Trypacidin, a Spore-Borne Toxin from Aspergillus fumigatus, Is Cytotoxic to Lung Cells

Inhalation of Aspergillus fumigatus conidia can cause severe aspergillosis in immunosuppressed people. A. fumigatus produces a large number of secondary metabolites, some of which are airborne by conidia and whose toxicity to the respiratory tract has not been investigated. We found that spores of A. fumigatus contain five main compounds, tryptoquivaline F, fumiquinazoline C, questin, monomethylsulochrin and trypacidin. Fractionation of culture extracts using RP-HPLC and LC-MS showed that samples containing questin, monomethylsulochrin and trypacidin were toxic to the human A549 lung cell line. These compounds were purified and their structure verified using NMR in order to compare their toxicity against A549 cells. Trypacidin was the most toxic, decreasing cell viability and triggering cell lysis, both effects occurring at an IC50 close to 7 µM. Trypacidin toxicity was also observed in the same concentration range on human bronchial epithelial cells. In the first hour of exposure, trypacidin initiates the intracellular formation of nitric oxide (NO) and hydrogen peroxide (H2O2). This oxidative stress triggers necrotic cell death in the following 24 h. The apoptosis pathway, moreover, was not involved in the cell death process as trypacidin did not induce apoptotic bodies or a decrease in mitochondrial membrane potential. This is the first time that the toxicity of trypacidin to lung cells has been reported

SCRINSHOT enables spatial mapping of cell states in tissue sections with single-cell resolution.

Changes in cell identities and positions underlie tissue development and disease progression. Although single-cell mRNA sequencing (scRNA-Seq) methods rapidly generate extensive lists of cell states, spatially resolved single-cell mapping presents a challenging task. We developed SCRINSHOT (Single-Cell Resolution IN Situ Hybridization On Tissues), a sensitive, multiplex RNA mapping approach. Direct hybridization of padlock probes on mRNA is followed by circularization with SplintR ligase and rolling circle amplification (RCA) of the hybridized padlock probes. Sequential detection of RCA-products using fluorophore-labeled oligonucleotides profiles thousands of cells in tissue sections. We evaluated SCRINSHOT specificity and sensitivity on murine and human organs. SCRINSHOT quantification of marker gene expression shows high correlation with published scRNA-Seq data over a broad range of gene expression levels. We demonstrate the utility of SCRINSHOT by mapping the locations of abundant and rare cell types along the murine airways. The amenability, multiplexity, and quantitative qualities of SCRINSHOT facilitate single-cell mRNA profiling of cell-state alterations in tissues under a variety of native and experimental conditions

Clathrin-Mediated Albumin Clearance in Alveolar Epithelial Cells of Murine Precision-Cut Lung Slices

A hallmark of acute respiratory distress syndrome (ARDS) is an accumulation of protein-rich alveolar edema that impairs gas exchange and leads to worse outcomes. Thus, understanding the mechanisms of alveolar albumin clearance is of high clinical relevance. Here, we investigated the mechanisms of the cellular albumin uptake in a three-dimensional culture of precision-cut lung slices (PCLS). We found that up to 60% of PCLS cells incorporated labeled albumin in a time- and concentration-dependent manner, whereas virtually no uptake of labeled dextran was observed. Of note, at a low temperature (4 °C), saturating albumin receptors with unlabeled albumin and an inhibition of clathrin-mediated endocytosis markedly decreased the endocytic uptake of the labeled protein, implicating a receptor-driven internalization process. Importantly, uptake rates of albumin were comparable in alveolar epithelial type I (ATI) and type II (ATII) cells, as assessed in PCLS from a SftpcCreERT2/+: tdTomatoflox/flox mouse strain (defined as EpCAM+CD31−CD45−tdTomatoSPC−T1α+ for ATI and EpCAM+CD31−CD45−tdTomatoSPC+T1α− for ATII cells). Once internalized, albumin was found in the early and recycling endosomes of the alveolar epithelium as well as in endothelial, mesenchymal, and hematopoietic cell populations, which might indicate transcytosis of the protein. In summary, we characterize albumin uptake in alveolar epithelial cells in the complex setting of PCLS. These findings may open new possibilities for pulmonary drug delivery that may improve the outcomes for patients with respiratory failure

Sestrin-2, a repressor of PDGFRβ signalling, promotes cigarette-smoke-induced pulmonary emphysema in mice and is upregulated in individuals with COPD

