Response Of Lung Epithelial Cells Research Articles

Silver nanoparticles reduce the apoptosis induced by tumor necrosis factor-α

ABSTRACTSilver nanoparticles (AgNPs) are widely known to have anti-inflammatory properties, but the exact mechanism underlying this anti-inflammatory effect is not clearly understood. Tumor necrosis factor-α (TNFα) is a major pro-inflammatory cytokine that is expressed in the early stage of cell inflammation and induces apoptosis by several known pathways. Our study aimed to investigate the effect of AgNPs on the response of lung epithelial cells to TNFα and the molecular mechanism of this response. Lung epithelial cell line NCI-H292 cells were exposed to AgNPs (5 µg/mL) and/or TNFα (20 ng/mL) for 24 h, then cellular uptake was analyzed using flow cytometry. Our results showed that AgNPs were taken up by cells in a dose-dependent manner and that the cellular uptake ratio of AgNPs was significantly increased in the presence of TNFα. Apoptosis assays indicated that exposure to AgNPs significantly decreased the apoptotic effect of TNFα. Confocal microscopy was used to localize the tumor necrosis factor receptor 1 (TNFR1) and revealed that TNFR1 localized on the surface of cells exposed to TNFα. In contrast, TNFR1 localized inside cells exposed to both AgNPs and TNFα, with very few receptors scattered on the cell membrane. The results indicated that AgNPs reduced the cell surface TNFR1 expression level. The results suggested that the reduction of surface TNFR1 reduced cellular response to TNFα, resulting in an anti-apoptotic effect.

IL-17A and GDF15 are able to induce epithelial-mesenchymal transition of lung epithelial cells in response to cigarette smoke.

Smoking is one of the primary causes of chronic obstructive pulmonary disease (COPD). Sustained active epithelial-mesenchymal transition (EMT) in COPD may explain the core pathophysiology of airway fibrosis and why lung cancer is so common among smokers. Interleukin (IL)-17A and growth/differentiation factor (GDF)15 have been reported to be biomarkers of COPD; however, the role of IL-17A and GDF15 in EMT remains unclear. The aim of the present study was to investigate the role of IL-17A and GDF15 in the pathogenesis of COPD. It was demonstrated that IL-17A and GDF15 are upregulated in patients with COPD, particularly those with a history of smoking. The results also revealed that IL-17A and GDF15 expression was negatively correlated with the epithelial marker epithelial-cadherin and positively correlated with the mesenchymal marker vimentin. Furthermore, treatment with cigarette smoke extract or IL-17A induced GDF15 expression. Combined treatment with IL-17A and GDF15 induced EMT in human small epithelial HSAEpiC cells in vitro. Collectively, the results of the present study suggest that IL-17A and GDF15-induced EMT serves an important role in the pathology of COPD.

Cigarette smoke dampens antiviral signaling in small airway epithelial cells by disrupting TLR3 cleavage.

Cigarette smokers and people exposed to second-hand smoke are at an increased risk for pulmonary viral infections, and yet the mechanism responsible for this heightened susceptibility is not understood. To understand the effect of cigarette smoke on susceptibility to viral infection, we used an air-liquid interface culture system and exposed primary human small airway epithelial cells (SAEC) to whole cigarette smoke, followed by treatment with the viral mimetic polyinosinic polycytidylic acid (poly I:C) or influenza A virus (IAV). We found that prior smoke exposure strongly inhibited production of proinflammatory (interleukin-6 and interleukin-8) and antiviral [interferon-γ-induced protein 10 (IP-10) and interferons] mediators in SAECs in response to poly I:C and IAV infection. Impaired antiviral responses corresponded to increased infection with IAV. This was associated with a decrease in phosphorylation of the key antiviral transcription factor interferon response factor 3 (IRF3). Here, we found that cigarette smoke exposure inhibited activation of Toll-like receptor 3 (TLR3) by impairing TLR3 cleavage, which was required for downstream phosphorylation of IRF3 and production of IP-10. These results identify a novel mechanism by which cigarette smoke exposure impairs antiviral responses in lung epithelial cells, which may contribute to increased susceptibility to respiratory infections.

