Meta-analysis of the accuracy for RASSF1A methylation in bronchial aspirates for the diagnosis of lung cancer.

OBJECTIVE: To establish the diagnostic accuracy of RASSF1A (Ras association domain family 1 isoform) methylation using bronchial aspirates as an auxiliary method for diagnosing lung cancer through a systematic review and meta-analysis. METHODS: Studies published prior to October 30, 2022, were retrieved from the Embase, PubMed, Web of Science, and Wan Fang databases using the keywords "lung cancer", "RASSF1A", "methylation", and "bronchial aspirates". A fixed or random effect model was used to calculate the combined sensitivity, specificity, positive likelihood ratios (LR), negative LR, diagnostic odds ratio (DOR), along with the respective 95% confidence intervals (CIs) and the area under the curve (AUC) with Q index. The threshold effect was defined by using the Spearman correlation coefficient, and the Deeks funnel plot was generated to evaluate publication bias. RESULTS: Among the 12 trials that met the inclusion criteria, a total of 2388 participants were involved. The pooled results for the diagnosis of lung cancer were as follows, when compared to the pathological diagnosis: sensitivity of 0.47 (95% CI: 0.45-0.50), specificity of 0.96 (95% CI: 0.95-0.97), positive LR of 12.18 (95% CI: 8.96-16.55), negative LR of 0.56 (95% CI: 0.52-0.61), DOR of 24.05 (95% CI: 17.29-33.47), and AUC of 0.78 (Q index = 0.72), respectively. The sensitivity of the RASSF1A methylation assay was relatively low in a detailed subgroup analysis, fluctuating between 0.39 and 0.90, indicating a limitation in its diagnostic value for lung cancer. The RASSF1A methylation assay, on the other hand, demonstrated excellent specificity, suggesting a high exclusion value. Of note, the diagnostic sensitivity, specificity, DOR, and AUC for small cell lung cancer were 0.90 (0.84-0.94), 0.95 (0.94-0.97), 249.5 (103.94-598.8), and 0.98, respectively, showing that RASSF1A methylation was a promising biomarker for diagnosing small cell lung cancer with both high diagnostic and exclusion value. Furthermore, RASSF1A methylation using bronchial washings and bronchial aspirates showed a high AUC of 0.998 and 0.93, respectively, indicating excellent diagnostic performance. CONCLUSIONS: The methylation of RASSF1A in bronchial aspirates demonstrated a high level of diagnostic accuracy and has the potential to be a valuable supplementary diagnostic method, especially for identifying small cell lung cancer.

Select amino acids recover cytokine-altered ENaC function in human bronchial epithelial cells.

The airway epithelium plays a pivotal role in regulating mucosal immunity and inflammation. Epithelial barrier function, homeostasis of luminal fluid, and mucociliary clearance are major components of mucosal defense mechanisms. The epithelial sodium channel (ENaC) is one of the key players in controlling airway fluid volume and composition, and characteristic cytokines cause ENaC and barrier dysfunctions following pulmonary infections or allergic reactions. Given the limited understanding of the requisite duration and magnitude of cytokines to affect ENaC and barrier function, available treatment options for restoring normal ENaC activity are limited. Previous studies have demonstrated that distinct amino acids can modulate epithelial ion channel activities and barrier function in intestines and airways. Here, we have investigated the time- and concentration-dependent effect of representative cytokines for Th1- (IFN-γ and TNF-α), Th2- (IL-4 and IL-13), and Treg-mediated (TGF-ß1) immune responses on ENaC activity and barrier function in human bronchial epithelial cells. When cells were exposed to Th1 and Treg cytokines, ENaC activity decreased gradually while barrier function remained largely unaffected. In contrast, Th2 cytokines had an immediate and profound inhibitory effect on ENaC activity that was subsequently followed by epithelial barrier disruption. These functional changes were associated with decreased membrane protein expression of α-, ß-, and γ-ENaC, and decreased mRNA levels of ß- and γ-ENaC. A proprietary blend of amino acids was developed based on their ability to prevent Th2 cytokine-induced ENaC dysfunction. Exposure to the select amino acids reversed the inhibitory effect of IL-13 on ENaC activity by increasing mRNA levels of ß- and γ-ENaC, and protein expression of γ-ENaC. This study indicates the beneficial effect of select amino acids on ENaC activity in an in vitro setting of Th2-mediated inflammation suggesting these amino acids as a novel therapeutic approach for correcting this condition.

