Integrative epigenomic analysis in differentiated human primary bronchial epithelial cells exposed to cigarette smoke.
ABSTRACT: Cigarette smoke (CS) is one of the major risk factors for many pulmonary diseases, including chronic obstructive pulmonary disease (COPD) and lung cancer. The first line of defense for CS exposure is the bronchial epithelial cells. Elucidation of the epigenetic changes during CS exposure is key to gaining a mechanistic understanding into how mature and differentiated bronchial epithelial cells respond to CS. Therefore, we performed epigenomic profiling in conjunction with transcriptional profiling in well-differentiated human bronchial epithelial (HBE) cells cultured in air-liquid interface (ALI) exposed to the vapor phase of CS. The genome-wide enrichment of histone 3 lysine 27 acetylation was detected by chromatin immunoprecipitation followed by next generation sequencing (ChIP-Seq) in HBE cells and suggested the plausible binding of specific transcription factors related to CS exposure. Additionally, interrogation of ChIP-Seq data with gene expression profiling of HBE cells after CS exposure for different durations (3?hours, 2 days, 4 days) suggested that earlier epigenetic changes (3?hours after CS exposure) may be associated with later gene expression changes induced by CS exposure (4 days). The integration of epigenetics and gene expression data revealed signaling pathways related to CS-induced epigenetic changes in HBE cells that may identify novel regulatory pathways related to CS-induced COPD.
Project description:Epithelial-mesenchymal transition (EMT) is a process associated with airway remodeling in chronic obstructive pulmonary disease (COPD), which leads to progressive pulmonary destruction. Panax ginseng is a traditional herbal medicine that has been shown to improve pulmonary function and exercise capacity in patients with COPD. Ginsenoside Rg1 is one of the main active components and was shown to inhibit oxidative stress and inflammation. The present study investigated the hypothesis that ginsenoside Rg1 attenuates EMT in COPD rats induced by cigarette smoke (CS) and human bronchial epithelial (HBE) cells exposed to cigarette smoke extract (CSE). Our data showed that CS or CSE exposure increased expression of the mesenchymal marker ?-smooth muscle actin (?-SMA) and decreased expression of the epithelial marker epithelial cadherin (E-cad) in both lung tissues and HBE cells, which was markedly suppressed by ginsenoside Rg1. Importantly, CS-induced upregulation of TGF-?1/Smad pathway components, including TGF-?1, TGF-?R1, phospho-Smad2, and phospho-Smad3, was also inhibited by ginsenoside Rg1. Additionally, ginsenoside Rg1 mimicked the effect of SB525334, a TGF-?R1-Smad2/3 inhibitor, on suppression of EMT in CSE-induced HBE cells. Collectively, we concluded that ginsenoside Rg1 alleviates CS-induced pulmonary EMT, in both COPD rats and HBE cells, via inhibition of the TGF-?1/Smad pathway.
Project description:Jianpiyifei II granules (JPYF II), a herbal formula, are used for the treatment of chronic obstructive pulmonary disease (COPD) in Guangdong Provincial Hospital of Chinese Medicine. The protective effects of JPYF II against bronchial epithelial cell apoptosis in mice exposed to cigarette smoke (CS) and apoptosis of human bronchial epithelial cell lines (BEAS-2B and 16-HBE) stimulated with cigarette smoke extract (CSE) were investigated. Mice were exposed to CS generated from four cigarettes/day for 30 days and administered a dose of JPYF II (0.75, 1.5, and 3 g/kg/d) from the 3rd week of CS exposure. In mice exposed to CS, JPYF II significantly inhibited CS-induced apoptosis and overexpression of endoplasmic reticulum (ER) stress-related markers in bronchial epithelial cells of the lung tissues. In CSE-stimulated BEAS-2B and 16-HBE cells, JPYF II attenuated apoptosis and cell cycle arrest in the G0/G1 phase. Mechanistically, CSE initially induced intracellular reactive oxygen species (ROS) production, which then triggered ER stress, leading to the release of Ca2+ from ER inositol trisphosphate receptor (IP3R)-mediated stores and finally cell death. Treatment with JPYF II resulted in a significant reduction in CSE-induced apoptosis through interruption of the ROS-ER stress-Ca2+ signaling pathway. Therefore, the results of this study have revealed the underlying mechanism of action of JPYF II in the treatment of COPD.
