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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.

Background Cigarette smoke is considered a risk factor for lung and colorectal cancer. A convincing link between epithelial-to-mesenchymal transition (EMT) with colorectal cancer progression and therapeutic resistance has emerged. Deregulated expression of E-Cadherin and Claudin-1 and increased miR-21 expression and invasiveness represent hallmarks of EMT. The effects of cigarette smoke exposure on EMT in colorectal adenocarcinoma cells are largely unknown. Aim The aim of the study is to evaluate the effect of cigarette smoke extract (CSE) on miR-21, Claudin-1 and E-Cadherin, molecules associated to EMT in colorectal cancer cells. Methods A human colorectal adenocarcinoma cell line (Caco-2) was treated with CSE at different concentration (5% and 10%) and for different time points (3 h and 24 h). Metabolic activity (by MTS assay), cell necrosis/cell apoptosis (evaluating Propidium Iodide/Annexin V expression by flow cytometry), miR-21, Claudin-1 and E-Cadherin gene expression were evaluated by Real time PCR. Cell permeability, actin polymerization and cancer cell migration was assessed by Trans-Epitelial Electrical Resistance (TEER), Phalloidin expression and matrigel system, respectively. Results CSE at all the tested concentrations and at all time points reduced cell necrosis. CSE at 10% increased miR-21 and reduced the metabolic activity, cell necrosis, Claudin-1 and E-cadherin mRNA at 3 h. Cell permeability, actin polymerization and cancer cell migration were all increased upon CSE exposure. Conclusion These results showed that CSE increasing miR-21, Claudin-1 and E-Cadherin and enhancing the aggressiveness of cancer cells, may concur to colorectal cancer progression.

Background Cigarette smoke exposure, increasing Toll-like receptor 4 (TLR4) and reactive oxygen species (ROS), promotes inflammatory responses in airway epithelial cells. Chronic inflammation, microRNA (miRNA), and oxidative stress are associated with cancer development. Aims The present study was aimed to explore whether cigarette smoke exposure, altering miR-21 expression, promoted inflammatory responses and tumorigenesis processes in airway epithelial cells. Methods Airway normal and cancer epithelial cells (16HBE and A549) were exposed to cigarette smoke extracts (CSE) or with/without agomiR-21, and then it was assessed: a) miR-21 expression; b) signal transducer and activator of transcription 3 (STAT3) nuclear protein expression and ERK1/2 activation; c) IL-8 gene expression and protein release. An antagonist of TLR4 (CLI-095) and the antioxidant flavonoid, apigenin, were also included to evaluate miR-21 expression in CSE exposed cells. Results It was demonstrated that: a) A549 cells constitutively expressed higher levels of miR-21 and IL-8; b) CSE increased STAT3 nuclear expression in 16HBE; c) in both cell lines, CSE and agomiR-21 increased: miR-21 expression; ERK1/2 activation and IL-8 gene expression and protein release; d) TLR4 inhibition counteracted the effects of CSE on miR-21 in A549; e) apigenin reduced miR-21 and IL-8 gene expression in both cell lines. Conclusions Data herein provided identified for the first time new mechanisms supporting the crucial role of cigarette smoke-induced miR-21 expression in the amplification of inflammatory responses and in tumorigenesis processes within the airways.

The integrity of the respiratory epithelium is crucial for airway homeostasis. Tobacco smoke exposure and recurrent infections of the airways play a crucial role in the progression and in the decline of the respiratory function in chronic obstructive pulmonary disease (COPD). The aim of this study was to detect differentially expressed proteins in a bronchial epithelial cell line (16-HBE) stimulated with cigarette smoke extract (CSE) and lipopolysaccharide (LPS), a constituent of gram-negative bacteria, alone and/or in combination, by using two-dimensional electrophoresis (2DE) analysis coupled with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Western blot analysis was applied to confirm the expression of significantly modulated proteins. Flow cytometry and immunofluorescence were used to assess F-actin polimerization by phalloidin method. Fourteen proteins, with significant (p < 0.05) changes in intensity, were identified at various experimental points: 6 were up-regulated and 8 were down-regulated. As expected, bioinformatic analysis revealed that most of these proteins are involved in anti-oxidant and immune responses and in cytoskeleton stability. Western blot analysis confirmed that: Proteasome activator complex subunit 2 (PSME2), Peroxiredoxin-6 (PRDX6), Annexin A5 (ANXA5) and Heat shock protein beta-1 (HSPB1) were reduced and Coactosin-like protein (COTL-1) was increased by co-exposure of CSE and LPS. Furthermore, LPS and CSE increased actin polimerization. In conclusion, although further validation studies are needed, our findings suggest that, CSE and LPS could contribute to the progressive deterioration of lung function, altering the expression of proteins involved in metabolic processes and cytoskeleton rearrangement in bronchial epithelial cells.

