Dynamic Hippo pathway activity underlies mesenchymal differentiation during lung alveolar morphogenesis.

Alveologenesis, the final stage in lung development, substantially remodels the distal lung, expanding the alveolar surface area for efficient gas exchange. Secondary crest myofibroblasts (SCMF) exist transiently in the neonatal distal lung and are crucial for alveologenesis. However, the pathways that regulate SCMF function, proliferation and temporal identity remain poorly understood. To address this, we purified SCMFs from reporter mice, performed bulk RNA-seq and found dynamic changes in Hippo-signaling components during alveologenesis. We deleted the Hippo effectors Yap/Taz from Acta2-expressing cells at the onset of alveologenesis, causing a significant arrest in alveolar development. Using single cell RNA-seq, we identified a distinct cluster of cells in mutant lungs with altered expression of marker genes associated with proximal mesenchymal cell types, airway smooth muscle and alveolar duct myofibroblasts. In vitro studies confirmed that Yap/Taz regulates myofibroblast-associated gene signature and contractility. Together, our findings show that Yap/Taz is essential for maintaining functional myofibroblast identity during postnatal alveologenesis.

Dysregulated alveolar epithelial cell progenitor function and identity in Hermansky-Pudlak syndrome pulmonary fibrosis.

Hermansky-Pudlak syndrome (HPS) is a genetic disorder of endosomal protein trafficking associated with pulmonary fibrosis in specific subtypes, including HPS-1 and HPS-2. Single mutant HPS1 and HPS2 mice display increased fibrotic sensitivity while double mutant HPS1/2 mice exhibit spontaneous fibrosis with aging, which has been attributed to HPS mutations in alveolar epithelial type II (AT2) cells. We utilized HPS mouse models and human lung tissue to investigate mechanisms of AT2 cell dysfunction driving fibrotic remodeling in HPS. Starting at 8 weeks of age, HPS mice exhibited progressive loss of AT2 cell numbers. HPS AT2 cell was impaired ex vivo and in vivo. Incorporating AT2 cell lineage tracing in HPS mice, we observed aberrant differentiation with increased AT2-derived alveolar epithelial type I cells. Transcriptomic analysis of HPS AT2 cells revealed elevated expression of genes associated with aberrant differentiation and p53 activation. Lineage tracing and modeling studies demonstrated that HPS AT2 cells were primed to persist in a Krt8+ reprogrammed transitional state, mediated by p53 activity. Intrinsic AT2 progenitor cell dysfunction and p53 pathway dysregulation are novel mechanisms of disease in HPS-related pulmonary fibrosis, with the potential for early targeted intervention before the onset of fibrotic lung disease.

Circadian regulation of lung repair and regeneration.

Optimal lung repair and regeneration are essential for recovery from viral infections, including influenza A virus (IAV). We have previously demonstrated that acute inflammation and mortality induced by IAV is under circadian control. However, it is not known whether the influence of the circadian clock persists beyond the acute outcomes. Here, we utilize the UK Biobank to demonstrate an association between poor circadian rhythms and morbidity from lower respiratory tract infections, including the need for hospitalization and mortality after discharge; this persists even after adjusting for common confounding factors. Furthermore, we use a combination of lung organoid assays, single-cell RNA sequencing, and IAV infection in different models of clock disruption to investigate the role of the circadian clock in lung repair and regeneration. We show that lung organoids have a functional circadian clock and the disruption of this clock impairs regenerative capacity. Finally, we find that the circadian clock acts through distinct pathways in mediating lung regeneration - in tracheal cells via the Wnt/ß-catenin pathway and through IL-1ß in alveolar epithelial cells. We speculate that adding a circadian dimension to the critical process of lung repair and regeneration will lead to novel therapies and improve outcomes.

Engineering and Modeling the Lung Mesenchyme.

The structure of the mammalian lung controls the flow of air through the airways and into the distal alveolar region where gas exchange occurs. Specialized cells in the lung mesenchyme produce the extracellular matrix (ECM) and growth factors required for lung structure. Historically, characterizing the mesenchymal cell subtypes was challenging due to their ambiguous morphology, overlapping expression of protein markers, and limited cell-surface molecules needed for isolation. The recent development of single-cell RNA sequencing (scRNA-seq) complemented with genetic mouse models demonstrated that the lung mesenchyme comprises transcriptionally and functionally heterogeneous cell-types. Bioengineering approaches that model tissue structure clarify the function and regulation of mesenchymal cell types. These experimental approaches demonstrate the unique abilities of fibroblasts in mechanosignaling, mechanical force generation, ECM production, and tissue regeneration. This chapter will review the cell biology of the lung mesenchyme and experimental approaches to study their function.

