Small Airway Epithelium Research Articles

The pH-sensitive translocation of V-ATPase in the small airway of cystic fibrosis pigs.

In contrast to pig large airways, the pH of airway surface liquid (ASL) in pig small airways is regulated by CFTR mediated HCO-3 secretion and the vacuolar-type H+ ATPase (V-ATPase) proton secretion. We hypothesized that in Cystic Fibrosis (CF), the ASL pH of small airways is acidic, and the V-ATPase is internalized. We quantified proton secretion during the addition of an alkaline test solutions by measuring changes in a pH dependent fluorescent dye generated by porcine small airway epithelia in the absence and presence of bafilomycin A1. The pH-dependent translocation of V-ATPase in ex vivo and in vivo preparations was measured using immunolocalization of V-ATPase. We found that bafilomycin sensitive proton secretion stopped when the ASL pH was less than 7.10. In non-CF pigs and mice, we found that V-ATPase was localized in the apical membrane, and internalized when the lungs were instilled with a pH 6.8 solution.Studies where we immediately fixed lungs from pigs revealed apical V-ATPase detection in non-CF piglets and less apical detection in CF piglets. Our data suggest that V-ATPase in small airways is internalized when the ASL pH is acidic. The decrease in apical localization of V-ATPase in CF pigs is consistent with an acidic ASL pH.

Inflammation in the COVID-19 airway is due to inhibition of CFTR signaling by the SARS-CoV-2 spike protein

SARS-CoV-2-contributes to sickness and death in COVID-19 patients partly by inducing a hyper-proinflammatory immune response in the host airway. This hyper-proinflammatory state involves activation of signaling by NFκB, and unexpectedly, ENaC, the epithelial sodium channel. Post-infection inflammation may also contribute to "Long COVID"/PASC. Enhanced signaling by NFκB and ENaC also marks the airway of patients suffering from cystic fibrosis, a life-limiting proinflammatory genetic disease due to inactivating mutations in the CFTR gene. We therefore hypothesized that inflammation in the COVID-19 airway might similarly be due to inhibition of CFTR signaling by SARS-CoV-2 spike protein, and therefore activation of both NFκB and ENaC signaling. We used western blot and electrophysiological techniques, and an organoid model of normal airway epithelia, differentiated on an air–liquid-interface (ALI). We found that CFTR protein expression and CFTR cAMP-activated chloride channel activity were lost when the model epithelium was exposed to SARS-CoV-2 spike proteins. As hypothesized, the absence of CFTR led to activation of both TNFα/NFκB signaling and α and γ ENaC. We had previously shown that the cardiac glycoside drugs digoxin, digitoxin and ouabain blocked interaction of spike protein and ACE2. Consistently, addition of 30 nM concentrations of the cardiac glycoside drugs, prevented loss of both CFTR protein and CFTR channel activity. ACE2 and CFTR were found to co-immunoprecipitate in both basal cells and differentiated epithelia. Thus spike-dependent CFTR loss might involve ACE2 as a bridge between Spike and CFTR. In addition, spike exposure to the epithelia resulted in failure of endosomal recycling to return CFTR to the plasma membrane. Thus, failure of CFTR recovery from endosomal recycling might be a mechanism for spike-dependent loss of CFTR. Finally, we found that authentic SARS-CoV-2 virus infection induced loss of CFTR protein, which was rescued by the cardiac glycoside drugs digitoxin and ouabain. Based on experiments with this organoid model of small airway epithelia, and comparisons with 16HBE14o- and other cell types expressing normal CFTR, we predict that inflammation in the COVID-19 airway may be mediated by inhibition of CFTR signaling by the SARS-CoV-2 spike protein, thus inducing a cystic fibrosis-like clinical phenotype. To our knowledge this is the first time COVID-19 airway inflammation has been experimentally traced in normal subjects to a contribution from SARS-CoV-2 spike-dependent inhibition of CFTR signaling.

Platelet Activating Factor Receptor and Intercellular Adhesion Molecule-1 Expression Increases in the Small Airway Epithelium and Parenchyma of Patients with Idiopathic Pulmonary Fibrosis: Implications for Microbial Pathogenesis.