SUMMARY Chronic obstructive pulmonary disease (COPD) is a leading cause of morbidity and mortality worldwide. COPD is caused by chronic exposure to cigarette smoke and/or other environmental pollutants that are believed to induce reactive oxygen species (ROS) that gradually disrupt signalling pathways responsible for maintaining lung integrity. Here we identify the antioxidant protein sestrin-2 (SESN2) as a repressor of PDGFRβ signalling, and PDGFRβ signalling as an upstream regulator of alveolar maintenance programmes. In mice, the mutational inactivation of Sesn2 prevents the development of cigarette-smoke-induced pulmonary emphysema by upregulating PDGFRβ expression via a selective accumulation of intracellular superoxide anions (O2−). We also show that SESN2 is overexpressed and PDGFRβ downregulated in the emphysematous lungs of individuals with COPD and to a lesser extent in human lungs of habitual smokers without COPD, implicating a negative SESN2-PDGFRβ interrelationship in the pathogenesis of COPD. Taken together, our results imply that SESN2 could serve as both a biomarker and as a drug target in the clinical management of COPD

Comparison of LC-MS analysis of the fractions obtained from flash chromatography and their effect on the A549 cell viability.

<p>Fractionation of bulk culture extracts using flash chromatography. To measure their toxicity, each 5 ml fraction was dried and solubilised in 500 µl in PBS-DMSO 40% and then diluted 10 fold in PBS-DMSO 12%. The final concentration of DMSO in the assay medium was 1.2%. The cells were exposed for 24 h before measuring the cell viability as described in the Materials and Methods. The effect of 1.2% DMSO was used as negative control. For each metabolite, the figures are arbitrary units indicating its elution profile with 100 corresponding to the maximum normalized level measured by the LC-MS sensor. nd means not detectable.</p

Sestrin-2, a repressor of PDGFRβ signalling, promotes cigarette-smoke-induced pulmonary emphysema in mice and is upregulated in individuals with COPD

Chronic obstructive pulmonary disease (COPD) is a leading cause of morbidity and mortality worldwide. COPD is caused by chronic exposure to cigarette smoke and/or other environmental pollutants that are believed to induce reactive oxygen species (ROS) that gradually disrupt signalling pathways responsible for maintaining lung integrity. Here we identify the antioxidant protein sestrin-2 (SESN2) as a repressor of PDGFRβ signalling, and PDGFRβ signalling as an upstream regulator of alveolar maintenance programmes. In mice, the mutational inactivation of Sesn2 prevents the development of cigarette-smoke-induced pulmonary emphysema by upregulating PDGFRβ expression via a selective accumulation of intracellular superoxide anions (O2−). We also show that SESN2 is overexpressed and PDGFRβ downregulated in the emphysematous lungs of individuals with COPD and to a lesser extent in human lungs of habitual smokers without COPD, implicating a negative SESN2-PDGFRβ interrelationship in the pathogenesis of COPD. Taken together, our results imply that SESN2 could serve as both a biomarker and as a drug target in the clinical management of COPD

Sestrin-2, a repressor of PDGFRβ signalling, promotes cigarette smokeinduced pulmonary emphysema in mice and is upregulated in patients with COPD

Chronic obstructive pulmonary disease (COPD) is a leading cause of morbidity and mortality worldwide. COPD is caused by chronic exposure to cigarette smoke and/or other environmental pollutants that are believed to induce reactive oxygen species (ROS) that gradually disrupt signalling pathways responsible for maintaining lung integrity. Here we identify the antioxidant protein Sestrin 2 (Sesn2) as a repressor of PDGFRβ signalling and PDGFRβ signalling as an upstream regulator of alveolar maintenance programs. In mice, the mutational inactivation of Sesn2 prevents the development of cigarette-smoke induced pulmonary emphysema by upregulating PDGFRβ expression via a selective accumulation of intracellular superoxide anions (O2-). We also show that SESN2 is overexpressed and PDGFRβ downregulated in the emphysematous lungs of patients with COPD and to a lesser extent in human lungs of habitual smokers without COPD, implicating a negative SESN2/PDGFRβ interrelationship in the pathogenesis of COPD. Taken together, our results imply that SESN2 could serve as both a biomarker and as a drug target in the clinical management of COPD