Search

Tissue regeneration requires coordinated and dynamic remodeling of stem and progenitor cells and the surrounding niche. Although the plasticity of epithelial cells has been well explored in many tissues, the dynamic changes occurring in niche cells remain elusive. Here, we show that, during lung repair after naphthalene injury, a population of PDGFRα + cells emerges in the non-cartilaginous conducting airway niche, which is normally populated by airway smooth muscle cells (ASMCs). This cell population, which we term "repair-supportive mesenchymal cells" (RSMCs), is distinct from conventional ASMCs, which have previously been shown to contribute to epithelial repair. Gene expression analysis on sorted lineage-labeled cells shows that RSMCs express low levels of ASMC markers, but high levels of the pro-regenerative marker Fgf10. Organoid co-cultures demonstrate an enhanced ability for RSMCs in supporting club-cell growth. Our study highlights the dynamics of mesenchymal cells in the airway niche and has implications for chronic airway-injury-associated diseases., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Lung cancer is the leading cause of cancer death for both men and women and accounts for almost 18.4% of all deaths due to cancer worldwide, with the global incidence increasing by approximately 0.5% per year. Lung cancer is regarded as a devastating type of cancer owing to its high prevalence, reduction in the health-related quality of life, frequently delayed diagnosis, low response rate, high toxicity, and resistance to available therapeutic options. The highly heterogeneous nature of this cancer with a proximal-to-distal distribution throughout the respiratory tract dramatically affects its diagnostic and therapeutic management. The diverse composition and plasticity of lung epithelial cells across the respiratory tract are regarded as significant factors underlying lung cancer heterogeneity. Therefore, definitions of the cells of origin for different types of lung cancer are urgently needed to understand lung cancer biology and to achieve early diagnosis and develop cell-targeted therapies. In the present review, we will discuss the current understanding of the cellular and molecular alterations in distinct lung epithelial cells that result in each type of lung cancer., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Influenza Virus (IV) pneumonia is associated with severe damage of the lung epithelium and respiratory failure. Apart from efficient host defense, structural repair of the injured epithelium is crucial for survival of severe pneumonia. The molecular mechanisms underlying stem/progenitor cell mediated regenerative responses are not well characterized. In particular, the impact of IV infection on lung stem cells and their regenerative responses remains elusive. Our study demonstrates that a highly pathogenic IV infects various cell populations in the murine lung, but displays a strong tropism to an epithelial cell subset with high proliferative capacity, defined by the signature EpCamhighCD24lowintegrin(α6)high. This cell fraction expressed the stem cell antigen-1, highly enriched lung stem/progenitor cells previously characterized by the signature integrin(β4)+CD200+, and upregulated the p63/krt5 regeneration program after IV-induced injury. Using 3-dimensional organoid cultures derived from these epithelial stem/progenitor cells (EpiSPC), and in vivo infection models including transgenic mice, we reveal that their expansion, barrier renewal and outcome after IV-induced injury critically depended on Fgfr2b signaling. Importantly, IV infected EpiSPC exhibited severely impaired renewal capacity due to IV-induced blockade of β-catenin-dependent Fgfr2b signaling, evidenced by loss of alveolar tissue repair capacity after intrapulmonary EpiSPC transplantation in vivo. Intratracheal application of exogenous Fgf10, however, resulted in increased engagement of non-infected EpiSPC for tissue regeneration, demonstrated by improved proliferative potential, restoration of alveolar barrier function and increased survival following IV pneumonia. Together, these data suggest that tropism of IV to distal lung stem cell niches represents an important factor of pathogenicity and highlight impaired Fgfr2b signaling as underlying mechanism. Furthermore, increase of alveolar Fgf10 levels may represent a putative therapy to overcome regeneration failure after IV-induced lung injury.

Acute respiratory distress syndrome (ARDS) is clinical syndrome characterized by decreased lung fluid reabsorption, causing alveolar edema. Defective alveolar ion transport undertaken in part by the Na(+)/K(+)-ATPase underlies this compromised fluid balance, although the molecular mechanisms at play are not understood. We describe here increased expression of FXYD1, FXYD3 and FXYD5, three regulatory subunits of the Na(+)/K(+)-ATPase, in the lungs of ARDS patients. Transforming growth factor (TGF)-β, a pathogenic mediator of ARDS, drove increased FXYD1 expression in A549 human lung alveolar epithelial cells, suggesting that pathogenic TGF-β signaling altered Na(+)/K(+)-ATPase activity in affected lungs. Lentivirus-mediated delivery of FXYD1 and FXYD3 allowed for overexpression of both regulatory subunits in polarized H441 cell monolayers on an air/liquid interface. FXYD1 but not FXYD3 overexpression inhibited amphotericin B-sensitive equivalent short-circuit current in Ussing chamber studies. Thus, we speculate that FXYD1 overexpression in ARDS patient lungs may limit Na(+)/K(+)-ATPase activity, and contribute to edema persistence., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Purpose of the Study: Acute respiratory distress syndrome (ARDS) represents a major cause of mortality in intensive care patients. Activation of peroxisome proliferator-activated receptor-α (PPAR-α) by fibrates, such as WY-14643 (WY), has been described to beneficially influence inflammation and experimental lung injury. The impact of PPAR-α activation on alveolar epithelial cells (AEC) has not been studied yet., Materials and Methods: To investigate the effect of PPAR-α activator WY in wild-type (WT) and in PPAR-α knockout (PPAR-α(-/-)) animals, mice were treated in different regimes: mice received chow enriched with or without WY for 14 days prior AEC isolation (in-vivo treatment). Furthermore, isolated AEC from both groups were subsequently cultured with or without WY (in-vitro treatment). AEC were stimulated with lipopolysaccharide (LPS). Cell culture supernatant and cell lysate were used for analysis of pro-inflammatory mediators., Results: AEC challenged with LPS showed a significantly increased generation of pro-inflammatory mediators. After in-vivo WY-exposure, AEC displayed significantly reduced concentration of TNF-α, MIP-2, and TxB2 after LPS stimulation. This beneficial effect was abrogated in PPAR-α(-/-) animals. Interestingly, sole in-vitro application of WY-14643 failed to reduce levels of pro-inflammatory mediators whereas we found an additive effect of a combined in-vivo and in-vitro PPAR-α activation. PGE2 concentration remained high after LPS challenge and was unaffected by WY treatment., Conclusion: PPAR-α activation by in-vivo exposure to fibrates reduced the inflammatory response in isolated AEC. These findings may facilitate further studies investigating the translation of pharmacological PPAR-α activation into clinical therapy of ARDS.