Inhaled Cryptococcus neoformans elicits allergic airway inflammation independent of Nuclear Factor Kappa B signalling in lung epithelial cells.

Pulmonary challenge with the ubiquitous fungus Cryptococcus neoformans results in allergic airway inflammation (AAI) characterized by robust recruitment of eosinophils and T cells producing type 2 cytokines to the lungs. Previous studies have demonstrated a critical role for Nuclear Factor Kappa B (NF-κB) activation within lung epithelial cells (LECs) in driving AAI in response to protein allergens, yet the role of LEC-intrinsic NF-κB in promoting AAI following exposure to C. neoformans is poorly understood. To investigate the role of LEC-intrinsic NF-κB in promoting AAI following C. neoformans challenge, we used IKK∆LEC mice, which lack canonical NF-κB activation specifically within LECs. IKK∆LEC and littermate control mice were intranasally challenged with 106 CFU of C. neoformans strain 52D, and lung tissues were collected at 7, 14 and 21 days post infection to assess the development of AAI. Notably, the absence of epithelial NF-κB signalling did not affect the magnitude or kinetics of lung eosinophilia when compared with the response in wild-type control mice. The total numbers of lung T cells producing the type 2 cytokines interleukin-5 and interleukin-13 were also unchanged in IKK∆LEC mice. Furthermore, IKK∆LEC mice showed no defect in the recruitment of protective interferon-γ-producing CD4 T cells to the lungs, fungal clearance, or host survival compared with control mice. Immunofluorescence imaging surprisingly revealed no evidence of nuclear localization of NF-κB in LECs in response to C. neoformans challenge, indicating that NF-κB is not activated within these cells. Taken together, these data strongly suggest that NF-κB signalling within LECs does not promote AAI observed in response to C. neoformans.

69 - Age related alteration of the antioxidant/ inflammatory axis in human lung epithelial cells in response to nanoparticle challenge

Silica nanoparticles with iron on their surface (SiFeP) induce the production of oxidants and pro-inflammatory cytokines in epithelial cells. Crosstalk between the nuclear factor erythroid-derived 2-like factor 2 (Nrf2) and NF-κB signaling was found in response to SiFeP exposure. However, little is known about the age-related change in interaction between antioxidants and pro-inflammatory cytokine production in response to SiFeP in elderly humans. In this study, levels of Nrf2-regulated antioxidant mRNA and protein and NFκB dependent cytokine mRNAs were measured in human bronchial epithelial (HBE) cells from young adult (28-29 years) and older (67-69 years) non-smokers following the exposure to 40 μg/ml SiFeP. At 6 h, mRNA induction of interleukin 6 in the cells from the elderly was 2 times as high as that in the young adult cells. After 24 h treatment, mRNA expression of two representative Nrf2-regulated genes, catalytic subunit of glutamate cysteine ligase (GCLC) and NAD(P)H quinone oxidoreductase 1 (NQO1), was higher in cells from the young adult donors compared with cells from the older donors. The induction of GCLC and NQO1 in young cells were 1.67 and 1.31 times higher than old cells respectively. In addition, Bach1, which is Nrf2 suppressor, was increased compared to negative control in the old cells while barely changed in young adult cells either after 6 h or 24 h treatment. These results suggested that the decreased inducibility of Nrf2 regulated genes in old cells might be related to elevated expression of Bach1 and enhanced activation of NF-κB signaling in response to SiFeP exposure compared to the young adult cells. This may partially explain the increased susceptibility to environmental respiratory disease in the elderly.