Background: Idiopathic pulmonary fibrosis (IPF) is an irreversible lung fibrotic disorder of unknown cause. It has been reported that bacterial and viral co-infections exacerbate disease pathogenesis. These pathogens use adhesion molecules such as platelet activating factor receptor (PAFR) and intercellular adhesion molecule-1 (ICAM-1) to gain cellular entry, causing infections. Methods: Immunohistochemical staining was carried out for lung resections from IPF patients (n = 11) and normal controls (n = 12). The quantification of PAFR and ICAM-1 expression is presented as a percentage in the small airway epithelium. Also, type 2 pneumocytes and alveolar macrophages were counted as cells per mm2 of the parenchymal area and presented as a percentage. All image analysis was done using Image Pro Plus 7.0 software. Results: PAFR expression significantly increased in the small airway epithelium (p < 0.0001), type 2 pneumocytes (p < 0.0001) and alveolar macrophages (p < 0.0001) compared to normal controls. Similar trend was observed for ICAM-1 expression in the small airway epithelium (p < 0.0001), type 2 pneumocytes (p < 0.0001) and alveolar macrophages (p < 0.0001) compared to normal controls. Furthermore, the proportion of positively expressed type 2 pneumocytes and alveolar macrophages was higher in IPF than in normal control. Conclusions: This is the first study to show PAFR and ICAM-1 expression in small airway epithelium, type 2 pneumocytes and alveolar macrophages in IPF. These findings could help intervene microbial impact and facilitate management of disease pathogenesis.

Inefficient antiviral response in reconstituted small-airway epithelium from chronic obstructive pulmonary disease patients following human parainfluenza virus type 3 infection

Chronic obstructive pulmonary disease (COPD) affects over 250 million individuals globally and stands as the third leading cause of mortality. Respiratory viral infections serve as the primary drivers of acute exacerbations, hastening the decline in lung function and worsening the prognosis. Notably, Human Parainfluenza Virus type 3 (HPIV-3) is responsible for COPD exacerbations with a frequency comparable to that of Respiratory Syncytial Virus and Influenza viruses. However, the impact of HPIV-3 on respiratory epithelium within the context of COPD remains uncharacterized.In this study, we employed in vitro reconstitution of lower airway epithelia from lung tissues sourced from healthy donors (n = 4) and COPD patients (n = 5), maintained under air–liquid interface conditions. Through a next-generation sequencing-based transcriptome analysis, we compared the cellular response to HPIV-3 infection.Prior to infection, COPD respiratory epithelia exhibited a pro-inflammatory profile, notably enriched in canonical pathways linked to antiviral response, B cell signaling, IL-17 signaling, and epithelial-mesenchymal transition, in contrast to non-COPD epithelia. Intriguingly, post HPIV-3 infection, only non-COPD epithelia exhibited significant enrichment in interferon signaling, pattern recognition receptors of viruses and bacteria, and other pathways involved in antiviral responses. This deficiency could potentially hinder immune cell recruitment essential for controlling viral infections, thus fostering prolonged viral presence and persistent inflammation.

Differential expression of mast cells in the small airways and alveolar septa of current smokers and patients with small airway disease and COPD.

COPD patients suffer from dysregulated and suppressed immune functionality, determined by their loss of degranulating capacity. Here we provide crucial information on the presence of degranulated mast cells (MCs) in COPD airways and demonstrate their relationship to lung physiology and airway remodelling. Small airway lung resections from non-smoking controls (NC), normal lung function smokers (NLFS), small airway disease (SAD), and mild-to-moderate COPD current smokers (COPD-CS) and ex-smokers (COPD-ES) were dual immuno-stained with MC tryptase and degranulation marker lysosome-associated membrane protein (LAMP)-1. Total MCs, degranulating MCs and non-MCs were enumerated in small airway epithelium and subepithelium, and in alveolar septa. In the small airway wall subepithelial areas, COPD-CS and COPD-ES patients had significantly lower MCs than the NC group (p<0.05), although the numbers were considerably higher in the small airway epithelium (p<0.01). Degranulating non-MCs were higher in SAD (p<0.05) than in COPD in the small airway subepithelium. In contrast, there were significant increases in total MCs (degranulated and non-degranulated) and degranulated non-MCs in the alveolar septum of COPD patients compared with the NC group (p<001). The lower numbers of MCs in the subepithelium correlated with lower forced expiratory volume in 1 s (FEV1)/forced vital capacity (FVC) and forced expiratory flow at 25-75% of FVC (FEF25-75%), higher smoking rates in COPD patients, and increased small airway wall thickness and extracellular matrix. The increase in MCs in the alveolar septum negatively correlated with FEF25-75%. This study is the first to assess the differential pattern of MC, degranulating MC and non-MC populations in the small airways and alveoli of COPD patients. The spatial positioning of the MCs within the airways showed variable correlations with lung function.

