Activation of the integrated stress response rewires cardiac metabolism in Barth syndrome.

Barth Syndrome (BTHS) is an inherited cardiomyopathy caused by defects in the mitochondrial transacylase TAFAZZIN (Taz), required for the synthesis of the phospholipid cardiolipin. BTHS is characterized by heart failure, increased propensity for arrhythmias and a blunted inotropic reserve. Defects in Ca2+-induced Krebs cycle activation contribute to these functional defects, but despite oxidation of pyridine nucleotides, no oxidative stress developed in the heart. Here, we investigated how retrograde signaling pathways orchestrate metabolic rewiring to compensate for mitochondrial defects. In mice with an inducible knockdown (KD) of TAFAZZIN, and in induced pluripotent stem cell-derived cardiac myocytes, mitochondrial uptake and oxidation of fatty acids was strongly decreased, while glucose uptake was increased. Unbiased transcriptomic analyses revealed that the activation of the eIF2α/ATF4 axis of the integrated stress response upregulates one-carbon metabolism, which diverts glycolytic intermediates towards the biosynthesis of serine and fuels the biosynthesis of glutathione. In addition, strong upregulation of the glutamate/cystine antiporter xCT increases cardiac cystine import required for glutathione synthesis. Increased glutamate uptake facilitates anaplerotic replenishment of the Krebs cycle, sustaining energy production and antioxidative pathways. These data indicate that ATF4-driven rewiring of metabolism compensates for defects in mitochondrial uptake of fatty acids to sustain energy production and antioxidation.

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

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.

Dynamic Immune Cell Recruitment After Murine Pulmonary Aspergillus fumigatus Infection under Different Immunosuppressive Regimens.

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.