Modulation of Bronchial Epithelial Barrier Integrity by Low Molecular Weight Components from Birch Pollen.

Pollen, in addition to allergens, comprise low molecular weight components (LMC) smaller than 3 kDa. Emerging evidence indicates the relevance of LMC in allergic immune responses. However, the interaction of birch pollen (BP)-derived LMC and epithelial cells has not been extensively studied. We investigated epithelial barrier modifications induced by exposure to BP LMC, using the human bronchial epithelial cell line 16HBE14o-. Epithelial cell monolayers were apically exposed to the major BP allergen Bet v 1, aqueous BP extract or BP-derived LMC. Barrier integrity after the treatments was monitored by measuring transepithelial electrical resistance at regular intervals and by using the xCELLigence Real-Time Cell Analysis system. The polarized release of cytokines 24 h following treatment was measured using a multiplex immunoassay. Epithelial barrier integrity was significantly enhanced upon exposure to BP LMC. Moreover, BP LMC induced the repair of papain-mediated epithelial barrier damage. The apical release of CCL5 and TNF-α was significantly reduced after exposure to BP LMC, while the basolateral release of IL-6 significantly increased. In conclusion, the results of our study demonstrate that BP-derived LMC modify the physical and immunological properties of bronchial epithelial cells and thus regulate airway epithelial barrier responses.

Comparison of a novel potentiator of CFTR channel activity to ivacaftor in ameliorating mucostasis caused by cigarette smoke in primary human bronchial airway epithelial cells.

BACKGROUND: Cystic Fibrosis causing mutations in the gene CFTR, reduce the activity of the CFTR channel protein, and leads to mucus aggregation, airway obstruction and poor lung function. A role for CFTR in the pathogenesis of other muco-obstructive airway diseases such as Chronic Obstructive Pulmonary Disease (COPD) has been well established. The CFTR modulatory compound, Ivacaftor (VX-770), potentiates channel activity of CFTR and certain CF-causing mutations and has been shown to ameliorate mucus obstruction and improve lung function in people harbouring these CF-causing mutations. A pilot trial of Ivacaftor supported its potential efficacy for the treatment of mucus obstruction in COPD. These findings prompted the search for CFTR potentiators that are more effective in ameliorating cigarette-smoke (CS) induced mucostasis. METHODS: Small molecule potentiators, previously identified in CFTR binding studies, were tested for activity in augmenting CFTR channel activity using patch clamp electrophysiology in HEK-293 cells, a fluorescence-based assay of membrane potential in Calu-3 cells and in Ussing chamber studies of primary bronchial epithelial cultures. Addition of cigarette smoke extract (CSE) to the solutions bathing the apical surface of Calu-3 cells and primary bronchial airway cultures was used to model COPD. Confocal studies of the velocity of fluorescent microsphere movement on the apical surface of CSE exposed airway epithelial cultures, were used to assess the effect of potentiators on CFTR-mediated mucociliary movement. RESULTS: We showed that SK-POT1, like VX-770, was effective in augmenting the cyclic AMP-dependent channel activity of CFTR. SK-POT-1 enhanced CFTR channel activity in airway epithelial cells previously exposed to CSE and ameliorated mucostasis on the surface of primary airway cultures. CONCLUSION: Together, this evidence supports the further development of SK-POT1 as an intervention in the treatment of COPD.

Analysis of Methylome of Different Forms of Basal Cell Hyperplasia and Squamous Cell Metaplasia of Bronchial Epithelium.

