Alveolar Epithelial Cell Line Research Articles

ZNF365 is overexpressed in Pulmonary Fibrosis

Idiopathic pulmonary fibrosis (IPF) is a chronic and progressive interstitial lung disease of unknown etiology characterized by the expansion of fibroblast population and excessive extracellular matrix (ECM) deposition. Transforming growth factor‐beta 1 (TGF‐β1) is one of the most potent mediators of the fibrotic response and induces fibroblast proliferation, and their differentiation to myofibroblasts, cells characterized by increased contractility, apoptosis resistance and the production of multiple ECM components (mainly collagens).Global gene expression of human lung fibroblasts stimulated with TGFβ performed in our laboratory demonstrated the overexpression of ZNF365, a zinc finger protein that belongs to the C2H2 domain family that has been poorly described in the literature. The purpose of this study is to evaluate ZNF365 expression in IPF lung tissue, the expression of its ortholog Zfp365 in mice lung tissue and its functional effects on human lung fibroblasts and epithelial cells in vitro.Expression of ZNF365 and Zfp365 were evaluated by qPCR and WB in human and mice samples, as well as in fibroblasts and epithelial cells stimulated with TGFβ. Localization of the protein was performed by immunohistochemistry in IPF lungs and in bleomycin‐induced lung fibrosis in mice. For silencing experiments, specific siRNAs and scrambled controls designed for ZNF365 were used. For overexpression, cells were stably transfected with ZNF365. Growth rate and proliferation were analyzed using WST‐1 and CyQUANT reagent, and apoptosis was measured by flow cytometry.We found that ZNF365 is overexpressed in IPF lung tissues and localized in alveolar and bronchial epithelial cells and in fibroblasts/myofibroblasts. The orthologous Zfp365 is overexpressed in lung tissue from bleomycin instilled mice, and the immunoreactive protein was observed in alveolar and airway epithelium. In vitro studies confirmed that ZNF365 is overexpressed in control and IPF fibroblasts stimulated with TGFβ. Likewise, TGFβ induced the upregulation of ZNF365 in a human alveolar epithelial cell line. Functional assays revealed that ZNF365 silencing in human lung fibroblasts and alveolar epithelial cells induced a significant reduction of growth rate and proliferation compared with the scrambled and WT controls. Preliminary data show that silencing of this protein increases senescence‐asociated β‐galactosidase activity and increases expression of the histone variant γH2A.X, another senescence marker. These results demonstrate that ZNF365 is upregulated in IPF and experimental lung fibrosis and may be involved in the regulation of proliferation and senescence of human lung cells.Support or Funding InformationSupported by PAPIIT 201520

Cyb5R3 in Alveolar Epithelial Cells Confers Protection Against Lung Fibrosis Through Suppression of TGF‐β Signaling

BackgoundAlterations in the redox homeostasis play a role in the pathogenesis of age‐related diseases such idiopathic pulmonary fibrosis (IPF). The most accepted theory in the pathogenesis of IPF is the injury and activation of alveolar epithelial type II cells (AECII) triggering excessive and chronic wound‐healing responses, mediated by growth factors such as TGF‐β. A mitochondrial membrane‐bound NADH‐dependent redox enzyme that contributes to stress protection and associates with healthspan and aging is cytochrome b5 reductase isoform 3 (Cyb5R3). Cyb5R3 is a flavoprotein recognized for its ability to reduce heme iron, a major target of reactive oxygen species. However, the link between Cyb5R3 expression levels in AECII with the modulation of the TGF‐β signaling pathways and the age‐related vulnerability to injury and fibrosis in the lung is unknown.MethodsLungs from young, old and IPF donors were lysated to test Cyb5R3 levels. Cyb5R3 flox mice (Cyb5R3 fl/fl or WT) and mice carrying a doxycycline‐inducible Cre‐recombinase under the control of a regulatory sequence of the SP‐C gene were bred. To promote recombination and generate a homozygous conditional type II lung epithelial cells (AECII) CYB5R3 knockout mice (AECII Cyb5R3 KO), mice were fed doxycycline chow (625 mg/kg, Envigo) for 2 weeks before injury or AECII isolation. Mice were infected intranasally with gammaherpesvirus 68 (MHV68) to evaluate inflammatory and fibrotic responses in the lung. Mouse alveolar epithelial cell line MLE12 was used to knockdown Cyb5R3 using adenovirus carrying scrambleshRNA or Cyb5R3shRNA constructs.ResultsAged donor and IPF total lung lysates show lower levels of Cyb5R3. In addition, isolated murine primary AECII showed that Cyb5R3 is significantly diminished in aging AECIIs (24 mo vs. 4 mo) associated with cellular oxidative stress and a senescence phenotype. Conditional deletion of Cyb5R3 in AECII show increased susceptibility to lung fibrosis. AECII Cyb5R3 KO mice showed higher collagen deposition with significant higher transcript levels of TIMP1 (tissue inhibitor of the MMPs) and osteopontin than Cyb5R3 WT mice. Since TGF‐β is a key modulator of the net balance between MMPs and TIMPs, we analyzed the role of Cyb5R3 in TGF‐β signaling. In mouse epithelial cells, deficiency of Cyb5R3 enhances TGF‐β signaling with upregulation of profibrotic and senescence genes by modulation of cGMP‐PKG and cAMPPKA pathways.ConclusionsAltogether, these data suggest a possible role for Cyb5R3 in the fragility of AECIIs and the age related susceptibility to lung fibrosis. New therapies designed to modulate Cyb5R3 expression and activity might lessen severity of pulmonary fibrosis, especially in the context of the aging lung.Support or Funding InformationThis work was funded by the Aging Institute & Vascular Medicine Institute at the University of Pittsburgh

