The persistent dynamic secrets of senescence. Schmitt Clemens A Nature cell biology While the beneficial versus detrimental implications of the senescence-associated secretome remain an issue of debate, time-resolved analyses of its composition, regulatory mechanisms and functional consequences have been largely missing. The dynamic activity of NOTCH is now shown to direct two distinct senescence phenotypes, by first promoting a pro-senescent TGF-β1-dependent secretome, followed by a second wave of pro-inflammatory, senescence-clearing cytokines. 10.1038/ncb3403
    Amplified canonical transforming growth factor-β signalling heat shock protein 90 in pulmonary fibrosis. Sibinska Zaneta,Tian Xia,Korfei Martina,Kojonazarov Baktybek,Kolb Janina Susanne,Klepetko Walter,Kosanovic Djuro,Wygrecka Malgorzata,Ghofrani Hossein Ardeschir,Weissmann Norbert,Grimminger Friedrich,Seeger Werner,Guenther Andreas,Schermuly Ralph T The European respiratory journal Interstitial lung fibroblast activation coupled with extracellular matrix production is a pathological signature of idiopathic pulmonary fibrosis (IPF), and is governed by transforming growth factor (TGF)-β/Smad signalling. We sought to define the role of heat shock protein (HSP)90 in profibrotic responses in IPF and to determine the therapeutic effects of HSP90 inhibition in a murine model of pulmonary fibrosis.We investigated the effects of HSP90 inhibition by applying 17-AAG (17-allylamino-17-demethoxygeldanamycin) to lung fibroblasts and A549 cells and by administering 17-DMAG (17-dimethylaminoethylamino-17-demethoxygeldanamycin) to mice with bleomycin-induced pulmonary fibrosis.HSP90 expression was increased in (myo)fibroblasts from fibrotic human and mouse lungs compared with controls. 17-AAG inhibited TGF-β1-induced extracellular matrix production and transdifferentiation of lung fibroblasts and epithelial-mesenchymal transition of A549 cells. The antifibrotic effects were associated with TGF-β receptor disruption and inhibition of Smad2/3 activation. Co-immunoprecipitation revealed that HSP90β interacted with TGF-β receptor II and stabilised TGF-β receptors. Furthermore, 17-DMAG improved lung function and decreased fibrosis and matrix metalloproteinase activity in the lungs of bleomycin-challenged mice.In conclusion, this is the first study to demonstrate that HSP90 inhibition blocks pulmonary fibroblast activation and ameliorates bleomycin-induced pulmonary fibrosis in mice. 10.1183/13993003.01941-2015
    Safety and Tolerability of Alveolar Type II Cell Transplantation in Idiopathic Pulmonary Fibrosis. Serrano-Mollar Anna,Gay-Jordi Gemma,Guillamat-Prats Raquel,Closa Daniel,Hernandez-Gonzalez Fernanda,Marin Pedro,Burgos Felip,Martorell Jaume,Sánchez Marcelo,Arguis Pedro,Soy Dolors,Bayas José M,Ramirez José,Tetley Teresa D,Molins Laureano,de la Bellacasa Jordi Puig,Rodríguez-Villar Camino,Rovira Irene,Fiblà Juan José,Xaubet Antoni, Chest BACKGROUND:Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal lung disease with limited response to currently available therapies. Alveolar type II (ATII) cells act as progenitor cells in the adult lung, contributing to alveolar repair during pulmonary injury. However, in IPF, ATII cells die and are replaced by fibroblasts and myofibroblasts. In previous preclinical studies, we demonstrated that ATII-cell intratracheal transplantation was able to reduce pulmonary fibrosis. The main objective of this study was to investigate the safety and tolerability of ATII-cell intratracheal transplantation in patients with IPF. METHODS:We enrolled 16 patients with moderate and progressive IPF who underwent ATII-cell intratracheal transplantation through fiberoptic bronchoscopy. We evaluated the safety and tolerability of ATII-cell transplantation by assessing the emergent adverse side effects that appeared within 12 months. Moreover, pulmonary function, respiratory symptoms, and disease extent during 12 months of follow-up were evaluated. RESULTS:No significant adverse events were associated with the ATII-cell intratracheal transplantation. After 12 months of follow-up, there was no deterioration in pulmonary function, respiratory symptoms, or disease extent. CONCLUSIONS:Our results support the hypothesis that ATII-cell intratracheal transplantation is safe and well tolerated in patients with IPF. This study opens the door to designing a clinical trial to elucidate the potential beneficial effects of ATII-cell therapy in IPF. 10.1016/j.chest.2016.03.021
    Localization of cystic fibrosis transmembrane conductance regulator mRNA in human fetal lung tissue by in situ hybridization. McCray P B,Wohlford-Lenane C L,Snyder J M The Journal of clinical investigation The fetal pulmonary epithelium secretes fluid. Cl transport is presumed to provide the driving force for net fluid secretion, although the cellular mechanisms have not been well identified in the fetus. The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP- and nucleoside triphosphate-regulated Cl channel; mutations in CFTR cause cystic fibrosis. We hypothesized that if CFTR is involved in fetal lung fluid transport, the fetal pulmonary epithelium should express CFTR mRNA. We used the technique of in situ hybridization with 3H-anti-sense and, as a control, 3H-sense CFTR cRNA probes to localize CFTR mRNA in human fetal lung tissue and cultured lung explants and determine when in gestation it is expressed. Epithelial cells of both first and second trimester lung tissues expressed CFTR mRNA. A decreasing gradient of CFTR mRNA expression was present from the proximal to the distal pulmonary epithelium. Cultured second trimester lung tissue explants expressed more CFTR mRNA than the uncultured starting tissue, suggesting CFTR gene expression increased during the five days in culture. Furthermore, alveolar type II cells in cultured explants expressed CFTR mRNA, suggesting that these cells are Cl-secretory and may be involved in lung fluid transport. These data confirm that CFTR mRNA is expressed in the human fetal pulmonary epithelium, consistent with the Cl-secretory properties of the fetal lung. 10.1172/JCI115901
    Early pulmonary inflammation in infants with cystic fibrosis. Khan T Z,Wagener J S,Bost T,Martinez J,Accurso F J,Riches D W American journal of respiratory and critical care medicine The mechanisms underlying the initiation of lung disease and early respiratory morbidity in cystic fibrosis (CF) are poorly understood. By identifying infants with CF through a statewide neonatal screening program, we investigated whether airway inflammation was present in these infants, with the goal of furthering our understanding of the early events in this lung disease. Bronchoalveolar lavage fluid (BALF) from 16 infants with CF (mean age, 6 mo) and 11 disease control infants (mean age, 12 mo) was examined for the following inflammatory parameters: (1) neutrophil count; (2) activity of free neutrophil elastase; (3) elastase/alpha 1-antiprotease inhibitor complexes; and (4) the level of interleukin-8 (IL-8). We also quantified the spontaneous level of expression of IL-8 mRNA transcripts by airway macrophages. Each index of airway inflammation was increased in the BALF of infants with CF as compared with control infants. In addition, both the number of neutrophils and IL-8 levels were increased in infants with CF who had negative cultures (n = 7) for common bacterial CF-related pathogens, as well as for common respiratory viruses and fungi at the time of bronchoalveolar lavage (BAL). These findings suggest that airway inflammation is already present in infants with CF who are as young as 4 wks. Furthermore, although many different cell types (e.g., epithelial cells) may express IL-8, airway macrophages appear to be a source of this chemokine, and may thus play a prominent role in early neutrophil influx into the lung. 10.1164/ajrccm/151.4.1075
    Inhibition of VEGF receptors causes lung cell apoptosis and emphysema. Kasahara Y,Tuder R M,Taraseviciene-Stewart L,Le Cras T D,Abman S,Hirth P K,Waltenberger J,Voelkel N F The Journal of clinical investigation Pulmonary emphysema, a significant global health problem, is characterized by a loss of alveolar structures. Because VEGF is a trophic factor required for the survival of endothelial cells and is abundantly expressed in the lung, we hypothesized that chronic blockade of VEGF receptors could induce alveolar cell apoptosis and emphysema. Chronic treatment of rats with the VEGF receptor blocker SU5416 led to enlargement of the air spaces, indicative of emphysema. The VEGF receptor inhibitor SU5416 induced alveolar septal cell apoptosis but did not inhibit lung cell proliferation. Viewed by angiography, SU5416-treated rat lungs showed a pruning of the pulmonary arterial tree, although we observed no lung infiltration by inflammatory cells or fibrosis. SU5416 treatment led to a decrease in lung expression of VEGF receptor 2 (VEGFR-2), phosphorylated VEGFR-2, and Akt-1 in the complex with VEGFR-2. Treatment with the caspase inhibitor Z-Asp-CH(2)-DCB prevented SU5416-induced septal cell apoptosis and emphysema development. These findings suggest that VEGF receptor signaling is required for maintenance of the alveolar structures and, further, that alveolar septal cell apoptosis contributes to the pathogenesis of emphysema. 10.1172/JCI10259
    Alveolar accumulation of hyaluronan and alveolar cellular response in bleomycin-induced alveolitis. Nettelbladt O,Scheynius A,Bergh J,Tengblad A,Hällgren R The European respiratory journal Hyaluronan (HA) accumulating in the alveolar interstitial tissue of rats injured by a single intratracheal instillation of bleomycin has been visualized histologically and assayed. HA was present already by Day 1 after bleomycin treatment, increased to a maximum value on Days 3 and 7 and then declined. A time-dependent relationship between this early connective tissue response and the invasion of inflammatory cells in the alveolar tissue was apparent. The dominating invading cells by Day 1 were granulocytes showing positive staining for the monoclonal antibody OX-42 reflecting the C3b receptor. The numbers of macrophages expressing class II antigens started to increase on Day 1, reaching a maximum on Days 3-7 and then declined. Macrophages were the dominating OX-42+ cells by Day 7. The appearance of W3/13+ cells ("pan-T-lymphocytes") showed a similar pattern to that for the class II expressing macrophages. The number of cells expressing CD4 antigen increased until Day 3 and levelled off on Day 30 whilst the largest number of cells expressing CD8 antigen was seen on Day 30. Few cells expressing B-cell phenotype outside lymph nodules were identified. Alveolar lining epithelial cells, probably epithelial type II cells, expressed class II antigens by Days 3-14. The time-related accumulation of HA and the appearance of T-cells, macrophages and granulocytes expressing signs of activation suggests that these cells may be involved in the early connective tissue response of the lung injured by bleomycin.
