Quercetin improves epithelial regeneration from airway basal cells of COPD patients.

BACKGROUND: Airway basal cells (BC) from patients with chronic obstructive pulmonary disease (COPD) regenerate abnormal airway epithelium and this was associated with reduced expression of several genes involved in epithelial repair. Quercetin reduces airway epithelial remodeling and inflammation in COPD models, therefore we examined whether quercetin promotes normal epithelial regeneration from COPD BC by altering gene expression. METHODS: COPD BC treated with DMSO or 1 µM quercetin for three days were cultured at air/liquid interface (ALI) for up to 4 weeks. BC from healthy donors cultured at ALI were used as controls. Polarization of cells was determined at 8 days of ALI. The cell types and IL-8 expression in differentiated cell cultures were quantified by flow cytometry and ELISA respectively. Microarray analysis was conducted on DMSO or 1 µM quercetin-treated COPD BC for 3 days to identify differentially regulated genes (DEG). Bronchial brushings obtained from COPD patients with similar age and disease status treated with either placebo (4 subjects) or 2000 mg/day quercetin (7 subjects) for 6 months were used to confirm the effects of quercetin on gene expression. RESULTS: Compared to placebo-, quercetin-treated COPD BC showed significantly increased transepithelial resistance, more ciliated cells, fewer goblet cells, and lower IL-8. Quercetin upregulated genes associated with tissue and epithelial development and differentiation in COPD BC. COPD patients treated with quercetin, but not placebo showed increased expression of two developmental genes HOXB2 and ELF3, which were also increased in quercetin-treated COPD BC with FDR < 0.001. Active smokers showed increased mRNA expression of TGF-ß (0.067) and IL-8 (22.0), which was reduced by 3.6 and 4.14 fold respectively after quercetin treatment. CONCLUSIONS: These results indicate that quercetin may improve airway epithelial regeneration by increasing the expression of genes involved in epithelial development/differentiation in COPD. TRIAL REGISTRATION: This study was registered at ClinicalTrials.gov on 6-18-2019. The study number is NCT03989271.

Identification of toxicity-induced biomarkers in human non-immune airway cells exposed to respiratory sensitizers: A mechanistic approach.

Occupational asthma covers a group of work-related diseases whose clinical manifestations include airway hyperresponsiveness and airflow limitation. Although the chemical respiratory allergy (CRA) induced by Low Molecular Weight (LMW) sensitizers is a major concern, especially in terms of the regulatory framework, to date there are no methods available for preclinically addressing this toxicological outcome, as its mechanistic background is not fully understood at molecular or cellular levels. This paper proposes a mechanistic study applying New Approach Methodologies (NAM) of the pro-inflammatory and functional effects triggered by LMW respiratory allergens in different respiratory tract cell lines, including bronchial epithelial (BEAS-2B), lung fibroblast (MRC-5), and endothelial cells (EA.hy926), and an analysis of the capacity of such chemicals to interact with the mucin protein, to address certain toxicodynamic aspects of such compounds. The results showed that some of the sensitizers evaluated interact with mucin, the main protein mucus component, but the toxicant-mucin complex formation does not seem to be a common feature of different chemical classes of allergens. At a cellular level, sensitizers promoted an increase in IL-8, IL-6, and IL-1ß production in the evaluated cell types. It also impaired the MUC1 expression by bronchial cells and activated endothelial cells, thereby increasing the ICAM-I surface expression. Taken together, our results showed that these aforementioned cell types participate in the CRA Adverse Outcome Pathway and must be considered when developing preclinical testing strategies, particularly investigating danger signal production after exposure to LMW sensitizers in different tissue compartments.

Lack of a transcriptional response of primary bronchial epithelial cells from patients with asthma and controls to IL-33.

