Human Lung Epithelial A549 Cells Research Articles

PM2.5-induced oxidative stress triggers autophagy in human lung epithelial A549 cells

Exposure to higher levels of air pollution particulate matter (PM) with an aerodynamic diameter of less than 2.5μm (PM2.5) links with an increased risk of cardiovascular and respiratory deaths and hospital admission as well as lung cancer. Although the mechanism underlying the correlation between PM2.5 exposure and adverse effects has not fully elucidated, PM2.5-induced oxidative stress has been considered as an important molecular mechanism of PM2.5-mediated toxicity. In this work, human lung epithelial A549 cells were used to further investigate the biological effects of PM2.5 on autophagy. The cell viability showed both time- and concentration-dependent decrease when exposure to PM2.5, which can be attributed to increase of the levels of extracellular lactate dehydrogenase (LDH) release and intracellular reactive oxygen species (ROS) generation in A549 cells. Moreover, PM2.5-induced oxidative damage in A549 cells was observed through the alteration of superoxide dismutase (SOD) and catalase (CAT) activities compared to the unexposed control cells. PM2.5-induced autophagy was indicated by an increase in microtubule-associated protein light chain-3 (LC3) puncta, and accumulation of LC3 in both time- and concentration-dependent manner. PM2.5-induced mRNA expression of autophagy-related protein Atg5 and Beclin1 was also observed compared with those of the unexposed control cells. These results suggest the possibility that PM2.5-induced oxidative stress probably plays a key role in autophagy in A549 cells, which may contribute to PM2.5-induced impairment of pulmonary function.

Cell-Cycle Changes and Oxidative Stress Response to Magnetite in A549 Human Lung Cells

In a recent study, magnetite was investigated for its potential to induce toxic effects and influence signaling pathways. It was clearly demonstrated that ROS formation leads to mitochondrial damage and genotoxic effects in A549 cells. On the basis of these findings, we wanted to elucidate the origin of magnetite-mediated ROS formation and its influence on the cell cycle of A549 and H1299 human lung epithelial cells. Concentration- and size-dependent superoxide formation, measured by electron paramagnetic resonance (EPR), was observed. Furthermore, we could show that the GSH level decreased significantly after exposure to magnetite particles, while catalase (CAT) activity was increased. These effects were also dependent on particle size, albeit less pronounced than those observed with EPR. We were able to show that incubation of A549 cells prior to particle treatment with diphenyleneiodonium (DPI), a NADPH-oxidase (NOX) inhibitor, leads to decreased ROS formation, but this effect was not observed for the NOX inhibitor apocynin. Soluble iron does not contribute considerably to ROS production. Analysis of cell-cycle distribution revealed a pronounced sub-G1 peak, which cannot be linked to increased cell death. Western blot analysis did not show activation of p53 but upregulation of p21 in A549. Here, we were unexpectedly able to demonstrate that exposure to magnetite leads to p21-mediated G1-like arrest. This has been reported previously only for low concentrations of microtubule stabilization drugs. Importantly, the arrested sub-G1 cells were viable and showed no caspase 3/7 activation.

Systems Analysis of a RIG-I Agonist Inducing Broad Spectrum Inhibition of Virus Infectivity

