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.

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.

PGF2α signaling drives fibrotic remodeling and fibroblast population dynamics in mice.

Idiopathic pulmonary fibrosis (IPF) is a chronic parenchymal lung disease characterized by repetitive alveolar cell injury, myofibroblast proliferation, and excessive extracellular matrix deposition for which unmet need persists for effective therapeutics. The bioactive eicosanoid, prostaglandin F2α, and its cognate receptor FPr (Ptgfr) are implicated as a TGF-ß1-independent signaling hub for IPF. To assess this, we leveraged our published murine PF model (IER-SftpcI73T) expressing a disease-associated missense mutation in the surfactant protein C (Sftpc) gene. Tamoxifen-treated IER-SftpcI73T mice developed an early multiphasic alveolitis and transition to spontaneous fibrotic remodeling by 28 days. IER-SftpcI73T mice crossed to a Ptgfr-null (FPr-/-) line showed attenuated weight loss and gene dosage-dependent rescue of mortality compared with FPr+/+ cohorts. IER-SftpcI73T/FPr-/- mice also showed reductions in multiple fibrotic endpoints for which administration of nintedanib was not additive. Single-cell RNA-Seq, pseudotime analysis, and in vitro assays demonstrated Ptgfr expression predominantly within adventitial fibroblasts, which were reprogrammed to an "inflammatory/transitional" cell state in a PGF2α /FPr-dependent manner. Collectively, the findings provide evidence for a role for PGF2α signaling in IPF, mechanistically identify a susceptible fibroblast subpopulation, and establish a benchmark effect size for disruption of this pathway in mitigating fibrotic lung remodeling.

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.

Disruption of proteostasis causes IRE1 mediated reprogramming of alveolar epithelial cells.

Disruption of alveolar type 2 cell (AEC2) protein quality control has been implicated in chronic lung diseases, including pulmonary fibrosis (PF). We previously reported the in vivo modeling of a clinical surfactant protein C (SP-C) mutation that led to AEC2 endoplasmic reticulum (ER) stress and spontaneous lung fibrosis, providing proof of concept for disruption to proteostasis as a proximal driver of PF. Using two clinical SP-C mutation models, we have now discovered that AEC2s experiencing significant ER stress lose quintessential AEC2 features and develop a reprogrammed cell state that heretofore has been seen only as a response to lung injury. Using single-cell RNA sequencing in vivo and organoid-based modeling, we show that this state arises de novo from intrinsic AEC2 dysfunction. The cell-autonomous AEC2 reprogramming can be attenuated through inhibition of inositol-requiring enzyme 1 (IRE1α) signaling as the use of an IRE1α inhibitor reduced the development of the reprogrammed cell state and also diminished AEC2-driven recruitment of granulocytes, alveolitis, and lung injury. These findings identify AEC2 proteostasis, and specifically IRE1α signaling through its major product XBP-1, as a driver of a key AEC2 phenotypic change that has been identified in lung fibrosis.

Human distal airways contain a multipotent secretory cell that can regenerate alveoli.

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.

mTORC1 activation in lung mesenchyme drives sex- and age-dependent pulmonary structure and function decline.

Lymphangioleiomyomatosis (LAM) is a rare fatal cystic lung disease due to bi-allelic inactivating mutations in tuberous sclerosis complex (TSC1/TSC2) genes coding for suppressors of the mechanistic target of rapamycin complex 1 (mTORC1). The origin of LAM cells is still unknown. Here, we profile a LAM lung compared to an age- and sex-matched healthy control lung as a hypothesis-generating approach to identify cell subtypes that are specific to LAM. Our single-cell RNA sequencing (scRNA-seq) analysis reveals novel mesenchymal and transitional alveolar epithelial states unique to LAM lung. This analysis identifies a mesenchymal cell hub coordinating the LAM disease phenotype. Mesenchymal-restricted deletion of Tsc2 in the mouse lung produces a mTORC1-driven pulmonary phenotype, with a progressive disruption of alveolar structure, a decline in pulmonary function, increase of rapamycin-sensitive expression of WNT ligands, and profound female-specific changes in mesenchymal and epithelial lung cell gene expression. Genetic inactivation of WNT signaling reverses age-dependent changes of mTORC1-driven lung phenotype, but WNT activation alone in lung mesenchyme is not sufficient for the development of mouse LAM-like phenotype. The alterations in gene expression are driven by distinctive crosstalk between mesenchymal and epithelial subsets of cells observed in mesenchymal Tsc2-deficient lungs. This study identifies sex- and age-specific gene changes in the mTORC1-activated lung mesenchyme and establishes the importance of the WNT signaling pathway in the mTORC1-driven lung phenotype.

