Human Lung Epithelial A549 Cells Research Articles

Aqueous areca nut extract induces oxidative stress in human lung epithelial A549 cells: Probable role of p21 in inducing cell death

Areca nut a well-known masticator used across globe. Habitual chewing of areca nut is associated with serious oral health effects. However, the role of areca nut in oxidative stress induction and cell death is less understood. Hence, in the present study we aimed to evaluate the toxic mechanism of areca nut extract on human lung epithelial A549 cells. Cells were treated with or without aqueous areca nut extract and cell viability was measured by MTT assay. Cells treated with areca nut extract show reduced viability in a dose dependent manner with the IC50 of 0.5% concentration. Areca nut extract induced the reactive oxygen species (ROS), lipid peroxidation followed by membrane damage with leakage of lactate dehydrogenase (LDH) enzyme. Cells with continuous exposure of areca nut extract depletes the free radical neutralizing anti-oxidant enzymes like superoxide dismutase (SOD), Glutathione peroxidase (GSH-Px) and Glutathione-S-transferase (GST). Further, the analysis of mRNA expression of apoptotic genes and cell cycle regulators show decreased expression of anti-apoptotic gene (Bcl-2), Cyclin E1, Cyclin D1, CDK4, Rb and p53 whereas induced expression of p21 and marginal increase of pro-apoptotic gene (Bax) confirms the toxic nature of areca nut. Thus, cell death due to areca nut exposure may be through different mechanism rather than the conventional apoptotic pathway, where p21 induction might be independent of p53 action, which possibly suggests that there may be a role of p21 in oxidative stress induced cell death. Further FACS analysis confirms cell death in areca nut treated cells.

In vitro cytotoxicity effect and antibacterial performance of human lung epithelial cells A549 activity of Zinc oxide doped TiO2 nanocrystals: Investigation of bio-medical application by chemical method

We report the synthesis of high quality ZnO doped TiO2 nanocrystals by chemical method at room temperature (RT), it can cause serious oxidative stress and DNA damage to human lung epithelial cells (A549) lines. Our aim in this study, to reduce the cytotoxicity effect of ZnO doped TiO2 nanocrystals are widely in biological fields. Several studies have been performed to understand the influence of ZnO doped titanium dioxide (TiO2-NPs) on cell function; however the effects of nanoparticle against to exposure on the cell membrane have been duly addressed fascinatingly so far. However, In this interaction, which may alter cell metabolism and integrity, it is one of the importance to understand the modifications of the cell membrane, mechanisms of pulmonary A549 cell lines nanoparticles were uptake and the molecular pathway during the initial cell responses are still unclear and much more investigative efforts are need to properly characterize the ZnO doped titanium dioxide nanoparticles were reported successfully. In particular of the epithelial cells, upon particles are exposed human pulmonary epithelial cells (A549) to various concentrations of composition, structure and morphology of the nanocrystals were analyzed by X-ray diffraction (XRD) and high resolution transmission electron microscopy (HRTEM). XRD assessed the crystal structure of the nanocrystals which identified peaks associated with (002), (100) and (101) planes of hexagonal wurtzite-type ZnO with lattice constants of a=b=3.249Å and c=5.219Å. The IR results showed high purity of products and indicated that the nanocrystals are made up of TiO and ZnO bonds. The Photoluminescence (PL) spectra are dominated by a strong narrow band edge emission tunable in the blue region of the visible spectra indicating a narrow size distribution of ZnO/TiO2 nanocrystals which exhibits antibacterial activity over a broad range of bacterial species and in particular against Stre. Mut where it out competes four other commonly used E.coli, Pse. Aug, Stre. Mut and Salm. sps, well as testing four different appropriate concentration from the results showed a significant gain in viable cell numbers of all four bacteria species, with 5mM and 10mM being the most effective and 2mM being the worst, where it provided an effective improvement from the growth mechanism has been also proposed to the current interest of these nanocrystals will discussed in detail.

Actin-Related Protein 2 (ARP2) and Virus-Induced Filopodia Facilitate Human Respiratory Syncytial Virus Spread.

