Three-dimensional lung parenchyma model for studies of Aspergillus fumigatus infection and antifungal treatment.

Aim: This work aims to standardize the three-dimensional hydroxyethyl-alginate-gelatin (HAG) scaffold as a model to evaluate Aspergillus fumigatus biofilm and antifungal treatments. Methods: The scaffold was characterized by physical, rheological and microscopic analyses; the antibiofilm action was evaluated by determination of cfu and metabolic activity. Results: The scaffold was non-toxic showing stability in aqueous media, swelling capacity, elasticity and had homogeneously distributed pores averaging 190 µm. The A. fumigatus biofilm established itself very well on the scaffold and treatment with amphotericin B and voriconazole reduced viable cells and metabolic activity. Conclusion: The HAG scaffold proved to be a model to mimic lung parenchyma, suitable for establishing a 3D biofilm culture of A. fumigatus and evaluating the efficacy of antifungals.

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Secretome profiling of human epithelial cells exposed to cigarette smoke extract and their effect on human lung microvascular endothelial cells.

Cigarette smoke (CS) is one of the leading causes of pulmonary diseases and can induce lung secretome alteration. CS exposure-induced damages to human pulmonary epithelial cells and microvascular endothelial cells have been extensively demonstrated; however, the effects of the secretome of lung epithelial cells exposed to CS extracts (CSE) on lung microvascular endothelial cells are not fully understood. In this study, we aimed to determine the effects of the secretome of lung epithelial cells exposed to CSE on lung microvascular endothelial cells. Human lung epithelial cells, A549, were exposed to CSE, and the secretome was collected. Human lung microvascular endothelial cells, HULEC-5a, were used to evaluate the effect of the secretome of A549 exposed to CSE. Secretome profile, endothelial cell death, inflammation, and permeability markers were determined. CSE altered the secretome expression of A549 cells, and secretome derived from CSE-exposed A549 cells caused respiratory endothelial cell death, inflammation, and moderately enhanced endothelial permeability. This study demonstrates the potential role of cellular interaction between endothelial and epithelial cells during exposure to CSE and provides novel therapeutic targets or beneficial biomarkers using secretome analysis for CSE-related respiratory diseases.

MPoMA protects against lung epithelial cell injury via p65 degradation.

Numerous cases of lung injury caused by viral infection were reported during the coronavirus disease-19 pandemic. While there have been significant efforts to develop drugs that block viral infection and spread, the development of drugs to reduce or reverse lung injury has been a lower priority. This study aimed to identify compounds from a library of compounds that prevent viral infection that could reduce and prevent lung epithelial cell damage. We investigated the cytotoxicity of the compounds, their activity in inhibiting viral spike protein binding to cells, and their activity in reducing IL-8 production in lung epithelial cells damaged by amodiaquine (AQ). We identified N-(4-(4-methoxyphenoxy)-3-methylphenyl)-N-methylacetamide (MPoMA) as a non-cytotoxic inhibitor against viral infection and AQ-induced cell damage. MPoMA inhibited the expression of IL-8, IL-6, IL-1ß, and fibronectin induced by AQ and protected against AQ-induced morphological changes. However, MPoMA did not affect basal IL-8 expression in lung epithelial cells in the absence of AQ. Further mechanistic analysis confirmed that MPoMA selectively promoted the proteasomal degradation of inflammatory mediator p65, thereby reducing intracellular p65 expression and p65-mediated inflammatory responses. MPoMA exerted potent anti-inflammatory and protective functions in epithelial cells against LPS-induced acute lung injury in vivo. These findings suggest that MPoMA may have beneficial effects in suppressing viral infection and preventing lung epithelial cell damage through the degradation of p65 and inhibition of the production of inflammatory cytokines.

Tobramycin Reduces Pulmonary Toxicity of Polymyxin B via Inhibiting the Megalin-Mediated Drug Uptake in the Human Lung Epithelial Cells.