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.

A census of the lung: CellCards from LungMAP.

The human lung plays vital roles in respiration, host defense, and basic physiology. Recent technological advancements such as single-cell RNA sequencing and genetic lineage tracing have revealed novel cell types and enriched functional properties of existing cell types in lung. The time has come to take a new census. Initiated by members of the NHLBI-funded LungMAP Consortium and aided by experts in the lung biology community, we synthesized current data into a comprehensive and practical cellular census of the lung. Identities of cell types in the normal lung are captured in individual cell cards with delineation of function, markers, developmental lineages, heterogeneity, regenerative potential, disease links, and key experimental tools. This publication will serve as the starting point of a live, up-to-date guide for lung research at https://www.lungmap.net/cell-cards/. We hope that Lung CellCards will promote the community-wide effort to establish, maintain, and restore respiratory health.

Genomic, epigenomic, and biophysical cues controlling the emergence of the lung alveolus.

The lung alveolus is the functional unit of the respiratory system required for gas exchange. During the transition to air breathing at birth, biophysical forces are thought to shape the emerging tissue niche. However, the intercellular signaling that drives these processes remains poorly understood. Applying a multimodal approach, we identified alveolar type 1 (AT1) epithelial cells as a distinct signaling hub. Lineage tracing demonstrates that AT1 progenitors align with receptive, force-exerting myofibroblasts in a spatial and temporal manner. Through single-cell chromatin accessibility and pathway expression (SCAPE) analysis, we demonstrate that AT1-restricted ligands are required for myofibroblasts and alveolar formation. These studies show that the alignment of cell fates, mediated by biophysical and AT1-derived paracrine signals, drives the extensive tissue remodeling required for postnatal respiration.

STAT3-BDNF-TrkB signalling promotes alveolar epithelial regeneration after lung injury.

Alveolar epithelial regeneration is essential for recovery from devastating lung diseases. This process occurs when type II alveolar pneumocytes (AT2 cells) proliferate and transdifferentiate into type I alveolar pneumocytes (AT1 cells). We used genome-wide analysis of chromatin accessibility and gene expression following acute lung injury to elucidate repair mechanisms. AT2 chromatin accessibility changed substantially following injury to reveal STAT3 binding motifs adjacent to genes that regulate essential regenerative pathways. Single-cell transcriptome analysis identified brain-derived neurotrophic factor (Bdnf) as a STAT3 target gene with newly accessible chromatin in a unique population of regenerating AT2 cells. Furthermore, the BDNF receptor tropomyosin receptor kinase B (TrkB) was enriched on mesenchymal alveolar niche cells (MANCs). Loss or blockade of AT2-specific Stat3, Bdnf or mesenchyme-specific TrkB compromised repair and reduced Fgf7 expression by niche cells. A TrkB agonist improved outcomes in vivo following lung injury. These data highlight the biological and therapeutic importance of the STAT3-BDNF-TrkB axis in orchestrating alveolar epithelial regeneration.

Defining the role of pulmonary endothelial cell heterogeneity in the response to acute lung injury.

Pulmonary endothelial cells (ECs) are an essential component of the gas exchange machinery of the lung alveolus. Despite this, the extent and function of lung EC heterogeneity remains incompletely understood. Using single-cell analytics, we identify multiple EC populations in the mouse lung, including macrovascular endothelium (maEC), microvascular endothelium (miECs), and a new population we have termed Car4-high ECs. Car4-high ECs express a unique gene signature, and ligand-receptor analysis indicates they are primed to receive reparative signals from alveolar type I cells. After acute lung injury, they are preferentially localized in regenerating regions of the alveolus. Influenza infection reveals the emergence of a population of highly proliferative ECs that likely arise from multiple miEC populations and contribute to alveolar revascularization after injury. These studies map EC heterogeneity in the adult lung and characterize the response of novel EC subpopulations required for tissue regeneration after acute lung injury.