The RIG-I like receptor pathway is stimulated during RNA virus infection by interaction between cytosolic RIG-I and viral RNA structures that contain short hairpin dsRNA and 5′ triphosphate (5′ppp) terminal structure. In the present study, an RNA agonist of RIG-I was synthesized in vitro and shown to stimulate RIG-I-dependent antiviral responses at concentrations in the picomolar range. In human lung epithelial A549 cells, 5′pppRNA specifically stimulated multiple parameters of the innate antiviral response, including IRF3, IRF7 and STAT1 activation, and induction of inflammatory and interferon stimulated genes - hallmarks of a fully functional antiviral response. Evaluation of the magnitude and duration of gene expression by transcriptional profiling identified a robust, sustained and diversified antiviral and inflammatory response characterized by enhanced pathogen recognition and interferon (IFN) signaling. Bioinformatics analysis further identified a transcriptional signature uniquely induced by 5′pppRNA, and not by IFNα-2b, that included a constellation of IRF7 and NF-kB target genes capable of mobilizing multiple arms of the innate and adaptive immune response. Treatment of primary PBMCs or lung epithelial A549 cells with 5′pppRNA provided significant protection against a spectrum of RNA and DNA viruses. In C57Bl/6 mice, intravenous administration of 5′pppRNA protected animals from a lethal challenge with H1N1 Influenza, reduced virus titers in mouse lungs and protected animals from virus-induced pneumonia. Strikingly, the RIG-I-specific transcriptional response afforded partial protection from influenza challenge, even in the absence of type I interferon signaling. This systems approach provides transcriptional, biochemical, and in vivo analysis of the antiviral efficacy of 5′pppRNA and highlights the therapeutic potential associated with the use of RIG-I agonists as broad spectrum antiviral agents.

Assessment of Nanoparticle Release from Polyamide 6- and Polypropylene-Silicon Composites and Cytotoxicity in Human Lung A549 Cells

The steady increase in application of nanoparticles in novel products over the past decade has triggered concerns about the impact of nanoparticles on human health. This study assessed the release of nanoparticles from polyamide 6 (PA6)- and polypropylene (PP)-silicon composites during drilling test and tested the toxicity of the released nanoparticles. The PA6 and PP polymers and their respective polymer–silicon composites, each containing 5 wt% of nano-fillers (silica nanoparticles, organically modified montmorrillonite) or larger fillers (glass fiber or foam glass crystal materials) were studied. It was shown that PA6-based materials generated up to ten times more nanoparticles than the PP-based materials. The polymer-silica nanocomposites generated the highest amount of nanoparticles, whilst the polymer-montmorrillonite composites and neat polymers generated the least. In human lung epithelial A549 cells, the toxicity for PA6-based nanoparticles appeared to be higher than PP-based nanoparticles. This study suggests that the assessment of nanoparticle release integrated with a nanoparticle toxicity study could reveal the potential risk of human exposure at certain stages of the product life cycle and the development of safe products.

The Antiviral Effector IFITM3 Disrupts Intracellular Cholesterol Homeostasis to Block Viral Entry

SummaryVesicle-membrane-protein-associated protein A (VAPA) and oxysterol-binding protein (OSBP) regulate intracellular cholesterol homeostasis, which is required for many virus infections. During entry, viruses or virus-containing vesicles can fuse with endosomal membranes to mediate the cytosolic release of virions, and alterations in endosomal cholesterol can inhibit this invasion step. We show that the antiviral effector protein interferon-inducible transmembrane protein 3 (IFITM3) interacts with VAPA and prevents its association with OSBP, thereby disrupting intracellular cholesterol homeostasis and inhibiting viral entry. By altering VAPA-OSBP function, IFITM3 induces a marked accumulation of cholesterol in multivesicular bodies and late endosomes, which inhibits the fusion of intraluminal virion-containing vesicles with endosomal membranes and thereby blocks virus release into the cytosol. Consequently, ectopic expression or depletion of the VAPA gene profoundly affects IFITM3-mediated inhibition of viral entry. Thus, IFITM3 disrupts intracellular cholesterol homeostasis to block viral entry, further underscoring the importance of cholesterol in virus infection.

ITRAQ-Based and Label-Free Proteomics Approaches for Studies of Human Adenovirus Infections

