The Punicalagin Compound Mitigates Bronchial Epithelial Cell Senescence Induced by Cigarette Smoke Extract through the PAR2/mTOR Pathway.

BACKGROUND: Tobacco smoke is an important inducer of airway epithelial cell aging. Punicalagin(PCG) is a natural anti-aging compound. The effect of PCG on tobacco smoke-induced airway epithelial cell senescence is unknown. OBJECTIVE: Our study investigated whether PCG can treat the human bronchial epithelial cell line (BEAS-2B) aging by inhibiting the protease-activated receptor 2 (PAR2)/m- TOR pathway. METHODS: Bioinformatics techniques were used to analyze the potential biological functions of PAR2. Molecular dynamics evaluated the binding ability of PCG and PAR2. The CCK8 assay was used to detect the cytotoxicity of CSE and PCG. The activity of the PAR2/mTOR pathway and the expression of the characteristic aging markers p16, p21, and SIRT1 are detected by qRT-PCR and Western blotting. Cell senescence was observed by Senescence-associated ß-galactosidase (SA-ß-gal) staining. The senescence-associated secretory phenotype (SASP): concentrations of interleukin IL-6, IL-8, and TNF- α were detected by ELISA. RESULTS: The GSE57148 bioinformatics analysis dataset showed that PAR2 regulates lung senescence through the mTOR signaling pathway. Molecular dynamics results found that PCG and PAR2 had a strong and stable binding force. CSE induces BEAS-2B cell senescence and activates the PAR2/mTOR pathway. Inhibition of PAR2 mitigated the senescence changes. In addition, PCG's pretreatment can significantly alleviate CSE-induced BEAS-2B cell senescence while inhibiting the PAR2/mTOR pathway. CONCLUSION: PCG has a therapeutic effect on the senescence of airway epithelial cells.

Cigarette smoke extract decreases human bone marrow mesenchymal stromal cell adipogenic differentiation.

BACKGROUND: Smoking and nicotine impose detrimental health effects including adipose tissue dysfunction. Despite extensive physiological evidence, the cellular mechanisms remain poorly understood, with few studies examining the effects of cigarette smoke extract (CSE) or nicotine on adipocyte differentiation. METHODS: Primary human bone marrow-derived mesenchymal stromal cells (MSCs) were exposed to CSE or nicotine (50-500 ng/ml) during adipogenic differentiation. Cell viability and metabolic activity were assessed via MTT assay. Lipid droplet accumulation was evaluated using Sudan III staining and quantitative image analysis. Adiponectin, IL6, and IL8 concentrations were measured after 35 days using ELISA. RESULTS: At these doses, CSE and nicotine do not immediately affect cell viability but inhibit undifferentiated cell proliferation. Notably, both agents at 50 ng/ml significantly increased lipid accumulation during adipogenesis, while higher CSE doses nearly completely inhibited this process. Additionally, CSE dose-dependently decreased adiponectin secretion and increased IL6 and IL8, indicating a shift towards an inflammatory state. Nicotine alone primarily increased IL6 secretion with less pronounced effects. CONCLUSION: The study highlights the complex impact of CSE and nicotine on adipocyte function during early differentiation from MSCs. Dose-dependent changes in lipid accumulation, cytokine, and adiponectin secretion induced by CSE and nicotine can partly explain smoking-related adipose tissue dysfunction.

Alterations in the molecular regulation of mitochondrial metabolism in human alveolar epithelial cells in response to cigarette- and heated tobacco product emissions.