Animal lungs are filled with tiny air sacks called alveoli, where the gas exchanges that keep organisms alive can take place. Small blood vessels known as capillaries come in close contact with the alveoli, allowing oxygen to be extracted from the air into the blood, and carbon dioxide to be released from the blood into the air. The cells that line the inside of these capillaries (known as pulmonary endothelial cells) are important actors in these exchanges. After having been damaged, for example by viruses like influenza, the lungs need to regenerate and create new capillaries. Yet, it was still unclear how pulmonary endothelial cells participate in the healing process, and if capillaries contain several populations of endothelial cells that play different roles. To investigate this question, Niethamer et al. used an approach called single-cell analytics to examine individual endothelial cells in the alveoli of mice infected with influenza. This revealed that different subtypes of endothelial cells exist in capillaries, and that some may be able to perform slightly different jobs during lung recovery. Niethamer et al. found that all subtypes could quickly multiply after injury to create more endothelial cells and re-establish gas exchanges. However, one newly identified group (called Car4-high ECs) was particularly primed to receive orders from damaged alveoli. These cells were also often found at the sites where the alveoli were most injured. Lung injuries are a major cause of death worldwide. Understanding how pulmonary endothelial cells work when the organ is both healthy and injured should help to find ways to boost repair, and to create therapies that could target these cells.

Cellular crosstalk in the development and regeneration of the respiratory system.

The respiratory system, including the peripheral lungs, large airways and trachea, is one of the most recently evolved adaptations to terrestrial life. To support the exchange of respiratory gases, the respiratory system is interconnected with the cardiovascular system, and this interconnective nature requires a complex interplay between a myriad of cell types. Until recently, this complexity has hampered our understanding of how the respiratory system develops and responds to postnatal injury to maintain homeostasis. The advent of new single-cell sequencing technologies, developments in cellular and tissue imaging and advances in cell lineage tracing have begun to fill this gap. The view that emerges from these studies is that cellular and functional heterogeneity of the respiratory system is even greater than expected and also highly adaptive. In this Review, we explore the cellular crosstalk that coordinates the development and regeneration of the respiratory system. We discuss both the classic cell and developmental biology studies and recent single-cell analysis to provide an integrated understanding of the cellular niches that control how the respiratory system develops, interacts with the external environment and responds to injury.

Dnmt1 is required for proximal-distal patterning of the lung endoderm and for restraining alveolar type 2 cell fate.

Lung endoderm development occurs through a series of finely coordinated transcriptional processes that are regulated by epigenetic mechanisms. However, the role of DNA methylation in regulating lung endoderm development remains poorly understood. We demonstrate that DNA methyltransferase 1 (Dnmt1) is required for early branching morphogenesis of the lungs and for restraining epithelial fate specification. Loss of Dnmt1 leads to an early branching defect, a loss of epithelial polarity and proximal endodermal cell differentiation, and an expansion of the distal endoderm compartment. Dnmt1 deficiency also disrupts epithelial-mesenchymal crosstalk and leads to precocious distal endodermal cell differentiation with premature expression of alveolar type 2 cell restricted genes. These data reveal an important requirement for Dnmt1 mediated DNA methylation in early lung development to promote proper branching morphogenesis, maintain proximal endodermal cell fate, and suppress premature activation of the distal epithelial fate.

Early lineage specification defines alveolar epithelial ontogeny in the murine lung.

During the stepwise specification and differentiation of tissue-specific multipotent progenitors, lineage-specific transcriptional networks are activated or repressed to orchestrate cell specification. The gas-exchange niche in the lung contains two major epithelial cell types, alveolar type 1 (AT1) and AT2 cells, and the timing of lineage specification of these cells is critical for the correct formation of this niche and postnatal survival. Integrating cell-specific lineage tracing studies, spatially specific mRNA transcript and protein expression, and single-cell RNA-sequencing analysis, we demonstrate that specification of alveolar epithelial cell fate begins concomitantly with the proximal-distal specification of epithelial progenitors and branching morphogenesis earlier than previously appreciated. By using a newly developed dual-lineage tracing system, we show that bipotent alveolar cells that give rise to AT1 and AT2 cells are a minor contributor to the alveolar epithelial population. Furthermore, single-cell assessment of the transcriptome identifies specified AT1 and AT2 progenitors rather than bipotent cells during sacculation. These data reveal a paradigm of organ formation whereby lineage specification occurs during the nascent stages of development coincident with broad tissue-patterning processes, including axial patterning of the endoderm and branching morphogenesis.

IL-17A Recruits Rab35 to IL-17R to Mediate PKCα-Dependent Stress Fiber Formation and Airway Smooth Muscle Contractility.

