Preventing carbon nanoparticle-induced lung inflammation reduces antigen-specific sensitization and subsequent allergic reactions in a mouse model.

BACKGROUND: Exposure of the airways to carbonaceous nanoparticles can contribute to the development of immune diseases both via the aggravation of the allergic immune response in sensitized individuals and by adjuvant mechanisms during the sensitization against allergens. The cellular and molecular mechanisms involved in these adverse pathways are not completely understood. We recently described that the reduction of carbon nanoparticle-induced lung inflammation by the application of the compatible solute ectoine reduced the aggravation of the allergic response in an animal system. In the current study we investigated the influence of carbon nanoparticles on the sensitization of animals to ovalbumin via the airways. Ectoine was used as a preventive strategy against nanoparticle-induced neutrophilic lung inflammation. METHODS: Balb/c mice were repetitively exposed to the antigen ovalbumin after induction of airway inflammation by carbon nanoparticles, either in the presence or in the absence of ectoine. Allergic sensitization was monitored by measurement of immunoglobulin levels and immune responses in lung and lung draining lymph nodes after challenge. Furthermore the role of dendritic cells in the effect of carbon nanoparticles was studied in vivo in the lymph nodes but also in vitro using bone marrow derived dendritic cells. RESULTS: Animals exposed to antigen in the presence of carbon nanoparticles showed increased effects with respect to ovalbumin sensitization, to the allergic airway inflammation after challenge, and to the specific TH2 response in the lymph nodes. The presence of ectoine during the sensitization significantly reduced these parameters. The number of antigen-loaded dendritic cells in the draining lymph nodes was identified as a possible cause for the adjuvant effect of the nanoparticles. In vitro assays indicate that the direct interaction of the particles with dendritic cells is not able to trigger CCR7 expression, while this endpoint is achieved by lung lavage fluid from nanoparticle-exposed animals. CONCLUSIONS: Using the intervention strategy of applying ectoine into the airways of animals we were able to demonstrate the relevance of neutrophilic lung inflammation for the adjuvant effect of carbon nanoparticles on allergic sensitization.

Recovery of neutrophil apoptosis by ectoine: a new strategy against lung inflammation.

The life span of neutrophilic granulocytes has a determining impact on the intensity and duration of neutrophil driven lung inflammation. Based on the compatible solute ectoine, we aimed to prevent anti-apoptotic reactions in neutrophils triggered by the inflammatory microenvironment in the lung. Neutrophils from chronic obstructive pulmonary disease patients and control individuals were exposed to inflammatory mediators and xenobiotics in the presence or absence of ectoine. The in vivo relevance of this approach was tested in xenobiotic-induced lung inflammation in rats. The reduction of apoptosis rates of ex vivo-exposed neutrophils from all study groups was significantly restored in the presence of ectoine. However, natural apoptosis rates not altered by inflammatory stimuli were not changed by ectoine. Mechanistic analyses demonstrated the preventive effect of ectoine on the induction of anti-apoptotic signalling. Neutrophilic lung inflammation induced by single or multiple expositions of animals to environmental particles was reduced after the therapeutic intervention with ectoine. Analyses of neutrophils from bronchoalveolar lavage indicate that the in vivo effect is due to the restoration of neutrophil apoptosis. Ectoine, a compound of the highly compliant group of compatible solutes, demonstrates a reproducible and robust effect on the resolution of lung inflammation.

Carbon nanoparticles induce ceramide- and lipid raft-dependent signalling in lung epithelial cells: a target for a preventive strategy against environmentally-induced lung inflammation.

BACKGROUND: Particulate air pollution in lung epithelial cells induces pathogenic endpoints like proliferation, apoptosis, and pro-inflammatory reactions. The activation of the epidermal growth factor receptor (EGFR) is a key event responsible for signalling events involving mitogen activated protein kinases specific for these endpoints. The molecular events leading to receptor activation however are not well understood. These events are relevant for the toxicological evaluation of inhalable particles as well as for potential preventive strategies in situations when particulate air pollution cannot be avoided. The current study therefore had the objective to elucidate membrane-coupled events leading to EGFR activation and the subsequent signalling cascade in lung epithelial cells. Furthermore, we aimed to identify the molecular target of ectoine, a biophysical active substance which we described to prevent carbon nanoparticle-induced lung inflammation. METHODS: Membrane signalling events were investigated in isolated lipid rafts from lung epithelial cells with regard to lipid and protein content of the signalling platforms. Using positive and negative intervention approaches, lipid raft changes, subsequent signalling events, and lung inflammation were investigated in vitro in lung epithelial cells (RLE-6TN) and in vivo in exposed animals. RESULTS: Carbon nanoparticle treatment specifically led to an accumulation of ceramides in lipid rafts. Detailed analyses demonstrated a causal link of ceramides and subsequent EGFR activation coupled with a loss of the receptor in the lipid raft fractions. In vitro and in vivo investigations demonstrate the relevance of these events for carbon nanoparticle-induced lung inflammation. Moreover, the compatible solute ectoine was able to prevent ceramide-mediated EGFR phosphorylation and subsequent signalling as well as lung inflammation in vivo. CONCLUSION: The data identify a so far unknown event in pro-inflammatory signalling and contribute to the understanding of particle cell interaction and therefore to risk identification and risk assessment of inhalable xenobiotics. Moreover, as this cellular reaction can be prevented by the well tolerated substance ectoine, a molecular preventive strategy for susceptible persons against airway inflammation is proposed.

