are not well understood due to analytical limitations. Here we present 58 quantitative light sheet fluorescence microscopy (LSFM) to describe fungal growth and the 59 local immune response in whole lungs at cellular resolution within its anatomical context. We 60 analyzed three very common murine models of pulmonary aspergillosis based on 61 immunosuppression with corticosteroids, chemotherapy-induced leukopenia or 62 myeloablative irradiation. LSFM uncovered distinct architectures of fungal growth and 63 degrees of tissue invasion in each model. Furthermore, LSFM revealed the spatial 64 distribution, interaction and activation of two key immune cell populations in antifungal 65 defense: alveolar macrophages and polymorphonuclear neutrophils. Interestingly, the 66 patterns of fungal growth correlated with the detected effects of the immunosuppressive 67 regimens on the local immune cell populations. Moreover, LSFM demonstrates that the 68 commonly used intranasal route of spore administration did not result in the desired intra-69 alveolar deposition, as more than 60% of fungal growth occurred outside of the alveolar 70 space. Hence, LSFM allows for more rigorous characterization of murine models of invasive 71 pulmonary aspergillosis and pinpointing their strengths and limitations. 72 IMPORTANCE 73The use of animal models of infection is essential to advance our understanding of complex 74 host-pathogen interactions that take place during Aspergillus fumigatus lung infections. As in 75 the case of humans, mice need to be immunosuppressed to become susceptible to invasive 76 pulmonary aspergillosis, the most serious infection caused by A. fumigatus. There are 77 several immunosuppressive regimens that are routinely used to investigate fungal growth 78 and/or immune responses in murine models of invasive pulmonary aspergillosis (IPA). 79However, the precise consequences that each immunosuppressive model has on the local 80 immune populations and for fungal growth are not completely understood. Here we 81 employed light sheet fluorescence microscopy (LSFM) to analyze whole lungs at cellular 82 resolution, to pin down the scenario commonly used IPA models. Our results will be ...
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