Histone deacetylase expression/activity may control inflammation, cell senescence, and responses to corticosteroids. Cigarette smoke exposure, increasing oxidative stress, may negatively affect deacetylase expression/activity. The effects of cigarette smoke extracts (CSE), carbocysteine, and beclomethasone dipropionate on chromatin remodeling processes in human bronchial epithelial cells are largely unknown. The present study was aimed to assess the effects of cigarette smoke, carbocysteine, and beclomethasone dipropionate on histone deacetylase 3 (HDAC3) expression/activity, N-CoR (nuclear receptor corepressor) expression, histone acetyltransferases (HAT) (p300/CBP) expression, p-CREB and IL-1 m-RNA expression, neutrophil chemotaxis. Increased p-CREB expression was observed in the bronchial epithelium of smokers. CSE increased p-CREB expression and decreased HDAC3 expression and activity and N-CoR m-RNA and protein expression. At the same time, CSE increased the expression of the HAT, p300/CBP. All these events increased acetylation processes within the cells and were associated to increased IL-1 m-RNA expression and neutrophil chemotaxis. The incubation of CSE exposed cells with carbocysteine and beclomethasone counteracted the effects of cigarette smoke on HDAC3 and N-CoR but not on p300/CBP. The increased deacetylation processes due to carbocysteine and beclomethasone dipropionate incubation is associated to reduced p-CREB, IL-1 m-RNA expression, neutrophil chemotaxis. These findings suggest a new role of combination therapy with carbocysteine and beclomethasone dipropionate in restoring deacetylation processes compromised by cigarette smoke exposure. J. Cell. Physiol. 232: 2851-2859, 2017. © 2016 Wiley Periodicals, Inc.

Notch-1 intervenes in the reparative processes of mucosa by controlling cell proliferation, differentiation and stem cell maintenance. Cigarette smoke alters airway epithelial homeostasis. The present study explored whether: Smokers showed altered Notch-1 expression; and whether in bronchial epithelial cells (16HBE): a) cigarette smoke extracts (CSE) altered the expression of Notch-1, of its ligand Jagged-1 (Jag-1) and the nuclear translocation of Notch-1; b) Notch-1 signaling activation as well as CSE modified Ki67, PCNA, p21, IL-33 expression, cell proliferation and repair processes. Notch-1 expression was assessed in the epithelium from large airway surgical samples from non-smoker and smoker subjects by immunohistochemistry.16HBE were cultured with/without CSE and Jag-1. A Notch-1 inhibitor (DAPT) was used as control. The expression of Notch-1, Jag-1, Ki67, PCNA, p21, IL-33 and cell proliferation (by CFSE) were all assessed by flow cytometry. Notch-1 nuclear expression was evaluated by immunofluorescence and western blot analysis. Repair processes were assessed by wound assay. Smokers had cytoplasmic but not nuclear Notch-1 expression. Although CSE increased Notch-1 expression, it counteracted Notch-1 signaling activation since it reduced Jag-1 expression and Notch-1 nuclear translocation. Notch-1 signaling activation by Jag-1 increased Ki67, PCNA and repair processes but reduced intracellular IL-33 and p21 expression without affecting cell proliferation. DAPT counteracted the effects of Notch-1 activation on PCNA and IL-33. CSE increased Ki67, PCNA, p21 and IL-33 expression but reduced cell proliferation and repair processes. In conclusion, cigarette smoke exposure, limiting Notch-1 signaling activation and hindering repair processes, amplifies injury processes in bronchial epithelial cells.

Background Cigarette smoke may accelerate cellular senescence by increasing oxidative stress. Altered proliferation and altered expression of anti-aging factors, including SIRT1 and FoxO3, characterise cellular senescence. The effects of carbocysteine on the SIRT1/FoxO3 axis and on downstream molecular mechanisms in human bronchial epithelial cells exposed to cigarette smoke are largely unknown. Aims Aim of this study was to explore whether carbocysteine modulated SIRT1/FoxO3 axis, and downstream molecular mechanisms associated to cellular senescence, in a bronchial epithelial cell line (16-HBE) exposed to cigarette smoke. Methods 16HBE cells were stimulated with/without cigarette smoke extracts (CSE) and carbocysteine. Flow cytometry and clonogenic assay were used to assess cell proliferation; western blot analysis was used for assessing nuclear expression of SIRT1 and FoxO3. The nuclear co-localization of SIRT1 and FoxO3 was assessed by fluorescence microscopy. Beta galactosidase (a senescence marker) and SIRT1 activity were assessed by specific staining and colorimetric assays, respectively. ChiP Assay and flow cytometry were used for assessing survivin gene regulation and protein expression, respectively. Results CSE decreased cell proliferation, the nuclear expression of SIRT1 and FoxO3 and increased beta galactosidase staining. CSE, reduced SIRT1 activity and FoxO3 localization on survivin promoter thus increasing survivin expression. In CSE stimulated bronchial epithelial cells carbocysteine reverted these phenomena by increasing cell proliferation, and SIRT1 and FoxO3 nuclear expression, and by reducing beta galactosidase staining and survivin expression. Conclusions The study shows for the first time that carbocysteine may revert some senescence processes induced by oxidative stress due to cigarette smoke exposure.