Quantitative Proteomic Approach Identifies Vpr Binding Protein as Novel Host Factor Supporting Influenza A Virus Infections in Human Cells

Influenza A virus (IAV) infections are a major cause for respiratory disease in humans, which affects all age groups and contributes substantially to global morbidity and mortality. IAV have a large natural host reservoir in avian species. However, many avian IAV strains lack adaptation to other hosts and hardly propagate in humans. While seasonal or pandemic IAV strains replicate efficiently in permissive human cells, many avian IAV cause abortive nonproductive infections in these hosts despite successful cell entry. However, the precise reasons for these differential outcomes are poorly defined. We hypothesized that the distinct course of an IAV infection with a given virus strain is determined by the differential interplay between specific host and viral factors. By using Spike-in SILAC mass spectrometry-based quantitative proteomics we characterized sets of cellular factors whose abundance is specifically up- or downregulated in the course of permissive versus nonpermissive IAV infection, respectively. This approach allowed for the definition and quantitative comparison of about 3500 proteins in human lung epithelial cells in response to seasonal or low-pathogenic avian H3N2 IAV. Many identified proteins were similarly regulated by both virus strains, but also 16 candidates with distinct changes in permissive versus nonpermissive infection were found. RNAi-mediated knockdown of these differentially regulated host factors identified Vpr binding protein (VprBP) as proviral host factor because its downregulation inhibited efficient propagation of seasonal IAV whereas overexpression increased viral replication of both seasonal and avian IAV. These results not only show that there are similar differences in the overall changes during permissive and nonpermissive influenza virus infections, but also provide a basis to evaluate VprBP as novel anti-IAV drug target.

Focal adhesion kinase signaling determines the fate of lung epithelial cells in response to TGF-β

Alveolar epithelial cell (AEC) injury and apoptosis are prominent pathological features of idiopathic pulmonary fibrosis (IPF). There is evidence of AEC plasticity in lung injury repair response and in IPF. In this report, we explore the role of focal adhesion kinase (FAK) signaling in determining the fate of lung epithelial cells in response to transforming growth factor-β1 (TGF-β1). Rat type II alveolar epithelial cells (RLE-6TN) were treated with or without TGF-β1, and the expressions of mesenchymal markers, phenotype, and function were analyzed. Pharmacological protein kinase inhibitors were utilized to screen for SMAD-dependent and -independent pathways. SMAD and FAK signaling was analyzed using siRNA knockdown, inhibitors, and expression of a mutant construct of FAK. Apoptosis was measured using cleaved caspase-3 and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. TGF-β1 induced the acquisition of mesenchymal markers, including α-smooth muscle actin, in RLE-6TN cells and enhanced the contraction of three-dimensional collagen gels. This phenotypical transition or plasticity, epithelial-myofibroblast plasticity (EMP), is dependent on SMAD3 and FAK signaling. FAK activation was found to be dependent on ALK5/SMAD3 signaling. We observed that TGF-β1 induces both EMP and apoptosis in the same cell culture system but not in the same cell. While blockade of SMAD signaling inhibited EMP, it had a minimal effect on apoptosis; in contrast, inhibition of FAK signaling markedly shifted to an apoptotic fate. The data support that FAK activation determines whether AECs undergo EMP vs. apoptosis in response to TGF-β1 stimulation. TGF-β1-induced EMP is FAK- dependent, whereas TGF-β1-induced apoptosis is favored when FAK signaling is inhibited.

Water extract of Helminthostachys zeylanica attenuates LPS-induced acute lung injury in mice by modulating NF-κB and MAPK pathways

Ethnopharmacological relevancePrevious studies showed that Helminthostachys zeylanica (L.) Hook. could reduce inflammatory responses in macrophage and brain astrocytes. Aim of the studyIn the present study, we evaluated whether an ethyl acetate extract (HZE) or a water extract (HZW) of H. zeylanica could reduce inflammatory responses in lung epithelial cells and ameliorate lipopolysaccharide (LPS)-induced acute lung injury in mice. MethodsHuman lung epithelial A549 cells were pre-treated with HZE or HZW (1–10μg/mL), then stimulated with LPS. BALB/c mice received oral HZW for 7 consecutive days, then an intratracheal instillation of LPS to induce lung injury. ResultsHZW reduced chemokine and proinflammatory cytokine production in LPS-activated A549 cells. HZW also suppressed ICAM-1 expression and reduced the adherence of acute monocytic leukemia cells to inflammatory A549 cells. HZE had less efficacy than HZW in suppressing inflammatory responses in A549 cells. In vivo, HZW significantly suppressed neutrophil infiltration and reduced the TNF-α and IL-6 levels in bronchoalveolar lavage fluid and serum from LPS-treated mice. HZW also modulated superoxide dismutase activity, glutathione, and myeloperoxidase activity in lung tissues from LPS-treated mice. HZW decreased the phosphorylation of mitogen-activated protein kinase and nuclear factor kappa B, and promoted heme oxygenase-1 expression in inflamed lung tissue from LPS-treated mice. ConclusionOur findings suggested that HZW reduced lung injury in mice by reducing oxidative stress and inflammatory responses. HZW also reduced inflammatory responses in human lung epithelial cells.

