The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) novel coronavirus 2019 (COVID-19) global pandemic has led to millions of cases and hundreds of thousands of deaths. While older adults ...appear at high risk for severe disease, hospitalizations and deaths due to SARS-CoV-2 among children have been relatively rare. Integrating single-cell RNA sequencing (scRNA-seq) of developing mouse lung with temporally resolved immunofluorescence in mouse and human lung tissue, we found that expression of SARS-CoV-2 Spike protein primer TMPRSS2 was highest in ciliated cells and type I alveolar epithelial cells (AT1), and TMPRSS2 expression increased with aging in mice and humans. Analysis of autopsy tissue from fatal COVID-19 cases detected SARS-CoV-2 RNA most frequently in ciliated and secretory cells in airway epithelium and AT1 cells in peripheral lung. SARS-CoV-2 RNA was highly colocalized in cells expressing TMPRSS2. Together, these data demonstrate the cellular spectrum infected by SARS-CoV-2 in lung epithelium and suggest that developmental regulation of TMPRSS2 may underlie the relative protection of infants and children from severe respiratory illness.
Bronchopulmonary dysplasia (BPD) is a leading complication of preterm birth that affects infants born in the saccular stage of lung development at <32 weeks of gestation. Although the mechanisms ...driving BPD remain uncertain, exposure to hyperoxia is thought to contribute to disease pathogenesis.
To determine the effects of hyperoxia on epithelial-mesenchymal interactions and to define the mediators of activated Wnt/β-catenin signaling after hyperoxia injury.
Three hyperoxia models were used: A three-dimensional organotypic coculture using primary human lung cells, precision-cut lung slices (PCLS), and a murine
hyperoxia model. Comparisons of normoxia- and hyperoxia-exposed samples were made by real-time quantitative PCR, RNA
hybridization, quantitative confocal microscopy, and lung morphometry.
Examination of an array of Wnt ligands in the three-dimensional organotypic coculture revealed increased mesenchymal expression of
. Inhibition of Wnt5A abrogated the BPD transcriptomic phenotype induced by hyperoxia. In the PCLS model, Wnt5A inhibition improved alveolarization following hyperoxia exposure, and treatment with recombinant Wnt5a reproduced features of the BPD phenotype in PCLS cultured in normoxic conditions. Chemical inhibition of NF-κB with BAY11-7082 reduced
expression in the PCLS hyperoxia model and
mouse hyperoxia model, with improved alveolarization in the PCLS model.
Increased mesenchymal Wnt5A during saccular-stage hyperoxia injury contributes to the impaired alveolarization and septal thickening observed in BPD. Precise targeting of Wnt5A may represent a potential therapeutic strategy for the treatment of BPD.
Emerging evidence indicates that early life events can increase the risk for developing chronic obstructive pulmonary disease (COPD). Using an inducible transgenic mouse model for NF-κB activation in ...the airway epithelium, we found that a brief period of inflammation during the saccular stage (P3-P5) but not alveolar stage (P10-P12) of lung development disrupted elastic fiber assembly, resulting in permanent reduction in lung function and development of a COPD-like lung phenotype that progressed through 24 months of age. Neutrophil depletion prevented disruption of elastic fiber assembly and restored normal lung development. Mechanistic studies uncovered a role for neutrophil elastase (NE) in downregulating expression of critical elastic fiber assembly components, particularly fibulin-5 and elastin. Further, purified human NE and NE-containing exosomes from tracheal aspirates of premature infants with lung inflammation downregulated elastin and fibulin-5 expression by saccular-stage mouse lung fibroblasts. Together, our studies define a critical developmental window for assembling the elastin scaffold in the distal lung, which is required to support lung structure and function throughout the lifespan. Although neutrophils play a well-recognized role in COPD development in adults, neutrophilic inflammation may also contribute to early-life predisposition to COPD.