c-Src-mediated activation of Erk1/2 is a reaction of epithelial cells to carbon nanoparticle treatment and may be a target for a molecular preventive strategy.

Owing to their specific physico/chemical properties, engineered as well as environmental nanoparticles can induce pathogenic endpoints in humans. Earlier studies demonstrated that pure carbon nanoparticles induce cell signaling events at the level of membrane receptor activation in lung epithelial cells. As a possible link between receptor activation and subsequent MAP-kinase signaling, the involvement of Src family kinases was investigated in cell lines of organs potentially exposed to environmental nanoparticles. Human cells from bronchus, intestine, and skin (keratinocytes) as well as rat lung epithelial cells showed similar time patterns for the activation of mitogen-activated protein kinases Erk1/2 as well as Src family kinases (SFK) when treated with carbon nanoparticles. Moreover, c-Src was identified as an integral part of the signaling mediating the transfer of information from membrane receptors to members of the proliferative signaling cascade in lung epithelial cells. Pretreatment of cells with the compatible solute ectoine, which is known to stabilize macromolecules, reduced the nanoparticle specific phosphorylation of SFK. Together with earlier in vivo and in vitro data, this demonstrates that compatible solutes prevent nanoparticle-induced signaling steps at the level of membrane-coupled signaling.

The compatible solute ectoine protects against nanoparticle-induced neutrophilic lung inflammation.

RATIONALE: Inflammatory reactions of the airways induced by nanoparticles of occupational and environmental origin contribute to organ-specific and systemic human diseases. Because this kind of exposure in modern societies is often unavoidable, a strategy of molecular prevention on an individual level could help to prevent inflammation-derived secondary diseases. OBJECTIVES: To test whether the compatible solute ectoine [(S)-2-methyl-1,4,5,6-tetrahydropyrimidine-4-carboxylic acid], which is known to reduce cell stress effects on a molecular level, prevents nanoparticle-induced lung inflammation. METHODS: Inflammatory parameters were studied in Fischer 344 rats treated with model carbon nanoparticles. The molecular effects of ectoin on proinflammatory signal transduction were demonstrated in the rat and in the human system using cultured lung epithelial cells. MEASUREMENTS AND MAIN RESULTS: Ectoine, given with or before the nanoparticles, dose-dependently reduced neutrophil inflammation in the lung. This preventive effect was not observed when lung inflammation was induced by bacterial lipopolysaccharide. Analyses of the underlying mode of action revealed that ectoine acted on lung epithelial cells. Ectoine administration inhibited nanoparticle-induced signaling, which is known to be responsible for proinflammatory reactions in rat lung epithelial cells in vitro as well as in vivo. These findings were corroborated and extended in experiments with cultured human bronchial epithelial cells in which ectoine inhibited nanoparticle-triggered cell signaling and IL-8 induction. CONCLUSIONS: Because compatible solutes are compliant natural products without known toxic potential, we propose that this group of substances may be used for the prevention of particle-induced airway inflammation in humans.

Reduction of neutrophilic lung inflammation by inhalation of the compatible solute ectoine: a randomized trial with elderly individuals.