    Human cystic fibrosis transmembrane conductance regulator directed to respiratory epithelial cells of transgenic mice. Whitsett J A,Dey C R,Stripp B R,Wikenheiser K A,Clark J C,Wert S E,Gregory R J,Smith A E,Cohn J A,Wilson J M Nature genetics Human cystic fibrosis transmembrane conductance regulator (CFTR) was expressed in transgenic mice under the control of transcriptional elements derived from the human surfactant protein C (SP-C) gene. The hCFTR mRNA was expressed in lungs and testes: in the lung, we found hCFTR mRNA in bronchiolar and alveolar epithelial cells, and CFTR protein in respiratory epithelial cells. While the level of expression of hCFTR mRNA varied, hCFTR mRNA and protein were detected in pulmonary epithelial cells of several lines. Lung weight, morphology, somatic growth and reproductive capacity were not altered by expression hCFTR in lung and testes of the transgenics. Our findings suggest that hCFTR can be safely transferred to lung epithelial cells for CF therapy. 10.1038/ng0992-13
    Apoptotic cell instillation after bleomycin attenuates lung injury through hepatocyte growth factor induction. Lee Ye-Ji,Moon Changsuk,Lee Seung Hae,Park Hyun-Jeong,Seoh Ju-Young,Cho Min-Sun,Kang Jihee Lee The European respiratory journal Apoptotic cell clearance by macrophages and neighbouring tissue cells induces hepatocyte growth factor (HGF) secretion. HGF plays a key role in alveolar epithelial regeneration and reconstruction after lung injury. Direct in vivo exposure to apoptotic cells enhances HGF production, resulting in attenuation of pulmonary injury. We investigated the direct effect of in vivo exposure to apoptotic cells in bleomycin-stimulated lungs (2 days old) on HGF induction. Furthermore, sequential changes of bleomycin-induced HGF production following apoptotic cell instillation related to the changes in inflammatory and fibrotic responses were assessed. At 2 h after apoptotic cell instillation into bleomycin-stimulated lungs, the levels of HGF mRNA and protein production, and apoptotic cell clearance by alveolar macrophages were enhanced. Furthermore, HGF induction persistently increased following apoptotic cell instillation up to 21 days after bleomycin treatment. Apoptotic cell instillation attenuated bleomycin-induced pro-inflammatory mediator production, inflammatory cell recruitment and total protein levels. Apoptotic cell instillation also induced antiapoptotic and antifibrotic effects. These anti-inflammatory and antiapoptotic effects could be reversed by co-administration of HGF-neutralising antibody. These findings indicate that in vivo exposure to apoptotic cells enhances transcriptional HGF production in bleomycin-stimulated lungs, resulting in attenuation of lung injury and fibrosis. 10.1183/09031936.00096711
    Vimentin regulates activation of the NLRP3 inflammasome. dos Santos Gimena,Rogel Micah R,Baker Margaret A,Troken James R,Urich Daniela,Morales-Nebreda Luisa,Sennello Joseph A,Kutuzov Mikhail A,Sitikov Albert,Davis Jennifer M,Lam Anna P,Cheresh Paul,Kamp David,Shumaker Dale K,Budinger G R Scott,Ridge Karen M Nature communications Activation of the NLRP3 inflammasome and subsequent maturation of IL-1β have been implicated in acute lung injury (ALI), resulting in inflammation and fibrosis. We investigated the role of vimentin, a type III intermediate filament, in this process using three well-characterized murine models of ALI known to require NLRP3 inflammasome activation. We demonstrate that central pathophysiologic events in ALI (inflammation, IL-1β levels, endothelial and alveolar epithelial barrier permeability, remodelling and fibrosis) are attenuated in the lungs of Vim(-/-) mice challenged with LPS, bleomycin and asbestos. Bone marrow chimeric mice lacking vimentin have reduced IL-1β levels and attenuated lung injury and fibrosis following bleomycin exposure. Furthermore, decreased active caspase-1 and IL-1β levels are observed in vitro in Vim(-/-) and vimentin-knockdown macrophages. Importantly, we show direct protein-protein interaction between NLRP3 and vimentin. This study provides insights into lung inflammation and fibrosis and suggests that vimentin may be a key regulator of the NLRP3 inflammasome. 10.1038/ncomms7574
    Platelet-derived growth factor in idiopathic pulmonary fibrosis. Antoniades H N,Bravo M A,Avila R E,Galanopoulos T,Neville-Golden J,Maxwell M,Selman M The Journal of clinical investigation Fibrosis is a complex process involving an inflammatory reaction, fibroblast proliferation, and abnormal accumulation of interstitial collagens. Mononuclear cells are usually present in lung fibrosis. Activated monocytes and macrophages in culture have been shown to produce several growth factors including platelet-derived growth factor (PDGF). PDGF is a potent mitogen and chemoattractant for fibroblasts and smooth muscle cells and a stimulator of collagen synthesis. We have studied the expression of c-sis/PDGF-2 mRNA in lung tissues derived from five patients with idiopathic pulmonary fibrosis (IPF) and from four control individuals without IPF. Northern blot analysis of specimens obtained from four patients with IPF revealed the expression of the c-sis/PDGF-2 protooncogene. A control lung tissue without IPF did not express the c-sis protooncogene. In situ hybridization extended these studies demonstrating the expression of the c-sis mRNA in the five specimens with IPF but not in the four control specimens without IPF. The expression of c-sis mRNA was localized primarily in the epithelial cells. Invading alveolar macrophages also expressed c-sis mRNA. The expression of c-sis mRNA was accompanied by the expression of PDGF-like proteins in lung specimens with IPF but not in control lung specimens. These findings demonstrate the in vivo expression of the c-sis/PDGF-2 protooncogene and the production of PDGF-like proteins in the epithelial cells and macrophages of the fibrotic tissue. This localized and sustained production of PDGF-like mitogen may constitute an important contributing factor in the abnormal fibroblast proliferation and collagen production, events associated with pulmonary fibrosis. 10.1172/JCI114808
    Inhibition of LTβR signalling activates WNT-induced regeneration in lung. Conlon Thomas M,John-Schuster Gerrit,Heide Danijela,Pfister Dominik,Lehmann Mareike,Hu Yan,Ertüz Zeynep,Lopez Martin A,Ansari Meshal,Strunz Maximilian,Mayr Christoph,Angelidis Ilias,Ciminieri Chiara,Costa Rita,Kohlhepp Marlene Sophia,Guillot Adrien,Günes Gizem,Jeridi Aicha,Funk Maja C,Beroshvili Giorgi,Prokosch Sandra,Hetzer Jenny,Verleden Stijn E,Alsafadi Hani,Lindner Michael,Burgstaller Gerald,Becker Lore,Irmler Martin,Dudek Michael,Janzen Jakob,Goffin Eric,Gosens Reinoud,Knolle Percy,Pirotte Bernard,Stoeger Tobias,Beckers Johannes,Wagner Darcy,Singh Indrabahadur,Theis Fabian J,de Angelis Martin Hrabé,O'Connor Tracy,Tacke Frank,Boutros Michael,Dejardin Emmanuel,Eickelberg Oliver,Schiller Herbert B,Königshoff Melanie,Heikenwalder Mathias,Yildirim Ali Önder Nature Lymphotoxin β-receptor (LTβR) signalling promotes lymphoid neogenesis and the development of tertiary lymphoid structures, which are associated with severe chronic inflammatory diseases that span several organ systems. How LTβR signalling drives chronic tissue damage particularly in the lung, the mechanism(s) that regulate this process, and whether LTβR blockade might be of therapeutic value have remained unclear. Here we demonstrate increased expression of LTβR ligands in adaptive and innate immune cells, enhanced non-canonical NF-κB signalling, and enriched LTβR target gene expression in lung epithelial cells from patients with smoking-associated chronic obstructive pulmonary disease (COPD) and from mice chronically exposed to cigarette smoke. Therapeutic inhibition of LTβR signalling in young and aged mice disrupted smoking-related inducible bronchus-associated lymphoid tissue, induced regeneration of lung tissue, and reverted airway fibrosis and systemic muscle wasting. Mechanistically, blockade of LTβR signalling dampened epithelial non-canonical activation of NF-κB, reduced TGFβ signalling in airways, and induced regeneration by preventing epithelial cell death and activating WNT/β-catenin signalling in alveolar epithelial progenitor cells. These findings suggest that inhibition of LTβR signalling represents a viable therapeutic option that combines prevention of tertiary lymphoid structures and inhibition of apoptosis with tissue-regenerative strategies. 10.1038/s41586-020-2882-8
    Heterozygosity for a surfactant protein C gene mutation associated with usual interstitial pneumonitis and cellular nonspecific interstitial pneumonitis in one kindred. Thomas Alan Q,Lane Kirk,Phillips John,Prince Melissa,Markin Cheryl,Speer Marcy,Schwartz David A,Gaddipati Radhika,Marney Annis,Johnson Joyce,Roberts Richard,Haines Jonathan,Stahlman Mildred,Loyd James E American journal of respiratory and critical care medicine Familial pulmonary fibrosis is a heterogeneous group of interstitial lung diseases of unknown cause that is associated with multiple pathologic subsets. Mutations in the surfactant protein C (SP-C) gene (SFTPC) are associated with familial desquamative and nonspecific interstitial pneumonitis. Genetic studies in familial usual interstitial pneumonitis have been inconclusive. Using a candidate gene approach, we found a heterozygous exon 5 + 128 T-->A transversion of SFTPC in a large familial pulmonary fibrosis kindred, including adults with usual interstitial pneumonitis and children with cellular nonspecific interstitial pneumonitis. The mutation is predicted to substitute a glutamine for a conserved leucine residue and may hinder processing of SP-C precursor protein. SP-C precursor protein displayed aberrant subcellular localization by immunostaining. Electron microscopy of affected lung revealed alveolar type II cell atypia, with numerous abnormal lamellar bodies. Mouse lung epithelial cells transfected with the SFTPC mutation were notable for similar electron microscopy findings and for exaggerated cellular toxicity. We show that an SFTPC mutation segregates with the pulmonary fibrosis phenotype in this kindred and may cause type II cellular injury. The presence of two different pathologic diagnoses in affected relatives sharing this mutation indicates that in this kindred, these diseases may represent pleiotropic manifestations of the same central pathogenesis. 10.1164/rccm.200112-123OC
    Aerosolized administration of N-acetylcysteine attenuates lung fibrosis induced by bleomycin in mice. Hagiwara S I,Ishii Y,Kitamura S American journal of respiratory and critical care medicine Reactive oxygen species (ROS) play an important role in the pathogenesis of pulmonary fibrosis. We previously demonstrated that N-acetylcysteine (NAC), an antioxidant, inhibited adhesion molecule expression and cytokine production in lung cells. When NAC is inhaled into the alveolar space, it is expected to directly interact with inflammatory cells and to elevate glutathione levels in the epithelial lining fluids. We therefore examined whether inhaled NAC inhibits lung fibrosis induced by bleomycin (BLM). Male ICR mice were given a single intravenous injection of BLM (150 mg/ kg). Thirty milliliters of NAC (70 mg/ml) or saline were inhaled twice a day for 28 d using an ultrasonic nebulizer. In the inflammatory phase (Day 7), NAC administration attenuated the cellular infiltration in both bronchoalveolar lavage fluid (BALF) and alveolar tissues. At Day 28, the fibrotic changes estimated by Aschroft's criteria and hydroxyproline content in the NAC inhalation group were significantly decreased compared with the BLM-only group (p < 0.05). CXC chemokines, macrophage inflammatory protein-2 (MIP-2), cytokine-induced neutrophil chemoattractant (KC), and CC chemokines, macrophage inflammatory protein-1alpha (MIP-1alpha), in BALF were mostly elevated on Day 7 in the BLM-only group; however, these elevations were significantly repressed by NAC inhalation (p < 0.05). Lipid hydroperoxide (LPO) was also quantified in BALF. LPO was markedly increased on Day 3 in the BLM-only group, and this increase was significantly decreased by NAC inhalation (p < 0.05). These results revealed that aerosolized NAC ameliorated acute pulmonary inflammation induced by BLM injection via the repression of chemokines and LPO production, resulting in the attenuation of subsequent lung fibrosis. These findings are limited to the BLM-induced lung fibrosis animal model. However, NAC inhalation will be expected to be a potential therapy for patients with other interstitial pneumonias because ROS are involved in the pathogenesis of lung injury in most interstitial pneumonia. 10.1164/ajrccm.162.1.9903129
    Gli1 mesenchymal stromal cells form a pathological niche to promote airway progenitor metaplasia in the fibrotic lung. Cassandras Monica,Wang Chaoqun,Kathiriya Jaymin,Tsukui Tatsuya,Matatia Peri,Matthay Michael,Wolters Paul,Molofsky Ari,Sheppard Dean,Chapman Hal,Peng Tien Nature cell biology Aberrant epithelial reprogramming can induce metaplastic differentiation at sites of tissue injury that culminates in transformed barriers composed of scar and metaplastic epithelium. While the plasticity of epithelial stem cells is well characterized, the identity and role of the niche has not been delineated in metaplasia. Here, we show that Gli1 mesenchymal stromal cells (MSCs), previously shown to contribute to myofibroblasts during scarring, promote metaplastic differentiation of airway progenitors into KRT5 basal cells. During fibrotic repair, Gli1 MSCs integrate hedgehog activation signalling to upregulate BMP antagonism in the progenitor niche that promotes metaplasia. Restoring the balance towards BMP activation attenuated metaplastic KRT5 differentiation while promoting adaptive alveolar differentiation into SFTPC epithelium. Finally, fibrotic human lungs demonstrate altered BMP activation in the metaplastic epithelium. These findings show that Gli1 MSCs integrate hedgehog signalling as a rheostat to control BMP activation in the progenitor niche to determine regenerative outcome in fibrosis. 10.1038/s41556-020-00591-9
    Oxidant-mediated epithelial cell injury in idiopathic pulmonary fibrosis. Cantin A M,North S L,Fells G A,Hubbard R C,Crystal R G The Journal of clinical investigation Lung inflammatory cells of patients with idiopathic pulmonary fibrosis (IPF) were evaluated for their ability to injure 51Cr-labeled AKD alveolar epithelial cells in the presence and absence of IPF alveolar epithelial lining fluid (ELF). The IPF cells were spontaneously releasing exaggerated amounts of superoxide (O.2) and hydrogen peroxide (H2O2) compared with normal (P less than 0.02). Cytotoxicity of the AKD cells was markedly increased when the IPF inflammatory cells were incubated with autologous ELF (P less than 0.02). The majority of IPF patients had ELF myeloperoxidase levels above normal (P less than 0.002). Incubation of IPF ELF with AKD cells in the presence of H2O2 caused increased cellular injury (P less than 0.01 compared with control), which was suppressed by methionine, a myeloperoxidase system scavenger. IPF patients with high concentrations of ELF myeloperoxidase deteriorated more rapidly than those with low ELF myeloperoxidase (P less than 0.05). Thus, IPF is characterized by an increased spontaneous production of oxidants by lung inflammatory cells, the presence of high concentrations of myeloperoxidase in the ELF of the lower respiratory tract, and a synergistic cytotoxic effect of alveolar inflammatory cells and ELF on lung epithelial cells, suggesting oxidants may play a role in causing the epithelial cell injury of this disorder. 10.1172/JCI113005
