Honokiol ameliorates cigarette smoke-induced damage of airway epithelial cells via the SIRT3/SOD2 signalling pathway

Cigarette smoking can cause damage of airway epithelial cells and contribute to chronic obstructive pulmonary disease (COPD). Honokiol is originally isolated from Magnolia obovata with multiple biological activities. Here, we investigated the protective effects of honokiol on cigarette smoke extract (CSE)-induced injury of BEAS-2B cells. BEAS-2B cells were treated with 300 mg/L CSE to construct an in vitro cell injury model, and cells were further treated with 2, 5 and 10 μM honokiol, then cell viability and LDH leakage were analysed by CCK-8 and LDH assay kits, respectively. Apoptosis was detected by flow cytometry analysis. ELISA was used to measure the levels of tumour necrosis factor (TNF)-ɑ, IL-1β, IL-6, IL-8 and MCP-1. The results showed that honokiol (0.5–20 μM) showed non-toxic effects on BEAS-2B cells. Treatment with honokiol (2, 5 and 10 μM) reduced CSE (300 mg/L)-induced decrease in cell viability and apoptosis in BEAS-2B cells. Honokiol also decreased CSE-induced inflammation through inhibiting expression and secretion of inflammatory cytokines, such as TNF-ɑ, IL-1β, IL-6, IL-8 and MCP-1. Moreover, honokiol repressed CSE-induced reactive oxygen species (ROS) production, decrease of ATP content and mitochondrial biogenesis, as well as mitochondrial membrane potential. Mechanistically, honokiol promoted the expression of SIRT3 and its downstream target genes, which are critical regulators of mitochondrial function and oxidative stress. Silencing of SIRT3 reversed the protective effects of honokiol on CSE-induced damage and mitochondrial dysfunction in BEAS-2B cells. These results indicated that honokiol attenuated CSE-induced damage of airway epithelial cells through regulating SIRT3/SOD2 signalling pathway.     

Klotho Reduction in Alveolar Macrophages Contributes to Cigarette Smoke Extract-induced Inflammation in Chronic Obstructive Pulmonary Disease

Abnormal inflammation and accelerated decline in lung function occur in patients with chronic obstructive pulmonary disease (COPD). Klotho, an anti-aging protein, has an anti-inflammatory function. However, the role of Klotho has never been investigated in COPD. The aim of this study is to investigate the possible role of Klotho by alveolar macrophages in airway inflammation in COPD. Klotho levels were assessed in the lung samples and peripheral blood mononuclear cells of non-smokers, smokers, and patients with COPD. The regulation of Klotho expression by cigarette smoke extract (CSE) was studied in vitro, and small interfering RNA (siRNA) and recombinant Klotho were employed to investigate the role of Klotho on CSE-induced inflammation. Klotho expression was reduced in alveolar macrophages in the lungs and peripheral blood mononuclear cells of COPD patients. CSE decreased Klotho expression and release from MH-S cells. Knockdown of endogenous Klotho augmented the expression of the inflammatory mediators, such as MMP-9, IL-6, and TNF-α, by MH-S cells. Exogenous Klotho inhibited the expression of CSE-induced inflammatory mediators. Furthermore, we showed that Klotho interacts with IκBα of the NF-κB pathway. Dexamethasone treatment increased the expression and release level of Klotho in MH-S cells. Our findings suggest that Klotho plays a role in sustained inflammation of the lungs, which in turn may have therapeutic implications in COPD.     

The role of uPAR in epithelial-mesenchymal transition in small airway epithelium of patients with chronic obstructive pulmonary disease

Background

Epithelial-mesenchymal transition (EMT) plays a crucial role in small airway fibrosis of patients with chronic obstructive pulmonary disease (COPD). Increasing evidence suggests that the urokinase plasminogen activator receptor (uPAR) is involved in the pathogenesis of COPD. Increased uPAR expression has been implicated in the promotion of EMT in numerous cancers; however the role of uPAR in EMT in small airway epithelial cells of patients with COPD remains unclear. In this study, we investigated the degree of EMT and uPAR expression in lung epithelium of COPD patients, and verified the effect of uPAR on cigarette smoke extract (CSE)-induced EMT in vitro.

