Airway Epithelial Cell Function Research Articles

Alcohol Decreases the Barrier Function of Airway Epithelial cells through the TGFß1 Pathway

Alcohol use disorder affects over 14 million adults and causes 88,000 deaths each year in the United States. Chronic alcohol use significantly increases people's risk of developing lung infections and acute respiratory distress syndrome (ARDS). This increased sensitivity to injury is a condition known as alcoholic lung syndrome, and it is caused by dysfunction of the lung immune system and alveolar epithelial barrier. However, little is known about how alcohol impacts epithelial cells in the conducting airway, i.e. the trachea and bronchi, which are the first line of defense against infectious pathogens in the lung. We hypothesize that alcohol exposure causes a decrease in barrier function of airway epithelial cells through a TGFß1-dependent mechanism. To test this, we cultured primary airway epithelial cells isolated from rats and humans on Transwell permeable supports and differentiated them in vitro at air liquid interface (ALI). We treated cells in vitro with 60mM or 100mM ethanol changed daily for 2 weeks and measured barrier function by transepithelial electrical resistance (TER). We found that ethanol caused a decrease in barrier function compared to control cells. However, cells treated with ethanol and a TGFß1 inhibitor had barrier function similar to control cells. Additionally, we cultured primary airway epithelial cells isolated from healthy and alcoholic patients on Transwell permeable supports and differentiated them in vitro to determine if in vivo exposure caused long-term changes to tight junction protein levels. It is known that the transmembrane protein junctional adhesion molecule A (JAM-A) regulates barrier function by controlling the paracellular flow of small molecules as part of the tight junction barrier. Our results show in response to alcohol exposure, cells have lower JAM-A protein expression compared to healthy cells. Rat airway cells exposed to ethanol in vitro also show a decrease in JAM-A at both the RNA and protein level. Together, these results indicate that chronic alcohol exposure detrimentally alters the conducting airway epithelial cell barrier potentially through the loss of JAM-A. Future directions include determining if other tight junction proteins are altered by alcohol and whether the alcohol-induced reduction in JAM-A is sufficient to cause barrier dysfunction in airway cells.

MR1 expression and surface translocation in airway epithelial cells from COPD or smoker lungs is altered following exposure to IFNγ or MAIT cells.

Abstract MAIT cells, a highly prevalent subset of T cells restricted by the MHC Class I-like molecule MR1, play a key role in early response to bacterial infection. Chronic obstructive pulmonary disease (COPD) pathogenesis is associated with airway inflammation, increased infiltration by CD8+ T lymphocytes, and infection-driven exacerbations. Cigarette smoke (CS), a leading cause of COPD, leads to impaired immune function of airway epithelial cells (AECs) and decreases surface expression of classical MHC Class I molecules. However, the impact of CS and COPD on MR1 expression and airway MAIT cell function is unknown. We have demonstrated that exposure to CS alters MAIT cell responses to primary AECs from healthy, COPD, or smoker lungs. Here, we examined whether CS impacts expression of MR1 mRNA or surface translocation of MR1 in these primary AEC populations as a possible mechanism for this dysregulation. Short-term treatment of AECs with CS or infection with S. pneumoniae (SP) did not alter MR1 expression in any of the donor AEC groups. However, incubating CS-treated or SP-infected AECs with MAIT cells increased MR1 expression. This upregulation may be mediated through IFNγ signaling pathways, as treatment with IFNγ alone resulted in MR1 upregulation. Our data further suggest MAIT cell- and IFNγ-mediated MR1 upregulation is altered in COPD cells. Additionally, we observed increased baseline surface expression of MR1 protein in smoker AECs and reduced surface translocation upon addition of exogenous ligand. COPD- and CS-mediated dysregulation of MR1 expression could impact MAIT cell function in airways, resulting in inflammation or reduced early response to bacteria, contributing to lower airway colonization and COPD exacerbations.

Attenuated lncRNA NKILA Enhances the Secretory Function of Airway Epithelial Cells Stimulated by Mycoplasma pneumoniae via NF-κB.

The secretory function of airway epithelial cells is important in the pathogenesis of Mycoplasma pneumoniae pneumonia (MPP). To investigate the regulatory function of NKILA (nuclear factor-κB (NF-κB) interacting long noncoding RNA (lncRNA)) in MPP, we first detected NKILA as well as the concentration of interleukin 8 (IL-8) and tumor necrosis factor-α (TNF-α) in bronchoalveolar lavage fluid of children with MPP. Then, NKILA was knocked down in epithelial cells to investigate its effect on their secretory function. The results suggested that NKILA was downregulated in children with MPP, while IL-8 and TNF-α levels increased. Knockdown of NKILA in vitro promoted the inflammatory effects of Mycoplasma pneumoniae (MP) in epithelial A549 and BEAS-2B cells. Knockdown of NKILA promoted inhibitor of κBα (IκBα) phosphorylation and degradation, and NF-κB p65 nuclear translocation. Furthermore, RNA immunoprecipitation showed that NKILA could physically bind to IκBα in MP-treated A549 cells. Collectively, our data demonstrated that attenuation of NKILA enhances the effects of MP-stimulated secretory functions of epithelial cells via regulation of NF-κB signaling.

