Cigarette Smoke Condensate Exposure Research Articles

MitoQ relieves mitochondrial dysfunction in UVA and cigarette smoke-induced Fuchs endothelial corneal dystrophy

Fuchs endothelial corneal dystrophy (FECD), a degenerative corneal condition, is characterized by the droplet-like accumulation of the extracellular matrix, known as guttae and progressive loss of corneal endothelial cells ultimately leading to visual distortion and glare. FECD can be influenced by environmental stressors and genetic conditions. However, the role of mitochondrial dysfunction for advancing FECD pathogenesis is not yet fully studied. Therefore, in the present study we sought to determine whether a combination of environmental stressors (ultraviolet-A (UVA) light and cigarette smoke condensate (CSC)) can induce mitochondrial dysfunction leading to FECD. We also investigated if MitoQ, a water-soluble antioxidant, can target mitochondrial dysfunction induced by UVA and CSC in human corneal endothelial cells mitigating FECD pathogenesis. We modeled the FECD by increasing exogenous oxidative stress with CSC (0.2%), UVA (25J/cm2) and a combination of UVA + CSC and performed a temporal analysis of their cellular and mitochondrial effects on HCEnC-21T immortalized cells in vitro before and after MitoQ (0.05 μM) treatment. Interestingly, we observed that a combination of UVA + CSC exposure increased mitochondrial ROS and fragmentation leading to a lower mitochondrial membrane potential and increased levels of cytochrome c release leading to apoptosis and cell death. MitoQ intervention successfully mitigated these effects and restored cell viability. The UVA + CSC model could be used to study stress induced mitochondrial dysfunction. Additionally, MitoQ can serve as a viable antioxidant in attenuating mitochondrial dysfunction, underscoring its potential as a molecular-focused treatment approach to combat FECD pathogenesis.

Abstract 1463: Characterizing inflammation-induced epigenetic and transcriptomic alterations driving NSCLC using lung organoid models

Abstract Chronic inflammation is a major driver of lung cancer and contributes to its pathogenesis by inducing genetic and epigenetic alterations. These, in turn, modulate the microenvironment to facilitate emergence of tumor promoting immune evasive mechanisms. The evolution of epigenetic events and their role, both alone, and in association with lung cancer specific genetic drivers in initiation of lung cancer is not well delineated. Specific pathological subtypes of non-small cell lung cancer (NSCLC) have distinct cells of origin, suggestive of cell type-specific changes in driving their development. We employed lung organoid models with chronic exposure to cigarette smoke condensate (CSC) to elucidate sequential, cell type-specific epigenetic and transcriptomic alterations and their role in co-operating with genetic events to drive development of NSCLC. We examined epigenomic and transcriptomic alterations over a 6-month exposure period by DNA methylation arrays, ATAC-seq, and RNA-seq. We observe that CSC exposure causes distinct morphological changes in organoid structure and composition, accompanied by an increase in proliferative potential. Using flow cytometry and immunofluorescent staining approaches, we demonstrate a chronic inflammation-driven shift in stem cell populations accompanied by reduction in differentiated cell types. Bulk and single-cell RNA sequencing approaches identified distinct temporal changes in transcriptional signatures in CSC exposed organoids suggestive of evolution of an immune evasive and pro-tumorigenic phenotype. Gene set enrichment analysis revealed a downregulation of interferon signaling and upregulation of Myc targets and oxidative phosphorylation pathways in CSC treated organoids. Notably, co-culturing organoids with key immune cell populations, demonstrate the ability of the CSC exposed organoids to modulate shifts in immune cells from a pro- to anti- inflammatory state. In accordance with this shift in immune cell phenotype, CSC treated organoids secreted decreased levels of pro-inflammatory cytokines as measured by multiplex ELISA assays. Genome wide DNA methylation analysis reveal that CSC methylated genes were associated with differentiation and cell fate commitment as well as transcription factor activity. Finally, we demonstrate that introduction of key NSCLC-specific driver events at the 6-month time point sensitize only CSC treated organoids to tumor formation in vivo, leading to the development of distinct genetic driver-specific subtypes of NSCLC. Altogether, our results demonstrate the ability of chronic inflammation to drive key cell type-specific transcriptomic and epigenetic changes leading to the development of distinct subtypes of NSCLC. Our findings will help identify molecular correlates for early detection and aid development of novel therapeutic strategies for disease interception. Citation Format: Na Wang, Kara Lombardo, Raksha Padaki, Ray-Whay Chiu Yen, Malcolm V. Brock, Leslie Cope, Hariharan Easwaran, Stephen B. Baylin, Michelle Vaz. Characterizing inflammation-induced epigenetic and transcriptomic alterations driving NSCLC using lung organoid models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1463.