Phagocytosis of alveolar macrophages is suppressed in a mouse model of lipopolysaccharide-induced acute lung injury

To investigate the changes in phagocytic function of alveolar macrophages (AMs) in mice with lipopolysaccharide (LPS)-induced acute lung injury (ALI) and explore the possible mechanism. Kunming mice were randomly divided into normal control group and ALI (induced by LPS instillation in the airway) model group. AMs were obtained from bronchoalveolar lavage fluid in both groups, and phagocytosis of the AMs was observed using flow cytometry and fluorescence microscopy. Western blotting and ELISA were used to detect the expression and secretion of IL-33 in the lung tissue of the mice. We also detected the secretion of IL-33 by an alveolar epithelial cell line MLE-12 in response to stimulation with different concentrations of LPS. The AMs from the normal control mice were treated with different concentrations of LPS and IL-33, and the changes in the phagocytic activity of the cells were observed. Compared with those in normal control group, the percentage of AMs phagocytosing fluorescent microspheres was significantly decreased, and the expression of IL-33 in lung tissue and IL-33 level in the bronchoalveolar lavage fluid were significantly increased in ALI mice (P < 0.01). LPS (100-1000 ng/mL) obviously promoted the secretion of IL-33 in cultured MLE-12 cells (P < 0.01). Both LPS (10-500 ng/mL) and IL-33 (100 ng/mL) significantly inhibited the phagocytic activity of the AMs from normal control mice (P < 0.01). The phagocytic activity of AMs is weakened in ALI mice possibly due to direct LPS stimulation and the inhibitory effect of the alarmin IL-33 produced by LPS-stimulated alveolar epithelial cells.

IRF7 Is Required for the Second Phase Interferon Induction during Influenza Virus Infection in Human Lung Epithelia.

Influenza A virus (IAV) infection is a major cause of morbidity and mortality. Retinoic acid-inducible protein I (RIG-I) plays an important role in the recognition of IAV in most cell types, and leads to the activation of interferon (IFN). We investigated mechanisms of RIG-I and IFN induction by IAV in the BCi-NS1.1 immortalized human airway basal cell line and in the A549 human alveolar epithelial cell line. We found that the basal expression levels of RIG-I and regulatory transcription factor (IRF) 7 were very low in BCi-NS1.1 cells. IAV infection induced robust RIG-I and IRF7, not IRF3, expression. siRNA against IRF7 and mitochondrial antiviral-signaling protein (MAVS), but not IRF3, significantly inhibited RIG-I mRNA expression and IFN induction by IAV infection. Most importantly, even without virus infection, IFN-β alone induced RIG-I, and siRNA against IRF7 did not inhibit RIG-I induction by IFN-β. Similar results were found in the alveolar basal epithelial A549 cell line. RIG-I and IRF7 expression in humans is highly inducible and greatly amplified by IFN produced from virus infected cells. IFN induction can be separated into two phases, that initially induced by the virus with basal RIG-I (the first phase), and that induced by the subsequent virus with amplified RIG-I from the first phase IFN (the second phase). The de novo synthesis of IRF7 is required for the second phase IFN induction during influenza virus infection in human lung bronchial and alveolar epithelial cells.