Amiodarone (AD) is a highly efficient antiarrhythmic drug with potentially serious side effects. Severe pulmonary toxicity is reported in patients receiving AD even at low doses and may cause interstitial pneumonia as well as lung fibrosis. Apoptosis of alveolar epithelial type II cells (AECII) has been suggested to play an important role in this disease. In the current study, we aimed to establish a murine model of AD-induced lung fibrosis and analyze surfactant homeostasis, lysosomal, and endoplasmic reticulum (ER) stress in this model. AD/vehicle was instilled intratracheally into C57BL/6 mice, which were sacrificed on days 7, 14, 21, and 28. Extent of lung fibrosis development was assessed by trichrome staining and hydroxyproline measurement. Cytotoxicity was assessed by lactate dehydrogenase assay. Phospholipids (PLs) were analyzed by mass spectrometry. Surfactant proteins (SP) and markers for apoptosis, lysosomal, and ER stress were studied by Western blotting and immunohistochemistry. AECII morphology was evaluated by electron microscopy. Extensive lung fibrosis and AECII hyperplasia were observed in AD-treated mice already at day 7. Surfactant PL and SP accumulated in AECII over time. In parallel, induction of apoptosis, lysosomal, and ER stress was encountered in AECII of mice lungs and in MLE12 cells treated with AD. In vitro, siRNA-mediated knockdown of cathepsin D did not alter the AD-induced apoptotic response. Our data suggest that mice exposed to intratracheal AD develop severe pulmonary fibrosis, exhibit extensive surfactant alterations and cellular stress, but AD-induced AECII apoptosis is not mediated primarily via cathepsin D., (© The Author 2014. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Tumors are often greatly dependent on signaling cascades promoting cell growth or survival and may become hypersensitive to inactivation of key components within these signaling pathways. Ras and RAF mutations found in human cancer confer constitutive activity to these signaling molecules thereby converting them into an oncogenic state. RAF dimerization is required for normal Ras-dependent RAF activation and is required for the oncogenic potential of mutant RAFs. Here we describe a new mouse model for lung tumor development to investigate the role of B-RAF in oncogenic C-RAF-mediated adenoma initiation and growth. Conditional elimination of B-RAF in C-RAF BxB-expressing embryonic alveolar epithelial type II cells did not block adenoma formation. However, loss of B-RAF led to significantly reduced tumor growth. The diminished tumor growth upon B-RAF inactivation was due to reduced cell proliferation in absence of senescence and increased apoptosis. Furthermore, B-RAF elimination inhibited C-RAF BxB-mediated activation of the mitogenic cascade. In line with these data, mutation of Ser-621 in C-RAF BxB abrogated in vitro the dimerization with B-RAF and blocked the ability to activate the MAPK cascade. Taken together these data indicate that B-RAF is an important factor in oncogenic C-RAF-mediated tumorigenesis., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

Alveolar type-II cells (ATII cells) are lung progenitor cells responsible for regeneration of alveolar epithelium during homeostatic turnover and in response to injury. Characterization of ATII cells will have a profound impact on our understanding and treatment of lung disease. The identification of novel ATII cell-surface proteins can be used for sorting and enrichment of these cells for further characterization. Here we combined a high-resolution mass spectrometry-based membrane proteomic approach using lungs of the SILAC mice with an Affymetrix microarray-based transcriptome analysis of ATII cells. We identified 16 proteins that are enriched in the membrane fraction of ATII cells and whose genes are highly expressed in these cells. Interestingly, we confirmed our data for two of these genes, integrin beta 2 and 6 (Itgb2 and Itgb6), by qRT-PCR expression analysis and Western blot analysis of protein extracts. Moreover, flow cytometry and immunohistochemistry in adult lung revealed that ITGB2 and ITGB6 are present in subpopulations of surfactant-associated-protein-C-positive cells, suggesting the existence of different types of ATII cells. Furthermore, analysis of the Itgb2(-/-) mice showed that Itgb2 is required for proper WNT signaling regulation in the lung.

