Gold nanowires-based sensor for quantification of H2O2 released by human airway epithelial cells.

Hydrogen peroxide (H2O2) is a biomarker relevant for oxidative stress monitoring. Most chronic airway diseases are characterized by increased oxidative stress. To date, the main methods for the detection of this analyte are expensive and time-consuming laboratory techniques such as fluorometric and colorimetric assays. There is a growing interest in the development of electrochemical sensors for H2O2 detection due to their low cost, ease of use, sensitivity and rapid response. In this work, an electrochemical sensor based on gold nanowire arrays has been developed. Thanks to the catalytic activity of gold against hydrogen peroxide reduction and the high surface area of nanowires, this sensor allows the quantification of this analyte in a fast, efficient and selective way. The sensor was obtained by template electrodeposition and consists of gold nanowires about 5 µm high and with an average diameter of about 200 nm. The high active surface area of this electrode, about 7 times larger than a planar gold electrode, ensured a high sensitivity of the sensor (0.98 µA µM-1cm-2). The sensor allows the quantification of hydrogen peroxide in the range from 10 µM to 10 mM with a limit of detection of 3.2 µM. The sensor has excellent properties in terms of reproducibility, repeatability and selectivity. The sensor was validated by quantifying the hydrogen peroxide released by human airways A549 cells exposed or not to the pro-oxidant compound rotenone. The obtained results were validated by comparing them with those obtained by flow cytometry after staining the cells with the fluorescent superoxide-sensitive Mitosox Red probe giving a very good concordance.

In-silico guided chemical exploration of KDM4A fragments hits.

BACKGROUND: Lysine demethylase enzymes (KDMs) are an emerging class of therapeutic targets, that catalyse the removal of methyl marks from histone lysine residues regulating chromatin structure and gene expression. KDM4A isoform plays an important role in the epigenetic dysregulation in various cancers and is linked to aggressive disease and poor clinical outcomes. Despite several efforts, the KDM4 family lacks successful specific molecular inhibitors. RESULTS: Herein, starting from a structure-based fragments virtual screening campaign we developed a synergic framework as a guide to rationally design efficient KDM4A inhibitors. Commercial libraries were used to create a fragments collection and perform a virtual screening campaign combining docking and pharmacophore approaches. The most promising compounds were tested in-vitro by a Homogeneous Time-Resolved Fluorescence-based assay developed for identifying selective substrate-competitive inhibitors by means of inhibition of H3K9me3 peptide demethylation. 2-(methylcarbamoyl)isonicotinic acid was identified as a preliminary active fragment, displaying inhibition of KDM4A enzymatic activity. Its chemical exploration was deeply investigated by computational and experimental approaches which allowed a rational fragment growing process. The in-silico studies guided the development of derivatives designed as expansion of the primary fragment hit and provided further knowledge on the structure-activity relationship. CONCLUSIONS: Our study describes useful insights into key ligand-KDM4A protein interaction and provides structural features for the development of successful selective KDM4A inhibitors.

Caspase-8 activation by cigarette smoke induces pro-inflammatory cell death of human macrophages exposed to lipopolysaccharide.

