Targeted inhibition of m6A demethylase FTO by FB23 attenuates allergic inflammation in the airway epithelium.

Epithelial cells play a crucial role in asthma, contributing to chronic inflammation and airway hyperresponsiveness. m6A modification, which involves key proteins such as the demethylase fat mass and obesity-associated protein (FTO), is crucial in the regulation of various diseases, including asthma. However, the role of FTO in epithelial cells and the development of asthma remains unclear. In this study, we investigated the demethylase activity of FTO using a small-molecule inhibitor FB23 in epithelial cells and allergic inflammation in vivo and in vitro. We examined the FTO-regulated transcriptome-wide m6A profiling by methylated RNA immunoprecipitation sequencing (MeRIP-seq) and RNA-seq under FB23 treatment and allergic inflammation conditions. Immunofluorescence staining was performed to assess the tissue-specific expression of FTO in asthmatic bronchial mucosa. We demonstrated that FB23 alleviated allergic inflammation in IL-4/IL-13-treated epithelial cells and house dust mite (HDM)-induced allergic airway inflammation mouse model. The demethylase activity of FTO contributed to the regulation of TNF-α signaling via NF-κB and epithelial-mesenchymal transition-related pathways under allergic inflammation conditions in epithelial cells. FTO was expressed in epithelial, submucosal gland, and smooth muscle cells in human bronchial mucosa. In conclusion, FB23-induced inhibition of FTO alleviates allergic inflammation in epithelial cells and HDM-induced mice, potentially through diverse cellular processes and epithelial-mesenchymal transition signaling pathways, suggesting that FTO is a potential therapeutic target in asthma management.

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.

PM2.5 Exposure Inhibits Transepithelial Anion Short-circuit Current by Downregulating P2Y2 Receptor/CFTR Pathway.

Fine particulate matter (PM2.5) can damage airway epithelial barriers. The anion transport system plays a crucial role in airway epithelial barriers. However, the detrimental effect and mechanism of PM2.5 on the anion transport system are still unclear. In this study, airway epithelial cells and ovalbumin (OVA)-induced asthmatic mice were used. In transwell model, the adenosine triphosphate (ATP)-induced transepithelial anion short-circuit current (Isc) and airway surface liquid (ASL) significantly decreased after PM2.5 exposure. In addition, PM2.5 exposure decreased the expression levels of P2Y2R, CFTR and cytoplasmic free-calcium, but ATP can increase the expressions of these proteins. PM2.5 exposure increased the levels of Th2-related cytokines of bronchoalveolar lavage fluid, lung inflammation, collagen deposition and hyperplasisa of goblet cells. Interestingly, the administration of ATP showed an inhibitory effect on lung inflammation induced by PM2.5. Together, our study reveals that PM2.5 impairs the ATP-induced transepithelial anion Isc through downregulating P2Y2R/CFTR pathway, and this process may participate in aggravating airway hyperresponsiveness and airway inflammation. These findings may provide important insights on PM2.5-mediated airway epithelial injury.

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.

SQSTM1 improves acute lung injury via inhibiting airway epithelium ferroptosis in a vitamin D receptor/autophagy-mediated manner.

Emerging evidence has reported that acute lung injury (ALI), characterized by inflammation and oxidative stress in airway epithelium, is regulated by programmed cell death. Ferroptosis, a regulated form of cell death spurred by uncontrolled lipid peroxidation, has been proven to implicate various diseases. Inhibiting ferroptosis represents a feasible strategy for ALI through the suppression of lipid peroxidation, while the mechanism remains to be further elucidated. Here, we identified Sequestosome 1 (SQSTM1) as a negative regulator of airway epithelium ferroptosis during ALI. SQSTM1 knockdown cells manifested higher sensitivity to ferroptosis. Mechanistically, SQSTM1 was found to directly interact with vitamin D receptor (VDR) through its nuclear receptor (NR) box motif, facilitating its nuclear translocation and initiating autophagy at the transcriptional level. To further validate these findings, an in vivo preventive model utilizing spermidine, a proven inducer of SQSTM1 was established. The results consistently demonstrated that spermidine supplementation significantly induced SQSTM1 and ameliorated ALI by mitigating airway epithelial ferroptosis. Notably, these effects were abrogated in the absence of SQSTM1. Taken together, this study identified SQSTM1 as a negative regulator of airway epithelium ferroptosis in a VDR-mediated autophagy manner, making it a potential therapeutic target for the treatment of ALI.