Abstract 4830: Predicting drug treatment response in African American lung cancer patients who smoke

Abstract Background: Lung cancer is the third most commonly diagnosed cancer in the US and the leading cause of cancer-related death. African Americans (AAs) have higher lung cancer incidence and mortality rates, and lower survival rates, when compared to their European American (EA) counterparts. It is widely known that cigarette smoking is the strongest risk factor for lung cancer development, but it also plays a significant role in clinical outcome. Cigarette smoking after lung cancer diagnosis (up to 30% patients) comes with several risks, including increased secondary tumors and mortality rates (by 20%), as well as poor treatment outcomes (higher radiation and chemotherapy failure and higher drug clearance). There are limited transcriptomic studies that describe molecular characterizations of AA smokers with lung cancer to predict treatment outcomes. Hypothesis: Cigarette smoking leads to greater drug resistance-related gene expression in AA lung cancer patients. Methods: mRNA expression changes in an AA (NCI-H2172) and EA (NCI-H1944) lung cancer cell line pair exposed to liquid cigarette smoke condensate (CSC) were profiled. The cell lines were matched based on age (62 years), sex (female), histology (lung adenocarcinoma, LUAD, the most common subtype), and culture properties (adherent). The CSC was derived from Murty Pharmaceuticals and prepared by smoking University of Kentucky's 3R4F Standard Research Cigarette on an FTC Smoke Machine. A total of 500,000 cells per well were treated for one hour under standard incubation conditions (37°C, pH 7.0, 5% CO2). Cells were pelleted, RNAs were isolated, treated with DNAse-I to remove contaminating genomic DNA, quantified, and sent for bulk mRNA-sequencing (Novogene Co). Three biological replicates and technical triplicates were performed. Partek Flow and Partek Genomics Suite workflows were used to identify population-specific differentially expressed gene (DEG) signatures and pathway enrichment after CSC exposure. DEG were profiled in LUAD tissues from current smokers in an AA and EA TCGA cohort (n = 18 AAs, 86 EAs). Results: Population-specific gene expression changes were discovered (n = 52 AA DEGs, n = 31 EA DEGs; P <0.001). All of the DEGs were mutually exclusive with no overlap. Some pathways were shared between both populations and others were unique. PI3K/Akt/mTOR signaling pathway enrichment was unique in AA LUAD cells and ECM receptor interactions were distinct in EA LUAD cells. AKT2 (which activates mTOR) had significantly higher expression in LUAD tumor tissues from current AA smokers compared to current EA smokers. Conclusion: Population-specific PI3K/Akt/mTOR pathway alterations may help explain drug resistance in LUAD tumors from AA patients. PI3K signaling is considered a significant cause of resistance to chemotherapy, targeted therapy, and immunotherapy. Future Directions: Expose AA and EA LUAD cell lines to CSC and PI3K/Akt/mTOR pathway inhibitors. Citation Format: Mia Ray, Isaiah Osei-Gyening, Kwabena Acheampong, Marisa Riebesell, Khadijah A. Mitchell. Predicting drug treatment response in African American lung cancer patients who smoke [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4830.

ABCG2 transporter reduces protein aggregation in cigarette smoke condensate-exposed A549 lung cancer cells.

Cigarette smoke-induced protein aggregation damages the lung cells in emphysema and COPD; however, lung cancer cells continue to thrive, evolving to persist in the toxic environment. Here, we showed that upon the cigarette smoke condensate exposure, A549 lung cancer cells exhibit better survival and reduced level of protein aggregation when compared to non-cancerous Beas-2B and H-6053 cells. Our data suggests that upregulation of efflux pumps in cancer cells assists in reducing smoke toxicity. Specifically, we demonstrated that inhibition of the ABCG2 transporter in A549 by febuxostat or its downregulation by shRNA-mediated RNA interference resulted in a significant increase in protein aggregation due to smoke exposure.

Korean Red Ginseng suppresses emphysematous lesions induced by cigarette smoke condensate through inhibition of macrophage-driven apoptosis pathways

BackgroundCigarette smoke is generally accepted as a major contributor to chronic obstructive pulmonary disease (COPD), which is characterized by emphysematous lesions. In this study, we investigated the protective effects of Korean Red Ginseng (KRG) against cigarette smoke condensate (CSC)-induced emphysema. MethodsMice were instilled with 50 mg/kg of CSC intranasally once a week for 4 weeks, KRG was administered to the mice once daily for 4 weeks at doses of 100 or 300 mg/kg, and dexamethasone (DEX, positive control) was administered to the mice once daily for 2 weeks at 3 mg/kg. ResultsKRG markedly decreased the macrophage population in bronchoalveolar lavage fluid and reduced emphysematous lesions in the lung tissues. KRG suppressed CSC-induced apoptosis as revealed by terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick-end labeling staining and Caspase 3 immunohistochemistry. Additionally, KRG effectively inhibited CSC-mediated activation of Bcl-2-associated X protein/Caspase 3 signaling, followed by the induction of cell survival signaling, including vascular endothelial growth factor/phosphoinositide 3-kinase/protein kinase B in vivo and in vitro. The DEX group also showed similar improved results in vivo and in vitro. ConclusionTaken together, KRG effectively inhibits macrophage-mediated emphysema induced by CSC exposure, possibly via the suppression of pro-apoptotic signaling, which results in cell survival pathway activation. These findings suggest that KRG has therapeutic potential for the prevention of emphysema in COPD patients.

