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Although alveolar epithelial cells type II (AEC2) are chronically injured in Idiopathic Pulmonary Fibrosis (IPF), they contribute to epithelial regeneration in IPF.We hypothezised that Notch signaling may contribute to AEC2 proliferation, de-differentiation characterized by loss of surfactant processing machinery and lung fibrosis in IPF Methods: We applied microarray analysis, kinome profiling, flow cytometry, immunofluorescence analysis, western blotting, qPCR, proliferation- and surface activity- analysis to study epithelial differentiation, proliferation and matrix deposition in vitro (AEC2 cell lines, primary murine/human AEC2), ex vivo (human IPF-derived precision cut lung slices; IPF-PCLS) and in vivo (bleomycin-, pepstatin-application, Notch1 ICD overexpression) Measurements and Main Results: We document here extensive surfactant protein (SP)-B/C processing defects in IPF AEC2, due to loss of Napsin A, resulting in increased intra-alveolar surface tension and alveolar collapse and induction of ER-stress in AEC2. In vivo pharmacological inhibition of Napsin A results in the development of AEC2 injury and overt lung fibrosis. We also demonstrate that Notch1 signaling is already activated early in IPF and determines AEC2 fate by inhibiting differentiation (reduced lamellar body compartment, reduced capacity to process hydrophobic SP) and by causing increased epithelial proliferation and development of lung fibrosis, putatively via altered JAK/Stat signaling in AEC2. Conversely, inhibition of Notch signaling in IPF-PCLS improved surfactant processing capacity of AEC2s and reversed fibrosis.Notch1 is a central regulator of AEC2 fate in IPF. It induces alveolar epithelial proliferation, loss of Napsin A and of surfactant proprotein processing and it contributes to fibroproliferation.

Cellular and molecular life sciences : CMLS 79(3), 151 (2022). doi:10.1007/s00018-022-04189-2, Published by Springer International Publishing AG, Cham (ZG)

Insufficient autophagy has been reported in idiopathic pulmonary fibrosis (IPF) lungs. Specific roles of autophagy-related proteins in lung fibrosis development remain largely unknown. Here, we investigated the role of autophagy marker protein microtubule-associated protein 1 light chain 3β (LC3B) in the development of lung fibrosis. LC3B-/- mice upon aging show smaller lamellar body profiles, increased cellularity, alveolar epithelial cell type II (AECII) apoptosis, surfactant alterations, and lysosomal and endoplasmic reticulum stress. Autophagosomal soluble N-ethylmaleimide-sensitive factor attachment protein receptor syntaxin 17 is increased in the AECII of aged LC3B-/- mice and patients with IPF. Proteasomal activity, however, remained unaltered in LC3B-/- mice. In vitro knockdown of LC3B sensitized mouse lung epithelial cells to bleomycin-induced apoptosis, but its overexpression was protective. In vivo, LC3B-/- mice displayed increased susceptibility to bleomycin-induced lung injury and fibrosis. We identified cathepsin A as a novel LC3B binding partner and its overexpression in vitro drives MLE12 cells to apoptosis. Additionally, cathepsin A is increased in the AECII of aged LC3B-/- mice and in the lungs of patients with IPF. Our study reveals that LC3B mediated autophagy plays essential roles in AECII by modulating the functions of proteins like cathepsin A and protects alveolar epithelial cells from apoptosis and subsequent lung injury and fibrosis.-Kesireddy, V. S., Chillappagari, S., Ahuja, S., Knudsen, L., Henneke, I., Graumann, J., Meiners, S., Ochs, M., Ruppert, C., Korfei, M., Seeger, W., Mahavadi, P. Susceptibility of microtubule-associated protein 1 light chain 3β (MAP1LC3B/LC3B) knockout mice to lung injury and fibrosis.

Background: Longitudinal response to treatment with pirfenidone is highly variable. This study evaluated the effectiveness of pirfenidone, as measured by longitutidal changes in FVC (% pred.), DLco (% pred), Borg dyspnoe scale, and six minute walking distance test, as well as survival analyses. Methods: Data were available for 122 eurIPFreg patients. Patients’ data from at least two subsequent records with/without antifibrotic treatment were analyzed by slope-changes in each individual patient, as well as in global analysis, where the data from all patients were used to fit a global segmented regression model. Results: The individual analysis showed a tremendous heterogeneity, and the mean FVC slope difference was not significantly different from baseline (p = 0.32). The global analysis revealed an average slope change of +1.48 (% p.a.) ± 0.28 (p Conclusions: We report the effectiveness of pirfenidone in European IPF cohort with outcome data extending up to 9 years. Global analyses demonstrated a positive effect of pirfenidone on the decline of the lung function over time.

Background: Despite novel treatment options, idiopathic pulmonary fibrosis (IPF) still shows a progressive and fatal course. In search for further parameters of disease progression, we studied the role of Tidal Volume (VT), alongside with established risk factors such as FVC or GAP index. Aim and Methods: We analyzed 266 IPF patients recruited in Giessen und Paris into the eurIPFreg for baseline data, change in VT or other lung function values, and intake of antifibrotics (AF) for a follow up period of up to ten years. The estimated effects of VT on the hazard ratio (HR) were adjusted by sex, age at t0, smoking status, AF, ideal body weight and GAP score at t0. Results: The cohort showed the following demographics: 80.6% males, mean age at t0 67.8 years, FVC 64.7% pred., 77.4% active or previous smokers. VT correlated significantly with VC % pred., FVC % pred., pH and pCO2. A survival analysis revealed a highly significant impact of GAP score on survival, higher in the group under AF. VT also exerted significant effects on survival; every 100ml increase in the baseline VT was associated with a predicted decrease of the HR by a factor of 0.84 and a correspondingly improved survival. There was no clear evidence for a protective effect of very low TV. Conclusions: In IPF subjects, multiple factors affect outcome, among these are GAP index, smoking, antifibrotic treatment, and also VT. Against our anticipation, very low VTs were not associated with improved outcome, most likely because baseline VT is impacted by FVC and, hence, the extent of fibrotic disease.