    Upregulation of alveolar epithelial active Na+ transport is dependent on beta2-adrenergic receptor signaling. Mutlu Gökhan M,Dumasius Vidas,Burhop James,McShane Pamela J,Meng Fan Jing,Welch Lynn,Dumasius Andrew,Mohebahmadi Nima,Thakuria Gloria,Hardiman Karen,Matalon Sadis,Hollenberg Steven,Factor Phillip Circulation research Alveolar epithelial beta-adrenergic receptor (betaAR) activation accelerates active Na+ transport in lung epithelial cells in vitro and speeds alveolar edema resolution in human lung tissue and normal and injured animal lungs. Whether these receptors are essential for alveolar fluid clearance (AFC) or if other mechanisms are sufficient to regulate active transport is unknown. In this study, we report that mice with no beta1- or beta2-adrenergic receptors (beta1AR-/-/beta2AR-/-) have reduced distal lung Na,K-ATPase function and diminished basal and amiloride-sensitive AFC. Total lung water content in these animals was not different from wild-type controls, suggesting that betaAR signaling may not be required for alveolar fluid homeostasis in uninjured lungs. Comparison of isoproterenol-sensitive AFC in mice with beta1- but not beta2-adrenergic receptors to beta1AR-/-/beta2AR-/- mice indicates that the beta2AR mediates the bulk of beta-adrenergic-sensitive alveolar active Na+ transport. To test the necessity of betaAR signaling in acute lung injury, beta1AR-/-/beta2AR-/-, beta1AR+/+/beta2AR-/-, and beta1AR+/+/beta2AR+/+ mice were exposed to 100% oxygen for up to 204 hours. beta1AR-/-/beta2AR-/- and beta1AR+/+/beta2AR-/- mice had more lung water and worse survival from this form of acute lung injury than wild-type controls. Adenoviral-mediated rescue of beta2-adrenergic receptor (beta2AR) function into the alveolar epithelium of beta1AR-/-/beta2AR-/- and beta1AR+/+/beta2AR-/- mice normalized distal lung beta2AR function, alveolar epithelial active Na+ transport, and survival from hyperoxia. These findings indicate that betaAR signaling may not be necessary for basal AFC, and that beta2AR is essential for the adaptive physiological response needed to clear excess fluid from the alveolar airspace of normal and injured lungs. 10.1161/01.RES.0000125623.56442.20
    Cytoskeletal protein modulation in pulmonary alveolar myofibroblasts during idiopathic pulmonary fibrosis. Possible role of transforming growth factor beta and tumor necrosis factor alpha. Kapanci Y,Desmouliere A,Pache J C,Redard M,Gabbiani G American journal of respiratory and critical care medicine Pulmonary biopsy specimens from ten cases of idiopathic pulmonary fibrosis (IPF) were examined using routine histological stains, including toluidine blue, and immunohistochemistry by means of specific antibodies against alpha-smooth muscle (alpha-SM) actin, desmin, keratin, TGF beta 1, and TNF alpha. The sections were compared with two cases of normal lung. As shown previously, normal alveolar interstitium did not contain alpha-SM actin positive myofibroblasts nor did the alveolar lining contain any significant number of TGF beta 1 or TNF alpha laden epithelial cells. In IPF, during the inflammatory stage, the alveolar myofibroblasts expressed alpha-SM actin and the regenerating type II alveolar epithelium staining strongly with TGF beta 1 and TNF alpha antibodies. The former cytokine was also detected in the interstitial matrix and fibroblastic cells as well as in the wall of vessels. At this stage, a manifest mast cell infiltration was noted. In very fibrotic and cystic alveolar tissue, i.e., at end stage fibrosis, the number of alpha-SM actin positive myofibroblasts as well as that of TNF alpha laden type II epithelial cells diminished, while TGF beta 1 positive cells persisted. Our findings demonstrate that during IPF alveolar type II epithelium constitutes, if not the site of synthesis, at least the main reservoir for TGF beta 1 and TNF alpha. These cytokines, besides their involvement in fibrogenesis, play probably an important role in the expression of alpha-SM actin by alveolar myofibroblasts. Our study suggests the possibility of an interaction between interstitial cells and alveolar epithelium, during IPF. 10.1164/ajrccm.152.6.8520791
    Platelet-derived Wnt antagonist Dickkopf-1 is implicated in ICAM-1/VCAM-1-mediated neutrophilic acute lung inflammation. Guo Yujie,Mishra Amarjit,Howland Emily,Zhao Chunling,Shukla Dhananjay,Weng Tingting,Liu Lin Blood Neutrophil infiltration represents the early acute inflammatory response in acute lung injury. The recruitment of neutrophils from the peripheral blood across the endothelial-epithelial barrier into the alveolar airspace is highly regulated by the adhesion molecules on alveolar epithelial cells (AECs). Wnt/β-catenin signaling is involved in the progression of inflammatory lung diseases including asthma, emphysema, and pulmonary fibrosis. However, the function of Wnt/β-catenin signaling in acute lung inflammation is unknown. Here, we identified platelet-derived Dickkopf-1 (Dkk1) as the major Wnt antagonist contributing to the suppression of Wnt/β-catenin signaling in AECs during acute lung inflammation. Intratracheal administration of Wnt3a or an antibody capable of neutralizing Dkk1 inhibited neutrophil influx into the alveolar airspace of injured lungs. Activation of Wnt/β-catenin signaling in AECs attenuated intercellular adhesion molecule 1 (ICAM-1)/vascular cell adhesion molecule 1 (VCAM-1)-mediated adhesion of both macrophages and neutrophils to AECs. Our results suggest a role for Wnt/β-catenin signaling in modulating the inflammatory response, and a functional communication between platelets and AECs during acute lung inflammation. Targeting Wnt/β-catenin signaling and the communication between platelets and AECs therefore represents potential therapeutic strategies to limit the damage of acute pulmonary inflammation. 10.1182/blood-2015-02-622233
    Distinct Mesenchymal Lineages and Niches Promote Epithelial Self-Renewal and Myofibrogenesis in the Lung. Zepp Jarod A,Zacharias William J,Frank David B,Cavanaugh Christina A,Zhou Su,Morley Michael P,Morrisey Edward E Cell The lung is an architecturally complex organ comprising a heterogeneous mixture of various epithelial and mesenchymal lineages. We use single-cell RNA sequencing and signaling lineage reporters to generate a spatial and transcriptional map of the lung mesenchyme. We find that each mesenchymal lineage has a distinct spatial address and transcriptional profile leading to unique niche regulatory functions. The mesenchymal alveolar niche cell is Wnt responsive, expresses Pdgfrα, and is critical for alveolar epithelial cell growth and self-renewal. In contrast, the Axin2+ myofibrogenic progenitor cell preferentially generates pathologically deleterious myofibroblasts after injury. Analysis of the secretome and receptome of the alveolar niche reveals functional pathways that mediate growth and self-renewal of alveolar type 2 progenitor cells, including IL-6/Stat3, Bmp, and Fgf signaling. These studies define the cellular and molecular framework of lung mesenchymal niches and reveal the functional importance of developmental pathways in promoting self-renewal versus a pathological response to tissue injury. 10.1016/j.cell.2017.07.034
    Epithelial stress and apoptosis underlie Hermansky-Pudlak syndrome-associated interstitial pneumonia. Mahavadi Poornima,Korfei Martina,Henneke Ingrid,Liebisch Gerhard,Schmitz Gerd,Gochuico Bernadette R,Markart Philipp,Bellusci Saverio,Seeger Werner,Ruppert Clemens,Guenther Andreas American journal of respiratory and critical care medicine RATIONALE:The molecular mechanisms underlying Hermansky-Pudlak syndrome-associated interstitial pneumonia (HPSIP) are poorly understood but, as in idiopathic pulmonary fibrosis, may be linked to chronic alveolar epithelial type II cell (AECII) injury. OBJECTIVES:We studied the development of fibrosis and the role of AECII injury in various murine models of HPS. METHODS:HPS1, HPS2, and HPS6 monomutant mice, and HPS1/2 and HPS1/6 double-mutant and genetic background mice, were killed at 3 and 9 months of age. Quantitative morphometry was undertaken in lung sections stained with hemalaun-eosin. The extent of lung fibrosis was assessed by trichrome staining and hydroxyproline measurement. Surfactant lipids were analyzed by electrospray ionization mass spectrometry. Surfactant proteins, apoptosis, and lysosomal and endoplasmic reticulum stress markers were studied by Western blotting and immunohistochemistry. Cell proliferation was measured by water-soluble tetrazolium salt-1 and bromodeoxyuridine assays. MEASUREMENTS AND MAIN RESULTS:Spontaneous and slowly progressive HPSIP was observed in HPS1/2 double mutants, but not in other HPS mutants, with subpleural onset at 3 months and full-blown fibrosis at 9 months. In these mice, extensive surfactant abnormalities were encountered in AECII and were paralleled by early lysosomal stress (cathepsin D induction), late endoplasmic reticulum stress (activating transcription factor-4 [ATF4], C/EBP homologous protein [CHOP] induction), and marked apoptosis. These findings were fully corroborated in human HPSIP. In addition, cathepsin D overexpression resulted in apoptosis of MLE-12 cells and increased proliferation of NIH 3T3 fibroblasts incubated with conditioned medium of the transfected cells. CONCLUSIONS:Extensively impaired surfactant trafficking and secretion underlie lysosomal and endoplasmic reticulum stress with apoptosis of AECII in HPSIP, thereby causing the development of HPSIP. 10.1164/rccm.200909-1414OC
    Conditional depletion of airway progenitor cells induces peribronchiolar fibrosis. Perl Anne-Karina T,Riethmacher Dieter,Whitsett Jeffrey A American journal of respiratory and critical care medicine 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 nonciliated epithelial cells play important roles in the repair of the bronchiolar epithelium after acute injury. Cellular processes underlying the repair and remodeling of the lung after 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-surfactant protein C-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:Colocalization 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 reepithelialization, and peribronchiolar fibrosis. Induction of proliferation in subepithelial fibroblasts supports the concept that chronic epithelial depletion caused peribronchiolar fibrosis. 10.1164/rccm.201005-0744OC
    The use of alveolar epithelial type I cell-selective markers to investigate lung injury and repair. McElroy M C,Kasper M The European respiratory journal Alveolar epithelial type I cells cover most of the internal surface area of the lungs. Ultrastructural studies demonstrate that alveolar epithelial type I cell damage is frequently observed in acute and chronic lung diseases. This article discusses the use of cell-selective proteins as markers for the investigation of injury and repair of the alveolar epithelium. The utility of proteins specific to alveolar epithelial type I cells as diagnostic markers of alveolar epithelial injury in acute lung injury is considered, and expression of proteins selective for alveolar epithelial type I cells in lungs following injury and in fibrosis are discussed. 10.1183/09031936.04.00096003
    Use of EpiAlveolar Lung Model to Predict Fibrotic Potential of Multiwalled Carbon Nanotubes. Barosova Hana,Maione Anna G,Septiadi Dedy,Sharma Monita,Haeni Laetitia,Balog Sandor,O'Connell Olivia,Jackson George R,Brown David,Clippinger Amy J,Hayden Patrick,Petri-Fink Alke,Stone Vicki,Rothen-Rutishauser Barbara ACS nano Expansion in production and commercial use of nanomaterials increases the potential human exposure during the lifecycle of these materials (production, use, and disposal). Inhalation is a primary route of exposure to nanomaterials; therefore it is critical to assess their potential respiratory hazard. Herein, we developed a three-dimensional alveolar model (EpiAlveolar) consisting of human primary alveolar epithelial cells, fibroblasts, and endothelial cells, with or without macrophages for predicting long-term responses to aerosols. Following thorough characterization of the model, proinflammatory and profibrotic responses based on the adverse outcome pathway concept for lung fibrosis were assessed upon repeated subchronic exposures (up to 21 days) to two types of multiwalled carbon nanotubes (MWCNTs) and silica quartz particles. We simulate occupational exposure doses for the MWCNTs (1-30 μg/cm) using an air-liquid interface exposure device (VITROCELL Cloud) with repeated exposures over 3 weeks. Specific key events leading to lung fibrosis, such as barrier integrity and release of proinflammatory and profibrotic markers, show the responsiveness of the model. Nanocyl induced, in general, a less pronounced reaction than Mitsui-7, and the cultures with human monocyte-derived macrophages (MDMs) showed the proinflammatory response at later time points than those without MDMs. In conclusion, we present a robust alveolar model to predict inflammatory and fibrotic responses upon exposure to MWCNTs. 10.1021/acsnano.9b06860
    The antifibrotic effects of plasminogen activation occur via prostaglandin E2 synthesis in humans and mice. Bauman Kristy A,Wettlaufer Scott H,Okunishi Katsuhide,Vannella Kevin M,Stoolman Joshua S,Huang Steven K,Courey Anthony J,White Eric S,Hogaboam Cory M,Simon Richard H,Toews Galen B,Sisson Thomas H,Moore Bethany B,Peters-Golden Marc The Journal of clinical investigation Plasminogen activation to plasmin protects from lung fibrosis, but the mechanism underlying this antifibrotic effect remains unclear. We found that mice lacking plasminogen activation inhibitor-1 (PAI-1), which are protected from bleomycin-induced pulmonary fibrosis, exhibit lung overproduction of the antifibrotic lipid mediator prostaglandin E2 (PGE2). Plasminogen activation upregulated PGE2 synthesis in alveolar epithelial cells, lung fibroblasts, and lung fibrocytes from saline- and bleomycin-treated mice, as well as in normal fetal and adult primary human lung fibroblasts. This response was exaggerated in cells from Pai1-/- mice. Although enhanced PGE2 formation required the generation of plasmin, it was independent of proteinase-activated receptor 1 (PAR-1) and instead reflected proteolytic activation and release of HGF with subsequent induction of COX-2. That the HGF/COX-2/PGE2 axis mediates in vivo protection from fibrosis in Pai1-/- mice was demonstrated by experiments showing that a selective inhibitor of the HGF receptor c-Met increased lung collagen to WT levels while reducing COX-2 protein and PGE2 levels. Of clinical interest, fibroblasts from patients with idiopathic pulmonary fibrosis were found to be defective in their ability to induce COX-2 and, therefore, unable to upregulate PGE2 synthesis in response to plasmin or HGF. These studies demonstrate crosstalk between plasminogen activation and PGE2 generation in the lung and provide a mechanism for the well-known antifibrotic actions of the fibrinolytic pathway. 10.1172/JCI38369