Abstract LB046: Characterizing the role of inflammation-induced epigenetic alterations in modulating the immune microenvironment during lung cancer initiation

Abstract One of the most important goals of developing timely detection and effective intervention strategies for lung cancer is to understand the biology and molecular mechanisms involved in early-stage evolution of this disease. Chronic inflammation is a major driver of lung cancer and contributes to its pathogenesis by inducing genetic and epigenetic abnormalities, as well as shifts in key immune cell populations. However, the evolution of these changes and the mechanisms underlying the early stages in development of pre-neoplasias and progression to invasive tumors have not been well delineated. Monolayer cell culture systems, while providing valuable insights into lung cancer development, lack the complexity of an intact organ system. This study aims to overcome these limitations by using a 3D lung organoid model to delineate the role of epigenetic alterations in driving initiation of non-small cell lung cancer. The stem cell enriched nature of the organoids makes it an ideal system to evaluate the implications of epigenetic alterations in these key cell types and their role in cancer initiation. To better identify the epigenetic alterations that play key roles in lung cancer initiation, cigarette smoke condensate (CSC) was applied to normal lung organoids to mimic chronic inflammatory exposure. Epigenomic and transcriptomic alterations were evaluated by genome-wide DNA methylation analysis, ATAC-seq, and RNA-seq. Flow cytometry and confocal microscopy were used to evaluate changes in composition of cell populations comprising the organoids. Long-term CSC exposure caused distinct morphological changes in organoid structure and cellular composition, accompanied by an increased proliferative potential. Analysis of changes in cell composition revealed increases in Krt14 expression in CSC treated organoids, leading to key shifts in basal stem cell populations from a TP63+KRT5+ to a TP63+KRT5+KRT14+ population. This was accompanied by reduction in differentiated cell types, as analyzed by qRT-PCR. Analysis of genome wide DNA methylation showed increases in promoter DNA methylation in key genes associated with lung tumorigenesis. Most importantly, co-culture studies involving culturing CSC treated and control organoids with key immune cells revealed that chronic CSC treatment caused modulation of the microenvironment from a pro- to an anti-inflammatory state. Our results suggest that chronic inflammation causes key shifts in populations of lung stem cells with an associated decrease in differentiation potential. These changes are accompanied by DNA methylation and gene expression changes suggestive of an increased tumorigenic potential. Finally, these changes in the CSC treated organoids are associated with the ability to modulate the function of key immune cells associated with lung tumorigenesis from a pro- to an anti-inflammatory phenotype. Results from our study, will aid in developing novel biomarker-based methodologies to distinguish and treat lung cancer in its early stages. Citation Format: Na Wang, Ray-Whay Chiu Yen, Hamza Khan, Malcolm V. Brock, Hariharan Easwaran, Stephen B. Baylin, Michelle Vaz. Characterizing the role of inflammation-induced epigenetic alterations in modulating the immune microenvironment during lung cancer initiation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB046.

Loranthus tanakae Franch. & Sav. Suppresses Inflammatory Response in Cigarette Smoke Condensate Exposed Bronchial Epithelial Cells and Mice.

Loranthus tanakae Franch. & Sav. found in China, Japan, and Korea is traditionally used for managing arthritis and respiratory diseases. In this study, we analyzed the components of L. tanakae 70% ethanol extract (LTE) and investigated the therapeutic effects of LTE on pulmonary inflammation using cells exposed to cigarette smoke condensate (CSC) and lipopolysaccharide (LPS) in vitro and in vivo in mice and performed a network analysis between components and genes based on a public database. We detected quercitrin, afzelin, rhamnetin 3-rhamnoside, and rhamnocitrin 3-rhamnoside in LTE, which induced a significant reduction in inflammatory mediators including interleukin (IL)-1β, IL-6, tumor necrosis factor (TNF)-α and inflammatory cells in CSC exposed H292 cells and in mice, accompanied by a reduction in inflammatory cell infiltration into lung tissue. In addition, LTE increased translocation into the nuclei of nuclear factor erythroid-2-related factor 2 (Nrf2). By contrast, the activation of nuclear factor (NF)-κB, induced by CSC exposure, decreased after LTE application. These results were consistent with the network pharmacological analysis. In conclusion, LTE effectively attenuated pulmonary inflammation caused by CSC+LPS exposure, which was closely involved in the enhancement of Nrf2 expression and suppression of NF-κB activation. Therefore, LTE may be a potential treatment option for pulmonary inflammatory diseases including chronic obstructive pulmonary disease (COPD).

The Sulfotransferase SULT1C2 Is Epigenetically Activated and Transcriptionally Induced by Tobacco Exposure and Is Associated with Patient Outcome in Lung Adenocarcinoma.