(1) Aim of the study: In spite of extensive research, up to 20% of interstitial lung diseases (ILD) patients cannot be safely classified. We analyzed clinical features, progression factors, and outcomes of unclassifiable ILD (uILD). (2) Methods: A total of 140 uILD subjects from the University of Giessen and Marburg Lung Center (UGMLC) were recruited between 11/2009 and 01/2019 into the European Registry for idiopathic pulmonary fibrosis (eurIPFreg) and followed until 01/2020. The diagnosis of uILD was applied only when a conclusive diagnosis could not be reached with certainty. (3) Results: In 46.4% of the patients, the uILD diagnosis was due to conflicting clinical, radiological, and pathological data. By applying the diagnostic criteria of usual interstitial pneumonia (UIP) based on computed tomography (CT), published by the Fleischner Society, 22.2% of the patients displayed a typical UIP pattern. We also showed that forced vital capacity (FVC) at baseline (p = 0.008), annual FVC decline ≥10% (p < 0.0001), smoking (p = 0.033), and a diffusing capacity of the lung for carbon monoxide (DLco) ≤55% of predicted value at baseline (p < 0.0001) were significantly associated with progressive disease. (4) Conclusions: The most important prognostic factors in uILD are baseline level and decline in lung function and smoking. The use of Fleischner diagnostic criteria allows further differentiation and accurate diagnosis.

Background The stress-regulated enzyme hemeoxygenase-1 (HO-1) contributes to the cell response towards inflammation and oxidative stress. We previously reported on curtailed HO-1 expression in cystic fibrosis (CF) bronchial epithelial (CFBE41o-) cells and CF-mice, but the molecular mechanisms for this are not known. Here, we compared healthy and CF bronchial epithelial cells for regulatory circuits controlling HO-1 protein levels. Methods In this study, we employed immunohistochemistry on CF and healthy lung sections to examine the BACH1 protein expression. Alteration of BACH1 protein levels in 16HBE14o- and CFBE41o- cells was achieved by using either siRNA-mediated knockdown of BACH1 or by increasing miRNA-155 levels. HO-1 luciferase reporter assay was chosen to examine the downstream affects after BACH1 modulation. Results Human CF lungs and cells showed increased levels of the HO-1 transcriptional repressor, BACH1, and increased miR-155 expression. Knockdown studies using BACH1 siRNA and overexpression of miR-155 did not significantly rescue HO-1 expression in CFBE41o- cells. Elevated BACH1 expression detected in CF cells was refractory to the inhibitory function of miR-155 and was instead due to increased protein stability. Conclusion We observed defects in the inhibitory activities of miR-155 and BACH1 on HO-1 expression in CF cells. Thus various defective regulatory loops account for dysregulated BACH1 expression in CF, which in turn may contribute to low HO-1 levels.

The main characteristic of idiopathic pulmonary fibrosis (IPF) is ER-stress and apoptosis in alveolar type-II cells (AECII), which in consequence cause chronic epithelial injury and progressive lung fibrosis. We developed double-transgenic mice expressing the proapoptotic ER-stress-factor Chop exclusively in AECII by using the “Tetracycline-On-system” (SP-C rtTA/tetO7-Chop). In response to 28 days doxycycline-feeding (Dox+), nuclear Chop-overexpression in AECII resulted in AECII-apoptosis, but not in development of lung fibrosis. We then infected Chop transgenic mice after 28 days transgene-induction (Dox+), as well as without transgene induction (Dox-), intranasally with murine gammaherpesvirus-68 (MHV68). At 15 days post infection, mice were sacrificed and analyzed. Lungs of MHV68-infected (Dox+)-Chop-mice, but not of infected (Dox-)-Chop-mice or uninfected (Dox+)- and (Dox-)-Chop-mice, evidently showed thickening of alveolar septae and fibrotic remodeling. In addition, ER-stress- and strong apoptosis-induction (caspase-3/-9-activation, PARP1-cleavage) was observed in lungs of both MHV68-infected (Dox+)- and (Dox-)-Chop-mice versus controls, presumably due to virus-induced lung injury, but was more pronounced in the infected (Dox+)-Chop-mice. Further, MHV68-infected (Dox+)- as well as (Dox-)-Chop-mice indicated upregulation of profibrotic protein-expression versus controls, but was “biggest” in the infected (Dox+)-Chop-mice. We conclude that our transgenic Chop-mouse-model resembles human IPF, since IPF-patients have been defined as (genetically) susceptible individuals prone to develop disease with second hits.