IL-33 and IL-1RL1 are well-replicated asthma genes that act in a single pathway toward type-2 immune responses. IL-33 is expressed by basal epithelial cells, and the release of IL-33 upon epithelial damage can activate innate lymphoid cells, T helper-2 cells, basophilic granulocytes, and mast cells through a receptor complex containing IL-1RL1. However, it is unknown how bronchial epithelial cells respond to IL-33, and whether this response is increased in the disease. We aimed to characterize the IL-33-driven transcriptomic changes in cultured primary bronchial epithelial cells from patients with asthma and healthy controls. Primary bronchial epithelial cells (PBECs) were obtained by bronchial brushing from six healthy control for air-liquid interface (ALI) cultures, whereas we selected eight healthy controls and seven patients with asthma for epithelial organoid cultures. We then stimulated the cultures for 24 h with recombinant IL-33 (rhIL33) at various concentrations with 1, 10, and 50 ng/mL for the ALI cultures and 20 ng/mL and 100 ng/mL for the organoid cultures, followed by RNA-sequencing and differential gene expression analysis. We did not detect any genome-wide significant differentially expressed genes after stimulation of PBECs with IL-33, irrespective of growth in three-dimensional (3-D) epithelial organoids or after differentiation in ALI cultures. These results were identical between PBECs obtained from patients with asthma or from healthy control subjects. We detected very low levels of IL-1RL1 gene expression in these airway epithelial cell cultures. We conclude that bronchial epithelial cells do not have a transcriptional response to IL-33, independent of their differentiation state. Hence, the airway epithelium acts as a source of IL-33 but does not seem to contribute to the response upon release of the alarmin after epithelial damage.NEW & NOTEWORTHY The IL-33/IL-1RL1 pathway stands as a formidable genetic predisposition for asthma, with ongoing clinical developments of various drugs designed to mitigate its influence in patients with asthma. The absence of a transcriptomic reaction to IL-33 within the bronchial epithelium holds significance in the pursuit of identifying biomarkers that can aid in pinpointing those individuals who would derive the greatest benefit from therapies targeting the IL-33 pathway.

Identification of host essential factors for recombinant AAV transduction of the polarized human airway epithelium.

IMPORTANCE: The essential steps of successful gene delivery by recombinant adeno-associated viruses (rAAVs) include vector internalization, intracellular trafficking, nuclear import, uncoating, double-stranded (ds)DNA conversion, and transgene expression. rAAV2.5T has a chimeric capsid of AAV2 VP1u and AAV5 VP2 and VP3 with the mutation A581T. Our investigation revealed that KIAA0319L, the multiple AAV serotype receptor, is not essential for vector internalization but remains critical for efficient vector transduction to human airway epithelia. Additionally, we identified that a novel gene WDR63, whose cellular function is not well understood, plays an important role in vector transduction of human airway epithelia but not vector internalization and nuclear entry. Our study also discovered the substantial transduction potential of rAAV2.5T in basal stem cells of human airway epithelia, underscoring its utility in gene editing of human airways. Thus, the knowledge derived from this study holds promise for the advancement of gene therapy in the treatment of pulmonary genetic diseases.

Electronic cigarette exposure disrupts airway epithelial barrier function and exacerbates viral infection.

The use of electronic cigarettes (e-cigs), especially among teenagers, has reached alarming and epidemic levels, posing a significant threat to public health. However, the short- and long-term effects of vaping on the airway epithelial barrier are unclear. Airway epithelial cells are the forefront protectors from viruses and pathogens. They contain apical junctional complexes (AJCs), which include tight junctions (TJs) and adherens junctions (AJs) formed between adjacent cells. Previously, we reported respiratory syncytial virus (RSV) infection, the leading cause of acute lower respiratory infection-related hospitalization in children and high-risk adults, induces a "leaky airway" by disrupting the epithelial AJC structure and function. We hypothesized chemical components of e-cigs disrupt airway epithelial barrier and exacerbate RSV-induced airway barrier dysfunction. Using confluent human bronchial epithelial (16HBE) cells and well-differentiated normal human bronchial epithelial (NHBE) cells, we found that exposure to extract and aerosol e-cig nicotine caused a significant decrease in transepithelial electrical resistance (TEER) and the structure of the AJC even at noncytotoxic concentrations. Western blot analysis of 16HBE cells exposed to e-cig nicotine extract did not reveal significant changes in AJC proteins. Exposure to aerosolized e-cig cinnamon or menthol flavors also induced barrier disruption and aggravated nicotine-induced airway barrier dysfunction. Moreover, preexposure to nicotine aerosol increased RSV infection and the severity of RSV-induced airway barrier disruption. Our findings demonstrate that e-cig exposure disrupts the airway epithelial barrier and exacerbates RSV-induced damage. Knowledge gained from this study will provide awareness of adverse e-cig respiratory effects and positively impact the mitigation of e-cig epidemic.NEW & NOTEWORTHY Electronic cigarette (e-cig) use, especially in teens, is alarming and at epidemic proportions, threatening public health. Our study shows that e-cig nicotine exposure disrupts airway epithelial tight junctions and increases RSV-induced barrier dysfunction. Furthermore, exposure to aerosolized flavors exaggerates e-cig nicotine-induced airway barrier dysfunction. Our study confirms that individual and combined components of e-cigs deleteriously impact the airway barrier and that e-cig exposure increases susceptibility to viral infection.

