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The human lung differs substantially from its mouse counterpart, resulting in a distinct distal airway architecture affected by disease pathology in chronic obstructive pulmonary disease. In humans, the distal branches of the airway interweave with the alveolar gas-exchange niche, forming an anatomical structure known as the respiratory bronchioles. Owing to the lack of a counterpart in mouse, the cellular and molecular mechanisms that govern respiratory bronchioles in the human lung remain uncharacterized. Here we show that human respiratory bronchioles contain a unique secretory cell population that is distinct from cells in larger proximal airways. Organoid modelling reveals that these respiratory airway secretory (RAS) cells act as unidirectional progenitors for alveolar type 2 cells, which are essential for maintaining and regenerating the alveolar niche. RAS cell lineage differentiation into alveolar type 2 cells is regulated by Notch and Wnt signalling. In chronic obstructive pulmonary disease, RAS cells are altered transcriptionally, corresponding to abnormal alveolar type 2 cell states, which are associated with smoking exposure in both humans and ferrets. These data identify a distinct progenitor in a region of the human lung that is not found in mouse that has a critical role in maintaining the gas-exchange compartment and is altered in chronic lung disease.

Premature birth disrupts normal lung development and places infants at risk for bronchopulmonary dysplasia (BPD), a disease disrupting lung health throughout the life of an individual and that is increasing in incidence. The TGF-[beta] superfamily has been implicated in BPD pathogenesis, however, what cell lineage it impacts remains unclear. We show that TGFbr2 is critical for alveolar epithelial (AT1) cell fate maintenance and function. Loss of TGFbr2 in AT1 cells during late lung development leads to AT1-AT2 cell reprogramming and altered pulmonary architecture, which persists into adulthood. Restriction of fetal lung stretch and associated AT1 cell spreading through a model of oligohydramnios enhances AT1-AT2 reprogramming. Transcriptomic and proteomic analyses reveal the necessity of TGFbr2 expression in AT1 cells for extracellular matrix production. Moreover, TGF-[beta] signaling regulates integrin transcription to alter AT1 cell morphology, which further impacts ECM expression through changes in mechanotransduction. These data reveal the cell intrinsic necessity of TGF-[beta] signaling in maintaining AT1 cell fate and reveal this cell lineage as a major orchestrator of the alveolar matrisome., Introduction Bronchopulmonary dysplasia (BPD) is a lung disease that disproportionately affects infants born at less than 28 weeks gestation, the threshold for extreme prematurity (1). Despite multiple medical advances including [...]

Lymphangioleiomyomatosis (LAM) is a progressive cystic lung disease caused by tuberous sclerosis complex 1/2 (TSC1/2) gene mutations in pulmonary mesenchymal cells, resulting in activation of the mechanistic target of rapamycin complex 1 (mTORC1). A subset of patients with LAM develop pulmonary vascular remodeling and pulmonary hypertension. Little, however, is known regarding how LAM cells communicate with endothelial cells (ECs) to trigger vascular remodeling. In endstage LAM lung explants, we identified EC dysfunction characterized by increased EC proliferation and migration, defective angiogenesis, and dysmorphic endothelial tube network formation. To model LAM disease, we used an mTORC1 gain-offunction mouse model with a Tsc2 KO ([Tsc2.sup.KO]) specific to lung mesenchyme ([Tbx4.sup.LME-Cre] [Tsc2.sup.fl/fl]), similar to the mesenchymespecific genetic alterations seen in human disease. As early as 8 weeks of age, ECs from mice exhibited marked transcriptomic changes despite an absence of morphological changes to the distal lung microvasculature. In contrast, 1-year-old [Tbx4.sup.LME-Cre] [Tsc2.sup.fl/fl] mice spontaneously developed pulmonary vascular remodeling with increased medial thickness. Single-cell RNA- Seq of 1-year-old mouse lung cells identified paracrine ligands originating from [Tsc2.sup.KO] mesenchyme, which can signal through receptors in arterial ECs. These ECs had transcriptionally altered genes including those in pathways associated with blood vessel remodeling. The proposed pathophysiologic mesenchymal ligand-EC receptor crosstalk highlights the importance of an altered mesenchymal cell/EC axis in LAM and other hyperactive mTORC1-driven diseases. Since ECs in patients with LAM and in [Tbx4.sup.LME-Cre] [Tsc2.sup.fl/fl] mice did not harbor TSC2 mutations, our study demonstrates that constitutively active mTORC1 lung mesenchymal cells orchestrated dysfunctional EC responses that contributed to pulmonary vascular remodeling., Introduction Lymphangioleiomyomatosis (LAM) is a destructive lung disease characterized by diffuse parenchymal cystic airspace enlargement, airflow obstruction, and chylothorax from lymphatic involvement (1-6). In addition, some patients with LAM develop [...]