Influenza viruses (IVs) cause pneumonia in humans with progression to lung failure. Pulmonary DCs are key players in the antiviral immune response, which is crucial to restore alveolar barrier function. The mechanisms of expansion and activation of pulmonary DC populations in lung infection remain widely elusive. Using mouse BM chimeric and cell-specific depletion approaches, we demonstrated that alveolar epithelial cell (AEC) GM-CSF mediates recovery from IV-induced injury by affecting lung DC function. Epithelial GM-CSF induced the recruitment of CD11b+ and monocyte-derived DCs. GM-CSF was also required for the presence of CD103+ DCs in the lung parenchyma at baseline and for their sufficient activation and migration to the draining mediastinal lymph nodes (MLNs) during IV infection. These activated CD103+ DCs were indispensable for sufficient clearance of IVs by CD8+ T cells and for recovery from IV-induced lung injury. Moreover, GM-CSF applied intratracheally activated CD103+ DCs, inducing increased migration to MLNs, enhanced viral clearance, and attenuated lung injury. Together, our data reveal that GM-CSF-dependent cross-talk between IV-infected AECs and CD103+ DCs is crucial for effective viral clearance and recovery from injury, which has potential implications for GM-CSF treatment in severe IV pneumonia.

Elevated CO(2) levels (hypercapnia) occur in patients with respiratory diseases and impair alveolar epithelial integrity, in part, by inhibiting Na,K-ATPase function. Here, we examined the role of c-Jun N-terminal kinase (JNK) in CO(2) signaling in mammalian alveolar epithelial cells as well as in diptera, nematodes and rodent lungs. In alveolar epithelial cells, elevated CO(2) levels rapidly induced activation of JNK leading to downregulation of Na,K-ATPase and alveolar epithelial dysfunction. Hypercapnia-induced activation of JNK required AMP-activated protein kinase (AMPK) and protein kinase C-ζ leading to subsequent phosphorylation of JNK at Ser-129. Importantly, elevated CO(2) levels also caused a rapid and prominent activation of JNK in Drosophila S2 cells and in C. elegans. Paralleling the results with mammalian epithelial cells, RNAi against Drosophila JNK fully prevented CO(2)-induced downregulation of Na,K-ATPase in Drosophila S2 cells. The importance and specificity of JNK CO(2) signaling was additionally demonstrated by the ability of mutations in the C. elegans JNK homologs, jnk-1 and kgb-2 to partially rescue the hypercapnia-induced fertility defects but not the pharyngeal pumping defects. Together, these data provide evidence that deleterious effects of hypercapnia are mediated by JNK which plays an evolutionary conserved, specific role in CO(2) signaling in mammals, diptera and nematodes.

Nitric oxide (NO) is an important regulator of Na(+) reabsorption by pulmonary epithelial cells and therefore of alveolar fluid clearance. The mechanisms by which NO affects epithelial ion transport are poorly understood and vary from model to model. In this study, the effects of NO on sodium reabsorption by H441 cell monolayers were studied in an Ussing chamber. Two NO donors, (Z)-1-[N-(3-aminopropyl)-N-(n-propyl)amino]diazen-1-ium-1,2-diolate and diethylammonium (Z)-1-(N,N-diethylamino)diazen-1-ium-1,2-diolate, rapidly, reversibly, and dose-dependently reduced amiloride-sensitive, short-circuit currents across H441 cell monolayers. This effect was neutralized by the NO scavenger hemoglobin and was not observed with inactive NO donors. The effects of NO were not blocked by 8-bromoguanosine-3',5'-cyclic monophosphate or by soluble guanylate cyclase inhibitors (methylene blue and 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one) and were therefore independent of soluble guanylate cyclase signaling. NO targeted apical, highly selective, amiloride-sensitive Na(+) channels in basolaterally permeabilized H441 cell monolayers. NO had no effect on the activity of the human epithelial sodium channel heterologously expressed in Xenopus oocytes. NO decreased Na(+)/K(+)-ATPase activity in apically permeabilized H441 cell monolayers. The inhibition of Na(+)/K(+)-ATPase activity by NO was reversed by mercury and was mimicked by N-ethylmaleimide, which are agents that reverse and mimic, respectively, the reaction of NO with thiol groups. Consistent with these data, S-NO groups were detected on the Na(+)/K(+)-ATPase α subunit in response to NO-donor application, using a biotin-switch approach coupled to a Western blot. These data demonstrate that, in the H441 cell model, NO impairs Na(+) reabsorption by interfering with the activity of highly selective Na(+) channels and the Na(+)/K(+)-ATPase.

Vascular endothelial growth factor-A (VEGF-A) responsive effects mediated via the receptors fetal liver kinase-1 (flk-1) and fms-like tyrosine kinase (flt-1), are key processes of pulmonary vascular development. Flk-1 has been shown to be involved in early embryonic lung epithelial to endothelial crosstalk and branching morphogenesis. Recent reports suggested a role of VEGF-A in lung epithelial cell function. Based on these observations, we hypothesize that epithelial flk-1 has a unique function in pulmonary development. Thus, the aim of this study is to elucidate spatiotemporal expression of flk-1 during lung development with respect to the epithelial system. Embryonic lungs were screened for flk-1 messenger RNA and protein at daily intervals, including postnatal stages. From Embryonic Day (ED) 12.5 through ED 15.5, flk-1 expression was restricted to the early vascular primitive network, while from ED 16.5 on flk-1 was detectable in the epithelial system and persisted there postnatally. At postnatal stages, flk-1 expression was increasingly restricted to individual cells in the alveolar septa. Isolation and in vitro cultivation of alveolar epithelial cells confirmed flk-1 expression and showed VEGF secretion into the supernatant. To our knowledge, this is the first murine study characterizing epithelial flk-1 expression at different stages throughout lung organogenesis until birth and at postnatal stages. To confirm epithelial flk-1 expression, we performed reporter gene analysis of the flk-1 promoter in vivo. Investigations on transgenic mouse strains, containing either a complete or incomplete flk-1 promoter driving expression of the lacZ reporter gene, suggested differential flk-1 regulation in endothelial and epithelial cells.