Project description:Rationale: Aberrant bronchial epithelium-fibroblast communication is essential for the airway remodeling that contributes to chronic obstructive pulmonary disease (COPD). Exosomes have emerged as novel mediators of intercellular communication, but their role in cigarette smoke (CS)-induced COPD is unknown. Here, we investigated the role of exosomal miR-21 in the dysfunctional epithelium-fibroblast cross-talk caused by CS. Methods: Normal or CS extract (CSE)-treated human bronchial epithelial (HBE) cells were co-cultured with bronchial fibroblasts (MRC-5 cells). Exosomes were obtained from culture media or serum by use of commercial kits. The size distribution and concentration of exosomes were analyzed by nanoparticle tracking analysis using a ZetaView particle tracker from ParticleMetrix. Inhibition of miR-21 levels by tail vein injection of antagomir-21 into mice exposed to CS was used to demonstrate the role of miR-21 in airway remodeling leading to COPD in animals. Results: For MRC-5 cells, co-culture with CSE-treated HBE cells or with exosomes derived from CSE-treated HBE cells resulted in the myofibroblast differentiation phenotype. Exosomal miR-21 was responsible for myofibroblast differentiation through hypoxia-inducible factor 1? (HIF-1?) signaling by targeting the von Hippel-Lindau protein (pVHL); HIF-1? transcriptionally regulated the ?-SMA gene. For mice, downregulation of miR-21 prevented CS-induced airway remodeling. The levels of exosomal miR-21 were high in sera of smokers and COPD patients and inversely correlated with FEV1/FVC. Conclusion: We demonstrate that CS triggers the modification of exosome components and identify miR-21 derived from bronchial epithelial cells as a mediator of myofibroblast differentiation through the pVHL/HIF-1? signaling pathway, which has potential value for diagnosis and treatment of COPD.
Project description:Chronic obstructive pulmonary disease (COPD) is a common inflammatory lung disease characterized by inflammatory cells activation and production of inflammatory mediators. Methyl-CpG-binding domain protein 2 (MBD2) plays an important role in diverse immunological disorders by regulating immune cell functions, such as differentiation and mediator secretion. However, the role of MBD2 in COPD remains unknown.MBD2 protein expression in lung tissues of patients with COPD and cigarette smoke (CS)-exposed mice were evaluated by Western blot and immunohistochemistry. The role of MBD2 in cigarette smoke extract (CSE)-induction of inflammatory mediator expression in the human bronchial epithelial (HBE) cell line was assessed by silencing MBD2 expression in vitro. The involvement of signaling pathways in mediation of inflammation was tested with signaling inhibitors.Compared with controls, MBD2 expression was distinctly reduced in the bronchial epithelium of both patients with COPD and CS-exposed mice. Moreover, MBD2 expression was decreased in HBE after CSE stimulation in vitro. Moreover, MBD2 knockdown enhanced interleukin (IL)-6 and IL-8 expression in HBE in the presence and absence of CSE treatment by the ERK signaling pathway.MBD2 protein expression was reduced in the airway epithelium of COPD. In HBE, this reduced expression was associated with increased levels of IL-6 and IL-8 mediated by the ERK pathway. These results suggest that MBD2 could contribute to chronic airway inflammation in COPD.
