Temporal effects of Sprouty on lung morphogenesis

AbstractParacrine signaling mediated by FGF-10 and the FGF-R2IIIb receptor is required for formation of the lung. To determine the temporal requirements for FGF signaling during pulmonary morphogenesis, Sprouty-4 (Spry-4), an intracellular FGF receptor antagonist, was expressed in epithelial cells of the fetal lung under control of a doxycycline-inducible system. Severe defects in lobulation and severe lung hypoplasia were observed when Spry-4 was expressed throughout fetal lung development (E6.5–E18.5) or from E6.5 until E13.5. Effects of Spry-4 on branching were substantially reversed by removal of doxycycline from the dam at E12.5, but not at E13.5. In contrast, when initiated late in development (E12.5 to birth), Spry-4 caused less severe pulmonary hypoplasia. Expression of Spry-4 from E16.5 to E18.5 reduced lung growth and resulted in perinatal death due to respiratory failure. Expression of Spry-4 during the saccular and alveolar stages, from E18.5 to postnatal day 21, caused mild emphysema. These findings demonstrate that the embryonic-pseudoglandular stage is a critical time period during which Spry-sensitive pathways are required for branching morphogenesis, lobulation, and formation of the peripheral lung parenchyma

Cumulative Effects of Neonatal Hyperoxia on Murine Alveolar Structure and Function

Background Bronchopulmonary dysplasia (BPD) results from alveolar simplification and abnormal development of alveolar and capillary structure. Survivors of BPD display persistent deficits in airflow and membrane and vascular components of alveolar gas diffusion. Despite being the defining feature of BPD, various neonatal hyperoxia models of BPD have not routinely assessed pulmonary gas diffusion. Methods To simulate the most commonly-utilized neonatal hyperoxia models, we exposed neonatal mice to room air or ≥90% hyperoxia during key stages of distal lung development: through the first 4 (saccular), 7 (early alveolar), or 14 (bulk alveolar) postnatal days, followed by a period of recovery in room air until 8 weeks of age when alveolar septation is essentially complete. We systematically assessed and correlated the effects of neonatal hyperoxia on the degree of alveolar–capillary structural and functional impairment. We hypothesized that the degree of alveolar–capillary simplification would correlate strongly with worsening diffusion impairment. Results Neonatal hyperoxia exposure, of any duration, resulted in alveolar simplification and impaired pulmonary gas diffusion. Mean Linear Intercept increased in proportion to the length of hyperoxia exposure while alveolar and total lung volume increased markedly only with prolonged exposure. Surprisingly, despite having a similar effect on alveolar surface area, only prolonged hyperoxia for 14 days resulted in reduced pulmonary microvascular volume. Estimates of alveolar and capillary structure, in general, correlated poorly with assessment of gas diffusion. Conclusion Our results help define the physiological and structural consequences of commonly-employed neonatal hyperoxia models of BPD and informtheir clinical utility

An obligatory role for club cells in preventing obliterative bronchiolitis in lung transplants

Obliterative bronchiolitis (OB) is a poorly understood airway disease characterized by the generation of fibrotic bronchiolar occlusions. In the lung transplant setting, OB is a pathological manifestation of bronchiolitis obliterans syndrome (BOS), which is a major impediment to long-term recipient survival. Club cells play a key role in bronchiolar epithelial repair, but whether they promote lung transplant tolerance through preventing OB remains unclear. We determined if OB occurs in mouse orthotopic lung transplants following conditional transgene-targeted club cell depletion. In syngeneic lung transplants club cell depletion leads to transient epithelial injury followed by rapid club cell-mediated repair. In contrast, allogeneic lung transplants develop severe OB lesions and poorly regenerate club cells despite immunosuppression treatment. Lung allograft club cell ablation also triggers the recognition of alloantigens, and pulmonary restricted self-antigens reported associated with BOS development. However, CD8+ T cell depletion restores club cell reparative responses and prevents OB. In addition, ex-vivo analysis reveals a specific role for alloantigen-primed effector CD8+ T cells in preventing club cell proliferation and maintenance. Taken together, we demonstrate a vital role for club cells in maintaining lung transplant tolerance and propose a new model to identify the underlying mechanisms of OB

FGF signaling is required for myofibroblast differentiation during alveolar regeneration

Normal alveolarization has been studied in rodents using detailed morphometric techniques and loss of function approaches for growth factors and their receptors. However, it remains unclear how these growth factors direct the formation of secondary septae. We have previously developed a transgenic mouse model in which expression of a soluble dominant-negative FGF receptor (dnFGFR) in the prenatal period results in reduced alveolar septae formation and subsequent alveolar simplification. Retinoic acid (RA), a biologically active derivative of vitamin A, can induce regeneration of alveoli in adult rodents. In this study, we demonstrate that RA induces alveolar reseptation in this transgenic mouse model and that realveolarization in adult mice is FGF dependent. Proliferation in the lung parenchyma, an essential prerequisite for lung regrowth was enhanced after 14 days of RA treatment and was not influenced by dnFGFR expression. During normal lung development, formation of secondary septae is associated with the transient presence of α-smooth muscle actin (αSMA)-positive interstitial myofibroblasts. One week after completion of RA treatment, αSMA expression was detected in interstitial fibroblasts, supporting the concept that RA-initiated realveolarization recapitulates aspects of septation that occur during normal lung development. Expression of dnFGFR blocked realveolarization with increased PDGF receptor-α (PDGFRα)-positive cells and decreased αSMA-positive cells. Taken together, our data demonstrate that FGF signaling is required for the induction of αSMA in the PDGFRα-positive myofibroblast progenitor and the progression of alveolar regeneration