Morphologically and karyotypically atypical cells of 'normal' human bronchial epithelial cell line (Beas-2B).

Beas-2B is an adenovirus 12-SV40-transfected cell line of "normal" human bronchial epithelial cells. This cell line was able to replace normal human bronchial epithelial cells, which are currently unavailable, and served as a model for related studies in numerous toxicology and cancer transformation experiments. In any experiment involving toxins or carcinogens, the basic morphology of Beas-2B should be well characterized prior to exposure, but this has never been properly reported. In this study, atypical cells of the Beas-2B cell line in early passage culture were observed using light and electron microscopy, and the cells were further investigated for abnormal karyotypes by flow cytometry. This Beas-2B cell line could be morphologically categorized into two cell types, A and B. Type A contains a large nucleus and abundant cytoplasm (type A > 95%) and type B contains a small nucleus with dense and scarce cytoplasm (type B < 5%). Both atypical cell types had atypical and multilobed/multinucleated cells, including a high percentage (>30%) of mitotic figures, and were Ki-67 positive (100%). Karyotyping also revealed that 40.4% of the cells had atypical karyotyped chromosomes. In light of these findings, this cell line is no longer a "normal" cell, and experiments performed using this cell line can be questioned for non-default results. Experimenters should consider this error in future experiments.

Evaluation of the pulmonary toxicity of PSNPs using a Transwell-based normal human bronchial epithelial cell culture system.

To reduce the nanoplastics (NPs) toxicity assessment error, we established a Transwell-based bronchial epithelial cell exposure system to assess the pulmonary toxicity of polystyrene NPs (PSNPs). Transwell exposure system was more sensitive than submerged culture for toxicity detection of PSNPs. PSNPs adhered to the BEAS-2B cell surface, were ingested by the cell, and accumulated in the cytoplasm. PSNPs induced oxidative stress and inhibited cell growth through apoptosis and autophagy. A noncytotoxic dose of PSNPs (1 ng/cm2) increased the expression levels of inflammatory factors (ROCK-1, NF-κB, NLRP3, ICAM-1, etc) in BEAS-2B cells, whereas a cytotoxic dose (1000 ng/cm2) induced apoptosis and autophagy, which might inhibit the activation of ROCK-1 and contribute to reducing inflammation. In addition, the noncytotoxic dose increased the expression levels of zonula occludens-2 (ZO-2) and α1-antitrypsin (α-AT) proteins in BEAS-2B cells. Therefore, in response to PSNP exposure, a compensatory increase in the activities of inflammatory factors, ZO-2, and α-AT may be triggered at low doses as a mechanism to preserve the survival of BEAS-2B cells. In contrast, exposure to a high dose of PSNPs elicits a noncompensatory response in BEAS-2B cells. Overall, these findings suggest that PSNPs may be harmful to human pulmonary health even at an ultralow concentration.

PPARδ Agonist GW501516 Suppresses the TGF-ß-Induced Profibrotic Response of Human Bronchial Fibroblasts from Asthmatic Patients.