Methods

The expression of EMT biomarkers and uPAR was assessed in lung epithelium specimens from non-smokers (n = 25), smokers (n = 25) and non-smokers with COPD (n = 10) and smokers with COPD (n = 18). The role of uPAR on CSE-induced EMT in human small airway epithelial cells (HSAEpiCs) was assessed by silencing uPAR expression in vitro.

Results

Markers of active EMT and uPAR expression were significantly increased in the small airway epithelium of patients with COPD compared with controls. We also observed a significant correlation between uPAR and vimentin expression in the small airway epithelium. In vitro, CSE-induced EMT in HSAEpiCs was associated with high expression of uPAR, and targeted silencing of uPAR using shRNA inhibited CSE-induced EMT. Finally, we demonstrate that the PI3K/Akt signaling pathway is required for uPAR-mediated EMT in HSAEpiCs.

Conclusions

A uPAR-dependent signaling pathway is required for CSE-induced EMT, which contributes to small airway fibrosis in COPD. We propose that increased uPAR expression in the small airway epithelium of patients with COPD participates in an active EMT process.     

Role of the Mycoplasma pneumoniae/Interleukin-8/Neutrophil Axis in the Pathogenesis of Pneumonia

Neutrophil infiltration is the characteristic pathological feature of M. pneumoniae pneumonia (MPP). This study aimed to explore the associations among neutrophil activity, clinical presentation, and role of the M. pneumoniae/interleukin-8 (IL-8)/neutrophil axis in the pathogenesis of MPP. A total of 42 patients with MPP were prospectively enrolled in the study. Neutrophil activity, including matrix metalloproteinase-9 (MMP-9), myeloperoxidase (MPO), and neutrophil elastase (NE), were measured. Clinical information was collected for all patients and control group. In vitro, IL-8 production was measured at different time points after M. pneumoniae infection of bronchial epithelial cells, and neutrophil activity was analyzed after IL-8 stimulation. The percentage of neutrophil in the bronchoalveolar lavage fluid was higher in the group of patients with high levels of M. pneumoniae DNA than in those with low levels of M. pneumoniae DNA (P < 0.05). IL-8, MMP-9, and NE in patients with MPP significantly increased compared with controls and decreased after treatment (P < 0.05). MPO and MMP-9 were associated with duration of fever (r = 0.332, P < 0.05) and length of stay (r = 0.342, P < 0.05), respectively. In vitro, M. pneumoniae induced IL-8 production by bronchial epithelial cells in a time dependent manner. MPO, MMP-9 and NE production by neutrophils significantly increased compared with medium controls after IL-8 stimulation. In summary, the M. pneumoniae/IL-8/neutrophil axis likely plays a vital role in the pathogenesis of MPP.     

Neutrophil elastase up-regulates interleukin-8 via toll-like receptor 4

Cystic fibrosis is characterised in the lungs by high levels of neutrophil elastase (NE). NE induces interleukin-8 (IL-8) expression via an IL-1 receptor-associated kinase signalling pathway. Here, we show that these events involve the cell surface membrane bound toll-like receptor 4 (TLR4). We demonstrate that human embryonic kidney (HEK)293 cells transfected with a TLR4 cDNA (HEK-TLR4) express TLR4 mRNA and protein and induce IL-8 promoter activity in response to NE. Treatment of both HEK-TLR4 and human bronchial epithelial cells with NE decreases TLR4 protein expression. Furthermore, a TLR4 neutralising antibody abrogates NE-induced IL-8 production, and induces tolerance to a secondary lipopolysaccharide stimulus. These data implicate TLR4 in NE induced IL-8 expression in bronchial epithelium.     

