SARS-CoV-2 Receptor ACE2 Is an Interferon-Stimulated Gene in Human Airway Epithelial Cells and Is Detected in Specific Cell Subsets across Tissues.

There is pressing urgency to understand the pathogenesis of the severe acute respiratory syndrome coronavirus clade 2 (SARS-CoV-2), which causes the disease COVID-19. SARS-CoV-2 spike (S) protein binds angiotensin-converting enzyme 2 (ACE2), and in concert with host proteases, principally transmembrane serine protease 2 (TMPRSS2), promotes cellular entry. The cell subsets targeted by SARS-CoV-2 in host tissues and the factors that regulate ACE2 expression remain unknown. Here, we leverage human, non-human primate, and mouse single-cell RNA-sequencing (scRNA-seq) datasets across health and disease to uncover putative targets of SARS-CoV-2 among tissue-resident cell subsets. We identify ACE2 and TMPRSS2 co-expressing cells within lung type II pneumocytes, ileal absorptive enterocytes, and nasal goblet secretory cells. Strikingly, we discovered that ACE2 is a human interferon-stimulated gene (ISG) in vitro using airway epithelial cells and extend our findings to in vivo viral infections. Our data suggest that SARS-CoV-2 could exploit species-specific interferon-driven upregulation of ACE2, a tissue-protective mediator during lung injury, to enhance infection.

CXCL12 defines lung endothelial heterogeneity and promotes distal vascular growth.

There is a growing amount of data uncovering the cellular diversity of the pulmonary circulation and mechanisms governing vascular repair after injury. However, the molecular and cellular mechanisms contributing to the morphogenesis and growth of the pulmonary vasculature during embryonic development are less clear. Importantly, deficits in vascular development lead to significant pediatric lung diseases, indicating a need to uncover fetal programs promoting vascular growth. To address this, we used a transgenic mouse reporter for expression of Cxcl12, an arterial endothelial hallmark gene, and performed single-cell RNA sequencing on isolated Cxcl12-DsRed+ endothelium to assess cellular heterogeneity within pulmonary endothelium. Combining cell annotation with gene ontology and histological analysis allowed us to segregate the developing artery endothelium into functionally and spatially distinct subpopulations. Expression of Cxcl12 is highest in the distal arterial endothelial subpopulation, a compartment enriched in genes for vascular development. Accordingly, disruption of CXCL12 signaling led to, not only abnormal branching, but also distal vascular hypoplasia. These data provide evidence for arterial endothelial functional heterogeneity and reveal conserved signaling mechanisms essential for pulmonary vascular development.

The emerging role of extracellular vesicles in bronchopulmonary dysplasia.

Extracellular vesicle (EV) biology in neonatal lung development and disease is a rapidly growing area of investigation. Although EV research in the neonatal population lags behind EV research in adult lung diseases, recent discoveries demonstrate promise in furthering our understanding of the pathophysiology of bronchopulmonary dysplasia and the potential use of EVs in the clinical setting, as both biomarkers and therapeutic agents. This review article explores some of the recent advances in this field and our evolving knowledge of the role of EVs in bronchopulmonary dysplasia.

A single-cell atlas of mouse lung development.

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.

AP-3-dependent targeting of flippase ATP8A1 to lamellar bodies suppresses activation of YAP in alveolar epithelial type 2 cells.

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.

Developmental trajectory of extracellular vesicle characteristics from the lungs of preterm infants.

Extracellular vesicles (EVs) are secreted lipid-enclosed particles that have emerged as potential biomarkers and therapeutic agents in lung disease, including bronchopulmonary dysplasia (BPD), a leading complication of preterm birth. Many unanswered questions remain about the content and cargo of EVs in premature infants and their role in lung development. To characterize EVs during human lung development, tracheal aspirates were collected from premature neonates between 22 and 35 wk gestational age and analyzed via nanoparticle tracking analysis, electron microscopy, and bead-based flow cytometry. EVs were detectable across late canalicular through saccular stages of lung development, demonstrating larger sizes earlier in gestation. EVs contained an abundance of the EV-enriched tetraspanins CD9, CD63, and CD81, as well as epithelial cell and immune cell markers. Increases in select surface proteins (CD24 and CD14) on EVs were associated with gestational age and with the risk of BPD. Finally, query of expression data obtained from epithelial cells in a single-cell atlas of murine lung development found that epithelial EV marker expression also changes with developmental time. Together, these data demonstrate an association between EV profile and lung development and provide a foundation for future functional classification of EVs, with the goal of determining their role in cell signaling during development and harnessing their potential as a new therapeutic target in BPD.