Insufficient autophagy has been reported in idiopathic pulmonary fibrosis (IPF) lungs. Specific roles of autophagy-related proteins in lung fibrosis development remain largely unknown. Here, we investigated the role of autophagy marker protein microtubule-associated protein 1 light chain 3β (LC3B) in the development of lung fibrosis. LC3B(−/−) mice upon aging show smaller lamellar body profiles, increased cellularity, alveolar epithelial cell type II (AECII) apoptosis, surfactant alterations, and lysosomal and endoplasmic reticulum stress. Autophagosomal soluble N-ethylmaleimide–sensitive factor attachment protein receptor syntaxin 17 is increased in the AECII of aged LC3B(−/−) mice and patients with IPF. Proteasomal activity, however, remained unaltered in LC3B(−/−) mice. In vitro knockdown of LC3B sensitized mouse lung epithelial cells to bleomycin-induced apoptosis, but its overexpression was protective. In vivo, LC3B(−/−) mice displayed increased susceptibility to bleomycin-induced lung injury and fibrosis. We identified cathepsin A as a novel LC3B binding partner and its overexpression in vitro drives MLE12 cells to apoptosis. Additionally, cathepsin A is increased in the AECII of aged LC3B(−/−) mice and in the lungs of patients with IPF. Our study reveals that LC3B mediated autophagy plays essential roles in AECII by modulating the functions of proteins like cathepsin A and protects alveolar epithelial cells from apoptosis and subsequent lung injury and fibrosis.—Kesireddy, V. S., Chillappagari, S., Ahuja, S., Knudsen, L., Henneke, I., Graumann, J., Meiners, S., Ochs, M., Ruppert, C., Korfei, M., Seeger, W., Mahavadi, P. Susceptibility of microtubule-associated protein 1 light chain 3β (MAP1LC3B/LC3B) knockout mice to lung injury and fibrosis.

Alveolar epithelial cell type II (AEC2) injury underlies idiopathic pulmonary fibrosis (IPF). Here we show increased Notch1 signaling in AEC2s in human IPF and IPF models, causing enhanced proliferation and de-differentiation of AEC2s. As a result, we observed defective surfactant protein (SP)-B/C processing, elevated alveolar surface tension, repetitive alveolar collapse and development of lung fibrosis. Similar changes were encountered upon pharmacological inhibition of SP-B/C processing in vivo by pepstatin A. Inhibition of Notch signaling in cultured human IPF precision cut lung slices improved surfactant processing capacity of AEC2s and reversed fibrosis. Notch1 therefore offers as novel therapeutic target.One sentence summaryNotch1 inhibition restores alveolar epithelial differentiation and surface tension and reverses matrix deposition in lung fibrosis

The immunomodulatory properties of adipokines have previously been reported in autoimmune disorders. Less is known about the role of adipokines in systemic sclerosis (SSc). Lung and gastrointestinal tract are frequently involved in SSc; therefore, these organs were analyzed for adipokine expression as well as pulmonary samples of patients suffering from idiopathic pulmonary fibrosis (IPF) as comparison.Gastric samples (antrum, corpus) of SSc were analyzed immunohistochemically for adiponectin, resistin and visfatin compared with non-SSc related gastritis. Inflammatory cells were quantified in gastric samples and correlated with adipokine expression. Lung samples of SSc, IPF and healthy controls were also analyzed. Protein levels of lung tissue lysates and bronchoalveolar lavages (BAL) in minor fibrotic stages were measured by ELISA.Lung sections of donor parenchyma showed significantly stronger adiponectin signals as IPF and SSc (donor vs. IPF: p 0.0001). In SSc and IPF, resistin and visfatin were increased within immune cell infiltrates, but overall no difference in expression for resistin or visfatin compared to controls was observed. In BAL and lung protein lysates of early stages of fibrosis, adiponectin and visfatin were not reduced in IPF and SSc compared to controls. In gastric samples collected by standard endoscopic gastric biopsy, adiponectin was also significantly reduced in SSc- compared to non-SSc gastritis (p = 0.049) while resistin and visfatin were comparable although deeper fibrotic layers were not included in the respective samples. Adiponectin-positive tissues showed higher amounts of CD4Adipokines are expressed in gastric and lung samples of patients with SSc and in lung samples affected by IPF. Prominently, adiponectin levels were reduced in fibrotic SSc gastritic tissue as well as in IPF and SSc lung tissue. Consequently, adiponectin expression seems to be associated with fibrotic progression in the context of SSc and IPF.

