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.
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.
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.
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 E
(PGE
) ...receptor EP
. However, in humans and mice, disrupted PGE
-EP
signaling in utero causes unexpected patency of the DA (PDA) after birth, suggesting another role for EP
during development. We used EP
-knockout (KO) mice and acute versus chronic pharmacological approaches to investigate EP
signaling in DA development and function. Expression analyses identified EP
as the primary EP receptor in the DA from midgestation to term; inhibitor studies verified EP
as the primary dilator during this period. Chronic antagonism recapitulated the EP
KO phenotype and revealed a narrow developmental window when EP
stimulation is required for postnatal DA closure. Myography studies indicate that despite reduced contractile properties, the EP
KO DA maintains an intact oxygen response. In newborns, hyperoxia constricted the EP
KO DA but survival was not improved, and permanent remodeling was disrupted. Vasomotion and increased nitric oxide (NO) sensitivity in the EP
KO DA suggest incomplete DA development. Analysis of DA maturity markers confirmed a partially immature EP
KO DA phenotype. Together, our data suggest that EP
signaling in late gestation plays a key developmental role in establishing a functional term DA. When disrupted in EP
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.
EP
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 EP
-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.
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.
Wnt/β-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 β-catenin phosphorylated at tyrosine 489 and epithelial cell cytosolic localization of β-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 β-catenin in the lung epithelium and surrounding mesenchyme in BPD and IPF, closely resembling the pattern observed in 18-week fetal lung. Nuclear β-catenin phosphorylated at tyrosine 489 associated with an increased expression of Wnt target gene AXIN2, suggesting that the observed β-catenin signature is of functional significance during normal development and injury repair. The association of specific modifications of β-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 β-catenin phosphorylation has potential as a therapeutic target for the treatment and prevention of both BPD and IPF.
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.
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.
Objective
The healthy alveolar epithelium is protected by a heparan sulfate rich, glycosaminoglycan layer called the epithelial glycocalyx. Our group found that the epithelial glycocalyx is shed in ...patients with acute respiratory distress syndrome (ARDS). In murine models of LPS‐ or bleomycin‐induced acute lung injury, sheddases (membrane‐bound enzymes that cleave extracellular potions of transmembrane proteins) are upregulated and associated with glycocalyx shedding and increased lung permeability. ARDS is commonly caused by viral infections including influenza A (IAV). In murine models, IAV causes massive and persistent glycocalyx shedding into the airspace but the mechanisms by which this occurs are unknown. The objective of this work is to determine the molecular processes underlying IAV‐induced shedding of the glycocalyx.
Hypothesis
We hypothesize that IAV causes glycocalyx shedding through induction of host sheddases.
Methods
We examined the literature and curated a list of sheddases associated with IAV with potential to cleave the glycocalyx (MMP‐7, ‐2, ‐9 and their inhibitors TIMP‐1 and ‐2). C57BL/6 mice were infected intranasally with A/PR/8/34 (H1N1) at 30,000 PFU/mouse and bronchoalveolar lavage and lung tissue were collected at day 1, 3, and 7 post infection. Sheddase expression was assessed by RT‐qPCR and RNAscope was used to localize lung sheddase expression in infected and uninfected lungs. MLE‐12 mouse lung epithelial cells were infected with viable or heat‐inactivated (56C for 30 min) A/PR/8/34 (H1N1) at a MOI of 1 and sheddase expression measured by RT‐qPCR.
Results
Mice infected with IAV develop significant lung inflammation (increased BAL inflammatory cells), lung permeability (increased BAL protein), and increased glycocalyx shedding. MMP‐7 is upregulated in infected vs. uninfected lungs at day 1 and 3 post infection, then returns to baseline levels by day 7. MMP‐7 is only expressed in cells that are directly infected by IAV. Expression of the MMP‐7 inhibitor TIMP‐1 is similar to uninfected lungs on day 1, but increases 50‐fold on day 3. In contrast, MMP‐2 and MMP‐9, as well as their inhibitor TIMP‐2 are not upregulated in the first 7 days after IAV infection. Preliminary studies in lung epithelial cells suggest that heat‐inactivated IAV fails to upregulate MMP‐7.
Conclusions
Together, these data suggest that localized IAV infection increases MMP‐7 in a murine model of IAV infection, but has no effect on several other sheddases. This suggests that MMP‐7 may modulate IAV‐induced glycocalyx shedding. Future studies will explore the mechanisms of IAV induced glycocalyx shedding which could provide molecular targets for clinical intervention in IAV‐ARDS pathogenesis.