Epithelial Yap/Taz are required for functional alveolar regeneration following acute lung injury.

A hallmark of idiopathic pulmonary fibrosis (IPF) and other interstitial lung diseases is dysregulated repair of the alveolar epithelium. The Hippo pathway effector transcription factors YAP and TAZ are implicated as essential for type 1 and type 2 alveolar epithelial cell (AT1 and AT2) differentiation in the developing lung, yet aberrant activation of YAP/TAZ is a prominent feature of the dysregulated alveolar epithelium in IPF. In these studies, we sought to define the functional role of YAP/TAZ activity during alveolar regeneration. We demonstrated that Yap and Taz were normally activated in AT2 cells shortly after injury, and deletion of Yap/Taz in AT2 cells led to pathologic alveolar remodeling, failure of AT2-to-AT1 cell differentiation, increased collagen deposition, exaggerated neutrophilic inflammation, and increased mortality following injury induced by a single dose of bleomycin. Loss of Yap/Taz activity prior to an LPS injury prevented AT1 cell regeneration, led to intraalveolar collagen deposition, and resulted in persistent innate inflammation. These findings establish that AT2 cell Yap/Taz activity is essential for functional alveolar epithelial repair and prevention of fibrotic remodeling.

Multiplatform Single-Cell Analysis Identifies Immune Cell Types Enhanced in Pulmonary Fibrosis.

Immune cells have been implicated in idiopathic pulmonary fibrosis (IPF), but the phenotypes and effector mechanisms of these cells remain incompletely characterized. We performed mass cytometry to quantify immune cell subsets in lungs of 12 patients with IPF and 15 organ donors without chronic lung disease and used existing single-cell RNA-sequencing data to investigate transcriptional profiles of immune cells overrepresented in IPF. Among myeloid cells, we found increased numbers of alveolar macrophages (AMØs) and dendritic cells (DCs) in IPF, as well as a subset of monocyte-derived DCs. In contrast, monocyte-like cells and interstitial macrophages were reduced in IPF. Transcriptomic profiling identified an enrichment for IFN-γ response pathways in AMØs and DCs from IPF, as well as antigen processing in DCs and phagocytosis in AMØs. Among T cells, we identified three subsets of memory T cells that were increased in IPF, including CD4+ and CD8+ resident memory T cells (TRM) and CD8+ effector memory cells. The response to the IFN-γ pathway was enriched in CD4 TRM and CD8 TRM cells in IPF, together with T cell activation and immune response-regulating signaling pathways. Increased AMØs, DCs, and memory T cells were present in IPF lungs compared with control subjects. In IPF, these cells possess an activation profile indicating increased IFN-γ signaling and upregulation of adaptive immunity in the lungs. Together, these studies highlight critical features of the immunopathogenesis of IPF.

Localized hypoxia links ER stress to lung fibrosis through induction of C/EBP homologous protein.

ER stress in type II alveolar epithelial cells (AECs) is common in idiopathic pulmonary fibrosis (IPF), but the contribution of ER stress to lung fibrosis is poorly understood. We found that mice deficient in C/EBP homologous protein (CHOP), an ER stress-regulated transcription factor, were protected from lung fibrosis and AEC apoptosis in 3 separate models where substantial ER stress was identified. In mice treated with repetitive intratracheal bleomycin, we identified localized hypoxia in type II AECs as a potential mechanism explaining ER stress. To test the role of hypoxia in lung fibrosis, we treated mice with bleomycin, followed by exposure to 14% O2, which exacerbated ER stress and lung fibrosis. Under these experimental conditions, CHOP-/- mice, but not mice with epithelial HIF (HIF1/HIF2) deletion, were protected from AEC apoptosis and fibrosis. In vitro studies revealed that CHOP regulates hypoxia-induced apoptosis in AECs via the inositol-requiring enzyme 1α (IRE1α) and the PKR-like ER kinase (PERK) pathways. In human IPF lungs, CHOP and hypoxia markers were both upregulated in type II AECs, supporting a conclusion that localized hypoxia results in ER stress-induced CHOP expression, thereby augmenting type II AEC apoptosis and potentiating lung fibrosis.

Successful Establishment of Primary Type II Alveolar Epithelium with 3D Organotypic Coculture.