The NLRP3 inflammasome is a multi-protein complex that initiates innate immunity responses when exposed to a wide range of stimuli, including pathogen-associated molecular patterns (PAMPs) and danger-associated molecular patterns (DAMPs). Inflammasome activation leads to the release of the pro-inflammatory cytokines interleukin (IL)-1β and IL-18 and to pyroptotic cell death. Over-activation of NLRP3 inflammasome has been associated with several chronic inflammatory diseases. A deep knowledge of NLRP3 inflammasome biology is required to better exploit its potential as therapeutic target and for the development of new selective drugs. To this purpose, in the past few years, several tools have been developed for the biological characterization of the multimeric inflammasome complex, the identification of the upstream signaling cascade leading to inflammasome activation, and the downstream effects triggered by NLRP3 activation. In this review, we will report cellular models and cellular, biochemical, and biophysical assays that are currently available for studying inflammasome biology. A special focus will be on those models/assays that have been used to identify NLRP3 inhibitors and their mechanism of action.

BACKGROUND: Allergic rhinitis is characterized by a remodeling of nasal epithelium. Since the Notch and TGF-β signaling pathways are known to be involved in cell differentiation and remodeling processes and leptin adipokine has already been identified as a marker for homeostasis in human bronchial and nasal epithelial cells of asthmatics, roles played by these pathways have been investigated for chronic allergic rhinitis. METHODS: The leptin/leptin receptor expression has been investigated in a study with 40 biopsies from allergic (AR, n = 18) and non-allergic (C, n = 22) inferior turbinates, using immunohistochemistry, immunofluorescence staining and RT-PCR. In addition, extracts from in vitro samples prepared from primary cells of inferior turbinates as well as in vitro cultured human nasal epithelial RPMI 2650 cells (ATCC-CCL-30) were also tested for leptin expression and activation of the Notch-1 pathway. RESULTS: With regards to AR, in vivo expression levels of both leptin and its receptor significantly decreased in comparison to C. Furthermore, leptin receptor mRNA was significantly reduced in AR as compared to C. Immunofluorescence showed an apparent co-expression of leptin receptor with Notch-1, which was not seen with TGF-β. In vitro, in primary turbinate epithelial cells, the expression of leptin receptor and Notch-1 significantly decreased in AR as compared to C. Moreover, in RPMI 2650 cells, leptin receptor expression was shown to be induced by Notch-1 ligand signaling. CONCLUSION: Thus, both the leptin and Notch-1 pathways appear to represent markers for epithelial homeostasis in allergic rhinitis. Copyright © 2019 Elsevier B.V. All rights reserved. KEYWORDS: Allergic rhinitis; Epithelium; Leptin; Notch

Inflammation and cellular senescence (also called inflammaging) are involved in the pathogenesis of premature lung aging, a key driver of chronic obstructive pulmonary disease (COPD). Downregulation of histone deacetylases and FoxO3 expression, activation of the ERK 1/2 pathway and IL-8 increase are hallmarks of lung inflammaging. The effects of Budesonide (BUD), Aclidinium (ACL) and Formoterol (FO) on lung inflammaging are unknown. This study was aimed to assess the effects of BUD, ACL and FO in bronchial epithelial cells exposed to cigarette smoke extract (CSE) by evaluating: a) Expression of TLR4 and survivin and LPS binding by flow cytometry; b) expression of HDAC2, HDAC3, SIRT1 and FoxO3 and activation of the ERK 1/2 pathway by western blot; c) IL-8 mRNA levels and release by Real Time-PCR and ELISA, respectively. Reported results show that CSE increased TLR4 and survivin, LPS binding, ERK 1/2 activation, IL-8 release and mRNA levels but decreased SIRT1, HDAC2, HDAC3 and FoxO3 nuclear expression. Combined therapy with BUD, ACL and FO counteracted the effects of CSE on LPS binding, FoxO3 nuclear expression, ERK 1/2 activation, survivin and IL-8 release and mRNA levels. These findings suggest a new role of combination therapy with BUD, ACL and FO in counteracting inflammaging processes induced by cigarette smoke exposure.