Cigarette smoke alters the secretome of lung epithelial cells

Cigarette smoke is the most relevant risk factor for the development of lung cancer and chronic obstructive pulmonary disease. Many of its more than 4500 chemicals are highly reactive, thereby altering protein structure and function. Here, we used subcellular fractionation coupled to label-free quantitative MS to globally assess alterations in the proteome of different compartments of lung epithelial cells upon exposure to cigarette smoke extract. Proteomic profiling of the human alveolar derived cell line A549 revealed the most pronounced changes within the cellular secretome with preferential downregulation of proteins involved in wound healing and extracellular matrix organization. In particular, secretion of secreted protein acidic and rich in cysteine, a matricellular protein that functions in tissue response to injury, was consistently diminished by cigarette smoke extract in various pulmonary epithelial cell lines and primary cells of human and mouse origin as well as in mouse ex vivo lung tissue cultures. Our study reveals a previously unrecognized acute response of lung epithelial cells to cigarette smoke that includes altered secretion of proteins involved in extracellular matrix organization and wound healing. This may contribute to sustained alterations in tissue remodeling as observed in lung cancer and chronic obstructive pulmonary disease.

MicroRNA-19b Mediates Lung Epithelial-Mesenchymal Transition via Phosphatidylinositol-3,4,5-Trisphosphate 3-Phosphatase in Response to Mechanical Stretch.

Lung epithelial-mesenchymal transition (EMT) plays an important role in ventilation-associated lung fibrosis, which may contribute to the poor outcome of patients with acute respiratory distress syndrome. Because microRNAs control and modulate normal physiological and pathophysiological processes, we investigated the role of microRNAs in the development of acute respiratory distress syndrome-associated EMT in response to mechanical stress. In the current study, primary human alveolar epithelial type II (AEII) cells were subjected to cyclic stretch that resulted in EMT profiles with decreased gene expression of cytokeratin-8, E-cadherin, and surfactant protein B, and increased expression of vimentin, α-smooth muscle actin, and N-cadherin. Microarray analysis revealed that the expression of microRNA-19b (miR-19b) was up-regulated in the AEII cells, and real-time polymerase chain reaction showed that the expression of miR-19b increased in both the AEII cells and the primary human small-airway epithelial cells. Overexpression of miR-19b in small-airway epithelial cells promoted the mechanical stretch-induced EMT phenotypes, whereas inhibition of miR-19b attenuated it. The inhibitory effect of miR-19b was attributed to enhanced signaling of phosphatidylinositol-3,4,5-trisphosphate 3-phosphatase (PTEN), leading to inactivation of the AKT pathway. Restoration of PTEN expression or inhibition of AKT phosphorylation suppressed the mechanical stretch-induced EMT phenotypes. We further demonstrated that the mechanical stretch-induced miR19 expression was regulated by the focal adhesion kinase-Rho pathway. In conclusion, we found that miR-19b plays a key role in the development of the EMT phenotype through down-regulation of PTEN in human lung epithelial cells in response to mechanical stretch. The miR-19b-PTEN signaling pathway may serve as a novel therapeutic target in the context of ventilator-associated lung fibrosis.

The life cycle stages of Pneumocystis murina have opposing effects on the immune response to this opportunistic, fungal pathogen.