The airway wall remodeling observed in asthma is associated with subepithelial fibrosis and enhanced activation of human bronchial fibroblasts (HBFs) in the fibroblast to myofibroblast transition (FMT), induced mainly by transforming growth factor-ß (TGF-ß). The relationships between asthma severity, obesity, and hyperlipidemia suggest the involvement of peroxisome proliferator-activated receptors (PPARs) in the remodeling of asthmatic bronchi. In this study, we investigated the effect of PPARδ ligands (GW501516 as an agonist, and GSK0660 as an antagonist) on the FMT potential of HBFs derived from asthmatic patients cultured in vitro. This report shows, for the first time, the inhibitory effect of a PPARδ agonist on the number of myofibroblasts and the expression of myofibroblast-related markers-α-smooth muscle actin, collagen 1, tenascin C, and connexin 43-in asthma-related TGF-ß-treated HBF populations. We suggest that actin cytoskeleton reorganization and Smad2 transcriptional activity altered by GW501516 lead to the attenuation of the FMT in HBF populations derived from asthmatics. In conclusion, our data demonstrate that a PPARδ agonist stimulates antifibrotic effects in an in vitro model of bronchial subepithelial fibrosis. This suggests its potential role in the development of a possible novel therapeutic approach for the treatment of subepithelial fibrosis during asthma.

Targeting calcium signaling by inositol trisphosphate receptors: A novel mechanism for the anti-asthmatic effects of Houttuynia cordata.

Asthma is a chronic inflammatory disease characterized by airway hypersensitivity and remodeling. The current treatments provide only short-term benefits and may have undesirable side effects; thus, alternative or supplementary therapy is needed. Because intracellular calcium (Ca2+) signaling plays an essential role in regulating the contractility and remodeling of airway smooth muscle cells, the targeting of Ca2+ signaling is a potential therapeutic strategy for asthma. Houttuynia cordata is a traditional Chinese herb that is used to treat asthma due to its anti-allergic and anti-inflammatory properties. We hypothesized that H. cordata might modulate intracellular Ca2+ signaling and could help relieve asthmatic airway remodeling. We found that the mRNA and protein levels of inositol trisphosphate receptors (IP3Rs) were elevated in interleukin-stimulated primary human bronchial smooth muscle cells and a house dust mite-sensitized model of asthma. The upregulation of IP3R expression enhanced intracellular Ca2+ release upon stimulation and contributed to airway remodeling in asthma. Intriguingly, pretreatment with H. cordata essential oil rectified the disruption of Ca2+ signaling, mitigated asthma development, and prevented airway narrowing. Furthermore, our analysis suggested that houttuynin/2-undecanone could be the bioactive component in H. cordata essential oil because we found similar IP3R suppression in response to the commercially available derivative sodium houttuyfonate. An in silico analysis showed that houttuynin, which downregulates IP3R expression, binds to the IP3 binding domain of IP3R and may mediate a direct inhibitory effect. In summary, our findings suggest that H. cordata is a potential alternative treatment choice that may reduce asthma severity by targeting the dysregulation of Ca2+ signaling.

Downregulation of Lysosome-Associated Membrane Protein-2A Contributes to the Pathogenesis of COPD.

Background: Macroautophagy plays an important role in the pathogenesis of chronic obstructive pulmonary disease (COPD), but the role of chaperone-mediated autophagy (CMA) has not been investigated. We investigated if and how CMA is involved in the pathogenesis of COPD. Methods: We measured the level of lysosome-associated membrane protein-2A (LAMP-2A), which is a critical component of CMA that functions as a receptor for cytosolic substrate proteins, in total lung tissues and primary human bronchial epithelial cells (HBECs) from healthy never smokers, smokers, and COPD patients. We assessed the effects of LAMP-2A knock-down on cigarette smoke extract (CSE)-induced aging, cell cycle arrest, and apoptosis in BEAS-2B cells and the expression levels of apoptosis hallmarks in primary HBECs and lung tissue sections. Results: We found that the protein levels of LAMP-2A in lung homogenates and primary HBECs from smokers and COPD patients were lower than those from never smokers. In addition, its level in primary HBECs was negatively correlated with years of smoking. CSE caused degradation of LAMP-2A protein via the lysosomal pathway by activating macroautophagy. Knock-down of LAMP-2A markedly enhanced CSE-induced expression of senescence markers such as p16, p21, p27, and p53. G2/M cell cycle arrest, up-regulation of cyclin B1, and apoptosis in BEAS-2B cells. Apoptosis was increased in CSE-treated primary HBECs and in lung tissues from smokers and COPD patients. Conclusion: Cigarette smoke-induced down-regulation of LAMP-2A is involved in acceleration of aging and apoptosis of lung epithelial cells, which might at least partially contribute to COPD pathogenesis.