Both isobaric tags for relative and absolute quantitation (iTRAQ) and label-free methods are widely used for quantitative proteomics. Here, we provide a detailed evaluation of these proteomics approaches based on large datasets from biological samples. iTRAQ-label-based and label-free quantitations were compared using protein lysate samples from noninfected human lung epithelial A549 cells and from cells infected for 24 h with human adenovirus type 3 or type 5. Either iTRAQ-label-based or label-free methods were used, and the resulting samples were analyzed by liquid chromatography (LC) and tandem mass spectrometry (MS/MS). To reduce a possible bias from quantitation software, we applied several software packages for each procedure. ProteinPilot and Scaffold Q+ software were used for iTRAQ-labeled samples, while Progenesis LC-MS and ProgenesisF-T2PQ/T3PQ were employed for label-free analyses. R 2 correlation coefficients correlated well between two software packages applied to the same datasets with values between 0.48 and 0.78 for iTRAQ-label-based quantitations and 0.5 and 0.86 for label-free quantitations. Analyses of label-free samples showed higher levels of protein up- or downregulation in comparison to iTRAQ-labeled samples. The concentration differences were further evaluated by Western blotting for four downregulated proteins. These data suggested that the label-free method was more accurate than the iTRAQ method.

A high-throughput microfluidic biochip to quantify bacterial adhesion to single host cells by real-time PCR assay

A high-throughput microfluidic poly-(dimethylsiloxane) biochip was developed to quantify bacterial adhesion to single host cells by real-time PCR assay. The biochip is simply structured with a two-dimensional array of 900 micro-wells, one inlet, and one outlet micro-channels. Isolation of single infected host cells into the individual micro-wells of the biochip was achieved by one-step vacuum-driven microfluidics. The adhered bacterial cells were then quantified by direct on-chip real-time PCR assay with single-bacterium-detection sensitivity. The performance of this microfluidic platform was demonstrated through profiling of the association of a common bacterial pathogen, Pseudomonas aeruginosa, to single host human lung epithelial A549 cells, revealing an adherence distribution that has not been previously reported. This microfluidic platform offers a simple and effective tool for biologists to analyze pathogen-host interaction at the single-cell level without the necessities of fluorescence labeling. The chip can similarly be used for other PCR-based applications requiring single-cell analysis.

Sodium tauroursodeoxycholate prevents paraquat-induced cell death by suppressing endoplasmic reticulum stress responses in human lung epithelial A549 cells

Paraquat is a commonly used herbicide; however, it is highly toxic to humans and animals. Exposure to paraquat causes severe lung damage, leading to pulmonary fibrosis. However, it has not been well clarified as how paraquat causes cellular damage, and there is no established standard therapy for paraquat poisoning. Meanwhile, endoplasmic reticulum stress (ERS) is reported to be one of the causative factors in many diseases, although mammalian cells have a defense mechanism against ERS-induced apoptosis (unfolded protein response). Here, we demonstrated that paraquat changed the expression levels of unfolded protein response-related molecules, resulting in ERS-related cell death in human lung epithelial A549 cells. Moreover, treatment with sodium tauroursodeoxycholate (TUDCA), a chemical chaperone, crucially rescued cells from death caused by exposure to paraquat. These results indicate that paraquat toxicity may be associated with ERS-related molecules/events. Through chemical chaperone activity, treatment with TUDCA reduced paraquat-induced ERS and mildly suppressed cell death. Our findings also suggest that TUDCA treatment represses the onset of pulmonary fibrosis caused by paraquat, and therefore chemical chaperones may have novel therapeutic potential for the treatment of paraquat poisoning.

Protein Kinase Regulated by dsRNA Downregulates the Interferon Production in Dengue Virus- and dsRNA-Stimulated Human Lung Epithelial Cells