Human respiratory syncytial virus (RSV) is an enveloped RNA virus that is the most important viral cause of acute pediatric lower respiratory tract illness worldwide, and lacks a vaccine or effective antiviral drug. The involvement of host factors in the RSV replicative cycle remains poorly characterized. A genome-wide siRNA screen in human lung epithelial A549 cells identified actin-related protein 2 (ARP2) as a host factor involved in RSV infection. ARP2 knockdown did not reduce RSV entry, and did not markedly reduce gene expression during the first 24 hr of infection, but decreased viral gene expression thereafter, an effect that appeared to be due to inhibition of viral spread to neighboring cells. Consistent with reduced spread, there was a 10-fold reduction in the release of infectious progeny virions in ARP2-depleted cells at 72 hr post-infection. In addition, we found that RSV infection induced filopodia formation and increased cell motility in A549 cells and that this phenotype was ARP2 dependent. Filopodia appeared to shuttle RSV to nearby uninfected cells, facilitating virus spread. Expression of the RSV F protein alone from a plasmid or heterologous viral vector in A549 cells induced filopodia, indicating a new role for the RSV F protein, driving filopodia induction and virus spread. Thus, this study identified roles for ARP2 and filopodia in RSV-induced cell motility, RSV production, and RSV cell-to-cell spread.

Responses of A549 human lung epithelial cells to cristobalite and α-quartz exposures assessed by toxicoproteomics and gene expression analysis.

In this study, we used cytotoxicity assays, proteomic and gene expression analyses to examine the difference in response of A549 cells to two silica particles that differ in physical properties, namely cristobalite (CR) and α‐quartz (Min‐U‐Sil 5, MI). Cytotoxicity assays such as lactate dehydrogenase release, 5‐bromo‐2′‐deoxyuridine incorporation and cellular ATP showed that both silica particles could cause cell death, decreased cell proliferation and metabolism in the A549 human lung epithelial cells. While cytotoxicity assays revealed little difference between CR and MI exposures, proteomic and gene expression analyses unveiled both similar and unique molecular changes in A549 cells. For instance, two‐dimensional gel electrophoresis data indicated that the expression of proteins in the cell death (e.g., ALDH1A1, HTRA2 and PRDX6) and cell proliferation (e.g., FSCN1, HNRNPAB and PGK1) pathways were significantly different between the two silica particles. Reverse transcription–polymerase chain reaction data provided additional evidence supporting the proteomic findings. Preliminary assessment of the physical differences between CR and MI suggested that the extent of surface interaction between particles and cells could explain some of the observed biological effects. However, the differential dose–response curves for some other genes and proteins suggest that other physical attributes of particulate matter can also contribute to particulate matter‐related cellular toxicity. Our results demonstrated that toxicoproteomic and gene expression analyses are sensitive in distinguishing subtle toxicity differences associated with silica particles of varying physical properties compared to traditional cytotoxicity endpoints. Copyright © 2016 Her Majesty the Queen in Right of Canada. Journal of Applied Toxicology published by John Wiley & Sons, Ltd.

Water-soluble and organic extracts of airborne particulate matter induce micronuclei in human lung epithelial A549 cells

The in vitro genotoxic effects of organic and water-soluble fractions of airborne particulate matter (PM10) with the cytokinesis blocked micronucleus (MN) test in human alveolar carcinoma cells A549 were investigated. Samples were collected in three different sites of São Paulo State, Brazil, and fifteen soluble metals and the sixteen EPÁs priority polycyclic aromatic hydrocarbons (PAH) were chemically determined. PAHs prevailing were fluoranthene and benzo(ghi)perylene. In the water-soluble extracts, highest concentration of metals was found for zinc, iron, and copper in all places of collection. Although PM10 concentration in all samples was in the range of 33.5–110.1μg/m3, lower than 120μg/m3 (limit established by São Paulo State’s legislation for PM10 in 24h), MN results showed that of the 24 samples analyzed, five organic and seven water-soluble extracts presented a significant increase in MN frequency. The frequency of MN correlates with the total PAH concentration of the three sites investigated, and the concentration of PM10 is correlated with the biological effect in two of them. For the water-soluble fraction, all the sites presented a relation between the PM10 concentration and the MN frequency. Again, the genotoxic response showed a correlation with the total concentration of water-soluble metals in two of the three sites. Our results confirm the importance of the soluble fraction of PM10 to the genotoxic effect of airborne PM even at low concentration of water-soluble compounds. Thus, together with chemical analysis, the implementation of the MN protocol for both organic and water-soluble fraction biological monitoring could be used as a strategy in a routine air-quality monitoring program, complementing other usual analyses for air pollution control and protection of populations.