Mitochondrial abnormalities in lung epithelial cells have been associated with chronic obstructive pulmonary disease (COPD) pathogenesis. Cigarette smoke (CS) can induce alterations in the molecular pathways regulating mitochondrial function in lung epithelial cells. Recently, heated tobacco products (HTPs) have been marketed as harm reduction products compared with regular cigarettes. However, the effects of HTP emissions on human alveolar epithelial cell metabolism and on the molecular mechanisms regulating mitochondrial content and function are unclear. In this study, human alveolar epithelial cells (A549) were exposed to cigarette or HTP emissions in the form of liquid extracts. The oxygen consumption rate of differently exposed cells was measured, and mRNA and protein abundancy of key molecules involved in the molecular regulation of mitochondrial metabolism were assessed. Furthermore, we used a mitophagy detection probe to visualize mitochondrial breakdown over time in response to the extracts. Both types of extracts induced increases in basal-, maximal- and spare respiratory capacity, as well as in cellular ATP production. Moreover, we observed alterations in the abundancy of regulatory molecules controlling mitochondrial biogenesis and mitophagy. Mitophagy was not significantly altered in response to the extracts, as no significant differences compared to vehicle-treated cells were observed.

Cigarette Smoke-Induced Epithelial-to-Mesenchymal Transition: Insights into Cellular Mechanisms and Signaling Pathways.

This review delves into the molecular complexities underpinning the epithelial-to-mesenchymal transition (EMT) induced by cigarette smoke (CS) in human bronchial epithelial cells (HBECs). The complex interplay of pathways, including those related to WNT//ß-catenin, TGF-ß/SMAD, hypoxia, oxidative stress, PI3K/Akt, and NF-κB, plays a central role in mediating this transition. While these findings significantly broaden our understanding of CS-induced EMT, the research reviewed herein leans heavily on 2D cell cultures, highlighting a research gap. Furthermore, the review identifies a stark omission of genetic and epigenetic factors in recent studies. Despite these shortcomings, the findings furnish a consolidated foundation not only for the academic community but also for the broader scientific and industrial sectors, including large tobacco companies and manufacturers of related products, both highlighting areas of current understanding and identifying areas for deeper exploration. The synthesis herein aims to propel further research, hoping to unravel the complexities of the EMT in the context of CS exposure. This review not only expands our understanding of CS-induced EMT but also reveals critical limitations in current methodologies, primarily the reliance on 2D cell cultures, which may not adequately simulate more complex biological interactions. Additionally, it highlights a significant gap in the literature concerning the genetic and epigenetic factors involved in CS-induced EMT, suggesting an urgent need for comprehensive studies that incorporate these types of experiments.

Protein inhibitor of activated signal transducer and activator of transcription 2 is an oncoprotein in oral squamous cell carcinoma and related to cigarette smoking - An in vitro study.

Background/purpose: Oral cancer is one of the most prevalent malignant tumors in Taiwan. Due to the heterogeneity of oral cancer cells, the five-year survival rate of patients is only 50%. Post-translational modifications contribute to protein diversity and directly influence cell functions. The protein inhibitor of activated signal transducer and activator of transcription 2 (PIAS2) is known to undergo post-translational modifications, yet its impact on oral cancer remains unclear. Materials and methods: PIAS2 expression was modulated by transfecting cells with a PIAS2 expression vector or by knocking down PIAS2 using siRNA with low and high PIAS2 expression, respectively. These cells were subjected to invasion, migration, and proliferation assays to evaluate the effects of PIAS2. Changes in genotype, such as epithelial-mesenchymal transition (EMT) markers, were also examined. Additionally, the effect of cigarette smoke condensate (CSC) on PIAS2 expression in oral squamous cell carcinoma (OSCC) cells was investigated. Results: Overexpression of PIAS2 significantly increased the malignant behaviors of oral cancer cells. In YD38 and SAS cells with low PIAS2 expression, overexpression of PIAS2 enhanced proliferation, invasion, and migration. PIAS2 overexpression also affected EMT gene expression, suppressing E-cadherin and increasing fibronectin expression. Conversely, PIAS2 knockdown in OECM1 and SCC25 cells suppressed malignant behaviors and reversed EMT markers, increasing E-cadherin and decreasing fibronectin expression. Furthermore, a dose-dependent increase in PIAS2 expression was observed when OSCC cells were treated with CSC. Conclusion: PIAS2 functions as an oncogene in oral cancer, and cigarette smoking induces PIAS2 expression. Increased PIAS2 levels lead to enhanced malignancy in oral cancer.