Long non-coding RNA MEG3 regulates CSE-induced apoptosis and inflammation via regulating miR-218 in 16HBE cells

Chronic obstructive pulmonary disease (COPD) is a prevalent disease worldwide, mainly caused by cigarette smoking. Maternally expressed gene 3 (MEG3) functions as the lncRNA and is upregulated in COPD patients and human bronchial epithelial cells after fine particulate matter (PM2.5) treatment. However, the molecular mechanism of MEG3 in COPD remains unknown. The expression of MEG3 and miR-218 in COPD tissues and cigarette smoke extract (CSE)-treated 16HBE cells was detected by RT-qPCR. The effects of MEG3 and miR-218 on proliferation and apoptosis in (CSE)-treated 16HBE cells were analyzed by CCK-8 and flow cytometry assay, respectively. The protein levels of inflammatory cytokines (IL-1β IL-6 and TNF-α) were detected in 16HBE cells by ELISA. MEG3 and miR-218 binding interaction was predicted by LncBase Predicted v.2 and further confirmed by dual luciferase reporter assay and RNA Immunoprecipitation (RIP) assay. MEG3 was upregulated in COPD tissues and inversely related to FEV1%. MEG3 was upregulated in (CSE)-treated 16HBE cells, and knockdown of MEG3 mitigated CSE-repressed proliferation and CSE-triggered apoptosis or inflammation. MiR-218 was demonstrated as a target miRNA of MEG3. MiR-218 was downregulated in COPD tissues and (CSE)-treated or MEG3 overexpressed 16HBE cells. MiR-218 overexpression attenuated CSE-blocked proliferation and CSE-induced apoptosis or inflammation. Deficiency of MEG3 counteracted CSE-blocked proliferation CSE-induced apoptotic rate and inflammatory cytokine (IL-1β IL-6 and TNF-α) levels, while introduction of anti-miR-218 reversed these effects. MEG3 regulated CSE-inhibited proliferation and CSE-induced apoptosis or inflammation by targeting miR-218, providing a possible therapeutic target for treatment of CSE-induced COPD.     

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.     

STAT3 activation inhibits human bronchial epithelial cell apoptosis in response to cigarette smoke exposure

We have previously reported that cigarette smoke can induce DNA damage in human lung cells without leading to apoptosis or necrosis. In this study, we report that STAT3 is required for the survival of human bronchial epithelial cells (HBECs) following cigarette smoke-induced DNA damage. Cigarette smoke extract (CSE) exposure increases STAT3 phosphorylation (Tyr 705) and DNA binding activity in HBECs. CSE also stimulates IL-6 release and mRNA expression. Anti-IL-6 neutralizing antibody partially blocks STAT3 activation and renders the cells sensitive to CSE-induced DNA damage. Suppression of STAT3 by siRNA results in severe DNA damage and cell death in response to CSE exposure. These findings suggest that STAT3 mediates HBEC survival in response to CSE-induced DNA damage, at least in part, through the IL-6/STAT3 signaling pathway.     

Pulmonary proteases in the cystic fibrosis lung induce interleukin 8 expression from bronchial epithelial cells via a heme/meprin/epidermal growth factor receptor/Toll-like receptor pathway

A high intrapulmonary protease burden is characteristic of cystic fibrosis (CF), and the resulting dysregulation of the protease/anti-protease balance has serious implications for inflammation in the CF lung. Because of this inflammation, micro-bleeds can occur releasing hemoglobin into the lung. The aim of this study was to investigate the effect of the protease-rich environment of the CF lung on human hemoglobin and to assess the proinflammatory effect of heme on CF bronchial epithelium. Here, we show that the Pseudomonas proteases (Pseudomonas elastase and alkaline protease) and the neutrophil proteases (neutrophil elastase (NE) and proteinase-3) are capable of almost complete degradation of hemoglobin in vitro but that NE is the predominant protease that cleaves hemoglobin in vivo in CF bronchoalveolar lavage fluid. One of the effects of this is the release of heme, and in this study we show that heme stimulates IL-8 and IL-10 protein production from ΔF508 CFBE41o(-) bronchial epithelial cells. In addition, heme-induced IL-8 expression utilizes a novel pathway involving meprin, EGF receptor, and MyD88. Meprin levels are elevated in CF cell lines and bronchial brushings, thus adding to the proinflammatory milieu. Interestingly, α(1)-antitrypsin, in addition to its ability to neutralize NE and protease-3, can also bind heme and neutralize heme-induced IL-8 from CFBE41o(-) cells. This study illustrates the proinflammatory effects of micro-bleeds in the CF lung, the process by which this occurs, and a potential therapeutic intervention.     