Nanoparticles and Airway Epithelial Cells: Exploring the Impacts and Methodologies in Toxicity Assessment.

The production of nanoparticles has recently surged due to their varied applications in the biomedical, pharmaceutical, textile, and electronic sectors. However, this rapid increase in nanoparticle manufacturing has raised concerns about environmental pollution, particularly its potential adverse effects on human health. Among the various concerns, inhalation exposure to nanoparticles poses significant risks, especially affecting the respiratory system. Airway epithelial cells play a crucial role as the primary defense against inhaled particulate matter and pathogens. Studies have shown that nanoparticles can disrupt the airway epithelial barrier, triggering inflammatory responses, generating reactive oxygen species, and compromising cell viability. However, our understanding of how different types of nanoparticles specifically impact the airway epithelial barrier remains limited. Both in vitro cell culture and in vivo murine models are commonly utilized to investigate nanoparticle-induced cellular responses and barrier dysfunction. This review discusses the methodologies frequently employed to assess nanoparticle toxicity and barrier disruption. Furthermore, we analyze and compare the distinct effects of various nanoparticle types on the airway epithelial barrier. By elucidating the diverse responses elicited by different nanoparticles, we aim to provide insights that can guide future research endeavors in assessing and mitigating the potential risks associated with nanoparticle exposure.

Initiator cell death event induced by SARS-CoV-2 in the human airway epithelium.

Virus-induced cell death is a key contributor to COVID-19 pathology. Cell death induced by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is well studied in myeloid cells but less in its primary host cell type, angiotensin-converting enzyme 2 (ACE2)-expressing human airway epithelia (HAE). SARS-CoV-2 induces apoptosis, necroptosis, and pyroptosis in HAE organotypic cultures. Single-cell and limiting-dilution analysis revealed that necroptosis is the primary cell death event in infected cells, whereas uninfected bystanders undergo apoptosis, and pyroptosis occurs later during infection. Mechanistically, necroptosis is induced by viral Z-RNA binding to Z-DNA-binding protein 1 (ZBP1) in HAE and lung tissues from patients with COVID-19. The Delta (B.1.617.2) variant, which causes more severe disease than Omicron (B1.1.529) in humans, is associated with orders of magnitude-greater Z-RNA/ZBP1 interactions, necroptosis, and disease severity in animal models. Thus, Delta induces robust ZBP1-mediated necroptosis and more disease severity.

FGF receptors mediate cellular senescence in the cystic fibrosis airway epithelium.

The number of adults living with cystic fibrosis (CF) has already increased significantly because of drastic improvements in life expectancy attributable to advances in treatment, including the development of highly effective modulator therapy. Chronic airway inflammation in CF contributes to morbidity and mortality, and aging processes like inflammaging and cell senescence influence CF pathology. Our results show that single-cell RNA sequencing data, human primary bronchial epithelial cells from non-CF and CF donors, a CF bronchial epithelial cell line, and Cftr-knockout (Cftr-/-) rats all demonstrated increased cell senescence markers in the CF bronchial epithelium. This was associated with upregulation of fibroblast growth factor receptors (FGFRs) and mitogen-activated protein kinase (MAPK) p38. Inhibition of FGFRs, specifically FGFR4 and to some extent FGFR1, attenuated cell senescence and improved mucociliary clearance, which was associated with MAPK p38 signaling. Mucociliary dysfunction could also be improved using a combination of senolytics in a CF ex vivo model. In summary, FGFR/MAPK p38 signaling contributes to cell senescence in CF airways, which is associated with impaired mucociliary clearance. Therefore, attenuation of cell senescence in the CF airways might be a future therapeutic strategy improving mucociliary dysfunction and lung disease in an aging population with CF.

Exosome microRNA-125a-5p derived from epithelium promotes M1 macrophage polarization by targeting IL1RN in chronic obstructive pulmonary disease.

