Generation of human alveolar epithelial type I cells from pluripotent stem cells.

Alveolar epithelial type I cells (AT1s) line the gas exchange barrier of the distal lung and have been historically challenging to isolate or maintain in cell culture. Here, we engineer a human in vitro AT1 model system via directed differentiation of induced pluripotent stem cells (iPSCs). We use primary adult AT1 global transcriptomes to suggest benchmarks and pathways, such as Hippo-LATS-YAP/TAZ signaling, enriched in these cells. Next, we generate iPSC-derived alveolar epithelial type II cells (AT2s) and find that nuclear YAP signaling is sufficient to promote a broad transcriptomic shift from AT2 to AT1 gene programs. The resulting cells express a molecular, morphologic, and functional phenotype reminiscent of human AT1 cells, including the capacity to form a flat epithelial barrier producing characteristic extracellular matrix molecules and secreted ligands. Our results provide an in vitro model of human alveolar epithelial differentiation and a potential source of human AT1s.

TGF-ß controls alveolar type 1 epithelial cell plasticity and alveolar matrisome gene transcription in mice.

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-ß 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-ß 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-ß signaling in maintaining AT1 cell fate and reveal this cell lineage as a major orchestrator of the alveolar matrisome.

A Lung Cancer Mouse Model Database.

Lung cancer, the leading cause of cancer mortality, exhibits diverse histological subtypes and genetic complexities. Numerous preclinical mouse models have been developed to study lung cancer, but data from these models are disparate, siloed, and difficult to compare in a centralized fashion. Here we established the Lung Cancer Mouse Model Database (LCMMDB), an extensive repository of 1,354 samples from 77 transcriptomic datasets covering 974 samples from genetically engineered mouse models (GEMMs), 368 samples from carcinogen-induced models, and 12 samples from a spontaneous model. Meticulous curation and collaboration with data depositors have produced a robust and comprehensive database, enhancing the fidelity of the genetic landscape it depicts. The LCMMDB aligns 859 tumors from GEMMs with human lung cancer mutations, enabling comparative analysis and revealing a pressing need to broaden the diversity of genetic aberrations modeled in GEMMs. Accompanying this resource, we developed a web application that offers researchers intuitive tools for in-depth gene expression analysis. With standardized reprocessing of gene expression data, the LCMMDB serves as a powerful platform for cross-study comparison and lays the groundwork for future research, aiming to bridge the gap between mouse models and human lung cancer for improved translational relevance.

An injury-induced tissue niche shaped by mesenchymal plasticity coordinates the regenerative and disease response in the lung.

Severe lung injury causes basal stem cells to migrate and outcompete alveolar stem cells resulting in dysplastic repair and a loss of gas exchange function. This "stem cell collision" is part of a multistep process that is now revealed to generate an injury-induced tissue niche (iTCH) containing Keratin 5+ epithelial cells and plastic Pdgfra+ mesenchymal cells. Temporal and spatial single cell analysis reveals that iTCHs are governed by mesenchymal proliferation and Notch signaling, which suppresses Wnt and Fgf signaling in iTCHs. Conversely, loss of Notch in iTCHs rewires alveolar signaling patterns to promote euplastic regeneration and gas exchange. The signaling patterns of iTCHs can differentially phenotype fibrotic from degenerative human lung diseases, through apposing flows of FGF and WNT signaling. These data reveal the emergence of an injury and disease associated iTCH in the lung and the ability of using iTCH specific signaling patterns to discriminate human lung disease phenotypes.

Lung repair and regeneration: Advanced models and insights into human disease.

The respiratory system acts as both the primary site of gas exchange and an important sensor and barrier to the external environment. The increase in incidences of respiratory disease over the past decades has highlighted the importance of developing improved therapeutic approaches. This review will summarize recent research on the cellular complexity of the mammalian respiratory system with a focus on gas exchange and immunological defense functions of the lung. Different models of repair and regeneration will be discussed to help interpret human and animal data and spur the investigation of models and assays for future drug development.

TGF-ßR2 signaling coordinates pulmonary vascular repair after viral injury in mice and human tissue.