Accumulation of polymyxins in the lung epithelial cells can lead to increased mitochondrial oxidative stress and pulmonary toxicity. Aminoglycosides and polymyxins are used, via intravenous and pulmonary delivery, against multidrug-resistant Gram-negative pathogens. Our recent in vitro and animal studies demonstrated that the co-administration of polymyxins with aminoglycosides decreases polymyxin-induced pulmonary toxicity. The aim of this study was to investigate the in vitro transport and uptake of polymyxin B and tobramycin in human lung epithelial Calu-3 cells and the mechanism of reduced pulmonary toxicity resulting from this combination. Transport, intracellular localization, and accumulation of polymyxin B and tobramycin were investigated using doses of 30 mg/L polymyxin B, 70 mg/L tobramycin, and the combination of both. Adding tobramycin significantly (p < 0.05) decreased the polymyxin B-induced cytotoxicity in Calu-3 cells. The combination treatment significantly reduced the transport and uptake of polymyxin B and tobramycin in Calu-3 cells, compared to each drug alone, which supported the reduced pulmonary toxicity. We hypothesized that cellular uptake of polymyxin B and tobramycin shared a common transporter, megalin. We further investigated the megalin expression of Calu-3 cells using confocal microscopy and evaluated megalin activity using a megalin substrate, FITC-BSA, and a megalin inhibitor, sodium maleate. Both polymyxin B and tobramycin significantly inhibited FITC-BSA uptake by Calu-3 cells in a concentration-dependent manner. Sodium maleate substantially inhibited polymyxin B and tobramycin transport and cellular accumulation in the Calu-3 cell monolayer. Our study demonstrated that the significantly reduced uptake of polymyxin B and tobramycin in Calu-3 cells is attributed to the mechanism of action that determines that polymyxin B and tobramycin share a common transporter, megalin.

miR-29a-3p Regulates Autophagy by Targeting Akt3-Mediated mTOR in SiO2-Induced Lung Fibrosis.

Silicosis is a refractory pneumoconiosis of unknown etiology that is characterized by diffuse lung fibrosis, and microRNA (miRNA) dysregulation is connected to silicosis. Emerging evidence suggests that miRNAs modulate pulmonary fibrosis through autophagy; however, its underlying molecular mechanism remains unclear. In agreement with miRNA microarray analysis, the qRT-PCR results showed that miR-29a-3p was significantly decreased in the pulmonary fibrosis model both in vitro and in vivo. Increased autophagosome was observed via transmission electron microscopy in lung epithelial cell models and lung tissue of silicosis mice. The expression of autophagy-related proteins LC3α/ß and Beclin1 were upregulated. The results from using 3-methyladenine, an autophagy inhibitor, or rapamycin, an autophagy inducer, together with TGF-ß1, indicated that autophagy attenuates fibrosis by protecting lung epithelial cells. In TGF-ß1-treated TC-1 cells, transfection with miR-29a-3p mimics activated protective autophagy and reduced alpha-smooth muscle actin and collagen I expression. miRNA TargetScan predicted, and dual-luciferase reporter experiments identified Akt3 as a direct target of miR-29a-3p. Furthermore, Akt3 expression was significantly elevated in the silicosis mouse model and TGF-ß1-treated TC-1 cells. The mammalian target of rapamycin (mTOR) is a central regulator of the autophagy process. Silencing Akt3 inhibited the transduction of the mTOR signaling pathway and activated autophagy in TGF-ß1-treated TC-1 cells. These results show that miR-29a-3p overexpression can partially reverse the fibrotic effects by activating autophagy of the pulmonary epithelial cells regulated by the Akt3/mTOR pathway. Therefore, targeting miR-29a-3p may provide a new therapeutic strategy for silica-induced pulmonary fibrosis.

Study on Chemical Components from Derris taiwaniana and Their Lung Epithelial Cell Protect Effects.

The ethanol extract of roots of Derris taiwaniana gave two undescribed compounds, 3,3'-dimethoxy-5'-hydroxystilbene-4-O-ß-apiofuranosyl-(1→6)-ß-D-glucopyranoside (1) and 4',5-dihydroxy-3'-methoxyisoflavone-7-O-ß-apiofuranosyl-(1→6)-ß-D-glucopyranoside (2), along with thirty known components. Among them, compounds 14, 16-17, 23, 26-32 were isolated from this genus for the first time. Their structures were established based on physico-chemical properties and spectroscopic data, the lung epithelial cell protective effects were evaluated using NNK-induced MLE-12 cells. Among them, 2α,3α-epoxy-5,7,3',4'-tetrahydroxyflavan-(4ß-8-catechin) (30) showed the best significant protective effect, speculated to be the key component of D. taiwaniana that plays a protective role in lung epithelial cells.

The role of transforming growth factor-ß2 in cigarette smoke-induced lung inflammation and injury.