Lipidated brush-PEG polymers as low molecular weight pulmonary drug delivery platforms

ABSTRACT Objectives Nanomedicines are being actively developed as inhalable drug delivery systems. However, there is a distinct utility in developing smaller polymeric systems that can bind albumin in the lungs. We therefore examined the pulmonary pharmacokinetic behavior of a series of lipidated brush-PEG (5 kDa) polymers conjugated to 1C2, 1C12 lipid or 2C12 lipids. Methods The pulmonary pharmacokinetics, patterns of lung clearance and safety of polymers were examined in rats. Permeability through monolayers of primary human alveolar epithelia, small airway epithelia and lung microvascular endothelium were also investigated, along with lung mucus penetration and cell uptake. Results Polymers showed similar pulmonary pharmacokinetic behavior and patterns of lung clearance, irrespective of lipid molecular weight and albumin binding capacity, with up to 30% of the dose absorbed from the lungs over 24 h. 1C12-PEG showed the greatest safety in the lungs. Based on its larger size, 2C12-PEG also showed the lowest mucus and cell membrane permeability of the three polymers. While albumin had no significant effect on membrane transport, the cell uptake of C12-conjugated PEGs were increased in alveolar epithelial cells. Conclusion Lipidated brush-PEG polymers composed of 1C12 lipid may provide a useful and novel alternative to large nanomaterials as inhalable drug delivery systems.

Utilization of endogenous albumin trafficking pathways in the lungs has potential to modestly increase the lung interstitial access and absorption of drug delivery systems after inhaled administration

ABSTRACT Objectives Drug delivery systems typically show limited access to the lung interstitium and absorption after pulmonary delivery. The aim of this work was to undertake a proof-of-concept investigation into the potential of employing endogenous albumin and albumin absorption mechanisms in the lungs to improve lung interstitial access and absorption of inhaled drug delivery systems that bind albumin. Methods The permeability of human albumin (HSA) through monolayers of primary human alveolar epithelia, small airway epithelia, and microvascular endothelium were investigated. The pulmonary pharmacokinetics of bovine serum albumin (BSA) was also investigated in efferent caudal mediastinal lymph duct-cannulated sheep after inhaled aerosol administration. Results Membrane permeability coefficient values (Papp) of HSA increased in the order alveolar epithelia<small airway epithelia<microvascular endothelium, where the permeability of HSA through small airway and microvascular endothelia were approximately 4- and 28-fold higher than alveolar epithelia, respectively. Only 6.5% of the delivered BSA aerosol dose was absorbed from the lungs of sheep over 5 days, although half of the absorbed dose was absorbed via the lung lymph. Conclusion Drug delivery systems that bind endogenous albumin may show a modest increase in lung permeability and absorption after inhaled delivery compared to systems that do not efficiently bind albumin.

Serial Sampling of the Small Airway Epithelium to Identify Persistent Smoking-dysregulated Genes.