Alveolar type II (AT2) epithelial cells are uniquely specialized to produce surfactant in the lung and act as progenitor cells in the process of repair after lung injury. AT2 cell injury has been implicated in several lung diseases, including idiopathic pulmonary fibrosis and bronchopulmonary dysplasia. The inability to maintain primary AT2 cells in culture has been a significant barrier in the investigation of pulmonary biology. We have addressed this knowledge gap by developing a three-dimensional (3D) organotypic coculture using primary human fetal AT2 cells and pulmonary fibroblasts. Grown on top of matrix-embedded fibroblasts, the primary human AT2 cells establish a monolayer and have direct contact with the underlying pulmonary fibroblasts. Unlike conventional two-dimensional (2D) culture, the structural and functional phenotype of the AT2 cells in our 3D organotypic culture was preserved over 7 days of culture, as evidenced by the presence of lamellar bodies and by production of surfactant proteins B and C. Importantly, the AT2 cells in 3D cocultures maintained the ability to replicate, with approximately 60% of AT2 cells staining positive for the proliferation marker Ki67, whereas no such proliferation is evident in 2D cultures of the same primary AT2 cells. This organotypic culture system enables interrogation of AT2 epithelial biology by providing a reductionist in vitro model in which to investigate the response of AT2 epithelial cells and AT2 cell-fibroblast interactions during lung injury and repair.

Longitudinal assessment of neurocognitive function in rats after cardiopulmonary bypass: evidence for long-term deficits.

OBJECTIVE(S): Neurologic and neurocognitive dysfunction after cardiopulmonary bypass (CPB) have been shown in both clinical and experimental settings. Although short-term outcome has been evaluated in rats, the assessment of neurocognitive dysfunction with long-term follow-up has not been reported in experimental CPB models. The objective of this study was to evaluate the effects of CPB on long-term neurocognitive function in the rat. DESIGN: Prospective, interventional study. SETTING: A university research laboratory. PARTICIPANTS: Male Sprague-Dawley rats. INTERVENTIONS: Sprague-Dawley rats were randomized to either CPB (n = 19) or sham-operated groups (n = 17). On days 3, 7, and 14 and at 6 weeks after surgery, the rats were submitted to standardized neurologic testing (Neuroscore). In addition, the animals underwent cognitive testing in the Morris water maze (MWM), including basic, probe, and reversal trial protocols during the first 19 postoperative days (short-term cognitive outcome) and then repeated 6 weeks after surgery (long-term cognitive outcome). MEASUREMENTS AND MAIN RESULTS: The CPB group had worse Neuroscores (day 3, 5[2]; day 7, 7[2]; day 14, 5[1]; 6 weeks, 5[1]) compared with the sham group (day 3, 7[2]; day 7, 7[1]; day 14, 7[1]; 6 weeks, 7[1]) at all time points tested (p < 0.05). In the MWM, the CPB group showed both short-term and persistent long-term neurocognitive dysfunction. CONCLUSIONS: Compared with sham-operated controls, rats undergoing CPB showed worse neurologic and neurocognitive outcome early after surgery. Importantly, long-term deficits also persisted at 6 weeks after surgery.

Complex integration of matrix, oxidative stress, and apoptosis in genetic emphysema.

Alveolar enlargement, which is characteristic of bronchopulmonary dysplasia, congenital matrix disorders, and cigarette smoke-induced emphysema, is thought to result from enhanced inflammation and ensuing excessive matrix proteolysis. Although there is recent evidence that cell death and oxidative stress punctuate these diseases, the mechanistic link between abnormal lung extracellular matrix and alveolar enlargement is lacking. We hypothesized that the tight-skin (TSK) mouse, which harbors a spontaneous internal duplication in the microfibrillar glycoprotein fibrillin-1, might show whether matrix alterations are sufficient to promote oxidative stress and cell death, injury cascades central to the development of clinical emphysema. We observed no evidence of increased metalloprotease activation by histochemical and zymographic methods. We did find initial oxidative stress followed by increased apoptosis in the postnatal TSK lung. Both blunted antioxidant production and reduced extracellular superoxide dismutase activity were evident in the neonatal lung. High-dose antioxidant treatment with N-acetylcysteine improved airspace caliber and attenuated oxidative stress and apoptosis in neonatal and adult TSK mice. These data establish that an abnormal extracellular matrix without overt elastolysis is sufficient to confer susceptibility to postnatal normoxia, reminiscent of bronchopulmonary dysplasia. The resultant oxidative stress and apoptosis culminate in profound airspace enlargement. The TSK lung exemplifies the critical interplay between extracellular matrix, oxidative stress, and cell-death cascades that may contribute to genetic and acquired airspace enlargement.