Cigarette smoking impairs the lung innate immune response making smokers more susceptible to infections and severe symptoms. Dysregulation of cell death is emerging as a key player in chronic inflammatory conditions. We have recently reported that short exposure of human monocyte-derived macrophages (hMDMs) to cigarette smoke extract (CSE) altered the TLR4-dependent response to lipopolysaccharide (LPS). CSE caused inhibition of the MyD88-dependent inflammatory response and activation of TRIF/caspase-8/caspase-1 pathway leading to Gasdermin D (GSDMD) cleavage and increased cell permeability. Herein, we tested the hypothesis that activation of caspase-8 by CSE increased pro-inflammatory cell death of LPS-stimulated macrophages. To this purpose, we measured apoptotic and pyroptotic markers as well as the expression/release of pro-inflammatory mediators in hMDMs exposed to LPS and CSE, alone or in combination, for 6 and 24 h. We show that LPS/CSE-treated hMDMs, but not cells treated with CSE or LPS alone, underwent lytic cell death (LDH release) and displayed apoptotic features (activation of caspase-8 and -3/7, nuclear condensation, and mitochondrial membrane depolarization). Moreover, the negative regulator of caspase-8, coded by CFLAR gene, was downregulated by CSE. Activation of caspase-3 led to Gasdermin E (GSDME) cleavage. Notably, lytic cell death caused the release of the damage-associated molecular patterns (DAMPs) heat shock protein-60 (HSP60) and S100A8/A9. This was accompanied by an impaired inflammatory response resulting in inhibited and delayed release of IL6 and TNF. Of note, increased cleaved caspase-3, higher levels of GSDME and altered expression of cell death-associated genes were found in alveolar macrophages of smoker subjects compared to non-smoking controls. Overall, our findings show that CSE sensitizes human macrophages to cell death by promoting pyroptotic and apoptotic pathways upon encountering LPS. We propose that while the delayed inflammatory response may result in ineffective defenses against infections, the observed cell death associated with DAMP release may contribute to establish chronic inflammation. CS exposure sensitizes human macrophages to pro-inflammatory cell death. Upon exposure to LPS, CS inhibits the TLR4/MyD88 inflammatory response, downregulating the pro-inflammatory genes TNF and IL6 and the anti-apoptotic gene CFLAR, known to counteract caspase-8 activity. CS enhances caspase-8 activation through TLR4/TRIF, with a partial involvement of RIPK1, resulting on the activation of caspase-1/GSDMD axis leading to increased cell permeability and DAMP release through gasdermin pores [19]. At later timepoints caspase-3 becomes strongly activated by caspase-8 triggering apoptotic events which are associated with mitochondrial membrane depolarization, gasdermin E cleavage and secondary necrosis with consequent massive DAMP release.

Electrochemical Quantification of H2O2 Released by Airway Cells Growing in Different Culture Media.

Quantification of oxidative stress is a challenging task that can help in monitoring chronic inflammatory respiratory airway diseases. Different studies can be found in the literature regarding the development of electrochemical sensors for H2O2 in cell culture medium to quantify oxidative stress. However, there are very limited data regarding the impact of the cell culture medium on the electrochemical quantification of H2O2. In this work, we studied the effect of different media (RPMI, MEM, DMEM, Ham's F12 and BEGM/DMEM) on the electrochemical quantification of H2O2. The used electrode is based on reduced graphene oxide (rGO) and gold nanoparticles (AuNPs) and was obtained by co-electrodeposition. To reduce the electrode fouling by the medium, the effect of dilution was investigated using diluted (50% v/v in PBS) and undiluted media. With the same aim, two electrochemical techniques were employed, chronoamperometry (CH) and linear scan voltammetry (LSV). The influence of different interfering species and the effect of the operating temperature of 37 °C were also studied in order to simulate the operation of the sensor in the culture plate. The LSV technique made the sensor adaptable to undiluted media because the test time is short, compared with the CH technique, reducing the electrode fouling. The long-term stability of the sensors was also evaluated by testing different storage conditions. By storing the electrode at 4 °C, the sensor performance was not reduced for up to 21 days. The sensors were validated measuring H2O2 released by two different human bronchial epithelial cell lines (A549, 16HBE) and human primary bronchial epithelial cells (PBEC) grown in RPMI, MEM and BEGM/DMEM media. To confirm the results obtained with the sensor, the release of reactive oxygen species was also evaluated with a standard flow cytometry technique. The results obtained with the two techniques were very similar. Thus, the LSV technique permits using the proposed sensor for an effective oxidative stress quantification in different culture media and without dilution.

Cigarette Smoke Impairs Airway Epithelial Wound Repair: Role of Modulation of Epithelial-Mesenchymal Transition Processes and Notch-1 Signaling.