A novel role for PGE2-EP4 in the developmental programming of the mouse ductus arteriosus: consequences for vessel maturation and function.

The ductus arteriosus (DA) is a vascular shunt that allows oxygenated blood to bypass the developing lungs in utero. Fetal DA patency requires vasodilatory signaling via the prostaglandin E2 (PGE2) receptor EP4. However, in humans and mice, disrupted PGE2-EP4 signaling in utero causes unexpected patency of the DA (PDA) after birth, suggesting another role for EP4 during development. We used EP4-knockout (KO) mice and acute versus chronic pharmacological approaches to investigate EP4 signaling in DA development and function. Expression analyses identified EP4 as the primary EP receptor in the DA from midgestation to term; inhibitor studies verified EP4 as the primary dilator during this period. Chronic antagonism recapitulated the EP4 KO phenotype and revealed a narrow developmental window when EP4 stimulation is required for postnatal DA closure. Myography studies indicate that despite reduced contractile properties, the EP4 KO DA maintains an intact oxygen response. In newborns, hyperoxia constricted the EP4 KO DA but survival was not improved, and permanent remodeling was disrupted. Vasomotion and increased nitric oxide (NO) sensitivity in the EP4 KO DA suggest incomplete DA development. Analysis of DA maturity markers confirmed a partially immature EP4 KO DA phenotype. Together, our data suggest that EP4 signaling in late gestation plays a key developmental role in establishing a functional term DA. When disrupted in EP4 KO mice, the postnatal DA exhibits signaling and contractile properties characteristic of an immature DA, including impairments in the first, muscular phase of DA closure, in addition to known abnormalities in the second permanent remodeling phase.NEW & NOTEWORTHY EP4 is the primary EP receptor in the ductus arteriosus (DA) and is critical during late gestation for its development and eventual closure. The "paradoxical" patent DA (PDA) phenotype of EP4-knockout mice arises from a combination of impaired contractile potential, altered signaling properties, and a failure to remodel associated with an underdeveloped immature vessel. These findings provide new mechanistic insights into women who receive NSAIDs to treat preterm labor, whose infants have unexplained PDA.

Bronchopulmonary dysplasia is associated with polyhydramnios in a scan for novel perinatal risk factors.

BACKGROUND: The pathogenesis of bronchopulmonary dysplasia (BPD) is multifactorial, and there are limited data about prenatal exposures and risk of BPD. STUDY DESIGN: Our study performed parallel analyses using a logistic regression model in a cohort of 4527 infants with data from a curated registry and using a phenome wide association study (PheWAS) based on ICD9/10-based phecodes. We examined 20 prenatal exposures from a neonatal intensive care unit (NICU) curated registry database related to pregnancy and maternal health as well as 94 maternal diagnosis phecodes with a PheWAS analysis. RESULT: In both the curated registry and PheWAS analyses, polyhydramnios was associated with an increased risk of BPD (OR 5.70, 95% CI 2.78-11.44, p = 1.37 × 10-6). CONCLUSION: Our data suggest that polyhydramnios may be a clinical indicator of premature infants at increased risk for bronchopulmonary dysplasia. Combining curated registry data with PheWAS analysis creates a valuable tool to generate hypotheses. IMPACT: Polyhydramnios was significantly associated with bronchopulmonary dysplasia in both a curated registry and by ICD coding analysis with a phenome wide association study (PheWAS). Preterm polyhydramnios may be a clinical indicator of infants at increased risk for developing bronchopulmonary dysplasia after preterm birth. Combining curated registry with PheWAS analysis creates a valuable tool to generate hypotheses about perinatal risk factors and morbidities associated with preterm birth.

Mildred T. Stahlman: a centenarian who pioneered modern scientific neonatal intensive care.

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.

Racing against time: leveraging preclinical models to understand pulmonary susceptibility to perinatal acetaminophen exposures.

