Lung Mesenchyme Research Articles

Impaired Myofibroblast Proliferation is a Central Feature of Pathologic Post-Natal Alveolar Simplification.

Premature infants with bronchopulmonary dysplasia (BPD) have impaired alveolar gas exchange due to alveolar simplification and dysmorphic pulmonary vasculature. Advances in clinical care have improved survival for infants with BPD, but the overall incidence of BPD remains unchanged because we lack specific therapies to prevent this disease. Recent work has suggested a role for increased transforming growth factor-beta (TGFβ) signaling and myofibroblast populations in BPD pathogenesis, but the functional significance of each remains unclear. Here, we utilize multiple murine models of alveolar simplification and comparative single-cell RNA sequencing to identify shared mechanisms that could contribute to BPD pathogenesis. Single-cell RNA sequencing reveals a profound loss of myofibroblasts in two models of BPD and identifies gene expression signatures of increased TGFβ signaling, cell cycle arrest, and impaired proliferation in myofibroblasts. Using pharmacologic and genetic approaches, we find no evidence that increased TGFβ signaling in the lung mesenchyme contributes to alveolar simplification. In contrast, this is likely a failed compensatory response, since none of our approaches to inhibit TGFb signaling protect mice from alveolar simplification due to hyperoxia while several make simplification worse. In contrast, we find that impaired myofibroblast proliferation is a central feature in several murine models of BPD, and we show that inhibiting myofibroblast proliferation is sufficient to cause pathologic alveolar simplification. Our results underscore the importance of impaired myofibroblast proliferation as a central feature of alveolar simplification and suggest that efforts to reverse this process could have therapeutic value in BPD.

The perspective for next-generation lung replacement therapies: functional whole lung generation by blastocyst complementation.

Blastocyst complementation represents a promising frontier in next-generation lung replacement therapies. This review aims to elucidate the future prospects of lung blastocyst complementation within clinical settings, summarizing the latest studies on generating functional lungs through this technique. It also explores and discusses host animal selection relevant to interspecific chimera formation, a challenge integral to creating functional human lungs via blastocyst complementation. Various gene mutations have been utilized to create vacant lung niches, enhancing the efficacy of donor cell contribution to the complemented lungs in rodent models. By controlling the lineage to induce gene mutations, chimerism in both the lung epithelium and mesenchyme has been improved. Interspecific blastocyst complementation underscores the complexity of developmental programs across species, with several genes identified that enhance chimera formation between humans and other mammals. While functional lungs have been generated via intraspecies blastocyst complementation, the generation of functional interspecific lungs remains unrealized. Addressing the challenges of controlling the host lung niche and selecting host animals relevant to interspecific barriers between donor human and host cells is critical to enabling the generation of functional humanized or entire human lungs in large animals.

Defective mesenchymal Bmpr1a-mediated BMP signaling causes congenital pulmonary cysts.

Abnormal lung development can cause congenital pulmonary cysts, the mechanisms of which remain largely unknown. Although the cystic lesions are believed to result directly from disrupted airway epithelial cell growth, the extent to which developmental defects in lung mesenchymal cells contribute to abnormal airway epithelial cell growth and subsequent cystic lesions has not been thoroughly examined. In the present study using genetic mouse models, we dissected the roles of bone morphogenetic protein (BMP) receptor 1a (Bmpr1a)-mediated BMP signaling in lung mesenchyme during prenatal lung development and discovered that abrogation of mesenchymal Bmpr1a disrupted normal lung branching morphogenesis, leading to the formation of prenatal pulmonary cystic lesions. Severe deficiency of airway smooth muscle cells and subepithelial elastin fibers were found in the cystic airways of the mesenchymal Bmpr1a knockout lungs. In addition, ectopic mesenchymal expression of BMP ligands and airway epithelial perturbation of the Sox2-Sox9 proximal-distal axis were detected in the mesenchymal Bmpr1a knockout lungs. However, deletion of Smad1/5, two major BMP signaling downstream effectors, from the lung mesenchyme did not phenocopy the cystic abnormalities observed in the mesenchymal Bmpr1a knockout lungs, suggesting that a Smad-independent mechanism contributes to prenatal pulmonary cystic lesions. These findings reveal for the first time the role of mesenchymal BMP signaling in lung development and a potential pathogenic mechanism underlying congenital pulmonary cysts.