Group 3 innate lymphoid cells secret neutrophil chemoattractants and are insensitive to glucocorticoid via aberrant GR phosphorylation.

BACKGROUND: Patients with neutrophil-mediated asthma have poor response to glucocorticoids. The roles and mechanisms of group 3 innate lymphoid cells (ILC3s) in inducing neutrophilic airway inflammation and glucocorticoid resistance in asthma have not been fully clarified. METHODS: ILC3s in peripheral blood were measured by flow cytometry in patients with eosinophilic asthma (EA) and non-eosinophilic asthma (NEA). ILC3s were sorted and cultured in vitro for RNA sequencing. Cytokines production and signaling pathways in ILC3s after IL-1ß stimulation and dexamethasone treatment were determined by real-time PCR, flow cytometry, ELISA and western blot. RESULTS: The percentage and numbers of ILC3s in peripheral blood was higher in patients with NEA compared with EA, and negatively correlated with blood eosinophils. IL-1ß stimulation significantly enhanced CXCL8 and CXCL1 production in ILC3s via activation of p65 NF-κB and p38/JNK MAPK signaling pathways. The expression of neutrophil chemoattractants from ILC3s was insensitive to dexamethasone treatment. Dexamethasone significantly increased phosphorylation of glucocorticoid receptor (GR) at Ser226 but only with a weak induction at Ser211 residues in ILC3s. Compared to human bronchial epithelial cell line (16HBE cells), the ratio of p-GR S226 to p-GR S211 (p-GR S226/S211) was significantly higher in ILC3s at baseline and after dexamethasone treatment. In addition, IL-1ß could induce Ser226 phosphorylation and had a crosstalk effect to dexamethasone via NF-κB pathway. CONCLUSIONS: ILC3s were elevated in patients with NEA, and associated with neutrophil inflammation by release of neutrophil chemoattractants and were glucocorticoid (GC) resistant. This paper provides a novel cellular and molecular mechanisms of neutrophil inflammation and GC-resistance in asthma. Trial registration The study has been prospectively registered in the World Health Organization International Clinical Trials Registry Platform (ChiCTR1900027125).

DOCK2 Promotes Asthma Development by Eliciting Airway Epithelial-Mesenchymal Transition.

Epithelial-mesenchymal transition (EMT) contributes to airway remodeling, a predominant feature of asthma. DOCK2 (dedicator of cytokinesis 2) is an innate immune signaling molecule involved in vascular remodeling. However, it is unknown if DOCK2 plays a role in airway remodeling during asthma development. In this study, we found that DOCK2 is highly induced in both normal human bronchial epithelial cells treated with house dust mite (HDM) extract and human asthmatic airway epithelium. DOCK2 is also upregulated by TGF-ß1 (transforming growth factor ß1) during EMT of human bronchial epithelial cells. Importantly, knockdown of DOCK2 inhibits, and overexpression of DOCK2 promotes, TGF-ß1-induced EMT. Consistently, DOCK2 deficiency suppresses the EMT of airway epithelium, attenuates the subepithelial fibrosis, and improves pulmonary function in HDM-induced asthmatic lungs. These data suggest that DOCK2 plays an important role in EMT and asthma development. Mechanistically, DOCK2 interacts with transcription factor FoxM1 (forkhead box M1), which enhances FoxM1 binding to mesenchymal marker gene promoters and further promotes mesenchymal marker gene transcription and expression, leading to EMT. Taken together, our study identifies DOCK2 as a novel regulator for airway EMT in an HDM-induced asthma model, thus providing a potential therapeutic target for treatment of asthma.

Ionocyte-Specific Regulation of Cystic Fibrosis Transmembrane Conductance Regulator.