The cyst cell wall β-glucans of Pneumocystis have been shown to stimulate immune responses in lung epithelial cells, dendritic cells, and alveolar macrophages. Little is known about how the trophic life forms, which do not have a fungal cell wall, interact with these innate immune cells. Here, we report differences in the responses of both neonatal and adult mice to the trophic and cystic life cycle stages of Pneumocystis murina The adult and neonatal immune responses to infection with Pneumocystis murina trophic forms were less robust than the response to infection with a physiologically normal mixture of cysts and trophic forms. Cysts promoted the recruitment of nonresident innate immune cells and T and B cells into the lungs. Cysts, but not trophic forms, stimulated increased IFN-γ cytokine concentrations in the alveolar spaces, and an increase in IFN-γ-producing CD4+ T cells. In vitro, bone marrow-derived dendritic cells (BMDCs) stimulated with cysts produced the proinflammatory cytokines IL-1β and IL-6. In contrast, trophic forms suppressed β-glucan-, LTA-, and LPS-induced IL-1β, IL-6, and TNFα production by BMDCs and antigen presentation to CD4+ T cells. The negative effects of trophic forms were not due to ligation of mannose receptor. Our results indicate that optimal innate and adaptive immune responses to Pneumocystis species are dependent on stimulation with the cyst life cycle stage. Conversely, trophic forms suppress β-glucan-induced proinflammatory responses in vitro, suggesting that the trophic forms dampen cyst-induced inflammation in vivo.

Exposure to common respiratory bacteria alters the airway epithelial response to subsequent viral infection.

BackgroundColonization of the airways with potential pathogenic bacteria is observed in a number of chronic respiratory diseases, such as COPD or cystic fibrosis. Infections with respiratory viruses are known triggers of exacerbations of these diseases. We here investigated if pre-exposure to bacteria alters the response of lung epithelial cells to subsequent viral infection.MethodsBronchial epithelial cells (BEAS-2B cells and primary bronchial epithelial cells) were exposed to heat-inactivated Haemophilus influenzae, Pseudomonas aeruginosa or Streptococcus pneumoniae and subsequently infected with respiratory syncytial virus (RSV), type 2 human adenovirus or influenza B. Levels of pro-inflammatory cytokines, viral replication and expression of pattern recognition receptors were determined in culture supernatants and/or cell lysates.ResultsExposure of BEAS-2B cells to H. influenzae before and during RSV-infection synergistically increased the release of IL-6 (increase above calculated additive effect at 72 h: 56 % ± 3 %, mean ± SEM) and IL-8 (53 % ± 12 %). This effect was sustained even when bacteria were washed away before viral infection and was neither associated with enhanced viral replication, nor linked to increased expression of key pattern recognition receptors. P. aeruginosa enhanced the release of inflammatory cytokines to a similar extent, yet only if bacteria were also present during viral infection. S. pneumoniae did not enhance RSV-induced cytokine release. Surprisingly, adenovirus infection significantly reduced IL-6 release in cells exposed to either of the three tested bacterial strains by on average more than 50 %. Infection with influenza B on the other hand did not affect cytokine production in BEAS-2B cells exposed to the different bacterial strains.ConclusionPre-exposure of epithelial cells to bacteria alters the response to subsequent viral infection depending on the types of pathogen involved. These findings highlight the complexity of microbiome interactions in the airways, possibly contributing to the susceptibility to exacerbations and the natural course of airway diseases.Electronic supplementary materialThe online version of this article (doi:10.1186/s12931-016-0382-z) contains supplementary material, which is available to authorized users.

Glutathione S-transferase pi modulates NF-κB activation and pro-inflammatory responses in lung epithelial cells

