Increase in markers of airway inflammation after ozone exposure can be observed also in stable treated asthmatics with minimal functional response to ozone

Background

The discrepancy between functional and inflammatory airway response to ozone has been reported in normal subjects, but few data are available for stable asthmatics regularly treated with inhaled corticosteroids.

Methods

Twenty-three well controlled, regularly treated, mild-to-moderate asthmatic patients underwent two sequential randomised exposures to either filtered air or ozone (0.3 ppm for 2 hours) in a challenge chamber. Pulmonary function (PF) was monitored, and patients with FEV1 decrease greater than 10% from pre-challenge value were considered as responders. Immediately after each exposure, exhaled breath condensate (EBC) was collected to measure malondialdehyde (MDA). Six hours after each exposure, PF and EBC collection were repeated, and sputum was induced to measure inflammatory cell counts and soluble mediators (IL-8 and neutrophil elastase). The response to ozone was also evaluated according to the presence of polymorphism in oxidative stress related NQO1 and GSTM1 genes.

Results

After ozone exposure, sputum neutrophils significantly increased in responders (n = 8), but not in nonresponders (n = 15). Other markers of neutrophil activation in sputum supernatant and MDA in EBC significantly increased in all patients, but only in nonresponders the increase was significant. In nonresponders, sputum eosinophils also significantly increased after ozone. There was a positive correlation between ozone-induced FEV1 fall and increase in sputum neutrophils. No difference in functional or inflammatory response to ozone was observed between subjects with or without the combination of NQO1wt- GSTM1null genotypes.

Conclusions

Markers of neutrophilic inflammation and oxidative stress increase also in asthmatic subjects not responding to ozone. A greater functional response to ozone is associated with greater neutrophil airway recruitment in asthmatic subjects.     

Impact of ozone exposure on the response to glucocorticoid in a mouse model of asthma: involvements of p38 MAPK and MKP-1

Background

Molecular mechanisms involved in the oxidative stress induced glucocorticoids insensitivity remain elusive. The mitogen-activated protein kinase phosphatase (MKP) 1 mediates a part of glucocorticoids action and can be modified by exogenous oxidants. Whether oxidant ozone (O₃) can affect the function of MKP-1 and hence blunt the response to corticotherapy is not clear.

Methods

Here we employed a murine model of asthma established with ovalbumin (OVA) sensitization and challenge to evaluate the influence of O₃ on the inhibitory effect of dexamethasone on AHR and airway inflammation, and by administration of SB239063, a selective p38 MAPK inhibitor, to explore the underlying involvements of the activation of p38 MAPK and the expression of MKP-1.

Results

Ozone exposure not only aggravated the pulmonary inflammation and AHR, but also decreased the inhibitory effects of dexamethasone, accompanied by the elevated oxidative stress, airway neutrophilia, enhanced phosphorylation of p38 MAPK, and upregulated expression of IL-17. Administration of SB239063 caused significant inhibition of the p38 MAPK phosphorylation, alleviation of the airway neutrophilia, and decrement of the ozone-induced IL-17 expression, and partly restored the ozone-impaired effects of dexamethasone. Ozone exposure not only decreased the protein expression of MKP-1, but also diminished the dexamethasone-mediated induction process of MKP-1 mRNA and protein expression.

Conclusions

The glucocorticoids insensitivity elicited by ozone exposure on current asthma model may involve the enhanced phosphorylation of p38 MAPK and disturbed expression of MKP-1.

Electronic supplementary material

The online version of this article (doi:10.1186/s12931-014-0126-x) contains supplementary material, which is available to authorized users.     