BACKGROUND: Compatible solutes are natural substances that are known to stabilize cellular functions. Preliminary ex vivo and in vivo studies demonstrated that the compatible solute ectoine restores natural apoptosis rates of lung neutrophils and contributes to the resolution of lung inflammation. Due to the low toxicity and known compatibility of the substance, an inhalative application as an intervention strategy for humans suffering from diseases caused by neutrophilic inflammation, like COPD, had been suggested. As a first approach to test the feasibility and efficacy of such a treatment, we performed a population-based randomized trial. OBJECTIVE: The objective of the study was to test whether the daily inhalation of the registered ectoine-containing medical device (Ectoin® inhalation solution) leads to a reduction of neutrophilic cells and interleukin-8 (IL-8) levels in the sputum of persons with mild symptoms of airway disease due to lifelong exposure to environmental air pollution. METHODS: A double-blinded placebo-controlled trial was performed to study the efficacy and safety of an ectoine-containing therapeutic. Prior to and after both inhalation periods, lung function, inflammatory parameters in sputum, serum markers, and quality-of-life parameters were determined. RESULTS: While the other outcomes revealed no significant effects, sputum parameters were changed by the intervention. Nitrogen oxides (nitrate and nitrite) were significantly reduced after ectoine inhalation with a mean quotient of 0.65 (95% confidence interval 0.45-0.93). Extended analyses considering period effects revealed that the percentage of neutrophils in sputum was significantly lower after ectoine inhalation than in the placebo group (P=0.035) even after the washout phase. CONCLUSION: The current study is the first human trial in which the effects of inhaled ectoine on neutrophilic lung inflammation were investigated. Besides demonstrating beneficial effects on inflammatory sputum parameters, the study proves the feasibility of the therapeutic approach in an aged study group.

The compatible solute ectoine reduces the exacerbating effect of environmental model particles on the immune response of the airways.

Exposure of humans to particulate air pollution has been correlated with the incidence and aggravation of allergic airway diseases. In predisposed individuals, inhalation of environmental particles can lead to an exacerbation of immune responses. Previous studies demonstrated a beneficial effect of the compatible solute ectoine on lung inflammation in rats exposed to carbon nanoparticles (CNP) as a model of environmental particle exposure. In the current study we investigated the effect of such a treatment on airway inflammation in a mouse allergy model. Ectoine in nonsensitized animals significantly reduced the neutrophilic lung inflammation after CNP exposure. This effect was accompanied by a reduction of inflammatory factors in the bronchoalveolar lavage. Reduced IL-6 levels in the serum also indicate the effects of ectoine on systemic inflammation. In sensitized animals, an aggravation of the immune response was observed when animals were exposed to CNP prior to antigen provocation. The coadministration of ectoine together with the particles significantly reduced this exacerbation. The data indicate the role of neutrophilic lung inflammation in the exacerbation of allergic airway responses. Moreover, the data suggest to use ectoine as a preventive treatment to avoid the exacerbation of allergic airway responses induced by environmental air pollution.

Signalling-dependent adverse health effects of carbon nanoparticles are prevented by the compatible solute mannosylglycerate (firoin) in vitro and in vivo.

The inhalation of combustion-derived nanoparticles leads to adverse health effects in the airways. In this context the induction of membrane-coupled signalling is considered as causative for changes in tissue homeostasis and pro-inflammatory reactions. The identification of these molecular cell reactions allowed to seek for strategies which interfere with these adverse effects. In the current study, we investigated the structurally different compatible solutes mannosylglycerate (firoin) from thermophilic bacteria and ectoine from halophilic bacteria for their capability to reduce signalling pathways triggered by carbon nanoparticles in target cells in the lung. The pre-treatment of lung epithelial cells with both substances decreased the particle-specific activation of mitogen-activated protein kinases and also the endpoints proliferation and apoptosis. Firoin applied into the lungs of animals, like ectoine, led to a significant reduction of the neutrophilic lung inflammation induced by particle exposure. The pro-inflammatory effect of carbon nanoparticles on human neutrophil granulocytes ex vivo was significantly reduced by both substances via the reduction of the anti-apoptotic membrane-dependent signalling. The data of this study together with earlier studies demonstrate that two structurally non-related compatible solutes are able to prevent pathogenic reactions of the airways to carbon nanoparticles by interfering with signalling events. The findings highlight the preventive or therapeutic potential of compatible solutes for adverse health effects caused by particle exposure of the airways.

Unhealthy diet and ultrafine carbon black particles induce senescence and disease associated phenotypic changes.