Defective mesenchymal Bmpr1a-mediated BMP signaling causes congenital pulmonary cysts

Abnormal lung development can cause congenital pulmonary cysts, the mechanisms of which remain largely unknown. Although the cystic lesions are believed to result directly from disrupted airway epithelial cell growth, the extent to which developmental defects in lung mesenchymal cells contribute to abnormal airway epithelial cell growth and subsequent cystic lesions has not been thoroughly examined. In the present study using genetic mouse models, we dissected the roles of bone morphogenetic protein (BMP) receptor 1a (Bmpr1a)-mediated BMP signaling in lung mesenchyme during prenatal lung development and discovered that abrogation of mesenchymal Bmpr1a disrupted normal lung branching morphogenesis, leading to the formation of prenatal pulmonary cystic lesions. Severe deficiency of airway smooth muscle cells and subepithelial elastin fibers were found in the cystic airways of the mesenchymal Bmpr1a knockout lungs. In addition, ectopic mesenchymal expression of BMP ligands and airway epithelial perturbation of the Sox2-Sox9 proximal-distal axis were detected in the mesenchymal Bmpr1a knockout lungs. However, deletion of Smad1/5, two major BMP signaling downstream effectors, from the lung mesenchyme did not phenocopy the cystic abnormalities observed in the mesenchymal Bmpr1a knockout lungs, suggesting that a Smad-independent mechanism contributes to prenatal pulmonary cystic lesions. These findings reveal for the first time the role of mesenchymal BMP signaling in lung development and a potential pathogenic mechanism underlying congenital pulmonary cysts.

Unraveling the Impact of Dab1 Gene Silencing on the Expression of Autophagy Markers in Lung Development.

The purpose of this study was to evaluate the effects of Dab1 gene silencing on the immunoexpression of light chain 3 beta (Lc3b), glucose regulating protein 78 (Grp78), heat shock cognate 71 (Hsc70), mammalian target of rapamycin (mTOR) and lysosomal-associated membrane protein 2A (Lamp2a) in the lung tissue of developing yotari (Dab1-/-) and wild-type (wt) mice. The lung epithelium and mesenchyme of the embryos at gestational days E13.5 and E15.5 were examined using immunofluorescence and semi-quantitative methods. In the pulmonary mesenchyme and epithelium, Grp78 and Lc3b of moderate fluorescence reactivity was demonstrated in wt mice for both evaluated time points, while yotari mice exhibited only epithelial reactivity for the same markers. Mild punctate expression of Hsc70 was observed for both genotypes. A significant difference was present when analyzing mTOR expression, where wt mice showed strong perinuclear staining in the epithelium. According to our data, Dab1 gene silencing may result in autophagy abnormalities, which could then cause respiratory system pathologies via defective lung cell degradation by lysosome-dependent cell elimination.

Rescuing lung development through embryonic inhibition of histone acetylation.

A major barrier to the impact of genomic diagnosis in patients with congenital malformations is the lack of understanding regarding how sequence variants contribute to disease pathogenesis and whether this information could be used to generate patient-specific therapies. Congenital diaphragmatic hernia (CDH) is among the most common and severe of all structural malformations; however, its underlying mechanisms are unclear. We identified loss-of-function sequence variants in the epigenomic regulator gene SIN3A in two patients with complex CDH. Tissue-specific deletion of Sin3a in mice resulted in defects in diaphragm development, lung hypoplasia, and pulmonary hypertension, the cardinal features of CDH and major causes of CDH-associated mortality. Loss of SIN3A in the lung mesenchyme resulted in reduced cellular differentiation, impaired cell proliferation, and increased DNA damage. Treatment of embryonic Sin3a mutant mice with anacardic acid, an inhibitor of histone acetyltransferase, reduced DNA damage, increased cell proliferation and differentiation, improved lung and pulmonary vascular development, and reduced pulmonary hypertension. These findings demonstrate that restoring the balance of histone acetylation can improve lung development in the Sin3a mouse model of CDH.

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.

