Swertiamarin attenuates paraquat-induced pulmonary epithelial-like cell apoptosis via NOX4-mediated regulation of redox and mitochondrial function

Abstract The aim of the present study was to investigate the effect of swertiamarin (STM) in attenuating paraquat (PQ)-induced human lung alveolar epithelial-like cell (A549) apoptosis and the underlying mechanisms. A549 cells were pretreated with different concentrations of STM for 2 hr and then cultured with or without PQ (700 µM) for 24 hr. Cell survival was determined using the CCK8 assay. Morphological changes, MDA content, inflammatory factors, fibrogenesis parameters, apoptosis rates, redox status and mitochondrial membrane potential (MMP) were evaluated. The expression of several genes involved in the modulation of redox status was measured by Western blotting. Cell viability and MMP were decreased, but the apoptosis rate and DCFH oxidation were elevated by PQ exposure. STM pretreatment notably increased cell viability and MMP and reduced the apoptosis rate and DCFH oxidation. Furthermore, TLR4- NOX4 signaling was significantly inhibited by STM. The downregulation of NOX4 by siRNA exerted the same protective effects as STM. This study provides the first evidence that STM attenuates PQ-induced pulmonary epithelial-like cell apoptosis via NOX4-mediated regulation of redox and mitochondrial function

Characterization of the COPD alveolar niche using single-cell RNA sequencing.

Chronic obstructive pulmonary disease (COPD) is a leading cause of death worldwide, however our understanding of cell specific mechanisms underlying COPD pathobiology remains incomplete. Here, we analyze single-cell RNA sequencing profiles of explanted lung tissue from subjects with advanced COPD or control lungs, and we validate findings using single-cell RNA sequencing of lungs from mice exposed to 10 months of cigarette smoke, RNA sequencing of isolated human alveolar epithelial cells, functional in vitro models, and in situ hybridization and immunostaining of human lung tissue samples. We identify a subpopulation of alveolar epithelial type II cells with transcriptional evidence for aberrant cellular metabolism and reduced cellular stress tolerance in COPD. Using transcriptomic network analyses, we predict capillary endothelial cells are inflamed in COPD, particularly through increased CXCL-motif chemokine signaling. Finally, we detect a high-metallothionein expressing macrophage subpopulation enriched in advanced COPD. Collectively, these findings highlight cell-specific mechanisms involved in the pathobiology of advanced COPD.

Stimulation of surfactant exocytosis in primary alveolar type II cells by A. fumigatus.

Aspergillus fumigatus is an opportunistic fungal pathogen with small airborne spores (conidia) that may escape clearance by upper airways and directly impact the alveolar epithelium. Consequently, innate alveolar defense mechanisms are being activated, including professional phagocytosis by alveolar macrophages, recruitment of circulating neutrophils and probably enhanced secretion of pulmonary surfactant by the alveolar type II (AT II) cells. However, no data are available in support of the latter hypothesis. We therefore used a coculture model of GFP-Aspergillus conidia with primary rat AT II cells and studied fungal growth, cellular Ca2+ homeostasis, and pulmonary surfactant exocytosis by live cell video microscopy. We observed all stages of fungal development, including reversible attachment, binding and internalization of conidia as well as conidial swelling, formation of germ tubes and outgrowth of hyphae. In contrast to resting conidia, which did not provoke immediate cellular effects, metabolically active conidia, fungal cellular extracts (CE) and fungal culture filtrates (CF) prepared from swollen conidia caused a Ca2+-independent exocytosis. Ca2+ signals of greatly varying delays, durations and amplitudes were observed by applying CE or CF obtained from hyphae of A. fumigatus, suggesting compounds secreted by filamentous A. fumigatus that severely interfere with AT II cell Ca2+ homeostasis. The mechanisms underlying the stimulatory effects, with respect to exocytosis and Ca2+ signaling, are unclear and need to be identified.

Single-cell multiomic profiling of human lungs reveals cell-type-specific and age-dynamic control of SARS-CoV2 host genes.