Nuclear Factor kappa B (NF-κB) is a transcription factor family critical in the activation of pro- inflammatory responses. The NF-κB pathway is regulated by oxidant-induced post-translational modifications. Protein S-glutathionylation, or the conjugation of the antioxidant molecule, glutathione to reactive cysteines inhibits the activity of inhibitory kappa B kinase beta (IKKβ), among other NF-κB proteins. Glutathione S-transferase Pi (GSTP) is an enzyme that has been shown to catalyze protein S-glutathionylation (PSSG) under conditions of oxidative stress. The objective of the present study was to determine whether GSTP regulates NF-κB signaling, S-glutathionylation of IKK, and subsequent pro-inflammatory signaling. We demonstrated that, in unstimulated cells, GSTP associated with the inhibitor of NF-κB, IκBα. However, exposure to LPS resulted in a rapid loss of association between IκBα and GSTP, and instead led to a protracted association between IKKβ and GSTP. LPS exposure also led to increases in the S-glutathionylation of IKKβ. SiRNA-mediated knockdown of GSTP decreased IKKβ-SSG, and enhanced NF-κB nuclear translocation, transcriptional activity, and pro-inflammatory cytokine production in response to lipopolysaccharide (LPS). TLK117, an isotype-selective inhibitor of GSTP, also enhanced LPS-induced NF-κB transcriptional activity and pro-inflammatory cytokine production, suggesting that the catalytic activity of GSTP is important in repressing NF-κB activation. Expression of both wild-type and catalytically-inactive Y7F mutant GSTP significantly attenuated LPS- or IKKβ-induced production of GM-CSF. These studies indicate a complex role for GSTP in modulating NF-κB, which may involve S-glutathionylation of IKK proteins, and interaction with NF-κB family members. Our findings suggest that targeting GSTP is a potential avenue for regulating the activity of this prominent pro-inflammatory and immunomodulatory transcription factor.

ATP8B1 Deficiency Enhances Hyperoxia-Induced Lipid Oxidation and Apoptotic Response in Lung Epithelial Cells

ATP8B1 Deficiency Enhances Hyperoxia-Induced Lipid Oxidation and Apoptotic Response in Lung Epithelial Cells

Ethanol, ethyl and sodium pyruvate decrease the inflammatory responses of human lung epithelial cells via Akt and NF-κB in vitro but have a low impact on hepatocellular cells.

Increases in pro-inflammatory cytokine levels and tissue-infiltrating leukocytes have been closely linked to increased systemic and local inflammation, which result in organ injury. Previously, we demonstrated the beneficial and hepatoprotective anti-inflammatory effects of acute ethanol (EtOH) ingestion in an in vivo model of acute inflammation. Due to its undesirable side-effects, however, EtOH does not represent a therapeutic option for treatment of acute inflammation. Therefore, in this study, we compared the effects of acute EtOH exposure with ethyl pyruvate (EtP) as an alternative anti-inflammatory drug in an in vitro model of hepatic and pulmonary inflammation. Human hepatocellular carcinoma cells Huh7 and alveolar epithelial cells A549 were stimulated with either interleukin (IL) IL-1β (1 ng/ml, 24 h) or tumor necrosis factor (TNF) (10 ng/ml, 4 h), and then treated with EtP (2.5-10 mM), sodium pyruvate (NaP, 10 mM) or EtOH (85-170 mM) for 1 h. IL-6 or IL-8 release from Huh7 or A549 cells, respectively, was measured by an enzyme‑linked immunosorbent assay. Neutrophil adhesion to cell monolayers and CD54 expression were also analyzed. Bcl-2 and Bax gene expression was determined by RT-qPCR, and western blot analysis was performed to determine the mechanisms involved. Treating A549 cells with either EtOH or EtP significantly reduced the IL-1β- or TNF‑induced IL-8 release, whereas treating Huh7 cells did not significantly alter IL-6 release. Similarly, neutrophil adhesion to stimulated A549 cells was significantly reduced by EtOH or EtP, whereas for Huh7 cells the tendency for reduced neutrophil adhesion rates by EtOH or EtP was not significant. CD54 expression was noticeably reduced in A549 cells, but this was not the case in Huh7 cells after treatment. The Bax/Bcl-2 ratio was dose‑dependently decreased by EtOH and by high-dose EtP in A549 cells, indicating a reduction in apoptosis, whereas this effect was not observed in Huh7 cells. The underlying mechanisms involve reduced phosphorylation of Akt and nuclear factor-κB (NF-κB) p65. We noted that as with EtP, EtOH reduced the inflammatory response in lung epithelial cells under acute inflammatory conditions. However, due to the low impact which EtP and EtOH had on the hepatocellular cells, our data suggest that both substances exerted different effects depending on the cellular entity. The possible underlying mechanisms involved the downregulation of Akt and the transcription factor NF-κB, but further research on this subject is required.