Abstract 18338: TBX4 Loss-of-Function Within the Embryonic Lung Mesenchyme Disrupts Postnatal Lung Development and Increases Susceptibility to Pulmonary Hypertension

Introduction: The T-box 4 (TBX4) transcription factor is recognized as a risk gene in pulmonary arterial hypertension (PAH). TBX4 is expressed within the lung mesenchyme in the developing lung and is required for lung branching morphogenesis. Genetic studies report that PAH cases with TBX4 mutations display an earlier onset of the disease. In addition, studies have reported carriers of TBX4 mutations with diffused alveolar development and pulmonary hypertension (PH). Currently, there is a lack of evidence on the effect of TBX4 loss-of-function (LoF) on later stages of lung development and predisposition to lung disease. Hypothesis: Lung mesenchyme Tbx4 deletion within the developing lung disrupts essential gene regulatory networks leading to abnormal postnatal lung development and susceptibility to pulmonary hypertension. Methods: We have generated Tbx4 conditional knockout ( Tbx4-CKO ) mice in which Cre recombinase removes the exon 5 of Tbx4 within the embryonic lung mesenchyme, creating a null allele. To examine alveolarization, we harvested postnatal day 14 (P14) and 36 (P36) lungs and calculated mean linear intercept (MLI). RNA-seq was performed at both stages to identify disrupted gene networks in Tbx4-CKO lungs. RVSP was measured by right heart catheterization in six-month-old mice to evaluate for PH. Results: Tbx4-CKO lungs show increasing alveolar simplification at P14 and P36, as confirmed by increases in MLI of 24.8% and 34.4%, respectively. Tbx4-CKO mice show a statistically significant increase of 10.4% in RVSP, and preliminary analysis of vessel wall thickness suggests mild vascular remodeling in their lungs. RNA-seq analysis shows enrichment of pathways (canonical WNT, VEGF, and BMP signaling) and genes ( Lgr5 , Tnc , Wnt3a , Areg , Gdf2 , and Bmper ) relevant to lung alveologenesis, angiogenesis, and PH. Conclusions: TBX4 LoF affects lung alveolar development, similar to observations made in human cases with TBX4 mutations. The clinical presentation of such cases is broad, but our mice show features of bronchopulmonary dysplasia, including alveolar simplification and PH. Further analysis of our RNA-seq data and planned single-cell analyses should provide insight into the role of TBX4 in alveolarization and PH risk.

Conditional blastocyst complementation of a defective Foxa2 lineage efficiently promotes the generation of the whole lung.

Millions suffer from incurable lung diseases, and the donor lung shortage hampers organ transplants. Generating the whole organ in conjunction with the thymus is a significant milestone for organ transplantation because the thymus is the central organ to educate immune cells. Using lineage-tracing mice and human pluripotent stem cell (PSC)-derived lung-directed differentiation, we revealed that gastrulating Foxa2 lineage contributed to both lung mesenchyme and epithelium formation. Interestingly, Foxa2 lineage-derived cells in the lung mesenchyme progressively increased and occupied more than half of the mesenchyme niche, including endothelial cells, during lung development. Foxa2 promoter-driven, conditional Fgfr2 gene depletion caused the lung and thymus agenesis phenotype in mice. Wild-type donor mouse PSCs injected into their blastocysts rescued this phenotype by complementing the Fgfr2-defective niche in the lung epithelium and mesenchyme and thymic epithelium. Donor cell is shown to replace the entire lung epithelial and robust mesenchymal niche during lung development, efficiently complementing the nearly entire lung niche. Importantly, those mice survived until adulthood with normal lung function. These results suggest that our Foxa2 lineage-based model is unique for the progressive mobilization of donor cells into both epithelial and mesenchymal lung niches and thymus generation, which can provide critical insights into studying lung transplantation post-transplantation shortly.

The lung mesenchyme in development, regeneration, and fibrosis.

Mesenchymal cells are uniquely located at the interface between the epithelial lining and the stroma, allowing them to act as a signaling hub among diverse cellular compartments of the lung. During embryonic and postnatal lung development, mesenchyme-derived signals instruct epithelial budding, branching morphogenesis, and subsequent structural and functional maturation. Later during adult life, the mesenchyme plays divergent roles wherein its balanced activation promotes epithelial repair after injury while its aberrant activation can lead to pathological remodeling and fibrosis that are associated with multiple chronic pulmonary diseases, including bronchopulmonary dysplasia, idiopathic pulmonary fibrosis, and chronic obstructive pulmonary disease. In this Review, we discuss the involvement of the lung mesenchyme in various morphogenic, neomorphogenic, and dysmorphogenic aspects of lung biology and health, with special emphasis on lung fibroblast subsets and smooth muscle cells, intercellular communication, and intrinsic mesenchymal mechanisms that drive such physiological and pathophysiological events throughout development, homeostasis, injury repair, regeneration, and aging.