In vitro evidence of antioxidant and anti-inflammatory effects of a new nutraceutical formulation explains benefits in a clinical setting of COPD patients.

Background and Aim: Increased oxidative stress within the airways is associated to epithelial damage and amplification of inflammatory responses that in turn contribute to Chronic Obstructive Pulmonary Disease (COPD) progression. This study was aimed to identify whether a new formulation of N-acetylcisteine (NAC), carnitine, curcumin and B2 vitamin could counteract oxidative stress and downstream pro-inflammatory events promoted by cigarette smoke extract (CSE) exposure in primary bronchial epithelial cells (PBEC), both submerged/undifferentiated (S-PBEC) and cultured at the air-liquid interface (ALI-PBEC). Methods: PBEC were exposed to CSE with/without the new formulation or NAC alone and ROS production, IL-8 and IL-6 gene expression and protein release were evaluated. Results: CSE increased ROS, IL-8 and IL-6 gene expression and protein release and the new formulation counteracted these effects. NAC alone was not effective on IL-8 and IL-6 release. The effects of a similar nutraceutical formulation were evaluated in COPD patients treated for six months. The results showed that the treatment reduced the concentration of IL-8 in nasal wash and improved quality of life. Conclusion: The tested formulation, exerting antioxidant and anti-inflammatory effects, can preserve airway epithelial homeostasis and improve clinical symptoms in COPD.

Long-term persistent exposure to cigarette smoke induces AhR driven corneal endothelial dysfunction in mice.

Epidemiological studies show cigarette smoking enhances corneal endothelial dysfunction, but mechanisms remain unclear. Our study reveals that prolonged smoke exposure activates the aryl hydrocarbon receptor (AhR), increasing CYP1B1 expression and accelerating senescence and fibrosis in corneal endothelium, potentially reflecting adaptive responses to maintain corneal resilience. Although these molecular modifications indicate early endothelial dysfunction, no pathological changes were observed. The findings indicate that while chronic cigarette smoke exposure triggers initial molecular alterations and endothelial dysfunction, the progression to Fuchs endothelial corneal dystrophy likely requires additional environmental or genetic factors beyond smoke exposure alone.

Decoding Oral Carcinogenesis and Tumor Progression in Whole Cigarette Smoke Exposure: A Systematic Review.

This systematic review aims to highlight the molecular mechanisms by which whole cigarette smoke affects oral carcinogenesis and its progression in human oral cells, based on evidence from original research articles published in the literature. A literature search was conducted using three databases: Web of Science, Scopus, and PubMed from May to June 2024. The articles were screened, and the data were extracted according to the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) guidelines (2020). The included studies were subsequently evaluated using the Systematic Review Center for Laboratory Animal Experimentation (SYRCLE) tool for bias factors. From the 14 included studies, two types of cell lines were frequently utilized: human oral mucosal epithelial cells or oral squamous cell carcinoma cells. In these cell lines, one of three forms of exposure was applied: cigarette smoke, its extract, or condensate. The mechanism of oral carcinogenesis and tumor progression includes aberrations in the heme metabolic pathway, modulation of miRNA-145, NOD1 and BiP expression, MMP-2, MMP-9, and cathepsin modulation, abnormal TSPO binding, RIP2-mediated NF-κB activation, MZF1-mediated VEGF binding, and activation of the RAGE signaling pathway. In conclusion, cigarette smoke significantly influences the development and progression of oral squamous cell carcinoma, based on the evidence highlighted in human oral cells. While previous studies have focused on specific carcinogens and pathways, this review added to our understanding of the overall impact of whole cigarette smoke on oral carcinogenesis at the molecular and cellular levels.

Cigarette smoke extract induces malignant transformation and DNA damage via c-MET phosphorylation in human bronchial epithelial cells.