The respiratory system, which includes the trachea, airways, and distal alveoli, is a complex multi-cellular organ that intimately links with the cardiovascular system to accomplish gas exchange. In this review and as members of the NIH/NHLBI-supported Progenitor Cell Translational Consortium, we discuss key aspects of lung repair and regeneration. We focus on the cellular compositions within functional niches, cell-cell signaling in homeostatic health, the responses to injury, and new methods to study lung repair and regeneration. We also provide future directions for an improved understanding of the cell biology of the respiratory system, as well as new therapeutic avenues.

Alveolar epithelium plays a pivotal role in protecting the lungs from inhaled infectious agents. Therefore, the regenerative capacity of the alveolar epithelium is critical for recovery from these insults in order to rebuild the epithelial barrier and restore pulmonary functions. Here, we show that sublethal infection of mice with Streptococcus pneumoniae, the most common pathogen of community-acquired pneumonia, led to exclusive damage in lung alveoli, followed by alveolar epithelial regeneration and resolution of lung inflammation. We show that surfactant protein C-expressing (SPC-expressing) alveolar epithelial type II cells (AECIIs) underwent proliferation and differentiation after infection, which contributed to the newly formed alveolar epithelium. This increase in AECII activities was correlated with increased nuclear expression of Yap and Taz, the mediators of the Hippo pathway. Mice that lacked Yap/Taz in AECIIs exhibited prolonged inflammatory responses in the lung and were delayed in alveolar epithelial regeneration during bacterial pneumonia. This impaired alveolar epithelial regeneration was paralleled by a failure to upregulate IκBa, the molecule that terminates NF-κB-mediated inflammatory responses. These results demonstrate that signals governing resolution of lung inflammation were altered in Yap/Taz mutant mice, which prevented the development of a proper regenerative niche, delaying repair and regeneration of alveolar epithelium during bacterial pneumonia.

BackgroundAcute respiratory distress syndrome (ARDS) is a severe form of lung injury characterized by damage to the epithelial barrier with subsequent pulmonary edema and hypoxic respiratory failure. ARDS is a significant medical problem in intensive care units with associated high care costs. There are many potential causes of ARDS; however, alveolar injury associated with mechanical ventilation, termed ventilator-induced lung injury (VILI), remains a well-recognized contributor. It is thus critical to understand the mechanism of VILI. Based on our published preliminary data, we hypothesized that the endoplasmic reticulum (ER) stress response molecule Protein Kinase R-like Endoplasmic Reticulum Kinase (PERK) plays a role in transmitting mechanosensory signals the alveolar epithelium.MethodsER stress signal responses to mechanical stretch were studied in ex-vivo ventilated pig lungs. To explore the effect of PERK inhibition on VILI, we ventilated live rats and compared lung injury parameters to non-ventilated controls. The effect of stretch-induced epithelial ER Ca2+ signaling on PERK was studied in stretched alveolar epithelial monolayers. To confirm the activation of PERK in human disease, ER stress signaling was compared between ARDS and non-ARDS lungs.ResultsOur studies revealed increased PERK-specific ER stress signaling in response to overstretch. PERK inhibition resulted in dose-dependent improvement of alveolar inflammation and permeability. Our data indicate that stretch-induced epithelial ER Ca2+ release is an activator of PERK. Experiments with human lung tissue confirmed PERK activation by ARDS.ConclusionOur study provides evidences that PERK is a mediator stretch signals in the alveolar epithelium.