Lung cancer is the leading cause of cancer-related death. Tobacco exposure is associated with 80–90% of lung cancer cases. The SULT1C2 sulfotransferase modifies xenobiotic compounds to enhance secretion but can also render these compounds carcinogenic. To determine if SULT1C2 contributes to tobacco-related carcinogenesis in the lung, we analyzed the expression and epigenetic state of SULT1C2 in human lung adenocarcinoma (LUAD) samples and in LUAD cell lines exposed to cigarette smoke condensate (CSC). SULT1C2 expression was significantly positively correlated to overall LUAD patient survival in smokers, was elevated in LUAD tumors compared to adjacent non-tumor lung, and was significantly correlated with levels of patient exposure to tobacco smoke. SULT1C2 promoter DNA methylation was inversely correlated with expression in LUAD, and hypomethylation of the SULT1C2 promoter was observed in Asian patients, as compared to Caucasians. In vitro analysis of LUAD cell lines indicates that CSC stimulates expression of SULT1C2 in a dose-dependent and cell-line-specific manner. In vitro methylation of the SULT1C2 promoter significantly decreased transcriptional activity of a reporter plasmid, and SULT1C2 expression was activated by the DNA demethylating agent 5-Aza-2′-deoxycytidine in a cell line in which the SULT1C2 promoter was hypermethylated. An aryl hydrocarbon receptor (AHR) binding site was detected spanning critical methylation sites upstream of SULT1C2. CSC exposure significantly increased AHR binding to this predicted binding site in the SULT1C2 promoter in multiple lung cell lines. Our data suggest that CSC exposure leads to activation of the AHR transcription factor, increased binding to the SULT1C2 promoter, and upregulation of SULT1C2 expression and that this process is inhibited by DNA methylation at the SULT1C2 locus. Additionally, our results suggest that the level of SULT1C2 promoter methylation and gene expression in normal lung varies depending on the race of the patient, which could in part reflect the molecular mechanisms of racial disparities seen in lung cellular responses to cigarette smoke exposure.

Abstract 2663: Determining the role of altered redox metabolism in lung tumorigenesis

Abstract The purpose of this study is to determine the pathways by which short-term cigarette smoke exposure induces expression of the cystine-glutamate antiporter xCT (SLC7A11) and its downstream implications in carcinogenesis. BEAS-2B lung epithelial cells and their tumorigenic counterpart, B39-TL cells, were exposed to cigarette smoke condensate (CSC) for 3-15 days. Cells were also exposed to a range of inhibitors. Erastin and buthionine sulfoximine (BSO) are inhibitors of various steps in the glutathione synthesis pathway, found to be hyperactive in xCT-overexpressing cells. Cells were also exposed to the drug mito-2-hydroxybenzoic acid (mito-2-HOBA), which scavenges isolevuglandins, reactive γ-ketoaldehydes implicated in oxidative stress and carcinogenesis. Expression of xCT and related proteins was measured using Western blotting. Glutathione levels were detected using the luciferase-based GSH-Glo assay (Promega). Oxidative stress was detected using dihydroethidium (DHE) fluorescence. It was found that CSC exposure significantly increased the expression of xCT and SLC1A5, a transporter that potentially works in synergy with xCT, and that tumorigenic B39-TL cells had significantly increased xCT and SLC1A5 expression compared to BEAS-2B cells. In addition, BEAS-2B cells exposed to CSC and a glutathione synthesis inhibitor such as erastin or BSO showed faster depletion of glutathione than cells treated with only inhibitor. Finally, it was found that BEAS-2B cells treated for 3 days with CSC did not show increased levels of superoxide compared to control cells. However, cells treated with CSC and mito-2-HOBA together showed significantly increased superoxide. These data suggest that CSC induces an antioxidant response which is ablated by mito-2-HOBA. This antioxidant response seems to at least partially rely on glutathione synthesis fueled by xCT, as evidenced by inhibitor studies. The response also seems to be activated by isolevuglandins, because removing these molecules significantly raises oxidative stress. The mechanistic link between CSC exposure, isolevuglandin generation, and xCT/glutathione induction will be clarified in further experiments. Citation Format: Alex Camai. Determining the role of altered redox metabolism in lung tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2663.