AMP-activated protein kinase deficiency reduces ozone-induced lung injury and oxidative stress in mice

Background

Acute ozone exposure causes lung oxidative stress and inflammation leading to lung injury. At least one mechanism underlying the lung toxicity of ozone involves excessive production of reactive oxygen and nitrogen intermediates such as peroxynitrite. In addition and beyond its major prooxidant properties, peroxynitrite may nitrate tyrosine residues altering phosphorylation of many protein kinases involved in cell signalling. It was recently proposed that peroxynitrite activates 5''-AMP-activated kinase (AMPK), which regulates metabolic pathways and the response to cell stress. AMPK activation as a consequence of ozone exposure has not been previously evaluated. First, we tested whether acute ozone exposure in mice would impair alveolar fluid clearance, increase lung tissue peroxynitrite production and activate AMPK. Second, we tested whether loss of AMP-activated protein kinase alpha1 subunit in mouse would prevent enhanced oxidative stress and lung injury induced by ozone exposure.

Methods

Control and AMPKα1 deficient mice were exposed to ozone at a concentration of 2.0 ppm for 3 h in glass cages. Evaluation was performed 24 h after ozone exposure. Alveolar fluid clearance (AFC) was evaluated using fluorescein isothiocyanate tagged albumin. Differential cell counts, total protein levels, cytokine concentrations, myeloperoxidase activity and markers of oxidative stress, i.e. malondialdehyde and peroxynitrite, were determined in bronchoalveolar lavage (BAL) and lung homogenates (LH). Levels of AMPK-Thr¹⁷² phosphorylation and basolateral membrane Na(+)-K(+)-ATPase abundance were determined by Western blot.

Results

In control mice, ozone exposure induced lung inflammation as evidence by increased leukocyte count, protein concentration in BAL and myeloperoxidase activity, pro-inflammatory cytokine levels in LH. Increases in peroxynitrite levels (3 vs 4.4 nM, p = 0.02) and malondialdehyde concentrations (110 vs 230 μmole/g wet tissue) were detected in LH obtained from ozone-exposed control mice. Ozone exposure consistently increased phosphorylated AMPK-Thr¹⁷² to total AMPK ratio by 80% in control mice. Ozone exposure causes increases in AFC and basolateral membrane Na(+)-K(+)-ATPase abundance in control mice which did not occur in AMPKα1 deficient mice.

Conclusions

Our results collectively suggest that AMPK activation participates in ozone-induced increases in AFC, inflammation and oxidative stress. Further studies are needed to understand how the AMPK pathway may provide a novel approach for the prevention of ozone-induced lung injury.     

Model-based analysis of avoidance of ozone stress by stomatal closure in Siebold's beech (Fagus crenata)

Background and Aims

Resistance of plants to ozone stress can be classified as either avoidance or tolerance. Avoidance of ozone stress may be explained by decreased stomatal conductance during ozone exposure because stomata are the principal interface for entry of ozone into plants. In this study, a coupled photosynthesis–stomatal model was modified to test whether the presence of ozone can induce avoidance of ozone stress by stomatal closure.

Methods

The response of Siebold''s beech (Fagus crenata), a representative deciduous tree species, to ozone was studied in a free-air ozone exposure experiment in Japan. Photosynthesis and stomatal conductance were measured under ambient and elevated ozone. An optimization model of stomata involving water, CO₂ and ozone flux was tested using the leaf gas exchange data.

Key Results

The data suggest that there are two phases in the avoidance of ozone stress via stomatal closure for Siebold''s beech: (1) in early summer ozone influx is efficiently limited by a reduction in stomatal conductance, without any clear effect on photosynthetic capacity; and (2) in late summer and autumn the efficiency of ozone stress avoidance was decreased because the decrease in stomatal conductance was small and accompanied by an ozone-induced decline of photosynthetic capacity.

Conclusions

Ozone-induced stomatal closure in Siebold''s beech during early summer reduces ozone influx and allows the maximum photosynthetic capacity to be reached, but is not sufficient in older leaves to protect the photosynthetic system.     