BACKGROUND: The interplay between airway epithelium and macrophages plays a pivotal role in Chronic Obstructive Pulmonary Disease (COPD) pathogenesis. Exosomes, which transport miRNA cargo, have emerged as novel mediators of intercellular communication. MicroRNA-125a-5p (miR-125a-5p) has been implicated in macrophage polarization.This study aims to investigate the role of exosomal miR-125a-5p in the dysfunctional epithelium-macrophage cross-talk in cigarette smoke (CS)-induced COPD. METHODS: In cell models, THP-1 monocytic cells were differentiated into macrophages (M0). Human bronchial epithelial cells treated with CS extract (CSE) were co-cultured with M0. Exosomes were isolated from culture media using commercial kits and characterized using nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM). Exosomes labeled with PKH26 red fluorescent cell linker kits were incubated with macrophages. Luciferase reporter assay was used to confirm the target gene of miR-125a-5p. In mouse experiments, inhibiting miR-125a-5p was utilized to examine its role in macrophage polarization. Furthermore, the underlying mechanism was explored. RESULTS: In vitro results indicated that CSE treatment led to upregulation of miR-125a-5p in HBE cells, and exosomes contained miR-125a-5p. PKH26-labeled exosomes were internalized by macrophages. Co-culture experiments between bronchial epithelial cells and miR-125a-5p mimic resulted in significant increase in M1 macrophage markers (TNF-α, iNOS-2, IL-1ß) and decrease in M2 markers (IL-10 and Arg-1). In COPD mouse models, miR-125a-5p inhibitor reduced levels of TNF-α, IL-1ß, and IL-6. Luciferase assays revealed that miR-125a-5p inhibitors enhanced the relative luciferase activity of IL1RN. Mechanistic experiments demonstrated that HBE-derived exosomes transfected with miR-125a-5p mimics promoted upregulation of MyD88, TRAF6, p65, iNOS-2, and downregulation of Arg-1. CONCLUSION: This study suggests that exosomal miR-125a-5p may act as a mediator in the cross-talk between airway epithelium and macrophage polarization in COPD. Exosomal miR-125a-5p targeting IL1RN may promote M1 macrophage polarization via the MyD88/NF-κB pathway.

The disruptive effects of COPD exacerbation-associated factors on epithelial repair responses.

Introduction: Exacerbations of chronic obstructive pulmonary disease (COPD) increase mortality risk and can lead to accelerated loss of lung function. The increased inflammatory response during exacerbations contributes to worsening of airflow limitation, but whether it also impacts epithelial repair is unclear. Therefore, we studied the effect of the soluble factor micro-environment during COPD exacerbations on epithelial repair using an exacerbation cocktail (EC), composed of four factors that are increased in COPD lungs during exacerbations (IL-1ß, IL-6, IL-8, TNF-α). Methods: Mouse organoids (primary CD31-CD45-Epcam+ cells co-cultured with CCL206 fibroblasts) were used to study epithelial progenitor behavior. Mature epithelial cell responses were evaluated using mouse precision cut lung slices (PCLS). The expression of epithelial supportive factors was assessed in CCL206 fibroblasts and primary human lung fibroblasts. Results: EC exposure increased the number and size of organoids formed, and upregulated Lamp3, Muc5ac and Muc5b expression in day 14 organoids. In PCLS, EC imparted no effect on epithelial marker expression. Pre-treatment of CCL206 fibroblasts with EC was sufficient to increase organoid formation. Additionally, the expression of Il33, Tgfa and Areg was increased in CCL206 fibroblasts from EC treated organoids, but these factors individually did not affect organoid formation or size. However, TGF-α downregulated Foxj1 expression and upregulated Aqp5 expression in day 14 organoids. Conclusions: EC exposure stimulates organoid formation and growth, but it alters epithelial differentiation. EC changes the epithelial progenitor support function of fibroblasts which contributes to observed effects on epithelial progenitors.

Peroxidase-mediated mucin cross-linking drives pathologic mucus gel formation in IL-13-stimulated airway epithelial cells.

Mucus plugs occlude airways to obstruct airflow in asthma. Studies in patients and in mouse models show that mucus plugs occur in the context of type 2 inflammation, and studies in human airway epithelial cells (HAECs) show that IL-13-activated cells generate pathologic mucus independently of immune cells. To determine how HAECs autonomously generate pathologic mucus, we used a magnetic microwire rheometer to characterize the viscoelastic properties of mucus secreted under varying conditions. We found that normal HAEC mucus exhibited viscoelastic liquid behavior and that mucus secreted by IL-13-activated HAECs exhibited solid-like behavior caused by mucin cross-linking. In addition, IL-13-activated HAECs shows increased peroxidase activity in apical secretions, and an overlaid thiolated polymer (thiomer) solution shows an increase in solid behavior that was prevented by peroxidase inhibition. Furthermore, gene expression for thyroid peroxidase (TPO), but not lactoperoxidase (LPO), was increased in IL-13-activated HAECs and both TPO and LPO catalyze the formation of oxidant acids that cross-link thiomer solutions. Finally, gene expression for TPO in airway epithelial brushings was increased in patients with asthma with high airway mucus plug scores. Together, our results show that IL-13-activated HAECs autonomously generated pathologic mucus via peroxidase-mediated cross-linking of mucin polymers.