    Alveolar epithelial type II cells from embryonic stem cells: knights in shining armour? Fehrenbach H The European respiratory journal 10.1183/09031936.00162111
    PINK1 deficiency impairs mitochondrial homeostasis and promotes lung fibrosis. Bueno Marta,Lai Yen-Chun,Romero Yair,Brands Judith,St Croix Claudette M,Kamga Christelle,Corey Catherine,Herazo-Maya Jose D,Sembrat John,Lee Janet S,Duncan Steve R,Rojas Mauricio,Shiva Sruti,Chu Charleen T,Mora Ana L The Journal of clinical investigation Although aging is a known risk factor for idiopathic pulmonary fibrosis (IPF), the pathogenic mechanisms that underlie the effects of advancing age remain largely unexplained. Some age-related neurodegenerative diseases have an etiology that is related to mitochondrial dysfunction. Here, we found that alveolar type II cells (AECIIs) in the lungs of IPF patients exhibit marked accumulation of dysmorphic and dysfunctional mitochondria. These mitochondrial abnormalities in AECIIs of IPF lungs were associated with upregulation of ER stress markers and were recapitulated in normal mice with advancing age in response to stimulation of ER stress. We found that impaired mitochondria in IPF and aging lungs were associated with low expression of PTEN-induced putative kinase 1 (PINK1). Knockdown of PINK1 expression in lung epithelial cells resulted in mitochondria depolarization and expression of profibrotic factors. Moreover, young PINK1-deficient mice developed similarly dysmorphic, dysfunctional mitochondria in the AECIIs and were vulnerable to apoptosis and development of lung fibrosis. Our data indicate that PINK1 deficiency results in swollen, dysfunctional mitochondria and defective mitophagy, and promotes fibrosis in the aging lung. 10.1172/JCI74942
    Regulator of telomere length 1 () mutations are associated with heterogeneous pulmonary and extra-pulmonary phenotypes. Borie Raphael,Bouvry Diane,Cottin Vincent,Gauvain Clement,Cazes Aurélie,Debray Marie-Pierre,Cadranel Jacques,Dieude Philippe,Degot Tristan,Dominique Stephane,Gamez Anne Sophie,Jaillet Madeleine,Juge Pierre-Antoine,Londono-Vallejo Arturo,Mailleux Arnaud,Mal Hervé,Boileau Catherine,Menard Christelle,Nunes Hilario,Prevot Gregoire,Quetant Sebastien,Revy Patrick,Traclet Julie,Wemeau-Stervinou Lidwine,Wislez Marie,Kannengiesser Caroline,Crestani Bruno The European respiratory journal Regulator of telomere length 1 () mutations have been evidenced in 5-9% of familial pulmonary fibrosis; however, the phenotype of patients with interstitial lung disease (ILD) and mutations is poorly understood.Whole exome sequencing was performed in 252 probands with ILD and we included all patients with ILD and mutation. expression was evaluated by immunochemistry in the lungs of controls, as well as in and telomerase reverse transcriptase () mutation carriers.We identified 35 subjects from 17 families. Median age at diagnosis of ILD was 53.1 years (range 28.0-80.6). The most frequent pulmonary diagnoses were idiopathic pulmonary fibrosis (n=20, 57%), secondary ILD (n=7, 20%) and unclassifiable fibrosis or interstitial pneumonia with autoimmune features (n=7, 20%). The median transplant-free and overall survival periods were 39.2 months and 45.3 months, respectively. Forced vital capacity at diagnosis was the only factor associated with decreased transplant-free survival. Extra-pulmonary manifestations were less frequent as compared to other telomere-related gene mutation carriers. A systematic analysis of the literature identified 110 patients with ILD and mutations (including this series) and confirmed the heterogeneity of the pulmonary phenotype, the prevalence of non-idiopathic diseases and the low prevalence of extra-pulmonary manifestations.Immunohistochemistry showed that was expressed by bronchial and alveolar epithelial cells, as well as by alveolar macrophages and lymphocytes, but not by fibroblasts. 10.1183/13993003.00508-2018
    Lung injury and fibrosis induced by a mutant form of surfactant protein C. Blackwell Timothy S The Journal of clinical investigation Although mutant forms of the gene encoding surfactant protein C (SFTPC) have been linked to interstitial lung disease, the mechanisms by which the most common of these mutations, SFTPCI73T, results in lung fibrosis are uncertain. In this issue of the JCI, Nureki et al. developed a knockin mouse model and showed that SFTPCI73T is expressed by alveolar type II (AT2) epithelial cells in the lungs. These mice developed an age-related fibrotic phenotype when the mutant allele was expressed at low levels and acute lung inflammation/injury followed by lung fibrosis when mutant SFTPCI73T expression was enhanced. This work provides important information regarding the impact of AT2 cell dysfunction on fibrotic remodeling in the lungs. 10.1172/JCI122727
    Diminished prostaglandin E2 contributes to the apoptosis paradox in idiopathic pulmonary fibrosis. Maher Toby M,Evans Iona C,Bottoms Stephen E,Mercer Paul F,Thorley Andrew J,Nicholson Andrew G,Laurent Geoffrey J,Tetley Teresa D,Chambers Rachel C,McAnulty Robin J American journal of respiratory and critical care medicine RATIONALE:Patients with idiopathic pulmonary fibrosis (IPF), a progressive disease with a dismal prognosis, exhibit an unexplained disparity of increased alveolar epithelial cell (AEC) apoptosis but reduced fibroblast apoptosis. OBJECTIVES:To examine whether the failure of patients with IPF to up-regulate cyclooxygenase (COX)-2, and thus the antifibrotic mediator prostaglandin (PG)E(2), accounts for this imbalance. METHODS:Fibroblasts and primary type II AECs were isolated from control and fibrotic human lung tissue. The effects of COX-2 inhibition and exogenous PGE(2) on fibroblast and AEC sensitivity to Fas ligand (FasL)-induced apoptosis were assessed. MEASUREMENTS AND MAIN RESULTS:IPF lung fibroblasts are resistant to FasL-induced apoptosis compared with control lung fibroblasts. Inhibition of COX-2 in control lung fibroblasts resulted in an apoptosis-resistant phenotype. Administration of PGE(2) almost doubled the rate of FasL-induced apoptosis in fibrotic lung fibroblasts compared with FasL alone. Conversely, in primary fibrotic lung type II AECs, PGE(2) protected against FasL-induced apoptosis. In human control and, to a greater extent, fibrotic lung fibroblasts, PGE(2) inhibits the phosphorylation of Akt, suggesting that regulation of this prosurvival protein kinase is an important mechanism by which PGE(2) modulates cellular apoptotic responses. CONCLUSIONS:The observation that PGE(2) deficiency results in increased AEC but reduced fibroblast sensitivity to apoptosis provides a novel pathogenic insight into the mechanisms driving persistent fibroproliferation in IPF. 10.1164/rccm.200905-0674OC
    Early alveolar epithelial dysfunction promotes lung inflammation in a mouse model of Hermansky-Pudlak syndrome. Atochina-Vasserman Elena N,Bates Sandra R,Zhang Peggy,Abramova Helen,Zhang Zhenguo,Gonzales Linda,Tao Jian-Qin,Gochuico Bernadette R,Gahl William,Guo Chang-Jiang,Gow Andrew J,Beers Michael F,Guttentag Susan American journal of respiratory and critical care medicine RATIONALE:The pulmonary phenotype of Hermansky-Pudlak syndrome (HPS) in adults includes foamy alveolar type 2 cells, inflammation, and lung remodeling, but there is no information about ontogeny or early disease mediators. OBJECTIVES:To establish the ontogeny of HPS lung disease in an animal model, examine disease mediators, and relate them to patients with HPS1. METHODS:Mice with mutations in both HPS1/pale ear and HPS2/AP3B1/pearl (EPPE mice) were studied longitudinally. Total lung homogenate, lung tissue sections, and bronchoalveolar lavage (BAL) were examined for phospholipid, collagen, histology, cell counts, chemokines, surfactant protein D (SP-D), and S-nitrosylated SP-D. Isolated alveolar epithelial cells were examined for expression of inflammatory mediators, and chemotaxis assays were used to assess their importance. Pulmonary function test results and BAL from patients with HPS1 and normal volunteers were examined for clinical correlation. MEASUREMENTS AND MAIN RESULTS:EPPE mice develop increased total lung phospholipid, followed by a macrophage-predominant pulmonary inflammation, and lung remodeling including fibrosis. BAL fluid from EPPE animals exhibited early accumulation of both SP-D and S-nitrosylated SP-D. BAL fluid from patients with HPS1 exhibited similar changes in SP-D that correlated inversely with pulmonary function. Alveolar epithelial cells demonstrated expression of both monocyte chemotactic protein (MCP)-1 and inducible nitric oxide synthase in juvenile EPPE mice. Last, BAL from EPPE mice and patients with HPS1 enhanced migration of RAW267.4 cells, which was attenuated by immunodepletion of SP-D and MCP-1. CONCLUSIONS:Inflammation is initiated from the abnormal alveolar epithelial cells in HPS, and S-nitrosylated SP-D plays a significant role in amplifying pulmonary inflammation. 10.1164/rccm.201011-1882OC
    Intratracheal transplantation of alveolar type II cells reverses bleomycin-induced lung fibrosis. Serrano-Mollar Anna,Nacher Maria,Gay-Jordi Gemma,Closa Daniel,Xaubet Antoni,Bulbena Oriol American journal of respiratory and critical care medicine RATIONALE:Transplantation of stem cells has been proposed as a strategy for repair of lung fibrosis. Nevertheless, many studies have yielded controversial results that currently limit the potential use of these cells as an efficient treatment. Alveolar type II cells are the progenitor cells of the pulmonary epithelium and usually proliferate after epithelial cell injury. During lung fibrosis, however, the altered regeneration process leads to uncontrolled fibroblast proliferation. OBJECTIVES:To investigate whether intratracheal transplantation of isolated alveolar type II cells can halt and reverse the fibrotic process in an experimental model of bleomycin-induced lung fibrosis in rats. METHODS:Lung fibrosis was induced in syngeneic female Lewis rats by a single intratracheal instillation of bleomycin (2.5 U/kg). Animals were transplanted with alveolar type II cells from male animals at a dose of 2.5 x 10(6) cells per animal 3, 7, and 15 days after endotracheal bleomycin instillation. Animals were killed 21 days after the induction of lung fibrosis. MEASUREMENTS AND MAIN RESULTS:Lung fibrosis was assessed by histologic study and determination of hydroxyproline content. Engraftment of transplanted cells was measured by real-time polymerase chain reaction for the Y chromosome and by fluorescence in situ hybridization for the Y chromosome. Transplantation of alveolar type II cells into damaged lung 3, 7, or 15 days after bleomycin instillation led to reduced collagen deposition, and reduction in the severity of pulmonary fibrosis. CONCLUSIONS:This study demonstrates the potential role of alveolar type II cell transplantation in designing future therapies for lung fibrosis. 10.1164/rccm.200610-1491OC
    Recombinant Acid Ceramidase Reduces Inflammation and Infection in Cystic Fibrosis. Gardner Aaron I,Haq Iram J,Simpson A John,Becker Katrin A,Gallagher John,Saint-Criq Vinciane,Verdon Bernard,Mavin Emily,Trigg Alexandra,Gray Michael A,Koulman Albert,McDonnell Melissa J,Fisher Andrew J,Kramer Elizabeth L,Clancy John P,Ward Christopher,Schuchman Edward H,Gulbins Erich,Brodlie Malcolm American journal of respiratory and critical care medicine In cystic fibrosis the major cause of morbidity and mortality is lung disease characterized by inflammation and infection. The influence of sphingolipid metabolism is poorly understood with a lack of studies using human airway model systems. To investigate sphingolipid metabolism in cystic fibrosis and the effects of treatment with recombinant human acid ceramidase on inflammation and infection. Sphingolipids were measured using mass spectrometry in fully differentiated cultures of primary human airway epithelial cells and cocultures with . activity assays, Western blotting, and quantitative PCR were used to investigate function and expression of ceramidase and sphingomyelinase. Effects of treatment with recombinant human acid ceramidase on sphingolipid profile and inflammatory mediator production were assessed in cell cultures and murine models. Ceramide is increased in cystic fibrosis airway epithelium owing to differential function of enzymes regulating sphingolipid metabolism. Sphingosine, a metabolite of ceramide with antimicrobial properties, is not upregulated in response to by cystic fibrosis airway epithelia. Tumor necrosis factor receptor 1 is increased in cystic fibrosis epithelia and activates NF-κB signaling, generating inflammation. Treatment with recombinant human acid ceramidase, to decrease ceramide, reduced both inflammatory mediator production and susceptibility to infection. Sphingolipid metabolism is altered in airway epithelial cells cultured from people with cystic fibrosis. Treatment with recombinant acid ceramidase ameliorates the two pivotal features of cystic fibrosis lung disease, inflammation and infection, and thus represents a therapeutic approach worthy of further exploration. 10.1164/rccm.202001-0180OC
    A transitional stem cell state in the lung. Verheyden Jamie M,Sun Xin Nature cell biology 10.1038/s41556-020-0561-5
    Increased alveolar soluble annexin V promotes lung inflammation and fibrosis. Buckley Susan,Shi Wei,Xu Wei,Frey Mark R,Moats Rex,Pardo Annie,Selman Moises,Warburton David The European respiratory journal The causes underlying the self-perpetuating nature of idiopathic pulmonary fibrosis (IPF), a progressive and usually lethal disease, remain unknown. We hypothesised that alveolar soluble annexin V contributes to lung fibrosis, based on the observation that human IPF bronchoalveolar lavage fluid (BALF) containing high annexin V levels promoted fibroblast involvement in alveolar epithelial wound healing that was reduced when annexin V was depleted from the BALF. Conditioned medium from annexin V-treated alveolar epithelial type 2 cells (AEC2), but not annexin V per se, induced proliferation of human fibroblasts and contained pro-fibrotic, IPF-associated proteins, as well as pro-inflammatory cytokines that were found to correlate tightly (r>0.95) with annexin V levels in human BALF. ErbB2 receptor tyrosine kinase in AECs was activated by annexin V, and blockade reduced the fibrotic potential of annexin V-treated AEC-conditioned medium. In vivo, aerosol delivery of annexin V to mouse lung induced inflammation, fibrosis and increased hydroxyproline, with activation of Wnt, transforming growth factor-β, mitogen-activated protein kinase and nuclear factor-κB signalling pathways, as seen in IPF. Chronically increased alveolar annexin V levels, as reflected in increased IPF BALF levels, may contribute to the progression of IPF by inducing the release of pro-fibrotic mediators. 10.1183/09031936.00002115