Long‐term exposure to cigarette smoke influences characteristics in human gingival fibroblasts

Periodontal disease is a chronic inflammatory disease caused by periodontopathic bacteria accumulated in the gingival sulcus and periodontal pocket. Cigarette smoking is a well-established risk factor for periodontal disease, and periodontal tissues in smokers are chronically exposed to cigarette smoke on a long-term basis. In this study, we investigated the effects of long-term exposure to nicotine or cigarette smoke condensate (CSC) on cellular functions of human gingival fibroblasts (HGFs). In vitro-maintained HGFs were divided into two groups. The HGFs of the short-term and the long-term culture groups were cultured for 4 and 25days, respectively, in the presence or absence of nicotine, which is one of the main components of cigarette smoke, or CSC. The cellular proliferation and migration capacities of HGFs exposed to nicotine or CSC were evaluated by WST-1 and wound healing assays. The effects of exposure to nicotine or CSC on the expression of various extracellular matrix (ECM) components, inflammatory cytokines, and senescence-related genes were examined by real-time polymerase chain reaction and enzyme-linked immunosorbent assay. The cellular senescence of HGFs exposed to nicotine or CSC was detected by the senescence-associated β-galactosidase (SA-β-gal) assay. To explore the senescence-associated microRNA (miRNA), we extracted miRNA from the HGFs and the expression profiles were examined by miRNA array. In short-term culture, no significant changes were observed. Long-term exposure of HGFs to nicotine or CSC significantly suppressed their cellular proliferation and migration and upregulated type Ⅰ collagen, type Ⅲ collagen, interleukin (IL)-6, IL-8, p16, p21, and p53mRNA expression, and IL-6 and IL-8 protein expression. Furthermore, long-term nicotine or CSC exposure significantly increased the percentage of SA-β-gal-positive HGFs. In addition, long-term nicotine or CSC exposure reduced miR-29b and miR-199a expression to less than 50% of that in the unstimulated HGFs. These data suggest that long-term smoking habits may reduce wound healing ability, modulate ECM protein homeostasis, stimulate the inflammatory response, and accelerate cellular senescence in HGFs, and consequently accelerate the progression of periodontal diseases.

Functional Analysis of the Enhancer at cg05575921, a DNA Methylation Site that is Tobacco Smoke‐Responsive and Highly Associated with Lung Cancer Risk

This project aims to investigate the molecular role of epigenetic changes from tobacco smoke exposure in the development of lung cancer, the largest cause of cancer-related deaths. The epigenome consists of the heritable chemical groups on the genome that regulate gene expression. One well-studied epigenetic mark is DNA methylation, which occurs at CpG dinucleotides and is usually associated with gene silencing. Environmental and lifestyle factors such as smoking can induce heritable alterations to the epigenome and can thereby influence disease risk. However, the mechanisms by which tobacco smoke exposure leads to increased lung cancer risk are not fully understood. Past studies have linked hypomethylation of cg05575921, a CpG in the aryl hydrocarbon receptor repressor (AHRR) gene, a regulator of the xenobiotic response pathway, as a strong indicator of lung cancer risk. Tobacco smoke exposure results in hypomethylation of cg05575921 in blood and lung tissue. Our previous research showed that cg05575921 flanks a tobacco smoke-inducible enhancer linked to the induction of AHRR expression. Our guiding hypothesis is that hypomethylation of cg05575921 is a byproduct of enhancer activation by tobacco smoke, and that it is the enhancer activity that has functional consequences, not the loss of methylation at cg05575921. To evaluate the functional role of this enhancer, we used CRISPR/Cas9 gene editing technology to generate monoclonal A549 cell lines with targeted DNA deletions of either the enhancer region, the CpG site adjacent to it, or a negative control region lacking regulatory marks. Resulting clones will be treated with cigarette smoke condensate (CSC) and analyzed for AHRR expression through whole-transcriptome mRNA sequencing (RNA-seq). Initial findings show a high rate of successful deletions in transfected cells, bolstering the technique's potential for studying enhancers’ roles in cancerous transformation. Thus far, clones in the enhancer deletion group have been screened via PCR for the desired deletion, further characterized via Sanger sequencing across deletion sites and heterozygous SNPs, exposed to CSC for 48 hours, and RNA has been subjected to RNA-seq. Analysis of RNA-seq data is ongoing. CpG deletion clones have been screened and sequenced and are ready to undergo CSC exposure. Negative control deletion clones have been generated and are growing in preparation for screening. Our analyses promise to shed light on the mechanism underlying the link between cg05575921 hypomethylation and lung cancer risk.

Molecular insights of cigarette smoke condensate-activated NLRP3 inflammasome in THP-1 cells in a stage-specific atherogenesis