Squamous cell lung cancer (SCLC) occurs as a result of dysregenerative changes in the bronchial epithelium: basal cell hyperplasia (BCH), squamous cell metaplasia (SM), and dysplasia. We previously suggested that combinations of precancerous changes detected in the small bronchi of patients with SCLC may reflect various "scenarios" of the precancerous process: isolated BCH→stopping at the stage of hyperplasia, BCH+SM→progression of hyperplasia into metaplasia, SM+dysplasia→progression of metaplasia into dysplasia. In this study, DNA methylome of various forms of precancerous changes in the bronchial epithelium of SCLC patients was analyzed using the genome-wide bisulfite sequencing. In BCH combined with SM, in contrast to isolated BCH, differentially methylated regions were identified in genes of the pathogenetically significant MET signaling pathway (RNMT, HPN). Differentially methylated regions affecting genes involved in inflammation regulation (IL-23, IL-23R, IL12B, IL12RB1, and FIS1) were detected in SM combined with dysplasia in comparison with SM combined with BCH. The revealed changes in DNA methylation may underlie various "scenarios" of the precancerous process in the bronchial epithelium.

Expression of ISG60 is induced by TLR3 signaling in BEAS­2B bronchial epithelial cells: Possible involvement in CXCL10 expression.

Viral infections in the respiratory tract are common, and, in recent years, severe acute respiratory syndrome coronavirus 2 outbreaks have highlighted the effect of viral infections on antiviral innate immune and inflammatory reactions. Specific treatments for numerous viral respiratory infections have not yet been established and they are mainly treated symptomatically. Therefore, understanding the details of the innate immune system underlying the airway epithelium is crucial for the development of new therapies. The present study aimed to investigate the function and expression of interferon (IFN)­stimulated gene (ISG)60 in non­cancerous bronchial epithelial BEAS­2B cells exposed to a Toll­like receptor 3 agonist. BEAS­2B cells were treated with a synthetic TLR3 ligand, polyinosinic­polycytidylic acid (poly IC). The mRNA and protein expression levels of ISG60 were analyzed using reverse transcription­quantitative PCR and western blotting, respectively. The levels of C­X­C motif chemokine ligand 10 (CXCL10) were examined using an enzyme­linked immunosorbent assay, and the effects of knockdown of IFN­ß, ISG60 and ISG56 were examined using specific small interfering RNAs. Notably, ISG60 expression was increased in proportion to poly IC concentration, and recombinant human IFN­ß also induced ISG60 expression. By contrast, knockdown of IFN­ß and ISG56 decreased ISG60 expression, and ISG60 knockdown reduced CXCL10 and ISG56 expression. These findings suggested that ISG60 is partly implicated in CXCL10 expression and that ISG60 may serve a role in the innate immune response of bronchial epithelial cells. The present study highlights ISG60 as a potential target for new therapeutic strategies against viral infections in the airway.

Reduced sialylation of airway mucin impairs mucus transport by altering the biophysical properties of mucin.

Mucus stasis is a pathologic hallmark of muco-obstructive diseases, including cystic fibrosis (CF). Mucins, the principal component of mucus, are extensively modified with hydroxyl (O)-linked glycans, which are largely terminated by sialic acid. Sialic acid is a negatively charged monosaccharide and contributes to the biochemical/biophysical properties of mucins. Reports suggest that mucin sialylation may be altered in CF; however, the consequences of reduced sialylation on mucus clearance have not been fully determined. Here, we investigated the consequences of reduced sialylation on the charge state and conformation of the most prominent airway mucin, MUC5B, and defined the functional consequences of reduced sialylation on mucociliary transport (MCT). Reduced sialylation contributed to a lower charged MUC5B form and decreased polymer expansion. The inhibition of total mucin sialylation de novo impaired MCT in primary human bronchial epithelial cells and rat airways, and specific α-2,3 sialylation blockade was sufficient to recapitulate these findings. Finally, we show that ST3 beta-galactoside alpha-2,3-sialyltransferase (ST3Gal1) expression is downregulated in CF and partially restored by correcting CFTR via Elexacaftor/Tezacaftor/Ivacaftor treatment. Overall, this study demonstrates the importance of mucin sialylation in mucus clearance and identifies decreased sialylation by ST3Gal1 as a possible therapeutic target in CF and potentially other muco-obstructive diseases.

Chloride intracellular channel 4 participates in the regulation of lipopolysaccharide-induced inflammatory responses in human bronchial epithelial cells.