Project description:BACKGROUND:CD147 is expressed in many tissues and is involved in many inflammatory diseases. Emerging evidence suggests that the overproduction of mucus is a malignant factor in chronic obstructive pulmonary disease (COPD), which results in severe airway obstruction and repeated airway infections. However, it is still unclear whether CD147 is involved in mucus production in COPD. METHODS:We determined the expression levels of CD147 and MUC5AC by immunohistochemistry in 42 human lung specimens from three groups (non-smokers without COPD, smokers without COPD and smokers with COPD). For the in vitro experiment, human bronchial epithelial (HBE) cells were treated with cigarette smoke (CS) extract to establish a mucus secretion model; then, CD147 and MUC5AC production were detected by RT-PCR, Western blotting and ELISA. To determine how CD147 is involved in MUC5AC secretion, HBE cells were transfected with small interfering RNA to silence CD147 and pretreated with inhibitors of MMP9 and p38 MAPK, which are common signaling molecules involved in MUC5AC secretion; then, MUC5AC expression was evaluated. RESULTS:Compared with the expression levels in the non-smokers and smokers without COPD, CD147 and MUC5AC expression levels were higher in the smokers with COPD. In the in vitro experiment, CD147 and MUC5AC expression levels were significantly increased after CS extract incubation compared with those after no treatment. Silencing CD147 by siRNA decreased the CS extract-induced MUC5AC secretion and MMP9 and phosphorylated p38 MAPK production. In addition, inhibiting MMP9 or p38 MAPK decreased the CS extract-induced MUC5AC secretion. CONCLUSIONS:In lung specimens, CD147 and MUC5AC expression levels were increased in COPD patients. Increased CD147 levels induced by CS extract could stimulate MUC5AC secretion through the MMP9 and p38 MAPK signaling pathway in HBE cells. Therefore, the regulation of CD147 could be a promising target for mucus hypersecretion in COPD.
Project description:Cigarette smoking is a major risk factor for the inflammatory disease, chronic obstructive pulmonary disease (COPD). The mechanism by which cigarette smoke (CS) induces chronic lung inflammation is still largely unknown. We hypothesize that immunogenic airway epithelial cell death is involved in the initiation of the inflammatory response. We previously identified CFLAR, the gene encoding the cell death regulator protein c-FLIP, to be associated with CS-induced release of damage-associated molecular patterns (DAMPs). Here, we investigated the effect of CS on expression levels of CFLAR in bronchial biopsies from smokers and non-smokers and CFLAR transcript isoform-expression in a dataset of air-liquid interface-differentiated bronchial epithelial cells. Furthermore, CFLAR was down-regulated by siRNA in lung epithelial A549 cells, followed by investigation of the effects on apoptosis, necrosis and DAMP release. CS exposure significantly decreased CFLAR expression in bronchial epithelial cells. Moreover, we observed a shift in relative abundance of the isoforms c-FLIPS and c-FLIPL transcripts in bronchial biopsies of current smokers compared to non-smokers, consistent with a shift towards necroptosis. In vitro, down-regulation of CFLAR increased apoptosis at baseline as well as CS extract-induced necrosis and DAMP release. In conclusion, CS exposure decreases CFLAR expression, which might increase susceptibility to immunogenic cell death.
Project description:Cigarette smoking is the main risk factor associated with chronic obstructive pulmonary disease (COPD), and contributes to COPD development and progression by causing epithelial injury and inflammation. Whereas it is known that cigarette smoke (CS) may affect the innate immune function of airway epithelial cells and epithelial repair, this has so far not been explored in an integrated design using mucociliary differentiated airway epithelial cells. In this study, we examined the effect of whole CS exposure on wound repair and the innate immune activity of mucociliary differentiated primary bronchial epithelial cells, upon injury induced by disruption of epithelial barrier integrity or by mechanical wounding. Upon mechanical injury CS caused a delayed recovery in the epithelial barrier integrity and wound closure. Furthermore CS enhanced innate immune responses, as demonstrated by increased expression of the antimicrobial protein RNase 7. These differential effects on epithelial repair and innate immunity were both mediated by CS-induced oxidative stress. Overall, our findings demonstrate modulation of wound repair and innate immune responses of injured airway epithelial cells that may contribute to COPD development and progression.
Project description:PURPOSE:The aim of this study was to investigate the effect of resveratrol (RSV) on cigarette smoke extract (CSE)-induced cell apoptosis and mitochondrial dysfunction in vitro, as well as changes in the MFN2 expression level. METHODS:Cultured human bronchial epithelial (HBE) cells were initially treated with CSE to induce apoptosis, followed by incubation either with or without RSV. Numerous techniques were used to evaluate the outcomes of the present study, including a cell counting kit-8 assay, real-time quantitative polymerase chain reaction (real-time qPCR), western blotting, JC-1 fluorescence, Hoechst 33342 staining, Annexin V-PI flow cytometry apoptosis analyses, and siRNA technology. RESULTS:A 24 h incubation in 3.5% CSE induced apoptosis in HBE cells, and pretreatment of HBE cells with RSV (20 ?M) significantly suppressed the CSE-induced apoptosis, prevented the CSE-induced decrease in MFN2 levels, suppressed BAX translocation to the mitochondria, and prevented mitochondrial membrane potential loss and cytochrome C release. However, following the transfection of MFN2 siRNA, the anti-apoptotic effects of RSV were significantly attenuated. CONCLUSION:The results of the present study demonstrated that RSV may protect bronchial epithelial cells from CS-induced apoptosis in vitro by preventing mitochondrial dysfunction, and MFN2 may be associated with the anti-apoptotic functions of RSV in HBE cells.