The differences between sinonasal respiratory epithelial adenomatoid hamartoma and nasal polyps: insights into immunopathology.

BACKGROUND: Respiratory epithelial adenomatoid hamartoma (REAH) is a benign lesion commonly occurring in the nasal cavity and sinuses. It is often accompanied by nasal polyps (NP). While the histological features of these two conditions have been studied, there is limited knowledge about their differences in the underlying immunopathology. METHODS: Nasal tissue specimens were collected from 8 patients with concurrent REAH and NP and 10 controls. The expression levels of inflammatory cytokines, tight junctions (TJ), and epithelial-mesenchymal transition (EMT)-related factors in the tissues were analyzed. The mRNA expression of the aforementioned factors was measured using qRT-PCR, while the expression of TJ and EMT-related proteins was analyzed through Western blotting and immunohistochemistry. RESULTS: Compared to the control group, levels of inflammatory cytokines (IFN-α, IL-5, IL-17A, IL-31, IL-33, and TNF-α) and EMT-related factors (α-SMA, COL1A1, MMP9, TGF-ß1, and Vimentin) were significantly increased in both REAH and NP tissues. Conversely, E-Cadherin and TJ-related factors (Claudin-4 and Occludin) significantly decreased. When comparing REAH with NP, it was observed that the expression of IL-4, IL-5, and IL-33 was lower in REAH, while TNF-ɑ; was higher. Regarding TJ-related factors, the expression of Occludin was lower in REAH. Furthermore, in terms of EMT-related factors, except for E-Cadherin, the expressions of ɑ-SMA, COL1A1, CTGF, MMP9, TGF-ß11, and Vimentin were higher in REAH. CONCLUSION: REAH and NP exhibit different immunopathological mechanisms. NP demonstrates a more severe inflammatory response, whereas REAH is characterized by a more pronounced TJ and EMT breakdown than NP.

Airway hillocks are injury-resistant reservoirs of unique plastic stem cells.

Airway hillocks are stratified epithelial structures of unknown function1. Hillocks persist for months and have a unique population of basal stem cells that express genes associated with barrier function and cell adhesion. Hillock basal stem cells continually replenish overlying squamous barrier cells. They exhibit dramatically higher turnover than the abundant, largely quiescent classic pseudostratified airway epithelium. Hillocks resist a remarkably broad spectrum of injuries, including toxins, infection, acid and physical injury because hillock squamous cells shield underlying hillock basal stem cells from injury. Hillock basal stem cells are capable of massive clonal expansion that is sufficient to resurface denuded airway, and eventually regenerate normal airway epithelium with each of its six component cell types. Hillock basal stem cells preferentially stratify and keratinize in the setting of retinoic acid signalling inhibition, a known cause of squamous metaplasia2,3. Here we show that mouse hillock expansion is the cause of vitamin A deficiency-induced squamous metaplasia. Finally, we identify human hillocks whose basal stem cells generate functional squamous barrier structures in culture. The existence of hillocks reframes our understanding of airway epithelial regeneration. Furthermore, we show that hillocks are one origin of 'squamous metaplasia', which is long thought to be a precursor of lung cancer.

Green tea extract suppresses airway inflammation via oxidative stress-driven MAPKs/MMP-9 signaling in asthmatic mice and human airway epithelial cells.