BackgroundDengue virus (DENV) is found in the tropical and subtropical regions and affects millions of people annually. Currently, no specific vaccine or antiviral treatment against dengue virus is available. Innate immunity has been shown to be important for host resistance to DENV infection. Although protein kinase regulated by double-stranded RNA (PKR) has been found to promote the innate signaling in response to infection by several viruses, its role in the innate response to DENV infection is still unclear. Our study aimed to investigate the role of PKR in DENV-induced innate immune responses.Methodology/Principal FindingsBy RNAi, silencing of PKR significantly enhanced the expression of interferon (IFN)-β in DENV infected human lung epithelial A549 cells. Western blot and immunofluorescence microscopy data showed that PKR knockdown upregulated the activation of innate signaling cascades including p38 and JNK mitogen-activated protein kinases (MAPKs), interferon regulatory factor-3 and NF-κB, following DENV2 infection. Likewise, a negative regulatory effect of PKR on the IFN production was also observed in poly(IC) challenged cells. Moreover, the PKR knockdown-mediated IFN induction was attenuated by RIG-I or IPS-1 silencing. Finally, overexpression of a catalytically inactive PKR mutant (K296R), but not of a mutant lacking dsRNA binding activity (K64E) or the double mutant (K64EK296R), reversed the IFN induction mediated by PKR knockdown, suggesting that the dsRNA binding activity is required for PKR to downregulate IFN production.Conclusions/SignificancePKR acts as a negative regulator of IFN induction triggered by DENVs and poly(IC), and this regulation relies on its dsRNA binding activity. These findings reveal a novel regulatory role for PKR in innate immunity, suggesting that PKR might be a promising target for anti-DENV treatments.

Genotoxic and inflammatory effects of organic extracts from traffic-related particulate matter in human lung epithelial A549 cells: The role of quinones

Traffic-related particulate matter (PM) is associated with adverse health effects. Quinones present in the traffic-related PM are hypothesized to contribute to these harmful effects through reactive oxygen species (ROS) generation. However, the impacts of the traffic-related PM and quinones on inflammatory processes and genotoxic damages are less well known. In present study we aimed to examine the genotoxic and inflammatory impacts of organic extracts from traffic-related PM (oTRP) in human lung epithelial A549 cells, and reveal the contributions from quinones. Significant cytotoxicity and DNA damage were caused by oTRP. The pro-inflammatory genes, interleukin-6 (Il-6), interleukin-8 (Il-8) and tumor necrosis factor (Tnf), and two aromatic hydrocarbon receptor-regulated genes, Cyp1a1 and 1b1, were significantly up-regulated by oTRP. A concomitant increase in ROS was observed, suggesting that oTRP may mediate genotoxic and inflammatory effects through oxidative stress pathway. Second, the effects from two typical airborne quinones, 9,10-anthraquinone (AQ) and 1,4-naphthroquinone (NQ) were compared. NQ, but not AQ, induced significant DNA damage in A549 cells. NQ up-regulated Il-8, Tnf, and Mcp-1 genes, while AQ induced the expression of Rantes gene. These results suggest that the NQ and AQ may participate in the pro-inflammatory responses through releasing different types of cytokines/chemokines.

Multi-step regulation of STAT1 phosphorylation in response to double-stranded RNA