The airway epithelium is located at the interactional boundary between the external and internal environments of the organism and is often exposed to harmful environmental stimuli. Inflammatory response that occurs after airway epithelial stress is the basis of many lung and systemic diseases. Chloride intracellular channel 4 (CLIC4) is abundantly expressed in epithelial cells. The purpose of this study was to investigate whether CLIC4 is involved in the regulation of lipopolysaccharide (LPS)-induced inflammatory response in airway epithelial cells and to clarify its potential mechanism. Our results showed that LPS induced inflammatory response and decreased CLIC4 levels in vivo and in vitro. CLIC4 silencing aggravated the inflammatory response in epithelial cells, while overexpression of CLIC4 combined with LPS exposure significantly decreased the inflammatory response compared with cells exposed to LPS without CLIC4 overexpression. By labeling intracellular chloride ions with chloride fluorescent probe MQAE, we showed that CLIC4 mediated intracellular chloride ion-regulated LPS-induced cellular inflammatory response.

ALKBH5 Modulates Asthma Progression by Downregulating N6-methyladenosine Methylation.

Asthma is a chronic respiratory disease that is characterized by airway inflammation, excessive mucus production, and airway remodeling. Prevention and treatment for asthma is an urgent issue in clinical studies. In recent years, N6-methyladenosine methylation (m6A) has emerged as a promising regulatory approach involved in multiple diseases. ALKBH5 (alkB homolog 5) is a demethylase widely studied in disease pathologies. This work aimed to explore the regulatory mechanisms underlying the ALKBH5-regulated asthma. We established an interleukin-13 (IL-13)-stimulated cell model to mimic the in vitro inflammatory environment of asthma. ALKBH5 knockdown in bronchial epithelial cells was performed using siRNAs, and the knockdown efficacy was analyzed by quantitative PCR (qPCR). Cell viability and proliferation were measured by cell counting kit 8 (CCK-8) and colony formation assay. The ferroptosis was assessed by measuring the total iron, Fe2+, lipid reactive oxygen species (ROS), malondialdehyde (MDA), and superoxide dismutase (SOD) levels. The enrichment of N6-methyladenosine methylation (m6A) modification was detected by the MeRIP assay. Knockdown of ALKBH5 significantly elevated the survival and colony formation ability of bronchial epithelial cells in the IL-13 induction model. The levels of total iron, Fe2+, lipid ROS, and MDA were remarkedly elevated, and the SOD level was reduced in IL-13-induced bronchial epithelial cells, and depletion of ALKBH5 reversed these effects. Knockdown of ALKBH5 elevated the enrichment of m6A modification and expression of glutathione peroxidase 4 (GPX4). Knockdown of GPX4 abolished the pro-proliferation and anti-ferroptosis effects of siALKBH5. Knockdown of ALKBH5 improved the proliferation of bronchial epithelial cells and alleviated cell ferroptosis.

N-acetyltransferase metabolism and DNA damage following exposure to 4,4'-oxydianiline in human bronchial epithelial cells.

Waterpipe smoking is increasingly popular and understanding how chemicals found in hookah smoke may be harmful to human bronchial epithelial cells is of great importance. 4,4'-Oxydianiline (ODA), is an aromatic amine which is present at comparatively high levels in hookah smoke. The metabolism and the subsequent toxicity of ODA in human bronchial epithelial cells remains unknown. Given that ODA is an aromatic amine, we hypothesized that ODA is N-acetylated and induces DNA damage following exposure to immortalized human bronchial epithelial cells (BEP2D cells). We measured the N-acetylation of ODA to mono-acetyl-ODA and the N-acetylation of mono-acetyl-ODA to diacetyl-ODA by BEP2D cells following separation and quantitation by high performance liquid chromatography. For ODA, the apparent KM in cells was 12.4 ± 3.7 µM with a Vmax of 0.69 ± 0.03 nmol/min/106 cells, while for mono-acetyl-ODA, the apparent KM was 111.2 ± 48.3 µM with a Vmax of 17.8 ± 5.7 nmol/min/106 cells ODA exposure for 24 h resulted in DNA damage to BEP2D cells following concentrations as low as 0.1 µM as measured by yH2Ax protein expression These results demonstrate that ODA, the most prevalent aromatic amine identified in hookah smoke, is N-acetylated and induces DNA damage in human bronchial epithelial cells.

Loss of CFTR Reverses Senescence Hallmarks in SARS-CoV-2 Infected Bronchial Epithelial Cells.