Project description:COPD is a prevalent lung disease with significant impacts on public health. Affected airways exhibit pulmonary neutrophilia and consequent secretion of pro-inflammatory cytokines and proteases, which result in lung emphysema. Probiotics act as nonspecific modulators of the innate immune system that improve several inflammatory responses. To investigate the effect of Lactobacillus rhamnosus (Lr) on cigarette smoke (CS)-induced COPD C57Bl/6 mice were treated with Lr during the week before COPD induction and three times/week until euthanasia. For in vitro assays, murine bronchial epithelial cells as well as human bronchial epithelial cells exposed to cigarette smoke extract during 24 hours were treated with Lr 1 hour before CSE addition. Lr treatment attenuated the inflammatory response both in the airways and lung parenchyma, reducing inflammatory cells infiltration and the production of pro-inflammatory cytokines and chemokines. Also, Lr-treated mice presented with lower metalloproteases in lung tissue and lung remodeling. In parallel to the reduction in the expression of TLR2, TLR4, TLR9, STAT3, and NF-?B in lung tissue, Lr increased the levels of IL-10 as well as SOCS3 and TIMP1/2, indicating the induction of an anti-inflammatory environment. Similarly, murine bronchial epithelial cells as well as human bronchial epithelial cells (BEAS) exposed to CSE produced pro-inflammatory cytokines and chemokines, which were inhibited by Lr treatment in association with the production of anti-inflammatory molecules. Moreover, the presence of Lr also modulated the expression of COPD-associated transcription found into BALF of COPD mice group, i.e., Lr downregulated expression of NF-?B and STAT3, and inversely upregulated increased expression of SOCS3. Thus, our findings indicate that Lr modulates the balance between pro- and anti-inflammatory cytokines in human bronchial epithelial cells upon CS exposure and it can be a useful tool to improve the lung inflammatory response associated with COPD.
Project description:Aldehydes in cigarette smoke (CS) impair mitochondrial function and reduce ciliary beat frequency (CBF), leading to diminished mucociliary clearance (MCC). However, the effects of aldehyde e-cigarette flavorings on CBF are unknown. The purpose of this study was to investigate whether cinnamaldehyde, a flavoring agent commonly used in e-cigarettes, disrupts mitochondrial function and impairs CBF on well-differentiated human bronchial epithelial (hBE) cells. To this end, hBE cells were exposed to diluted cinnamon-flavored e-liquids and vaped aerosol and assessed for changes in CBF. hBE cells were subsequently exposed to various concentrations of cinnamaldehyde to establish a dose-response relationship for effects on CBF. Changes in mitochondrial oxidative phosphorylation and glycolysis were evaluated by Seahorse Extracellular Flux Analyzer, and adenine nucleotide levels were quantified by HPLC. Both cinnamaldehyde-containing e-liquid and vaped aerosol rapidly yet transiently suppressed CBF, and exposure to cinnamaldehyde alone recapitulated this effect. Cinnamaldehyde impaired mitochondrial respiration and glycolysis in a dose-dependent manner, and intracellular ATP levels were significantly but temporarily reduced following exposure. Addition of nicotine had no effect on the cinnamaldehyde-induced suppression of CBF or mitochondrial function. These data indicate that cinnamaldehyde rapidly disrupts mitochondrial function, inhibits bioenergetic processes, and reduces ATP levels, which correlates with impaired CBF. Because normal ciliary motility and MCC are essential respiratory defenses, inhalation of cinnamaldehyde may increase the risk of respiratory infections in e-cigarette users.