Background Idiopathic pulmonary fibrosis (IPF) is a devastating lung disease with a poor prognosis. Pirfenidone is the first antifibrotic agent to be approved for IPF-treatment as it is able to slow down disease progression. However, there is no curative treatment other than lung transplantation. Because epigenetic alterations are associated with IPF, histone deacetylase (HDAC)-inhibitors have recently been proven to attenuate fibrotic remodeling in vitro and in vivo. This study compared the effects of pirfenidone with the pan-HDAC-inhibitor panobinostat/LBH589, a FDA-approved drug for the treatment of multiple myeloma, head-to-head on survival, fibrotic activity and proliferation of primary IPF-fibroblasts in vitro. Methods Primary fibroblasts from six IPF-patients were incubated for 24h with vehicle (0.25% DMSO), panobinostat (LBH589, 85 nM) or pirfenidone (2.7 mM), followed by assessment of proliferation and expression analyses for profibrotic and anti-apoptosis genes, as well as for ER stress and apoptosis-markers. In addition, the expression status of all HDAC enzymes was examined. Results Treatment of IPF-fibroblasts with panobinostat or pirfenidone resulted in a downregulated expression of various extracellular matrix (ECM)-associated genes, as compared to vehicle-treated cells. In agreement, both drugs decreased protein level of phosphorylated (p)-STAT3, a transcription factor mediating profibrotic responses, in treated IPF-fibroblasts. Further, an increase in histone acetylation was observed in response to both treatments, but was much more pronounced and excessive in panobinostat-treated IPF-fibroblasts. Panobinostat, but not pirfenidone, led to a significant suppression of proliferation in IPF-fibroblasts, as indicated by WST1- and BrdU assay and markedly diminished levels of cyclin-D1 and p-histone H3. Furthermore, panobinostat-treatment enhanced α-tubulin-acetylation, decreased the expression of survival-related genes Bcl-XL and BIRC5/survivin, and was associated with induction of ER stress and apoptosis in IPF-fibroblasts. In contrast, pirfenidone-treatment maintained Bcl-XL expression, and was neither associated with ER stress-induction nor any apoptotic signaling. Pirfenidone also led to increased expression of HDAC6 and sirtuin-2, and enhanced α-tubulin-deacetylation. But in line with its ability to increase histone acetylation, pirfenidone reduced the expression of HDAC enzymes HDAC1, -2 and -9. Conclusions We conclude that, beside other antifibrotic mechanisms, pirfenidone reduces profibrotic signaling also through STAT3 inactivation and weak epigenetic alterations in IPF-fibroblasts, and permits survival of (altered) fibroblasts. The pan-HDAC-inhibitor panobinostat reduces profibrotic phenotypes while inducing cell cycle arrest and apoptosis in IPF-fibroblasts, thus indicating more efficiency than pirfenidone in inactivating IPF-fibroblasts. We therefore believe that HDAC-inhibitors such as panobinostat can present a novel therapeutic strategy for IPF.

Background Activation and differentiation of fibroblasts into contractile protein-expressing myofibroblasts and their acquired apoptosis-resistant phenotype are critical factors towards the development of idiopathic pulmonary fibrosis (IPF), a fatal disease characterised by distorted pulmonary structure and excessive extracellular matrix (ECM) deposition. The molecular mechanisms underlying these processes in IPF remain incompletely understood. We investigated the possible implication of aberrant overexpression and activity of histone deacetylases (HDACs) in IPF. Methods We analysed lung tissues from patients with sporadic IPF (n=26) and non-diseased control lungs (n=16) for expression of class I and II HDACs. Primary IPF fibroblasts were treated with HDAC inhibitors (HDACi) LBH589 or valproic acid (VPA). Results Compared to control lungs, protein levels of class I (HDAC1, HDAC2, HDAC3, HDAC8) and class II HDACs (HDAC4, HDAC 5, HDAC 7, HDAC 9) were significantly elevated in IPF lungs. Using immunohistochemistry, strong induction of nearly all HDAC enzymes was observed in myofibroblasts of fibroblast foci and in abnormal bronchiolar basal cells at sites of aberrant re-epithelialisation in IPF lungs, but not in controls. Treatment of primary IPF fibroblasts with the pan-HDACi LBH589 resulted in significantly reduced expression of genes associated with ECM synthesis, proliferation and cell survival, as well as in suppression of HDAC7, and was paralleled by induction of endoplasmic reticulum stress and apoptosis. The profibrotic and apoptosis-resistant phenotype of IPF fibroblasts was also partly attenuated by the class I HDACi VPA. Conclusions Aberrant overexpression of HDACs in basal cells of IPF lungs may contribute to the bronchiolisation process in this disease. Similarly, generation and apoptosis resistance of IPF fibroblasts are mediated by enhanced activity of HDAC enzymes. Therefore, pan-HDAC inhibition by LBH589 may present a novel therapeutic option for patients with IPF.

Amiodarone (AD) is an iodinated benzofuran derivative, especially known for its antiarrhythmic properties. It exerts serious side-effects even in patients receiving low doses. AD is well-known to induce apoptosis of type II alveolar epithelial cells (AECII), a mechanism that has been suggested to play an important role in AD-induced lung fibrosis. The precise molecular mechanisms underlying this disease are, however, still unclear. Because of its amphiphilic nature, AD becomes enriched in the lysosomal compartments, affecting the general functions of these organelles. Hence, in this study, we aimed to assess the role of autophagy, a lysosome-dependent homeostasis mechanism, in driving AECII apoptosis in response to AD. In vitro, AD-treated MLE12 and primary AECII cells showed increased proSP-C and LC3B positive vacuolar structures and underwent LC3B-dependent apoptosis. In addition, AD-induced autophagosome-lysosome fusion and increased autophagy flux were observed. In vivo, in C57BL/6 mice, LC3B was localised at the limiting membrane of lamellar bodies, which were closely connected to the autophagosomal structures in AECIIs. Our data suggest that AD causes activation of macroautophagy in AECIIs and extensive autophagy-dependent apoptosis of alveolar epithelial cells. Targeting the autophagy pathway may therefore represent an attractive treatment modality in AD-induced lung fibrosis.