Directed differentiation of mouse pluripotent stem cells into functional lung-specific mesenchyme

While the generation of many lineages from pluripotent stem cells has resulted in basic discoveries and clinical trials, the derivation of tissue-specific mesenchyme via directed differentiation has markedly lagged. The derivation of lung-specific mesenchyme is particularly important since this tissue plays crucial roles in lung development and disease. Here we generate a mouse induced pluripotent stem cell (iPSC) line carrying a lung-specific mesenchymal reporter/lineage tracer. We identify the pathways (RA and Shh) necessary to specify lung mesenchyme and find that mouse iPSC-derived lung mesenchyme (iLM) expresses key molecular and functional features of primary developing lung mesenchyme. iLM recombined with engineered lung epithelial progenitors self-organizes into 3D organoids with juxtaposed layers of epithelium and mesenchyme. Co-culture increases yield of lung epithelial progenitors and impacts epithelial and mesenchymal differentiation programs, suggesting functional crosstalk. Our iPSC-derived population thus provides an inexhaustible source of cells for studying lung development, modeling diseases, and developing therapeutics.

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.

IPSC-derived mesenchymal cells that support alveolar organoid development

SummaryMesenchymal cells are necessary for organ development. In the lung, distal tip fibroblasts contribute to alveolar and airway epithelial cell differentiation and homeostasis. Here, we report a method for generating human induced pluripotent stem cell (iPSC)-derived mesenchymal cells (iMESs) that can induce human iPSC-derived alveolar and airway epithelial lineages in organoids via epithelial-mesenchymal interaction, without the use of allogenic fetal lung fibroblasts. Through a transcriptome comparison of dermal and lung fibroblasts with their corresponding reprogrammed iPSC-derived iMESs, we found that iMESs had features of lung mesenchyme with the potential to induce alveolar type 2 (AT2) cells. Particularly, RSPO2 and RSPO3 expressed in iMESs directly contributed to AT2 cell induction during organoid formation. We demonstrated that the total iPSC-derived alveolar organoids were useful for characterizing responses to the influenza A (H1N1) virus and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, demonstrating their utility for disease modeling.

Transcriptional Profiling of Insulin-like Growth Factor Signaling Components in Embryonic Lung Development and Idiopathic Pulmonary Fibrosis.

Insulin-like growth factor (IGF) signaling controls the development and growth of many organs, including the lung. Loss of function of Igf1 or its receptor Igf1r impairs lung development and leads to neonatal respiratory distress in mice. Although many components of the IGF signaling pathway have shown to be dysregulated in idiopathic pulmonary fibrosis (IPF), the expression pattern of such components in different cellular compartments of the developing and/or fibrotic lung has been elusive. In this study, we provide a comprehensive transcriptional profile for such signaling components during embryonic lung development in mice, bleomycin-induced pulmonary fibrosis in mice and in human IPF lung explants. During late gestation, we found that Igf1 is upregulated in parallel to Igf1r downregulation in the lung mesenchyme. Lung tissues derived from bleomycin-treated mice and explanted IPF lungs revealed upregulation of IGF1 in parallel to downregulation of IGF1R, in addition to upregulation of several IGF binding proteins (IGFBPs) in lung fibrosis. Finally, treatment of IPF lung fibroblasts with recombinant IGF1 led to myogenic differentiation. Our data serve as a resource for the transcriptional profile of IGF signaling components and warrant further research on the involvement of this pathway in both lung development and pulmonary disease.