    Matrix metalloproteinase-19 is a key regulator of lung fibrosis in mice and humans. Yu Guoying,Kovkarova-Naumovski Elisabetha,Jara Paul,Parwani Anil,Kass Daniel,Ruiz Victor,Lopez-Otín Carlos,Rosas Ivan O,Gibson Kevin F,Cabrera Sandra,Ramírez Remedios,Yousem Samuel A,Richards Thomas J,Chensny Lara J,Selman Moisés,Kaminski Naftali,Pardo Annie American journal of respiratory and critical care medicine RATIONALE:Idiopathic pulmonary fibrosis (IPF) is a devastating disease characterized by epithelial phenotypic changes and fibroblast activation. Based on the temporal heterogeneity of IPF, we hypothesized that hyperplastic alveolar epithelial cells regulate the fibrotic response. OBJECTIVES:To identify novel mediators of fibrosis comparing the transcriptional signature of hyperplastic epithelial cells and conserved epithelial cells in the same lung. METHODS:Laser capture microscope and microarrays analysis were used to identify differentially expressed genes in IPF lungs. Bleomycin-induced lung fibrosis was evaluated in Mmp19-deficient and wild-type (WT) mice. The role of matrix metalloproteinase (MMP)-19 was additionally studied by transfecting the human MMP19 in alveolar epithelial cells. MEASUREMENTS AND MAIN RESULTS:Laser capture microscope followed by microarray analysis revealed a novel mediator, MMP-19, in hyperplastic epithelial cells adjacent to fibrotic regions. Mmp19(-/-) mice showed a significantly increased lung fibrotic response to bleomycin compared with WT mice. A549 epithelial cells transfected with human MMP19 stimulated wound healing and cell migration, whereas silencing MMP19 had the opposite effect. Gene expression microarray of transfected A549 cells showed that PTGS2 (prostaglandin-endoperoxide synthase 2) was one of the highly induced genes. PTGS2 was overexpressed in IPF lungs and colocalized with MMP-19 in hyperplastic epithelial cells. In WT mice, PTGS2 was significantly increased in bronchoalveolar lavage and lung tissues after bleomycin-induced fibrosis, but not in Mmp19(-/-) mice. Inhibition of Mmp-19 by siRNA resulted in inhibition of Ptgs2 at mRNA and protein levels. CONCLUSIONS:Up-regulation of MMP19 induced by lung injury may play a protective role in the development of fibrosis through the induction of PTGS2. 10.1164/rccm.201202-0302OC
    Syndecan-2 exerts antifibrotic effects by promoting caveolin-1-mediated transforming growth factor-β receptor I internalization and inhibiting transforming growth factor-β1 signaling. Shi Yuanyuan,Gochuico Bernadette R,Yu Guoying,Tang Xiaomeng,Osorio Juan C,Fernandez Isis E,Risquez Cristobal F,Patel Avignat S,Shi Ying,Wathelet Marc G,Goodwin Andrew J,Haspel Jeffrey A,Ryter Stefan W,Billings Eric M,Kaminski Naftali,Morse Danielle,Rosas Ivan O American journal of respiratory and critical care medicine RATIONALE:Alveolar transforming growth factor (TGF)-β1 signaling and expression of TGF-β1 target genes are increased in patients with idiopathic pulmonary fibrosis (IPF) and in animal models of pulmonary fibrosis. Internalization and degradation of TGF-β receptor TβRI inhibits TGF-β signaling and could attenuate development of experimental lung fibrosis. OBJECTIVES:To demonstrate that after experimental lung injury, human syndecan-2 confers antifibrotic effects by inhibiting TGF-β1 signaling in alveolar epithelial cells. METHODS:Microarray assays were performed to identify genes differentially expressed in alveolar macrophages of patients with IPF versus control subjects. Transgenic mice that constitutively overexpress human syndecan-2 in macrophages were developed to test the antifibrotic properties of syndecan-2. In vitro assays were performed to determine syndecan-2-dependent changes in epithelial cell TGF-β1 signaling, TGF-β1, and TβRI internalization and apoptosis. Wild-type mice were treated with recombinant human syndecan-2 during the fibrotic phase of bleomycin-induced lung injury. MEASUREMENTS AND MAIN RESULTS:We observed significant increases in alveolar macrophage syndecan-2 levels in patients with IPF. Macrophage-specific overexpression of human syndecan-2 in transgenic mice conferred antifibrotic effects after lung injury by inhibiting TGF-β1 signaling and downstream expression of TGF-β1 target genes, reducing extracellular matrix production and alveolar epithelial cell apoptosis. In vitro, syndecan-2 promoted caveolin-1-dependent internalization of TGF-β1 and TβRI in alveolar epithelial cells, which inhibited TGF-β1 signaling and epithelial cell apoptosis. Therapeutic administration of human syndecan-2 abrogated lung fibrosis in mice. CONCLUSIONS:Alveolar macrophage syndecan-2 exerts antifibrotic effects by promoting caveolin-1-dependent TGF-β1 and TβRI internalization and inhibiting TGF-β1 signaling in alveolar epithelial cells. Hence, molecules that facilitate TβRI degradation via endocytosis represent potential therapies for pulmonary fibrosis. 10.1164/rccm.201303-0434OC
    Muc5b overexpression causes mucociliary dysfunction and enhances lung fibrosis in mice. Hancock Laura A,Hennessy Corinne E,Solomon George M,Dobrinskikh Evgenia,Estrella Alani,Hara Naoko,Hill David B,Kissner William J,Markovetz Matthew R,Grove Villalon Diane E,Voss Matthew E,Tearney Guillermo J,Carroll Kate S,Shi Yunlong,Schwarz Marvin I,Thelin William R,Rowe Steven M,Yang Ivana V,Evans Christopher M,Schwartz David A Nature communications The gain-of-function MUC5B promoter variant rs35705950 is the dominant risk factor for developing idiopathic pulmonary fibrosis (IPF). Here we show in humans that MUC5B, a mucin thought to be restricted to conducting airways, is co-expressed with surfactant protein C (SFTPC) in type 2 alveolar epithelia and in epithelial cells lining honeycomb cysts, indicating that cell types involved in lung fibrosis in distal airspace express MUC5B. In mice, we demonstrate that Muc5b concentration in bronchoalveolar epithelia is related to impaired mucociliary clearance (MCC) and to the extent and persistence of bleomycin-induced lung fibrosis. We also establish the ability of the mucolytic agent P-2119 to restore MCC and to suppress bleomycin-induced lung fibrosis in the setting of Muc5b overexpression. Our findings suggest that mucociliary dysfunction might play a causative role in bleomycin-induced pulmonary fibrosis in mice overexpressing Muc5b, and that MUC5B in distal airspaces is a potential therapeutic target in humans with IPF. 10.1038/s41467-018-07768-9
    Cell cycle dependence of ACE-2 explains downregulation in idiopathic pulmonary fibrosis. Uhal Bruce D,Dang MyTrang,Dang Vinh,Llatos Roger,Cano Esteban,Abdul-Hafez Amal,Markey Jonathan,Piasecki Christopher C,Molina-Molina Maria The European respiratory journal Alveolar epithelial type II cells, a major source of angiotensin-converting enzyme (ACE)-2 in the adult lung, are normally quiescent but actively proliferate in lung fibrosis and downregulate this protective enzyme. It was, therefore, hypothesised that ACE-2 expression might be related to cell cycle progression. To test this hypothesis, ACE-2 mRNA levels, protein levels and enzymatic activity were examined in fibrotic human lungs and in the alveolar epithelial cell lines A549 and MLE-12 studied at postconfluent (quiescent) versus subconfluent (proliferating) densities. ACE-2 mRNA, immunoreactive protein and enzymatic activity were all high in quiescent cells, but were severely downregulated or absent in actively proliferating cells. Upregulation of the enzyme in cells that were progressing to quiescence was completely inhibited by the transcription blocker actinomycin D or by SP600125, an inhibitor of c-Jun N-terminal kinase (JNK). In lung biopsy specimens obtained from patients with idiopathic pulmonary fibrosis, immunoreactive enzyme was absent in alveolar epithelia that were positive for proliferation markers, but was robustly expressed in alveolar epithelia devoid of proliferation markers. These data explain the loss of ACE-2 in lung fibrosis and demonstrate cell cycle-dependent regulation of this protective enzyme by a JNK-mediated transcriptional mechanism. 10.1183/09031936.00015612
    WNT1-inducible signaling protein-1 mediates pulmonary fibrosis in mice and is upregulated in humans with idiopathic pulmonary fibrosis. Königshoff Melanie,Kramer Monika,Balsara Nisha,Wilhelm Jochen,Amarie Oana Veronica,Jahn Andreas,Rose Frank,Fink Ludger,Seeger Werner,Schaefer Liliana,Günther Andreas,Eickelberg Oliver The Journal of clinical investigation 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. 10.1172/JCI33950
    Inhibition and role of let-7d in idiopathic pulmonary fibrosis. Pandit Kusum V,Corcoran David,Yousef Hanadie,Yarlagadda Manohar,Tzouvelekis Argyris,Gibson Kevin F,Konishi Kazuhisa,Yousem Samuel A,Singh Mandal,Handley Daniel,Richards Thomas,Selman Moises,Watkins Simon C,Pardo Annie,Ben-Yehudah Ahmi,Bouros Demosthenes,Eickelberg Oliver,Ray Prabir,Benos Panayiotis V,Kaminski Naftali American journal of respiratory and critical care medicine RATIONALE:Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, and usually lethal fibrotic lung disease characterized by profound changes in epithelial cell phenotype and fibroblast proliferation. OBJECTIVES:To determine changes in expression and role of microRNAs in IPF. METHODS:RNA from 10 control and 10 IPF tissues was hybridized on Agilent microRNA microarrays and results were confirmed by quantitative real-time polymerase chain reaction and in situ hybridization. SMAD3 binding to the let-7d promoter was confirmed by chromatin immunoprecipitation, electrophoretic mobility shift assay, luciferase assays, and reduced expression of let-7d in response to transforming growth factor-beta. HMGA2, a let-7d target, was localized by immunohistochemistry. In mice, let-7d was inhibited by intratracheal administration of a let-7d antagomir and its effects were determined by immunohistochemistry, immunofluorescence, quantitative real-time polymerase chain reaction, and morphometry. MEASUREMENTS AND MAIN RESULTS:Eighteen microRNAs including let-7d were significantly decreased in IPF. Transforming growth factor-beta down-regulated let-7d expression, and SMAD3 binding to the let-7d promoter was demonstrated. Inhibition of let-7d caused increases in mesenchymal markers N-cadherin-2, vimentin, and alpha-smooth muscle actin (ACTA2) as well as HMGA2 in multiple epithelial cell lines. let-7d was significantly reduced in IPF lungs and the number of epithelial cells expressing let-7d correlated with pulmonary functions. HMGA2 was increased in alveolar epithelial cells of IPF lungs. let-7d inhibition in vivo caused alveolar septal thickening and increases in collagen, ACTA2, and S100A4 expression in SFTPC (pulmonary-associated surfactant protein C) expressing alveolar epithelial cells. CONCLUSIONS:Our results indicate a role for microRNAs in IPF. The down-regulation of let-7d in IPF and the profibrotic effects of this down-regulation in vitro and in vivo suggest a key regulatory role for this microRNA in preventing lung fibrosis. Clinical trial registered with www.clinicaltrials.gov (NCT 00258544). 10.1164/rccm.200911-1698OC
    β-catenin in the alveolar epithelium protects from lung fibrosis after intratracheal bleomycin. Tanjore Harikrishna,Degryse Amber L,Crossno Peter F,Xu Xiaochuan C,McConaha Melinda E,Jones Brittany R,Polosukhin Vasiliy V,Bryant Andrew J,Cheng Dong-Sheng,Newcomb Dawn C,McMahon Frank B,Gleaves Linda A,Blackwell Timothy S,Lawson William E American journal of respiratory and critical care medicine RATIONALE:Alveolar epithelial cells (AECs) play central roles in the response to lung injury and the pathogenesis of pulmonary fibrosis. OBJECTIVES:We aimed to determine the role of β-catenin in alveolar epithelium during bleomycin-induced lung fibrosis. METHODS:Genetically modified mice were developed to selectively delete β-catenin in AECs and were crossed to cell fate reporter mice that express β-galactosidase (βgal) in cells of AEC lineage. Mice were given intratracheal bleomycin (0.04 units) and assessed for AEC death, inflammation, lung injury, and fibrotic remodeling. Mouse lung epithelial cells (MLE12) with small interfering RNA knockdown of β-catenin underwent evaluation for wound closure, proliferation, and bleomycin-induced cytotoxicity. MEASUREMENTS AND MAIN RESULTS:Increased β-catenin expression was noted in lung parenchyma after bleomycin. Mice with selective deletion of β-catenin in AECs had greater AEC death at 1 week after bleomycin, followed by increased numbers of fibroblasts and enhanced lung fibrosis as determined by semiquantitative histological scoring and total collagen content. However, no differences in lung inflammation or protein levels in bronchoalveolar lavage were noted. In vitro, β-catenin-deficient AECs showed increased bleomycin-induced cytotoxicity as well as reduced proliferation and impaired wound closure. Consistent with these findings, mice with AEC β-catenin deficiency showed delayed recovery after bleomycin. CONCLUSIONS:β-Catenin in the alveolar epithelium protects against bleomycin-induced fibrosis. Our studies suggest that AEC survival and wound healing are enhanced through β-catenin-dependent mechanisms. Activation of the developmentally important β-catenin pathway in AECs appears to contribute to epithelial repair after epithelial injury. 10.1164/rccm.201205-0972OC