SARS-CoV-2 infection has been recently shown to induce cellular senescence in vivo. A senescence-like phenotype has been reported in cystic fibrosis (CF) cellular models. Since the previously published data highlighted a low impact of SARS-CoV-2 on CFTR-defective cells, here we aimed to investigate the senescence hallmarks in SARS-CoV-2 infection in the context of a loss of CFTR expression/function. We infected WT and CFTR KO 16HBE14o-cells with SARS-CoV-2 and analyzed both the p21 and Ki67 expression using immunohistochemistry and viral and p21 gene expression using real-time PCR. Prior to SARS-CoV-2 infection, CFTR KO cells displayed a higher p21 and lower Ki67 expression than WT cells. We detected lipid accumulation in CFTR KO cells, identified as lipolysosomes and residual bodies at the subcellular/ultrastructure level. After SARS-CoV-2 infection, the situation reversed, with low p21 and high Ki67 expression, as well as reduced viral gene expression in CFTR KO cells. Thus, the activation of cellular senescence pathways in CFTR-defective cells was reversed by SARS-CoV-2 infection while they were activated in CFTR WT cells. These data uncover a different response of CF and non-CF bronchial epithelial cell models to SARS-CoV-2 infection and contribute to uncovering the molecular mechanisms behind the reduced clinical impact of COVID-19 in CF patients.

Wnt5a-mediated autophagy contributes to the epithelial-mesenchymal transition of human bronchial epithelial cells during asthma.

BACKGROUND: The epithelial-mesenchymal transition (EMT) of human bronchial epithelial cells (HBECs) is essential for airway remodeling during asthma. Wnt5a has been implicated in various lung diseases, while its role in the EMT of HBECs during asthma is yet to be determined. This study sought to define whether Wnt5a initiated EMT, leading to airway remodeling through the induction of autophagy in HBECs. METHODS: Microarray analysis was used to investigate the expression change of WNT5A in asthma patients. In parallel, EMT models were induced using 16HBE cells by exposing them to house dust mites (HDM) or interleukin-4 (IL-4), and then the expression of Wnt5a was observed. Using in vitro gain- and loss-of-function approaches via Wnt5a mimic peptide FOXY5 and Wnt5a inhibitor BOX5, the alterations in the expression of the epithelial marker E-cadherin and the mesenchymal marker protein were observed. Mechanistically, the Ca2+/CaMKII signaling pathway and autophagy were evaluated. An autophagy inhibitor 3-MA was used to examine Wnt5a in the regulation of autophagy during EMT. Furthermore, we used a CaMKII inhibitor KN-93 to determine whether Wnt5a induced autophagy overactivation and EMT via the Ca2+/CaMKII signaling pathway. RESULTS: Asthma patients exhibited a significant increase in the gene expression of WNT5A compared to the healthy control. Upon HDM and IL-4 treatments, we observed that Wnt5a gene and protein expression levels were significantly increased in 16HBE cells. Interestingly, Wnt5a mimic peptide FOXY5 significantly inhibited E-cadherin and upregulated α-SMA, Collagen I, and autophagy marker proteins (Beclin1 and LC3-II). Rhodamine-phalloidin staining showed that FOXY5 resulted in a rearrangement of the cytoskeleton and an increase in the quantity of stress fibers in 16HBE cells. Importantly, blocking Wnt5a with BOX5 significantly inhibited autophagy and EMT induced by IL-4 in 16HBE cells. Mechanistically, autophagy inhibitor 3-MA and CaMKII inhibitor KN-93 reduced the EMT of 16HBE cells caused by FOXY5, as well as the increase in stress fibers, cell adhesion, and autophagy. CONCLUSION: This study illustrates a new link in the Wnt5a-Ca2+/CaMKII-autophagy axis to triggering airway remodeling. Our findings may provide novel strategies for the treatment of EMT-related diseases.

The Modulation of Respiratory Epithelial Cell Differentiation by the Thickness of an Electrospun Poly-ε-Carprolactone Mesh Mimicking the Basement Membrane.