Source apportionment of Beijing air pollution during a severe winter haze event and associated pro-inflammatory responses in lung epithelial cells

Abstract Air pollution is a leading risk factor for the disease burden in China and globally. Few epidemiologic studies have characterized the particulate matter (PM) components and sources that are most responsible for adverse health outcomes, particularly in developing countries. In January 2013, a severe haze event occurred over 25 days in urban Beijing, China. Ambient fine particulate matter (PM2.5) was collected at a central urban site in Beijing from January 16-31, 2013. We analyzed the samples for water soluble ions, metals, elemental carbon (EC), organic carbon (OC), and individual organic molecular markers including n-alkanes, hopanes, PAHs and sterols. Chemical components were used to quantify the source contributions to PM2.5 using the chemical mass balance (CMB) model by the conversion of the OC estimates combined with inorganic secondary components (e.g. NH4+, SO42−, NO3-). Water extracts of PM were exposed to lung epithelial cells, and supernatants recovered from cell cultures were assayed for the pro-inflammatory cytokines by a quantitative ELLSA method. Linear regression models were used to estimate the associations between PM sources and components with pro-inflammatory responses in lung epithelial cells following 24-hrs and 48-hrs of exposure. The largest contributors to PM2.5 during the monitoring period were inorganic secondary ions (53.2% and 54.0% on haze and non-haze days, respectively). Other organic matter (OM) contributed to a larger proportion of PM2.5 during haze days (16.9%) compared with non-haze days (12.9%), and coal combustion accounted for 10.9% and 8.7% on haze and non-haze days, respectively. We found PM2.5 mass and specific sources (e.g. coal combustion, traffic emission, dust, other OM, and inorganic secondary ions) were highly associated with inflammatory responses of lung epithelial cells. Our results showed greater responses in the exposure to 48-hr PM2.5 mass and its sources compared to 24-hr PM exposure, and that secondary and coal combustion sources play an important role in short-term inflammation and require cost-effective policy to control their contributions to air pollution.

307 - GSTP Modulates NF-kB Activation and Pro-Inflammatory Responses in Lung Epithelial Cells

307 - GSTP Modulates NF-kB Activation and Pro-Inflammatory Responses in Lung Epithelial Cells

MyD88 in lung resident cells governs airway inflammatory and pulmonary function responses to organic dust treatment.

Inhalation of organic dusts within agriculture environments contributes to the development and/or severity of airway diseases, including asthma and chronic bronchitis. MyD88 KO (knockout) mice are nearly completely protected against the inflammatory and bronchoconstriction effects induced by acute organic dust extract (ODE) treatments. However, the contribution of MyD88 in lung epithelial cell responses remains unclear. In the present study, we first addressed whether ODE-induced changes in epithelial cell responses were MyD88-dependent by quantitating ciliary beat frequency and cell migration following wounding by electric cell-substrate impedance sensing. We demonstrate that the normative ciliary beat slowing response to ODE is delayed in MyD88 KO tracheal epithelial cells as compared to wild type (WT) control. Similarly, the normative ODE-induced slowing of cell migration in response to wound repair was aberrant in MyD88 KO cells. Next, we created MyD88 bone marrow chimera mice to investigate the relative contribution of MyD88-dependent signaling in lung resident (predominately epithelial cells) versus hematopoietic cells. Importantly, we demonstrate that ODE-induced airway hyperresponsiveness is MyD88-dependent in lung resident cells, whereas MyD88 action in hematopoietic cells is mainly responsible for ODE-induced TNF-α release. MyD88 signaling in lung resident and hematopoietic cells are necessary for ODE-induced IL-6 and neutrophil chemoattractant (CXCL1 and CXCL2) release and neutrophil influx. Collectively, these findings underscore an important role for MyD88 in lung resident cells for regulating ciliary motility, wound repair and inflammatory responses to ODE, and moreover, show that airway hyperresponsiveness appears uncoupled from airway inflammatory consequences to organic dust challenge in terms of MyD88 involvement.Electronic supplementary materialThe online version of this article (doi:10.1186/s12931-015-0272-9) contains supplementary material, which is available to authorized users.