    Identification of periplakin as a new target for autoreactivity in idiopathic pulmonary fibrosis. Taillé Camille,Grootenboer-Mignot Sabine,Boursier Céline,Michel Laurence,Debray Marie-Pierre,Fagart Jérôme,Barrientos Lorena,Mailleux Arnaud,Cigna Natacha,Tubach Florence,Marchal-Sommé Joëlle,Soler Paul,Chollet-Martin Sylvie,Crestani Bruno American journal of respiratory and critical care medicine RATIONALE:Injury to alveolar epithelial cells is central to the pathophysiology of idiopathic pulmonary fibrosis (IPF). An abnormal autoimmune response directed against antigens of the alveolar epithelium may contribute to the disease. OBJECTIVES:To detect circulating autoantibodies (autoAbs) directed against epithelial structures. METHODS:We performed immunoblot by separating human placental amnion extract or alveolar epithelial cell (A549 cell line) proteins on polyacrylamide gels, blotting on nitrocellulose membranes, and incubating with serum from patients with IPF (n = 40) or healthy subjects (n = 40). Proteomic analysis and mass spectrometry characterized the target protein. Inhibition experiments performed with the correspondent recombinant protein confirmed our results. MEASUREMENTS AND MAIN RESULTS:We identified IgG autoAbs recognizing a 200-kD protein in the serum of patients with IPF. Proteomic analysis identified this protein as human periplakin (PPL), a component of desmosomes. Anti-PPL Abs were found by immunoblot in both serum and bronchoalveolar lavage in patients with IPF: 16/40 (40%) of them were positive versus none of the control subjects. Immunohistochemistry revealed that PPL was strongly expressed in bronchial and alveolar epithelium, but that PPL exhibited changes in intracellular localization among normal and fibrotic alveolar epithelium. In an alveolar epithelial wound repair assay, an anti-PPL IgG decreased cell migration. Recombinant PPL induced bronchoalveolar lavage T lymphocyte proliferation. Patients with IPF with anti-PPL Abs had a more severe respiratory disease, despite no difference in survival. CONCLUSIONS:We found a new circulating autoAb directed against PPL in patients with IPF, associated with a more severe disease. 10.1164/rccm.201001-0076OC
    Epithelial endoplasmic reticulum stress and apoptosis in sporadic idiopathic pulmonary fibrosis. Korfei Martina,Ruppert Clemens,Mahavadi Poornima,Henneke Ingrid,Markart Philipp,Koch Miriam,Lang Gyoergy,Fink Ludger,Bohle Rainer-Maria,Seeger Werner,Weaver Timothy E,Guenther Andreas American journal of respiratory and critical care medicine RATIONALE:The molecular pathomechanisms underlying idiopathic pulmonary fibrosis (IPF) are elusive, but chronic epithelial injury has recently been suggested as key event. OBJECTIVES:We investigated the possible implication of endoplasmic reticulum (ER) stress-mediated apoptosis in sporadic IPF. METHODS:We analyzed peripheral explanted lung tissues from patients with sporadic IPF (n = 24), chronic obstructive pulmonary disease (COPD) (n = 9), and organ donors (n = 12) for expression of major ER stress mediators and apoptosis markers by means of immunoblotting, semiquantitative reverse transcription-polymerase chain reaction, immunohistochemistry, and the TUNEL method. MEASUREMENTS AND MAIN RESULTS:Compared with COPD and donor lungs, protein levels of ER stress mediators, such as processed p50 activating transcription factor (ATF)-6 and ATF-4 and the apoptosis-inductor CHOP (C/EBP-homologous protein), as well as transcript levels of spliced X-box binding protein (XBP)-1, were significantly elevated in lung homogenates and type II alveolar epithelial cells (AECIIs) of IPF lungs. Proapoptotic, oligomeric forms of Bax, which play a key role in ER stress-mediated apoptosis downstream of CHOP induction, as well as caspase-3 cleavage, could be detected in IPF lungs. By means of immunohistochemistry, exclusive induction of active ATF-6, ATF-4, and CHOP in AECIIs was encountered in IPF but not in COPD or donor lungs. Immunoreactivity was most prominent in the epithelium near dense zones of fibrosis and fibroblast foci, where these ER stress markers colocalized with markers of apoptosis (TUNEL, cleaved caspase-3). CONCLUSIONS:Severe ER stress response in the AECIIs of patients with sporadic IPF may underlie the apoptosis of this cell type and development of fibrosis in this disease. 10.1164/rccm.200802-313OC
    Idiopathic pulmonary fibrosis. King Talmadge E,Pardo Annie,Selman Moisés Lancet (London, England) Idiopathic pulmonary fibrosis is a devastating, age-related lung disease of unknown cause that has few treatment options. This disease was once thought to be a chronic inflammatory process, but current evidence indicates that the fibrotic response is driven by abnormally activated alveolar epithelial cells (AECs). These cells produce mediators that induce the formation of fibroblast and myofibroblast foci through the proliferation of resident mesenchymal cells, attraction of circulating fibrocytes, and stimulation of the epithelial to mesenchymal transition. The fibroblast and myofibroblast foci secrete excessive amounts of extracellular matrix, mainly collagens, resulting in scarring and destruction of the lung architecture. The mechanisms that link idiopathic pulmonary fibrosis with ageing and aberrant epithelial activation are unknown; evidence suggests that the abnormal recapitulation of developmental pathways and epigenetic changes have a role. In this Seminar, we review recent data on the clinical course, therapeutic options, and underlying mechanisms thought to be involved in the pathogenesis of idiopathic pulmonary fibrosis. 10.1016/S0140-6736(11)60052-4
    Deranged fatty acid composition causes pulmonary fibrosis in Elovl6-deficient mice. Sunaga Hiroaki,Matsui Hiroki,Ueno Manabu,Maeno Toshitaka,Iso Tatsuya,Syamsunarno Mas Rizky A A,Anjo Saki,Matsuzaka Takashi,Shimano Hitoshi,Yokoyama Tomoyuki,Kurabayashi Masahiko Nature communications Despite the established role of alveolar type II epithelial cells for the maintenance of pulmonary function, little is known about the deregulation of lipid composition in the pathogenesis of pulmonary fibrosis. The elongation of long-chain fatty acids family member 6 (Elovl6) is a rate-limiting enzyme catalysing the elongation of saturated and monounsaturated fatty acids. Here we show that Elovl6 expression is significantly downregulated after an intratracheal instillation of bleomycin (BLM) and in human lung with idiopathic pulmonary fibrosis. Elovl6-deficient (Elovl6⁻/⁻) mice treated with BLM exhibit severe fibroproliferative response and derangement of fatty acid profile compared with wild-type mice. Furthermore, Elovl6 knockdown induces a change in fatty acid composition similar to that in Elovl6⁻/⁻ mice, resulting in induction of apoptosis, TGF-β1 expression and reactive oxygen species generation. Our findings demonstrate a previously unappreciated role for Elovl6 in the regulation of lung homeostasis, and in pathogenesis and exacerbation of BLM-induced pulmonary fibrosis. 10.1038/ncomms3563
    Age-driven developmental drift in the pathogenesis of idiopathic pulmonary fibrosis. Selman Moisés,López-Otín Carlos,Pardo Annie The European respiratory journal Idiopathic pulmonary fibrosis (IPF) is a progressive and usually lethal disease of unknown aetiology. A growing body of evidence supports that IPF represents an epithelial-driven process characterised by aberrant epithelial cell behaviour, fibroblast/myofibroblast activation and excessive accumulation of extracellular matrix with the subsequent destruction of the lung architecture. The mechanisms involved in the abnormal hyper-activation of the epithelium are unclear, but we propose that recapitulation of pathways and processes critical to embryological development associated with a tissue specific age-related stochastic epigenetic drift may be implicated. These pathways may also contribute to the distinctive behaviour of IPF fibroblasts. Genomic and epigenomic studies have revealed that wingless/Int, sonic hedgehog and other developmental signalling pathways are reactivated and deregulated in IPF. Moreover, some of these pathways cross-talk with transforming growth factor-β activating a profibrotic feedback loop. The expression pattern of microRNAs is also dysregulated in IPF and exhibits a similar expression profile to embryonic lungs. In addition, senescence, a process usually associated with ageing, which occurs early in alveolar epithelial cells of IPF lungs, likely represents a conserved programmed developmental mechanism. Here, we review the major developmental pathways that get twisted in IPF, and discuss the connection with ageing and potential therapeutic approaches. 10.1183/13993003.00398-2016
    Club cells inhibit alveolar epithelial wound repair via TRAIL-dependent apoptosis. Akram Khondoker M,Lomas Nicola J,Spiteri Monica A,Forsyth Nicholas R The European respiratory journal Club cells (Clara cells) participate in bronchiolar wound repair and regeneration. Located in the bronchioles, they become activated during alveolar injury in idiopathic pulmonary fibrosis (IPF) and migrate into the affected alveoli, a process called alveolar bronchiolisation. The purpose of this migration and the role of club cells in alveolar wound repair is controversial. This study was undertaken to investigate the role of club cells in alveolar epithelial wound repair and pulmonary fibrosis. A direct-contact co-culture in vitro model was used to evaluate the role of club cells (H441 cell line) on alveolar epithelial cell (A549 cell line) and small airway epithelial cell (SAEC) wound repair. Immunohistochemistry was conducted on lung tissue samples from patients with IPF to replicate the in vitro findings ex vivo. Our study demonstrated that club cells induce apoptosis in alveolar epithelial cells and SAECs through a tumour necrosis factor-related apoptosis-inducing ligand (TRAIL)-dependent mechanism resulting in significant inhibition of wound repair. Furthermore, in IPF lungs, TRAIL-expressing club cells were detected within the affected alveolar epithelia in areas of established fibrosis, together with widespread alveolar epithelial cell apoptosis. From these findings, we hypothesise that the extensive pro-fibrotic remodelling associated with IPF could be driven by TRAIL-expressing club cells inducing apoptosis in alveolar epithelial cells through a TRAIL-dependent mechanism. 10.1183/09031936.00213411
    Revealing the pathogenic and aging-related mechanisms of the enigmatic idiopathic pulmonary fibrosis. an integral model. Selman Moisés,Pardo Annie American journal of respiratory and critical care medicine A growing body of evidence indicates that aberrant activation of alveolar epithelial cells and fibroblasts in an aging lung plays a critical role in the pathogenesis of idiopathic pulmonary fibrosis (IPF). However, the biopathological processes linking aging with IPF and the mechanisms responsible for the abnormal activation of epithelial cells and fibroblasts have not been elucidated. Many of the hallmarks of aging (e.g., genomic instability, telomere attrition, epigenetic alterations, mitochondrial dysfunction, and cellular senescence) have been proposed as essential mechanisms for the development of IPF; however, these disturbances are not restricted to IPF and also occur in other aging-related lung disorders, primarily chronic obstructive pulmonary disease (COPD). Therefore, an unanswered question is why a current/former smoker of about 60 years of age with shorter telomeres, alveolar epithelial senescence, excessive oxidative stress, and mitochondrial dysfunction develops IPF and not COPD; in other words, what makes old lungs specifically susceptible to develop IPF? In this Perspective, we propose an integral model in which the combination of some gene variants and/or gene expression in the aging lung results in the loss of epithelial integrity and consequently in the failure of the alveoli to correctly respond to injury and to face the stress associated with mechanical stretch. Afterward, a distinctive epigenetic "reprogramming" that affects both epithelial cells and fibroblasts provokes, among others, the recapitulation of developmental pathways and the aberrant activation and miscommunication between both cell types, resulting in the exaggerated production and accumulation of extracellular matrix and the subsequent destruction of the lung architecture. 10.1164/rccm.201312-2221PP
    Epithelial Pten controls acute lung injury and fibrosis by regulating alveolar epithelial cell integrity. Miyoshi Kahori,Yanagi Shigehisa,Kawahara Kohichi,Nishio Miki,Tsubouchi Hironobu,Imazu Yoshifumi,Koshida Ryusuke,Matsumoto Nobuhiro,Taguchi Akiko,Yamashita Shu-ichi,Suzuki Akira,Nakazato Masamitsu American journal of respiratory and critical care medicine RATIONALE:Injury to alveolar epithelial cells (AECs) and to their repair process is integral to the pathogenesis of acute lung injury (ALI) and idiopathic pulmonary fibrosis (IPF). The mechanisms regulating the integrity of AECs and their intrinsic regulators remain unclear. Pten is a tumor suppressor, and its function in epithelial cells during organ fibrosis is unknown. OBJECTIVES:To determine the role of epithelial Pten in ALI and lung fibrosis. METHODS:Bronchioalveolar epithelium-specific Pten-deleted SP-C-rtTA/(tetO)(7)-Cre/Pten(Δ/Δ) (SOPten(Δ/Δ)) mice were studied by structural, biochemical, and physiologic analyses and compared with wild-type mice. Further mechanistic studies were performed in vivo, in vitro, and on samples from patients with IPF. MEASUREMENTS AND MAIN RESULTS:SOPten(Δ/Δ) mice demonstrated exacerbated alveolar flooding and subsequent augmented lung scarring with enhanced disassembly of tight junctions (TJs) of AECs and degradation of basement membranes. The induction of dominant negative PTEN gene in lung epithelial cells led to augmented transforming growth factor-1-induced disruptions of TJs. Epithelial-derived myofibroblasts were increased in the epithelium-specific Pten-deficient mice. The lungs of bleomycin-treated SOPten(Δ/Δ) mice showed increased pAkt, pS6K, Snail, and matrix metalloproteinase expressions and decreased claudin-4, E-cadherin, and laminin-β1 expressions. Akt inactivation definitively saved SOPten(Δ/Δ) mice through amelioration of ALI and retention of AEC integrity. We detected a reduction of PTEN expression and AKT hyperactivation in the AECs of human IPF lungs. CONCLUSIONS:Our results highlight epithelial Pten as a crucial gatekeeper controlling ALI and lung fibrosis by modulating AEC integrity, and the Pten/PI3K/Akt pathway as a potential therapeutic target in these intractable diseases. 10.1164/rccm.201205-0851OC