Role for TAK1 in cigarette smoke-induced proinflammatory signaling and IL-8 release by human airway smooth muscle cells

Chronic obstructive pulmonary disease (COPD) is an inflammatory disease, characterized by a progressive decline in lung function. Airway smooth muscle (ASM) mass may be increased in COPD, contributing to airflow limitation and proinflammatory cytokine production. Cigarette smoke (CS), the major risk factor of COPD, causes ASM cell proliferation, as well as interleukin-8 (IL-8)-induced neutrophilia. In various cell types, transforming growth factor-β-activated kinase 1 (TAK1) plays a crucial role in MAP kinase and NF-κB activation, as well as IL-8 release induced by IL-1β, TNF-α, and lipopolysaccharide. The role of TAK1 in CS-induced IL-8 release is not known. The aim of this study was to investigate the role of TAK1 in CS-induced NF-κB and MAP kinase signaling and IL-8 release by human ASM cells. Stimulation of these cells with CS extract (CSE) increased IL-8 release and ERK-1/2 phosphorylation, as well as Iκ-Bα degradation and p65 NF-κB subunit phosphorylation. CSE-induced ERK-1/2 phosphorylation and Iκ-Bα degradation were both inhibited by pretreatment with the specific TAK1 inhibitor LL-Z-1640-2 (5Z-7-oxozeaenol; 100 nM). Similarly, expression of dominant-negative TAK1 inhibited CSE-induced ERK-1/2 phosphorylation. In addition, inhibitors of TAK1 and the NF-κB (SC-514; 50 μM) and ERK-1/2 (U-0126; 3 μM) signaling inhibited the CSE-induced IL-8 release by ASM cells. These data indicate that TAK1 plays a major role in CSE-induced ERK-1/2 and NF-κB signaling and in IL-8 release by human ASM cells. Furthermore, they identify TAK1 as a novel target for the inhibition of CS-induced inflammatory responses involved in the development and progression of COPD.     

PARK2-mediated mitophagy is involved in regulation of HBEC senescence in COPD pathogenesis

Cigarette smoke (CS)-induced mitochondrial damage with increased reactive oxygen species (ROS) production has been implicated in COPD pathogenesis by accelerating senescence. Mitophagy may play a pivotal role for removal of CS-induced damaged mitochondria, and the PINK1 (PTEN-induced putative kinase 1)-PARK2 pathway has been proposed as a crucial mechanism for mitophagic degradation. Therefore, we sought to investigate to determine if PINK1-PARK2-mediated mitophagy is involved in the regulation of CS extract (CSE)-induced cell senescence and in COPD pathogenesis. Mitochondrial damage, ROS production, and cell senescence were evaluated in primary human bronchial epithelial cells (HBEC). Mitophagy was assessed in BEAS-2B cells stably expressing EGFP-LC3B, using confocal microscopy to measure colocalization between TOMM20-stained mitochondria and EGFP-LC3B dots as a representation of autophagosome formation. To elucidate the involvement of PINK1 and PARK2 in mitophagy, knockdown and overexpression experiments were performed. PINK1 and PARK2 protein levels in lungs from patients were evaluated by means of lung homogenate and immunohistochemistry. We demonstrated that CSE-induced mitochondrial damage was accompanied by increased ROS production and HBEC senescence. CSE-induced mitophagy was inhibited by PINK1 and PARK2 knockdown, resulting in enhanced mitochondrial ROS production and cellular senescence in HBEC. Evaluation of protein levels demonstrated decreased PARK2 in COPD lungs compared with non-COPD lungs. These results suggest that PINK1-PARK2 pathway-mediated mitophagy plays a key regulatory role in CSE-induced mitochondrial ROS production and cellular senescence in HBEC. Reduced PARK2 expression levels in COPD lung suggest that insufficient mitophagy is a part of the pathogenic sequence of COPD.     