Over the past decade, clinicians have increasingly prescribed acetaminophen (APAP) for patients in the neonatal intensive care unit (NICU). Acetaminophen has been shown to reduce postoperative opiate burden, and may provide similar efficacy for closure of the patent ductus arteriosus (PDA) as nonsteroidal anti-inflammatory drugs (NSAIDs). Despite these potential benefits, APAP exposures have spread to increasingly less mature infants, a highly vulnerable population for whom robust pharmacokinetic and pharmacodynamic data for APAP are lacking. Concerningly, preclinical studies suggest that perinatal APAP exposures may result in unanticipated adverse effects that are unique to the developing lung. In this review, we discuss the clinical observations linking APAP exposures to adverse respiratory outcomes and the preclinical data demonstrating a developmental susceptibility to APAP-induced lung injury. We show how clinical observations linking perinatal APAP exposures to pulmonary injury have been taken to the bench to produce important insights into the potential mechanisms underlying these findings. We argue that the available data support a more cautious approach to APAP use in the NICU until large randomized controlled trials provide appropriate safety and efficacy data.

Hyperoxia Injury in the Developing Lung Is Mediated by Mesenchymal Expression of Wnt5A.

Rationale: 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.Objectives: To determine the effects of hyperoxia on epithelial-mesenchymal interactions and to define the mediators of activated Wnt/ß-catenin signaling after hyperoxia injury.Methods: Three hyperoxia models were used: A three-dimensional organotypic coculture using primary human lung cells, precision-cut lung slices (PCLS), and a murine in vivo hyperoxia model. Comparisons of normoxia- and hyperoxia-exposed samples were made by real-time quantitative PCR, RNA in situ hybridization, quantitative confocal microscopy, and lung morphometry.Measurements and Main Results: Examination of an array of Wnt ligands in the three-dimensional organotypic coculture revealed increased mesenchymal expression of WNT5A. 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 Wnt5a expression in the PCLS hyperoxia model and in vivo mouse hyperoxia model, with improved alveolarization in the PCLS model.Conclusions: 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.

A Shared Pattern of ß-Catenin Activation in Bronchopulmonary Dysplasia and Idiopathic Pulmonary Fibrosis.

Wnt/ß-catenin signaling is necessary for normal lung development, and abnormal Wnt signaling contributes to the pathogenesis of both bronchopulmonary dysplasia (BPD) and idiopathic pulmonary fibrosis (IPF), fibrotic lung diseases that occur during infancy and aging, respectively. Using a library of human normal and diseased human lung samples, we identified a distinct signature of nuclear accumulation of ß-catenin phosphorylated at tyrosine 489 and epithelial cell cytosolic localization of ß-catenin phosphorylated at tyrosine 654 in early normal lung development and fibrotic lung diseases BPD and IPF. Furthermore, this signature was recapitulated in murine models of BPD and IPF. Image analysis of immunofluorescence colocalization demonstrated a consistent pattern of elevated nuclear phosphorylated ß-catenin in the lung epithelium and surrounding mesenchyme in BPD and IPF, closely resembling the pattern observed in 18-week fetal lung. Nuclear ß-catenin phosphorylated at tyrosine 489 associated with an increased expression of Wnt target gene AXIN2, suggesting that the observed ß-catenin signature is of functional significance during normal development and injury repair. The association of specific modifications of ß-catenin during normal lung development and again in response to lung injury supports the widely held concept that repair of lung injury involves the recapitulation of developmental programs. Furthermore, these observations suggest that ß-catenin phosphorylation has potential as a therapeutic target for the treatment and prevention of both BPD and IPF.

Successful Establishment of Primary Type II Alveolar Epithelium with 3D Organotypic Coculture.

Alveolar type II (AT2) epithelial cells are uniquely specialized to produce surfactant in the lung and act as progenitor cells in the process of repair after lung injury. AT2 cell injury has been implicated in several lung diseases, including idiopathic pulmonary fibrosis and bronchopulmonary dysplasia. The inability to maintain primary AT2 cells in culture has been a significant barrier in the investigation of pulmonary biology. We have addressed this knowledge gap by developing a three-dimensional (3D) organotypic coculture using primary human fetal AT2 cells and pulmonary fibroblasts. Grown on top of matrix-embedded fibroblasts, the primary human AT2 cells establish a monolayer and have direct contact with the underlying pulmonary fibroblasts. Unlike conventional two-dimensional (2D) culture, the structural and functional phenotype of the AT2 cells in our 3D organotypic culture was preserved over 7 days of culture, as evidenced by the presence of lamellar bodies and by production of surfactant proteins B and C. Importantly, the AT2 cells in 3D cocultures maintained the ability to replicate, with approximately 60% of AT2 cells staining positive for the proliferation marker Ki67, whereas no such proliferation is evident in 2D cultures of the same primary AT2 cells. This organotypic culture system enables interrogation of AT2 epithelial biology by providing a reductionist in vitro model in which to investigate the response of AT2 epithelial cells and AT2 cell-fibroblast interactions during lung injury and repair.