Disruption of pulmonary vascular homeostasis is a central feature of viral pneumonia, wherein endothelial cell (EC) death and subsequent angiogenic responses are critical determinants of the outcome of severe lung injury. A more granular understanding of the fundamental mechanisms driving reconstitution of lung endothelium is necessary to facilitate therapeutic vascular repair. Here, we demonstrated that TGF-ß signaling through TGF-ßR2 (transforming growth factor-ß receptor 2) is activated in pulmonary ECs upon influenza infection, and mice deficient in endothelial Tgfbr2 exhibited prolonged injury and diminished vascular repair. Loss of endothelial Tgfbr2 prevented autocrine Vegfa (vascular endothelial growth factor α) expression, reduced endothelial proliferation, and impaired renewal of aerocytes thought to be critical for alveolar gas exchange. Angiogenic responses through TGF-ßR2 were attributable to leucine-rich α-2-glycoprotein 1, a proangiogenic factor that counterbalances canonical angiostatic TGF-ß signaling. Further, we developed a lipid nanoparticle that targets the pulmonary endothelium, Lung-LNP (LuLNP). Delivery of Vegfa mRNA, a critical TGF-ßR2 downstream effector, by LuLNPs improved the impaired regeneration phenotype of EC Tgfbr2 deficiency during influenza injury. These studies defined a role for TGF-ßR2 in lung endothelial repair and demonstrated efficacy of an efficient and safe endothelial-targeted LNP capable of delivering therapeutic mRNA cargo for vascular repair in influenza infection.

Airway epithelial cell identity and plasticity are constrained by Sox2 during lung homeostasis, tissue regeneration, and in human disease.

Maintenance of the cellular boundary between airway and alveolar compartments during homeostasis and after injury is essential to prohibit pathological plasticity which can reduce respiratory function. Lung injury and disease can induce either functional alveolar epithelial regeneration or dysplastic formation of keratinized epithelium which does not efficiently contribute to gas exchange. Here we show that Sox2 preserves airway cell identity and prevents fate changes into either functional alveolar tissue or pathological keratinization following lung injury. Loss of Sox2 in airway epithelium leads to a loss of airway epithelial identity with a commensurate gain in alveolar and basal cell identity, in part due to activation of Wnt signaling in secretory cells and increased Trp63 expression in intrapulmonary basal-like progenitors. In idiopathic pulmonary fibrosis, loss of SOX2 expression correlates with increased WNT signaling activity in dysplastic keratinized epithelium. SOX2-deficient dysplastic epithelial cells are also observed in COVID-19 damaged lungs. Thus, Sox2 provides a molecular barrier that suppresses airway epithelial plasticity to prevent acquisition of alveolar or basal cell identity after injury and help guide proper epithelial fate and regeneration.

Hyperactive mTORC1 in lung mesenchyme induces endothelial cell dysfunction and pulmonary vascular remodeling.

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 end-stage 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-of-function mouse model with a Tsc2 KO (Tsc2KO) specific to lung mesenchyme (Tbx4LME-Cre Tsc2fl/fl), similar to the mesenchyme-specific 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 Tbx4LME-Cre Tsc2fl/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 Tsc2KO 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 Tbx4LME-Cre Tsc2fl/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.

Endothelial SARS-CoV-2 infection is not the underlying cause of COVID-19-associated vascular pathology in mice.

Endothelial damage and vascular pathology have been recognized as major features of COVID-19 since the beginning of the pandemic. Two main theories regarding how severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) damages endothelial cells and causes vascular pathology have been proposed: direct viral infection of endothelial cells or indirect damage mediated by circulating inflammatory molecules and immune mechanisms. However, these proposed mechanisms remain largely untested in vivo. In the present study, we utilized a set of new mouse genetic tools developed in our lab to test both the necessity and sufficiency of endothelial human angiotensin-converting enzyme 2 (hACE2) in COVID-19 pathogenesis. Our results demonstrate that endothelial ACE2 and direct infection of vascular endothelial cells do not contribute significantly to the diverse vascular pathology associated with COVID-19.