Induction of interleukin 8 (IL-8) production by Pseudomonas nitrite reductase in human alveolar macrophages and epithelial cells.

Interleukin-8 (IL-8) participates in the generation of dense neutrophil accumulations in bronchopulmonary infections caused by Pseudomonas aeruginosa (P. aeruginosa). We have recently reported that nitrite reductase, a bifunctional enzyme located in the periplasmic space of P. aeruginosa, induces IL-8 generation in bronchial epithelial cells (K. Oishi et al. Infect. Immun. 65: 2648-2655, 1997). We examined whether or not Pseudomonas nitrite reductase (PNR) could also stimulate human alveolar macrophages (AM) and pulmonary type II epithelial-like cells (A549) to induce IL-8 production and mRNA expression as well as the production of TNF alpha and IL-1beta. We demonstrated a time- and dose-dependent IL-8 protein synthesis and IL-8 mRNA expression, but no TNF alpha or IL-1beta production, by A549 cells in response to PNR. New protein translation was not required for PNR-mediated IL-8 mRNA expression in the same cells. Furthermore, simultaneous stimulation of PNR with serial doses of TNF alpha or IL-1beta resulted in additive IL-8 production in A549 cells. In adherent AM, PNR enhanced IL-8 protein synthesis and IL-8 mRNA expression in a time-dependent fashion. PNR similarly induced a time-dependent production of TNF alpha and IL-1beta by human adherent AM. Neutralization of TNF alpha or IL-1beta did not influence the levels of IL-8 production in adherent AM culture. We also evaluated whether the culture supernatants of the A549 cells or AM stimulated with PNR could similarly mediate neutrophil migration in vitro. When anti-human IL-8 immunoglobulin G was used for neutralizing neutrophil chemotactic factor (NCF) activities in the culture supernatants of these cells stimulated with 5 microg/ml of PNR, the mean percent reduction of NCF activities were 49-59% in A549 cells and 24-34% in AM. Our present data support that PNR directly stimulates AM and pulmonary epithelial cells to produce IL-8. PNR also mediates neutrophil migration, in part, through IL-8 production from AM and pulmonary epithelial cells. These data suggest the contribution of PNR to the pathogenesis of bronchopulmonary infections due to P. aeruginosa.     

Vasorin/ATIA Promotes Cigarette Smoke–Induced Transformation of Human Bronchial Epithelial Cells by Suppressing Autophagy-Mediated Apoptosis

BACKGROUND: Escaping cell death pathways is an important event during carcinogenesis. We previously identified anti-TNFα-induced apoptosis (ATIA, also known as vasorin) as an antiapoptotic factor that suppresses reactive oxygen species (ROS) production. However, the role of vasorin in lung carcinogenesis has not been investigated. METHODS: Vasorin expression was examined in human lung cancer tissues with immunohistochemistry and database analysis. Genetic and pharmacological approaches were used to manipulate protein expression and autophagy activity in human bronchial epithelial cells (HBECs). ROS generation was measured with fluorescent indicator, apoptosis with release of lactate dehydrogenase, and cell transformation was assessed with colony formation in soft agar. RESULTS: Vasorin expression was increased in human lung cancer tissues and cell lines, which was inversely associated with lung cancer patient survival. Cigarette smoke extract (CSE) and benzo[a]pyrene diol epoxide (BPDE)–induced vasorin expression in HBECs. Vasorin knockdown in HBECs significantly suppressed CSE-induced transformation in association with enhanced ROS accumulation and autophagy. Scavenging ROS attenuated autophagy and cytotoxicity in vasorin knockdown cells, suggesting that vasorin potentiates transformation by impeding ROS-mediated CSE cytotoxicity and improving survival of the premalignant cells. Suppression of autophagy effectively inhibited CSE-induced apoptosis, suggesting that autophagy was pro-apoptotic in CSE-treated cells. Importantly, blocking autophagy strongly potentiated CSE-induced transformation. CONCLUSION: These results suggest that vasorin is a potential lung cancer–promoting factor that facilitates cigarette smoke–induced bronchial epithelial cell transformation by suppressing autophagy-mediated apoptosis, which could be exploited for lung cancer prevention.     