    Inhibiting eicosanoid degradation exerts antifibrotic effects in a pulmonary fibrosis mouse model and human tissue. Bärnthaler Thomas,Theiler Anna,Zabini Diana,Trautmann Sandra,Stacher-Priehse Elvira,Lanz Ilse,Klepetko Walter,Sinn Katharina,Flick Holger,Scheidl Stefan,Thomas Dominique,Olschewski Horst,Kwapiszewska Grazyna,Schuligoi Rufina,Heinemann Akos The Journal of allergy and clinical immunology BACKGROUND:Idiopathic pulmonary fibrosis (IPF) is a disease with high 5-year mortality and few therapeutic options. Prostaglandin (PG) E exhibits antifibrotic properties and is reduced in bronchoalveolar lavage from patients with IPF. 15-Prostaglandin dehydrogenase (15-PGDH) is the key enzyme in PGE metabolism under the control of TGF-β and microRNA 218. OBJECTIVE:We sought to investigate the expression of 15-PGDH in IPF and the therapeutic potential of a specific inhibitor of this enzyme in a mouse model and human tissue. METHODS:In vitro studies, including fibrocyte differentiation, regulation of 15-PGDH, RT-PCR, and Western blot, were performed using peripheral blood from healthy donors and patients with IPF and A549 cells. Immunohistochemistry, immunofluorescence, 15-PGDH activity assays, and in situ hybridization as well as ex vivo IPF tissue culture experiments were done using healthy donor and IPF lungs. Therapeutic effects of 15-PGDH inhibition were studied in the bleomycin mouse model of pulmonary fibrosis. RESULTS:We demonstrate that 15-PGDH shows areas of increased expression in patients with IPF. Inhibition of this enzyme increases PGE levels and reduces collagen production in IPF precision cut lung slices and in the bleomycin model. Inhibitor-treated mice show amelioration of lung function, decreased alveolar epithelial cell apoptosis, and fibroblast proliferation. Pulmonary fibrocyte accumulation is also decreased by inhibitor treatment in mice, similar to PGE that inhibits fibrocyte differentiation from blood of healthy donors and patients with IPF. Finally, microRNA 218-5p, which is downregulated in patients with IPF, suppressed 15-PGDH expression in vivo and in vitro. CONCLUSIONS:These findings highlight the role of 15-PGDH in IPF and suggest 15-PGDH inhibition as a promising therapeutic approach. 10.1016/j.jaci.2019.11.032
    Time for a change: is idiopathic pulmonary fibrosis still idiopathic and only fibrotic? Wolters Paul J,Blackwell Timothy S,Eickelberg Oliver,Loyd James E,Kaminski Naftali,Jenkins Gisli,Maher Toby M,Molina-Molina Maria,Noble Paul W,Raghu Ganesh,Richeldi Luca,Schwarz Marvin I,Selman Moises,Wuyts Wim A,Schwartz David A The Lancet. Respiratory medicine Idiopathic pulmonary fibrosis (IPF) is a progressive, irreversible, and typically fatal lung disease characterised by subpleural fibrosis, subepithelial fibroblast foci, and microscopic honeycombing. Although understanding of the pathogenic mechanisms continues to evolve, evidence indicates that distal airway and alveolar epithelial cells are central drivers of the disease. In this Viewpoint, we review the history of naming and classifications used to define the disease now referred to as IPF, in the context of understanding the clinical presentation, causes, and pathogenesis of the disease. We aim to generate discussion on whether, given the substantial progress made in understanding the clinical, genetic, cellular, and molecular mechanisms involved in the development of IPF, a change of name should be considered. To initiate this discussion, we offer new suggestions to update the name of this disease and new approaches to classify all forms of pulmonary fibrosis. 10.1016/S2213-2600(18)30007-9
    The mitochondrial cardiolipin remodeling enzyme lysocardiolipin acyltransferase is a novel target in pulmonary fibrosis. Huang Long Shuang,Mathew Biji,Li Haiquan,Zhao Yutong,Ma Shwu-Fan,Noth Imre,Reddy Sekhar P,Harijith Anantha,Usatyuk Peter V,Berdyshev Evgeny V,Kaminski Naftali,Zhou Tong,Zhang Wei,Zhang Yanmin,Rehman Jalees,Kotha Sainath R,Gurney Travis O,Parinandi Narasimham L,Lussier Yves A,Garcia Joe G N,Natarajan Viswanathan American journal of respiratory and critical care medicine RATIONALE:Lysocardiolipin acyltransferase (LYCAT), a cardiolipin-remodeling enzyme regulating the 18:2 linoleic acid pattern of mammalian mitochondrial cardiolipin, is necessary for maintaining normal mitochondrial function and vascular development. We hypothesized that modulation of LYCAT expression in lung epithelium regulates development of pulmonary fibrosis. OBJECTIVES:To define a role for LYCAT in human and murine models of pulmonary fibrosis. METHODS:We analyzed the correlation of LYCAT expression in peripheral blood mononuclear cells (PBMCs) with the outcomes of pulmonary functions and overall survival, and used the murine models to establish the role of LYCAT in fibrogenesis. We studied the LYCAT action on cardiolipin remodeling, mitochondrial reactive oxygen species generation, and apoptosis of alveolar epithelial cells under bleomycin challenge. MEASUREMENTS AND MAIN RESULTS:LYCAT expression was significantly altered in PBMCs and lung tissues from patients with idiopathic pulmonary fibrosis (IPF), which was confirmed in two preclinical murine models of IPF, bleomycin- and radiation-induced pulmonary fibrosis. LYCAT mRNA expression in PBMCs directly and significantly correlated with carbon monoxide diffusion capacity, pulmonary function outcomes, and overall survival. In both bleomycin- and radiation-induced pulmonary fibrosis murine models, hLYCAT overexpression reduced several indices of lung fibrosis, whereas down-regulation of native LYCAT expression by siRNA accentuated fibrogenesis. In vitro studies demonstrated that LYCAT modulated bleomycin-induced cardiolipin remodeling, mitochondrial membrane potential, reactive oxygen species generation, and apoptosis of alveolar epithelial cells, potential mechanisms of LYCAT-mediated lung protection. CONCLUSIONS:This study is the first to identify modulation of LYCAT expression in fibrotic lungs and offers a novel therapeutic approach for ameliorating lung inflammation and pulmonary fibrosis. 10.1164/rccm.201310-1917OC
    Contributions of alveolar epithelial cell quality control to pulmonary fibrosis. Katzen Jeremy,Beers Michael F The Journal of clinical investigation Epithelial cell dysfunction has emerged as a central component of the pathophysiology of diffuse parenchymal diseases including idiopathic pulmonary fibrosis (IPF). Alveolar type 2 (AT2) cells represent a metabolically active lung cell population important for surfactant biosynthesis and alveolar homeostasis. AT2 cells and other distal lung epithelia, like all eukaryotic cells, contain an elegant quality control network to respond to intrinsic metabolic and biosynthetic challenges imparted by mutant protein conformers, dysfunctional subcellular organelles, and dysregulated telomeres. Failed AT2 quality control components (the ubiquitin-proteasome system, unfolded protein response, macroautophagy, mitophagy, and telomere maintenance) result in diverse cellular endophenotypes and molecular signatures including ER stress, defective autophagy, mitochondrial dysfunction, apoptosis, inflammatory cell recruitment, profibrotic signaling, and altered progenitor function that ultimately converge to drive downstream fibrotic remodeling in the IPF lung. As this complex network becomes increasingly better understood, opportunities will emerge to identify targets and therapeutic strategies for IPF. 10.1172/JCI139519
    Expression of mutant Sftpc in murine alveolar epithelia drives spontaneous lung fibrosis. Nureki Shin-Ichi,Tomer Yaniv,Venosa Alessandro,Katzen Jeremy,Russo Scott J,Jamil Sarita,Barrett Matthew,Nguyen Vivian,Kopp Meghan,Mulugeta Surafel,Beers Michael F The Journal of clinical investigation Epithelial cell dysfunction is postulated as an important component in the pathogenesis of idiopathic pulmonary fibrosis (IPF). Mutations in the surfactant protein C (SP-C) gene (SFTPC), an alveolar type II (AT2) cell-restricted protein, have been found in sporadic and familial IPF. To causally link these events, we developed a knockin mouse model capable of regulated expression of an IPF-associated isoleucine-to-threonine substitution at codon 73 (I73T) in Sftpc (SP-CI73T). Tamoxifen-treated SP-CI73T cohorts developed rapid increases in SftpcI73T mRNA and misprocessed proSP-CI73T protein accompanied by increased early mortality (days 7-14). This acute phase was marked by diffuse parenchymal lung injury, tissue infiltration by monocytes, polycellular alveolitis, and elevations in bronchoalveolar lavage and AT2 mRNA content of select inflammatory cytokines. Resolution of alveolitis (2-4 weeks), commensurate with a rise in TGF-β1, was followed by aberrant remodeling marked by collagen deposition, AT2 cell hyperplasia, α-smooth muscle actin-positive (α-SMA-positive) cells, and restrictive lung physiology. The translational relevance of the model was supported by detection of multiple IPF biomarkers previously reported in human cohorts. These data provide proof of principle that mutant SP-C expression in vivo causes spontaneous lung fibrosis, strengthening the role of AT2 cell dysfunction as a key upstream driver of IPF pathogenesis. 10.1172/JCI99287
    Is Progression of Pulmonary Fibrosis due to Ventilation-induced Lung Injury? Albert Richard K,Smith Bradford,Perlman Carrie E,Schwartz David A American journal of respiratory and critical care medicine 10.1164/rccm.201903-0497PP
    Senolytic drugs target alveolar epithelial cell function and attenuate experimental lung fibrosis . Lehmann Mareike,Korfei Martina,Mutze Kathrin,Klee Stephan,Skronska-Wasek Wioletta,Alsafadi Hani N,Ota Chiharu,Costa Rita,Schiller Herbert B,Lindner Michael,Wagner Darcy E,Günther Andreas,Königshoff Melanie The European respiratory journal Idiopathic pulmonary fibrosis (IPF) is a devastating lung disease with poor prognosis and limited therapeutic options. The incidence of IPF increases with age, and ageing-related mechanisms such as cellular senescence have been proposed as pathogenic drivers. The lung alveolar epithelium represents a major site of tissue injury in IPF and senescence of this cell population is probably detrimental to lung repair. However, the potential pathomechanisms of alveolar epithelial cell senescence and the impact of senolytic drugs on senescent lung cells and fibrosis remain unknown. Here we demonstrate that lung epithelial cells exhibit increased and expression as well as senescence-associated β-galactosidase activity in experimental and human lung fibrosis tissue and primary cells.Primary fibrotic mouse alveolar epithelial type (AT)II cells secreted increased amounts of senescence-associated secretory phenotype (SASP) factors , as analysed using quantitative PCR, mass spectrometry and ELISA. Importantly, pharmacological clearance of senescent cells by induction of apoptosis in fibrotic ATII cells or three-dimensional lung tissue cultures reduced SASP factors and extracellular matrix markers, while increasing alveolar epithelial markersThese data indicate that alveolar epithelial cell senescence contributes to lung fibrosis development and that senolytic drugs may be a viable therapeutic option for IPF. 10.1183/13993003.02367-2016
    Caveolin-1-derived peptide limits development of pulmonary fibrosis. Marudamuthu Amarnath Satheesh,Bhandary Yashodhar Prabhakar,Fan Liang,Radhakrishnan Vijay,MacKenzie BreAnne,Maier Esther,Shetty Shwetha Kumari,Nagaraja M R,Gopu Venkadesaperumal,Tiwari Nivedita,Zhang Yajie,Watts Alan B,Williams Robert O,Criner Gerald J,Bolla Sudhir,Marchetti Nathaniel,Idell Steven,Shetty Sreerama Science translational medicine Idiopathic pulmonary fibrosis (IPF) is a fatal fibrotic lung disease with a median 5-year survival of ~20%. Current U.S. Food and Drug Administration-approved pharmacotherapies slow progression of IPF, providing hope that even more effective treatments can be developed. Alveolar epithelial progenitor type II cell (AEC) apoptosis and proliferation, and accumulation of activated myofibroblasts or fibrotic lung fibroblasts (fLfs) contribute to the progression of IPF. Full-length caveolin-1 scaffolding domain peptide (CSP; amino acids 82 to 101 of Cav1: DGIWKASFTTFTVTKYWFYR) inhibits AEC apoptosis and fLf activation and expansion and attenuates PF in bleomycin (BLM)-induced lung injury in mice. Like full-length CSP, a seven-amino acid deletion fragment of CSP, CSP7 (FTTFTVT), demonstrated antifibrotic effects in murine models of lung fibrosis. When CSP7 was administered during the fibrotic phase in three preclinical models [single-dose BLM, repeated-dose BLM, and adenovirus expressing constitutively active transforming growth factor-β1 (Ad-TGF-β1)-induced established PF], CSP7 reduced extracellular matrix (ECM) markers characteristic of PF, increased AEC survival, and improved lung function. CSP7 is amenable to both systemic (intraperitoneal) or direct lung delivery in a nebulized or dry powder form. Furthermore, CSP7 treatment of end-stage human IPF lung tissue explants attenuated ECM production and promoted AEC survival. Ames testing for mutagenicity and in vitro human peripheral blood lymphocyte and in vivo mouse micronucleus transformation assays indicated that CSP7 is not carcinogenic. Together, these findings support the further development of CSP7 as an antifibrotic treatment for patients with IPF or other interstitial lung diseases. 10.1126/scitranslmed.aat2848
    Ineffectual Type 2-to-Type 1 Alveolar Epithelial Cell Differentiation in Idiopathic Pulmonary Fibrosis: Persistence of the KRT8 Transitional State. Jiang Peng,Gil de Rubio Rafael,Hrycaj Steven M,Gurczynski Stephen J,Riemondy Kent A,Moore Bethany B,Omary M Bishr,Ridge Karen M,Zemans Rachel L American journal of respiratory and critical care medicine 10.1164/rccm.201909-1726LE
    Long-term expanding human airway organoids for disease modeling. Sachs Norman,Papaspyropoulos Angelos,Zomer-van Ommen Domenique D,Heo Inha,Böttinger Lena,Klay Dymph,Weeber Fleur,Huelsz-Prince Guizela,Iakobachvili Nino,Amatngalim Gimano D,de Ligt Joep,van Hoeck Arne,Proost Natalie,Viveen Marco C,Lyubimova Anna,Teeven Luc,Derakhshan Sepideh,Korving Jeroen,Begthel Harry,Dekkers Johanna F,Kumawat Kuldeep,Ramos Emilio,van Oosterhout Matthijs Fm,Offerhaus G Johan,Wiener Dominique J,Olimpio Eduardo P,Dijkstra Krijn K,Smit Egbert F,van der Linden Maarten,Jaksani Sridevi,van de Ven Marieke,Jonkers Jos,Rios Anne C,Voest Emile E,van Moorsel Coline Hm,van der Ent Cornelis K,Cuppen Edwin,van Oudenaarden Alexander,Coenjaerts Frank E,Meyaard Linde,Bont Louis J,Peters Peter J,Tans Sander J,van Zon Jeroen S,Boj Sylvia F,Vries Robert G,Beekman Jeffrey M,Clevers Hans The EMBO journal Organoids are self-organizing 3D structures grown from stem cells that recapitulate essential aspects of organ structure and function. Here, we describe a method to establish long-term-expanding human airway organoids from broncho-alveolar resections or lavage material. The pseudostratified airway organoids consist of basal cells, functional multi-ciliated cells, mucus-producing secretory cells, and CC10-secreting club cells. Airway organoids derived from cystic fibrosis (CF) patients allow assessment of CFTR function in an organoid swelling assay. Organoids established from lung cancer resections and metastasis biopsies retain tumor histopathology as well as cancer gene mutations and are amenable to drug screening. Respiratory syncytial virus (RSV) infection recapitulates central disease features, dramatically increases organoid cell motility via the non-structural viral NS2 protein, and preferentially recruits neutrophils upon co-culturing. We conclude that human airway organoids represent versatile models for the study of hereditary, malignant, and infectious pulmonary disease. 10.15252/embj.2018100300