Cigarette smoke, a complex mixture produced by tobacco combustion, contains a variety of carcinogens and can trigger DNA damage. Overactivation of c-MET, a receptor tyrosine kinase, may cause cancer and cellular DNA damage, but the underlying mechanisms are unknown. In this work, we investigated the mechanisms of cigarette smoke extract (CSE) induced malignant transformation and DNA damage in human bronchial epithelial cells (BEAS-2B). The results demonstrated that CSE treatment led to up-regulated mRNA expression of genes associated with the c-MET signaling pathway, increased expression of the DNA damage sensor protein γ-H2AX, and uncontrolled proliferation in BEAS-2B cells. ATR, ATR, and CHK2, which are involved in DNA damage repair, as well as the phosphorylation of c-MET and a group of kinases (ATM, ATR, CHK1, CHK2) involved in the DNA damage response were all activated by CSE. In addition, CSE activation promotes the phosphorylation modification of ATR, CHK1 proteins associated with DNA damage repair. The addition of PHA665752, a specific inhibitor of c-MET, or knock-down with c-MET both attenuated DNA damage, while overexpression of c-MET exacerbated DNA damage. Thus, c-MET phosphorylation may be involved in CSE-induced DNA damage, providing a potential target for intervention in the prevention and treatment of smoking-induced lung diseases.

Characterization of cigarette smokeomics by in situ solid-phase microextraction and confined-space direct analysis in real time mass spectrometry.

Characterization of chemical composition in cigarette smoke is essential for establishing smoke-related exposure estimates. Currently used methods require complex sample preparation with limited capability for obtaining accurate chemical information. We have developed an in situ solid-phase microextraction (SPME) method for online processing of smoke aerosols and directly coupling the SPME probes with confined-space direct analysis in real time (cDART) ion source for high-resolution mass spectrometry (MS) analysis. In a confined space, the substances from SPME probes can be efficiently desorbed and ionized using the DART ion source, and the diffusion and evaporation of volatile species into the open air can be largely avoided. Using SPME-cDART-MS, mainstream smoke (MSS) and side-stream smoke (SSS) can be investigated and the whole analytical protocol can be accomplished in a few min. More than five hundred substances and several classes of compounds were detected and identified. The relative contents of 13 tobacco alkaloids were compared between MSS and SSS. Multivariate data analysis unveiled differences between different types of cigarette smoke and also discovered the characteristic ions. The method is reliable with good reproducibility and repeatability, and has the potential to be quantitative. This study provides a simple and high-efficiency method for smokeomics profiling of complex aerosol samples with in situ online extraction of volatile samples, and direct integration of extracted probes with a modified ambient ionization technique.

SMAD4 promotes EMT in COPD airway remodeling induced by cigarette smoke through interaction with O-GlcNAc transferase.

Cigarette smoke (CS) is a prevalent chemical indoor air contaminant known to be the primary cause of EMT during airway remodeling in COPD. While some evidence indicates the involvement of SMAD4 in EMT across certain diseases, its specific role in CS-induced EMT in airway remodeling associated with COPD is not established. In our research, we observed a substantial upregulation in SMAD4 expression, O-GlcNAcylation and EMT in patients with COPD, as well as in vitro and in vivo COPD models induced by CS, than those of the controls. Downregulation of SMAD4 resulted in a reduction in CS-induced EMT in vitro and in vivo. As a post-translational modification of proteins, O-GlcNAcylation is dynamically controlled by the duo of enzymes: O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT) and O-GlcNAcase (OGA). We further discovered the enhancement of O-GlcNAcylation levels induced by CS was due to an elevated OGT expression, as the expression of OGA remained unchanged. Using an OGT inhibitor (OSMI-1) counteracted the effects of SMAD4 on EMT. Whereas, overexpressing OGT increased SMAD4 expression and promoted EMT. OGT-mediated SMAD4 O-GlcNAcylation shielded SMAD4 from proteasomal degradation by reducing its ubiquitination, thereby aiding in SMAD4 stabilization in response to EMT induced by CS. Overall, this research uncovers a fresh pathway for CS-induced EMT in the airway remodeling of COPD and offers valuable insights.