Analysis of influenza virus-induced perturbation in autophagic flux and its modulation during Vitamin D3 mediated anti-apoptotic signaling

Vitamin D3/Calcitriol supplementation in humans is associated with reduced incidence and severity during influenza A virus (IAV) infection. Apoptosis in response to IAV infection is a major contributor to host cell death and tissue damage; however, its modulation by Vitamin D3 remains unclear. In this study, we demonstrate the efficacy of Vitamin D3 in preventing apoptosis induction by pandemic influenza A (H1N1)pdm09 virus in human alveolar cells (A549). Human alveolar epithelial cell line A549 was used to assess the cytotoxic effects of IAV infection. Immunoblotting and fluorescence microscopy were used to study apoptosis and autophagy. The results of the present study demonstrate that IAV induces apoptosis by subversion of host autophagy via down-regulating components of autophagic machinery involved in autophagosome-lysosome fusion and lysosomal activity. Vitamin D3 restores the autophagic flux inhibited by IAV by upregulating the expression of Syntaxin-17 (STX17) and V-type proton ATPase subunit (ATP6V0A2) thereby causing a concomitant decrease in cellular apoptosis via a Vitamin D3 receptor (VDR) dependent mechanism. The present study suggests that Vitamin D3 is a potentially useful agent for limiting IAV-induced cellular injury via its pro-autophagic action.

Internalized FGF-2-Loaded Nanoparticles Increase Nuclear ERK1/2 Content and Result in Lung Cancer Cell Death.

Innovative cancer treatments, which improve adjuvant therapy and reduce adverse events, are desperately needed. Nanoparticles provide controlled intracellular biomolecule delivery in the absence of activating external cell surface receptors. Prior reports suggest that intracrine signaling, following overexpression of basic fibroblast growth factor (FGF-2) after viral transduction, has a toxic effect on diseased cells. Herein, the research goals were to (1) encapsulate recombinant FGF-2 within stable, alginate-based nanoparticles (ABNs) for non-specific cellular uptake, and (2) determine the effects of ABN-mediated intracellular delivery of FGF-2 on cancer cell proliferation/survival. In culture, human alveolar adenocarcinoma basal epithelial cell line (A549s) and immortalized human bronchial epithelial cell line (HBE1s) internalized ABNs through non-selective endocytosis. Compared to A549s exposed to empty (i.e., blank) ABNs, the intracellular delivery of FGF-2 via ABNs significantly increased the levels of lactate dehydrogenase, indicating that FGF-2-ABN treatment decreased the transformed cell integrity. Noticeably, the nontransformed cells were not significantly affected by FGF-2-loaded ABN treatment. Furthermore, FGF-2-loaded ABNs significantly increased nuclear levels of activated-extracellular signal-regulated kinase ½ (ERK1/2) in A549s but had no significant effect on HBE1 nuclear ERK1/2 expression. Our novel intracellular delivery method of FGF-2 via nanoparticles resulted in increased cancer cell death via increased nuclear ERK1/2 activation.

Microvesicles derived from human Wharton\u2019s jelly mesenchymal stem cells enhance autophagy and ameliorate acute lung injury via delivery of miR-100

ObjectivesMicrovesicles (MVs) derived from human Wharton’s jelly mesenchymal stem cells (MSC-MVs) were demonstrated to ameliorate acute lung injury (ALI). We have previously found that MSC-MV-transferred hepatocyte growth factor was partly involved in their therapeutic effects. Since MSC-MVs also contained a substantial quantity of miR-100, which plays an important role in lung cancer and injury, we speculated that miR-100 might similarly account for a part of the therapeutic effects of MSC-MVs.MethodsMSCs were transfected with miR-100 inhibitor to downregulate miR-100 in MSC-MVs. A rat model of ALI and cell injury in rat type II alveolar epithelial cell line (L2) was induced by bleomycin (BLM). A co-culture model of alveolar epithelial cells and MSC-MVs was utilized to examine the therapeutic role of MSC-MVs and mechanism.ResultsMSC-MV treatment attenuated BLM-induced apoptosis and inflammation in BLM-treated L2 cells and ameliorated BLM-induced lung apoptosis, inflammation, and fibrosis in BLM-induced ALI rats. The beneficial effect of MSC-MVs was partly eliminated when miR-100 was knocked down in MSCs. Moreover, MSC-MV-transferred miR-100 mediated the therapeutic effect of MSC-MVs in ALI through enhancing autophagy by targeting mTOR.ConclusionMSC-MVs enhance autophagy and ameliorate ALI partially via delivery of miR-100.