Enhanced transforming growth factor (TGF) -beta signaling contributes to idiopathic pulmonary fibrosis (IPF), a progressive and fatal disease characterized by alveolar epithelial type II (ATII) cell hyperplasia, (myo)fibroblast accumulation, and excessive extracellular matrix deposition. TGF-beta is a potent inducer of lung fibrosis, and it regulates the ATII cell phenotype; however, direct TGF-beta target genes controlling the ATII cell phenotype remain elusive. Here, we identified the transgelin (tagln) gene as a novel immediate target of TGF-beta/Smad3-dependent gene expression in ATII cells using a Smad3 chromatin immunoprecipitation (ChIP) screen. Direct ChIP confirmed the rapid and specific binding of Smad3 to the tagln promoter. Luciferase assays demonstrated transactivation of the tagln promoter by activin-like kinase (Alk) 5-mediated TGF-beta signaling. TGF-beta treatment resulted in rapid up-regulation of tagln, but not tagln2, mRNA and protein expression, assessed by reverse transcription-polymerase chain reaction (RT-PCR), Western blotting, and immunofluorescence. In vivo, tagln expression was significantly increased in ATII cells of mice during bleomycin-induced lung fibrosis, as well as in lung specimen obtained from IPF patients, as assessed by RT-PCR and immunohistochemistry. Knockdown of tagln using siRNA inhibited TGF-beta-induced migration of lung epithelial A549 cells, as well as primary ATII cells. We thus identified tagln as a novel target of TGF-beta/Smad3-dependent gene expression in ATII cells. Increased ATII cell expression of tagln in experimental and idiopathic pulmonary fibrosis may contribute to TGF-beta-dependent ATII cell injury, repair, and migration in lung fibrosis.

Influenza A virus pneumonia is characterized by severe lung injury and high mortality. Early infection elicits a strong recruitment of monocytes from the peripheral blood across the endo-/epithelial barrier into the alveolar air space. However, it is currently unclear which of the infected resident lung cell populations, alveolar epithelial cells or alveolar macrophages, elicit monocyte recruitment during influenza A virus infection. In the current study, we investigated whether influenza A virus infection of primary alveolar epithelial cells and resident alveolar macrophages would elicit a basal-to-apical monocyte transepithelial migration in vitro. We found that infection of alveolar epithelial cells with the mouse-adapted influenza A virus strain PR/8 strongly induced the release of monocyte chemoattractants CCL2 and CCL5 followed by a strong monocyte transepithelial migration, and this monocytic response was strictly dependent on monocyte CCR2 but not CCR5 chemokine receptor expression. Analysis of the adhesion molecule pathways demonstrated a role of ICAM-1, VCAM-1, integrin-associated protein (CD47), and junctional adhesion molecule-c on the epithelial cell surface interacting with monocyte beta(1) and beta(2) integrins and integrin-associated protein in the monocyte transmigration process. Importantly, addition of influenza A virus-infected alveolar macrophages further enhanced monocyte transmigration across virus-infected epithelium in a TNF-alpha-dependent manner. Collectively, the data show an active role for virus-infected alveolar epithelium in the regulation of CCL2/CCR2-dependent monocyte transepithelial migration during influenza infection that is essentially dependent on both classical beta(1) and beta(2) integrins but also junctional adhesion molecule pathways.

Hypoxia sensing and related signaling events, including activation of hypoxia-inducible factor 1 (HIF-1), represent key features in cell physiology and lung function. Using cultured A549 cells, we investigated the role of NAD(P)H oxidase 1 (Nox1), suggested to be a subunit of a low-output NAD(P)H oxidase complex, in hypoxia signaling. Nox1 expression was detected on both the mRNA and protein levels. Upregulation of Nox1 mRNA and protein occurred during hypoxia, accompanied by enhanced reactive oxygen species (ROS) generation. A549 cells, which were transfected with a Nox1 expression vector, revealed an increase in ROS generation accompanied by activation of HIF-1-dependent target gene expression (heme oxygenase 1 mRNA, hypoxia-responsive-element reporter gene activity). In A549 cells stably overexpressing Nox1, accumulation of HIF-1alpha in normoxia and an additional increase in hypoxia were noted. Interference with ROS metabolism by the flavoprotein inhibitor diphenylene iodonium (DPI) and catalase inhibited HIF-1 induction. This suggests that H2O2 links Nox1 and HIF-1 activation. We conclude that hypoxic upregulation of Nox1 and subsequently augmented ROS generation may activate HIF-1-dependent pathways.