Diet and pollution are environmental factors known to compromise "healthy aging" of the cardiovascular and respiratory systems. The molecular consequences of this permanent burden in these cells are still unknown. Therefore, this study investigates the impact of unhealthy diet on aging-related signaling pathways of human, primary cardiovascular cells and of airborne particles on lung epithelial and human endothelial cells. Nutrition health reports have shown that the diet in industrialized countries contains more than 100mg/dl low density lipoprotein (LDL) and a high fraction of added sugars, especially fructose. Several studies demonstrated that ultrafine particles can enter the circulation and thus may interact with endothelial cells directly. Both, dietary compounds and pollution derived particles, have been shown to increase the risk for cardiovascular diseases. To simulate an unhealthy diet, we supplemented cell culture media of human primary endothelial cells, smooth muscle cells and cardiomyocytes with LDL and replaced 1/3 of glucose with fructose. We observed hypertrophy in cardiomyocytes, enhanced proliferation in smooth muscle cells and increased senescence, loss of endothelial nitric oxide synthase and increased nuclear FoxO3A in endothelial cells. With respect to pollution we have used ultrafine carbon black particles (ufCB), one of the major constituents of industrial and exhaust emissions, in concentrations our lungs and vessels are constantly exposed to. These concentrations of ufCB increased reactive oxygen species in lung epithelial and vascular endothelial cells and reduced the S-NO content, a marker for NO-bioavailability, in endothelial cells. NO increases activation of Telomerase Reverse Transcriptase (TERT), an enzyme essential for telomere maintenance. TERT is required for proper endothelial cell function and is inactivated by Src kinase under conditions of oxidative stress. ufCB significantly increased Src kinase activation and reduced Telomerase activity in endothelial and lung epithelial cells. As a consequence, ufCB increased senescence of endothelial cells. To investigate whether ufCB show also effects in vivo, we instilled ufCB in concentrations not inducing inflammation into mice. Indeed, eNOS expression was reduced in the abdominal aorta of animals treated with ufCB. Thus, a combination of fructose and LDL in the diet and ufCB, as a major constituent of air pollution, seem to accelerate respiratory and cardiovascular cellular changes, which may compromise "healthy aging" and can lead to cardiovascular and pulmonary diseases.

The interleukin-6-type cytokine oncostatin M induces aryl hydrocarbon receptor expression in a STAT3-dependent manner in human HepG2 hepatoma cells.

The aryl hydrocarbon receptor (AHR) is a ligand-dependent transcription factor that mediates the toxicity of dioxins, polycyclic aromatic hydrocarbons and related environmental pollutants. Besides drug metabolism, several studies have provided evidence that the AHR and its downstream targets trigger important developmental, physiological and pathophysiological processes. However, in contrast to the molecular mechanisms of AHR-dependent signaling pathways, the transcriptional regulation of the AHR gene itself is as yet only marginally understood. We found that the pleiotropic interleukin (IL)-6-type cytokine oncostatin M (OSM) is an inducer of AHR mRNA and protein expression in human HepG2 hepatocarcinoma cells. Analyses of the human AHR promoter revealed the existence of a putative signal transducer and activator of transcription (STAT)-binding element 5'-upstream of the transcription start site. By means of site-directed mutagenesis, inhibitor experiments and electrophoretic mobility shift assays, we demonstrated that this STAT motif is recognized by STAT3 to regulate basal and cytokine-inducible AHR expression in HepG2 cells. The identification of the AHR as a downstream target of IL-6-type cytokine-stimulated STAT3 signaling may contribute to a better understanding of the multiple facets of AHR during development, physiology and disease.

Reactive oxygen species as mediators of membrane-dependent signaling induced by ultrafine particles.

Cell-membrane-dependent proliferative signal transduction activated by ultrafine carbon particles in lung epithelial cells involves the specific induction of Akt and ERK1/2 phosphorylation. Particle-induced generation of reactive oxygen species (ROS) and oxidative stress are regarded as initial molecular mechanisms leading to the induction of diverse cellular responses. Therefore, we aimed to analyze the ROS dependence of the induced activation of the Akt/ERK1/2 signaling pathway upon exposure to ultrafine particulate matter (UPM). For this, ultrafine carbon black (ufCB) and ferric sulfate (FS) were used as a model representing the carbonaceous core and a nonparticulate Fenton-reactive transition metal salt often found in combustion-derived UPM. Cell-free as well as intracellular particle-induced ROS generation was assessed and related to the induced Akt and ERK1/2 phosphorylation by inhibiting oxidative stress with catalase, superoxide dismutase, and N-acetylcysteine. We show here that the activation of this signal transduction pathway was mainly due to intracellular, rather than extracellular, ROS production induced by both ufCB and FS. Further inhibitor studies on the role of cell membrane receptors pointed to the epidermal growth factor receptor as a common mediator for particle- as well as transition metal-induced signaling, whereas integrin-dependent Akt and ERK1/2 activation seems to be particle-specific.

Carbon nanoparticle-induced lung epithelial cell proliferation is mediated by receptor-dependent Akt activation.