Calcium is not required for triggering volume restoration in hypotonically challenged A549 epithelial cells.

Maintenance of cell volume is a fundamental housekeeping function in eukaryotic cells. Acute cell swelling activates a regulatory volume decrease (RVD) process with poorly defined volume sensing and intermediate signaling mechanisms. Here, we analyzed the putative role of Ca2+ signaling in RVD in single substrate-adherent human lung epithelial A549 cells. Acute cell swelling was induced by perfusion of the flow-through imaging chamber with 50% hypotonic solution at a defined fluid turnover rate. Changes in cytosolic Ca2+ concentration ([Ca2+]i) and cell volume were monitored simultaneously with ratiometric Fura-2 fluorescence and 3D reconstruction of stereoscopic single-cell images, respectively. Hypotonic challenge caused a progressive swelling peaking at ∼20min and followed, during the next 20min, by RVD of 60±7% of the peak volume increase. However, at the rate of swelling used in our experiments, these processes were not accompanied by a measurable increment of [Ca2+]i. Loading with intracellular Ca2+ chelator BAPTA slightly delayed peak of swelling but did not prevent RVD in 82% of cells. Further, electrophysiology whole-cell patch-clamp experiments showed that BAPTA did not block activation of volume-regulated anion channel (VRAC) measured as swelling-induced outwardly rectifying 5-nitro-2-(3-phenylpropyl-amino) benzoic acid sensitive current. Together, our data suggest that intracellular Ca2+-mediated signaling is not essential for VRAC activation and subsequent volume restoration in A549 cells.

Typical airborne quinones modulate oxidative stress and cytokine expression in lung epithelial A549 cells

ABSTRACTQuinones that exist in ambient particulate matter (PM) are hypothesized to be associated with adverse health effects through the generation of reactive oxygen species (ROS). However, the impacts of the quinones on the inflammatory processes have yet to be clearly understood. In this study, we examined the oxidative potentials and biological effects of typical airborne quinones in the human lung epithelial A549 cells. Significant change of redox status, loss of mitochondrial membrane potentials (△Ψ) and increase of superoxide dismutase (SOD) activity were induced by exposure to quinones. Some pro-inflammatory genes including interleukin-6 (IL-6), interleukin-8 (IL-8), tumor necrosis factor (TNF-α) and monocyte chemoattractant protein-1 (MCP-1); two aromatic hydrocarbon receptor-regulated genes, cytochromes P450 1A1 (Cyp1a1) and cytochromes P450 1B1 (Cyp1b1); and oxidative stress-related gene heme oxygenase-1 (HO-1) were up-regulated after quinones treatment. Among these quinones, 1,2-naphthoquinone (1,2-NQ) up-regulated expressions of IL-6, IL-8, TNF-α, Cyp1a1, and HO-1; 2-methoxy-1,4-naphthoquinone (MNQ) up-regulated MCP-1, Cyp1b1, Cyp1a1, and HO-1; 2-methylanthraquinone (MAQ) up-regulated IL-6, IL-8, TNF-α, MCP-1, Cyp1b1, and Cyp1a1; acenaphthenequinone (ACQ) up-regulated IL-8, TNF-α, MCP-1, Cyp1b1, and Cyp1a1. These results suggested that all these five quinones had a considerable pro-inflammatory potential by inducing oxidative stress and releasing different types of cytokines/chemokines.