Prolonged cigarette smoke exposure alters mitochondrial structure and function in airway epithelial cells

Background

Cigarette smoking is the major risk factor for COPD, leading to chronic airway inflammation. We hypothesized that cigarette smoke induces structural and functional changes of airway epithelial mitochondria, with important implications for lung inflammation and COPD pathogenesis.

Methods

We studied changes in mitochondrial morphology and in expression of markers for mitochondrial capacity, damage/biogenesis and fission/fusion in the human bronchial epithelial cell line BEAS-2B upon 6-months from ex-smoking COPD GOLD stage IV patients to age-matched smoking and never-smoking controls.

Results

We observed that long-term CSE exposure induces robust changes in mitochondrial structure, including fragmentation, branching and quantity of cristae. The majority of these changes were persistent upon CSE depletion. Furthermore, long-term CSE exposure significantly increased the expression of specific fission/fusion markers (Fis1, Mfn1, Mfn2, Drp1 and Opa1), oxidative phosphorylation (OXPHOS) proteins (Complex II, III and V), and oxidative stress (Mn-SOD) markers. These changes were accompanied by increased levels of the pro-inflammatory mediators IL-6, IL-8, and IL-1β. Importantly, COPD primary bronchial epithelial cells (PBECs) displayed similar changes in mitochondrial morphology as observed in long-term CSE-exposure BEAS-2B cells. Moreover, expression of specific OXPHOS proteins was higher in PBECs from COPD patients than control smokers, as was the expression of mitochondrial stress marker PINK1.

Conclusion

The observed mitochondrial changes in COPD epithelium are potentially the consequence of long-term exposure to cigarette smoke, leading to impaired mitochondrial function and may play a role in the pathogenesis of COPD.     

Inflammatory and Repair Pathways Induced in Human Bronchoalveolar Lavage Cells with Ozone Inhalation

Background

Inhalation of ambient levels of ozone causes airway inflammation and epithelial injury.

Methods

To examine the responses of airway cells to ozone-induced oxidative injury, 19 subjects (7 with asthma) were exposed to clean air (0ppb), medium (100ppb), and high (200ppb) ambient levels of ozone for 4h on three separate occasions in a climate-controlled chamber followed by bronchoscopy with bronchoalveolar lavage (BAL) 24h later. BAL cell mRNA expression was examined using Affymetrix GeneChip Microarray. The role of a differentially expressed gene (DEG) in epithelial injury was evaluated in an in vitro model of injury [16HBE14o- cell line scratch assay].

Results

Ozone exposure caused a dose-dependent up-regulation of several biologic pathways involved in inflammation and repair including chemokine and cytokine secretion, activity, and receptor binding; metalloproteinase and endopeptidase activity; adhesion, locomotion, and migration; and cell growth and tumorigenesis regulation. Asthmatic subjects had 1.7- to 3.8-fold higher expression of many DEGs suggestive of increased proinflammatory and matrix degradation and remodeling signals. The most highly up-regulated gene was osteopontin, the protein level of which in BAL fluid increased in a dose-dependent manner after ozone exposure. Asthmatic subjects had a disproportionate increase in non-polymerized osteopontin with increasing exposure to ozone. Treatment with polymeric, but not monomeric, osteopontin enhanced the migration of epithelial cells and wound closure in an α9β1 integrin-dependent manner.

Conclusions

Expression profiling of BAL cells after ozone exposure reveals potential regulatory genes and pathways activated by oxidative stress. One DEG, osteopontin, promotes epithelial wound healing in an in vitro model of injury.     

Ionizing Radiation-Induced Oxidative Stress Alters miRNA Expression

Background

MicroRNAs (miRNAs) are small, highly conserved, non-coding RNA that alter protein expression and regulate multiple intracellular processes, including those involved in the response to cellular stress. Alterations in miRNA expression may occur following exposure to several stress-inducing anticancer agents including ionizing radiation, etoposide, and hydrogen peroxide (H₂O₂).