CFTR (cystic fibrosis transmembrane conductance regulator) is a tightly regulated anion channel that mediates chloride and bicarbonate conductance in many epithelia and in other tissues, but whether its regulation varies depending on the cell type has not been investigated. Epithelial CFTR expression is highest in rare cells called ionocytes. We studied CFTR regulation in control and ionocyte-enriched cultures by transducing bronchial basal cells with adenoviruses that encode only eGFP or FOXI1 (forkhead box I1) + eGFP as separate polypeptides. FOXI1 dramatically increased the number of transcripts for ionocyte markers ASCL3 (Achaete-Scute Family BHLH Transcription Factor 3), BSND, ATP6V1G3, ATP6V0D2, KCNMA1, and CFTR without altering those for secretory (SCGB1A1), basal (KRT5, KRT6, TP63), goblet (MUC5AC), or ciliated (FOXJ1) cells. The number of cells displaying strong FOXI1 expression was increased 7-fold, and there was no evidence for a broad increase in background immunofluorescence. Total CFTR mRNA and protein levels increased 10-fold and 2.5-fold, respectively. Ionocyte-enriched cultures displayed elevated basal current, increased adenylyl cyclase 5 expression, and tonic suppression of CFTR activity by the phosphodiesterase PDE1C, which has not been shown previously to regulate CFTR activity. The results indicate that CFTR regulation depends on cell type and identifies PDE1C as a potential target for therapeutics that aim to increase CFTR function specifically in ionocytes.

A lncRNA MALAT1 is a positive regulator of RhoA protein expression in bronchial smooth muscle cells.

AIMS: Augmented smooth muscle contractility of the airways associated with an increased expression of RhoA, a monomeric GTPase responsible for Ca2+ sensitization of contraction, is one of the causes of airway hyperresponsiveness. However, the mechanism of the altered properties of airway smooth muscle cells, including the RhoA upregulation, is not fully understood. This study aims to define functional role of a long non-coding RNA MALAT1 in the RhoA expression and development of bronchial smooth muscle (BSM) hyper-contractility. MAIN METHODS: Cultured human BSM cells were transfected with MALAT1 antisense oligonucleotide (AS), miR-133a-3p mimic, and/or inhibitor, and then stimulated with interleukin-13 (IL-13). In animal experiments, the ovalbumin (OA)-sensitized mice were repeatedly challenged with aerosolized OA to induce asthmatic reaction. KEY FINDINGS: Treatment of the cells with IL-13 induced an increase in RhoA protein. Either MALAT1 AS or miR-133a-3p mimic transfection inhibited the IL-13-induced upregulation of RhoA. The inhibitory effect of MALAT1 AS was abolished by co-transfection with miR-133a-3p inhibitor. In BSMs of the murine asthma model, upregulations of Malat1 and RhoA protein were observed concomitantly with downregulation of miR-133a-3p. SIGNIFICANCE: These findings suggest that MALAT1 positively regulates RhoA protein expression by inhibiting miR-133a-3p in BSM cells, and that its upregulation causes the RhoA upregulation, resulting in an augmented BSM contractility.

Heterogeneity of Airway Smooth Muscle Remodeling in Asthma.

Rationale: Ventilatory defects in asthma are heterogeneous and may represent the distribution of airway smooth muscle (ASM) remodeling. Objectives: To determine the distribution of ASM remodeling in mild-severe asthma. Methods: The ASM area was measured in nine airway levels in three bronchial pathways in cases of nonfatal (n = 30) and fatal asthma (n = 20) and compared with control cases without asthma (n = 30). Correlations of ASM area within and between bronchial pathways were calculated. Asthma cases with 12 large and 12 small airways available (n = 42) were classified on the basis of the presence or absence of ASM remodeling (more than two SD of mean ASM area of control cases, n = 86) in the large or small airway or both. Measurements and Main Results: ASM remodeling varied widely within and between cases of nonfatal asthma and was more widespread and confluent and more marked in fatal cases. There were weak correlations of ASM between levels within the same or separate bronchial pathways; however, predictable patterns of remodeling were not observed. Using mean data, 44% of all asthma cases were classified as having no ASM remodeling in either the large or small airway despite a three- to 10-fold increase in the number of airways with ASM remodeling and 81% of asthma cases having ASM remodeling in at least one large and small airway. Conclusions: ASM remodeling is related to asthma severity but is heterogeneous within and between individuals and may contribute to the heterogeneous functional defects observed in asthma. These findings support the need for patient-specific targeting of ASM remodeling.