Rationale: The small airway epithelium (beyond the sixth generation), the initiation site of smoking-induced airway disorders, is highly sensitive to the stress of smoking. Because of variations over time in smoking habits, the small airway epithelium transcriptome is dynamic, fluctuating not only among smokers but also within each smoker. Objectives: To perform accurate assessment of the smoking-related dysregulation of the human small airway epithelium despite the variation of smoking within the same individual and of the effects of smoking cessation on the dysregulated transcriptome. Methods: We conducted serial sampling of the same smokers and nonsmoker control subjects over time to identify persistent smoking dysregulation of the biology of the small airway epithelium over 1 year. We conducted serial sampling of smokers who quit smoking, before and after smoking cessation, to assess the effect of smoking cessation on the smoking-dysregulated genes. Measurements and Main Results: Repeated measures ANOVA of the small airway epithelium transcriptome sampled four times in the same individuals over 1 year enabled the identification of 475 persistent smoking-dysregulated genes. Most genes were normalized after 12 months of smoking cessation; however, 53 (11%) genes, including CYP1B1, PIR, ME1, and TRIM16, remained persistently abnormally expressed. Dysregulated pathways enriched with the nonreversible genes included xenobiotic metabolism signaling, bupropion degradation, and nicotine degradation. Conclusions: Analysis of repetitive sampling of the same individuals identified persistent smoking-induced dysregulation of the small airway epithelium transcriptome and the effect of smoking cessation. These results help identify targets for the development of therapies that can be applicable to smoking-related airway diseases.

Transforming growth factor-β1 and SMAD signalling pathway in the small airways of smokers and patients with COPD: potential role in driving fibrotic type-2 epithelial mesenchymal transition.

COPD is a common disease characterized by respiratory airflow obstruction. TGF-β1 and SMAD pathway is believed to play a role in COPD pathogenesis by driving epithelial mesenchymal transition (EMT). We investigated TGF-β1 signalling and pSmad2/3 and Smad7 activity in resected small airway tissue from patients with; normal lung function and a smoking history (NLFS), current smokers and ex-smokers with COPD GOLD stage 1 and 2 (COPD-CS and COPD-ES) and compared these with normal non-smoking controls (NC). Using immunohistochemistry, we measured activity for these markers in the epithelium, basal epithelium, and reticular basement membrane (RBM). Tissue was also stained for EMT markers E-cadherin, S100A4 and vimentin. The Staining of pSMAD2/3 was significantly increased in the epithelium, and RBM of all COPD groups compared to NC (p <0.0005). There was a less significant increase in COPD-ES basal cell numbers compared to NC (p= 0.02). SMAD7 staining showed a similar pattern (p <0.0001). All COPD group levels of TGF-β1 in the epithelium, basal cells, and RBM cells were significantly lower than NC (p <0.0001). Ratio analysis showed a disproportionate increase in SMAD7 levels compared to pSMAD2/3 in NLFS, COPD-CS and COPD-ES. pSMAD negatively correlated with small airway calibre (FEF25-75%; p= 0.03 r= -0.36). EMT markers were active in the small airway epithelium of all the pathological groups compared to patients with COPD. Activation of the SMAD pathway via pSMAD2/3 is triggered by smoking and active in patients with mild to moderate COPD. These changes correlated to decline in lung function. Activation of the SMADs in the small airways is independent of TGF-β1, suggesting factors other than TGF-β1 are driving these pathways. These factors may have implications for small airway pathology in smokers and COPD through the process of EMT, however more mechanistic work is needed to prove these correlations.

Peroxinredoxin 6 reduction accelerates cigarette smoke extract‑induced senescence by regulating autophagy in BEAS‑2B cells.

Cigarette smoke (CS)-induced accelerated senescence and insufficient autophagy has been implicated in the pathogenesis of chronic obstructive pulmonary disease (COPD). Peroxiredoxin (PRDX) 6 is a protein with prevalent antioxidant capacity. Previous studies indicate that PRDX6 could activate autophagy and alleviate senescence in other diseases. The present study investigated whether PRDX6-regulated autophagy was involved in the regulation of CS extract (CSE)-induced BEAS-2B cell senescence via the knockdown of PRDX6 expression. Furthermore, the present study evaluated the mRNA levels of PRDX6, autophagy and senescence-associated genes in the small airway epithelium from patients with COPD by analyzing the GSE20257 dataset from the Gene Expression Omnibus database. The results demonstrated that CSE reduced PRDX6 expression levels and transiently induced the activation of autophagy, followed by the accelerated senescence of BEAS-2B cells. Knockdown of PRDX6 induced autophagy degradation and accelerated senescence in CSE-treated BEAS-2B cells. Furthermore, autophagy inhibition by 3-Methyladenine increased P16 and P21 expression levels, while autophagy activation by rapamycin reduced P16 and P21 expression levels in CSE-treated BEAS-2B cells. The GSE20257 dataset revealed that patients with COPD had lower PRDX6, sirtuin (SIRT) 1 and SIRT6 mRNA levels, and higher P62 and P16 mRNA levels compared with non-smokers. P62 mRNA was significantly correlated with P16, P21 and SIRT1, which indicated that insufficient autophagic clearance of damaged proteins could be involved in accelerated cell senescence in COPD. In conclusion, the present study demonstrated a novel protective role for PRDX6 in COPD. Furthermore, a reduction in PRDX6 could accelerate senescence by inducing autophagy impairment in CSE-treated BEAS-2B cells.