Idiopathic pulmonary fibrosis (IPF) and bleomycin-induced pulmonary fibrosis are associated with surfactant system dysfunction, alveolar collapse (derecruitment), and collapse induration (irreversible collapse). These events play undefined roles in the loss of lung function. The purpose of this study was to quantify how surfactant inactivation, alveolar collapse, and collapse induration lead to degradation of lung function. Design-based stereology and invasive pulmonary function tests were performed 1, 3, 7, and 14 days after intratracheal bleomycin-instillation in rats. The number and size of open alveoli was correlated to mechanical properties. Active surfactant subtypes declined by Day 1, associated with a progressive alveolar derecruitment and a decrease in compliance. Alveolar epithelial damage was more pronounced in closed alveoli compared with ventilated alveoli. Collapse induration occurred on Day 7 and Day 14 as indicated by collapsed alveoli overgrown by a hyperplastic alveolar epithelium. This pathophysiology was also observed for the first time in human IPF lung explants. Before the onset of collapse induration, distal airspaces were easily recruited, and lung elastance could be kept low after recruitment by positive end-expiratory pressure (PEEP). At later time points, the recruitable fraction of the lung was reduced by collapse induration, causing elastance to be elevated at high levels of PEEP. Surfactant inactivation leading to alveolar collapse and subsequent collapse induration might be the primary pathway for the loss of alveoli in this animal model. Loss of alveoli is highly correlated with the degradation of lung function. Our ultrastructural observations suggest that collapse induration is important in human IPF.

Autophagy is a fundamental biological process of every cell that aims to maintain cellular homeostasis by degrading unnecessary macromolecules and non-functional organelles. Dysfunctional autophagy was reported in idiopathic pulmonary fibrosis (IPF), a disease with a remarkable age-related onset which may be triggered by chronic lung alveolar epithelial cell type II (AECII) injury and apoptosis. The principle role of autophagy in cellular homeostasis has been well defined, but remains to be studied in the evolution of several forms of lung fibrosis. Amiodarone (AD) and bleomycin are two lung fibrosis inducing agents which trigger disturbed autophagy within the AECII. In this study, we aimed to identify the role of the autophagy marker protein, LC3B in the development of these two forms of lung fibrosis. First, we performed knock down of LC3B in mouse lung epithelial cells and treated them with AD or bleomycin. LC3B knockdown rescued AD induced apoptosis but on the contrary, increased susceptibility to bleomycin induced injury and apoptosis. A systematic analysis of the LC3B-/- mice lungs revealed that aged LC3B-/- mice showed lung collagen deposition, increased cellularity, decreased alveolar spaces, increased apoptosis of AECII paralleled with surfactant alterations, increased lysosomal and endoplasmic reticulum stress providing clues on the importance of this distal autophagy protein in lung fibrosis development. Further, we were able to identify novel LC3B interaction partners within the AECII by mass spectrometric analysis which are being further confirmed. In conclusion, our studies reveal that autophagy has a pathomechanistic but context-specific role in the development of lung fibrosis.

Background Hermansky-Pudlak Syndrome (HPS) is a rare hereditary multisystem disorder, characterized by oculocutaneous albinism, bleeding diathesis, pulmonary fibrosis, neutropenia and granulomatous colitis. Based on the responsible genes, nine types of HPS are known; among these types 1, 2, and 4 have been associated with pulmonary fibrosis. Aims and objectives Investigate the early pulmonary phenotype in infants and young children. Methods All patients categorized as HPS in the kids lung register were searched. Diagnosis was based on clinical symptoms and confirmed by genetics. Between 01/2001 and 02/2016 6 children were identified and analysed retrospectively. Results 5 children had HPS-2, 1 child HPS-1. Age at diagnosis ranged between 0.4 - 8.8 years (mean 2,7). There was a predominance of females (5/6), a relation not noted in literature. All children had at least one typical extrapulmonary sign of HPS. The girl with HPS-1 had no pulmonary symptoms, all children with HPS-2 had a pulmonary phenotype: recurrent pneumothoraces (3), dyspnoea (3), partial respiratory insufficiency (3), chronic cough (2). Chest CT was performed in all children with HPS-2, which showed ground glass opacity (3), honeycombing (2) and a reticular pattern (2). In a familial case one child died at the age of 6 years due to respiratory insufficiency. All others are alive. Conclusion Sporadic and familial cases with HPS can present in early infancy and may have a pulmonary phenotype which can determine the disease´s course. The treatment of life limiting respiratory diseases in children with HPS must be considered, e.g. when clinical trials on novel therapeutic interventions are performed for adults.