Event Abstract Back to Event Multi-step regulation of STAT1 phosphorylation in response to double-stranded RNA Tomoh Matsumiya1*, Junichi Dempoya1, Fei Xing1, Ryo Hayakari1, Hidemi Yoshida1 and Tadaatsu Imaizumi1 1 Hirosaki University Graduate School of Medicine, Japan Pattern recognition receptors which sense non-self RNA trigger to produce type I IFN. Type I IFN binds to its cognate receptor, IFNAR, resulting in the activation of STAT1. Therefore, it has long been thought that dsRNA-introduced STAT1 phosphorylation is through transactivation of type I IFN signaling. This study asked whether the phosphorylation of STAT1 is absolutely type I IFN-dependent. A synthetic dsRNA polyinosinic-polycytidylic acid (polyI:C) induces STAT1 phosphorylation in A549 human lung epithelial cells. We found the polyI:C induced STAT1 phosphorylation in a predominantly IFN-dependent manner. However, we also found that polyI:C was able to stimulate STAT1 phosphorylation in type I IFN receptor-deficient U5A cells, suggesting this phosphorylation is type I IFN-independent. Retinoic acid-inducible gene-I (RIG-I) was involved in the initial STAT1 phosphorylation in response to polyI:C; however, little contribution of RIG-I was observed in the late STAT1 phosphorylation. Taken together, our results showed comprehensive regulation in which dsRNA induces STAT1 phosphorylation, indicating importance of STAT1 with such a tight regulation in innate immune system. References Dempoya J, Matsumiya T, Imaizumi T, Hayakari R, Xing F, Yoshida H, Okumura K, Satoh K. Double-Stranded RNA Induces Biphasic STAT1 Phosphorylation by both Type I Interferon (IFN)-Dependent and Type I IFN-Independent Pathways. Journal of Virology 86: 12760-9, 2012 Keywords: RIG-I-like receptors, STAT1, type I IFN, dsRNA, Innate immune system Conference: 15th International Congress of Immunology (ICI), Milan, Italy, 22 Aug - 27 Aug, 2013. Presentation Type: Abstract Topic: Innate immunity Citation: Matsumiya T, Dempoya J, Xing F, Hayakari R, Yoshida H and Imaizumi T (2013). Multi-step regulation of STAT1 phosphorylation in response to double-stranded RNA. Front. Immunol. Conference Abstract: 15th International Congress of Immunology (ICI). doi: 10.3389/conf.fimmu.2013.02.00660 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 12 Jun 2013; Published Online: 22 Aug 2013. * Correspondence: Dr. Tomoh Matsumiya, Hirosaki University Graduate School of Medicine, Hirosaki, 036-8562, Japan, tomo1027@cc.hirosaki-u.ac.jp Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Tomoh Matsumiya Junichi Dempoya Fei Xing Ryo Hayakari Hidemi Yoshida Tadaatsu Imaizumi Google Tomoh Matsumiya Junichi Dempoya Fei Xing Ryo Hayakari Hidemi Yoshida Tadaatsu Imaizumi Google Scholar Tomoh Matsumiya Junichi Dempoya Fei Xing Ryo Hayakari Hidemi Yoshida Tadaatsu Imaizumi PubMed Tomoh Matsumiya Junichi Dempoya Fei Xing Ryo Hayakari Hidemi Yoshida Tadaatsu Imaizumi Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

In vitro tests to assess toxic effects of airborne PM10 samples. Correlation with metals and chlorinated dioxins and furans

Inhalation is an important exposure pathway to airborne pollutants such as heavy metals, polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs) and particulate matter. Chronic exposure to those chemicals, which form part of complex environmental mixtures, may mean important human health risks. In the present study, the suitability of different in vitro tests to evaluate the toxic effects of air PM10 pollutants is investigated. In addition, it is also assessed how to distinguish the contribution of chemical pollutants to toxicity. Sixty-three air samples were collected in various areas of Catalonia (Spain), and the levels of ecotoxicity, cytotoxicity and genotoxicity were evaluated. Aqueous acidic extractions of quartz fiber filters, where PM10 had been retained, were performed. The photo-luminescent bacteria Vibrio fischeri (Microtox®) bioassay was performed to assess ecotoxicity. Moreover, MTT and Comet Assays, both using human lung epithelial cells A549 as target cells, were applied to assess the cytotoxicity and genotoxicity of air samples, respectively. The results show that Microtox® is an excellent screening test to perform a first evaluation of air quality, as it presented a significant correlation with chemical contaminants, contrasting with MTT Assay. Although none of the samples exhibited genotoxicity, a high correlation was found between this in vitro test and carcinogenic agents. Urban samples from traffic-impacted areas would be significantly more toxic. Finally, environmental temperature was identified as a key parameter, as higher values of ecotoxicity were found in winter.