Among the anti-neutrophil cytoplasmic Abs (ANCA), those targeting proteinase 3 (PR3) have a high sensitivity and specificity for Wegener's granulomatosis (WG). A pathogenetic role for these autoantibodies has been proposed due to their capacity of activating neutrophils in vitro. Recently, PR3 was also detected in human renal tubular epithelial cells (TEC). In the present study, the effect of murine monoclonal anti-PR3 Abs (anti-PR3) and purified c-ANCA targeting PR3 from WG serum on isolated human renal tubular cell signaling and inflammatory mediator release was characterized. Priming of TEC with TNF-alpha resulted in surface expression of PR3, as quantified in immunofluorescence studies and by flow cytometry. Moreover, PR3 was immunoprecipitated on surface-labeled TEC. Primed TEC responded to anti-PR3 with a dose- and time-dependent activation of phosphoinositide hydrolysis, resulting in a remarkable accumulation of inositolphosphates. Control IgG was entirely ineffective, whereas PR3-ANCA reproduced the phosphoinositide response. The signaling response was accompanied by a pronounced release of superoxidanion into the cell supernatant. Moreover, large amounts of PGE(2) and, to a lesser extent, of thromboxane B(2), the stable metabolite of TxA(2), were secreted from anti-PR3-stimulated TEC. In parallel, a rise in intracellular cAMP levels was observed, which was blocked by the cyclooxygenase inhibitor indomethacin. We conclude that anti-PR3 Abs directly target renal TECs, thereby provoking pronounced activation of the phosphoinositide-related signal transduction pathway. Associated metabolic events such as the release of reactive oxygen species and lipid mediators may directly contribute to the development of renal lesions and loss of kidney function in WG.

Staphylococcus aureus alpha-toxin is a pore-forming bacterial exotoxin that has been implicated as a significant virulence factor in human staphylococcal diseases. In primary cultures of rat pneumocyte type II cells and the human A549 alveolar epithelial cell line, purified alpha-toxin provoked rapid-onset phosphatidylinositol (PtdIns) hydrolysis as well as liberation of nitric oxide and the prostanoids PGE(2), PGI(2), and thromboxane A(2). In addition, sustained upregulation of proinflammatory interleukin (IL)-8 mRNA expression and protein secretion occurred. "Priming" with low-dose IL-1beta markedly enhanced the IL-8 response to alpha-toxin, which was then accompanied by IL-6 appearance. The cytokine response was blocked by the intracellular Ca(2+)-chelating reagent 1,2-bis(2-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid, the protein kinase C inhibitor bis-indolyl maleimide I, as well as two independent inhibitors of nuclear factor-kappaB activation, pyrrolidine dithiocarbamate and caffeic acid phenethyl ester. We conclude that alveolar epithelial cells are highly reactive target cells of staphylococcal alpha-toxin. alpha-Toxin pore-associated transmembrane Ca(2+) flux and PtdIns hydrolysis-related signaling with downstream activation of protein kinase C and nuclear translocation of nuclear factor-kappaB are suggested to represent important underlying mechanisms. Such reactivity of the alveolar epithelial cells may be relevant for pathogenic sequelae in staphylococcal lung disease.

Introduction: Acute respiratory distress syndrome (ARDS) is a common complication of influenza virus (IV) infection. During ARDS, alveolar protein concentrations often reach 40-90% of plasma levels, causing severe impairment of gas exchange and promoting deleterious alveolar remodeling. Protein clearance from the alveolar space is at least in part facilitated by the multi-ligand receptor megalin through clathrin-mediated endocytosis. Methods: To investigate whether IV infection impairs alveolar protein clearance, we examined albumin uptake and megalin expression in MLE-12 cells and alveolar epithelial cells (AEC) from murine precision-cut lung slices (PCLS) and in vivo, under IV infection conditions by flow cytometry and western blot. Transcriptional levels from AEC and broncho-alveolar lavage (BAL) cells were analyzed in an in-vivo mouse model by RNAseq. Results: IV significantly downregulated albumin uptake, independently of activation of the TGF-ß1/GSK3ß axis that has been previously implicated in the regulation of megalin function. Decreased plasma membrane abundance, total protein levels, and mRNA expression of megalin were associated with this phenotype. In IV-infected mice, we identified a significant upregulation of matrix metalloproteinase (MMP)-14 in BAL fluid cells. Furthermore, the inhibition of this protease partially recovered total megalin levels and albumin uptake. Discussion: Our results suggest that the previously described MMP-driven shedding mechanisms are potentially involved in downregulation of megalin cell surface abundance and clearance of excess alveolar protein. As lower alveolar edema protein concentrations are associated with better outcomes in respiratory failure, our findings highlight the therapeutic potential of a timely MMP inhibition in the treatment of IV-induced ARDS. [ABSTRACT FROM AUTHOR]

A hallmark of acute respiratory distress syndrome (ARDS) is an accumulation of protein-rich alveolar edema that impairs gas exchange and leads to worse outcomes. Thus, understanding the mechanisms of alveolar albumin clearance is of high clinical relevance. Here, we investigated the mechanisms of the cellular albumin uptake in a three-dimensional culture of precision-cut lung slices (PCLS). We found that up to 60% of PCLS cells incorporated labeled albumin in a time- and concentration-dependent manner, whereas virtually no uptake of labeled dextran was observed. Of note, at a low temperature (4 °C), saturating albumin receptors with unlabeled albumin and an inhibition of clathrin-mediated endocytosis markedly decreased the endocytic uptake of the labeled protein, implicating a receptor-driven internalization process. Importantly, uptake rates of albumin were comparable in alveolar epithelial type I (ATI) and type II (ATII) cells, as assessed in PCLS from a SftpcCreERT2/+: tdTomatoflox/flox mouse strain (defined as EpCAM+CD31−CD45−tdTomatoSPC−T1α+ for ATI and EpCAM+CD31−CD45−tdTomatoSPC+T1α− for ATII cells). Once internalized, albumin was found in the early and recycling endosomes of the alveolar epithelium as well as in endothelial, mesenchymal, and hematopoietic cell populations, which might indicate transcytosis of the protein. In summary, we characterize albumin uptake in alveolar epithelial cells in the complex setting of PCLS. These findings may open new possibilities for pulmonary drug delivery that may improve the outcomes for patients with respiratory failure. [ABSTRACT FROM AUTHOR]