Cigarette smoke (CS) induces oxidative stress and chronic inflammation in airway epithelium. It is a major risk factor for respiratory diseases, characterized by epithelial injury. The impact of CS on airway epithelial repair, which involves epithelial-mesenchymal transition (EMT) and the Notch-1 pathway, is incompletely understood. In this study, we used primary bronchial epithelial cells (PBECs) to evaluate the effect of CS on epithelial repair and these mechanisms. The effect of CS and/or TGF-beta1 on wound repair, various EMT and Notch-1 pathway markers and epithelial cell markers (TP63, SCGB1A) was assessed in PBECs cultured submerged, at the air-liquid interface (ALI) alone and in co-culture with fibroblasts. TGF-beta1 increased epithelial wound repair, activated EMT (shown by decrease in E-cadherin, and increases in vimentin, SNAIL1/SNAIL2/ZEB1), and increased Notch-1 pathway markers (NOTCH1/JAGGED1/HES1), MMP9, TP63, SCGB1A1. In contrast, CS decreased wound repair and vimentin, NOTCH1/JAGGED1/HES1, MMP9, TP63, SCGB1A1, whereas it activated the initial steps of the EMT (decrease in E-cadherin and increases in SNAIL1/SNAIL2/ZEB1). Using combined exposures, we observed that CS counteracted the effects of TGF-beta1. Furthermore, Notch signaling inhibition decreased wound repair. These data suggest that CS inhibits the physiological epithelial wound repair by interfering with the normal EMT process and the Notch-1 pathway.

Cigarette smoke promotes inflammasome-independent activation of caspase-1 and -4 leading to gasdermin D cleavage in human macrophages.

Mechanisms and consequences of gasdermin D (GSDMD) activation in cigarette smoke (CS)-associated inflammation and lung disease are unknown. GSDMD is a downstream effector of caspase-1, -8, and -4. Upon cleavage, GSDMD generates pores into cell membranes. Different degrees of GSDMD activation are associated with a range of physiological outputs ranging from cell hyperactivation to pyroptosis. We have previously reported that in human monocyte-derived macrophages CS extract (CSE) inhibits the NLRP3 inflammasome and shifts the response to lipopolysaccharide (LPS) towards the TLR4-TRIF axis leading to activation of caspase-8, which, in turn, activates caspase-1. In the present work, we investigated whether other ASC-dependent inflammasomes could be involved in caspase activation by CSE and whether caspase activation led to GSDMD cleavage and other downstream effects. Presented results demonstrate that CSE promoted ASC-independent activation of caspase-1 leading to GSDMD cleavage and increased cell permeability, in the absence of cell death. GSDMD cleavage was strongly enhanced upon stimulation with LPS+CSE, suggesting a synergistic effect between the two stimuli. Noteworthy, CSE promoted LPS internalization leading to caspase-4 activation, thus contributing to increased GSDMD cleavage. Caspase-dependent GSDMD cleavage was associated with mitochondrial superoxide generation. Increased cleaved GSDMD was found in lung macrophages of smokers compared to ex-smokers and non-smoking controls. Our findings revealed that ASC-independent activation of caspase-1, -4, and -8 and GSDMD cleavage upon exposure to CS may contribute to macrophage dysfunction and feed the chronic inflammation observed in the smokers' lung.

Cellular and Molecular Signatures of Oxidative Stress in Bronchial Epithelial Cell Models Injured by Cigarette Smoke Extract.

Exposure of the airways epithelium to environmental insults, including cigarette smoke, results in increased oxidative stress due to unbalance between oxidants and antioxidants in favor of oxidants. Oxidative stress is a feature of inflammation and promotes the progression of chronic lung diseases, including Chronic Obstructive Pulmonary Disease (COPD). Increased oxidative stress leads to exhaustion of antioxidant defenses, alterations in autophagy/mitophagy and cell survival regulatory mechanisms, thus promoting cell senescence. All these events are amplified by the increase of inflammation driven by oxidative stress. Several models of bronchial epithelial cells are used to study the molecular mechanisms and the cellular functions altered by cigarette smoke extract (CSE) exposure, and to test the efficacy of molecules with antioxidant properties. This review offers a comprehensive synthesis of human in-vitro and ex-vivo studies published from 2011 to 2021 describing the molecular and cellular mechanisms evoked by CSE exposure in bronchial epithelial cells, the most used experimental models and the mechanisms of action of cellular antioxidants systems as well as natural and synthetic antioxidant compounds.

Cigarette smoke upregulates Notch-1 signaling pathway and promotes lung adenocarcinoma progression.