The topology of the basement membrane (BM) affects cell physiology and pathology, and BM thickening is associated with various chronic lung diseases. In addition, the topology of commercially available poly (ethylene terephthalate) (PET) membranes, which are used in preclinical in vitro models, differs from that of the human BM, which has a fibrous and elastic structure. In this study, we verified the effect of BM thickness on the differentiation of normal human bronchial epithelial (NHBE) cells. To evaluate whether the thickness of poly-ε-carprolactone (PCL) mesh affects the differentiation of NHBE cells, cells were grown on thin- (6-layer) and thick-layer (80-layer) meshes consisting of electrospun PCL nanofibers using an air-liquid interface (ALI) cell culture system. It was found that the NHBE cells formed a normal pseudostratified epithelium composed of ciliated, goblet, and basal cells on the thin-layer PCL mesh; however, goblet cell hyperplasia was observed on the thick-layer PCL mesh. Differentiated NHBE cells cultured on the thick-layer PCL mesh also demonstrated increased epithelial-mesenchymal transition (EMT) compared to those cultured on the thin-layer PCL mesh. In addition, expression of Sox9, nuclear factor (NF)-κB, and oxidative stress-related markers, which are also associated with goblet cell hyperplasia, was increased in the differentiated NHBE cells cultured on the thick-layer PCL mesh. Thus, the use of thick electrospun PCL mesh led to NHBE cells differentiating into hyperplastic goblet cells via EMT and the oxidative stress-related signaling pathway. Therefore, the topology of the BM, for example, thickness, may affect the differentiation direction of human bronchial epithelial cells.

[Aloperine suppresses TLR4/NF-κB/NLRP3 signaling to ameliorate cigarette smoke-induced injury to human bronchial epithelial cells].

Objective To explore the effects of aloperine (Alo) on cigarette smoke-induced injury in human bronchial epithelial cells and its potential mechanism. Methods After human bronchial epithelial 16HBE cells were co-treated by 100 mL/L cigarette smoke extract (CSE) and various concentrations (50,100 and 200 µmol/L) of Alo, cell viability was assessed using CCK-8 assay. Lactate dehydrogenase (LDH) activity was measured with a related kit. Cell apoptosis was evaluated using the terminal-deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay (TUNEL) and Western blot analysis. The levels of inflammatory factors were detected by ELISA. Oxidative stress levels were assessed using 2'7'-dichlorofluorescin diacetate (DCFH-DA) staining. The expression of Toll-like receptor 4 (TLR4)/nuclear factor-kappaB (NF-κB)/NLR family pyrin domain containing 3 (NLRP3) signaling-associated proteins was measured by Western blot analysis. After cells were co-treated with 100 mL/L CSE and 200 µmol/L Alo, the aforementioned assays were applied to evaluate the effects of TLR4 overexpression on the TLR4/NF-κB/NLRP3 signaling, LDH activity, apoptosis, inflammatory response and oxidative stress in cells. Results CSE exposure might inhibit 16HBE cell viability, increase LDH activity, apoptosis, inflammatory response and oxidative stress levels and activate TLR4/NF-κB/NLRP3 signaling. Treatment with Alo promoted cell viability, decreased LDH activity, cell apoptosis, inflammation and oxidative stress levels, and inactivated TLR4/NF-κB/NLRP3 signaling. Furthermore, TLR4 overexpression might reverse the protective role of Alo treatment in CSE-induced injury in 16HBE cells. Conclusion Alo may ameliorate CSE-induced injury in human bronchial epithelial cells via inhibiting TLR4/NF-κB/NLRP3 signaling.

Polyhexamethylene guanidine phosphate induces pyroptosis via reactive oxygen species-regulated mitochondrial dysfunction in bronchial epithelial cells.

Pyroptosis is a form of programmed cell death characterized by gasdermin (GSDM)-mediated pore formation in the cell membrane, resulting in the release of pro-inflammatory cytokines and cellular lysis. Increasing evidence has shown that pyroptosis is responsible for the progression of various pulmonary disorders. The inhalation of polyhexamethylene guanidine (PHMG) causes severe lung inflammation and pulmonary toxicity; however, the underlying mechanisms are unknown. Therefore, in this study, we investigate the role of pyroptosis in PHMG-induced pulmonary toxicity. We exposed bronchial epithelial cells, BEAS-2B, to PHMG phosphate (PHMG-p) and evaluated cell death type, reactive oxygen species (ROS) levels, and relative expression levels of pyroptosis-related proteins. Our data revealed that PHMG-p reduced viability and induced morphological alterations in BEAS-2B cells. Exposure to PHMG-p induced excessive accumulation of mitochondrial ROS (mtROS) in BEAS-2B cells. PHMG-p activated caspase-dependent apoptosis as well as NLRP3/caspase-1/GSDMD-mediated- and caspase-3/GSDME-mediated pyroptosis through mitochondrial oxidative stress in BEAS-2B cells. Notably, PHMG-p reduced mitochondrial respiratory function and induced the translocation of Bax and cleaved GSDM into the mitochondria, leading to mitochondrial dysfunction. Our results enhanced our understanding of PHMG-p-induced lung toxicity by demonstrating that PHMG-p induces pyroptosis via mtROS-induced mitochondrial dysfunction in bronchial epithelial cells.