Chronic WNT/β-catenin signaling induces cellular senescence in lung epithelial cells

The rapid expansion of the elderly population has led to the recent epidemic of age-related diseases, including increased incidence and mortality of chronic lung diseases, such as Idiopathic Pulmonary Fibrosis (IPF). Cellular senescence is a major hallmark of aging and has a higher occurrence in IPF. The lung epithelium represents a major site of tissue injury, cellular senescence and aberrant activity of developmental pathways such as the WNT/β-catenin pathway in IPF. The potential impact of WNT/β-catenin signaling on alveolar epithelial senescence in general as well as in IPF, however, remains elusive. Here, we characterized alveolar epithelial cells of aged mice and assessed the contribution of chronic WNT/β-catenin signaling on alveolar epithelial type (AT) II cell senescence. Whole lungs from old (16–24 months) versus young (3 months) mice had relatively less epithelial (EpCAM+) but more inflammatory (CD45+) cells, as assessed by flow cytometry. Compared to young ATII cells, old ATII cells showed decreased expression of the ATII cell marker Surfactant Protein C along with increased expression of the ATI cell marker Hopx, accompanied by increased WNT/β-catenin activity. Notably, when placed in an organoid assay, old ATII cells exhibited decreased progenitor cell potential. Chronic canonical WNT/β-catenin activation for up to 7 days in primary ATII cells as well as alveolar epithelial cell lines induced a robust cellular senescence, whereas the non-canonical ligand WNT5A was not able to induce cellular senescence. Moreover, chronic WNT3A treatment of precision-cut lung slices (PCLS) further confirmed ATII cell senescence. Simultaneously, chronic but not acute WNT/β-catenin activation induced a profibrotic state with increased expression of the impaired ATII cell marker Keratin 8. These results suggest that chronic WNT/β-catenin activity in the IPF lung contributes to increased ATII cell senescence and reprogramming. In the fibrotic environment, WNT/β-catenin signaling thus might lead to further progenitor cell dysfunction and impaired lung repair.

Role of plasminogen activator inhibitor-1 in methotrexate-induced epithelial-mesenchymal transition in alveolar epithelial A549 cells

There is increasing evidence that epithelial-mesenchymal transition (EMT) contributes to the development of organ fibrosis. We demonstrated that methotrexate (MTX) clearly induced EMT through the transforming growth factor (TGF)-β-related signaling pathway in human alveolar epithelial cell line, A549. However, critical factors associated with MTX-induced EMT have not yet been identified. In our study, we attempted to identify factors playing a crucial role in MTX-induced EMT in A549 cells. We focused on plasminogen activator inhibitor-1 (PAI-1) as the possible target for the prevention of MTX-induced EMT-related lung injury. Comprehensive gene expression analysis by microarray revealed that mRNA expression level of PAI-1 was clearly increased by MTX treatment. In addition, using several cloned A549 cells, we found a good correlation between MTX-induced increase in mRNA expression levels of α-smooth muscle actin (SMA), a representative EMT marker, and PAI-1. Furthermore, MTX upregulated mRNA and protein expression levels of PAI-1 in A549 cells; this upregulation was canceled by co-treatment with SB431542, a TGF-β-related signaling pathway inhibitor. Notably, tiplaxtinin, a PAI-1 inhibitor, and knockdown of urokinase-type plasminogen activator receptor (uPAR) prevented MTX-induced EMT in A549 cells. These findings indicate that MTX may induce EMT via upregulation of PAI-1 expression and interaction of PAI-1 with uPAR in A549 cells.

The extracytoplasmic function sigma factor \u03c3VreI is active during infection and contributes to phosphate starvation-induced virulence of Pseudomonas aeruginosa

The extracytoplasmic function sigma factor σVreI of the human pathogen Pseudomonas aeruginosa promotes transcription of potential virulence determinants, including secretion systems and secreted proteins. Its activity is modulated by the VreR anti-σ factor that inhibits the binding of σVreI to the RNA polymerase in the absence of a (still unknown) inducing signal. The vreI-vreR genes are expressed under inorganic phosphate (Pi) starvation, a physiological condition often encountered in the host that increases P. aeruginosa pathogenicity. However, whether or not σVreI is active in vivo during infection and contributes to the Pi starvation-induced virulence of this pathogen has not been analyzed yet. Using zebrafish embryos and a human alveolar basal epithelial cell line as P. aeruginosa hosts, we demonstrate in this work that σVreI is active during infection and that lack of σVreI considerably reduces the Pi starvation-induced virulence of this pathogen. Surprisingly, lack of the σVreI inhibitor, the VreR anti-σ factor, also diminishes the virulence of P. aeruginosa. By transcriptomic analyses we show that VreR modulates gene expression not only in a σVreI-dependent but also in a σVreI-independent manner. This includes potential virulence determinants and transcriptional regulators that could be responsible for the reduced virulence of the ΔvreR mutant.