BackgroundSmoking induces excessive inflammation which is associated with all the stages of atherosclerosis. Earlier, we reported Nod-like receptor protein 3 (NLRP3) inflammasome activation as a pro-atherosclerotic property of cigarette smoking. In the present study, we aimed to explore the underlying detailed upstream mechanism and the cellular status of putative downstream molecules of cigarette smoke condensate (CSC)-activated NLRP3 inflammasome in atherosclerotic disease. Methods and resultsTHP-1 monocytes, macrophages and foam cells represent crucial stages of atherogenesis as initiation, progression and development. To determine the upstream molecular regulators of smoking-induced NLRP3 inflammasome in atherogenesis, Myeloid differentiation primary response 88/Nuclear Factor kappa-light-chain-enhancer of activated B cells (MyD88/NF-κB) and Suppressor of cytokine signaling 3/Signal transduction and activator of transcription 3 (SOCS3/STAT3) pathways were elucidated. Stage-specific THP-1 cells were treated with MyD88 and SOCS3/STAT3 inhibitors. The results showed that MyD88 inhibition markedly attenuated the expression of NLRP3 markers (NLRP3, caspase-1, Interleukin (IL)-1β and IL-18), IL-6, SOCS3 and NF-κB. Moreover, the secretory levels of pro-cytokines were also significantly reduced in culture media. In contrast, no changes were observed with SOCS3/STAT3 inhibitor. Further, ac-vyad-cmk, an inflammasome inhibitor was used to explore the downstream targets of CSC-activated NLRP3 inflammasome in atherosclerotic process. The transcriptional profiling of 25 atherosclerotic markers was carried out using ExProfile™ Custom Gene qPCR Arrays. CSC exposure upregulated the expression of 17 genes and downregulated 4 genes in a stage-specific manner. Inhibitory experiments showed aberrant changes in CSC-regulated genes. Altogether, 15 molecules were common in all three stages. ConclusionThe findings may suggest that MyD88/NF-κB pathway is an upstream regulator of NLRP3 inflammasome underlying smoking-induced atherosclerosis. Notably, 15 atherosclerotic molecules associated with endothelial dysfunction, scavenger receptors, cholesterol esterification and matrix-metalloproteins were found downstream to CSC-activated NLRP3 inflammasome.

Differential packaging of inflammatory cytokines/ chemokines and oxidative stress modulators in U937 and U1 macrophages-derived extracellular vesicles upon exposure to tobacco constituents.

Smoking, which is highly prevalent in HIV-infected populations, has been shown to exacerbate HIV replication, in part via the cytochrome P450 (CYP)-induced oxidative stress pathway. Recently, we have shown that extracellular vesicles (EVs), derived from tobacco- and/or HIV-exposed macrophages, alter HIV replication in macrophages by cell-cell interactions. We hypothesize that cigarette smoke condensate (CSC) and/or HIV-exposed macrophage-derived EVs carry relatively high levels of pro-oxidant and pro-inflammatory cargos and/or low levels of antioxidant and anti-inflammatory cargos, which are key mediators for HIV pathogenesis. Therefore, in this study, we investigated differential packaging of pro- and anti-inflammatory cytokines/chemokines and pro- and anti-oxidant contents in EVs after CSC exposure to myeloid cells (uninfected U937 and HIV-infected U1 cells). Our results showed that relatively long to short exposures with CSC increased the expression of cytokines in EVs isolated from HIV-infected U1 macrophages. Importantly, pro-inflammatory cytokines, especially IL-6, were highly packaged in EVs isolated from HIV-infected U1 macrophages upon both long and short-term CSC exposures. In general, anti-inflammatory cytokines, particularly IL-10, had a lower packaging in EVs, while packaging of chemokines was mostly increased in EVs upon CSC exposure in both HIV-infected U1 and uninfected U937 macrophages. Moreover, we observed higher expression of CYPs (1A1 and 1B1) and lower expression of antioxidant enzymes (SOD-1 and catalase) in EVs from HIV-infected U1 macrophages than in uninfected U937 macrophages. Together, they are expected to increase oxidative stress factors in EVs derived from HIV-infected U1 cells. Taken together, our results suggest packaging of increased level of oxidative stress and inflammatory elements in the EVs upon exposure to tobacco constituents and/or HIV to myeloid cells, which would ultimately enhance HIV replication in macrophages via cell-cell interactions.

Exposure of cigarette smoke condensate activates NLRP3 inflammasome in vitro and in vivo: A connotation of innate immunity and atherosclerosis

ObjectiveSmoking is known to have detrimental effects on cardiovascular system. However, the potential molecular basis of smoking-induced atherosclerosis remains unclear. NLRP3 inflammasome is implicated in perpetuation of inflammatory response in atherosclerosis. Therefore, we aimed to explore the cytotoxic effects of cigarette smoke condensate (CSC) on the activation of NLRP3 inflammasome in vitro and in vivo. MethodsFor in vitro study, the pro-atherogenic effects of CSC were evaluated in THP-1 monocytes with different dose concentrations (0.1, 1, 5, 10 and 20 µg/ml) for varied time periods (6, 12, 24 and 48 h). For in vivo study, 30 male C57BL/6J mice were employed. 6 mice were sacrificed for baseline investigations. 24 mice were randomly divided into four groups: Group-I:Control mice, Group-II:CSC model, Group-III:High‐fat diet(HFD) model, and Group-IV:HFD + CSC model for 14 weeks (n = 6/group). The group-II and IV mice were injected with 720 µg CSC/20 g body weight intraperitoneally (6 days/week). ResultsIn vitro, higher dosage of CSC (20 µg/ml) was toxic to cells as significant decline in cell viability and proliferation was observed. Furthermore, the mRNA expression of NLRP3 inflammasome and its pro-cytokine levels were significantly augmented on CSC exposure in a dose-dependent manner but impeded in time-dependent manner. In vivo, CSC and HFD independently augmented the expression of NLRP3 inflammasome (~4–10 fold-change) along with pro-cytokine levels in Group-II and III vs Group-I mice whereas, HFD + CSC treatment demonstrated synergistic effects in Group-IV. ConclusionOur data suggest that CSC activates NLRP3 inflammasome in vitro and in vivo and collectively with HFD has synergistic effects in vivo that may promote atherosclerosis.