Direct Comparison of Mononucleated and Binucleated Cardiomyocytes Reveals Molecular Mechanisms Underlying Distinct Proliferative Competencies.

The mammalian heart is incapable of regenerating a sufficient number of cardiomyocytes to ameliorate the loss of contractile muscle after acute myocardial injury. Several reports have demonstrated that mononucleated cardiomyocytes are more responsive than are binucleated cardiomyocytes to pro-proliferative stimuli. We have developed a strategy to isolate and characterize highly enriched populations of mononucleated and binucleated cardiomyocytes at various times of development. Our results suggest that an E2f/Rb transcriptional network is central to the divergence of these two populations and that remnants of the differences acquired during the neonatal period remain in adult cardiomyocytes. Moreover, inducing binucleation by genetically blocking the ability of cardiomyocytes to complete cytokinesis leads to a reduction in E2f target gene expression, directly linking the E2f pathway with nucleation. These data identify key molecular differences between mononucleated and binucleated mammalian cardiomyocytes that can be used to leverage cardiomyocyte proliferation for promoting injury repair in the heart.

The NANCI-Nkx2.1 gene duplex buffers Nkx2.1 expression to maintain lung development and homeostasis.

A subset of long noncoding RNAs (lncRNAs) is spatially correlated with transcription factors (TFs) across the genome, but how these lncRNA-TF gene duplexes regulate tissue development and homeostasis is unclear. We identified a feedback loop within the NANCI (Nkx2.1-associated noncoding intergenic RNA)-Nkx2.1 gene duplex that is essential for buffering Nkx2.1 expression, lung epithelial cell identity, and tissue homeostasis. Within this locus, Nkx2.1 directly inhibits NANCI, while NANCI acts in cis to promote Nkx2.1 transcription. Although loss of NANCI alone does not adversely affect lung development, concurrent heterozygous mutations in both NANCI and Nkx2.1 leads to persistent Nkx2.1 deficiency and reprogramming of lung epithelial cells to a posterior endoderm fate. This disruption in the NANCI-Nkx2.1 gene duplex results in a defective perinatal innate immune response, tissue damage, and progressive degeneration of the adult lung. These data point to a mechanism in which lncRNAs act as rheostats within lncRNA-TF gene duplex loci that buffer TF expression, thereby maintaining tissue-specific cellular identity during development and postnatal homeostasis.

The tumor suppressor FLCN mediates an alternate mTOR pathway to regulate browning of adipose tissue.

Noncanonical mechanistic target of rapamycin (mTOR) pathways remain poorly understood. Mutations in the tumor suppressor folliculin (FLCN) cause Birt-Hogg-Dubé syndrome, a hamartomatous disease marked by mitochondria-rich kidney tumors. FLCN functionally interacts with mTOR and is expressed in most tissues, but its role in fat has not been explored. We show here that FLCN regulates adipose tissue browning via mTOR and the transcription factor TFE3. Adipose-specific deletion of FLCN relieves mTOR-dependent cytoplasmic retention of TFE3, leading to direct induction of the PGC-1 transcriptional coactivators, drivers of mitochondrial biogenesis and the browning program. Cytoplasmic retention of TFE3 by mTOR is sensitive to ambient amino acids, is independent of growth factor and tuberous sclerosis complex (TSC) signaling, is driven by RagC/D, and is separable from canonical mTOR signaling to S6K. Codeletion of TFE3 in adipose-specific FLCN knockout animals rescues adipose tissue browning, as does codeletion of PGC-1ß. Conversely, inducible expression of PGC-1ß in white adipose tissue is sufficient to induce beige fat gene expression in vivo. These data thus unveil a novel FLCN-mTOR-TFE3-PGC-1ß pathway-separate from the canonical TSC-mTOR-S6K pathway-that regulates browning of adipose tissue.