Notch-1 pathway plays an important role in lung carcinoma, stem cell regulation, cellular communication, growth and differentiation. Cigarette smoke is involved in the regulation of Notch signaling. However, current data regarding the impact of cigarette smoke on the Notch pathway in lung cancer progression are limited. The present study aimed to explore whether cigarette smoke exposure altered Notch-1 pathway in ex-vivo (surgical samples of lung parenchyma from non-smoker and smoker patients with lung adenocarcinoma) and in vitro (adenocarcinoma A549 cell line) approaches. The expression of Notch-1, Jagged-1 and CD133 in surgical samples was evaluated by immunohistochemistry. A549 were exposed to cigarette smoke extracts (2.5 % and 5 % CSE for 6, 24 and 48 h) and the expression of Notch-1, Jagged-1 and Hes-1 was evaluated by Real-Time PCR and Western Blot (nuclear fractions). Expression and localization of Notch-1, Hes-1, CD133 and ABCG2 were assessed by immunofluorescence. The expression of survivin and Ki-67 was assessed by flow cytometry following CSE exposure and inhibition of Notch-1 signaling. Smokers lung parenchyma exhibited higher expression of Notch-1. CSE exposure increased Notch-1 and Hes-1 gene and nuclear protein expression in A549. Immunofluorescence confirmed higher expression of nuclear Hes-1 in CSE-stimulated A549 cells. CSE increased both survivin and Ki-67 expression and this effect was reverted by inhibition of the Notch-1 pathway. In conclusion, these data show that cigarette smoke may promote adenocarcinoma progression by activating the Notch-1 pathway thus supporting its role as hallmark of lung cancer progression and as a new target for lung cancer treatment.

Cigarette smoke extract reduces FOXO3a promoting tumor progression and cell migration in lung cancer.

Lung cancer is the leading cause of cancer death worldwide, and the carcinogens in tobacco smoke play a role in its progression and metastasis. The related molecular events are largely unknown. FOXO3a is a transcription factor considered a tumor suppressor. Its inhibition leads to cell transformation, tumor progression and metastasis. The aim of this study was to investigate, in different types of lung cancer cell lines (A549, COLO 699 N, SK-MES-1), the effects of cigarette smoke on mitochondrial status and cell metabolism and on key pathways involved in tumor progression and cell migration, looking at the role of FOXO3a in these mechanisms. The different lung cancer cells were exposed to cigarette smoke extract (CSE) and TGF-ß1. Reactive oxygen species (ROS), mitochondrial superoxide, intracellular ATP, extracellular lactate, FOXO3a, p21, survivin, epithelial-to-mesenchymal transition (EMT) markers (E-cadherin, SNAIL1), MMP-9 and cellular migration were assessed by flow-cytometry, fluorimetry, western blot analysis, Real-Time PCR and scratch test. Our results showed that exposure to CSE: (i) increased ROS, mitochondrial superoxide, lactate release while reducing intracellular ATP; (ii) decreased FOXO3a and increased survivin and p21 in the cytoplasm; (iii) decreased E-cadherin, increased SNAIL1 and MMP-9 and promoted cell migration like TGF-ß1 did. These effects could be partly explained by downregulation of FOXO3a, as demonstrated by silencing experiments. These data suggest that cigarette smoke induces oxidative stress and mitochondrial damage leading to metabolic reprogramming associated with increased glycolytic flux. This is accompanied with a downregulation of FOXO3a contributing to EMT processes and cell migration therefore promoting tumor progression.

miR-1207-5p Can Contribute to Dysregulation of Inflammatory Response in COVID-19 via Targeting SARS-CoV-2 RNA.

The present study focuses on the role of human miRNAs in SARS-CoV-2 infection. An extensive analysis of human miRNA binding sites on the viral genome led to the identification of miR-1207-5p as potential regulator of the viral Spike protein. It is known that exogenous RNA can compete for miRNA targets of endogenous mRNAs leading to their overexpression. Our results suggest that SARS-CoV-2 virus can act as an exogenous competing RNA, facilitating the over-expression of its endogenous targets. Transcriptomic analysis of human alveolar and bronchial epithelial cells confirmed that the CSF1 gene, a known target of miR-1207-5p, is over-expressed following SARS-CoV-2 infection. CSF1 enhances macrophage recruitment and activation and its overexpression may contribute to the acute inflammatory response observed in severe COVID-19. In summary, our results indicate that dysregulation of miR-1207-5p-target genes during SARS-CoV-2 infection may contribute to uncontrolled inflammation in most severe COVID-19 cases.