Hydrogen Sulfide Attenuates Particulate Matter-Induced Emphysema and Airway Inflammation Through Nrf2-Dependent Manner.

PurposeTo investigate whether hydrogen sulfide provide protective effects on atmosphere particulate matter (PM)-induced emphysema and airway inflammation and its mechanism.MethodsWild type C57BL/6 and Nrf2 knockout mice were exposed to PM (200 µg per mouse). Hydrogen sulfide or propargylglycine were administered by intraperitoneal injection respectively 30 min before PM exposure, mice were anesthetized 29th day after administration. Mice emphysema, airway inflammation, and oxidative stress were evaluated, the expression of NLRP3, active caspase-1, and active caspase-3 were detected. Alveolar epithelial A549 cells line were transfected with control small interfering RNA (siRNA) or Nrf2 siRNA and then incubated with or without hydrogen sulfide for 30 min before exposed to fine particulate matter for 24 h, cell viability, terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick-end labeling (TUNEL) assay, the secretion of interleukin (IL)-1β, ASC speck formation, the expression level of NLRP3, active caspase-1, and active caspase-3 were measured.ResultsPM significantly increased mice emphysema and airway inflammation measured by mean linear intercept, alveolar destroy index and total cell, neutrophil counts, cytokines IL-6, tumor necrosis factor (TNF)-α, CXCL1, IL-1β in bronchoalveolar lavage fluid. PM-induced mice emphysema and airway inflammation was greatly attenuated by hydrogen sulfide, while propargylglycine aggravated that. PM-induced oxidative stress was reduced by hydrogen sulfide as evaluated by 8-OHdG concentrations in lung tissues. The expression of NLRP3, active caspase-1, and active caspase-3 enhanced by PM were also downregulated by hydrogen sulfide in mice lung. The protective effect of hydrogen sulfide on emphysema, airway inflammation, inhibiting oxidative stress, NLRP3 inflammasome formation, and anti-apoptosis was inhibited by Nrf2 knockout in mice. Similarly, hydrogen sulfide attenuated the secretion of IL-1β, NLRP3 expression, caspase-1 activation, ASC speck formation, and apoptosis caused by fine particulate matter exposure in A549 cells but not in Nrf2 silenced cells.ConclusionHydrogen sulfide played a protect role in PM-induced mice emphysema and airway inflammation by inhibiting NLRP3 inflammasome formation and apoptosis via Nrf2-dependent pathway.

Depression of lncRNA NEAT1 Antagonizes LPS-Evoked Acute Injury and Inflammatory Response in Alveolar Epithelial Cells via HMGB1-RAGE Signaling.

Sepsis-evoked acute lung injury (ALI) and its extreme manifestation, acute respiratory distress syndrome (ARDS), constitute a major cause of mortality in intensive care units. High levels of the long noncoding RNA nuclear paraspeckle assembly transcript 1 (NEAT1) have been positively correlated with increased severity and unfavorable prognoses in patients with sepsis. Nevertheless, the function and molecular mechanism of NEAT1 in ALI remain elusive. In the current study, high levels of NEAT1 were confirmed in lipopolysaccharide- (LPS-) induced ALI mice models and in LPS-stimulated cells from the alveolar epithelial A549 cell line. Intriguingly, cessation of NEAT1 led to increased cell viability and decreased lactate dehydrogenase release, apoptosis, and caspase-3/9 activity, which conferred protection against LPS-induced injury in these cells. NEAT1 inhibition also restrained LPS-evoked transcripts and production of inflammatory cytokines IL-6, IL-1β, and TNF-α. A mechanism analysis corroborated the activation of high-mobility group box1 (HMGB1)/receptors for advanced glycation end products (RAGE) and NF-κB signaling in LPS-treated A549 cells. NEAT1 suppression reversed the activation of this pathway. Notably, reactivating HMGB1/RAGE signaling via HMGB1 overexpression blunted the anti-injury and anti-inflammation effects of NEAT1 knockdown. These findings suggest that NEAT1 may aggravate the progression of ALI and ARDS by inducing alveolar epithelial cell injury and inflammation via HMGB1/RAGE signaling, implying a promising treatment target for these conditions.