IL-17A is a critical proinflammatory cytokine for the pathogenesis of asthma including neutrophilic pulmonary inflammation and airway hyperresponsiveness. In this study, by cell type-specific deletion of IL-17R and adaptor Act1, we demonstrated that IL-17R/Act1 exerts a direct impact on the contraction of airway smooth muscle cells (ASMCs). Mechanistically, IL-17A induced the recruitment of Rab35 (a small monomeric GTPase) and DennD1C (guanine nucleotide exchange factor [GEF]) to the IL-17R/Act1 complex in ASMCs, resulting in activation of Rab35. Rab35 knockdown showed that IL-17A-induced Rab35 activation was essential for protein kinase Cα (PKCα) activation and phosphorylation of fascin at Ser39 in ASMCs, allowing F-actin to interact with myosin to form stress fibers and enhance the contraction induced by methacholine. PKCα inhibitor or Rab35 knockdown indeed substantially reduced IL-17A-induced stress fiber formation in ASMCs and attenuated IL-17A-enhanced, methacholine-induced contraction of airway smooth muscle. Taken together, these data indicate that IL-17A promotes airway smooth muscle contraction via direct recruitment of Rab35 to IL-17R, followed by PKCα activation and stress fiber formation.

Regeneration of the lung alveolus by an evolutionarily conserved epithelial progenitor.

Functional tissue regeneration is required for the restoration of normal organ homeostasis after severe injury. Some organs, such as the intestine, harbour active stem cells throughout homeostasis and regeneration; more quiescent organs, such as the lung, often contain facultative progenitor cells that are recruited after injury to participate in regeneration. Here we show that a Wnt-responsive alveolar epithelial progenitor (AEP) lineage within the alveolar type 2 cell population acts as a major facultative progenitor cell in the distal lung. AEPs are a stable lineage during alveolar homeostasis but expand rapidly to regenerate a large proportion of the alveolar epithelium after acute lung injury. AEPs exhibit a distinct transcriptome, epigenome and functional phenotype and respond specifically to Wnt and Fgf signalling. In contrast to other proposed lung progenitor cells, human AEPs can be directly isolated by expression of the conserved cell surface marker TM4SF1, and act as functional human alveolar epithelial progenitor cells in 3D organoids. Our results identify the AEP lineage as an evolutionarily conserved alveolar progenitor that represents a new target for human lung regeneration strategies.

IL-17A-Induced PLET1 Expression Contributes to Tissue Repair and Colon Tumorigenesis.

This study identifies a novel mechanism linking IL-17A with colon tissue repair and tumor development. Abrogation of IL-17A signaling in mice attenuated tissue repair of dextran sulfate sodium (DSS)-induced damage in colon epithelium and markedly reduced tumor development in an azoxymethane/DSS model of colitis-associated cancer. A novel IL-17A target gene, PLET1 (a progenitor cell marker involved in wound healing), was highly induced in DSS-treated colon tissues and tumors in an IL-17RC-dependent manner. PLET1 expression was induced in LGR5+ colon epithelial cells after DSS treatment. LGR5+PLET1+ marks a highly proliferative cell population with enhanced expression of IL-17A target genes. PLET1 deficiency impaired tissue repair of DSS-induced damage in colon epithelium and reduced tumor formation in an azoxymethane/DSS model of colitis-associated cancer. Our results suggest that IL-17A-induced PLET1 expression contributes to tissue repair and colon tumorigenesis.

Distinct Mesenchymal Lineages and Niches Promote Epithelial Self-Renewal and Myofibrogenesis in the Lung.

The lung is an architecturally complex organ comprising a heterogeneous mixture of various epithelial and mesenchymal lineages. We use single-cell RNA sequencing and signaling lineage reporters to generate a spatial and transcriptional map of the lung mesenchyme. We find that each mesenchymal lineage has a distinct spatial address and transcriptional profile leading to unique niche regulatory functions. The mesenchymal alveolar niche cell is Wnt responsive, expresses Pdgfrα, and is critical for alveolar epithelial cell growth and self-renewal. In contrast, the Axin2+ myofibrogenic progenitor cell preferentially generates pathologically deleterious myofibroblasts after injury. Analysis of the secretome and receptome of the alveolar niche reveals functional pathways that mediate growth and self-renewal of alveolar type 2 progenitor cells, including IL-6/Stat3, Bmp, and Fgf signaling. These studies define the cellular and molecular framework of lung mesenchymal niches and reveal the functional importance of developmental pathways in promoting self-renewal versus a pathological response to tissue injury.

Emergence of a Wave of Wnt Signaling that Regulates Lung Alveologenesis by Controlling Epithelial Self-Renewal and Differentiation.

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.