AIMS: Transforming growth factor-ß2 (TGF-ß2) plays an important role in pleiotropic functions and has been reported to be involved in the pathogenesis of chronic obstructive lung disease. The role of TGF-ß2 in regulating cigarette smoke (CS)-induced lung inflammation and injury has not been investigated, and its underlying mechanism remains unclear. MAIN METHODS: Primary bronchial epithelial cells (PBECs) were treated with cigarette smoke extract (CSE), and the signaling pathway of TGF-ß2 regulating lung inflammation was investigated. Mice were exposed to CS and treated with TGF-ß2 i.p. or bovine whey protein extract containing TGF-ß2 p.o., and the role of TGF-ß2 in alleviating lung inflammation/injury was studied. KEY FINDINGS: In vitro, we demonstrated that TGF-ß2 attenuated CSE-induced IL-8 production from PBECs through the TGF-ß receptor I (TGF-ßRI), Smad3, and mitogen-activated protein kinase signaling pathways. Selective TGF-ßRI inhibitor (LY364947) and antagonist of Smad3 (SIS3) abolished the effect of TGF-ß2 on alleviating CSE-induced IL-8 production. In vivo, CS exposure for 4 weeks in mice increased the levels of total protein, inflammatory cell counts, and monocyte chemoattractant protein-1 in bronchoalveolar fluid and induced lung inflammation/injury, as revealed by immunohistochemistry. Administration of TGF-ß2 through intraperitoneal injection or oral feeding with bovine whey protein extract containing TGF-ß2 significantly reduced CS-induced lung inflammation and injury. SIGNIFICANCE: We concluded that TGF-ß2 reduced CSE-induced IL-8 production through the Smad3 signaling pathway in PBECs and alleviated lung inflammation/injury in CS-exposed mice. The anti-inflammatory effect of TGF-ß2 on CS-induced lung inflammation in humans deserves further clinical study.

Epigenetic cues regulating airway development in human lung organoids: Polycomb repressive complex 2 and altered chromatin accessibility determine cell fate.

Today, human organoids are becoming an integrated part of genomics and epigenomics, as they provide a platform that can be used for the definite study of molecular and cellular mechanisms occurring at different stages of development, particularly organogenesis, within the human body. Airway development is a complex process heavily influenced by epigenetic regulatory mechanisms in response to environmental changes, and as such, human lung organoids are an indispensable asset for further exploration of these mechanisms as a mode of transition from human in vitro to human ex vivo studies. Cultured primarily in compounds mimicking the extracellular matrix, such as Matrigel, these lung organoids have helped us to come to a better understanding of the role of polycomb repressive complex 2 (PRC2) and enhancer of zeste homolog 2 (EZH2) in lung epithelial cell differentiation and airway development, which was first reported in the FASEB journal in 2019. The following is an extended account of how the histone methylation-regulating PRC2 comes to play in the molding of the human bronchial tree, along with further epigenetic insights based on more recently developed human lung organoids.

Validation of Prognostic Club Cell Secretory Protein (CC16) Cut-point in an Independent ALTA Cohort.

Background: Club cell secretory protein (CC16) has demonstrated utility as a lung-specific biomarker in predicting mortality in acute respiratory distress syndrome (ARDS). These findings have been observed in pre-clinical trials and a re-analysis of a large, randomized controlled trial of ARDS (Fluid and Catheter Treatment Trial (FACTT)). Objectives: The purpose of this study was to validate previous findings by evaluating CC16 level as a mortality predictor in patients from the albuterol to treat acute lung injury (ALTA) trial. Design and Method: In this secondary biomarker analysis, plasma CC16 level was measured from 100 ALTA subjects using enzyme-linked immunosorbent assay (ELISA). The rate of mortality was assessed in patients with high (⩾45 ng/mL) versus low CC16 (<45 ng/mL) levels. This cut-off level was applied based on our previous analysis from FACTT trial. Significance was assessed using Kaplan-Meier curves and a log-rank test. Results: Subjects were an average of 50 years old and 46% of them were females. Patients with high CC16 levels had higher 90-day mortality compared to those with low CC16 levels, (37.73% vs 8.95%, P < .001). Other clinical outcomes including ICU-free days, ventilator-free days, and organ failure free days were significantly different between the groups (All P < .05). Conclusion: In this validation study, we demonstrated that ARDS patients with high plasma CC16 concentration had a higher mortality rate than those with low CC16 levels, confirming previous findings that CC16 levels are associated with ARDS mortality.