The tracheal epithelium is a primary target for pulmonary diseases as it provides a conduit for air flow between the environment and the lung lobes. The cellular and molecular mechanisms underlying airway epithelial cell proliferation and differentiation remain poorly understood. Hedgehog (HH) signaling orchestrates communication between epithelial and mesenchymal cells in the lung, where it modulates stromal cell proliferation, differentiation and signaling back to the epithelium. Here, we reveal a previously unreported autocrine function of HH signaling in airway epithelial cells. Epithelial cell depletion of the ligand sonic hedgehog (SHH) or its effector smoothened (SMO) causes defects in both epithelial cell proliferation and differentiation. In cultured primary human airway epithelial cells, HH signaling inhibition also hampers cell proliferation and differentiation. Epithelial HH function is mediated, at least in part, through transcriptional activation, as HH signaling inhibition leads to downregulation of cell type-specific transcription factor genes in both the mouse trachea and human airway epithelial cells. These results provide new insights into the role of HH signaling in epithelial cell proliferation and differentiation during airway development. [ABSTRACT FROM AUTHOR]

The lung tumor microenvironment plays a critical role in the tumorigenesis and metastasis of lung cancer, resulting from the crosstalk between cancer cells and microenvironmental cells. Therefore, comprehensive identification and characterization of cell populations in the complex lung structure is crucial for development of novel targeted anti-cancer therapies. Here, a hierarchical clustering approach with multispectral flow cytometry was established to delineate the cellular landscape of murine lungs under steady-state and cancer conditions. Fluorochromes were used multiple times to be able to measure 24 cell surface markers with only 13 detectors, yielding a broad picture for whole-lung phenotyping. Primary and metastatic murine lung tumor models were included to detect major cell populations in the lung, and to identify alterations to the distribution patterns in these models. In the primary tumor models, major altered populations included CD324+ epithelial cells, alveolar macrophages, dendritic cells, and blood and lymph endothelial cells. The number of fibroblasts, vascular smooth muscle cells, monocytes (Ly6C+ and Ly6C–) and neutrophils were elevated in metastatic models of lung cancer. Thus, the proposed clustering approach is a promising method to resolve cell populations from complex organs in detail even with basic flow cytometers. [ABSTRACT FROM AUTHOR]

Idiopathic pulmonary fibrosis (IPF) is a fatal disease characterized by type-II alveolar epithelial cell (AECII) injury and fibroblast hyperproliferation. Severe AECII endoplasmic reticulum (ER) stress is thought to underlie IPF, but is yet incompletely understood. We studied the regulation of C/EBP homologous protein (CHOP), a proapoptotic ER-stress-related transcription factor (TF) in AECII-like cells. Interestingly, single or combined overexpression of the active ER stress transducers activating transcription factor-4 (Atf4) and activating transcription factor-6 (p50Atf6) or spliced x-box-binding protein-1 (sXbp1) in MLE12 cells did not result in a substantial Chop induction, as compared to the ER stress inducer thapsigargin. Employing reporter gene assays of distinct CHOP promoter fragments, we could identify that, next to the conventional amino acid (AARE) and ER stress response elements (ERSE) within the CHOP promoter, activator protein-1 (AP-1) and c-Ets-1 TF binding sites are necessary for CHOP induction. Serial deletion and mutation analyses revealed that both AP-1 and c-Ets-1 motifs act in concert to induce CHOP expression. In agreement, CHOP promoter activity was greatly enhanced upon combined versus single overexpression of AP-1 and c-Ets-1. Moreover, combined overexpression of AP-1 and c-Ets-1 in MLE12 cells alone in the absence of any other ER stress inducer was sufficient to induce Chop protein expression. Further, AP-1 and c-Ets-1 were upregulated in AECII under ER stress conditions and in human IPF. Finally, Chop overexpression in vitro resulted in AECII apoptosis, lung fibroblast proliferation, and collagen-I production. We propose that CHOP activation by AP-1 and c-Ets-1 plays a key role in AECII maladaptive ER stress responses and consecutive fibrosis, offering new therapeutic prospects in IPF. Key messages: Overexpression of active ER stress sensors Atf4, Atf6, and Xbp1 does not induce Chop. AP-1 and c-Ets-1 TFs are necessary for induction of the ER stress factor Chop. AP-1 and c-Ets-1 alone induce Chop expression in the absence of any ER stress inducers. AP-1 and c-Ets-1 are induced in AECII under ER stress conditions and in human IPF. Chop expression alone triggers AECII apoptosis and consecutive profibrotic responses. [ABSTRACT FROM AUTHOR]

Hermansky-Pudlak syndrome (HPS) is a rare autosomal recessive disorder, and some patients with HPS develop pulmonary fibrosis, known as HPS-associated interstitial pneumonia (HPSIP). We have previously reported that HPSIP is associated with severe surfactant accumulation, lysosomal stress, and alveolar epithelial cell type II (AECII) apoptosis. Here, we hypothesized that defective autophagy might result in excessive lysosomal stress in HPSIP. Key autophagy proteins, including LC3B lipidation and p62, were increased in HPS1/2 mice lungs. Electron microscopy demonstrated a preferable binding of LC3B to the interior of lamellar bodies in the AECII of HPS1/2 mice, whereas in wild-type mice it was present on the limiting membrane in addition to the interior of the lamellar bodies. Similar observations were noted in human HPS1 lung sections. In vitro knockdown of HPS1 revealed increased LC3B lipidation and p62 accumulation, associated with an increase in proapoptotic caspases. Overexpression of LC3B decreased the HPS1 knockdown- induced p62 accumulation, whereas rapamycin treatment did not show the same effect. We conclude that loss of HPS1 protein results in impaired autophagy that is restored by exogenous LC3B and that defective autophagy might therefore play a critical role in the development and progression of HPSIP. [ABSTRACT FROM AUTHOR]