Glycyrrhizin suppresses epithelial-mesenchymal transition by inhibiting high-mobility group box1 via the TGF-β1/Smad2/3 pathway in lung epithelial cells.

BackgroundEpithelial-mesenchymal transition (EMT) plays an important role in fibrosis, chronic inflammation, tumor metastasis, etc. Glycyrrhizin, an active component extracted from licorice plant, has been reported to treat a variety of inflammatory reactions through inhibiting high-mobility group box1 (HMGB1), which has been suggested to be a significant mediator in EMT process. However, whether glycyrrhizin affects the EMT process or not remains unclear.MethodsHuman alveolar epithelial cell line A549 and normal human bronchial epithelial cell line BEAS-2B were treated with extrinsic TGF-β1 to induce EMT. Elisa was used to detect HMGB1 concentrations in cell supernatant. RNA interference and lentivirus infection experiments were performed to investigate the involvement of HMGB1 in EMT process. Cell Counting Kit-8 (CCK-8) was used to detect the viability of A549 and BEAS-2B cells treated with glycyrrhizin. Finally, the effects of glycyrrhizin on EMT changes, as well as the underlying mechanisms, were evaluated via Western blot, immunofluorescence and transwell assays.ResultsOur results showed that HMGB1 expression was increased by TGF-β1, and knockdown of HMGB1 expression reversed TGF-β1-induced EMT in A549 and BEAS-2B cells. Ectopic HMGB1 expression or TGF-β1 treatment caused a significant increase in HMGB1 release. Notably, we found that glycyrrhizin treatment effectively suppressed TGF-β1-induced EMT process by inhibiting HMGB1. Also, glycyrrhizin significantly inhibited the migration of both A549 and BEAS-2B cells promoted by TGF-β1. Mechanistically, HMGB1 overexpression could activate Smad2/3 signaling in A549 and BEAS-2B cells. Glycyrrhizin significantly blocked the phosphorylation of Smad2/3 stimulated either by TGF-β1 or by ectopic HMGB1 in A549 and BEAS-2B cells.ConclusionsHMGB1 is a vital mediator of EMT changes induced by TGF-β1 in lung epithelial cells. Importantly, glycyrrhizin can effectively block Smad2/3 signaling pathway through inhibiting HMGB1, thereby suppressing the EMT progress.

Blocking TG2 attenuates bleomycin-induced pulmonary fibrosis in mice through inhibiting EMT

BackgroundEpithelial-mesenchymal transformation (EMT) is a central mechanism for the occurrence and development of pulmonary fibrosis. Therefore, to identify the key target molecules regulating the EMT process is considered as an important direction for the prevention and treatment of pulmonary fibrosis. Transglutaminase 2 (TG2) has been recently found to play an important role in the regulation of inflammation and the generation of extracellular matrix. Here, our study focuses on the roles of TG2 in pulmonary fibrosis and EMT. Methodsat first, the expression of TG2 and the EMT-related markers like E-cadherin, Vimentin, and α-SMA were detected with Western Blotting, immunohistochemistry and other methods in the mice with pulmonary fibrosis induced by bleomycin. Further, MLE 12 cells were used to study the effects on EMT of the inhibition of TG2 in vitro. Finally, GK921, an inhibitor against TG2, was used to show its function in both prevention and treatment of pulmonary fibrosis induced by bleomycin in mice. Resultsbleomycin succeeded to induce pulmonary fibrosis in mice, with increased TG2 expression, EMT and Akt activation. Knock-down of TG2 by siRNA technique in MLE 12 cell (a mouse alveolar epithelial cell line) and GK921 (an inhibitor of TG2) all inhibited the EMT process, however SC79, an activator of Akt rescued above inhibition. Finally, GK921 alleviated pulmonary fibrosis in mice induced by bleomycin. ConclusionBlocking TG2 reduces bleomycin-induced pulmonary fibrosis in mice via inhibiting EMT.