Methamphetamine Reduces Human Influenza A Virus Replication

Methamphetamine (meth) is a highly addictive psychostimulant that is among the most widely abused illicit drugs, with an estimated over 35 million users in the world. Several lines of evidence suggest that chronic meth abuse is a major factor for increased risk of infections with human immunodeficiency virus and possibly other pathogens, due to its immunosuppressive property. Influenza A virus infections frequently cause epidemics and pandemics of respiratory diseases among human populations. However, little is known about whether meth has the ability to enhance influenza A virus replication, thus increasing severity of influenza illness in meth abusers. Herein, we investigated the effects of meth on influenza A virus replication in human lung epithelial A549 cells. The cells were exposed to meth and infected with human influenza A/WSN/33 (H1N1) virus. The viral progenies were titrated by plaque assays, and the expression of viral proteins and cellular proteins involved in interferon responses was examined by Western blotting and immunofluorescence staining. We report the first evidence that meth significantly reduces, rather than increases, virus propagation and the susceptibility to influenza infection in the human lung epithelial cell line, consistent with a decrease in viral protein synthesis. These effects were apparently not caused by meth’s effects on enhancing virus-induced interferon responses in the host cells, reducing viral biological activities, or reducing cell viability. Our results suggest that meth might not be a great risk factor for influenza A virus infection among meth abusers. Although the underlying mechanism responsible for the action of meth on attenuating virus replication requires further investigation, these findings prompt the study to examine whether other structurally similar compounds could be used as anti-influenza agents.

Inhibitory effect of butein on tumor necrosis factor-α-induced expression of cell adhesion molecules in human lung epithelial cells via inhibition of reactive oxygen species generation, NF-κB activation and Akt phosphorylation

Cell adhesion molecules play an important role in inflammatory response, angiogenesis and tumor progression. Butein (tetrahydroxychalcone) is a small molecule from natural sources, known to be a potential therapeutic drug with anti-inflammatory, anticancer and antioxidant activities. In the present study, we investigated the inhibitory effect of butein on tumor necrosis factor (TNF)-α-induced adhesion molecule expression and its molecular mechanism of action. Butein significantly decreased TNF-α-induced monocyte (U937) cell adhesion to lung epithelial cells in a dose-dependent manner. Butein also inhibited the protein and mRNA expression of intercellular cell adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) in TNF-α-stimulated A549 human lung epithelial cells in a dose-dependent manner. Butein inhibited TNF-α-induced reactive oxygen species (ROS) generation and nuclear factor-κB (NF-κB) activation in A549 cells; it also inhibited the phosphorylation of MAPKs and Akt, suggesting that the MAPK/Akt signaling pathway may be involved in the butein-mediated inhibition of TNF-α-induced leukocyte adhesion to A549 cells. Collectively, our results suggest that butein affects cell adhesion through the inhibition of TNF-α-induced ICAM-1 and VCAM-1 expression by inhibiting the NF-κB/MAPK/Akt signaling pathway and ROS generation, thereby, elucidating the role of butein in the anti-inflammatory response.

The TspanC8 Subgroup of Tetraspanins Interacts with A Disintegrin and Metalloprotease 10 (ADAM10) and Regulates Its Maturation and Cell Surface Expression

A disintegrin and metalloprotease 10 (ADAM10) is a ubiquitous transmembrane metalloprotease that cleaves the extracellular regions from over 40 different transmembrane target proteins, including Notch and amyloid precursor protein. ADAM10 is essential for embryonic development and is also important in inflammation, cancer, and Alzheimer disease. However, ADAM10 regulation remains poorly understood. ADAM10 is compartmentalized into membrane microdomains formed by tetraspanins, which are a superfamily of 33 transmembrane proteins in humans that regulate clustering and trafficking of certain other transmembrane "partner" proteins. This is achieved by specific tetraspanin-partner interactions, but it is not clear which tetraspanins specifically interact with ADAM10. The aims of this study were to identify which tetraspanins interact with ADAM10 and how they regulate this metalloprotease. Co-immunoprecipitation identified specific ADAM10 interactions with Tspan5, Tspan10, Tspan14, Tspan15, Tspan17, and Tspan33/Penumbra. These are members of the largely unstudied TspanC8 subgroup of tetraspanins, all six of which promoted ADAM10 maturation. Different cell types express distinct repertoires of TspanC8 tetraspanins. Human umbilical vein endothelial cells express relatively high levels of Tspan14, the knockdown of which reduced ADAM10 surface expression and activity. Mouse erythrocytes express predominantly Tspan33, and ADAM10 expression was substantially reduced in the absence of this tetraspanin. In contrast, ADAM10 expression was normal on Tspan33-deficient mouse platelets in which Tspan14 is the major TspanC8 tetraspanin. These results define TspanC8 tetraspanins as essential regulators of ADAM10 maturation and trafficking to the cell surface. This finding has therapeutic implications because focusing on specific TspanC8-ADAM10 complexes may allow cell type- and/or substrate-specific ADAM10 targeting.