    Apoptosis in lung injury and fibrosis. Drakopanagiotakis F,Xifteri A,Polychronopoulos V,Bouros D The European respiratory journal Pulmonary fibrosis is characterised by fibroblast accumulation and alveolar epithelium denudation. Increased apoptosis of alveolar epithelial cells and decreased apoptosis of fibroblasts may play an important role in the pathogenesis of disease. Inflammatory cells can modulate apoptosis of other cell types, both by removal of apoptotic debris and by cytokine production, thus preserving a pro-fibrotic environment. In the present review, some of the mechanisms by which apoptosis may contribute to the pathogenesis of idiopathic pulmonary fibrosis are described. 10.1183/09031936.00176807
    Telomere dysfunction causes alveolar stem cell failure. Alder Jonathan K,Barkauskas Christina E,Limjunyawong Nathachit,Stanley Susan E,Kembou Frant,Tuder Rubin M,Hogan Brigid L M,Mitzner Wayne,Armanios Mary Proceedings of the National Academy of Sciences of the United States of America Telomere syndromes have their most common manifestation in lung disease that is recognized as idiopathic pulmonary fibrosis and emphysema. In both conditions, there is loss of alveolar integrity, but the underlying mechanisms are not known. We tested the capacity of alveolar epithelial and stromal cells from mice with short telomeres to support alveolar organoid colony formation and found that type 2 alveolar epithelial cells (AEC2s), the stem cell-containing population, were limiting. When telomere dysfunction was induced in adult AEC2s by conditional deletion of the shelterin component telomeric repeat-binding factor 2, cells survived but remained dormant and showed all the hallmarks of cellular senescence. Telomere dysfunction in AEC2s triggered an immune response, and this was associated with AEC2-derived up-regulation of cytokine signaling pathways that are known to provoke inflammation in the lung. Mice uniformly died after challenge with bleomycin, underscoring an essential role for telomere function in AEC2s for alveolar repair. Our data show that alveoloar progenitor senescence is sufficient to recapitulate the regenerative defects, inflammatory responses, and susceptibility to injury that are characteristic of telomere-mediated lung disease. They suggest alveolar stem cell failure is a driver of telomere-mediated lung disease and that efforts to reverse it may be clinically beneficial. 10.1073/pnas.1504780112
    The mitochondria in lung fibrosis: friend or foe? Malsin Elizabeth S,Kamp David W Translational research : the journal of laboratory and clinical medicine Idiopathic pulmonary fibrosis (IPF) and other forms of lung fibrosis are age-associated diseases with increased deposition of mesenchymal collagen that promotes respiratory malfunction and eventual death from respiratory failure. Our understanding of the pathobiology underlying pulmonary fibrosis is incomplete and current therapies available to slow or treat lung fibrosis are limited. Evidence reviewed herein demonstrates key involvement of mitochondrial dysfunction in diverse pulmonary cell populations, including alveolar epithelial cells (AEC), fibroblasts, and macrophages and/or immune cells that collectively advances the development of pulmonary fibrosis. The mitochondria have an important role in regulating whether fibrogenic stimuli results in the return of normal healthy function ("friend") or the development of pulmonary fibrosis ("foe"). In particular, we summarize the evidence suggesting that AEC mitochondrial dysfunction is important in mediating lung fibrosis signaling via mechanisms involving imbalances in the levels of reactive oxygen species, endoplasmic reticulum stress response, mitophagy, apoptosis and/or senescence, and inflammatory signaling. Further, we review the emerging evidence suggesting that dysfunctional mitochondria in AECs and other cell types play crucial roles in modulating nearly all aspects of the 9 hallmarks of aging in the context of pulmonary fibrosis as well as some novel molecular pathways that have recently been identified. Finally, we discuss the potential translational aspects of these studies as well as the key knowledge gaps necessary for better informing our understanding of the pathobiology of the mitochondria in mediating pulmonary fibrosis. We reason that targeting deficient mitochondria-derived pathways may provide innovative future treatment strategies that are urgently needed for lung fibrosis. 10.1016/j.trsl.2018.05.005
    Mitochondria dysfunction and metabolic reprogramming as drivers of idiopathic pulmonary fibrosis. Bueno Marta,Calyeca Jazmin,Rojas Mauricio,Mora Ana L Redox biology Idiopathic pulmonary fibrosis (IPF) is a devastating lung disease of unknown etiology. It is characterized by deposition of extracellular matrix proteins, like collagen and fibronectin in the lung interstitium leading to respiratory failure. Our understanding of the pathobiology underlying IPF is still incomplete; however, it is accepted that aging is a major risk factor in the disease while growing evidence suggests that the mitochondria plays an important role in the initiation and progression of pulmonary fibrosis. Mitochondria dysfunction and metabolic reprogramming had been identified in different IPF lung cells (alveolar epithelial cells, fibroblasts, and macrophages) promoting low resilience and increasing susceptibility to activation of profibrotic responses. Here we summarize changes in mitochondrial numbers, biogenesis, turnover and associated metabolic adaptations that promote disrepair and fibrosis in the lung. Finally, we highlight new possible therapeutic approaches focused on ameliorate mitochondrial dysfunction. 10.1016/j.redox.2020.101509
    A pneumocyte-macrophage paracrine lipid axis drives the lung toward fibrosis. Romero Freddy,Shah Dilip,Duong Michelle,Penn Raymond B,Fessler Michael B,Madenspacher Jennifer,Stafstrom William,Kavuru Mani,Lu Bo,Kallen Caleb B,Walsh Kenneth,Summer Ross American journal of respiratory cell and molecular biology Lipid-laden macrophages, or "foam cells," are observed in the lungs of patients with fibrotic lung disease, but their contribution to disease pathogenesis remains unexplored. Here, we demonstrate that fibrosis induced by bleomycin, silica dust, or thoracic radiation promotes early and sustained accumulation of foam cells in the lung. In the bleomycin model, we show that foam cells arise from neighboring alveolar epithelial type II cells, which respond to injury by dumping lipids into the distal airspaces of the lungs. We demonstrate that oxidized phospholipids accumulate within alveolar macrophages (AMs) after bleomycin injury and that murine and human AMs treated with oxidized phosphatidylcholine (oxPc) become polarized along an M2 phenotype and display enhanced production of transforming growth factor-β1. The direct instillation of oxPc into the mouse lung induces foam cell formation and triggers a severe fibrotic reaction. Further, we show that reducing pulmonary lipid clearance by targeted deletion of the lipid efflux transporter ATP-binding cassette subfamily G member 1 increases foam cell formation and worsens lung fibrosis after bleomycin. Conversely, we found that treatment with granulocyte-macrophage colony-stimulating factor attenuates fibrotic responses, at least in part through its ability to decrease AM lipid accumulation. In summary, this work describes a novel mechanism leading to foam cell formation in the mouse lung and suggests that strategies aimed at blocking foam cell formation might be effective for treating fibrotic lung disorders. 10.1165/rcmb.2014-0343OC
    Lung surfactant metabolism: early in life, early in disease and target in cell therapy. Lopez-Rodriguez Elena,Gay-Jordi Gemma,Mucci Adele,Lachmann Nico,Serrano-Mollar Anna Cell and tissue research Lung surfactant is a complex mixture of lipids and proteins lining the alveolar epithelium. At the air-liquid interface, surfactant lowers surface tension, avoiding alveolar collapse and reducing the work of breathing. The essential role of lung surfactant in breathing and therefore in life, is highlighted by surfactant deficiency in premature neonates, which causes neonatal respiratory distress syndrome and results in early death after birth. In addition, defects in surfactant metabolism alter lung homeostasis and lead to disease. Special attention should be paid to two important key cells responsible for surfactant metabolism: alveolar epithelial type II cells (AE2C) and alveolar macrophages (AM). On the one hand, surfactant deficiency coming from abnormal AE2C function results in high surface tension, promoting alveolar collapse and mechanical stress in the epithelium. This epithelial injury contributes to tissue remodeling and lung fibrosis. On the other hand, impaired surfactant catabolism by AM leads to accumulation of surfactant in air spaces and the associated altered lung function in pulmonary alveolar proteinosis (PAP). We review here two recent cell therapies that aim to recover the activity of AE2C or AM, respectively, therefore targeting the restoring of surfactant metabolism and lung homeostasis. Applied therapies successfully show either transplantation of healthy AE2C in fibrotic lungs, to replace injured AE2C cells and surfactant, or transplantation of bone marrow-derived macrophages to counteract accumulation of surfactant lipid and proteinaceous material in the alveolar spaces leading to PAP. These therapies introduce an alternative treatment with great potential for patients suffering from lung diseases. 10.1007/s00441-016-2520-9
    Alveolar Dynamics and Beyond - The Importance of Surfactant Protein C and Cholesterol in Lung Homeostasis and Fibrosis. Sehlmeyer Kirsten,Ruwisch Jannik,Roldan Nuria,Lopez-Rodriguez Elena Frontiers in physiology Surfactant protein C (SP-C) is an important player in enhancing the interfacial adsorption of lung surfactant lipid films to the alveolar air-liquid interface. Doing so, surface tension drops down enough to stabilize alveoli and the lung, reducing the work of breathing. In addition, it has been shown that SP-C counteracts the deleterious effect of high amounts of cholesterol in the surfactant lipid films. On its side, cholesterol is a well-known modulator of the biophysical properties of biological membranes and it has been proven that it activates the inflammasome pathways in the lung. Even though the molecular mechanism is not known, there are evidences suggesting that these two molecules may interplay with each other in order to keep the proper function of the lung. This review focuses in the role of SP-C and cholesterol in the development of lung fibrosis and the potential pathways in which impairment of both molecules leads to aberrant lung repair, and therefore impaired alveolar dynamics. From molecular to cellular mechanisms to evidences in animal models and human diseases. The evidences revised here highlight a potential SP-C/cholesterol axis as target for the treatment of lung fibrosis. 10.3389/fphys.2020.00386
    Air Space Distension Precedes Spontaneous Fibrotic Remodeling and Impaired Cholesterol Metabolism in the Absence of Surfactant Protein C. Ruwisch Jannik,Sehlmeyer Kirsten,Roldan Nuria,Garcia-Alvarez Begoña,Perez-Gil Jesus,Weaver Timothy E,Ochs Matthias,Knudsen Lars,Lopez-Rodriguez Elena American journal of respiratory cell and molecular biology Surfactant protein (SP)-C deficiency is found in samples from patients with idiopathic pulmonary fibrosis, especially in familial forms of this disease. We hypothesized that SP-C may contribute to fibrotic remodeling in aging mice and alveolar lipid homeostasis. For this purpose, we analyzed lung function, alveolar dynamics, lung structure, collagen content, and expression of genes related to lipid and cholesterol metabolism of aging SP-C knockout mice. In addition, experiments with an alveolar macrophage cell line exposed to lipid vesicles with or without cholesterol and/or SP-C were performed. Alveolar dynamics showed progressive alveolar derecruitment with age and impaired oxygen saturation. Lung structure revealed that decreasing volume density of alveolar spaces was accompanied by increasing of the ductal counterparts. Simultaneously, septal wall thickness steadily increased, and fibrotic wounds appeared in lungs from the age of 50 weeks. This remarkable phenotype is unique to the 129Sv strain, which has an increased absorption of cholesterol, linking the accumulation of cholesterol and the absence of SP-C to a fibrotic remodeling process. The findings of this study suggest that overall loss of SP-C results in an age-dependent, complex, heterogeneous phenotype characterized by a combination of overdistended air spaces and fibrotic wounds that resembles combined emphysema and pulmonary fibrosis in patients with idiopathic pulmonary fibrosis. Addition of SP-C to cholesterol-laden lipid vesicles enhanced the expression of cholesterol metabolism and transport genes in an alveolar macrophage cell line, identifying a potential new lipid-protein axis involved in lung remodeling. 10.1165/rcmb.2019-0358OC
    Mitochondrial Dysfunction in Pulmonary Fibrosis. Rangarajan Sunad,Bernard Karen,Thannickal Victor J Annals of the American Thoracic Society The aging of the human population has resulted in an unprecedented increase in the incidence and prevalence of age-related diseases, including those of the lung. Idiopathic pulmonary fibrosis is a disease of aging, and is characterized by a progressive decline in lung function and high mortality. Recent studies suggest that mitochondrial dysfunction, which can accompany aging phenotypes, may contribute to the pathogenesis of idiopathic pulmonary fibrosis. In this review, we explore current evidence for mitochondrial dysfunction in alveolar epithelial cells, fibroblasts, and immune cells that participate in the fibrotic process. Further, the fates of these cell populations and the potential to target mitochondrial dysfunction as a therapeutic strategy are discussed. 10.1513/AnnalsATS.201705-370AW
    Lipids - two sides of the same coin in lung fibrosis. Mamazhakypov Argen,Schermuly Ralph T,Schaefer Liliana,Wygrecka Malgorzata Cellular signalling Idiopathic pulmonary fibrosis (IPF) is characterized by progressive extracellular matrix deposition in the lung parenchyma leading to the destruction of lung structure, respiratory failure and premature death. Recent studies revealed that the pathogenesis of IPF is associated with alterations in the synthesis and the activity of lipids, lipid regulating proteins and cell membrane lipid transporters and receptors in different lung cells. Furthermore, deregulated lipid metabolism was found to contribute to the profibrotic phenotypes of lung fibroblasts and alveolar epithelial cells. Consequently, several pharmacological agents, targeting lipids, lipid mediators, and lipoprotein receptors, was successfully tested in the animal models of lung fibrosis and entered early phase clinical trials. In this review, we highlight new therapeutic options to counteract disturbed lipid hemostasis in the maladaptive lung remodeling. 10.1016/j.cellsig.2019.04.007