TRIM47 promotes HDM-induced bronchial epithelial pyroptosis by regulating NEMO ubiquitination to activate NF-κB/NLRP3 signaling.

Asthma is an inflammatory disease. Airway epithelial cell pyroptosis and cytokine secretion promote asthma progression. Tripartite motif 47 (TRIM47) belongs to the E3 ubiquitin ligase family and is associated with apoptosis and inflammation in a range of diseases. However, the role of TRIM47 in asthma has not been explored. In this study, the human bronchial epithelial cell line BEAS-2B was treated with house dust mite (HDM) and TRIM47 expression was detected by RT-qPCR and Western blot. After transfection with TRIM47 interfering and overexpressing plasmids, the synthesis and secretion of cytokines, as well as pyroptosis-related indicators, were examined. Nuclear factor kappa-B (NF-κB) pathway proteins and nod-like receptor protein 3 (NLRP3) inflammasome were measured to explore the mechanism of TRIM47 action. In addition, the effect of TRIM47 on the level of NF-κB essential modulator (NEMO) ubiquitination was detected by an immunoprecipitation assay. The results showed that TRIM47 was upregulated in HDM-induced BEAS-2B cells and that TRIM47 mediated HDM-induced BEAS-2B cell pyroptosis and cytokine secretion. Mechanistically, TRIM47 promoted the K63-linked ubiquitination of NEMO and facilitated NF-κB/NLRP3 pathway activation. In conclusion, TRIM47 may promote cytokine secretion mediating inflammation and pyroptosis in bronchial epithelial cells by activating the NF-κB/NLRP3 pathway. Therefore, TRIM47 may be a potential therapeutic target for HDM-induced asthma.

Mitigation of acute lung injury by human bronchial epithelial cell-derived extracellular vesicles via ANXA1-mediated FPR signaling.

Acute lung injury (ALI) is characterized by respiratory failure resulting from the disruption of the epithelial and endothelial barriers as well as immune system. In this study, we evaluated the therapeutic potential of airway epithelial cell-derived extracellular vesicles (EVs) in maintaining lung homeostasis. We isolated human bronchial epithelial cell-derived EVs (HBEC-EVs), which endogenously express various immune-related surface markers and investigated their immunomodulatory potential in ALI. In ALI cellular models, HBEC-EVs demonstrated immunosuppressive effects by reducing the secretion of proinflammatory cytokines in both THP-1 macrophages and HBECs. Mechanistically, these effects were partially ascribed to nine of the top 10 miRNAs enriched in HBEC-EVs, governing toll-like receptor-NF-κB signaling pathways. Proteomic analysis revealed the presence of proteins in HBEC-EVs involved in WNT and NF-κB signaling pathways, pivotal in inflammation regulation. ANXA1, a constituent of HBEC-EVs, interacts with formyl peptide receptor (FPR)2, eliciting anti-inflammatory responses by suppressing NF-κB signaling in inflamed epithelium, including type II alveolar epithelial cells. In a mouse model of ALI, intratracheal administration of HBEC-EVs reduced lung injury, inflammatory cell infiltration, and cytokine levels. Collectively, these findings suggest the therapeutic potential of HBEC-EVs, through their miRNAs and ANXA1 cargo, in mitigating lung injury and inflammation in ALI patients.

Nanoplastics Penetrate Human Bronchial Smooth Muscle and Small Airway Epithelial Cells and Affect Mitochondrial Metabolism.