Development and characterization of a human cell line-based transactivation assay to assess thyroid EDCs

Thyroid hormones (THs) are one of the most important hormones, playing key roles in the regulation of various physiological functions. Although THs have important function in human, in vitro test methods based on human cells are currently insufficient to effectively screen and test TH-related endocrine disrupting chemicals (EDCs).We established a TH agonist TA assay using the adenocarcinomic human alveolar basal epithelial cell line A549 to test and screen potential TH agonists. To establish the TH agonist TA assay, a TRE-secNluc-IRES-EGFP reporter cassette was constructed and transfected into the A549 cell line using a retrovirus. We evaluated the TH agonistic properties of several chemicals which were tested by existing thyroid agonists testing method (OECD GD 207). Comparing the results of the TH agonist TA assay with the OECD GD 207, T3, T4, tiratricol, and tetrac (natural TH and 3,3′,5,5′-tetraiodothyroacetic acid derivatives), which are TH agonists according to the OECD GD 207, also tested positive in the TH agonist TA assay using the A549 cell line. These results suggested that the TH agonist TA assay developed in this study using a human cell line can provide the information, such as accuracy and specificity to TH agonistic properties of chemicals.

Co-expression of Hsp70 Protein and Autophagy Marker Protein LC3 in A549 Cells and THP1 Cells Exposed to Nanoparticles of Air Pollution.

The ability of air particulate matter (PM) to cause reactive oxygen species-driven protein damage is associated with both COPD and lung cancer, but the mechanisms are unsettled. In this study, we investigated the co-expression of Hsp70 and the autophagy marker protein LC3 in A549 cells (alveolar epithelial cell line) and THP-1 cells (monocyte/macrophage cells) grown in media supplemented with 100μg/mL of four types of PM: carbon black (CB), urban dust (UD), nanoparticulate CB (NPCB), and nanoparticulate CB coated with benzo(a)pyrene (NPCB-BaP). Fluorescent monoclonal antibodies and flow cytometry were used to assess the expression and co-expression of HSP70 and LC3 proteins. Hsp70 expression was significantly increased by all PM, while LC3 was decreased by CB in A549 cells, unchanged by CB and UD in THP-1 cells and increased by NPCB and NPCB-BaP in both cell types. All PMs increased the Hsp70/LC3 ratio in binary scatterplots; the relationship was positive and linear, which may reflect chaperone-dependent autophagy. The UD was the only PM type that affected the slopes of the spatial trend lines and altered binary patterns of Hsp70/LC3 distribution in THP1 cells. These findings provide an insight into the molecular mechanisms regulating proteostasis in PM-exposed cells through the chaperone-autophagy system in the cytoplasm.

LncRNA GAS5 suppresses inflammatory responses and apoptosis of alveolar epithelial cells by targeting miR-429/DUSP1

Acute lung injury (ALI) is a life-threatening syndrome characterized by excessive inflammation and apoptosis of alveolar epithelial cells. This study firstly investigated the role and mechanism of long non-coding RNA (lncRNA) growth arrest-specific 5 (GAS5) in regulating lipopolysaccharide (LPS)-induced inflammatory response and apoptosis of murine alveolar epithelial cell line MLE-12. The expression of GAS5, miR-429, and dual-specificity phosphatase 1 (DUSP1) were examined using quantitative Real-Time PCR (qRT-PCR) and western blot. The inflammatory responses were evaluated by detecting the levels of pro-inflammatory cytokines using ELISA. Cell apoptosis was assessed by TUNEL assay. The interactions among GAS5, miR-429, and DUSP1 were examined using luciferase reporter assay. The results showed that GAS5 and DUSP1 expression were decreased, whereas miR-429 was increased in lung tissues from LPS-induced ALI mice and LPS-treated MLE-12 cells. Furthermore, GAS5 overexpression decreased cell inflammatory responses and apoptosis in LPS-treated MLE-12 cells, which was reversed by miR-429 mimic and DUSP1 knockdown. Mechanistically, GAS5 acted as a competitive endogenous RNA by sponging miR-429 to facilitate DUSP1 expression. Our findings suggest that GAS5 suppresses inflammatory responses and apoptosis of alveolar epithelial cell MLE-12 by targeting miR-429/DUSP1 axis.