Cigarette smoke inhibits the NLRP3 inflammasome and leads to caspase-1 activation via the TLR4-TRIF-caspase-8 axis in human macrophages.

The NLRP3 inflammasome is formed by the sensor NLRP3, the adaptor ASC, and pro-caspase-1. Assembly and activation of the inflammasome trigger caspase-1-dependent cleavage of pro-IL-1ß and pro-IL-18 into their secreted forms. Cigarette smoke is a risk factor for chronic inflammatory diseases and is associated with macrophage dysfunction. The impact of cigarette smoke on NLRP3-dependent responses in macrophages is largely unknown. Herein, we investigated the effects of cigarette smoke extract (CSE) on the NLRP3 inflammasome in human monocyte-derived macrophages (MDMs) and THP-1 cells stimulated with lipopolysaccharide (LPS) and LPS plus the NLRP3 inflammasome activator ATP. We found that CSE inhibited the release of IL-1ß and IL-18 as well as the expression of NLRP3 acting mainly at the transcriptional level. Interestingly, we found that CSE increased the caspase-1 activity via an NLRP3-independent and TLR4-TRIF-caspase-8-dependent pathway. Activation of caspase-1 by CSE led to a reduction of the basal glycolytic flux and impaired glycolytic burst in response to LPS. Overall, our findings unveil novel pathways leading to immune-metabolic alterations in human macrophages exposed to cigarette smoke. These mechanisms may contribute to macrophage dysfunction and increased risk of infection in smokers.

Electrophilic derivatives of omega-3 fatty acids counteract lung cancer cell growth.

PURPOSE: 17-oxo-DHA is an electrophilic keto-derivative of the omega-3 fatty acid docosahexaenoic acid (DHA) endogenously generated by cyclooxygenase-2 and a cellular dehydrogenase. 17-oxo-DHA displays anti-inflammatory and cytoprotective actions. DHA, alone or in combination with standard chemotherapy, displays antitumor activity. However, the effects of electrophilic keto-derivatives of DHA on cancer growth have never been evaluated. We investigated whether 17-oxo-DHA, alone or in combination with gemcitabine, displayed antitumor effects. Furthermore, we evaluated whether the enzyme 15-prostaglandin dehydrogenase (15-PGDH) was required for transducing the antitumor effects of DHA. METHODS: A panel of five histologically different human non-small cell lung cancer (NSCLC) cell lines was used. Cells were treated with 17-oxo-DHA and gemcitabine, alone or in combination, and apoptosis, proliferation, Fas and FasL expression (mRNA and protein) and active caspase-3/7 and -8 were assessed. Furthermore, an inhibitor of 15-PGDH was used to test the involvement of this enzyme in mediating the antitumor effects of DHA. RESULTS: 17-oxo-DHA (50 µM, 72 h) significantly reduced proliferation, increased cell apoptosis, Fas and FasL expression as well as active caspase-8 and -3/7. When 17-oxo-DHA was given in combination with gemcitabine, stronger effects were observed compared to gemcitabine alone. The enzyme 15-PGDH was required for DHA to promote its full anti-apoptotic effect suggesting that enzymatically generated keto-derivatives of DHA mediate its antitumor actions. CONCLUSIONS: Data herein provided, demonstrate that 17-oxo-DHA displays antitumor effects in NSCLC cell lines. Of note, the combination of 17-oxo-DHA plus gemcitabine, resulted in stronger anticancer effects compared to gemcitabine alone.

SIRT1/FoxO3 axis alteration leads to aberrant immune responses in bronchial epithelial cells.