The COPD GWAS gene ADGRG6 instructs function and injury response in human iPSC-derived type II alveolar epithelial cells.

Emphysema and chronic obstructive pulmonary disease (COPD) most commonly result from the effects of environmental exposures in genetically susceptible individuals. Genome-wide association studies have implicated ADGRG6 in COPD and reduced lung function, and a limited number of studies have examined the role of ADGRG6 in cells representative of the airway. However, the ADGRG6 locus is also associated with DLCO/VA, an indicator of gas exchange efficiency and alveolar function. Here, we sought to evaluate the mechanistic contributions of ADGRG6 to homeostatic function and disease in type 2 alveolar epithelial cells. We applied an inducible CRISPR interference (CRISPRi) human induced pluripotent stem cell (iPSC) platform to explore ADGRG6 function in iPSC-derived AT2s (iAT2s). We demonstrate that ADGRG6 exerts pleiotropic effects on iAT2s including regulation of focal adhesions, cytoskeleton, tight junctions, and proliferation. Moreover, we find that ADGRG6 knockdown in cigarette smoke-exposed iAT2s alters cellular responses to injury, downregulating apical complexes in favor of proliferation. Our work functionally characterizes the COPD GWAS gene ADGRG6 in human alveolar epithelium.

Endothelial SARS-CoV-2 infection is not the underlying cause of COVID19-associated vascular pathology in mice.

Endothelial damage and vascular pathology have been recognized as major features of COVID-19 since the beginning of the pandemic. Two main theories regarding how Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) damages endothelial cells and causes vascular pathology have been proposed: direct viral infection of endothelial cells or indirect damage mediated by circulating inflammatory molecules and immune mechanisms. However, these proposed mechanisms remain largely untested in vivo. Here, we utilized a set of new mouse genetic tools 1 developed in our lab to test both the necessity and sufficiency of endothelial human angiotensin-converting enzyme 2 (hACE2) in COVID19 pathogenesis. Our results demonstrate that endothelial ACE2 and direct infection of vascular endothelial cells does not contribute significantly to the diverse vascular pathology associated with COVID-19.

DOT1L regulates lung developmental epithelial cell fate and adult alveolar stem cell differentiation after acute injury.

AT2 cells harbor alveolar stem cell activity in the lung and can self-renew and differentiate into AT1 cells during homeostasis and after injury. To identify epigenetic pathways that control the AT2-AT1 regenerative response in the lung, we performed an organoid screen using a library of pharmacological epigenetic inhibitors. This screen identified DOT1L as a regulator of AT2 cell growth and differentiation. In vivo inactivation of Dot1l leads to precocious activation of both AT1 and AT2 gene expression during lung development and accelerated AT1 cell differentiation after acute lung injury. Single-cell transcriptome analysis reveals the presence of a new AT2 cell state upon loss of Dot1l, characterized by increased expression of oxidative phosphorylation genes and changes in expression of critical transcription and epigenetic factors. Taken together, these data demonstrate that Dot1l controls the rate of alveolar epithelial cell fate acquisition during development and regeneration after acute injury.

Guided construction of single cell reference for human and mouse lung.

Accurate cell type identification is a key and rate-limiting step in single-cell data analysis. Single-cell references with comprehensive cell types, reproducible and functionally validated cell identities, and common nomenclatures are much needed by the research community for automated cell type annotation, data integration, and data sharing. Here, we develop a computational pipeline utilizing the LungMAP CellCards as a dictionary to consolidate single-cell transcriptomic datasets of 104 human lungs and 17 mouse lung samples to construct LungMAP single-cell reference (CellRef) for both normal human and mouse lungs. CellRefs define 48 human and 40 mouse lung cell types catalogued from diverse anatomic locations and developmental time points. We demonstrate the accuracy and stability of LungMAP CellRefs and their utility for automated cell type annotation of both normal and diseased lungs using multiple independent methods and testing data. We develop user-friendly web interfaces for easy access and maximal utilization of the LungMAP CellRefs.

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.

Atf3 defines a population of pulmonary endothelial cells essential for lung regeneration.