Stenotrophomonas maltophiliaouter membrane vesicles elicit a potent inflammatory responsein vitroandin vivo

Stenotrophomonas maltophilia has become one of the most prevalent opportunistic pathogens in hospitalized patients. This microorganism secretes outer membrane vesicles (OMVs), but the pathogenesis of S. maltophilia as it relates to OMVs has not been characterized. This study investigated the cytotoxic activity of S. maltophilia OMVs and their ability to induce inflammatory responses both invitro and invivo Stenotrophomonas maltophilia ATCC 13637 and two clinical isolates were found to secrete spherical OMVs during in vitro culture. OMVs from S. maltophilia ATCC 13637 were cytotoxic to human lung epithelial A549 cells. Stenotrophomonas maltophilia OMVs stimulated the expression of proinflammatory cytokine and chemokine genes, including interleukin (IL)-1β, IL-6, IL-8, tumor necrosis factor-α and monocyte chemoattractant protein-1, in A549 cells. Early inflammatory responses such as congestion and neutrophilic infiltrations and profound expression of proinflammatory cytokine and chemokine genes were observed in the lungs of mice injected with S. maltophilia OMVs, and were similar to responses elicited by the bacteria. Our data demonstrate that S. maltophilia OMVs are important secretory nanocomplexes that elicit a potent inflammatory response that might contribute to S. maltophilia pathogenesis during infection.

Comparative genotoxicity of silver nanoparticles in human liver HepG2 and lung epithelial A549 cells.

With the rapid expanding of human exposure to silver nanoparticles (AgNPs), genotoxicity screening of nanosilver is necessary to ensure consumer safety. Here, we assessed one key DNA damage responsive pathway activated by GADD45a gene after 24 h of AgNPs exposure in stable luciferase reporter cell systems based on two widely used in vitro cell models, human liver HepG2 and lung epithelial A549 cells. The comet assay and micronucleus test were also conducted to confirm the genetic damage induced by AgNPs. Our results showed that AgNPs produced a strong dose-dependent increase in transcriptional activation of GADD45a promoter indicated by luciferase activity accompanying by the significant decreases in cell viability. Surprisingly, in HepG2-luciferase cells, the relative luciferase activity was greater than 4.5× the control level after being treated with 200 μg ml-1 AgNPs. These results were generally in line with the positive and dose-dependent responses in cytotoxicity, DNA strand breaks indicated by Olive tail moment, tail DNA (%) and tail length, and chromosome damage indicated by induction of micronuclei, nucleoplasmic bridges, and nuclear buds. Additionally, compared with the A549-luciferase cells, the HepG2-luciferase cells seemed to be more susceptible to AgNPs as higher levels of genotoxicity were induced. We concluded that our GADD45a promoter-driven luciferase reporter gene cell system, together with the comet assay and micronucleus test, can be used as valuable tools for rapid screening of genotoxic potential of nanosilver. Copyright © 2016 John Wiley & Sons, Ltd.

Novel cationic peptide TP359 down-regulates the expression of outer membrane biogenesis genes in Pseudomonas aeruginosa: a potential TP359 anti-microbial mechanism.

BackgroundAntimicrobial peptides (AMPs) are a class of antimicrobial agents with broad-spectrum activities. Several reports indicate that cationic AMPs bind to the negatively charged bacterial membrane causing membrane depolarization and damage. However, membrane depolarization and damage may be insufficient to elicit cell death, thereby suggesting that other mechanism(s) of action could be involved in this phenomenon. In this study, we investigated the antimicrobial activity of a novel antimicrobial peptide, TP359, against two strains of Pseudomonas aeruginosa, as well as its possible mechanisms of action.ResultsTP359 proved to be bactericidal against P. aeruginosa as confirmed by the reduced bacteria counts, membrane damage and cytoplasmic membrane depolarization. In addition, it was non-toxic to mouse J774 macrophages and human lung A549 epithelial cells. Electron microscopy analysis showed TP359 bactericidal effects by structural changes of the bacteria from viable rod-shaped cells to those with cell membrane damages, proceeding into the efflux of cytoplasmic contents and emergence of ghost cells. Gene expression analysis on the effects of TP359 on outer membrane biogenesis genes underscored marked down-regulation, particularly of oprF, which encodes a major structural and outer membrane porin (OprF) in both strains studied, indicating that the peptide may cause deregulation of outer membrane genes and reduced structural stability which could lead to cell death.ConclusionOur data shows that TP359 has potent antimicrobial activity against P aeruginosa. The correlation between membrane damage, depolarization and reduced expression of outer membrane biogenesis genes, particularly oprF may suggest the bactericidal mechanism of action of the TP359 peptide.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-016-0808-2) contains supplementary material, which is available to authorized users.