Methodology/Principal Findings

Normal human fibroblasts were exposed to radiation, H₂O₂, or etoposide at doses determined by clonogenic cell survival curves. Total RNA was extracted and miRNA expression was determined by microarray. Time course and radiation dose responses were determined using RT-PCR for individual miRNA species. Changes in miRNA expression were observed for 17 miRNA species following exposure to radiation, 23 after H₂O₂ treatment, and 45 after etoposide treatment. Substantial overlap between the miRNA expression changes between agents was observed suggesting a signature miRNA response to cell stress. Changes in the expression of selected miRNA species varied in response to radiation dose and time. Finally, production of reactive oxygen species (ROS) increased with increasing doses of radiation and pre-treatment with the thiol antioxidant cysteine decreased both ROS production and the miRNA response to radiation.

Conclusions

These results demonstrate a common miRNA expression signature in response to exogenous genotoxic agents including radiation, H₂O₂, and etoposide. Additionally, pre-treatment with cysteine prevented radiation-induced alterations in miRNA expression which suggests that miRNAs are responsive to oxidative stress. Taken together, these results imply that miRNAs play a role in cellular defense against exogenous stress and are involved in the generalized cellular response to genotoxic oxidative stress.     

Transcript and Protein Analysis Reveals Better Survival Skills of Monocyte-Derived Dendritic Cells Compared to Monocytes during Oxidative Stress

Background

Dendritic cells (DCs), professional antigen-presenting cells with the unique ability to initiate primary T-cell responses, are present in atherosclerotic lesions where they are exposed to oxidative stress that generates cytotoxic reactive oxygen species (ROS). A large body of evidence indicates that cell death is a major modulating factor of atherogenesis. We examined antioxidant defence systems of human monocyte-derived (mo)DCs and monocytes in response to oxidative stress.

Methods

Oxidative stress was induced by addition of tertiary-butylhydroperoxide (tert-BHP, 30 min). Cellular responses were evaluated using flow cytometry and confocal live cell imaging (both using 5-(and-6)-chloromethyl-2,7-dichlorodihydrofluorescein diacetate, CM-H₂DCFDA). Viability was assessed by the neutral red assay. Total RNA was extracted for a PCR profiler array. Five genes were selected for confirmation by Taqman gene expression assays, and by immunoblotting or immunohistochemistry for protein levels.

Results

Tert-BHP increased CM-H₂DCFDA fluorescence and caused cell death. Interestingly, all processes occurred more slowly in moDCs than in monocytes. The mRNA profiler array showed more than 2-fold differential expression of 32 oxidative stress–related genes in unstimulated moDCs, including peroxiredoxin-2 (PRDX2), an enzyme reducing hydrogen peroxide and lipid peroxides. PRDX2 upregulation was confirmed by Taqman assays, immunoblotting and immunohistochemistry. Silencing PRDX2 in moDCs by means of siRNA significantly increased CM-DCF fluorescence and cell death upon tert-BHP-stimulation.

Conclusions

Our results indicate that moDCs exhibit higher intracellular antioxidant capacities, making them better equipped to resist oxidative stress than monocytes. Upregulation of PRDX2 is involved in the neutralization of ROS in moDCs. Taken together, this points to better survival skills of DCs in oxidative stress environments, such as atherosclerotic plaques.     

Association between Exposure to Environmental Tobacco Smoke and Biomarkers of Oxidative Stress Among Patients Hospitalised with Acute Myocardial Infarction

Objective

To determine whether exposure to environmental tobacco smoke was associated with oxidative stress among patients hospitalised for acute myocardial infarction.

Design

An existing cohort study of 1,261 patients hospitalised for acute myocardial infarction.

Setting

Nine acute hospitals in Scotland.

Participants

Sixty never smokers who had been exposed to environmental tobacco smoke (admission serum cotinine ≥3.0 ng/mL) were compared with 60 never smokers who had not (admission serum cotinine ≤0.1 ng/mL).