Patient-derived cell models for personalized medicine approaches in cystic fibrosis.

Cystic fibrosis is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) channel that perturb anion transport across the epithelia of the airways and other organs. To treat cystic fibrosis, strategies that target mutant CFTR have been developed such as correctors that rescue folding and enhance transfer of CFTR to the apical membrane, and potentiators that increase CFTR channel activity. While there has been tremendous progress in development and approval of CFTR therapeutics for the most common (F508del) and several other CFTR mutations, around 10-20% of people with cystic fibrosis have rare mutations that are still without an effective treatment. In the current decade, there was an impressive evolution of patient-derived cell models for precision medicine. In cystic fibrosis, these models have played a crucial role in characterizing the molecular defects in CFTR mutants and identifying compounds that target these defects. Cells from nasal, bronchial, and rectal epithelia are most suitable to evaluate treatments that target CFTR. In vitro assays using cultures grown at an air-liquid interface or as organoids and spheroids allow the diagnosis of the CFTR defect and assessment of potential treatment strategies. An overview of currently established cell culture models and assays for personalized medicine approaches in cystic fibrosis will be provided in this review. These models allow theratyping of rare CFTR mutations with available modulator compounds to predict clinical efficacy. Besides evaluation of individual personalized responses to CFTR therapeutics, patient-derived culture models are valuable for testing responses to developmental treatments such as novel RNA- and DNA-based therapies.

The Impact of Corticosteroids on Human Airway Smooth Muscle Contractility and Airway Hyperresponsiveness: A Systematic Review.

Classically, the effects elicited by corticosteroids (CS) are mediated by the binding and activation of cytosolic glucocorticoid receptors (GR). However, several of the non-genomic effects of CS seem to be mediated by putative non-classic membrane receptors characterized by pharmacological properties that are different from those of classic cytosolic GR. Since pre-clinical findings suggest that inhaled CS (ICS) may also regulate the bronchial contractile tone via putative CS membrane-associate receptors, the aim of this review was to systematically report and discuss the impact of CS on human airway smooth muscle (ASM) contractility and airway hyperresponsiveness (AHR). Current evidence indicates that CS have significant genomic/non-genomic beneficial effects on human ASM contractility and AHR, regardless of their anti-inflammatory effects. CS are effective in reducing either the expression, synthesis or activity of α-actin, CD38, inositol phosphate, myosin light chain kinase, and ras homolog family member A in response to several pro-contractile stimuli; overall these effects are mediated by the genomic action of CS. Moreover, CS elicited a strong bronchorelaxant effect via the rapid activation of the Gsα-cyclic-adenosine-monophosphate-protein-kinase-A pathway in hyperresponsive airways. The possibility of modulating the dose of the ICS in a triple ICS/long-acting ß2-adrenoceptor agonist/long-acting muscarinic antagonist fixed-dose combination supports the use of a Triple MAintenance and Reliever Therapy (TriMART) in those asthmatic patients at Step 3-5 who may benefit from a sustained bronchodilation and have been suffering from an increased parasympathetic tone.

Different Molecular Forms of TFF3 in the Human Respiratory Tract: Heterodimerization with IgG Fc Binding Protein (FCGBP) and Proteolytic Cleavage in Bronchial Secretions.

The polypeptide TFF3 belongs to the trefoil factor family (TFF) of lectins. TFF3 is typically secreted from mucous epithelia together with mucins. Both intestinal and salivary TFF3 mainly exist as disulfide-linked heterodimers with IgG Fc binding protein (FCGBP). Here, we investigated bronchial tissue specimens, bronchial secretions, and bronchoalveolar lavage (BAL) fluid from patients with a chronic obstructive pulmonary disease (COPD) background by fast protein liquid chromatography and proteomics. For the first time, we identified different molecular forms of TFF3 in the lung. The high-molecular mass form represents TFF3-FCGBP oligomers, whereas the low-molecular mass forms are homodimeric and monomeric TFF3 with possibly anti-apoptotic activities. In addition, disulfide-linked TFF3 heterodimers with an Mr of about 60k and 30k were detected in both bronchial secretions and BAL fluid. In these liquids, TFF3 is partly N-terminally truncated probably by neutrophil elastase cleavage. TFF3-FCGBP is likely involved in the mucosal innate immune defense against microbial infections. We discuss a hypothetical model how TFF3 might control FCGBP oligomerization. Furthermore, we did not find indications for interactions of TFF3-FCGBP with DMBT1gp340 or the mucin MUC5AC, glycoproteins involved in mucosal innate immunity. Surprisingly, bronchial MUC5AC appeared to be degraded when compared with gastric MUC5AC.