The influence of pyroptosis-related genes on the development of chronic obstructive pulmonary disease

Increasing evidences have demonstrated that pyroptosis exerts key roles in the occurrence, development of chronic obstructive pulmonary disease. However, the mechanisms of pyroptosis in COPD remain largely unknown. In our research, Statistics were performed using R software and related packages in this study. Series matrix files of small airway epithelium samples were downloaded from the GEO database. Differential expression analysis with FDR < 0.05 was performed to identify COPD-associated pyroptosis-related genes. 8 up-regulated genes (CASP4, CASP5, CHMP7, GZMB, IL1B, AIM2, CASP6, GSDMC) and 1 down-regulated genes (PLCG1) was identified as COPD-associated pyroptosis-related genes. Twenty-six COPD key genes was identified by WGCNA analysis. PPI analysis and gene correlation analysis showed their relationship clearly. KEGG and GO analysis have revealed the main pyroptosis-related mechanism of COPD. The expression of 9 COPD-associated pyroptosis-related genes in different grades was also depicted. The immune environment of COPD was also explored. Furthermore, the relationship of pyroptosis-related genes and the expression of immune cells was also be shown in the end. In the end, we concluded that pyroptosis influences the development of COPD. This study may provide new insight into the novel therapeutic targets for COPD clinical treatment.

9 Understanding how CeO2 Nanoparticles Modulate Bleomycin-Induced Inflammatory and Fibrotic Events in Both in Vivo and in Vitro Models

Abstract Cerium oxide nanoparticles (CeO2NPs) from some diesel fuel additives and other applications have been detected in ambient air. Concerns have been raised over their potential human health impact in situations of inadvertent exposure. Oxidative mechanisms have been suggested as a common feature for pulmonary injury in response to airborne particulate matter, including engineered nanomaterials. To understand in depth how CeO2NPs may influence oxidative stress induced pulmonary inflammation and fibrotic events, we used both in vivo and in vitro bleomycin-induced lung injury models. Male Sprague-Dawley rats were intratracheally instilled with bleomycin or saline (control) followed by nose-only inhalation exposure to nano-sized CeO2NP aerosols (mass concentration 1.8 mg/m3) or water (controls) for 3 hours per day for 4 days per week for one or two weeks. At 3 days post exposure, animals were sacrificed and bronchoalveolar lavage (BAL) fluid, lung histopathology and global mRNA expression analysed. Bleomycin exposure resulted in an increase in total BAL cells, fibrotic staining and significant induction of inflammatory and oxidative stress on mRNA sequencing analysis. Modifications of these responses by one-week exposure to CeO2NPs included attenuation of fibrotic staining and gene expression markers of lung function, inflammation and epithelial-mesenchymal transition (EMT). CeO2NP alone resulted in increased inflammatory responses but did not appear to cause fibrotic changes. Interpretation of these responses at a cellular level was further explored using 3D human small airway epithelium cultures (SmallAirTM) in an aerosol exposure air-liquid-interface system. This also indicated that some bleomycin-induced cellular responses could be attenuated by exposure to CeO2NP aerosols.

Anoikis resistance of small airway epithelium is involved in the progression of chronic obstructive pulmonary disease.