Calcitriol [1, 25[OH]2 D3] pre- and post-treatment suppresses inflammatory response to influenza A (H1N1) infection in human lung A549 epithelial cells

Influenza viruses infect airway epithelial cells, causing respiratory distress. Immune defense is maintained by chemokine/cytokine secretions from airway epithelial cells. While moderate inflammatory response protects from ill effects, hyper-inflammatory response promotes the pathogenesis. High circulating levels of vitamin D are known to mitigate effects of infectious diseases, including respiratory infectious diseases. The question whether and how vitamin D treatment pre-/post-viral exposure modulates inflammatory response is not clear. The present study was undertaken to understand autophagy/apoptosis balance and chemokine/cytokine response to influenza A (H1N1) infection by pre- and post-1, 25-dihydroxyvitamin D3 (1,25[OH]2 D3)[calcitriol] treatment of human lung A549 epithelial cells. Influenza A (H1N1) virus was propagated in A549 cell line, titrated using hemagglutination assay, and was used to assess effect of calcitriol. After confirming that 100nM of calcitriol fails to clear virus, A549 cells were either pre-treated (16h) with 100nM or post-treated with 30nM of 1,25[OH]2 D3 of virus inoculation (1h). Cells after incubation at 37°C under 5% CO2 for 48h were collected and subjected to RNA and protein extraction. Measurements of viability, influenza M protein, and molecular parameters of cell death and inflammatory response were performed. We report that treatment of these cells with 100/30nM of 1,25[OH]2 D3 prior to/or post-H1N1 exposure does not affect viral clearance but significantly reduces autophagy and restores increased apoptosis seen on H1N1 infection back to its constitutive level. However, it significantly decreases the levels of H1N1-induced TNF-α (tumor necrosis factor-alpha), IFN-β (interferon-beta), and IFN-stimulated gene-15 (ISG15). 1,25[OH]2 D3 treatment prior to/or post-H1N1 infection significantly down-regulates IL-8 as well as IL-6 RNA levels. These results demonstrate that calcitriol treatment suppresses the H1N1-induced transcription of the chemokines RANTES and IL-8 in epithelial cells. The findings provide support for the initiation of vitamin D supplementation program to VDD populations in reducing the severity of influenza.

Assessment of nanoparticle release and associated health effect of polymer-silicon composites

Little information is currently available on possible release of nanomaterials or/and nanoparticles (NP) from conventional and novel products and associated health effect. This study aimed to assess the possible release of NP during the application stage of conventional and nanoproducts. NP release was monitored during physical processing of polymer-silicon composites, and the toxicity of both the released NP and the raw silica nanomaterials that were used as fillers in the nanocomposites was assessed in vitro using human lung epithelial A549 cells. This study suggests that 1) NP can be released from the conventional and novel polymer-silicon composites under certain application scenario; 2) the level of NP release from polymer composites could be altered by different reinforcement materials; e.g. nanostructured MMT could reduce the release while SiO2 NP could increase the release; 3) working with polymer composites under certain conditions could risk inhalation of high level of polymer NP; 4) raw nanomaterials appeared to be toxic in the chosen in vitro system. Further study of the effect of novel filler materials on NP release from final polymer products and the effect of released NP on environment and human health will inform design of safe materials and minimization of negative impact on the environment and human health.