Viral inhibition of IL-1- and neutrophil elastase-induced inflammatory responses in bronchial epithelial cells

Previously, we elucidated the intracellular mechanisms by which neutrophil elastase (NE) up-regulates inflammatory gene expression in bronchial epithelial cells. In this study, we examine the effects of both IL-1 and NE on inflammatory gene expression in 16HBE14o- bronchial epithelial cells and investigate approaches to abrogate these inflammatory responses. IL-1 induced IL-8 protein production in time- and dose-dependent fashions, an important observation given that IL-8 is a potent neutrophil chemoattractant and a key inflammatory mediator. IL-1 and NE were shown to activate the p38 MAPK pathway in 16HBE14o- cells. Western blot analysis demonstrated IL-1R-associated kinase 1 (IRAK-1) degradation in response to stimulation with both IL-1 and NE. In addition, the expression of dominant negative IRAK-1 (IRAK-1delta), IRAK-2delta, or IRAK-4delta inhibited IL-1- and NE-induced NF-kappaB-linked reporter gene expression. Dominant negative versions of the intracellular adaptor proteins MyD88 (MyD88delta) and MyD88 adaptor-like (Mal P/H) abrogated NE-induced NF-kappaB reporter gene expression. In contrast, only MyD88delta was found to inhibit IL-1-induced NF-kappaB reporter activity. We also investigated the vaccinia virus proteins, A46R and A52R, which have been shown to antagonize IL-1 signaling. Transfection with A46R or A52R cDNA inhibited IL-1- and NE-induced NF-kappaB and IL-8R gene expression and IL-8 protein production in primary and transformed bronchial epithelial cells. Furthermore, cytokine array studies demonstrated that IL-1 and NE can up-regulate the expression of IL-6, oncostatin M, epithelial cell-derived neutrophil activating peptide-78, growth-related oncogene family members, vascular endothelial growth factor, and GM-CSF, with induction of these proteins inhibited by the viral proteins. These findings identify vaccinia virus proteins as possible therapeutic agents for the manifestations of several inflammatory lung diseases.     

Cigarette smoke enhances human rhinovirus-induced CXCL8 production via HuR-mediated mRNA stabilization in human airway epithelial cells

Background

Human rhinovirus (HRV) triggers exacerbations of asthma and chronic obstructive pulmonary disease (COPD). Cigarette smoking is the leading risk factor for the development of COPD and 25% of asthmatics smoke. Smoking asthmatics have worse symptoms and more frequent hospitalizations compared to non-smoking asthmatics. The degree of neutrophil recruitment to the airways correlates with disease severity in COPD and during viral exacerbations of asthma. We have previously shown that HRV and cigarette smoke, in the form of cigarette smoke extract (CSE), each induce expression of the neutrophil chemoattractant and activator, CXCL8, in human airway epithelial cells. Additionally, we demonstrated that the combination of HRV and CSE induces expression of levels of CXCL8 that are at least additive relative to induction by each stimulus alone, and that enhancement of CXCL8 expression by HRV+CSE is regulated, at least in part, via mRNA stabilization. Here we further investigate the mechanisms by which HRV+CSE enhances CXCL8 expression.

Methods

Primary human bronchial epithelial cells were cultured and treated with CSE alone, HRV alone or the combination of the two stimuli. Stabilizing/destabilizing proteins adenine/uridine-rich factor-1 (AUF-1), KH-type splicing regulatory protein (KHSRP) and human antigen R (HuR) were measured in cell lysates to determine expression levels following treatment. siRNA knockdown of each protein was used to assess their contribution to the induction of CXCL8 expression following treatment of cells with HRV and CSE.