Role of Mitophagy in Cigarette Smoke-induced Lung Epithelial Cell Injury In Vitro.

BACKGROUND: Mitochondria mediate airway inflammatory responses to cigarette smoke (CS). Removal of damaged or defective mitochondrial (mitophagy) may prevent the detrimental impact of CS extract (CSE) on airway and lung epithelial cells. METHODS: We studied the effect of a mitophagy activator (Urolithin A, UA) and a mitophagy inhibitor (Liensinine diperchlorate, Ld) on CSE-exposed alveolar (A549) and airway (BEAS-2B) epithelial cell proliferation, intracellular and mitochondrial ROS, inflammatory response, mitochondrial membrane potential (Δψm), mitochondrial morphology, mitochondrial complex activities, and protein levels of mitochondrial fission (DRP1, MFF) and mitophagy (SQSTM1/p62, LC3B). In both cell types, CSE exposure led to increased intracellular and mitochondrial oxidative stress, decreased Δψm and resulted in structural disruption of the mitochondrial network. CSE increased the expression of DRP1, MFF and SQSTM1/p62 while decreasing LC3B-II/I protein expression ratio. CSE also increased inflammatory (IL-1ß, IL-6, IL-18, CXCL1, CXCL8) and necroptosis factors (RIPK1, RIPK3, MLKL) mRNA expression. RESULTS: Pre-treatment with UA attenuated CSE-induced oxidative stress, inflammatory and necroptosis gene expression and restored mitochondrial structure and function. UA also prevented CSE-evoked increases in DRP1, MFF and SQSTM1/p62 protein expression and increased LC3B-II/I ratio. Conversely, pre-treatment with Ld aggravated CSE-induced cellular and mitochondrial responses. CONCLUSION: In conclusion, mitophagy mediates CSE-induced damage and inflammation of lung epithelial cells and may represent a therapeutic target in CS-driven diseases.

OXR1 signaling pathway as a possible mechanism of elastase-induced oxidative damage in pulmonary cells: the protective role of ellagic acid.

BACKGROUND AND OBJECTIVE: Oxidative stress is a process that occurs through free radicals on the cell membranes which causes damage to the cell and intracellular organelles, especially mitochondria membranes. H2O2 induced oxidative stress in human cells is of interest in toxicological research since oxidative stress plays a main role in the etiology of several pathological conditions. Neutrophil Elastase (Serine proteinase) is involved in the pathology process of emphysema as a respiratory disease through lung inflammation, and destruction of alveolar walls. The present study investigated the direct oxidative stress effects of Elastase in comparison with H2O2 on human lung epithelial cells (A549 cells) concerning the generation of reactive oxygen species (ROS) and modulation of oxidation resistance 1 (OXR1) and its downstream pathway using the well-known antioxidant Ellagic acid as an activator of antioxidant genes. MATERIALS AND METHODS: The human pulmonary epithelial cells (A549) were divided into the nine groups including Negative control, Positive control (H2O2), Elastase (15, 30, and 60 mU/mL), Ellagic acid (10 µmol/L), and Elastase + Ellagic acid. Cytotoxicity, ROS generation, oxidative stress profile, level of reactive metabolites, and gene expression of OXR1 and its downstream genes were measured in all groups. RESULTS: The obtained data demonstrated that Elastase exposure caused oxidative stress damage in a dose-depended manner which was associated with decreases in antioxidant defense system genes. Conversely, treatment with Ellagic acid as a potent antioxidant showed improved antioxidant enzyme activity and content which was in line with the upregulation of OXR1 signaling pathway genes. CONCLUSIONS: The present findings can highlight the novel mechanism underlying the oxidative stress induced by Neutrophil Elastase through OXR1 and related genes. Moreover, the benefit of Ellagic acid on cytoprotection, resulting from its antioxidant properties was documented.

Club Cell Secretory Protein-Derived Acute Respiratory Distress Syndrome Phenotypes Predict 90-Day Mortality: A Reanalysis of the Fluids and Catheter Treatment Trial.