Following acute injury, the capillary vascular bed in the lung must be repaired to reestablish gas exchange with the external environment. Little is known about the transcriptional and signaling factors that drive pulmonary endothelial cell (EC) proliferation and subsequent regeneration of pulmonary capillaries, as well as their response to stress. Here, we show that the transcription factor Atf3 is essential for the regenerative response of the mouse pulmonary endothelium after influenza infection. Atf3 expression defines a subpopulation of capillary ECs enriched in genes involved in endothelial development, differentiation, and migration. During lung alveolar regeneration, this EC population expands and increases the expression of genes involved in angiogenesis, blood vessel development, and cellular response to stress. Importantly, endothelial cell-specific loss of Atf3 results in defective alveolar regeneration, in part through increased apoptosis and decreased proliferation in the endothelium. This leads to the general loss of alveolar endothelium and persistent morphological changes to the alveolar niche, including an emphysema-like phenotype with enlarged alveolar airspaces lined with regions that lack vascular investment. Taken together, these data implicate Atf3 as an essential component of the vascular response to acute lung injury that is required for successful lung alveolar regeneration.

TGFß controls alveolar type 1 epithelial cell plasticity and alveolar matrisome gene transcription.

Premature birth disrupts normal lung development and places infants at risk for bronchopulmonary dysplasia (BPD), a disease increasing in incidence which disrupts lung health throughout the lifespan. The TGFß superfamily has been implicated in BPD pathogenesis, however, what cell lineage it impacts remains unclear. We show that Tgfbr2 is critical for 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 analysis reveal the necessity of Tgfbr2 expression in AT1 cells for extracellular matrix production. Moreover, TGFß 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 TGFß signaling in maintaining AT1 cell fate and reveal this cell lineage as a major orchestrator of the alveolar matrisome.

Temporal and spatial staging of lung alveolar regeneration is determined by the grainyhead transcription factor Tfcp2l1.

Alveolar epithelial type 2 (AT2) cells harbor the facultative progenitor capacity in the lung alveolus to drive regeneration after lung injury. Using single-cell transcriptomics, software-guided segmentation of tissue damage, and in vivo mouse lineage tracing, we identified the grainyhead transcription factor cellular promoter 2-like 1 (Tfcp2l1) as a regulator of this regenerative process. Tfcp2l1 loss in adult AT2 cells inhibits self-renewal and enhances AT2-AT1 differentiation during tissue regeneration. Conversely, Tfcp2l1 blunts the proliferative response to inflammatory signaling during the early acute injury phase. Tfcp2l1 temporally regulates AT2 self-renewal and differentiation in alveolar regions undergoing active regeneration. Single-cell transcriptomics and lineage tracing reveal that Tfcp2l1 regulates cell fate dynamics across the AT2-AT1 differentiation and restricts the inflammatory program in murine AT2 cells. Organoid modeling shows that Tfcp2l1 regulation of interleukin-1 (IL-1) receptor expression controlled these cell fate dynamics. These findings highlight the critical role Tfcp2l1 plays in balancing epithelial cell self-renewal and differentiation during alveolar regeneration.

Biophysical forces mediated by respiration maintain lung alveolar epithelial cell fate.

Lungs undergo mechanical strain during breathing, but how these biophysical forces affect cell fate and tissue homeostasis are unclear. We show that biophysical forces through normal respiratory motion actively maintain alveolar type 1 (AT1) cell identity and restrict these cells from reprogramming into AT2 cells in the adult lung. AT1 cell fate is maintained at homeostasis by Cdc42- and Ptk2-mediated actin remodeling and cytoskeletal strain, and inactivation of these pathways causes a rapid reprogramming into the AT2 cell fate. This plasticity induces chromatin reorganization and changes in nuclear lamina-chromatin interactions, which can discriminate AT1 and AT2 cell identity. Unloading the biophysical forces of breathing movements leads to AT1-AT2 cell reprogramming, revealing that normal respiration is essential to maintain alveolar epithelial cell fate. These data demonstrate the integral function of mechanotransduction in maintaining lung cell fate and identifies the AT1 cell as an important mechanosensor in the alveolar niche.