Burkholderia pseudomallei Differentially Regulates Host Innate Immune Response Genes for Intracellular Survival in Lung Epithelial Cells.

BackgroundBurkholderia pseudomallei, the causative agent of melioidosis poses a serious threat to humankind. B. pseudomallei secretes numerous virulence proteins that alter host cell functions to escape from intracellular immune sensors. However, the events underlying disease pathogenesis are poorly understood.MethodsWe determined the ability of B. pseudomallei to invade and survive intracellularly in A549 human lung epithelial cells, and also investigated the early transcriptional responses using an Illumina HumanHT-12 v4 microarray platform, after three hours of exposure to live B. pseudomallei (BCMS) and its secreted proteins (CCMS).ResultsWe found that the ability of B. pseudomallei to invade and survive intracellularly correlated with increase of multiplicity of infection and duration of contact. Activation of host carbohydrate metabolism and apoptosis as well as suppression of amino acid metabolism and innate immune responses both by live bacteria and its secreted proteins were evident. These early events might be linked to initial activation of host genes directed towards bacterial dissemination from lungs to target organs (via proposed in vivo mechanisms) or to escape potential sensing by macrophages.ConclusionUnderstanding the early responses of A549 cells toward B. pseudomallei infection provide preliminary insights into the likely pathogenesis mechanisms underlying melioidosis, and could contribute to development of novel intervention strategies to combat B. pseudomallei infections.

The ORF3 Protein of Genotype 1 Hepatitis E Virus Suppresses TLR3-induced NF-κB Signaling via TRADD and RIP1.

Hepatitis E virus (HEV) genotype 1 infection is common and can emerge as outbreaks in developing areas, thus posing a threat to public health. However, due to the absence of feasible animal models, the mechanism of HE pathogenesis remains obscure. The HEV pathogenic mechanism has been suggested to be mediated by the immune system and not by direct viral duplication. We firstly discovered that the open reading frame 3 (ORF3) protein of genotype 1 HEV downregulates TLR3-mediated NF-κB signaling in Human A549 Lung Epithelial Cells (A549 cells) which were exposed to different TLR agonists associated with viral nucleic acids. Additionally, we identified the P2 domain of ORF3 as being responsible for this inhibition. Intriguingly, tumor necrosis factor receptor 1-associated death domain protein (TRADD) expression and receptor-interacting protein kinase 1 (RIP1) K63-ubiquitination were reduced in the presence of both ORF3 and Poly(I:C). Furthermore, we found that Lys377 of RIP1 acts as the functional ubiquitination site for ORF3-associated inhibition. Overall, we found that ORF3 protein downregulates TLR3-mediated NF-κB signaling via TRADD and RIP1. Our findings provide a new perspective on the cellular response in HEV infection and expand our understanding of the molecular mechanisms of HEV pathogenesis in innate immunity.

Rapid induction and persistence of paracrine-induced cellular antiviral states arrest viral infection spread in A549 cells

The virus/host interaction is a complex interplay between pro- and anti-viral factors that ultimately determines the spread or halt of virus infections in tissues. This interplay develops over multiple rounds of infection. The purpose of this study was to determine how cellular-level processes combine to impact the spatial spread of infection. We measured the kinetics of virus replication (VSV), antiviral paracrine signal upregulation and secretion, spatial spread of virus and paracrine antiviral signaling, and inhibition of virus production in antiviral-exposed A549 human lung epithelial cells. We found that initially infected cells released antiviral signals 4-to-7h following production of virus. However, the subsequent rapid dissemination of signal and fast induction of a robust and persistent antiviral state ultimately led to a suppression of infection spread. This work shows how cellular responses to infection and activation of antiviral responses can integrate to ultimately control infection spread across host cell populations.

Selective Functionalization of Antimycin A Through an N-Transacylation Reaction.

Acylation of 3-(N-formylamino)salicylic acids resulted in transacylation with loss of the formyl moiety. The reaction proceeds through a bis-N-acylated intermediate, which undergoes facile deformylation. This transacylation reaction has been employed for the site-specific functionalization of the mitochondrial poison antimycin A, affording several novel derivatives. The selective cytotoxicity of some of these derivatives toward cultured A549 human lung epithelial adenocarcinoma cells, in comparison with WI-38 normal human lung fibroblasts, illustrates one application of this transacylation reaction.