Club cell secretory protein (CC16) is a protein with potential utility as a lung-specific biomarker for acute respiratory distress syndrome. The purpose of this study was to characterize CC16 in plasma from patients enrolled in the Fluid and Catheter Treatment Trial (FACTT) to determine the prognostic value for patient outcomes in our subgroup of FACTT patients. DESIGN: A secondary biomarker analysis of a prospective randomized-controlled trial. The primary outcome was area under the receiver operating characteristic (AUROC) of CC16 for prediction of 90-day mortality. Secondary outcomes included differences in mortality, length of stay, and ventilator-free days (VFDs) between patients with high and low CC16. Statistical analyses were performed with IBM SPSS Statistics. SETTING: Single-center laboratory analysis. SUBJECTS: Plasma samples from 68 FACTT subjects and 20 healthy controls. INTERVENTIONS: CC16 was measured in patient plasma samples by enzyme-linked immunosorbent assay. MEASUREMENTS AND MAIN RESULTS: Subjects were an average of 48 years old (sd, 16.7 yr old) and 51.5% male. AUROC analysis of CC16 on day 1 showed an area under the ROC curve of 0.78 for prediction of mortality (odds ratio, 1.011; 95% CI, 1.003-1.021) with an optimal cutoff value of 45 ng/mL. Patients in the low CC16 group (<45 ng/mL) had lower mortality (7.5 vs 50.0%; p < 0.001) and similar VFD (11.9 vs 13.2; p = 0.638). When stratified by CC16 concentration, there was no difference between mortality in the fluid liberal (36.4 vs 58.8%; p = 0.256) or conservative (4.3 vs 11.8%; p = 0.366) groups. CONCLUSIONS: CC16 demonstrated an acceptable AUROC for prediction of patient mortality with a cut point of 45 ng/mL. Patients with high CC16 on day 1 had worse outcomes compared with those with low CC16, suggesting a prognostic role for this lung-specific biomarker.

BMI1 Silencing Induces Mitochondrial Dysfunction in Lung Epithelial Cells Exposed to Hyperoxia.

Acute Lung Injury (ALI), characterized by bilateral pulmonary infiltrates that restrict gas exchange, leads to respiratory failure. It is caused by an innate immune response with white blood cell infiltration of the lungs, release of cytokines, an increase in reactive oxygen species (ROS), oxidative stress, and changes in mitochondrial function. Mitochondrial alterations, changes in respiration, ATP production and the unbalancing fusion and fission processes are key events in ALI pathogenesis and increase mitophagy. Research indicates that BMI1 (B cell-specific Moloney murine leukemia virus integration site 1), a protein of the Polycomb repressive complex 1, is a cell cycle and survival regulator that plays a role in mitochondrial function. BMI1-silenced cultured lung epithelial cells were exposed to hyperoxia to determine the role of BMI1 in mitochondrial metabolism. Its expression significantly decreases in human lung epithelial cells (H441) following hyperoxic insult, as determined by western blot, Qrt-PCR, and functional analysis. This decrease correlates with an increase in mitophagy proteins, PINK1, Parkin, and DJ1; an increase in the expression of tumor suppressor PTEN; changes in the expression of mitochondrial biomarkers; and decreases in the oxygen consumption rate (OCR) and tricarboxylic acid enzyme activity. Our bioinformatics analysis suggested that the BMI1 multifunctionality is determined by its high level of intrinsic disorder that defines the ability of this protein to bind to numerous cellular partners. These results demonstrate a close relationship between BMI1 expression and mitochondrial health in hyperoxia-induced acute lung injury (HALI) and indicate that BMI1 is a potential therapeutic target to treat ALI and Acute Respiratory Distress Syndrome.

In silico Drug Screening Approach Using L1000-Based Connectivity Map and Its Application to COVID-19.

Conventional drug screening methods search for a limited number of small molecules that directly interact with the target protein. This process can be slow, cumbersome and has driven the need for developing new drug screening approaches to counter rapidly emerging diseases such as COVID-19. We propose a pipeline for drug repurposing combining in silico drug candidate identification followed by in vitro characterization of these candidates. We first identified a gene target of interest, the entry receptor for the SARS-CoV-2 virus, angiotensin converting enzyme 2 (ACE2). Next, we employed a gene expression profile database, L1000-based Connectivity Map to query gene expression patterns in lung epithelial cells, which act as the primary site of SARS-CoV-2 infection. Using gene expression profiles from 5 different lung epithelial cell lines, we computationally identified 17 small molecules that were predicted to decrease ACE2 expression. We further performed a streamlined validation in the normal human epithelial cell line BEAS-2B to demonstrate that these compounds can indeed decrease ACE2 surface expression and to profile cell health and viability upon drug treatment. This proposed pipeline combining in silico drug compound identification and in vitro expression and viability characterization in relevant cell types can aid in the repurposing of FDA-approved drugs to combat rapidly emerging diseases.