WNT/RYK signaling functions as an antiinflammatory modulator in the lung mesenchyme

A number of inflammatory lung diseases, including chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis, and pneumonia, are modulated by WNT/β-catenin signaling. However, the underlying molecular mechanisms remain unclear. Here, starting with a forward genetic screen in mouse, we identify the WNT coreceptor Related to receptor tyrosine kinase (RYK) acting in mesenchymal tissues as a cell survival and antiinflammatory modulator. Ryk mutant mice exhibit lung hypoplasia and inflammation as well as alveolar simplification due to defective secondary septation, and deletion of Ryk specifically in mesenchymal cells also leads to these phenotypes. By analyzing the transcriptome of wild-type and mutant lungs, we observed the up-regulation of proapoptotic and inflammatory genes whose expression can be repressed by WNT/RYK signaling invitro. Moreover, mesenchymal Ryk deletion at postnatal and adult stages can also lead to lung inflammation, thus indicating a continued role for WNT/RYK signaling in homeostasis. Our results indicate that RYK signaling through β-catenin and Nuclear Factor kappa B (NF-κB) is part of a safeguard mechanism against mesenchymal cell death, excessive inflammatory cytokine production, and inflammatory cell recruitment and accumulation. Notably, RYK expression is down-regulated in the stromal cells of pneumonitis patient lungs. Altogether, our data reveal that RYK signaling plays critical roles as an antiinflammatory modulator during lung development and homeostasis and provide an animal model to further investigate the etiology of, and therapeutic approaches to, inflammatory lung diseases.

Activation of PI3K/p110α in the Lung Mesenchyme Affects Branching Morphogenesis and Club Cell Differentiation.

Epithelial–mesenchymal interaction is required for normal growth, morphogenetic patterning, and cellular differentiation in developing lungs. Various signaling pathways have been defined in establishing the patterning of this branched organ. The phosphoinositide-3-kinase (PI3K) signaling plays an important role in disease pathogenesis but remains largely uncharacterized in embryonic development. In this study, we activated a specific catalytic subunit of PI3K catalytic enzymes, Class IA p110α (p110α), in the embryonic lung mesenchyme using the Dermo1-Cre mouse. Activation of p110α promoted branching morphogenesis and blocked club cell differentiation in both proximal and distal airways. Mechanistically, the LIM homeodomain gene Islet-1 (Isl1), fibroblast growth factor 10 (Fgf10), and SRY (sex-determining region Y)-box9 (Sox9) were found to be downstream targets of p110α. The significantly increased expressions of Isl1, Fgf10, and Sox9 resulted in the stimulation of branching in mutant lungs. Activation of p110α-mediated signaling also increased the expression of phosphatase and tensin homolog deleted on chromosome 10 (Pten) and hairy/enhancer of split 1 (Hes1), which in turn blocked club cell differentiation. Thus, the signaling pathway by which PI3K/p110α-regulated epithelial–mesenchymal interactions may entail Isl1–Fgf10–Sox9 and Pten–Hes1 networks, which consequently regulate branching morphogenesis and club cell differentiation, respectively.

Metastatic Sarcomatous Tumor to Mandible Identifies Pleuropulmonary Blastoma Tumor Predisposition Syndrome

<h3>Introduction</h3> Pleuropulmonary blastoma (PPB) affects infants and young children and may occur as a cystic (Type I), partially cystic-partially solid (Type 2), or solid (Type 3) tumor. PPB is an embryonal tumor derived from lung and pleural mesenchyme. This malignant tumor is composed of sarcomatous elements, most commonly a mixture of spindle cell sarcoma with focal rhabdomyosarcomatous elements. However, it may have a wide variety of sarcomatous components. The tumor is characterized by DICER1 mutation, which may be somatic (tumoral) or constitutional. Constitutional DICER1 mutation indicates a predisposition for the development of other tumors and may have implications for the child and other family members. <h3>Clinical Presentation and Pathology Findings</h3> This 18 month-old male presented with a rapidly enlarging, expansile left mandibular mass. A biopsy was performed which showed a predominantly spindle cell sarcoma (vimentin-positive) with focal areas of readily identified rhabdomyoblasts (positive for desmin; negative for myogenin and MyoD1. Molecular genetic testing of the tumor identified a DICER 1 mutation and was negative for rearrangements associated with rhabdomyosarcoma (FOXO1, FUS-TFCP2, EWSR1-TFCP2, NCOA2, VGLL2). Radiologic workup identified a predominantly solid pulmonary mass. Lobectomy was performed and diagnosis of PPB was confirmed. Sarcoma-based oncologic management was carried out. Subsequent genetic testing identified DICER1 constitutional mutation. <h3>Conclusion</h3> PPB presenting as a metastatic disease is not unusual, with the involvement of various sites and organs (most common sites: brain, bone). Although exceedingly rare, PPB should be considered in an infant or young child when encountering a possible metastatic sarcomatous tumor and prompts radiologic evaluation for a possible primary lung tumor. Molecular genetics plays an essential role in identifying risk for PPB Tumor Predisposition Syndrome. Oncologic management is sarcomabased and children with isolated lung tumors have variable outcomes (Type I - 85%, Type 2 - 60%, Type 3 - 40%).