STUDY QUESTION What is the underlying mechanism of Sertoli cell (SC) resistance to cell death? SUMMARY ANSWER High expression of prosurvival B-cell lymphoma-2 (BCL2) proteins and inhibition of apoptosis and autophagy prolongs SC survival upon exposure to stress stimuli. WHAT IS KNOWN ALREADY In human and in experimental models of orchitis, tolerogenic SC survive stress conditions, while germ cells undergo massive apoptosis. In general, non-dividing highly differentiated cells tend to resist stress conditions for a longer time by favoring activation of prosurvival mechanisms and inhibition of cell death pathways. STUDY DESIGN, SIZE, DURATION In this cross sectional study, conditions stimulating apoptosis and autophagy were used to induce cell death in primary rat SC. Primary rat peritubular cells (PTC) and immortalized rat 93RS2 SC were used as controls. Each cell isolation was counted as one experiment (n = 1), and each experiment was repeated three to six times. PARTICIPANTS/MATERIALS, SETTING, METHODS Testis biopsy samples from infertile or subfertile patients and testis samples from rats with experimental autoimmune orchitis were used for immunohistological analysis. Primary SC were isolated from 19-day-old male Wistar rats. To maintain cell purity, cells were cultured in serum-free medium for apoptosis experiments and in medium supplemented with 1% serum for autophagy analyses. To induce apoptosis, cells were stimulated with staurosporine, borrelidin, cisplatin and etoposide for 4 or 24 h. Caspase three activation was examined by immunoblotting and enzymatic activity assay. Mitochondrial membrane potential was measured using tetramethylrhodamine methyl ester followed by flow cytometric analysis. Cytochrome c release was monitored by immunofluorescence. Cell viability was determined using the methylthiazole tetrazolium assay. To monitor autophagy flux, cells were deprived of nutrients using Hank's balanced salt solution for 1, 2 and 3 h. Formation of autophagosomes was analyzed by using immunoblotting, immunofluorescence labeling and ultrastructural analyses. Relative mRNA levels of genes involved in the regulation of apoptosis and autophagy were evaluated. Extracellular high mobility group box protein one was measured as a marker of necrosis using ELISA. MAIN RESULTS AND THE ROLE OF CHANCE SC survive the inflammatory conditions in vivo in human testis and in experimental autoimmune orchitis. Treatment with apoptosis inducing chemotherapeutics did not cause caspase three activation in isolated rat SC. Moreover, mitochondrial membrane potential and mitochondrial localization of cytochrome c were not changed by treatment with staurosporine, suggesting a premitochondrial blockade of apoptosis in SC. Expression levels of prosurvival BCL2 family members were significantly higher in SC compared to PTC at both mRNA and protein levels. Furthermore, after nutrient starvation, autophagy signaling was initiated in SC as observed by decreased levels of phosphorylated UNC- 51-like kinase -1 (ULK1). However, levels of light chain 3 II (LC3 II) and sequestosome1 (SQSTM1) remained unchanged, indicating blockade of the autophagy flux. Lysosomal activity was intact in SC as shown by accumulation of LC3 II following administration of lysosomal protease inhibitors, indicating that inhibition of autophagy flux occurs at a preceding stage. LARGE SCALE DATA N/A. LIMITATIONS, REASONS FOR CAUTION In this study, we have used primary SC from prepubertal rats. Caution should be taken when translating our results to adult animals, where crosstalk with other testicular cells and hormonal factors may also play a role in regulating survival of SC. WIDER IMPLICATIONS OF THE FINDINGS Our results suggest that inhibition of autophagy and apoptosis following exposure to extrinsic stress stimuli promotes SC survival, and is a possible mechanism to explain the robustness of SC in response to stress. Cell death resistance in SC is crucial for the recovery of spermatogenesis after chemotherapy treatment in cancer patients. Additionally, understanding the molecular mechanisms of SC survival unravels valuable target proteins, such as BCL2, that may be manipulated therapeutically to control cell viability depending on the context of the disease. STUDY FUNDING AND COMPETING INTEREST(S) This study was funded by the Deutsche Forschungsgemeinschaft (DFG) Grant BH93/1-1, and by the International Research Training Group between Justus Liebig University of Giessen and Monash University, Melbourne (GRK 1871/1) funded by the DFG and Monash University. The support of the Medical Faculty of Justus-Liebig University of Giessen is gratefully acknowledged. The authors declare no conflict of interest.

The activation of Endoplasmic Reticulum (ER) stress and Unfolded Protein Response (UPR) was first observed in patients with familial interstitial pneumonia (FIP) carrying mutations in the C-terminal BRICHOS domain of surfactant protein C (SFTPC). Here, aggresome formation and severe ER stress was demonstrated in type-II alveolar epithelial cells (AECII), which specifically express this very hydrophobic surfactant protein. In subsequent studies, FIP-patients with mutations in the gene encoding surfactant protein A2 (SFTPA2) were discovered, whose overexpression in epithelial cells in vitro also resulted in significant induction of ER stress. Moreover, prominent ER stress in AECII was also observed in FIP-patients not carrying the SFTPC/SFTPA2 mutations, as well as in patients with the more common sporadic forms of IP. Additionally, cases of adult-onset FIP with mutations in Telomerase genes and other telomereassociated components were reported. These mutations were associated with telomere shortening, which is a potential cause for triggering a persistent DNA damage response and replicative senescence in affected cells. Moreover, shortened telomeres were observed directly in the AECII of FIP-patients, and even sporadic IP cases, in the absence of any gene mutations. Here, we try to figure out the possible origins of ER stress in sporadic IP cases and non-SFTPC/SFTPA2-associated FIP.

Background Neutrophil elastase (NE) rapidly degrades gel-forming airway mucins in cystic fibrosis (CF) sputum. We hypothesized that KRP-109, a small molecule NE inhibitor, would inhibit CF mucin degradation in vitro . Methods Sputa were collected from CF patients ( n =5) chronically or intermittently infected with Pseudomonas aeruginosa (P.a.) . Mucin degradation was analyzed using western blot. Protease inhibitor studies were performed using alpha1-proteinase inhibitor (A1-PI Prolastin®) and KRP-109. Elastase activity assays were performed using spectrophotometry. Results There were significant differences in the amount of active NE in different CF sputum samples. KRP-109 decreased the NE driven mucin degradation in vitro . Pseudomonas elastases appeared to blunt elastase inhibition by A1-PI or KRP-109. Conclusion Inhibitors of neutrophil and Pseudomonas -derived elastases might rescue mucus clearance and reverse airway obstruction in CF.