Impact of blaNDM-1 on fitness and pathogenicity of Escherichia coli and Klebsiella pneumoniae

The objective of this study was to determine whether acquisition of New Delhi metallo-β-lactamase-1 (NDM-1) has an impact on the fitness and virulence of Escherichia coli and Klebsiella pneumoniae. Growth kinetics and the cost of fitness of NDM-1 plasmid carriage were assessed in isogenic E. coli J53 and K. pneumoniae PRZ in vitro by pairwise competition assays. The pathogenicity of NDM-1-expressing E. coli and K. pneumoniae strains and their isogenic controls was analysed in vivo using a Galleria mellonella infection model. The cytotoxicity of NDM-1 was assessed in A549 human lung epithelial cells using the lactate dehydrogenase (LDH) assay. No differences in growth kinetics were recorded between NDM-1-expressing strains and controls (P = 0.92). A reduction in fitness of NDM-1-carrying strains was observed both for E. coli J53 and K. pneumoniae PRZ [selection rate constant (s) = −1.27 ± 0.27 for E. coli J53 and −0.19 ± 0.14 for K. pneumoniae PRZ; P < 0.0001]. Survival of G. mellonella larvae infected with NDM-1-expressing strains and controls was similar for E. coli J53 and K. pneumoniae PRZ. Cytotoxicity in A549 cells was not affected by NDM-1 expression (P > 0.05). The presence of blaNDM-1 did not increase the virulence or cytotoxicity of isogenic strains. However, there was a considerable cost of fitness incurred by carriage of the pNDM-1 plasmid. Interestingly, the cost of fitness was significantly higher in E. coli J53 compared with K. pneumoniae PRZ.

Proteomic changes in human lung epithelial cells (A549) in response to carbon black and titanium dioxide exposures

This study combined cytotoxicity assays with proteomic analysis to characterize the unique biological responses of the A549 human lung epithelial cell line to two physicochemically distinct respirable particles titanium dioxide (TiO2) and carbon black (CB). Cellular LDH, ATP, BrdU incorporation and resazurin reduction indicated that CB was more potent than TiO2. Proteomic analysis was done using 2D-GE and MALDI-TOF-TOF-MS. Proteomic changes reflected common and particle-specific responses. Particle-specific proteomic responses were associated with cell death (necrosis and apoptosis), viability and proliferation pathways. Our results suggested that these pathways were consistent with the cytotoxicity data. For instance, increased expressions of anti-proliferative proteins LMNA and PA2G4 were in agreement with the decreased BrdU incorporation in A549 cells after exposure to CB. Similarly, increased expression of HSPA5 that is associated with ATPase activity was consistent with decreased cellular ATP levels in these cells. These findings reveal that proteomic changes can explain the cellular cytotoxicity characteristics of the particles. In essence, our results demonstrate that the in vitro toxicoproteomic approach is a promising tool to gain insight into molecular mechanisms underlying particle exposure-specific cytotoxicity. Biological significanceIn this study we have shown that toxicoproteomics is a sensitive and informative method to resolve the toxicity characteristics of particles with different physicochemical properties. This approach can be useful in the investigation of molecular mechanisms underpinning cellular cytotoxic responses elicited by particle exposures. Thus, the toxicoproteomic approach can be valuable in assessing the risk associated with particle exposures in vitro.

The South Pacific epidemic strain of Zika virus replicates efficiently in human epithelial A549 cells leading to IFN-β production and apoptosis induction

Zika virus (ZIKV) is an emerging flavivirus since the first epidemics in South Pacific in 2007. The recent finding that ZIKV is now circulating in Western Hemisphere and can be associated to severe human diseases, warrants the need for its study. Here we evaluate the susceptibility of human lung epithelial A549 cells to South Pacific epidemic strain of ZIKV isolated in 2013. We showed that ZIKV growth in A549 cells is greatly efficient. ZIKV infection resulted in the secretion of IFN-β followed by the expression of pro-inflammatory cytokines such as IL-1β, and transcriptional activity of IFIT genes. At the maximum of virus progeny production, ZIKV triggers mitochondrial apoptosis through activation of caspases-3 and -9. Whereas at early infection times, the rapid release of IFN-β which exerts an antiviral effect against ZIKV might delay apoptosis in infected cells.