Posttranslational modification of ß-catenin is associated with pathogenic fibroblastic changes in bronchopulmonary dysplasia.

Bronchopulmonary dysplasia (BPD) is a common complication of premature birth. The histopathology of BPD is characterized by an arrest of alveolarization with fibroblast activation. The Wnt/ß-catenin signaling pathway is important in early lung development. When Wnt signaling is active, phosphorylation of ß-catenin by tyrosine kinases at activating sites, specifically at tyrosine 489 (Y489), correlates with nuclear localization of ß-catenin. We examined fetal lung tissue, lung tissue from term newborns, and lung tissue from infants who died with BPD; we found nuclear ß-catenin phosphorylation at Y489 in epithelial and mesenchymal cells in fetal tissue and BPD tissue, but not in the lungs of term infants. Using a 3D human organoid model, we found increased nuclear localization of ß-catenin phosphorylated at Y489 (p-ß-cateninY489) after exposure to alternating hypoxia and hyperoxia compared with organoids cultured in normoxia. Exogenous stimulation of the canonical Wnt pathway in organoids was sufficient to cause nuclear localization of p-ß-cateninY489 in normoxia and mimicked the pattern of α-smooth muscle actin (α-SMA) expression seen with fibroblastic activation from oxidative stress. Treatment of organoids with a tyrosine kinase inhibitor prior to cyclic hypoxia-hyperoxia inhibited nuclear localization of p-ß-cateninY489 and prevented α-SMA expression by fibroblasts. Posttranslational phosphorylation of ß-catenin is a transient feature of normal lung development. Moreover, the persistence of p-ß-cateninY489 is a durable marker of fibroblast activation in BPD and may play an important role in BPD disease pathobiology.

A three-dimensional human model of the fibroblast activation that accompanies bronchopulmonary dysplasia identifies Notch-mediated pathophysiology.

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.

Distinct Spatiotemporally Dynamic Wnt-Secreting Niches Regulate Proximal Airway Regeneration and Aging.

Our understanding of dynamic interactions between airway basal stem cells (ABSCs) and their signaling niches in homeostasis, injury, and aging remains elusive. Using transgenic mice and pharmacologic studies, we found that Wnt/ß-catenin within ABSCs was essential for proliferation post-injury in vivo. ABSC-derived Wnt ligand production was dispensable for epithelial proliferation. Instead, the PDGFRα+ lineage in the intercartilaginous zone (ICZ) niche transiently secreted Wnt ligand necessary for ABSC proliferation. Strikingly, ABSC-derived Wnt ligand later drove early progenitor differentiation to ciliated cells. We discovered additional changes in aging, as glandular-like epithelial invaginations (GLEIs) derived from ABSCs emerged exclusively in the ICZ of aged mice and contributed to airway homeostasis and repair. Further, ABSC Wnt ligand secretion was necessary for GLEI formation, and constitutive activation of ß-catenin in young mice induced their formation in vivo. Collectively, these data underscore multiple spatiotemporally dynamic Wnt-secreting niches that regulate functionally distinct phases of airway regeneration and aging.

Age-determined expression of priming protease TMPRSS2 and localization of SARS-CoV-2 infection in the lung epithelium.

The SARS-CoV-2 novel coronavirus global pandemic (COVID-19) has led to millions of cases and hundreds of thousands of deaths around the globe. While the elderly 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 the developing mouse lung with temporally-resolved RNA-in-situ hybridization (ISH) 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 was increased with aging in mice and humans. Analysis of autopsy tissue from fatal COVID-19 cases revealed SARS-CoV-2 RNA was detected most frequently in ciliated and secretory cells in the airway epithelium and AT1 cells in the 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 the lung epithelium, and suggest that developmental regulation of TMPRSS2 may underlie the relative protection of infants and children from severe respiratory illness.