Interleukin-22 regulates interferon lambda expression in a mice model of pseudomonas aeruginosa pneumonia

BackgroundInterleukin (IL)-22 is a cytokine involved in tissue protection and repair following lung pathologies. Interferon (IFN)-λ cytokines displayed similar properties during viral infection and a synergy of action between these two players has been documented in the intestine. We hypothesize that during Pseudomonas aeruginosa challenge, IL-22 up-regulates IFN-λ and that IFN-λ exhibits protective functions during Pseudomonas aeruginosa acute pneumonia model in mice. MethodsUsing an in vitro human alveolar epithelial cell line A549, we assessed the ability of IL-22 to enhance IFN-λ expression during infection. IFN-λ protective function was evaluated in an acute mouse pneumonia model. ResultsWe first demonstrated in murine lungs that only type-II alveolar cells express IL-22 receptor and that IL-22 treatment of A549 cell line up-regulates IFN-λ expression. In a murine acute pneumonia model, IL-22 administration maintained significant IFN-λ levels in the broncho-alveolar fluids whereas IL-22 neutralization abolished IFN-λ up-regulation. In vivo administration of IFN-λ during Pseudomonas aeruginosa pneumonia improves mice outcome by dampening neutrophil recruitment and decreasing epithelium damages. DiscussionWe show here that IL-22 regulates IFN-λ levels during Pseudomonas aeruginosa pneumonia.

A composite Gelatin/hyaluronic acid hydrogel as an ECM mimic for developing mesenchymal stem cell-derived epithelial tissue patches.

Here we report fabrication of Gelatin-based biocomposite films and their application in developing epithelial patches. The films were loaded with an epithelial cell growth factor cocktail and used as an extracellular matrix mimic for in vitro regeneration of organized respiratory epithelium using Calu-3 cell line and mesenchymal stem cells (MSCs). Our data show differentiation of Calu-3 cells on composite films as evidenced by tight junction protein expression and barrier formation. The films also supported attachment, migration, and proliferation of alveolar basal epithelial cell line A549. We also show the suitability of the composite films as a biomimetic scaffold and growth factor delivery platform for differentiation of human MSCs to epithelial cells. MSCs differentiation to the epithelial lineage was confirmed by staining for epithelial and stem cell specific markers. Our data show that the MSCs acquire the epithelial characteristics after 2 weeks with significant reduction in vimentin, increase in pan cytokeratin expression, and morphological changes. However, despite the expression of epithelial lineage markers, these cells did not form fully functional tight junctions as evidenced by low expression of junctional protein ZO1. Further optimisation of culture conditions and growth factor cocktail is required to enhance tight junction formation in MSCs-derived epithelial cells on the composite hydrogels. Nevertheless, our data clearly highlight the possibility of using MSCs in epithelial tissue engineering and the applicability of the composite hydrogels as transferrable extracellular matrix mimics and delivery platforms with potential applications in regenerative medicine and in vitro modelling of barrier tissues.

Idebenone has preventative and therapeutic effects on pulmonary fibrosis via preferential suppression of fibroblast activity

Alveolar epithelial injury induced by reactive oxygen species (ROS) and abnormal collagen production by activated fibroblasts (myofibroblasts) is involved in the onset and exacerbation of idiopathic pulmonary fibrosis (IPF). Compared with alveolar epithelial cells, lung fibroblasts, especially myofibroblasts, exhibit an apoptosis-resistance phenotype (apoptosis paradox) that appears to be involved in IPF pathogenesis. Thus, we screened for chemicals eliciting preferential cytotoxicity of LL29 cells (lung fibroblasts from an IPF patient) compared with A549 cells (human lung alveolar epithelial cell line) from medicines already in clinical use. We identified idebenone, a synthetic analogue of coenzyme Q10 (CoQ10, an antioxidant) that has been used clinically as a brain metabolic stimulant. Idebenone induced cell growth inhibition and cell death in LL29 cells at a lower concentration than in A549 cells, a feature that was not observed for other antioxidant molecules (such as CoQ10) and two IPF drugs (pirfenidone and nintedanib). Administration of idebenone prevented bleomycin-induced pulmonary fibrosis and increased pulmonary ROS levels. Importantly, idebenone also improved pulmonary fibrosis and lung function when administered after the development of fibrosis, whereas administration of CoQ10 similarly prevented bleomycin-induced pulmonary fibrosis, but had no effect after its development. Administration of idebenone, but not CoQ10, suppressed bleomycin-induced increases in lung myofibroblasts. In vitro, treatment of LL29 cells with idebenone, but not CoQ10, suppressed TGF-β–induced collagen production. These results suggest that in addition to antioxidant activity, idebenone exerts inhibitory activity on the function of lung fibroblasts, with the former activity being preventative and the latter therapeutic for bleomycin-induced fibrosis. Thus, we propose that idebenone may be more therapeutically beneficial for IPF patients than current treatments.