Airway Smooth Muscle Cell Mitochondria Damage and Mitophagy in COPD via ERK1/2 MAPK.

Chronic obstructive pulmonary disease (COPD) is characterized by irreversible deterioration of the airway wall. Cigarette smoking is the major trigger, and in vitro studies showed that cigarette smoke extract (CSE) induced mitophagy in airway epithelial cells via oxidative stress, but this mechanism was not studied in airway smooth muscle cells (ASMCs). Primary ASMCs isolated from COPD patients or non-disease donors were investigated for CSE-induced remodeling and mitochondria structure. Proteins were assessed by Western blots for remodeling: collagen type-I, α-smooth muscle actin (α-SMA) and fibronectin; autophagy: beclin-1, protein62 (p62), light chain (LC)3A/B; mitochondria activity: mitochondrially encoded cytochrome c oxidase II & -IV (MTCO2, MTCO4), peroxisome proliferator activated receptor gamma coactivator 1α (PGC-1α); lysosomes: early endosome antigen 1, lysosome activated membrane protein 1; and cell signaling: extracellular signal regulated kinase (ERK1/2). Lysotracker and Mitotracker were used to monitor mitochondria morphology and organelle co-localization. Compared with controls, untreated COPD ASMCs showed lower collagen type-I and α-SMA expressions, but increased fibronectin levels. CSE further downregulated collagen type-I and α-SMA expression, but upregulated fibronectin. CSE decreased PGC-1α, MTCO2, and MTCO4, but increased beclin-1, p62, and LC3. CSE upregulated mitophagy and lysosomes activity via ERK1/2 phosphorylation. In vitro, cigarette smoke induced the deterioration of ASMCs, which might explain the tissue loss and structural remodeling in COPD bronchi. The results suggest that preventing exceeded mitophagy in ASMCs might present a novel therapeutic target for COPD.

DNMT3A-mediated high expression of circ_0057504 promotes benzo[a]pyrene-induced DNA damage via the NONO-SFPQ complex in human bronchial epithelial cells.

Benzo[a]pyrene (B[a]P) is a class I carcinogen and hazardous environmental pollutant with genetic toxicity. Understanding the molecular mechanisms underlying genetic deterioration and epigenetic alterations induced by environmental contaminants may contribute to the early detection and prevention of cancer. However, the role and regulatory mechanisms of circular RNAs (circRNAs) in the B[a]P-induced DNA damage response (DDR) have not been elucidated. In this study, human bronchial epithelial cell lines (16HBE and BEAS-2B) were exposed to various concentrations of B[a]P, and BALB/c mice were treated with B[a]P intranasally. B[a]P exposure was found to induce DNA damage and upregulate circular RNA hsa_circ_0057504 (circ_0057504) expression in vitro and in vivo. In addition, B[a]P upregulated TMEM194B mRNA and circ_0057504 expression through inhibition of DNA methyltransferase 3 alpha (DNMT3A) expression in vitro. Modulation (overexpression or knockdown) of circ_0057504 expression levels using a lentiviral system in human bronchial epithelial cells revealed that circ_0057504 promoted B[a]P-induced DNA damage. RNA pull-down and western blot assays showed that circ_0057504 interacted with non-POU domain-containing octamer-binding (NONO) and splicing factor proline and glutamine rich (SFPQ) proteins and regulated formation of the NONO-SFPQ protein complex. Thus, our findings indicate that circ_0057504 acts as a novel regulator of DNA damage in human bronchial epithelial cells exposed to B[a]P. The current study reveals novel insights into the role of circRNAs in the regulation of genetic damage, and describes the effect and regulatory mechanisms of circ_0057504 on B[a]P genotoxicity.