Conditional depletion of airway progenitor cells induces peribronchiolar fibrosis

RATIONALE: The respiratory epithelium has a remarkable capacity to respond to acute injury. In contrast, repeated epithelial injury is often associated with abnormal repair, inflammation and fibrosis. There is increasing evidence that non-ciliated epithelial cells play important roles in the repair of the bronchiolar epithelium following acute injury. Cellular processes underlying the repair and remodeling of the lung following chronic epithelial injury are poorly understood. OBJECTIVES: To identify cell processes mediating epithelial regeneration and remodeling after acute and chronic Clara cell depletion. METHODS: A transgenic mouse model was generated to conditionally express diphtheria toxin A to ablate Clara cells in the adult lung. Epithelial regeneration and peribronchiolar fibrosis were assessed after acute and chronic Clara cell depletion. MEASUREMENTS AND MAIN RESULTS: Acute Clara cell ablation caused squamous metaplasia of ciliated cells and induced proliferation of residual progenitor cells. Ciliated cells in the bronchioles and pro-SPC expressing cells in the bronchiolar alveolar duct junctions did not proliferate. Epithelial cell proliferation occurred at multiple sites along the airways and was not selectively associated with regions around neuroepithelial bodies. Chronic Clara cell depletion resulted in ineffective repair and caused peribronchiolar fibrosis. CONCLUSIONS: Co-localization of proliferation and cell type specific markers demonstrate that Clara cells are critical airway progenitor cells. Continuous depletion of Clara cells resulted in persistent squamous metaplasia, lack of normal re-epithelialization and peribronchiolar fibrosis. Induction of proliferation in subepithelial fibroblasts, supports the concept that chronic epithelial depletion caused peribronchiolar fibrosis

Signaling pathways in the epithelial origins of pulmonary fibrosis

Pulmonary fibrosis complicates a number of disease processes and leads to substantial morbidity and mortality. Idiopathic pulmonary fibrosis (IPF) is perhaps the most pernicious and enigmatic form of the greater problem of lung fibrogenesis with a median survival of three years from diagnosis in affected patients. In this review, we will focus on the pathology of IPF as a model of pulmonary fibrotic processes, review possible cellular mechanisms, review current treatment approaches and review two transgenic mouse models of lung fibrosis to provide insight into processes that cause lung fibrosis. We will also summarize the potential utility of signaling pathway inhibitors as a future treatment in pulmonary fibrosis. Finally, we will present data demonstrating a minimal contribution of epithelial-mesenchymal transition in the development of fibrotic lesions in the transforming growth factor-alpha transgenic model of lung fibrosis

Conditional Recombination Reveals Distinct Subsets of Epithelial Cells in Trachea, Bronchi, and Alveoli

To identify relationships amongst tracheal and alveolar epithelial cells during lung development, we used conditional systems controlled by the rat CCSP and human SFTPC gene promoters to express Cre-recombinase in the developing mouse lung, thereby permanently labeling cells by expression of alkaline phosphatase or green fluorescent protein. When controlled by the rat CCSP promoter, continuous exposure of the fetus to doxycycline caused widespread recombination in conducting airway epithelial cells, including cells of the trachea, bronchi, and bronchioles before birth, and in both conducting and peripheral airways after birth. Neuroepithelial cells, identified by CGRP staining, were never labeled. Recombination and permanent labeling were observed in both ciliated and nonciliated respiratory epithelial cells, demonstrating their derivation from common progenitor cells during lung morphogenesis. Remarkable dorsal–ventral and cephalo–caudal labeling patterns, established before birth, were identified by recombination controlled by the rat CCSP gene promoter. In the trachea, subsets of epithelial cells labeled by the CCSP promoter were organized horizontally along the dorsal–ventral axis of the trachea, where selective labeling of cells juxtaposed to tracheal and bronchial cartilage was observed. In sharp contrast, recombination controlled by the human SFTPC gene promoter identified related cells that were organized in linear patterns along the cephalo–caudal axis of the conducting airways. Conditional expression of Cre-recombinase in the respiratory epithelium provides a useful model for the study of gene expression and function in the mouse respiratory tract and in the lung

Single-cell RNA sequencing identifies diverse roles of epithelial cells in idiopathic pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is a lethal interstitial lung disease characterized by airway remodeling, inflammation, alveolar destruction, and fibrosis. We utilized single-cell RNA sequencing (scRNA-seq) to identify epithelial cell types and associated biological processes involved in the pathogenesis of IPF. Transcriptomic analysis of normal human lung epithelial cells defined gene expression patterns associated with highly differentiated alveolar type 2 (AT2) cells, indicated by enrichment of RNAs critical for surfactant homeostasis. In contrast, scRNA-seq of IPF cells identified 3 distinct subsets of epithelial cell types with characteristics of conducting airway basal and goblet cells and an additional atypical transitional cell that contributes to pathological processes in IPF. Individual IPF cells frequently coexpressed alveolar type 1 (AT1), AT2, and conducting airway selective markers, demonstrating “indeterminate” states of differentiation not seen in normal lung development. Pathway analysis predicted aberrant activation of canonical signaling via TGF-β, HIPPO/YAP, P53, WNT, and AKT/PI3K. Immunofluorescence confocal microscopy identified the disruption of alveolar structure and loss of the normal proximal-peripheral differentiation of pulmonary epithelial cells. scRNA-seq analyses identified loss of normal epithelial cell identities and unique contributions of epithelial cells to the pathogenesis of IPF. The present study provides a rich data source to further explore lung health and disease