Results

We show that total expression of stabilizing/de-stabilizing proteins linked to CXCL8 regulation, including AUF-1, KHSRP and HuR, are not altered by CSE, HRV or the combination of the two stimuli. Importantly, however, siRNA-mediated knock-down of HuR, but not AUF-1 or KHSRP, abolishes the enhancement of CXCL8 by HRV+CSE. Data were analyzed using one-way ANOVA with student Newman-Keuls post hoc analysis and values of p≤ 0.05 were considered significant.

Conclusions

Induction of CXCL8 by the combination of HRV and CSE is regulated by mRNA stabilization involving HuR. Thus, targeting the HuR pathway may be an effective method of dampening CXCL8 production during HRV-induced exacerbations of lower airway disease, particularly in COPD patients and asthmatic patients who smoke.     

Silencing FUNDC1 alleviates chronic obstructive pulmonary disease by inhibiting mitochondrial autophagy and bronchial epithelium cell apoptosis under hypoxic environment

Chronic obstructive pulmonary disease (COPD) is a major global epidemic with increasing incidence worldwide. The pathogenesis of COPD is involved with mitochondrial autophagy. Recently, it has been reported that FUN14 domain containing 1 (FUNDC1) is a mediator of mitochondrial autophagy. Therefore, we hypothesized that FUNDC1 was involved in cigarette smoke (CS)-induced COPD progression by regulating mitochondrial autophagy. In vitro cigarette smoke extract (CSE)-treated human bronchial epithelial cell (hBEC) Beas-2B cell line and in vivo CS-induced COPD mouse models were developed, in which FUNDC1 expression was measured. Next, whether FUNDC1 interacted with dynamin-related protein 1 (DRP1) in COPD was investigated. The functional mechanism of FUNDC1 in COPD was evaluated through gain- or loss-of-function studies. Then, pulmonary function, mitochondrial transmembrane potential (MTP) and mucociliary clearance (MCC) were examined. Levels of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) and expression of autophagy-specific markers (light chain 3 [LC3] II, LC3 I, and Tom20) were measured. Finally, apoptosis and mitochondrial autophagy were assessed. FUNDC1 was highly expressed in CSE-treated hBECs and COPD mice. Meanwhile, FUNDC1 was proved to interact with DRP1 in CSE-treated cells. Moreover, in CSE-treated hBECs, silencing FUNDC1 was observed to reduce levels of IL-6 and TNF-α, and MTP but increase MCC, and inhibit CSE-induced mitochondrial autophagy and Beas-2B cell apoptosis, which was consistent with the trend in COPD mouse models. In addition, pulmonary function of COPD mouse models was increased in response to FUNDC1 silencing. Finally, silencing of DRP1 also inhibited mitochondrial autophagy and Beas-2B cell apoptosis. Collectively, FUNDC1 silencing could suppress the progression of COPD by inhibiting mitochondrial autophagy and hBEC apoptosis through interaction with DRP1, highlighting a potential therapeutic target for COPD treatment.     

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.     

Cilomilast counteracts the effects of cigarette smoke in airway epithelial cells

Cigarette smoke extracts (CSE) alter TLR4 expression and activation in bronchial epithelial cells. Cilomilast, a phosphodiesterase-4 inhibitor, inhibits cigarette smoke-induced neutrophilia.This study was aimed to explore whether cilomilast, in a human bronchial epithelial cell line (16-HBE), counteracted CSE effects. In particular, TLR4 expression, IP-10 and IL-8 release, lymphocyte and neutrophil chemotactic activity and ERK and IkBa phosphorylation in CSE and LPS-stimulated 16-HBE were assessed.CSE increased TLR4 expression, reduced IP-10 release and lymphocyte chemotactic activity and increased IL-8 release and neutrophil chemotactic activity. Cilomilast reduced TLR4 expression, IL-8 release and neutrophil chemotactic activity as well as it increased IP-10 release and lymphocyte chemotactic activity. All these cilomilast mediated effects were associated with a reduced ERK1/2 and with an increased IkBa phosphorylation.In conclusion, the present study provides compelling evidences that cilomilast may be considered a possible valid therapeutic option in controlling inflammatory processes present in smokers.