Intervention

None.

Main outcome measures

Three biomarkers of oxidative stress (protein carbonyl, malondialdehyde (MDA) and oxidised low-density lipoprotein (ox-LDL)) were measured on admission blood samples and adjusted for potential confounders.

Results

After adjusting for baseline differences in age, sex and socioeconomic status, exposure to environmental tobacco smoke was associated with serum concentrations of both protein carbonyl (beta coefficient 7.96, 95% CI 0.76, 15.17, p = 0.031) and MDA (beta coefficient 10.57, 95% CI 4.32, 16.81, p = 0.001) but not ox-LDL (beta coefficient 2.14, 95% CI −8.94, 13.21, p = 0.703).

Conclusions

Exposure to environmental tobacco smoke was associated with increased oxidative stress. Further studies are requires to explore the role of oxidative stress in the association between environmental tobacco smoke and myocardial infarction.     

The ion channel transient receptor potential melastatin-2 does not play a role in inflammatory mouse models of chronic obstructive pulmonary diseases

Background

There is strong evidence that oxidative stress is associated with the pathogenesis of chronic obstructive pulmonary disease (COPD). The transient receptor potential melastatin-2 (TRPM2) is an oxidative stress sensing channel that is expressed in a number of inflammatory cells and therefore it has been suggested that inhibition of TRPM2 could lead to a beneficial effect in COPD patients. In this study, we have investigated the role of TRPM2 in a variety of mouse models of oxidative stress and COPD using TRPM2-deficent mice.

Methods

Mice were exposed to ozone (3 ppm for 4 h) or lipopolysaccharide (LPS, 0.3 mg/kg, intranasaly). In another model, mice were exposed to tobacco smoke (750 μg/l total wet particulate matter) for 30 min twice a day on three consecutive days. For the exacerbation model, the smoke exposure on the morning of day 3 animals was replaced with intranasal administration of LPS (0.3 mg/kg). Animals were killed 3 and 24 h after the challenge (ozone and LPS model) or 18 h after the last tobacco smoke exposure. In vitro neutrophil chemotaxis and monocyte activation were also studied using cells isolated from wild type and TRPM2-deficient animals. Statistical significance for the in vivo data (P < 0.05) was determined using analysis of variance with Kruskal-Wallis and Dunns multiple comparison test.

Results

In all models studied, no difference in the bronchoalveolar lavage inflammation could be evidenced when comparing wild type and TRPM2-deficient mice. In addition, no difference could be seen in the lung inflammation as assessed by the measurement of various cytokines/chemokines. Similarly in various in vitro cellular activation assays using isolated neutrophils and monocytes no significant differences could be observed when comparing wild type and TRPM2-deficient mice.

Discussion

We have shown, in all the models tested, no difference in the development of airway inflammation or cell activation between TRPM2-deficient mice and their wild type counterparts. These results would suggest that inhibiting TRPM2 activity in COPD would have no anti-inflammatory effect.     

Role of oxygen and the OxyR protein in the response to iron limitation in Rhodobacter sphaeroides

Background

High intracellular levels of unbound iron can contribute to the production of reactive oxygen species (ROS) via the Fenton reaction, while depletion of iron limits the availability of iron-containing proteins, some of which have important functions in defence against oxidative stress. Vice versa increased ROS levels lead to the damage of proteins with iron sulphur centres. Thus, organisms have to coordinate and balance their responses to oxidative stress and iron availability. Our knowledge of the molecular mechanisms underlying the co-regulation of these responses remains limited. To discriminate between a direct cellular response to iron limitation and indirect responses, which are the consequence of increased levels of ROS, we compared the response of the α-proteobacterium Rhodobacter sphaeroides to iron limitation in the presence or absence of oxygen.