Anoikis resistance is recognized as a crucial step in the metastasis of cancer cells. Most epithelial tumors are distinguished by the ability of epithelial cells to abscond anoikis when detached from the extracellular matrix. However, no study has investigated the involvement of anoikis in the small airway epithelium (SAE) of chronic obstructive pulmonary disease (COPD). Anoikis-related genes (ANRGs) exhibiting differential expression in COPD were identified using microarray datasets obtained from the Gene Expression Omnibus (GEO) database. Unsupervised clustering was performed to classify COPD patients into anoikis-related subtypes. Gene Ontology (GO) analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, gene set enrichment analysis (GSEA), and gene set variation analysis (GSVA) were used to annotate the functions between different subtypes. Differential expression analysis and weighted gene co-expression network analysis (WGCNA) were leveraged to identify key molecules. The relative proportion of infiltrating immune cells in the SAE was quantified using the CIBERSORT and ssGSEA computational algorithms, and the correlation between key molecules and immune cell abundance was analyzed. The expression of key molecules in BEAS-2B cells exposed to cigarette smoke extract (CSE) was validated using qRT-PCR. A total of 25 ANRGs exhibited differential expression in the SAE of COPD patients, based on which two subtypes of COPD patients with distinct anoikis patterns were identified. COPD patients with anoikis resistance had more advanced GOLD stages and cigarette consumption. Functional annotations revealed a different immune status between COPD patients with pro-anoikis and anoikis resistance. Tenomodulin (TNMD) and long intergenic non-protein coding RNA 656 (LINC00656) were subsequently identified as key molecules involved in this process, and a close correlation between TNMD and the infiltrating immune cells was observed, such as activated CD4+ memory T cells, M1 macrophages, and activated NK cells. Further enrichment analyses clarified the relationship between TNMD and the inflammatory and apoptotic signaling pathway as the potential mechanism for regulating anoikis. In vitro experiments showed a dramatic upregulation of TNMD and LINC00656 in BEAS-2B cells when exposed to 3% CSE for 48 hours. TNMD contributes to the progression of COPD by inducing anoikis resistance in SAE, which is intimately associated with the immune microenvironment.

Differentially Expressed Genes Analysis in the Human Small Airway Epithelium of Healthy Smokers Shows Potential Risks of Disease Caused by Oxidative Stress and Inflammation and the Potentiality of Astaxanthin as an Anti-Inflammatory Agent.

Cigarette smoke (CS) was known for its effect of increasing oxidative stress that could trigger tissue injury and endothelial dysfunction mediated by free radicals and reactive oxygen species (ROS). ROS itself is a key signaling molecule that plays a role in the development of inflammatory disorders. Nuclear factor erythroid2 related factor2 (Nrf2) is the main regulator of antioxidant cellular response to cell and tissue-destroying components caused by CS. Nrf2 protein that is significantly activated in the smokers' small airway epithelium is followed by a series of gene expression changes in the same cells. This study aims to observe differentially expressed genes (DEGs) in the human small airway epithelium of smokers compared to genes whose expression changes due to astaxanthin (AST) treatment, an antioxidant compound that can modulate Nrf2. Gene expression data that was stored in the GEO browser (GSE 11952) was analyzed using GEO2R to search for DEG among smokers and nonsmokers subject. DEG was further compared to those genes whose expression changes due to astaxanthin treatment (AST) that were obtained from the Comparative Toxicogenomics Database (CTD; https://ctdbase.org/). DEG (p < 0.05) analysis result shows that there are 23 genes whose expression regulation is reversed compared to gene expression due to AST treatment. The gene function annotations of the 23 DEGs showed the involvement of some of these genes in chemical and oxidative stress, reactive oxygen species (ROS), and apoptotic signaling pathways. All of the genes were involved/associated with chronic bronchitis, adenocarcinoma of the lung, non-small-cell lung carcinoma, carcinoma, small cell lung carcinoma, type 2 diabetes mellitus, emphysema, ischemic stroke, lung diseases, and inflammation. Thus, AST treatment for smokers could potentially decrease the development of ROS and oxidative stress that leads to inflammation and health risks associated with smoking.

Metabolism-Related Gene TXNRD1 Regulates Inflammation and Oxidative Stress Induced by Cigarette Smoke through the Nrf2/HO-1 Pathway in the Small Airway Epithelium.