Three-axis classification of mouse lung mesenchymal cells reveals two populations of myofibroblasts.

The mesenchyme consists of heterogeneous cell populations that support neighboring structures and are integral to intercellular signaling, but are poorly defined morphologically and molecularly. Leveraging single-cell RNA-sequencing, 3D imaging and lineage tracing, we classify the mouse lung mesenchyme into three proximal-distal axes that are associated with the endothelium, epithelium and interstitium, respectively. From proximal to distal: the vascular axis includes vascular smooth muscle cells and pericytes that transition as arterioles and venules ramify into capillaries; the epithelial axis includes airway smooth muscle cells and two populations of myofibroblasts - ductal myofibroblasts, surrounding alveolar ducts and marked by CDH4, HHIP and LGR6, which persist post-alveologenesis, and alveolar myofibroblasts, surrounding alveoli and marked by high expression of PDGFRA, which undergo developmental apoptosis; and the interstitial axis, residing between the epithelial and vascular trees and sharing the marker MEOX2, includes fibroblasts in the bronchovascular bundle and the alveolar interstitium, which are marked by IL33/DNER/PI16 and Wnt2, respectively. Single-cell imaging reveals a distinct morphology of mesenchymal cell populations. This classification provides a conceptual and experimental framework applicable to other organs.

Human alveolar type 2 epithelium transdifferentiates into metaplastic KRT5+ basal cells.

Loss of alveolar type 2 cells (AEC2s) and ectopic appearance of basal cells in the alveoli characterize severe lung injuries such as idiopathic pulmonary fibrosis (IPF). Here we demonstrate that human alveolar type 2 cells (hAEC2s), unlike murine AEC2s, transdifferentiate into basal cells in response to fibrotic signaling in the lung mesenchyme in vitro and in vivo. Single cell analysis of normal hAEC2s and mesenchymal cells in organoid co-cultures revealed the emergence of pathologic fibroblasts and basloid cells previously described in IPF. TGFβ1 and anti-BMP signaling in the organoids promoted transdifferentiation. Trajectory and histologic analyses of both hAEC2-derived organoids and IPF epithelium indicated hAEC2s transdifferentiate into basal cells through alveolar-basal intermediates (ABIs) that accumulate in proximity to pathologic CTHRC1high/TGFB1high fibroblasts. Our study indicates that hAEC2-loss and expansion of alveolar metaplastic basal cells in severe human lung injuries are causally connected through a hAEC2-basal cell lineage trajectory driven by aberrant mesenchyme.

The Lung Elastin Matrix Undergoes Rapid Degradation Upon Adult Loss of Hox5 Function.

Hox genes encode transcription factors that are critical for embryonic skeletal patterning and organogenesis. The Hoxa5, Hoxb5, and Hoxc5 paralogs are expressed in the lung mesenchyme and function redundantly during embryonic lung development. Conditional loss-of-function of these genes during postnatal stages leads to severe defects in alveologenesis, specifically in the generation of the elastin network, and animals display bronchopulmonary dysplasia (BPD) or BPD-like phenotype. Here we show the surprising results that mesenchyme-specific loss of Hox5 function at adult stages leads to rapid disruption of the mature elastin matrix, alveolar enlargement, and an emphysema-like phenotype. As the elastin matrix of the lung is considered highly stable, adult disruption of the matrix was not predicted. Just 2 weeks after deletion, adult Hox5 mutant animals show significant increases in alveolar space and changes in pulmonary function, including reduced elastance and increased compliance. Examination of the extracellular matrix (ECM) of adult Tbx4rtTA; TetOCre; Hox5afafbbcc lungs demonstrates a disruption of the elastin network although the underlying fibronectin, interstitial collagen and basement membrane appear unaffected. An influx of macrophages and increased matrix metalloproteinase 12 (MMP12) are observed in the distal lung 3 days after Hox5 deletion. In culture, fibroblasts from Hox5 mutant lungs exhibit reduced adhesion. These findings establish a novel role for Hox5 transcription factors as critical regulators of lung fibroblasts at adult homeostasis.