Treatment of lung epithelial cells with different kinds of nano-sized particles leads to cell proliferation. Because bigger particles fail to induce this reaction, it is suggested that the special surface properties, due to the extremely small size of these kinds of materials, is the common principle responsible for this specific cell reaction. Here the activation of the protein kinase B (Akt) signaling cascade by carbon nanoparticles was investigated with regard to its relevance for proliferation. Kinetics and dose-response experiments demonstrated that Akt is specifically activated by nanoparticulate carbon particles in rat alveolar type II epithelial cells as well as in human bronchial epithelial cells. This pathway appeared to be dependent on epidermal growth factor receptor and beta(1)-integrins. The activation of Akt by these receptors is known to be a feature of adhesion-dependent signaling. However, intracellular proteins described in this context (focal adhesion kinase pp125(FAK) and integrin-linked kinase) were not activated, indicating a specific signaling mechanism. Inhibitor studies demonstrate that nanoparticle-induced proliferation is mediated by phosphoinositide 3-kinases and Akt. Moreover, overexpression of mutant Akt, as well as pretreatment with an Akt inhibitor, reduced nanoparticle-specific ERK1/2 phosphorylation, which is decisive for nanoparticle-induced proliferation. With this report, we describe the activation of a pathway by carbon nanoparticles that was so far known to be triggered by ligand receptor binding or on cell adhesion to extracellular matrix proteins.

Analysis of the transcriptional regulation and molecular function of the aryl hydrocarbon receptor repressor in human cell lines.

The aryl hydrocarbon receptor repressor (AhRR) is a member of the aryl hydrocarbon receptor (AhR) signaling cascade, which mediates dioxin toxicity and is involved in regulation of cell growth and differentiation. The AhRR was described as a feedback modulator, which counteracts AhR-dependent gene expression. We investigated the molecular mechanisms of transcriptional regulation of the human AhRR by cloning its regulatory DNA region located in intron I of the AhRR. By means of reporter gene analyses and generation of deletion variants, we identified a functional, 3-methylcholanthrene-sensitive xenobiotic response element (XRE) site. Chromatin immunoprecipitation analyses revealed that the AhRR binds to this XRE, displaying an autoregulatory loop of AhRR expression. In addition we show that an adjacent GC-box is of functional relevance for AhRR transcription, since blocking of this GC-box resulted in a decrease of constitutive and inducible AhRR gene activity. The differences in constitutive AhRR mRNA level observed in HepG2, primary fibroblast, and HeLa cells are directly correlated with CYP1A1 inducibility. We show that the nonresponsiveness of high AhRR-expressing cells toward AhR-agonists is associated with a constitutive binding of the AhRR to XRE sites of CYP1A1. Treatment with the histone deacetylase inhibitor sodium butyrate restored the responsiveness of CYP1A1 in these cell lines, due to the dissociation of AhRR from the XREs. Furthermore, transient AhRR mRNA silencing in untreated HeLa cells was accompanied by an increase of basal CYP1A1 expression, pointing to a constitutive role of the AhRR in regulation of CYP1A1. The functional relevance of the AhRR in high AhRR-expressing primary fibroblasts is discussed.

Ultrafine carbon particles induce apoptosis and proliferation in rat lung epithelial cells via specific signaling pathways both using EGF-R.

Apoptosis and proliferation are important causes of adverse health effects induced by inhaled ultrafine particles. The molecular mechanisms of particle cell interactions mediating these end points are therefore a major topic of current particle toxicology and molecular preventive medicine. Initial studies revealed that ultrafine particles induce apoptosis and proliferation in parallel in rat lung epithelial cells, dependent on time and dosage. With these end points, two antagonistic reactions seem to be induced by the same extracellular stimulus. It was therefore investigated whether proliferation is induced directly by the particles or as a compensation of particle-caused cell death. Experimental conditions excluding compensatory proliferation demonstrated that both end points are induced independently by specific signaling pathways. Events eliciting signaling cascades leading to apoptosis and proliferation were studied with specific inhibitors of membrane receptors. Epidermal growth factor receptor (EGF-R) kinase activity was identified as essential for apoptosis as well as for proliferation. As ultrafine particle-induced proliferation alone was dependent on the activation of beta1-integrins, these membrane receptors are suggested to mediate the specificity of EGF-R signaling concerning the decision as to whether apoptosis or proliferation is triggered. Accordingly, MAP kinase signaling downstream of EGF-R showed comparable specificity with regard to receptor-dependent induction of apoptosis and proliferation. As key mediators of signaling cascades, the activation of extracellular signal-regulated kinases 1 and 2 proved to be specific for proliferation in a beta1-integrin-dependent manner, whereas phosphorylation of c-Jun NH2-terminal kinases 1 and 2 was correlated with the induction of apoptosis.