Association between serum cotinine and muscle mass: results from NHANES 2011-2018.

PURPOSE: Recently, the detrimental effect of cigarette smoking on muscle metabolism has attracted much attention, but the relationship between cigarette smoking and muscle mass is poorly understood. Thus, this study investigated the association between exposure to cigarette smoke, defined based on serum cotinine, and muscle mass in the US population. METHODS: We utilized National Health and Nutrition Examination Survey (NHANES) data between 2011 and 2018 for analysis. Data on serum cotinine, muscle mass (quantified by appendicular skeletal muscle mass index, ASMI), and covariates were extracted and analyzed. Weighted multivariate linear regression analyses and smooth curve fittings were performed to investigate the association between serum cotinine and ASMI. Subgroup analyses were stratified by gender, race and smoking status. When nonlinearity was detected, the threshold effects were analyzed using a two-piecewise linear regression model. RESULTS: In total, 8004 participants were included for analysis. The serum level of cotinine was negatively associated with ASMI in the fully adjusted model. Furthermore, comparing participants in the highest vs. the lowest tertile of serum cotinine, we found that ASMI decreased by 0.135 Kg/m2. In subgroup analysis stratified by gender and race, the association between serum cotinine and ASMI remained significant in all genders and races. In addition, the association remained significant among current and former smokers, but not among those who never smoked. Smooth curve fittings showed nonlinear relationships between serum cotinine and ASMI, with the inflection points identified at 356 ng/mL. CONCLUSIONS: Our study revealed that serum cotinine was negatively related to muscle mass. This finding improves our understanding of the deleterious effects of cigarette smoking on muscle mass and highlights the importance of smoking cessation for muscle health.

Prenatal cigarette smoke exposure sensitizes acetaminophen-induced liver injury by modulating miR-34a-5p in male offspring mice.

Introduction: Cigarette smoke (CS) exacerbates the severity of diseases not only in lungs, but also in systemic organs having no direct contact with smoke. In addition, smoking during pregnancy can have severe health consequences for both the mother and the fetus. Therefore, our aim was to evaluate effects of prenatal exposure to CS on acetaminophen (APAP)-induced acute liver injury (ALI) in offspring. Methods: Female C57BL/6 mice on day 6 of gestation were exposed to mainstream CS (MSCS) at 0, 150, 300, or 600 µg/L for 2 h a day, 5 days a week for 2 weeks using a nose-only exposure system. At four weeks old, male offspring mice were injected intraperitoneally with a single dose of APAP at 300 mg/kg body weight to induce ALI. Results: Maternal MSCS exposure significantly amplified pathological effects associated with ALI as evidenced by elevated serum alanine aminotransferase levels, increased hepatocellular apoptosis, higher oxidative stress, and increased inflammation. Interestingly, maternal MSCS exposure reduced microRNA (miR)-34a-5p expression in livers of offspring. Moreover, treatment with a miR-34a-5p mimic significantly mitigated the severity of APAP-induced hepatotoxicity. Overexpression of miR-34a-5p completely abrogated adverse effects of maternal MSCS exposure in offspring with ALI. Mechanistically, miR-34a-5p significantly decreased expression levels of hepatocyte nuclear factor 4 alpha, leading to down-regulated expression of cytochrome P450 (CYP)1A2 and CYP3A11. Discussion: Prenatal exposure to MSCS can alter the expression of miRNAs, even in the absence of additional MSCS exposure, potentially increasing susceptibility to APAP exposure in male offspring mice.

Dendritic Cells Promote the Differentiation of ILCs into NCR-ILC3s in the Lungs of Mice Exposed to Cigarette Smoke.