Micro- and nanoplastic particles, including common forms like polyethylene and polystyrene, have been identified as relevant pollutants, potentially causing health problems in living organisms. The mechanisms at the cellular level largely remain to be elucidated. This study aims to visualize nanoplastics in bronchial smooth muscle (BSMC) and small airway epithelial cells (SAEC), and to assess the impact on mitochondrial metabolism. Healthy and asthmatic human BSMC and SAEC in vitro cultures were stimulated with polystyrene nanoplastics (PS-NPs) of 25 or 50 nm size, for 1 or 24 h. Live cell, label-free imaging by holotomography microscopy and mitochondrial respiration and glycolysis assessment were performed. Furthermore, 25 and 50 nm NPs were shown to penetrate SAEC, along with healthy and diseased BSMC, and they impaired bioenergetics and induce mitochondrial dysfunction compared to cells not treated with NPs, including changes in oxygen consumption rate and extracellular acidification rate. NPs pose a serious threat to human health by penetrating airway tissues and cells, and affecting both oxidative and glycolytic metabolism.

Airway epithelial CD47 plays a critical role in inducing influenza virus-mediated bacterial super-infection.

Respiratory viral infection increases host susceptibility to secondary bacterial infections, yet the precise dynamics within airway epithelia remain elusive. Here, we elucidate the pivotal role of CD47 in the airway epithelium during bacterial super-infection. We demonstrated that upon influenza virus infection, CD47 expression was upregulated and localized on the apical surface of ciliated cells within primary human nasal or bronchial epithelial cells. This induced CD47 exposure provided attachment sites for Staphylococcus aureus, thereby compromising the epithelial barrier integrity. Through bacterial adhesion assays and in vitro pull-down assays, we identified fibronectin-binding proteins (FnBP) of S. aureus as a key component that binds to CD47. Furthermore, we found that ciliated cell-specific CD47 deficiency or neutralizing antibody-mediated CD47 inactivation enhanced in vivo survival rates. These findings suggest that interfering with the interaction between airway epithelial CD47 and pathogenic bacterial FnBP holds promise for alleviating the adverse effects of super-infection.

The Cytochrome P450 2C8*3 Variant (rs11572080) Is Associated with Improved Asthma Symptom Control in Children and Altered Lipid Mediator Production and Inflammatory Response in Human Bronchial Epithelial Cells.

This study investigated an association between the cytochrome P450 (CYP) 2C8*3 polymorphism with asthma symptom control in children and changes in lipid metabolism and pro-inflammatory signaling by human bronchial epithelial cells (HBECs) treated with cigarette smoke condensate (CSC). CYP genes are inherently variable in sequence, and while such variations are known to produce clinically relevant effects on drug pharmacokinetics and pharmacodynamics, the effects on endogenous substrate metabolism and associated physiologic processes are less understood. In this study, CYP2C8*3 was associated with improved asthma symptom control among children: Mean asthma control scores were 3.68 (n = 207) for patients with one or more copies of the CYP2C8*3 allele versus 4.42 (n = 965) for CYP2C8*1/*1 (P = 0.0133). In vitro, CYP2C8*3 was associated with an increase in montelukast 36-hydroxylation and a decrease in linoleic acid metabolism despite lower mRNA and protein expression. Additionally, CYP2C8*3 was associated with reduced mRNA expression of interleukin-6 (IL-6) and C-X-C motif chemokine ligand 8 (CXCL-8) by HBECs in response to CSC, which was replicated using the soluble epoxide hydrolase inhibitor, 12-[[(tricyclo[3.3.1.13,7]dec-1-ylamino)carbonyl]amino]-dodecanoic acid. Interestingly, 9(10)- and 12(13)- dihydroxyoctadecenoic acid, the hydrolyzed metabolites of 9(10)- and 12(13)- epoxyoctadecenoic acid, increased the expression of IL-6 and CXCL-8 mRNA by HBECs. This study reveals previously undocumented effects of the CYP2C8*3 variant on the response of HBECs to exogenous stimuli. SIGNIFICANCE STATEMENT: These findings suggest a role for CYP2C8 in regulating the epoxyoctadecenoic acid:dihydroxyoctadecenoic acid ratio leading to a change in cellular inflammatory responses elicited by environmental stimuli that exacerbate asthma.