Exposure of cigarette smoke condensate activates NLRP3 inflammasome in THP-1 cells in a stage-specific manner: An underlying role of innate immunity in atherosclerosis.

BackgroundSmoking is known to affect all the phases of atherosclerosis, thus is considered as an independent and major risk factor. The underlying mechanism responsible for the atherogenic effects of smoking is still uncertain and a major concern. Recent evidence implicates NLRP3 inflammasome, an innate immunity component in the pathogenesis of atherosclerosis. Therefore, we hypothesized that NLRP3 inflammasome may be an associated pathway between smoking and atherosclerosis. Methods and resultsDifferentiation in monocytes, macrophages and foam cells are the key stages in atherosclerotic plaque development, best mimicked by THP-1 cells. Therefore, to determine whether cigarette smoke condensate (CSC) could induce differentiation of THP-1 monocytes into macrophages, morphological changes and the expression levels of the inflammatory surface markers, i.e. CD11b, CD14 and CD36 were analyzed. The results showed that CD14 and CD36 levels were significantly increased in CSC-treated THP-1 monocytes. Further, we investigated the effect of CSC exposure on the status of NLRP3 inflammasome markers, i.e. NLRP3, pro-caspase-1, caspase-1, pro-IL-18, pro-IL-1β, IL-1β and IL-18 in a stage-specific manner. For this, THP-1 monocytes, PMA-differentiated macrophages and oxidized-low density lipoprotein (ox-LDL)-induced macrophage foam cells were exposed to 10 μg/ml of CSC for 6 h. CSC exposure significantly upregulated the expression of NLRP3 inflammasome in CSC-treated cells at both transcriptional and translational levels. Moreover, downstream pro-cytokines, i.e. IL-1β and IL-18 levels were also significantly increased in culture supernatants of CSC-exposed cells. ConclusionThese observations suggest that CSC exposure may activate NLRP3 inflammasome in a stage-specific manner and may promote initiation and progression of atherosclerosis.

B24 The Role of Cigarette Smoke and miR520a in Pulmonary Frizzled 9 Expression

Lung cancer is the deadliest cancer, and for this reason treatment is highly researched. Alternatively, chemoprevention can be used to combat lung cancer in individuals who are at high risk of diagnosis, such as cigarette smokers. Frizzled 9 (Fzd9) is required in vitro for chemopreventive effects of iloprost, a prostacyclin analogue, in the lung. In non-small cell lung cancer (NSCLC) cell lines, Fzd9 activates PPARγ, leading to inhibition of transformed growth. Cigarette smoke exposure decreases Fzd9 expression. The goal of this study is to elucidate the relationship between cigarette smoke and miRNA regulation of Fzd9 expression. NSCLC cells exposed to cigarette smoke condensate (CSC) showed decreased Fzd9 3’ UTR activity, suggesting CSC regulates Fzd9 expression through miRNA. miRNA database analysis suggested miR-95, miR-106b, and miR-520a as potential regulators of Fzd9. Immortalized human bronchial epithelial cells (HBEC) and an Fzd9-positive NSCLC cell line (A549) transfected with miR-520a oligonucleotide mimic showed decreased Fzd9 3’UTR luciferase activity. miR-520a expression increased in HBEC and NSCLC cells after CSC exposure. We have tested a miR-520a inhibitor to use for future rescue experiments in an Fzd9-negative cell line (H322). Transient overexpression of miR-520a in HBEC and A549 did not affect cell viability or proliferation, so we made a stable miR-520a expressing HBEC line that we will use for longer-duration cell assays. miR-520a may play an important role in the regulation of Fzd9 by cigarette smoke, and future experiments will characterize this relationship and potentially impact the application of iloprost chemoprevention.

Cigarette Smoke Condensate Exposure Changes RNA Content of Extracellular Vesicles Released from Small Airway Epithelial Cells

Exposure to environmental tobacco smoke (ETS) is a known risk factor for the development of chronic lung diseases, cancer, and the exacerbation of viral infections. Extracellular vesicles (EVs) have been identified as novel mediators of cell–cell communication through the release of biological content. Few studies have investigated the composition/function of EVs derived from human airway epithelial cells (AECs) exposed to cigarette smoke condensate (CSC), as surrogates for ETS. Using novel high-throughput technologies, we identified a diverse range of small noncoding RNAs (sncRNAs), including microRNA (miRNAs), Piwi-interacting RNA (piRNAs), and transfer RNA (tRNAs) in EVs from control and CSC-treated SAE cells. CSC treatment resulted in significant changes in the EV content of miRNAs. A total of 289 miRNAs were identified, with five being significantly upregulated and three downregulated in CSC EVs. A total of 62 piRNAs were also detected in our EV preparations, with five significantly downregulated and two upregulated in CSC EVs. We used TargetScan and Gene Ontology (GO) analysis to predict the biological targets of hsa-miR-3913-5p, the most represented miRNA in CSC EVs. Understanding fingerprint molecules in EVs will increase our knowledge of the relationship between ETS exposure and lung disease, and might identify potential molecular targets for future treatments.