Inflammation and ageing are intertwined in chronic obstructive pulmonary disease (COPD). The histone deacetylase SIRT1 and the related activation of FoxO3 protect from ageing and regulate inflammation. The role of SIRT1/FoxO3 in COPD is largely unknown. This study evaluated whether cigarette smoke, by modulating the SIRT1/FoxO3 axis, affects airway epithelial pro-inflammatory responses. Human bronchial epithelial cells (16HBE) and primary bronchial epithelial cells (PBECs) from COPD patients and controls were treated with/without cigarette smoke extract (CSE), Sirtinol or FoxO3 siRNA. SIRT1, FoxO3 and NF-κB nuclear accumulation, SIRT1 deacetylase activity, IL-8 and CCL20 expression/release and the release of 12 cytokines, neutrophil and lymphocyte chemotaxis were assessed. In PBECs, the constitutive FoxO3 expression was lower in patients with COPD than in controls. Furthermore, CSE reduced FoxO3 expression only in PBECs from controls. In 16HBE, CSE decreased SIRT1 activity and nuclear expression, enhanced NF-κB binding to the IL-8 gene promoter thus increasing IL-8 expression, decreased CCL20 expression, increased the neutrophil chemotaxis and decreased lymphocyte chemotaxis. Similarly, SIRT1 inhibition reduced FoxO3 expression and increased nuclear NF-κB. FoxO3 siRNA treatment increased IL-8 and decreased CCL20 expression in 16HBE. In conclusion, CSE impairs the function of SIRT1/FoxO3 axis in bronchial epithelium, dysregulating NF-κB activity and inducing pro-inflammatory responses.

17-oxo-DHA displays additive anti-inflammatory effects with fluticasone propionate and inhibits the NLRP3 inflammasome.

Chronic obstructive pulmonary disease (COPD) is characterized by reduced lung function associated with increased local and systemic inflammatory markers, such as TNFα and IL-1ß. Glucocorticoids are used to treat this chronic disease, however their efficacy is low and new drugs are very much required. 17-oxo-DHA is a cyclooxygenase-2-dependent, electrophilic, α,ß-unsaturated keto-derivative of docosahexaenoic acid with anti-inflammatory properties. We evaluated the action of 17-oxo-DHA alone or in combination with the steroid fluticasone propionate (FP) in peripheral blood mononuclear cells (PBMCs) from COPD patients and healthy individuals exposed to lipopolysaccharide. We show that PBMCs from COPD patients released higher levels of TNFα and IL-1ß compared to controls. 17-oxo-DHA displayed strong anti-inflammatory effects. The addition of 17-oxo-DHA in combination with FP showed enhanced anti-inflammatory effects through the modulation of transcriptional and post-transcriptional mechanisms. 17-oxo-DHA, but not FP, was able to suppress the release of mature IL-1ß through inhibition of the NLRP3 inflammasome. Furthermore, 17-oxo-DHA inhibited inflammasome-dependent degradation of the glucocorticoid receptor (GR). Our findings suggest that 17-oxo-DHA in combination with FP or other steroids might achieve higher therapeutic efficacy than steroids alone. Combined treatment might be particularly relevant in those conditions where increased inflammasome activation may lead to GR degradation and steroid-unresponsive inflammation.

Endogenous Generation and Signaling Actions of Omega-3 Fatty Acid Electrophilic Derivatives.

Dietary omega-3 polyunsaturated fatty acids (PUFAs) are beneficial for a number of conditions ranging from cardiovascular disease to chronic airways disorders, neurodegeneration, and cancer. Growing evidence has shown that bioactive oxygenated derivatives are responsible for transducing these salutary effects. Electrophilic oxo-derivatives of omega-3 PUFAs represent a class of oxidized derivatives that can be generated via enzymatic and nonenzymatic pathways. Inflammation and oxidative stress favor the formation of these signaling species to promote the resolution of inflammation within a fine autoregulatory loop. Endogenous generation of electrophilic oxo-derivatives of omega-3 PUFAs has been observed in in vitro and ex vivo human models and dietary supplementation of omega-3 PUFAs has been reported to increase their formation. Due to the presence of an α,ß-unsaturated ketone moiety, these compounds covalently and reversibly react with nucleophilic residues on target proteins triggering the activation of cytoprotective pathways, including the Nrf2 antioxidant response, the heat shock response, and the peroxisome proliferator activated receptor γ (PPARγ) and suppressing the NF-κB proinflammatory pathway. The endogenous nature of electrophilic oxo-derivatives of omega-3 PUFAs combined with their ability to simultaneously activate multiple cytoprotective pathways has made these compounds attractive for the development of new therapies for the treatment of chronic disorders and acute events characterized by inflammation and oxidative stress.