The involvement of Group 3 innate lymphoid cells (ILC3s) and dendritic cells (DCs) in chronic lung inflammation has been increasingly regarded as the key to understand the inflammatory mechanisms of smoke-related chronic obstructive pulmonary disease (COPD). However, the mechanism underlying the engagement of both remains unclear. Our study aimed to explore NCR-ILC3 differentiation in the lungs of mice exposed to cigarette smoke (CS) and to further investigate whether DCs activated by CS exposure contribute to the differentiation of ILCs into NCR-ILC3s. The study involved both in vivo and in vitro experiments. In the former, the frequencies of lung NCR-ILC3s and NKp46-IL-17A+ ILCs and the expression of DCs, CD40, CD86, IL-23, and IL-1ß quantified by flow cytometry were compared between CS-exposed mice and air-exposed mice. In the latter, NKp46-IL-17A+ ILC frequencies quantified by flow cytometry were compared after two cocultures, one involving lung CD45+Lin-CD127+ ILCs sorted from air-exposed mice and DCs sifted by CD11c magnetic beads from CS-exposed mice and another including identical CD45+Lin-CD127+ ILCs and DCs from air-exposed mice. The results indicated significant increases in the frequencies of NCR-ILC3s and NKp46-IL-17A+ ILCs; in the expression of DCs, CD40, CD86, IL-23, and IL-1ß in CS-exposed mice; and in the frequency of NKp46-IL-17A+ ILCs after the coculture with DCs from CS-exposed mice. In conclusion, CS exposure increases the frequency of lung ILCs and NCR-ILC3s. CS-induced DC activation enhances the differentiation of ILCs into NCR-ILC3s, which likely acts as a mediating step in the involvement of NCR-ILC3s in chronic lung inflammation.

Mitochondrial free radicals contribute to cigarette smoke condensate-induced impairment of oxidative phosphorylation in the skeletal muscle in situ.

Oxidative stress plays a critical role in cellular dysfunction associated with cigarette smoke exposure and aging. Some chemicals from tobacco smoke have the potential to amplify mitochondrial ROS (mROS) production, which, in turn, may impair mitochondrial respiratory function. Accordingly, the present study tested the hypothesis that a mitochondria-targeted antioxidant (MitoTEMPO, MT) would attenuate the inhibitory effects of cigarette smoke on skeletal muscle respiratory capacity of middle-aged mice. Specifically, mitochondrial oxidative phosphorylation was assessed using high-resolution respirometry in permeabilized fibers from the fast-twitch gastrocnemius muscle of middle-aged C57Bl/6J mice. Before the assessment of respiration, tissues were incubated for 1hr with a control buffer (CON), cigarette smoke condensate (2 % dilution, SMOKE), or MitoTEMPO (10 µM) combined with cigarette smoke condensate (MT + SMOKE). Cigarette smoke condensate (CSC) decreased maximal-ADP stimulated respiration (CON: 60 ± 15 pmolO2.s-1.mg-1 and SMOKE: 33 ± 8 pmolO2.s-1.mg-1; p = 0.0001), and this effect was attenuated by MT (MT + SMOKE: 41 ± 7 pmolO2.s-1.mg-1; p = 0.02 with SMOKE). Complex-I specific respiration was inhibited by CSC, with no significant effect of MT (p = 0.35). Unlike CON, the addition of glutamate (ΔGlutamate) had an additive effect on respiration in fibers exposed to CSC (CON: 0.9 ± 1.1 pmolO2.s-1.mg-1 and SMOKE: 5.4 ± 3.7 pmolO2.s-1.mg-1; p = 0.008) and MT (MT + SMOKE: 8.2 ± 3.8 pmolO2.s-1.mg-1; p ≤ 0.01). Complex-II specific respiration was inhibited by CSC but was partially restored by MT (p = 0.04 with SMOKE). Maximal uncoupled respiration induced by FCCP was inhibited by CSC, with no significant effect of MT. These findings underscore that mROS contributes to cigarette smoke condensate-induced inhibition of mitochondrial respiration in fast-twitch gastrocnemius muscle fibers of middle-aged mice thus providing a potential target for therapeutic treatment of smoke-related diseases. In addition, this study revealed that CSC largely impaired muscle respiratory capacity by decreasing metabolic flux through mitochondrial pyruvate transporter (MPC) and/or the enzymes upstream of α-ketoglutarate in the Krebs cycle.