Lung epithelial lineages have been difficult to maintain in pure form in vitro, and lineage-specific reporters have proven invaluable for monitoring their emergence from cultured pluripotent stem cells (PSCs). However, reporter constructs for tracking proximal airway lineages generated from PSCs have not been previously available, limiting the characterization of these cells. Here, we engineer mouse and human PSC lines carrying airway secretory lineage reporters that facilitate the tracking, purification, and profiling of this lung subtype. Through bulk and single-cell-based global transcriptomic profiling, we find PSC-derived airway secretory cells are susceptible to phenotypic plasticity exemplified by the tendency to co-express both a proximal airway secretory program as well as an alveolar type 2 cell program, which can be minimized by inhibiting endogenous Wnt signaling. Our results provide global profiles of engineered lung cell fates, a guide for improving their directed differentiation, and a human model of the developing airway.

Alveologenesis is the culmination of lung development and involves the correct temporal and spatial signals to generate the delicate gas exchange interface required for respiration. Using a Wnt-signaling reporter system, we demonstrate the emergence of a Wnt-responsive alveolar epithelial cell sublineage, which arises during alveologenesis, called the axin2+ alveolar type 2 cell, or AT2Axin2. The number of AT2Axin2 cells increases substantially during late lung development, correlating with a wave of Wnt signaling during alveologenesis. Transcriptome analysis, in vivo clonal analysis, and ex vivo lung organoid assays reveal that AT2sAxin2 promote enhanced AT2 cell growth during generation of the alveolus. Activating Wnt signaling results in the expansion of AT2s, whereas inhibition of Wnt signaling inhibits AT2 cell development and shunts alveolar epithelial development toward the alveolar type 1 cell lineage. These findings reveal a wave of Wnt-dependent AT2 expansion required for lung alveologenesis and maturation.

Postnatal tissue quiescence is thought to be a default state in the absence of a proliferative stimulus such as injury. Although previous studies have demonstrated that certain embryonic developmental programs are reactivated aberrantly in adult organs to drive repair and regeneration, it is not well understood how quiescence is maintained in organs such as the lung, which displays a remarkably low level of cellular turnover. Here we demonstrate that quiescence in the adult lung is an actively maintained state and is regulated by hedgehog signalling. Epithelial-specific deletion of sonic hedgehog (Shh) during postnatal homeostasis in the murine lung results in a proliferative expansion of the adjacent lung mesenchyme. Hedgehog signalling is initially downregulated during the acute phase of epithelial injury as the mesenchyme proliferates in response, but returns to baseline during injury resolution as quiescence is restored. Activation of hedgehog during acute epithelial injury attenuates the proliferative expansion of the lung mesenchyme, whereas inactivation of hedgehog signalling prevents the restoration of quiescence during injury resolution. Finally, we show that hedgehog also regulates epithelial quiescence and regeneration in response to injury via a mesenchymal feedback mechanism. These results demonstrate that epithelial-mesenchymal interactions coordinated by hedgehog actively maintain postnatal tissue homeostasis, and deregulation of hedgehog during injury leads to aberrant repair and regeneration in the lung.

Respiratory disease is the third leading cause of death in the industrialized world. Consequently, the trachea, lungs, and cardiopulmonary vasculature have been the focus of extensive investigations. Recent studies have provided new information about the mechanisms driving lung development and differentiation. However, there is still much to learn about the ability of the adult respiratory system to undergo repair and to replace cells lost in response to injury and disease. This Review highlights the multiple stem/progenitor populations in different regions of the adult lung, the plasticity of their behavior in injury models, and molecular pathways that support homeostasis and repair.