Chronic obstructive pulmonary disease (COPD), a small airway disease, is regarded as a metabolic disorder. To further uncover the metabolic profile of COPD patients, it is necessary to identify metabolism-related differential genes in small airway epithelium (SAE) of COPD. Metabolism-related differential genes in SAE between COPD patients and nonsmokers were screened from GSE128708 and GSE20257 datasets. KEGG, GO, and PPI analyses were performed to evaluate the pathway enrichment, term enrichment, and protein interaction of candidate metabolism-related differential genes, respectively. RT-PCR was used to verify the mRNA expression of the top ten differential genes. Western blotting was used to evaluate the protein expression of TXNRD1. TXNRD1 inhibitor auranofin (AUR) was used to assess the impact of TXNRD1 on oxidative stress and inflammation induced by cigarette smoke extraction (CSE). Twenty-four metabolism-related differential genes were selected. ALDH3A1, AKR1C3, CYP1A1, AKC1C1, CPY1B1, and TXNRD1 in the top ten genes were significantly upregulated after CSE simulation for 24 h in human bronchial epithelial (16HBE) cells. Among them, CYP1A1 and TXNRD1 also have a significant upregulation in primary SAE after simulation of CSE for 24 h. The overexpression of protein TXNRD1 has also been detected in 16HBE cells, primary SAE stimulated with CSE, and mouse lung exposed to cigarette smoke (CS). Additionally, inhibition of TXNRD1 with 0.1 μM AUR alleviated the expression of IL-6 and reactive oxygen species (ROS) induced by CSE by activating the Nrf2/HO-1 pathway in 16HBE cells. This study identified twenty-four metabolism-related differential genes associated with COPD. TXNRD1 might participate in the oxidative stress and inflammation induced by CS by regulating the activation of the Nrf2/HO-1 pathway.

Oral epithelium response of electronic cigarette users to electronic cigarette.

Electronic cigarettes are increasing in popularity, but there is only little information on their biologic effects on the oral epithelium, the initial site exposed to electronic cigarette smoke. We assessed the oral epithelium response to electronic cigarettes by comparing the histology and RNA transcriptome (mRNA and miRNA) of healthy electronic cigarette vapers to nonsmokers. mRNA was assessed based on: (1) genome-wide; (2) genes previously identified as dysregulated in the oral epithelium of electronic cigarette vapers versus nonsmokers; (3) immune and inflammatory-related genes previously identified as dysregulated in the nasal epithelium of electronic cigarette vapers compared to nonsmokers; (4) genes previously identified as dysregulated in the small airway epithelium of nonsmokers following an acute exposure to electronic cigarette; and (5) genes related to the initial steps of COVID-19 infection. In addition, miRNA was assessed genome-wide. Comparisons were performed using analysis of variance, and Benajmini-Hochberg corrected p< 0.05 was considered significant. The histology of the epithelium, lamina propria and basal layer in electronic cigarette vapers appeared normal. Assessment of mRNA and miRNA, based on all gene lists, did not identify any genes significantly modified in the oral epithelium of electronic cigarette vapers in response to electronic cigarette use. An average history of 2 years of vaping results in no detectable histologic or transcriptome abnormalities in the buccal mucosa.

Identification of Small Airway Epithelium-Related Hub Genes in Chronic Obstructive Pulmonary Disease.