Alveolarization of the developing lung is an important step toward the switch from intrauterine life to breathing oxygen-rich air after birth. The distal airways structurally change to minimize the gas exchange path, and Type II pneumocytes increase the production of surfactants, which are required to reduce surface tension at the air-liquid interface in the alveolus. Hypoxia-inducible factor 2 alpha (Hif2 alpha) is an oxygen-regulated transcription factor expressed in endothelial and Type II cells, and its expression increases toward the end of gestation. We investigated the role of Hif2 alpha in Type II cells by conditionally expressing an oxygen-insensitive mutant of Hif2 alpha in airway epithelial cells during development. Newborn mice expressing the mutant Hif2 alpha were born alive but quickly succumbed to respiratory distress. Subsequent analysis of the lungs revealed dilated alveoli covered with enlarged, aberrant Type II cells and a diminished number of Type I cells. The Type II cells accumulated glycogen in part by increased glucose uptake via the up-regulation of the glucose transporter 1. Furthermore, the cells lacked two crucial enzymes involved in the metabolism of glycogen into surfactant lipids, lysophosphatidylcholine acyltransferase and ATP-binding cassette sub-family A member 3. We conclude that Hif2 alpha is a key regulator in alveolar maturation and the production of phospholipids by Type II cells.

Rationale Lung cancer is the most common malignancy in humans. Urokinase (uPA) plays a crucial role in carcinogenesis by facilitating tumor cell invasion and metastasis. Objectives We investigated the effect of the highly specific urokinase inhibitor CJ-463 (benzylsulfonyl-D-Ser-Ser-4-amidinobenzylamide) on tumor growth, metastasis formation, and tumor vascularization in the murine Lewis lung carcinoma (LLC) and a human small lung cancer model. Methods A quantity of 3 x 10(6) LLC cells were subcutaneously injected into the right flank of C57Bl6/N mice, uPA knock out, and uPA receptor knockout mice. Seven days later mice were randomized to receive intraperitoneally either saline (control group), CJ-463 (10 and 100 mg/kg, twice a day), or its ineffective stereoisomer (10 mg/kg, twice a day). Tumor volume was measured every second day and metastasis formation was monitored by volumetric-computed tomography. Twelve days after onset of treatment mice were killed and tumors were prepared for histologic examination. Measurements and main results Treatment with CJ-463 resulted in a significant inhibition of primary tumor growth, with the highest efficacy seen in the 100 mg/kg group. In addition, histological analysis of the lung revealed a significant reduction in lung micrometastasis in the 100 mg/kg group. Similarly, a reduced seeding of tumor cells into the lung after intravenous injection of LLC cells was observed in inhibitor-treated mice. In these mice, treatment with CJ-463 appeared not to significantly alter the relative extent of tumor vascularization. In vitro, proliferation of LLC cells remained unchanged upon inhibitor treatment. CJ-463 was found to similarly reduce tumor growth in uPA receptor knockout mice, but was ineffective in uPA knockout mice. Conclusions Our results suggest that synthetic low-molecular-weight uPA-inhibitors offer as novel agents for treatment of lung cancer.

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.We studied the development of fibrosis and the role of AECII injury in various murine models of HPS.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.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.Extensively impaired surfactant trafficking and secretion underlie lysosomal and endoplasmic reticulum stress with apoptosis of AECII in HPSIP, thereby causing the development of HPSIP.

Increasing evidence indicates that disequilibrium of the alveolar oxidant-antioxidant balance may play a role in the pathogenesis of chronic fibrosing lung diseases. Excessive production of oxidants and a differential regulation of antioxidant enzymes have been described under these conditions. We characterized for the first time numerous nonenzymatic low-molecular-weight antioxidants in bronchoalveolar lavage fluids from patients with different forms of lung fibrosis initiated either by injury to the alveolar epithelium (idiopathic pulmonary fibrosis, IPF) or by inflammation (chronic sarcoidosis/hypersensitivity pneumonitis). Footprints of oxidative stress accompanied by an increase in the majority of antioxidants assessed were observed in all patient groups: elevated levels of uric acid, ascorbic acid, retinol, and alpha-tocopherol were noted, whereas glutathione levels were unchanged. The expression of Nrf2, an important redox-sensitive transcriptional regulator of antioxidants, was increased in IPF lungs. Our findings were corroborated in the bleomycin model of lung fibrosis where--aside from uric acid--nonenzymatic antioxidants were elevated during the fibrotic phase. In conclusion, alveolar levels of nonenzymatic antioxidants are elevated in fibrosing lung diseases, but are incapable of restoring oxidative balance. This increase may be part of an adaptive response to oxidative stress. However, a leakage from the blood may also contribute to our findings.

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 lun...

SummaryIntraalveolar fibrin deposition is commonly observed during acute inflammatory and chronic interstitial lung diseases and may contribute to impairment of surfactant function and gas exchange.We recently described a chemically cross-linked chimeric protein consisting of surfactant protein (SP)-B and urokinase (uPA) for targeting alveolar fibrin under conditions such as acute respiratory distress syndrome (ARDS) or lung fibrosis.We now investigated the feasibility of a recombinant production of a fusion protein encoding mature SP-B and uPA, termed SPUC. Four different SPUC proteins (N-term SP-B/C-term uPA, N-term uPA/C-term SP-B, each +/− His-tag) were prepared by cloning the cDNA encoding mature SP-B and low-molecular-weight uPA into the expression vector pcDNA3.1. CHO-cells were transfected with the constructs and the supernatant and cell lysates were analyzed for expression of SPUC.Using a chromogenic substrate assay uPA activity was found in supernatants and lysates of transfected cells with highest activities related to the N-term uPA/C-term SP-B (+/− His-tag) construct in supernatants 48h after transfection.Casein enzymography showed an enzymatically active fusion proteins with a molecular weight of ∼ 42 kDa in the supernatant of cells transfected with the N-term uPA/C-term SP-B (+/− His-tag) construct,but only a minor activity with the N-term SP-B/C-term uPA construct. The N-term uPA/C-term SP-B construct was also shown to possess higher resistance towards inhibition by plasminogen activator inhibitor-1.We conclude that recombinant production of a fusion protein consisting of mature SP-B and uPA is feasible, when the SP-B moiety is fused to the C-terminus of urokinase.