Untargeted and targeted metabolomics reveal that adenosine nucleotides released in Actinobacillus pleuropneumoniae supernatant inhibit porcine reproductive and respiratory syndrome virus replication.

Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most devastating viruses in the swine industry and causes major economic losses. To date, there has not been an effective antiviral treatment for the disease. We have shown in previous studies that culture supernatant of Actinobacillus pleuropneumoniae (App), the causative agent of porcine pleuropneumonia, possesses antiviral activity in vitro against PRRSV, and we have clearly established that the antiviral activity was mediated by small molecular weight (i.e., <1 kDa), heat resistant metabolites present in the App supernatant ultrafiltrates. However, the identity of those metabolites remains unknown. The objective of the current study was to identify the active metabolites using untargeted and targeted mass spectrometry-based metabolomics and test their respective antiviral activity against PRRSV in the Jude Porcine Lung Epithelial Cell Line (SJPL). The results presented reveal very significant antiviral activity of App supernatant ultrafiltrates against PRRSV in SJPL cells. Consequently, we identified and quantified several adenosine nucleotide metabolites present in App supernatant ultrafiltrates using mass spectrometry-based metabolomics, and the concentrations detected were very high. SJPL cells infected with PRRSV and treated with 2'-adenosine monophosphate (2-AMP), 3'-adenosine monophosphate (3-AMP) or 5'-adenosine monophosphate (5-AMP) significantly reduced PRRSV infection. Interestingly, many antiviral drugs or prodrugs are adenosine analogs, and the mechanism of action was previously elucidated. Currently marketed nucleoside analog drugs could potentially be used to treat PRRSV infection.

Syndecan-4 assists Mycobacterium tuberculosis entry into lung epithelial cells by regulating the Cdc42, N-WASP, and Arp2/3 signaling pathways.

Syndecan-4 (SDC4) is a transmembrane heparin sulfate proteoglycan that regulates inflammatory responses, cell motility, cell adhesion and intracellular signaling. In this study, we found that overexpression of SDC4 promoted the infection efficiency of Mycobacterium tuberculosis (Mtb), whereas knockdown of SDC4 reduced the infection efficiency, suggesting that SDC4 assisted Mtb infection of epithelial cells. We also observed that Mtb infection affected the F-actin/G-actin ratio, which was also correlated with SDC4 expression levels. Analysis of the Cdc42, N-WASP, and Arp2/3 signaling pathways during Mtb infection revealed that knockdown of Cdc42 and N-WASP or the addition of ZCL278, Wiskostatin or CK636 (blockers of Cdc42, N-WASP, and Arp2/3, respectively) significantly exacerbated Mtb infection in lung epithelial cells. Taken together, our data indicate that SDC4 assists Mtb infection of epithelial cells by regulating the Cdc42, N-WASP, and Arp2/3 signaling pathways, which regulate the polymerization of the actin cytoskeleton.

Deducing the cellular mechanisms associated with the potential genotoxic impact of gold and silver engineered nanoparticles upon different lung epithelial cell lines in vitro.

Human ENP exposure is inevitable and the novel, size-dependent physicochemical properties that enable ENPs to be beneficial in innovative technologies are concomitantly causing heightened public concerns as to their potential adverse effects upon human health. This study aims to deduce the mechanisms associated with potential ENP mediated (geno)toxicity and impact upon telomere integrity, if any, of varying concentrations of both ∼16 nm (4.34 × 10-3 to 17.36 × 10-3 mg/mL) Gold (Au) and ∼14 nm (0.85 × 10-5 to 3.32 × 10-5 mg/mL) Silver (Ag) ENPs upon two commonly used lung epithelial cell lines, 16HBE14o- and A549. Following cytotoxicity analysis (via Trypan Blue and Lactate Dehydrogenase assay), two sub-lethal concentrations were selected for genotoxicity analysis using the cytokinesis-blocked micronucleus assay. Whilst both ENP types induced significant oxidative stress, Ag ENPs (1.66 × 10-5 mg/mL) did not display a significant genotoxic response in either epithelial cell lines, but Au ENPs (8.68 × 10-3 mg/mL) showed a highly significant 2.63-fold and 2.4-fold increase in micronucleus frequency in A549 and 16HBE14o- cells respectively. It is hypothesized that the DNA damage induced by acute 24-h Au ENP exposure resulted in a cell cycle stall indicated by the increased mononuclear cell fraction (>6.0-fold) and cytostasis level. Albeit insignificant, a small reduction in telomere length was observed following acute exposure to both ENPs which could indicate the potential for ENP mediated telomere attrition. Finally, from the data shown, both in vitro lung cell cultures (16HBE14o- and A549) are equally as suitable and reliable for the in vitro ENP hazard identification approach adopted in this study.