Respiratory failure associated with COVID-19 has placed focus on the lungs. Here, we present single-nucleus accessible chromatin profiles of 90,980 nuclei and matched single-nucleus transcriptomes of 46,500 nuclei in non-diseased lungs from donors of ~30 weeks gestation,~3 years and ~30 years. We mapped candidate cis-regulatory elements (cCREs) and linked them to putative target genes. We identified distal cCREs with age-increased activity linked to SARS-CoV-2 host entry gene TMPRSS2 in alveolar type 2 cells, which had immune regulatory signatures and harbored variants associated with respiratory traits. At the 3p21.31 COVID-19 risk locus, a candidate variant overlapped a distal cCRE linked to SLC6A20, a gene expressed in alveolar cells and with known functional association with the SARS-CoV-2 receptor ACE2. Our findings provide insight into regulatory logic underlying genes implicated in COVID-19 in individual lung cell types across age. More broadly, these datasets will facilitate interpretation of risk loci for lung diseases.

Isolation of Rat and Mouse Alveolar Type II Epithelial Cells.

The gas exchange surface of the lungs is lined by an epithelium consisting of alveolar type (AT) I and ATII cells. ATII cells function to produce surfactant, play a role in host defense and fluid and ion transport, and serve as progenitors. ATI cells are important for gas exchange and fluid and ion transport. Our understanding of the biology of these cells depends on the investigation of isolated cells. Here, we present methods for the isolation of mouse and rat ATII cells.

Intratracheal instillation of alveolar type II cells enhances recovery from acute lung injury in rats.

BACKGROUND: Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are characterized by excess production of inflammatory factors. Alveolar type II (ATII) cells help repair damaged lung tissue, rapidly proliferating and differentiating into alveolar type I cells after epithelial cell injury. In ALI, the lack of viable ATII favors progression to more severe lung injury. ATII cells regulate the immune response by synthesizing surfactant and other anti-inflammatory proteins and lipids. Cross-talk between ATII and other cells such as macrophages may also be part of the ATII function. The aim of this study was to test the anti-inflammatory and reparative effects of ATII cells in an experimental model of ALI. METHODS: In this study ATII cells (2.5 × 106 cells/animal) were intratracheally instilled in rats with HCl and lipopolysaccharide (LPS)-induced ALI and in healthy animals to check for side effects. The specific effect of ATII cells was compared with fibroblast transplantation. RESULTS: ATII cell transplantation promoted recovery of lung function, decrease mortality and lung inflammation of the animals with ALI. The primary mechanisms for benefit were paracrine effects of prostaglandin E2 (PGE2) and surfactant protein A (SPA) released from ATII cells that modulate alveolar macrophages to an anti-inflammatory phenotype. To our knowledge, these data are the first to provide evidence that ATII cells secrete PGE2 and SPA, reducing pro-inflammatory macrophage activation and ALI. CONCLUSION: ATII cells and their secreted molecules have shown an ability to resolve ALI, thereby highlighting a potential novel therapeutic target.

Fraction of MHCII and EpCAM expression characterizes distal lung epithelial cells for alveolar type 2 cell isolation.

BACKGOUND: Alveolar type 2 (AT2) cells play important roles in maintaining adult lung homeostasis. AT2 cells isolated from the lung have revealed the cell-specific functions of AT2 cells. Comprehensive molecular and transcriptional profiling of purified AT2 cells would be helpful for elucidating the underlying mechanisms of their cell-specific functions. To enable the further purification of AT2 cells, we aimed to discriminate AT2 cells from non-AT2 lung epithelial cells based on surface antigen expression via fluorescence activated cell sorting (FACS). METHODS: Single-cell suspensions obtained from enzymatically digested murine lungs were labeled for surface antigens (CD45/CD31/epithelial cell adhesion molecule (EpCAM)/ major histocompatibility complex class II (MHCII)) and for pro-surfactant protein C (proSP-C), followed by FACS analysis for surface antigen expression on AT2 cells. AT2 cells were sorted, and purity was evaluated by immunofluorescence and FACS. This newly developed strategy for AT2 cell isolation was validated in different strains and ages of mice, as well as in a lung injury model. RESULTS: FACS analysis revealed that EpCAM+ epithelial cells existed in 3 subpopulations based on EpCAM and MHCII expression: EpCAMmedMHCII+ cells (Population1:P1), EpCAMhiMHCII- cells (P2), and EpCAMlowMHCII- cells (P3). proSP-C+ cells were enriched in P1 cells, and the purity values of the sorted AT2 cells in P1 were 99.0% by immunofluorescence analysis and 98.0% by FACS analysis. P2 cells were mainly composed of ciliated cells and P3 cells were composed of AT1 cells, respectively, based on the gene expression analysis and immunofluorescence. EpCAM and MHCII expression levels were not significantly altered in different strains or ages of mice or following lipopolysaccharide (LPS)-induced lung injury. CONCLUSIONS: We successfully classified murine distal lung epithelial cells based on EpCAM and MHCII expression. The discrimination of AT2 cells from non-AT2 epithelial cells resulted in the isolation of pure AT2 cells. Highly pure AT2 cells will provide accurate and deeper insights into the cell-specific mechanisms of alveolar homeostasis.