Primary alveolar epithelial cells play a pivotal role in lung research, particularly when focusing on gas exchange, barrier function, and transepithelial transport processes. However, efficient transfection of primary alveolar epithelial cells continues to be a major challenge. In the present study, we applied nucleofection, a novel method of gene and oligonucleotide delivery to the nucleus of cells by electroporation, to achieve highly efficient transfection of primary alveolar epithelial type II (ATII) cells. To quantify the amount of ATII cells effectively transfected, we applied a plasmid expressing GFP and assessed the amount of GFP-expressing cells by flow cytometry. Analysis of the nucleofected ATII cells revealed a concentration-dependent transfection efficiency of up to 50% when using 3-8 μg plasmid DNA without affecting cell viability. Nucleofection of cultured A549 and H441 cells yielded similar transfection rates. Importantly, nucleofection of ATII cells did not interfere with the integrity of ATII monolayers even with use of relatively high concentrations of plasmid DNA. In subsequent studies, we also efficiently delivered small interfering RNAs to ATII cells by nucleofection, thereby silencing Akt and the multiligand receptor megalin, which has been recently shown to play a key role in removal of excess protein from the alveolar space, and effectively inhibited megalin-driven uptake and transcellular transport of albumin in ATII cells. Thus we report successful transfection of primary rat alveolar epithelial cells with both plasmids and oligonucleotides via nucleofection with high viability and consistently good transfection rates without impairing key physiological properties of the cells. [ABSTRACT FROM AUTHOR]

Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal degenerative lung disease of unknown etiology. Although in its final stages it implicates, in a reactive manner, all lung cell types, the initial damage involves the alveolar epithelial compartment, in particular the alveolar epithelial type 2 cells (AEC2s). AEC2s serve dual progenitor and surfactant secreting functions, both of which are deeply impacted in IPF. Thus, we hypothesize that the size of the surfactant processing compartment, as measured by LysoTracker incorporation, allows the identification of different epithelial states in the IPF lung. Flow cytometry analysis of epithelial LysoTracker incorporation delineates two populations (Lysohigh and Lysolow) of AEC2s that behave in a compensatory manner during bleomycin injury and in the donor/IPF lung. Employing flow cytometry and transcriptomic analysis of cells isolated from donor and IPF lungs, we demonstrate that the Lysohigh population expresses all classical AEC2 markers and is drastically diminished in IPF. The Lysolow population, which is increased in proportion in IPF, co-expressed AEC2 and basal cell markers, resembling the phenotype of the previously identified intermediate AEC2 population in the IPF lung. In that regard, we provide an in-depth flow-cytometry characterization of LysoTracker uptake, HTII-280, proSP-C, mature SP-B, NGFR, KRT5, and CD24 expression in human lung epithelial cells. Combining functional analysis with extracellular and intracellular marker expression and transcriptomic analysis, we advance the current understanding of epithelial cell behavior and fate in lung fibrosis. [ABSTRACT FROM AUTHOR]

Elevated CO2 levels (hypercapnia) occur in patients with respiratory diseases and impair alveolar epithelial integrity, in part, by inhibiting Na,K-ATPase function. Here, we examined the role of c-Jun N-terminal kinase (JNK) in CO2 signaling in mammalian alveolar epithelial cells as well as in diptera, nematodes and rodent lungs. In alveolar epithelial cells, elevated CO2 levels rapidly induced activation of JNK leading to downregulation of Na,K-ATPase and alveolar epithelial dysfunction. Hypercapnia-induced activation of JNK required AMP-activated protein kinase (AMPK) and protein kinase C-ß leading to subsequent phosphorylation of JNK at Ser-129. Importantly, elevated CO2 levels also caused a rapid and prominent activation of JNK in Drosophila S2 cells and in C. elegans. Paralleling the results with mammalian epithelial cells, RNAi against Drosophila JNK fully prevented CO2-induced downregulation of Na,K-ATPase in Drosophila S2 cells. The importance and specificity of JNK CO2 signaling was additionally demonstrated by the ability of mutations in the C. elegans JNK homologs, jnk-1 and kgb-2 to partially rescue the hypercapnia-induced fertility defects but not the pharyngeal pumping defects. Together, these data provide evidence that deleterious effects of hypercapnia are mediated by JNK which plays an evolutionary conserved, specific role in CO2 signaling in mammals, diptera and nematodes. [ABSTRACT FROM AUTHOR]

A correction to the article "Macrophage-expressed IFN-β Contributes to Apoptotic Alveolar Epithelial Cell Injury in Severe Influenza Virus Pneumonia" is presented.