Dual anti-oxidant and anti-inflammatory actions of the electrophilic cyclooxygenase-2-derived 17-oxo-DHA in lipopolysaccharide- and cigarette smoke-induced inflammation.

BACKGROUND: 17-Oxo-DHA is an endogenous electrophilic derivative of the omega-3 fatty acid docosahexaenoic acid (DHA) which is generated in activated macrophages by the action of cyclooxygenase-2. METHODS: The ability of 17-oxo-DHA to control inflammation and oxidative stress was tested in human macrophages (THP-1) and bronchial epithelial cell line (16HBE) stimulated with cigarette smoke extract (CSE) and lipopolysaccharide (LPS). All data were further confirmed using primary bronchial epithelial cells, alveolar macrophages and peripheral blood mononuclear cells. RESULTS: 17-Oxo-DHA was a strong inducer of the anti-oxidant response promoting Nrf2 nuclear accumulation, leading to the expression of heme oxygenase 1 and more than doubling glutathione levels. This resulted in suppression of CSE-induced ROS generation in macrophages. In macrophages, 17-oxo-DHA potently suppressed TNFα release in response to LPS, CSE and IL-1ß acting at transcriptional level via a mechanism independent of Nrf2. Externally supplemented 17-oxo-DHA displayed the same effects in the presence of the Cox-inhibitor indomethacin. The non-electrophilic 17-oxo-DHA precursor DHA did not show any biological actions, indicating that the electrophilic moiety was required for this compound to become bioactive. CONCLUSIONS: 17-Oxo-DHA promotes cytoprotective actions both in immune and structural cells. In immune cells, 17-oxo-DHA is effective in contrasting CSE- and LPS-induced oxidative damage and inflammation acting via multiple independent pathways. GENERAL SIGNIFICANCE: Herein we provide insights on how the novel endogenous electrophilic DHA-derivative 17-oxo-DHA promotes anti-oxidant and anti-inflammatory actions. Data herein reported indicate that 17-oxo-DHA is an attractive lead compound for the development of new treatments for cigarette smoke-related airway inflammatory disorders.

Comparative cytoprotective effects of carbocysteine and fluticasone propionate in cigarette smoke extract-stimulated bronchial epithelial cells.

Cigarette smoke extracts (CSE) induce oxidative stress, an important feature in chronic obstructive pulmonary disease (COPD), and oxidative stress contributes to the poor clinical efficacy of corticosteroids in COPD patients. Carbocysteine, an antioxidant and mucolytic agent, is effective in reducing the severity and the rate of exacerbations in COPD patients. The effects of carbocysteine on CSE-induced oxidative stress in bronchial epithelial cells as well as the comparison of these antioxidant effects of carbocysteine with those of fluticasone propionate are unknown. The present study was aimed to assess the effects of carbocysteine (10(-4) M) in cell survival and intracellular reactive oxygen species (ROS) production (by flow cytometry) as well as total glutathione (GSH), heme oxygenase-1 (HO-1), nuclear-related factor 2 (Nrf2) expression and histone deacetylase 2 (HDAC-2) expression/activation in CSE-stimulated bronchial epithelial cells (16-HBE) and to compare these effects with those of fluticasone propionate (10(-8) M). CSE, carbocysteine or fluticasone propionate did not induce cell necrosis (propidium positive cells) or cell apoptosis (annexin V-positive/propidium-negative cells) in 16-HBE. CSE increased ROS production, nuclear Nrf2 and HO-1 in 16-HBE. Fluticasone propionate did not modify intracellular ROS production, GSH and HDCA-2 but reduced Nrf2 and HO-1 in CSE-stimulated 16-HBE. Carbocysteine reduced ROS production and increased GSH, HO-1, Nrf2 and HDAC-2 nuclear expression/activity in CSE-stimulated cells and was more effective than fluticasone propionate in modulating the CSE-mediated effects. In conclusion, the present study provides compelling evidences that the use of carbocysteine may be considered a promising strategy in diseases associated with corticosteroid resistance.