Introduction: Idiopathic pulmonary fibrosis (IPF) is a fatal disease characterized by distorted pulmonary structure and the excessive deposition of extracellular matrix (ECM) proteins, such as collagen. Myofibroblasts are the primary collagen-producing cells in IPF lungs, and their accumulation within lesions called fibroblast foci (FF) is a hallmark of IPF. We investigated the therapeutic potential of pan-histone deacetylase inhibitors (HDACi), because we have discovered a significant overexpression of ClassI/II-HDAC enzymes in IPF fibroblasts/myofibroblasts. Methods: Isolated primary IPF fibroblasts were treated for 20h with the pan-HDACi panobinostat (LBH589, 85nmol) or the IPF drug pirfenidone (2.7 mM). Results: Treatment of primary IPF fibroblasts with the pan-HDACi LBH589 resulted in significantly reduced expression of genes associated with fibrogenesis (ACTA2, COL1A1, COL3A1, FN), cell survival (BIRC5), proliferation (CCND1), as well as in suppression of HDAC7, and was paralleled by induction of severe endoplasmic reticulum (ER) stress (ATF6, CHOP) and apoptosis (p21, PUMA, cleaved caspase-3). Pirfenidone treatment led also to a significant downregulation of COL1A1, COL3A1, and FN, but not of ACTA2. The profibrotic genes CNN1 and P4HTM were exclusively reduced by pirfenidone -, but not by LBH589 treatment. Importantly, pirfenidone therapy lead also to significant downregulation of the cancer-associated gene BIRC5, but was not associated with induction of ER stress and apoptosis. Finally, pirfenidone did not greatly affect expression of HDAC proteins. Discussion: We conclude that generation and apoptosis resistance of IPF fibroblasts are mediated due to enhanced activity of HDAC proteins, and LBH589/panobinostat can present a novel therapeutic option (in addition to pirfenidone) for IPF. *Presenting author

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

Chronic injury of alveolar epithelial type II cells (AE2 cells) represents a key event in the development of lung fibrosis in animal models and in humans, such as idiopathic pulmonary fibrosis (IPF). Intratracheal delivery of amiodarone to mice results in a profound injury and macroautophagy-dependent apoptosis of AE2 cells. Increased autophagy manifested in AE2 cells by disturbances of the intracellular surfactant. Hence, we hypothesized that ultrastructural alterations of the intracellular surfactant pool are signs of epithelial stress correlating with the severity of fibrotic remodeling. With the use of design-based stereology, the amiodarone model of pulmonary fibrosis in mice was characterized at the light and ultrastructural level during progression. Mean volume of AE2 cells, volume of lamellar bodies per AE2 cell, and mean size of lamellar bodies were correlated to structural parameters reflecting severity of fibrosis like collagen content. Within 2 wk amiodarone leads to an increase in septal wall thickness and a decrease in alveolar numbers due to irreversible alveolar collapse associated with alveolar surfactant dysfunction. Progressive hypertrophy of AE2 cells and increase in mean individual size and total volume of lamellar bodies per AE2 cell were observed. A high positive correlation of these AE2 cell-related ultrastructural changes and the deposition of collagen fibrils within septal walls were established. Qualitatively, similar alterations could be found in IPF samples with mild to moderate fibrosis. We conclude that ultrastructural alterations of AE2 cells including the surfactant system are tightly correlated with the progression of fibrotic remodeling.

Hemeoxygenase-1 (HO-1), an inducible heat shock protein, is upregulated in response to multiple cellular insults via oxidative stress, lipopolysaccharides (LPS), and hypoxia. In this study, we investigated in vitro the role of Toll-like receptor 4 (TLR4), hypoxia-inducible factor 1α (HIF-1α), and iron on HO-1 expression in cystic fibrosis (CF). Immunohistochemical analysis of TLR4, HO-1, ferritin, and HIF-1α were performed on lung sections of CFTR−/− and wild-type mice. CFBE41o- and 16HBE14o- cell lines were employed for in vitro analysis via immunoblotting, immunofluorescence, real-time PCR, luciferase reporter gene analysis, and iron quantification. We observed a reduced TLR4, HIF-1α, HO-1, and ferritin in CFBE41o- cell line and CF mice. Knockdown studies using TLR4-siRNA in 16HBE14o- revealed significant decrease of HO-1, confirming the role of TLR4 in HO-1 downregulation. Inhibition of HO-1 using tin protoporphyrin in 16HBE14o- cells resulted in increased iron levels, suggesting a probable role of HO-1 in iron accumulation. Additionally, sequestration of excess iron using iron chelators resulted in increased hypoxia response element response in CFBE41o- and 16HBE14o-, implicating a role of iron in HIF-1α stabilization and HO-1. To conclude, our in vitro results demonstrate that multiple regulatory factors, such as impaired TLR4 surface expression, increased intracellular iron, and decreased HIF-1α, downregulate HO-1 expression in CFBE41o- cells.