Pulmonary small airway epithelia are the primary site of cellular and histological alterations in chronic obstructive pulmonary disease (COPD), while the potential therapeutic hub genes of pulmonary epithelia are rarely identified to elucidate profound alterations in the progression of the disease. Microarray dataset of GSE11906 containing small airway epithelia from 34 healthy non-smokers and 33 COPD patients was applied to screen differentially expressed genes (DEGs). Weighted gene correlation network analysis (WGCNA) was further used to identify the hub genes related to clinical features. Moreover, single-cell RNA sequencing data from GSE173896 and GSE167295 dataset were applied to explore the expression and distribution of the hub genes. The expression levels of hub genes in epithelial cells stimulated by cigarette smoke extract (CSE) were detected by RT-qPCR. Ninety-eight DEGs correlated with clinical features of COPD were identified via limma and WGCNA. Eight hub genes (including AKR1C3, ALDH3A1, AKR1C1, CYP1A1, GPX2, CBR3, AKR1B1 and GSR) that might exert an antioxidant role in COPD process were identified. Single-cell transcriptomic analysis indicated that the expressions of AKRAC3, ALDH3A1, GPX2, CBR3 and AKR1B1 were significantly increased in the COPD group when compared with the normal group. Moreover, we found that the expression of ALDH3A1 was the most abundantly expressed in ciliated cells. RT-qPCR results indicated that the majority of candidate novel genes were significantly elevated when the epithelial cells were exposed to CSE. Through integrating limma, WGCNA, and protein-protein interaction (PPI) analysis, a total of eight candidate hub genes of pulmonary airway epithelia were identified in COPD. Moreover, single-cell transcriptomic analysis indicated that ALDH3A1 was enriched in ciliated cells, which may provide a new insight into the pathogenesis and treatment of COPD.

630 Cellular tropism of adeno-associated viral vector 4 (AAV4) in human large and small airway epithelia

630 Cellular tropism of adeno-associated viral vector 4 (AAV4) in human large and small airway epithelia

Integrated genomics approaches identify transcriptional mediators and epigenetic responses to Afghan desert particulate matter in small airway epithelial cells.

Military Deployment to Southwest Asia and Afghanistan and exposure to toxic airborne particulates have been associated with an increased risk of developing respiratory disease, collectively termed deployment-related respiratory diseases (DRRDs). Our knowledge about how particulates mediate respiratory disease is limited, precluding the appropriate recognition or management. Central to this limitation is the lack of understanding of how exposures translate into dysregulated cell identity with dysregulated transcriptional programs. The small airway epithelium is involved in both the pathobiology of DRRD and fine particulate matter deposition. To characterize small airway epithelial cell epigenetic and transcriptional responses to Afghan desert particulate matter (APM) and investigate the functional interactions of transcription factors that mediate these responses, we applied two genomics assays, the assay for transposase accessible chromatin with sequencing (ATAC-seq) and Precision Run-on sequencing (PRO-seq). We identified activity changes in a series of transcriptional pathways as candidate regulators of susceptibility to subsequent insults, including signal-dependent pathways, such as loss of cytochrome P450 or P53/P63, and lineage-determining transcription factors, such as GRHL2 loss or TEAD3 activation. We further demonstrated that TEAD3 activation was unique to APM exposure despite similar inflammatory responses when compared with wood smoke particle exposure and that P53/P63 program loss was uniquely positioned at the intersection of signal-dependent and lineage-determining transcriptional programs. Our results establish the utility of an integrated genomics approach in characterizing responses to exposures and identifying genomic targets for the advanced investigation of the pathogenesis of DRRD.

Secretory Cells Are the Primary Source of pIgR in Small Airways.

Loss of secretory IgA (SIgA) is common in chronic obstructive pulmonary disease (COPD) small airways and likely contributes to disease progression. We hypothesized that loss of SIgA results from reduced expression of pIgR (polymeric immunoglobulin receptor), a chaperone protein needed for SIgA transcytosis, in the COPD small airway epithelium. pIgR-expressing cells were defined and quantified at single-cell resolution in human airways using RNA in situ hybridization, immunostaining, and single-cell RNA sequencing. Complementary studies in mice used immunostaining, primary murine tracheal epithelial cell culture, and transgenic mice with secretory or ciliated cell-specific knockout of pIgR. SIgA degradation by human neutrophil elastase or secreted bacterial proteases from nontypeable Haemophilus influenzae was evaluated invitro. We found that secretory cells are the predominant cell type responsible for pIgR expression in human and murine airways. Loss of SIgA in small airways was not associated with a reduction in secretory cells but rather a reduction in pIgR protein expression despite intact PIGR mRNA expression. Neutrophil elastase and nontypeable H. influenzae-secreted proteases are both capable of degrading SIgA invitro and may also contribute to a deficient SIgA immunobarrier in COPD. Loss of the SIgA immunobarrier in small airways of patients with severe COPD is complex and likely results from both pIgR-dependent defects in IgA transcytosis and SIgA degradation.