TLR4 up-regulation and reduced Foxp3 expression in mechanically ventilated smokers with obstructive chronic bronchitis.

BACKGROUND: Chronic bronchitis (CB) is a risk factor in chronic obstructive pulmonary disease (COPD) for accelerated lung function decline and increased mortality. The lung and systemic inflammatory and immunological profile of COPD patients with CB which acutely experience respiratory failure upon a disease exacerbation is unknown. METHODS: In this study, we explored the expression of Foxp3 by western blot analysis, TLR4 by immunocytochemistry and the concentrations of IP-10 and IL-8 by ELISA in the mini-bronchoalveolar lavages (mini-BAL) and in the peripheral blood of patients with respiratory failure requiring intubation and mechanical ventilation. The recruited subjects were separated into three different groups: smokers with CB and COPD (COPD, n = 18), smokers with CB but without COPD (S, n = 8) and patients without CB and without COPD (C, n = 10). RESULTS: In mini-BAL of COPD group, Foxp3 and IP-10 were significantly reduced while TLR4 was significantly increased in comparison to C. TLR4 was also increased in mini-BAL of S. In COPD peripheral blood, Foxp3 was reduced in comparison to C but no significant differences were observed for TLR4 and for IP-10. No significant differences were observed for IL-8 concentrations in the mini-BAL and in the blood of the recruited patients. The mini-BAL TLR4 expression correlated with the Clinical Infective Pulmonary Score. CONCLUSIONS: In exacerbated COPD patients with respiratory failure, lung and systemic reduced immune regulatory events (low Foxp3 expression) and lung increased innate immunity responses (high TLR4 expression) occur. These events may contribute to the increased inflammatory events leading to respiratory failure.

Metabolome, transcriptome and metabolic flux analysis of arabinose fermentation by engineered Saccharomyces cerevisiae.

One of the challenges in strain improvement by evolutionary engineering is to subsequently determine the molecular basis of the improved properties that were enriched from the natural genetic variation during the selective conditions. This study focuses on Saccharomyces cerevisiae IMS0002 which, after metabolic and evolutionary engineering, ferments the pentose sugar arabinose. Glucose- and arabinose-limited anaerobic chemostat cultures of IMS0002 and its non-evolved ancestor were subjected to transcriptome analysis, intracellular metabolite measurements and metabolic flux analysis. Increased expression of the GAL-regulon and deletion of GAL2 in IMS0002 confirmed that the galactose transporter is essential for growth on arabinose. Elevated intracellular concentrations of pentose-phosphate-pathway intermediates and upregulation of TKL2 and YGR043c (encoding transketolase and transaldolase isoenzymes) suggested an involvement of these genes in flux-controlling reactions in arabinose fermentation. Indeed, deletion of these genes in IMS0002 caused a 21% reduction of the maximum specific growth rate on arabinose.

Cyclooxygenase-2 generates anti-inflammatory mediators from omega-3 fatty acids.

Electrophilic fatty acids are generated during inflammation by non-enzymatic reactions and can modulate inflammatory responses. We used a new mass spectrometry-based electrophile capture strategy to reveal the formation of electrophilic oxo-derivatives (EFOX) from the omega-3 fatty acids docosahexaenoic acid (DHA) and docosapentaenoic acid (DPA). These EFOX were generated by a cyclooxygenase-2 (COX-2)-catalyzed mechanism in activated macrophages. Modulation of COX-2 activity by aspirin increased the rate of EFOX production and their intracellular levels. Owing to their electrophilic nature, EFOX adducted to cysteine and histidine residues of proteins and activated Nrf2-dependent anti-oxidant gene expression. We confirmed the anti-inflammatory nature of DHA- and DPA-derived EFOX by showing that they can act as peroxisome proliferator-activated receptor-gamma (PPAR gamma) agonists and inhibit pro-inflammatory cytokine and nitric oxide production, all within biological concentration ranges. These data support the idea that EFOX are signaling mediators that transduce the beneficial clinical effects of omega-3 fatty acids, COX-2 and aspirin.