Macrophage migration inhibitory factor (MIF) is a pleiotropic proinflammatory cytokine involved in acute lung injury and other processes such as wound repair and tumor growth. MIF exerts pro-proliferative effects on a variety of cell types including monocytes/macrophages, B cells, and gastric epithelial cell lines through binding to the major histocompatibility complex type II-associated invariant chain, CD74. In acute lung injury, inflammatory damage of the alveolar epithelium leads to loss of type I alveolar epithelial cells (AEC-I), which are replaced by proliferation and differentiation of type II alveolar epithelial cells (AEC-Il). In this study we have investigated the potential of MIF to contribute to alveolar repair by stimulating alveolar epithelial cell proliferation. We show that murine AEC-Il, but not AEC-I, express high surface levels of CD74 in vivo. Culture of AEC-Il in vitro resulted in decreased mRNA levels for CD74 and loss of surface CD74 expression, which correlated with a transition of AEC-Il to an AEC-I-like phenotype. MIF stimulation of AEC-H induced rapid and prolonged phosphorylation of ERK 1/2 and Akt, increased expression of cyclins Dl and E, as well as AEC-Il proliferation. Corresponding MIF signaling and enhanced thymidine incorporation was observed after MIF stimulation of MLE-12 cells transfected to overexpress CD74. In contrast, MIF did not induce MAPK activation, gene transcription, or increased proliferation in differentiated AEC-I-like cells that lack CD74. These data suggest a previously unidentified role of MIF-CD74 interaction by inducing proliferation of AEC-II, which may contribute to alveolar repair. [ABSTRACT FROM AUTHOR]

Alveolar edema, impaired alveolar fluid clearance, and elevated CO2 levels (hypercapnia) are hallmarks of the acute respiratory distress syndrome (ARDS). This study investigated how hypercapnia affects maturation of the Na,K-ATPase (NKA), a key membrane transporter, and a cell adhesion molecule involved in the resolution of alveolar edema in the endoplasmic reticulum (ER). Exposure of human alveolar epithelial cells to elevated CO2 concentrations caused a significant retention of NKA-β in the ER and, thus, decreased levels of the transporter in the Golgi apparatus. These effects were associated with a marked reduction of the plasma membrane (PM) abundance of the NKA-α/β complex as well as a decreased total and ouabain-sensitive ATPase activity. Furthermore, our study revealed that the ER-retained NKA-β subunits were only partially assembled with NKA α-subunits, which suggests that hypercapnia modifies the ER folding environment. Moreover, we observed that elevated CO2 levels decreased intracellular ATP production and increased ER protein and, particularly, NKA-β oxidation. Treatment with α-ketoglutaric acid (α-KG), which is a metabolite that has been shown to increase ATP levels and rescue mitochondrial function in hypercapnia-exposed cells, attenuated the deleterious effects of elevated CO2 concentrations and restored NKA PM abundance and function. Taken together, our findings provide new insights into the regulation of NKA in alveolar epithelial cells by elevated CO2 levels, which may lead to the development of new therapeutic approaches for patients with ARDS and hypercapnia. [ABSTRACT FROM AUTHOR]

Idiopathic pulmonary fibrosis (IPF) is characterized by distorted lung architecture and loss of respiratory function. Enhanced (myo)fibroblast activation, ECM deposition, and alveolar epithelial type II (ATII) cell dysfunction contribute to IPF pathogenesis. However, the molecular pathways linking ATII cell dysfunction with the development of fibrosis are poorly understood. Here, we demonstrate, in a mouse model of pulmonary fibrosis, increased proliferation and altered expression of components of the WNT/beta-catenin signaling pathway in ATII cells. Further analysis revealed that expression of WNT1-inducible signaling protein-1 (WISP1), which is encoded by a WNT target gene, was increased in ATII cells in both a mouse model of pulmonary fibrosis and patients with IPF. Treatment of mouse primary ATII cells with recombinant WISP1 led to increased proliferation and epithelial-mesenchymal transition (EMT), while treatment of mouse and human lung fibroblasts with recombinant WISP1 enhanced deposition of ECM components. In the mouse model of pulmonary fibrosis, neutralizing mAbs specific for WISP1 reduced the expression of genes characteristic of fibrosis and reversed the expression of genes associated with EMT. More importantly, these changes in gene expression were associated with marked attenuation of lung fibrosis, including decreased collagen deposition and improved lung function and survival. Our study thus identifies WISP1 as a key regulator of ATII cell hyperplasia and plasticity as well as a potential therapeutic target for attenuation of pulmonary fibrosis. [ABSTRACT FROM AUTHOR]

The genetic, regulatory, and tissue-specific characterization of G-protein-coupled receptors is substantial since it contributes to the identification of the natural ligand which may influence basic physiological processes and cell function. Here we explored the genomic structure of a human orphan seven-transmembrane receptor which presents the human homologue of a receptor which has been controversially identified as a rat adrenomedullin receptor subtype. Based on the cDNA sequence a 3.4 kb genomic DNA fragment was isolated. Sequencing of the fragment and comparison studies revealed an intron of 544 bp in the 5′ untranslated region, followed by a second exon encoding the receptor protein of 404 amino acids. The gene is localized on chromosome 12q. The 5′ regulatory region contains several SP1, AP2, and CAAT sites as well as hypoxia responsive elements (HRE) both in the 5′ and 3′ regulatory region. RT-PCR with intron spanning primers demonstrated mRNA signals in various tissues, especially in lung. Characterizing the histological expression pattern in lung sections by nonisotopic in situ hybridization, a strong signal of receptor mRNA was identified in pulmonary epithelial cells of bronchi and alveoli. Analysis of the two human pulmonary epithelial cell lines, H23 and A549, showed significant mRNA induction of this receptor subtype in hypoxia. [Copyright &y& Elsevier]

Examines alveolar epithelial barrier functions in ventilated perfused rabbit lungs. Use of ultrasonic nebulization; Estimation on the volume of alveolar epithelial lining fluid; Determination of albumin clearance rates.