Long noncoding RNAs (lncRNAs) are thought to play important roles in regulating gene transcription, but few have well-defined expression patterns or known biological functions during mammalian development. Using a conservative pipeline to identify lncRNAs that have important biological functions, we identified 363 lncRNAs in the lung and foregut endoderm. Importantly, we show that these lncRNAs are spatially correlated with transcription factors across the genome. In-depth expression analyses of lncRNAs with genomic loci adjacent to the critical transcription factors Nkx2.1, Gata6, Foxa2 (forkhead box a2), and Foxf1 mimic the expression patterns of their protein-coding neighbor. Loss-of-function analysis demonstrates that two lncRNAs, LL18/NANCI (Nkx2.1-associated noncoding intergenic RNA) and LL34, play distinct roles in endoderm development by controlling expression of critical developmental transcription factors and pathways, including retinoic acid signaling. In particular, we show that LL18/NANCI acts upstream of Nkx2.1 and downstream from Wnt signaling to regulate lung endoderm gene expression. These studies reveal that lncRNAs play an important role in foregut and lung endoderm development by regulating multiple aspects of gene transcription, often through regulation of transcription factor expression.

Wnt/β-catenin signaling is a central regulator of adult stem cells. Variable sensitivity of Wnt reporter transgenes, β-catenin's dual roles in adhesion and signaling, and hair follicle degradation and inflammation resulting from broad deletion of epithelial β-catenin have precluded clear understanding of Wnt/β-catenin's functions in adult skin stem cells. By inducibly deleting β-catenin globally in skin epithelia, only in hair follicle stem cells, or only in interfollicular epidermis and comparing the phenotypes with those caused by ectopic expression of the Wnt/β-catenin inhibitor Dkk1, we show that this pathway is necessary for hair follicle stem cell proliferation. However, β-catenin is not required within hair follicle stem cells for their maintenance, and follicles resume proliferating after ectopic Dkk1 has been removed, indicating persistence of functional progenitors. We further unexpectedly discovered a broader role for Wnt/β-catenin signaling in contributing to progenitor cell proliferation in nonhairy epithelia and interfollicular epidermis under homeostatic, but not inflammatory, conditions.

Development of the pulmonary system is essential for terrestrial life. The molecular pathways that regulate this complex process are beginning to be defined, and such knowledge is critical to our understanding of congenital and acquired lung diseases. A recent workshop was convened by the National Heart, Lung, and Blood Institute to discuss the developmental principles that regulate the formation of the pulmonary system. Emerging evidence suggests that key developmental pathways not only regulate proper formation of the pulmonary system but are also reactivated upon postnatal injury and repair and in the pathogenesis of human lung diseases. Molecular understanding of early lung development has also led to new advances in areas such as generation of lung epithelium from pluripotent stem cells. The workshop was organized into four different topics, including early lung cell fate and morphogenesis, mechanisms of lung cell differentiation, tissue interactions in lung development, and environmental impact on early lung development. Critical points were raised, including the importance of epigenetic regulation of lung gene expression, the dearth of knowledge on important mesenchymal lineages within the lung, and the interaction between the developing pulmonary and cardiovascular system. This manuscript describes the summary of the discussion along with general recommendations to overcome the gaps in knowledge in lung developmental biology.

The pulmonary circulation is a highly specialized vascular bed that physically and functionally connects the heart and the lungs. The interdependence of these two organs is illustrated in embryonic development, when the lung endoderm protrudes into the surrounding mesoderm as the heart tube elongates and folds into structurally distinct chambers. The pulmonary vascular precursors then undergo highly stereotyped cellular maturation and patterning to form a multilayered vascular network that parallels the airways and links the arterial and venous poles of the heart. Upon the first breath, the mature pulmonary circulation is poised to receive the entire cardiac output for efficient gas exchange, and deliver oxygenated blood to the systemic circulation. Disruption of this developmental process can result in congenital defects such as the syndrome tetralogy of Fallot, or differentiation defects leading to persistent pulmonary hypertension of the newborn. Prior studies into the role of angiogenesis and vasculogenesis in pulmonary vascular development have not clearly yielded the identity of pulmonary vascular precursors, or the signals coordinating vascular maturation. We outline key questions on pulmonary vascular development that consider the role of heart-lung interaction in promoting the differentiation and patterning of the pulmonary vasculature.