Neonatology pioneer Mildred (Millie) T. Stahlman celebrated her 100th birthday on July 31, 2022. Her distinguished career at Vanderbilt University Medical Center in Nashville, TN, is reviewed to ...commemorate this milestone. Stahlman was arguably the first to establish a modern neonatal intensive care unit in 1961, successfully utilizing negative pressure ventilation and umbilical arterial and venous catheters to monitor blood gasses and pH levels. She received early invaluable training in newborn physiology at the Karolinska Institute in Stockholm, Sweden, under John Lind and Petter Karlberg, and at Vanderbilt under Elliot V. Newman. Stahlman also consulted with luminaries Geoffrey Dawes, Donald Barron, and L. Stanley James. As director of the Vanderbilt NICU, she trained 80 fellows from more than 20 countries. The latter 20 years of her career were highlighted by collaborations with Jeff Whitsett. She was the recipient of the AAP Virginia Apgar Award, the APS John Howland Medal, and served as a member of the Institute of Medicine.
Bronchopulmonary dysplasia (BPD) is a leading complication of premature birth and occurs primarily in infants delivered during the saccular stage of lung development. Histopathology shows decreased ...alveolarization and a pattern of fibroblast proliferation and differentiation to the myofibroblast phenotype. Little is known about the molecular pathways and cellular mechanisms that define BPD pathophysiology and progression. We have developed a novel three-dimensional human model of the fibroblast activation associated with BPD, and using this model we have identified the Notch pathway as a key driver of fibroblast activation and proliferation in response to changes in oxygen. Fetal lung fibroblasts were cultured on sodium alginate beads to generate lung organoids. After exposure to alternating hypoxia and hyperoxia, the organoids developed a phenotypic response characterized by increased α-smooth muscle actin (α-SMA) expression and other genes known to be upregulated in BPD and also demonstrated increased expression of downstream effectors of the Notch pathway. Inhibition of Notch with a γ-secretase inhibitor prevented the development of the pattern of cellular proliferation and α-SMA expression in our model. Analysis of human autopsy tissue from the lungs of infants who expired with BPD demonstrated evidence of Notch activation within fibrotic areas of the alveolar septae, suggesting that Notch may be a key driver of BPD pathophysiology.
Lamellar bodies (LBs) are lysosome-related organelles (LROs) of surfactant-producing alveolar type 2 (AT2) cells of the distal lung epithelium. Trafficking pathways to LBs have been understudied but ...are likely critical to AT2 cell homeostasis given associations between genetic defects of endosome to LRO trafficking and pulmonary fibrosis in Hermansky Pudlak syndrome (HPS). Our prior studies uncovered a role for AP-3, defective in HPS type 2, in trafficking Peroxiredoxin-6 to LBs. We now show that the P4-type ATPase ATP8A1 is sorted by AP-3 from early endosomes to LBs through recognition of a C-terminal dileucine-based signal. Disruption of the AP-3/ATP8A1 interaction causes ATP8A1 accumulation in early sorting and/or recycling endosomes, enhancing phosphatidylserine exposure on the cytosolic leaflet. This in turn promotes activation of Yes-activating protein, a transcriptional coactivator, augmenting cell migration and AT2 cell numbers. Together, these studies illuminate a mechanism whereby loss of AP-3-mediated trafficking contributes to a toxic gain-of-function that results in enhanced and sustained activation of a repair pathway associated with pulmonary fibrosis.
Lung organogenesis requires precise timing and coordination to effect spatial organization and function of the parenchymal cells. To provide a systematic broad-based view of the mechanisms governing ...the dynamic alterations in parenchymal cells over crucial periods of development, we performed a single-cell RNA-sequencing time-series yielding 102,571 epithelial, endothelial and mesenchymal cells across nine time points from embryonic day 12 to postnatal day 14 in mice. Combining computational fate-likelihood prediction with RNA in situ hybridization and immunofluorescence, we explore lineage relationships during the saccular to alveolar stage transition. The utility of this publicly searchable atlas resource (www.sucrelab.org/lungcells) is exemplified by discoveries of the complexity of type 1 pneumocyte function and characterization of mesenchymal Wnt expression patterns during the saccular and alveolar stages - wherein major expansion of the gas-exchange surface occurs. We provide an integrated view of cellular dynamics in epithelial, endothelial and mesenchymal cell populations during lung organogenesis.
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