Exposure to aerosol extract from heated tobacco products causes a drastic decrease of glutathione and protein carbonylation in human lung epithelial cells.

Heated tobacco products (HTPs) are an emerging class of tobacco goods that claim to have lower health risks than those of smoking combustible tobacco products. In this study, we exposed human lung epithelial cell lines to extracts prepared from HTP aerosols and combustible cigarette smoke to compare cytotoxicity. We focused on the effects of aldehydes present in the aerosols of HTPs at levels close to those in combustible cigarette smoke. Significant toxicity was confirmed for the HTP extract, albeit to a lesser extent than that with the combustible cigarette extract. When redox balance was evaluated by the oxidative loss of low-molecular-weight thiols in the cells, we found that total glutathione (GSH) contents and low-molecular-weight thiol levels were significantly decreased after exposure to the aerosol extract of HTPs. These results indicated that GSH is rapidly consumed during the detoxification of xenobiotics, such as aldehydes from tobacco extracts. Accordingly, exposure to the aerosol extract of HTPs resulted in the enhanced carbonylation of many proteins. In a simple comparison, the results for HTPs were significantly different from those obtained with combustible cigarette smoke, suggesting reduced toxicity of HTPs. However, we found significant and harmful effects after exposing lung epithelial cells to the aerosol extract of HTPs. Thus, a further comprehensive study is needed to clarify the lung damage induced via the long-term inhalation of aerosols from HTPs.

Bactericidal effects and stability of LL-37 and CAMA in the presence of human lung epithelial cells.

Cationic antimicrobial peptides (CAMPs) are important actors in host innate immunity and represent a promising alternative to combat antibiotic resistance. Here, the bactericidal activity of two CAMPs (LL-37 and CAMA) was evaluated against Pseudomonas aeruginosa (PA) in the presence of IB3-1 cells, a cell line derived from patients with cystic fibrosis. The two CAMPs exerted different effects on PA survival depending on the timing of their administration. We observed a greater bactericidal effect when IB3-1 cells were pretreated with sub-minimum bactericidal concentrations (Sub-MBCs) of the CAMPs prior to infection. These findings suggest that CAMPs induce the production of factors by IB3-1 cells that improve their bactericidal action. However, we observed no bactericidal effect when supra-minimum bactericidal concentrations (Supra-MBCs) of the CAMPs were added to IB3-1 cells at the same time or after infection. Western-blot analysis showed a large decrease in LL-37 levels in supernatants of infected IB3-1 cells and an increase in LL-37 binding to these cells after LL-37 administration. LL-37 induced a weak inflammatory response in the cells without being toxic. In conclusion, our findings suggest a potential prophylactic action of CAMPs. The bactericidal effects were low when the CAMPs were added after cell infection, likely due to degradation of CAMPs by bacterial or epithelial cell proteases and/or due to adherence of CAMPs to cells becoming less available for direct bacterial killing.

Research progress on mechanisms of malignant transformation of lung epithelial cells induced by PM2.5 exposure / 环境与职业医学

Fine particulate matter (PM2.5) has attracted more and more attention in the field of environmental pollution and public health. Previous studies have found that PM2.5 can be inhaled and deposit in the airway and alveoli, and even spread to the whole-body tissues and organs through blood, resulting in various toxic effects. The malignant transformation of lung epithelial cells associated with long-term exposure to PM2.5 may play an important role in the occurrence and development of lung cancer. This paper reviewed recent studies on the mechanisms of malignant transformation of lung epithelial cells associated with PM2.5 exposure, and discussed the main biological mechanisms, including epigenetics, tumor microenvironment, and other biological pathways. Besides, the potential research directions of malignant transformation of lung epithelial cells associated with PM2.5 exposure were proposed. This work aims to provide a scientific basis and reference for public health management and air quality assessment.