Functional analysis of a lung cancer risk haplotype in the IL1B gene regulatory region

Interleukin-1β (IL-1β), encoded by the IL1B gene, is a cytokine important in regulation of the inflammatory response. Elevated levels of IL1B expression have been associated with risk of gastric and lung cancer. We previously reported that a certain haplotype containing four single-nucleotide polymorphisms (SNPs) (-3893G, -1464G, -511C and -31T; GGCT) in the IL1B gene regulatory region was associated with lung cancer risk and increased expression of the IL1B gene in the lung. In the present study, we have cloned the two haplotypes that were either protective (ACTC) or increasing lung cancer risk (GGCT) in a luciferase reporter vector system. We also cloned the IL1B -3893 and -1464 SNPs that were found to be associated with risk of lung cancer. The haplotype associated with lung cancer risk showed higher transcriptional activity in the human lung epithelial A549 cell line in vitro. We also found that the IL1B -1464C allele increased transcriptional activity compared with the -1464G allele in the tumor necrosis factor α-stimulated cells, as well as specific transcription factor binding patterns to the IL1B -1464C allele. Interestingly, in vitro results showed a similar expression pattern as observed in the normal lung tissues of lung cancer patients reported earlier.

Host cell kinases, α5 and β1 integrins, and Rac1 signalling on the microtubule cytoskeleton are important for non-typable Haemophilus influenzae invasion of respiratory epithelial cells

Non-typable Haemophilus influenzae (NTHi) is a common commensal of the human nasopharynx, but causes opportunistic infection when the respiratory tract is compromised by infection or disease. The ability of NTHi to invade epithelial cells has been described, but the underlying molecular mechanisms are poorly characterized. We previously determined that NTHi promotes phosphorylation of the serine-threonine kinase Akt in A549 human lung epithelial cells, and that Akt phosphorylation and NTHi cell invasion are prevented by inhibition of phosphoinositide 3-kinase (PI3K). Because PI3K-Akt signalling is associated with several host cell networks, the purpose of the current study was to identify eukaryotic molecules important for NTHi epithelial invasion. We found that inhibition of Akt activity reduced NTHi internalization; differently, bacterial entry was increased by phospholipase Cγ1 inhibition but was not affected by protein kinase inhibition. We also found that α5 and β1 integrins, and the tyrosine kinases focal adhesion kinase and Src, are important for NTHi A549 cell invasion. NTHi internalization was shown to be favoured by activation of Rac1 guanosine triphosphatase (GTPase), together with the guanine nucleotide exchange factor Vav2 and the effector Pak1. Also, Pak1 might be associated with inactivation of the microtubule destabilizing agent Op18/stathmin, to facilitate microtubule polymerization and NTHi entry. Conversely, inhibition of RhoA GTPase and its effector ROCK increased the number of internalized bacteria. Src and Rac1 were found to be important for NTHi-triggered Akt phosphorylation. An increase in host cyclic AMP reduced bacterial entry, which was linked to protein kinase A. These findings suggest that NTHi finely manipulates host signalling molecules to invade respiratory epithelial cells.

Cytotoxicity evaluation of nanoclays in human epithelial cell line A549 using high content screening and real-time impedance analysis

Continuously expanding use of products containing nanoclays for wide range of applications have raised public concerns about health and safety. Although the products containing nanoclays may not be toxic, it is possible that nanomaterials may come in contact with humans during handling, manufacture, or disposal, and cause adverse health impact. This necessitates biocompatibility evaluation of the commonly used nanoclays. Here, we investigated the cytotoxic effects of platelet (Bentone MA, ME-100, Cloisite Na+, Nanomer PGV, and Delite LVF) and tubular (Halloysite, and Halloysite MP1) type nanoclays on cultured human lung epithelial cells A549. For the first time with this aim, we employed a cell-based automated high content screening in combination with real-time impedance sensing. We demonstrate varying degree of dose- and time-dependent cytotoxic effects of both nanoclay types. Overall, platelet structured nanoclays were more cytotoxic than tubular type. A low but significant level of cytotoxicity was observed at 25 μg/mL of the platelet-type nanoclays. A549 cells exposed to high concentration (250 μg/mL) of tubular structured nanoclays showed inhibited cell growth. Confocal microscopy indicated intracellular accumulation of nanoclays with perinuclear localization. Results indicate a potential hazard of nanoclay-containing products at significantly higher concentrations, which warrant their further biohazard assessment on the actual exposure in humans.