Although IL-33/ST2 axis is involved in the development of allergic diseases, its contribution in food allergy is still unknown. In this study, we assessed the serum levels of IL-33 and its s-ST2 receptor in 53 control patients (without allergic diseases), 47 peach (Pru p 3)-sensitized allergic patients (SAP), and in 68 non-Pru p 3-SAP. Basophil activation test (BAT) was used to assess the basophil activation due to allergen exposure before and after the addition of s-ST2 to the blood samples from 5 Pru p 3-SAP. IL-33 levels in Pru p 3-SAP were higher than in non-Pru p 3-SAP and in normal controls. Lower s-ST2 levels were found in Pru p 3-SAP than in non-Pru p 3-SAP. IL-33/s-ST2 ratio was higher in Pru p 3-SAP than in both non-Pru p 3-SAP and controls. Higher IL-33/s-ST2 ratio was observed in Pru p 3-SAP with severe than in those with mild systemic symptoms. BAT analysis in Pru p 3-SAP showed a decrease in basophil activation due to Pru p 3 exposure after the addition of s-ST2 to the blood samples. An imbalance in the baseline levels of IL-33/ST2 pathway is present in Pru p 3-SAP. The measurement of this pathway might be helpful to detect patients at a higher risk of developing severe systemic symptoms.

Rationale: Chronic inflammation and cellular senescence (also called inflammaging) are deeply involved in the pathogenesis of premature lung aging, an important contributing factor in driving chronic obstructive pulmonary disease (COPD). SIRT1, activating FoxO3 transcription factor protects against lung inflammaging. FoxO3, deacetylated by SIRT1, within the nucleus binds to the survivin promoter thus blocking the survivin gene transcription However, phosphorylation of FoxO3 promotes FoxO3 nuclear export and protein degradation. Aims: The aims of the study were to assess the effects of budesonide (BUD), aclidinium (ACL) and formoterol (FO) on inflammaging processes of bronchial epithelial cells exposed to cigarette smoke. Lipopolysaccharide (LPS) binding and survivin expression were assessed by flow-cytometry, ERK 1/2 pathway activation and nuclear expression of SIRT1, HDAC3, FoxO3 were assessed by western-blot analysis. Real time PCR was applied to assess IL-8 and telomerase mRNA expression. Results. Here, it was demonstrated that, in bronchial epithelial cells, Cigarette smoke extract (CSE) increased LPS binding, ERK 1/2 pathway activation, IL-8 mRNA and survivin expression but decreased SIRT1, HDAC3, FoxO3 nuclear and telomerase mRNA expression. A combination of BUD, ACL and FO is able to counteract the effects of CSE on the LPS binding, FoxO3 nuclear expression, ERK 1/2 pathway activation, survivin and IL-8 mRNA expression. Conclusions: These findings suggest a new effect of combination with BUD, ACL and FO in counteracting inflammaging processes induced by cigarette smoke exposure.

Background Cigarette smoke, the principal risk factor for chronic obstructive pulmonary disease (COPD), negatively influences the effectiveness of the immune system’s response to a pathogen. The antibiotic ceftaroline exerts immune-modulatory effects in bronchial epithelial cells exposed to cigarette smoke. Aims and methods The present study aims to assess the effects of ceftaroline on TLR2 and TLR4 expression, LPS binding and TNF-α and human beta defensin (HBD2) release in an undifferentiated and PMA-differentiated human monocyte cell line (THP-1) exposed or not to cigarette smoke extracts (CSE). TLR2, TLR4, and LPS binding were assessed by flow cytometry, TNF-α and HBD2 release were evaluated by ELISA. Results The constitutive expression of TLR2 and TLR4 and LPS binding were higher in differentiated compared to undifferentiated THP-1 cells. In undifferentiated THP-1 cells, CSE increased TLR2 and TLR4 protein levels, LPS binding and TNF-α release and reduced HBD2 release and ceftaroline counteracted all these effects. In differentiated THP-1, CSE did not significantly affect TLR2 and TLR4 expression and LPS binding but reduced HBD2 release and increased TNF-α release. Ceftaroline counteracted the effects of CSE on HBD2 release in differentiated THP-1. Conclusion Ceftaroline counteracts the effect of CSE in immune cells by increasing the effectiveness of the innate immune system. This effect may also assist in reducing pathogen activity and recurrent exacerbations in COPD patients.