Corrigendum: Cigarette smoke restricts the ability of mesenchymal cells to support lung epithelial organoid formation.

[This corrects the article DOI: 10.3389/fcell.2023.1165581.].

Transcriptomic analysis reveals distinct effects of cigarette smoke on murine airspace and bone-marrow derived macrophages.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is an inflammatory airway disease characterized by emphysema and chronic bronchitis and a leading cause of mortality worldwide. COPD is commonly associated with several comorbid diseases which contribute to exacerbated patient outcomes. Cigarette smoke (CS) is the most prominent risk factor for COPD development and progression and is known to be detrimental to numerous effector functions of lung resident immune cells, including phagocytosis and cytokine production. However, how CS mediates the various pathologies distant from the lung in COPD, and whether CS has a similar biological effect on systemic immune cells remains unknown. METHODS: C57BL/6 mice were exposed to 8 weeks of CS as an experimental model of COPD. Bone marrow cells were isolated from both CS-exposed and room air (RA) control mice and differentiated to bone marrow-derived macrophages (BMDMs). Airspace macrophages (AMs) were isolated from the same CS-exposed and RA mice and bulk RNA-Seq performed. The functional role of differentially expressed genes was assessed through gene ontology analyses. Ingenuity Pathway Analysis was used to determine the activation states of canonical pathways and upstream regulators enriched in differentially expressed genes in both cell types, and to compare the differences between the two cell types. RESULTS: CS induced transcriptomic changes in BMDMs, including an upregulation of genes in sirtuin signalling and oxidative phosphorylation pathways and a downregulation of genes involved in histone and lysine methylation. In contrast, CS induced decreased expression of genes involved in pathogen response, phagosome formation, and immune cell trafficking in AMs. Little overlap was observed in differentially expressed protein-coding genes in BMDMs compared to AMs and their associated pathways, highlighting the distinct effects of CS on immune cells in different compartments. CONCLUSIONS: CS exposure can induce transcriptomic remodelling in BMDMs which is distinct to that of AMs. Our study highlights the ability of CS exposure to affect immune cell populations distal to the lung and warrants further investigation into the functional effects of these changes and the ensuing role in driving multimorbid disease.

MicroRNA mediated suppression of airway lactoperoxidase by TGF-ß1 and cigarette smoke promotes airway inflammation.

Transforming Growth Factor Beta1 (TGF-ß1) signaling is upregulated in Chronic Obstructive Pulmonary disease (COPD), smokers, and people living with HIV. Cigarette smoking and HIV are also independent risk factors for COPD. Chronic inflammation is a hallmark of COPD. However, the underlying mechanisms remain unknown. Previous research has suggested that TGF-ß1 alters the airway epithelial microRNAome and transcriptome, potentially contributing to lung inflammation. The Lactoperoxidase (LPO) system is an integral component of innate immunity within the airway. LPO plays a crucial role in host defense by catalyzing the oxidation of thiocyanate to hypothiocyanite in the presence of hydrogen peroxide (H2O2), generating a potent antibacterial and antiviral agent. Additionally, the LPO system potentially aids in maintaining cellular redox balance by reducing the levels of H2O2, thus mitigating oxidative stress within the airway epithelium. LPO dysfunction can impair immune responses and exacerbate inflammatory processes in respiratory diseases.In this study, primary bronchial epithelial cells and bronchial cell lines were treated with TGF-ß1 and exposed to cigarette smoke to characterize the effect of these factors on LPO and their downstream effects. RT-qPCR and Western Blot were applied to quantify mRNA and proteins' expression. The levels of H2O2 were detected using the Amplex Red Assay. Magnetofection and transfection were applied to probe the effect of miR-449b-5p. Staining procedures using the MitoTracker Green and C12FDG dyes were used to establish mitochondria mass and senescence. The levels of pro-inflammatory cytokines were measured via Luminex assays.We found that TGF-ß1 and cigarette smoke suppressed airway LPO expression, increasing H2O2 levels. This increase in H2O2 had downstream effects on mitochondrial homeostasis, epithelial cellular senescence, and the pro-inflammatory cytokine response. We demonstrate for the first time that airway LPO is regulated by TGF-ß1-induced miRNA-mediated post-transcriptional silencing through miR-449b-5p in the lungs. Further, we identify and validate miR-449-5p as the candidate miRNA upregulated by TGF-ß1, which is involved in LPO suppression. This paper demonstrates a new mechanism by which TGF-ß1 can lead to altered redox status in the airway.