Results

One third of all genes with altered expression under iron limitation showed a response that was independent of oxygen availability. The other iron-regulated genes showed different responses in oxic or anoxic conditions and were grouped into six clusters based on the different expression profiles. For two of these clusters, induction in response to iron limitation under oxic conditions was dependent on the OxyR regulatory protein. An OxyR mutant showed increased ROS production and impaired growth under iron limitation.

Conclusion

Some R. sphaeroides genes respond to iron limitation irrespective of oxygen availability. These genes therefore reflect a “core iron response” that is independent of potential ROS production under oxic, iron-limiting conditions. However, the regulation of most of the iron-responsive genes was biased by oxygen availability. Most strikingly, the OxyR-dependent activation of a subset of genes upon iron limitation under oxic conditions, including many genes with a role in iron metabolism, revealed that elevated ROS levels were an important trigger for this response. OxyR thus provides a regulatory link between the responses to oxidative stress and to iron limitation in R. sphaeroides.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-794) contains supplementary material, which is available to authorized users.     

Study of Oxidative Stress in Human Lens Epithelial Cells Exposed to 1.8 GHz Radiofrequency Fields

Objectives

The aims of the present study were to determine oxidative stress and to explore possible reasons of reactive oxygen species (ROS) increase in human lens epithelial (HLE) B3 cells exposed to low intensity 1.8 GHz radiofrequency fields (RF).

Methods

The HLE B3 cells were divided into RF exposure and RF sham-exposure groups. The RF exposure intensity was at specific absorption rate (SAR) of 2, 3, or 4 W/kg. The ROS levels were measured by a fluorescent probe 2′7′-dichlorofluorescin diacetate (DCFH-DA) assay in the HLE B3 cells exposed to 1.8 GHz RF for 0.5, 1, and 1.5 h. Lipid peroxidation and cellular viability were detected by an MDA test and Cell Counting Kit-8 (CCK-8) assays, respectively, in the HLE B3 cells exposed to 1.8 GHz RF for 6, 12, and 24 h, respectively. The mRNA expression of SOD1, SOD2, CAT, and GPx1 genes and the expression of SOD1, SOD2, CAT, and GPx1 proteins was measured by qRT-PCR and Western blot assays in the HLE B3 cells exposed to 1.8 GHz RF for 1 h.

Results

The ROS and MDA levels significantly increased (P<0.05) in the RF exposure group and that the cellular viability, mRNA expression of four genes, and expression of four proteins significantly decreased (P<0.05) compared with the RF sham-exposure group.

Conclusions

Oxidative stress is present in HLE B3 cells exposed to 1.8 GHz low-intensity RF and that the increased production of ROS may be related to down-regulation of four antioxidant enzyme genes induced by RF exposure.     

Meningothelial cells react to elevated pressure and oxidative stress

Background

Meningothelial cells (MECs) are the cellular components of the meninges enveloping the brain. Although MECs are not fully understood, several functions of these cells have been described. The presence of desmosomes and tight junctions between MECs hints towards a barrier function protecting the brain. In addition, MECs perform endocytosis and, by the secretion of cytokines, are involved in immunological processes in the brain. However, little is known about the influence of pathological conditions on MEC function; e.g., during diseases associated with elevated intracranial pressure, hypoxia or increased oxidative stress.

Methods

We studied the effect of elevated pressure, hypoxia, and oxidative stress on immortalized human as well as primary porcine MECs. We used MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) bioreduction assays to assess the proliferation of MECs in response to treatment and compared to untreated control cells. To assess endocytotic activity, the uptake of fluorescently labeled latex beads was analyzed by fluorescence microscopy.

Results

We found that exposure of MECs to elevated pressure caused significant cellular proliferation and a dramatic decrease in endocytotic activity. In addition, mild oxidative stress severely inhibited endocytosis.

Conclusion

Elevated pressure and oxidative stress impact MEC physiology and might therefore influence the microenvironment of the subarachnoid space and thus the cerebrospinal fluid within this compartment with potential negative impact on neuronal function.