Adolescent diabetes induced by multiple parental exposures to cigarette smoke condensate

Parental exposure to cigarette smoke is closely related to the development of long-term metabolic diseases in the offspring. However, different exposure times at various developmental stages may cause these effects to vary. In this study, mice were exposed to cigarette smoke condensate (CSC) during the developmental time stages of paternal puberty or/and maternal pregnancy. The results showed that either paternal or maternal exposure to CSC could lead to increased low birth weight (LBW) and fetal growth restriction (FGR) of the offspring, but maternal factors were the leading ones. Moreover, maternal exposure during pregnancy could induce lipid metabolism abnormalities in the adulthood offspring. Most importantly, additional paternal CSC exposure further induced diabetes in adolescent offspring who experienced altered weight gain, blood lipids, and glucose metabolism. A preliminary analysis indicated that the offspring with metabolic abnormalities also had significant changes in their intestinal microbiota. In conclusion, this study showed that parental CSC exposure has an impact on the metabolic properties of the offspring, and multiple parental exposures to adverse factors may significantly increase the risk of long-term metabolic abnormalities.

Cigarette smoke condensate may disturb immune function with apoptotic cell death by impairing function of organelles in alveolar macrophages

Considering that cigarette smoke condensate (CSC) is primarily absorbed through the alveoli in the lungs, herein, we used a mouse alveolar macrophage cell line (MH-S cells). After 24 h exposure, CSC decreased dose-dependently cell viability accompanying an increase in intracellular ROS and NO level. CSC structurally or functionally damaged organelles including mitochondria, ER and lysosome and enhanced the expression of proteins related to apoptosis, ER stress and DNA damage accompanying an elevated proportion of annexin V-bound cells. Meanwhile, the expression of certain proteins related to mitochondrial dynamics (OPA1 and DRP1) and autophagy (ATG5) did not overall show significant dose-dependent change in cells exposed to CSC. More importantly, conversion of LC3-I to LC3B-II, a representative marker for autophagy, was also unclear. Considering that intracellular organelles work together in harmony to perform defense mechanism against foreign bodies, we investigated changes in immune response following CSC exposure. The level of IFN-γ and MIP-1α was elevated in CSC-exposed cells, whereas the MCP-1α level decreased. The expression of chemokine receptors (CD195 and CXCR2) and an adhesion molecule (CD54) increased by CSC treatment, the expression of certain antigen presentation-related proteins (MHC class II, CD40, and CD80) were also enhanced. Meanwhile, the expression of CD86, a co-stimulatory molecule for antigen presentation, dose-dependently decreased. In conclusion, we suggest that CSC may induce apoptotic cell death and disturbance in host defense mechanisms by impairing function of cellular components.

Bile acid and cigarette smoke enhance the aggressive phenotype of esophageal adenocarcinoma cells by downregulation of the mitochondrial uncoupling protein-2.

Limited information is available regarding mechanisms that link the known carcinogenic risk factors of gastro-esophageal reflux and cigarette smoking to metabolic alterations in esophageal adenocarcinoma (EAC). In the present study, we utilized a novel in-vitro model to examine whether bile acid and cigarette smoke increase the aggressiveness of EAC and whether these changes are associated with metabolic changes.EAC cells (EACC) were exposed to 10 μg/ml cigarette smoke condensate (CSC) and/or 100 μM of the oncogenic bile acid, deoxycholic acid (DCA), for 5 days. These exposure conditions were chosen given their lack of effect on proliferation or viability. DCA and CSC increased invasion, migration, and clonogenicity in EAC cells. These changes were associated with concomitant increases in ATP, ROS, and lactate production indicative of increased mitochondrial respiration as well as glycolytic activity. DCA and CSC exposure significantly decreased expression of uncoupling protein-2 (UCP2), a mitochondrial inner membrane protein implicated in regulation of the proton gradient. Knockdown of UCP2 in EACC phenocopied DCA and CSC exposure as evidenced by increased cell migration, invasion, and clonogenicity, whereas over-expression of UCP2 had an inverse effect. Furthermore, over-expression of UCP2 abrogated DCA and CSC-mediated increases in lactate and ATP production in EACC.DCA and CSC promote the aggressive phenotype of EACC with concomitant metabolic changes occurring via downregulation of UCP2. These results indicate that UCP2 is integral to the aggressive phenotype of EACC. This mechanism suggests that targeting alterations in cellular energetics may be a novel strategy for EAC therapy.