Color Modifications of a Maxillofacial Silicone Elastomer under the Effect of Cigarette Smoke.

Although it is known (from the observations of medical professionals) that cigarette smoke negatively affects maxillofacial prostheses, especially through staining/discoloration, systematic research in this regard is limited. Herein, the color modifications of M511 maxillofacial silicone, unpigmented and pigmented with red or skin tone pigments, covered with mattifiers, or with makeup and mattifiers, and directly exposed to cigarette smoke, were investigated by spectrophotometric measurements in the CIELab and RGB color systems. The changes in color parameters are comparatively discussed, showing that the base silicone material without pigmentation and coating undergoes the most significant modifications. Visible and clinically unacceptable changes occurred after direct exposure to only 20 cigarettes. By coating and application of makeup, the material is more resistant to color changes, which suggests that surface treatments provide increased protection to adsorption of the smoke components. The dynamic water vapor sorption (DVS) measurements indicate a decrease of the sorption capacity in pigmented versus unpigmented elastomers, in line with the changes in color parameters.

Short Term Exposure of Sheep Tracheal Epithelium to Cigarette Smoke Extract Reduces ENaC Current: A Pilot Study.

BACKGROUND/AIM: Cigarette smoke has been shown to induce a phenotype in humans known as "acquired cystic fibrosis". This occurs because the cystic fibrosis transmembrane conductance regulator (CFTR) functions are impaired systemically due to the deleterious effects of smoke components. Elucidation of cigarette smoke effects on the tracheal epithelium is important. The aim of this study was to develop an ex vivo sheep tracheal model to investigate tracheal ion function. In this model, the epithelial sodium channel (ENaC) is inhibited after exposure to cigarette smoke extract (CSE) as a proof of principle. MATERIALS AND METHODS: Tracheas were isolated from healthy sheep and the tracheal epithelium was surgically excised. Tissues were mounted in Ussing chambers and the short circuit current (Isc) was measured after incubation with 5% CSE in PBS or PBS alone for 30 min. The function of ENaC was investigated by the addition of amiloride (10-5M) apically. Western blot analysis was performed to assess differences in ENaC quantity after CSE exposure. Some specimens were stained with H&E for detection of histological alterations. RESULTS: The amiloride effect on normal epithelium led to a significant decrease in Isc [ΔI=33±5.92 µA/cm2; p<0.001 versus control experiments (ΔI=1.44±0.71 µA/cm2)]. After incubation with CSE, ENaC Isc was significantly reduced (ΔI=14.80±1.96 µA/cm2; p<0.001). No differences in αENaC expression were observed between CSE-exposed and normal tracheal epithelium. Histological images post CSE incubation revealed decreases in the height of the epithelium, with basal cell hyperplasia and loss of ciliated cells. CONCLUSION: Reduced ENaC inhibition by amiloride after CSE incubation could be due to alterations in the tracheal epithelium.