Water soluble and insoluble components of urban PM2.5 and their cytotoxic effects on epithelial cells (A549) in vitro

When PM2.5 enters human bodies, the water soluble (WS-PM2.5) and insoluble components (WIS-PM2.5) of PM2.5 would interact with cells and cause adverse effects. However, the knowledge about the individual toxicity contribution of these two components is limited. In this study, the physiochemical properties of PM2.5 were well characterized. The toxic effects of WS-PM2.5 and WIS-PM2.5, which include the cell viability, cell membrane damage, reactive oxygen species (ROS) generation and morphological changes, were examined with human lung epithelial A549 cells in vitro. The results indicated that WS-PM2.5 could induce the early response of ROS generation, multiplied mitochondria and multi-lamellar bodies in A549 cells, which might cause cell damage through oxidative stress. Meanwhile, WIS-PM2.5 was predominantly associated with the cell membrane disruption, which might lead to the cell damage through cell-particle interactions. Moreover, the synergistic cytotoxic effects of WS-PM2.5 and WIS-PM2.5 were observed at longer exposure time. These findings demonstrate the different cytotoxicity mechanisms of WS-PM2.5 and WIS-PM2.5, which suggest that not only the size and dosage of PM2.5 but also the solubility of PM2.5 should be taken into consideration when evaluating the toxicity of PM2.5.

Reconstitution of ST2 (IL-1R4) specific for IL-33 activity; no suppression by IL-1Ra though a common chain IL-1R3 (IL-1RAcP) shared with IL-1

Interleukin-33 (IL-33) receptors are composed of ST2 (also known as IL-1R4), a ligand binding chain, and IL-1 receptor accessory protein (IL-1RAcP, also known as IL-1R3), a signal transducing chain. IL-1R3 is a common receptor for IL-1α, and IL-1β, IL-33, and three IL-36 isoforms. A549 human lung epithelial cells are highly sensitive to IL-1α and IL-1β but not respond to IL-33. The lack of responsiveness to IL-33 is due to ST2 expression. ST2 was stably transfected into A549 cells to reconstitute its activity. RT-PCR and FACS analysis confirmed ST2 expression on the cell surface of A549/ST2 cells. Upon IL-33 stimulation, A549/ST2 cells induced IL-8 and IL-6 production in a dose dependent manner while A549/mock cells remained unresponsive. There was no difference in IL-1α and IL-1β activity in A549/ST2 cells compared to A549/mock cells despite the fact that IL-33 shares IL-1R3 with IL-1α/β. IL-33 activated inflammatory signaling molecules in a time- and dose-dependent manner. Anti-ST2 antibody and soluble recombinant ST2-Fc abolished IL-33-induced IL-6 and IL-8 production in A549/ST2 cells but the IL-1 receptor antagonist failed to block IL-33-induced cytokines. This result demonstrates for the first time the reconstitution of ST2 in A549 human lung epithelial cell line and verified its function in IL-33-mediated cytokine production and signal transduction.

Genetic characterization of an adapted pandemic 2009 H1N1 influenza virus that reveals improved replication rates in human lung epithelial cells

The 2009 influenza pandemic originated from a swine-origin H1N1 virus, which, although less pathogenic than anticipated, may acquire additional virulence-associated mutations in the future. To estimate the potential risk, we sequentially passaged the isolate A/Hamburg/04/2009 in A549 human lung epithelial cells. After passage 6, we observed a 100-fold increased replication rate. High-throughput sequencing of viral gene segments identified five dominant mutations, whose contribution to the enhanced growth was analyzed by reverse genetics. The increased replication rate was pinpointed to two mutations within the hemagglutinin (HA) gene segment (HA1 D130E, HA2 I91L), near the receptor binding site and the stem domain. The adapted virus also replicated more efficiently in mice in vivo. Enhanced replication rate correlated with increased fusion pH of the HA protein and a decrease in receptor affinity. Our data might be relevant for surveillance of pre-pandemic strains and development of high titer cell culture strains for vaccine production.