Human iPS cell-derived alveolar epithelium repopulates lung extracellular matrix.

The use of induced pluripotent stem cells (iPSCs) has been postulated to be the most effective strategy for developing patient-specific respiratory epithelial cells, which may be valuable for lung-related cell therapy and lung tissue engineering. We generated a relatively homogeneous population of alveolar epithelial type II (AETII) and type I (AETI) cells from human iPSCs that had phenotypic properties similar to those of mature human AETII and AETI cells. We used these cells to explore whether lung tissue can be regenerated in vitro. Consistent with an AETII phenotype, we found that up to 97% of cells were positive for surfactant protein C, 95% for mucin-1, 93% for surfactant protein B, and 89% for the epithelial marker CD54. Additionally, exposing induced AETII to a Wnt/ß-catenin inhibitor (IWR-1) changed the iPSC-AETII-like phenotype to a predominantly AETI-like phenotype. We found that of induced AET1 cells, more than 90% were positive for type I markers, T1α, and caveolin-1. Acellular lung matrices were prepared from whole rat or human adult lungs treated with decellularization reagents, followed by seeding these matrices with alveolar cells derived from human iPSCs. Under appropriate culture conditions, these progenitor cells adhered to and proliferated within the 3D lung tissue scaffold and displayed markers of differentiated pulmonary epithelium.

Disruption of sorting nexin 5 causes respiratory failure associated with undifferentiated alveolar epithelial type I cells in mice.

Sorting nexin 5 (Snx5) has been posited to regulate the degradation of epidermal growth factor receptor and the retrograde trafficking of cation-independent mannose 6-phosphate receptor/insulin-like growth factor II receptor. Snx5 has also been suggested to interact with Mind bomb-1, an E3 ubiquitin ligase that regulates the activation of Notch signaling. However, the in vivo functions of Snx5 are largely unknown. Here, we report that disruption of the Snx5 gene in mice (Snx5(-/-) mice) resulted in partial perinatal lethality; 40% of Snx5(-/-) mice died shortly after birth due to cyanosis, reduced air space in the lungs, and respiratory failure. Histological analysis revealed that Snx5(-/-) mice exhibited thickened alveolar walls associated with undifferentiated alveolar epithelial type I cells. In contrast, alveolar epithelial type II cells were intact, exhibiting normal surfactant synthesis and secretion. Although the expression levels of surfactant proteins and saturated phosphatidylcholine in the lungs of Snx5(-/-) mice were comparable to those of Snx5(+/+) mice, the expression levels of T1α, Aqp5, and Rage, markers for distal alveolar epithelial type I cells, were significantly decreased in Snx5 (-/-) mice. These results demonstrate that Snx5 is necessary for the differentiation of alveolar epithelial type I cells, which may underlie the adaptation to air breathing at birth.

Influenza A viruses target type II pneumocytes in the human lung.

BACKGROUND: Highly pathogenic avian H5N1 influenza viruses preferentially infect alveolar type II pneumocytes in human lung. However, it is unknown whether this cellular tropism contributes to high viral virulence because the primary target cells of other influenza viruses have not been systematically studied. METHODS: We provide the first comparison of the replication, tropism, and cytokine induction of human, highly pathogenic avian influenza A virus subtype H5N1 and other animal influenza A viruses in primary human lung organ cultures. RESULTS: Subytpe H5N1 and human-adapted subtype H1N1 and H3N2 viruses replicated efficiently in the lung tissue, whereas classic swine and low-pathogenicity avian viruses propagated only poorly. Nevertheless, all viruses examined were detected almost exclusively in type II pneumocytes, with a minor involvement of alveolar macrophages. Infection with avian viruses that have a low and high pathogenicity provoked a pronounced induction of cytokines and chemokines, while human and pandemic H1N1-2009 viruses triggered only weak responses. CONCLUSIONS: These findings show that differences in the pathogenic potential of influenza A viruses in the human lung cannot be attributed to a distinct cellular tropism. Rather, high or low viral pathogenicity is associated with a strain-specific capacity to productively replicate in type II pneumocytes and to cope with the induced cytokine response.