Introduction: The anti-inflammatory effect of green tea extract (GTE) has been confirmed in asthmatic mice, however, the pharmacological mechanism is not fully elucidated. Methods: To investigate the therapeutic efficacy of GTE in asthma and identify specific pathways, murine model of allergic asthma was established by ovalbumin (OVA) sensitization and the challenge for 4 weeks, with oral treatment using GTE and dexamethasone (DEX). Inflammatory cell counts, cytokines, OVA-specific IgE, airway hyperreactivity, and antioxidant markers in the lung were evaluated. Also, pulmonary histopathological analysis and western blotting were performed. In vitro, we established the model by stimulating the human airway epithelial cell line NCI-H292 using lipopolysaccharide, and treating with GTE and mitogen-activated protein kinases (MAPKs) inhibitors. Results: The GTE100 and GTE400 groups showed a decrease in airway hyperresponsiveness and the number of inflammatory cells in the bronchoalveolar lavage fluid (BALF) compared to the OVA group. GTE treatment also reduced interleukin (IL)-13, IL-5, and IL-4 levels in the BALF, and OVA-specific immunoglobulin E levels in the serum compared to those in the OVA group. GTE treatment decreased OVA-induced mucus secretion and airway inflammation. In addition, GTE suppressed the oxidative stress, and phosphorylation of MAPKs, which generally occurs after exposure to OVA. GTE administration also reduced matrix metalloproteinase-9 activity and protein levels. Conclusion: GTE effectively inhibited asthmatic respiratory inflammation and mucus hyperproduction induced by OVA inhalation. These results suggest that GTE has the potential to be used for the treatment of asthma.

Revisiting airway epithelial dysfunction and mechanisms in chronic obstructive pulmonary disease: the role of mitochondrial damage.

Chronic exposure to environmental hazards causes airway epithelial dysfunction, primarily impaired physical barriers, immune dysfunction, and repair or regeneration. Impairment of airway epithelial function subsequently leads to exaggerated airway inflammation and remodeling, the main features of chronic obstructive pulmonary disease (COPD). Mitochondrial damage has been identified as one of the mechanisms of airway abnormalities in COPD, which is closely related to airway inflammation and airflow limitation. In this review, we evaluate updated evidence for airway epithelial mitochondrial damage in COPD and focus on the role of mitochondrial damage in airway epithelial dysfunction. In addition, the possible mechanism of airway epithelial dysfunction mediated by mitochondrial damage is discussed in detail, and recent strategies related to airway epithelial-targeted mitochondrial therapy are summarized. Results have shown that dysregulation of mitochondrial quality and oxidative stress may lead to airway epithelial dysfunction in COPD. This may result from mitochondrial damage as a central organelle mediating abnormalities in cellular metabolism. Mitochondrial damage mediates procellular senescence effects due to mitochondrial reactive oxygen species, which effectively exacerbate different types of programmed cell death, participate in lipid metabolism abnormalities, and ultimately promote airway epithelial dysfunction and trigger COPD airway abnormalities. These can be prevented by targeting mitochondrial damage factors and mitochondrial transfer. Thus, because mitochondrial damage is involved in COPD progression as a central factor of homeostatic imbalance in airway epithelial cells, it may be a novel target for therapeutic intervention to restore airway epithelial integrity and function in COPD.

Akt-driven TGF-ß and DKK1 Secretion Impairs F508del Cystic Fibrosis Airway Epithelium Polarity.

Epithelial polarity is fundamental in maintaining barrier integrity and tissue protection. In cystic fibrosis (CF), apicobasal polarity of the airway epithelium is altered, resulting in increased apical fibronectin deposition and enhanced susceptibility to bacterial infections. Here, we evaluated the effect of highly effective modulator treatment (HEMT) on fibronectin apical deposition and investigated the intracellular mechanisms triggering the defect in polarity of the CF airway epithelium. To this end, primary cultures of CF (F508del variant) human airway epithelial cells (HAECs) and a HAEC line, Calu-3, knocked down for CFTR (CF transmembrane conductance regulator) were compared with control counterparts. We show that CFTR mutation in primary HAECs and CFTR knockdown cells promote the overexpression and oversecretion of TGF-ß1 and DKK1 when cultured at an air-liquid interface. These dynamic changes result in hyperactivation of the TGF-ß pathway and inhibition of the Wnt pathway through degradation of ß-catenin leading to imbalanced proliferation and polarization. The abnormal interplay between TGF-ß and Wnt signaling pathways is reinforced by aberrant Akt signaling. Pharmacological manipulation of TGF-ß, Wnt, and Akt pathways restored polarization of the F508del CF epithelium, a correction that was not achieved by HEMT. Our data shed new insights into the signaling pathways that fine-tune apicobasal polarization in primary airway epithelial cells and may provide an explanation to the mitigated efficacy of HEMT on lung infection in people with CF.