Pre- or post-treatment with ethanol and ethyl pyruvate results in distinct anti-inflammatory responses of human lung epithelial cells triggered by interleukin-6.

Increased local and systemic levels of interleukin (IL)-6 are associated with inflammatory processes, including neutrophil infiltration of the alveolar space, resulting in lung injury. Our previous study demonstrated the beneficial anti-inflammatory effects of acute exposure to ethanol (EtOH) in an acute in vivo model of inflammation. However, due to its side-effects, EtOH is not used clinically. In the present study, the effects of EtOH and ethyl pyruvate (EtP) as an alternative anti-inflammatory drug prior to and following application of an IL-6 stimulus on cultured A549 lung epithelial cells were compared, and it was hypothesized that treatment with EtOH and EtP reduces the inflammatory potential of the A549 cells. Time- and dose-dependent release of IL-8 from the A549 cells was observed following stimulation with IL-6. The release of IL-8 from the A549 cells was assessed following treatment with EtP (2.5-10 mM), sodium pyruvate (NaP; 10 mM) or EtOH (85-170 mM) for 1, 24 or 72 h, prior to and following IL-6 stimulation. The adhesion capacities of neutrophils to the treated A549 cells, and the expression levels of cluster of differentiation (CD)54 by the epithelial cells were measured. Treatment of the A549 cells with either EtOH or EtP significantly reduced the IL-6-induced release of IL-8. This effect was observed in the pre- and post-stimulatory conditions, which is of therapeutic importance. Similar data was revealed regarding the IL-6-induced neutrophil adhesion to the treated A549 cells, in which pre- and post-treatment with EtOH or EtP decreased the adhesion capacity, however, the results were dependent on the duration of incubation. Incubation durations of 1 and 24 h decreased the adhesion rates of neutrophils to the stimulated A549 cells, however, the reduction was only significant at 72 h post-treatment. The expression of CD54 was reduced only following treatment for 24 h with either EtOH or EtP, prior to IL-6 stimulation. Therefore, EtOH and EtP reduced the inflammatory response of lung epithelial cells, and the potential of EtP to mimic EtOH was observed in the pre- and post-treatment conditions.

Vapors produced by electronic cigarettes and e-juices with flavorings induce toxicity, oxidative stress, and inflammatory response in lung epithelial cells and in mouse lung.

Oxidative stress and inflammatory response are the key events in the pathogenesis of chronic airway diseases. The consumption of electronic cigarettes (e-cigs) with a variety of e-liquids/e-juices is alarmingly increasing without the unrealized potential harmful health effects. We hypothesized that electronic nicotine delivery systems (ENDS)/e-cigs pose health concerns due to oxidative toxicity and inflammatory response in lung cells exposed to their aerosols. The aerosols produced by vaporizing ENDS e-liquids exhibit oxidant reactivity suggesting oxidants or reactive oxygen species (OX/ROS) may be inhaled directly into the lung during a “vaping” session. These OX/ROS are generated through activation of the heating element which is affected by heating element status (new versus used), and occurs during the process of e-liquid vaporization. Unvaporized e-liquids were oxidative in a manner dependent on flavor additives, while flavors containing sweet or fruit flavors were stronger oxidizers than tobacco flavors. In light of OX/ROS generated in ENDS e-liquids and aerosols, the effects of ENDS aerosols on tissues and cells of the lung were measured. Exposure of human airway epithelial cells (H292) in an air-liquid interface to ENDS aerosols from a popular device resulted in increased secretion of inflammatory cytokines, such as IL-6 and IL-8. Furthermore, human lung fibroblasts exhibited stress and morphological change in response to treatment with ENDS/e-liquids. These cells also secrete increased IL-8 in response to a cinnamon flavored e-liquid and are susceptible to loss of cell viability by ENDS e-liquids. Finally, exposure of wild type C57BL/6J mice to aerosols produced from a popular e-cig increase pro-inflammatory cytokines and diminished lung glutathione levels which are critical in maintaining cellular redox balance. Thus, exposure to e-cig aerosols/juices incurs measurable oxidative and inflammatory responses in lung cells and tissues that could lead to unrealized health consequences.