Integration of transcriptome analysis with pathophysiological endpoints to evaluate cigarette smoke toxicity in an in vitro human airway tissue model

Exposure to cigarette smoke (CS) is a known risk factor in the pathogenesis of smoking-caused diseases, such as chronic obstructive pulmonary diseases (COPD) and lung cancer. To assess the effects of CS on the function and phenotype of airway epithelial cells, we developed a novel repeated treatment protocol and comprehensively evaluated the progression of key molecular, functional, and structural abnormalities induced by CS in a human in vitro air–liquid-interface (ALI) airway tissue model. Cultures were exposed to CS (diluted with 0.5 L/min, 1.0 L/min, and 4.0 L/min clean air) generated from smoking five 3R4F University of Kentucky reference cigarettes under the International Organization for Standardization (ISO) machine smoking regimen, every other day for 4 weeks (3 days per week, 40 min/day). By integrating the transcriptomics-based approach with the in vitro pathophysiological measurements, we demonstrated CS-mediated effects on oxidative stress, pro-inflammatory cytokines and matrix metalloproteinases (MMPs), ciliary function, expression and secretion of mucins, and squamous cell differentiation that are highly consistent with abnormalities observed in airways of smokers. Enrichment analysis on the transcriptomic profiles of the ALI cultures revealed key molecular pathways, such as xenobiotic metabolism, oxidative stress, and inflammatory responses that were perturbed in response to CS exposure. These responses, in turn, may trigger aberrant tissue remodeling, eventually leading to the onset of respiratory diseases. Furthermore, changes of a panel of genes known to be disturbed in smokers with COPD were successfully reproduced in the ALI cultures exposed to CS. In summary, findings from this study suggest that such an integrative approach may be a useful tool for identifying genes and adverse cellular events caused by inhaled toxicants, like CS.

Respiratory syncytial virus persistent infection causes acquired CFTR dysfunction in human bronchial epithelial cells.

Many studies have shown that respiratory syncytial virus persistent infection may be the main cause of chronic respiratory pathology.However, the mechanism is unclear. Cystic fibrosis transmembrane conduction regulator (CFTR) is an apical membrane chloride channel, which is very important for the regulation of epithelial fluid, chloride ion, and bicarbonate transport. CFTR dysfunction will lead to changes in bronchial secretions and impair mucus clearance, which is related to airway inflammation. In our previous study, we observed the down-regulation of CFTR in airway epithelial cells in respiratory syncytial virus (RSV) infected mouse model. In this study, we further investigated the expression and function of CFTR by constructing an airway epithelial cell model of RSV persistent infection. 16HBE14o- cells were infected with RSV at 0.01 multiplicity of infection (MOI). The expression of CFTR was detected by real-time RT-PCR, immunofluorescence, and Western blotting. The intracellular chloride concentration was measured by N-(ethoxycarbonylmethyl)-6-methoxyquinolium bromide (MQAE) and the chloride current was measured by whole-cell patch clamp recording. 16HBE14o- cells infected with RSV were survived to successive passages of the third generation (G3), while the expression and function of CFTR was progressively decreased upon RSV infection from the first generation (G1) to G3. Exposure of 16HBE14o- cells to RSV led to the gradual increase of TGF-β1 as well as phosphorylation of Smad2 following progressive RSV infection. Disruption of TGF-β1 signaling by SB431542 prevented Smad2 phosphorylation and rescued the expression of CFTR. RSV infection can lead to defective CFTR function in airway epithelial cells, which may be mediated via activation of TGF-β1 signaling pathway.

SARS-CoV-2 Infection of Airway Epithelial Cells.

Coronavirus disease 2019 caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been spreading worldwide since its outbreak in December 2019, and World Health Organization declared it as a pandemic on March 11, 2020. SARS-CoV-2 is highly contagious and is transmitted through airway epithelial cells as the first gateway. SARS-CoV-2 is detected by nasopharyngeal or oropharyngeal swab samples, and the viral load is significantly high in the upper respiratory tract. The host cellular receptors in airway epithelial cells, including angiotensin-converting enzyme 2 and transmembrane serine protease 2, have been identified by single-cell RNA sequencing or immunostaining. The expression levels of these molecules vary by type, function, and location of airway epithelial cells, such as ciliated cells, secretory cells, olfactory epithelial cells, and alveolar epithelial cells, as well as differ from host to host depending on age, sex, or comorbid diseases. Infected airway epithelial cells by SARS-CoV-2 in ex vivo experiments produce chemokines and cytokines to recruit inflammatory cells to target organs. Same as other viral infections, IFN signaling is a critical pathway for host defense. Various studies are underway to confirm the pathophysiological mechanisms of SARS-CoV-2 infection. Herein, we review cellular entry, host-viral interactions, immune responses to SARS-CoV-2 in airway epithelial cells. We also discuss therapeutic options related to epithelial immune reactions to SARS-CoV-2.

P133 Impact of specialised pro-resolving lipid mediators on cystic fibrosis airway epithelial cell functions

P133 Impact of specialised pro-resolving lipid mediators on cystic fibrosis airway epithelial cell functions

Butyrate and Propionate Restore the Cytokine and House Dust Mite Compromised Barrier Function of Human Bronchial Airway Epithelial Cells

Barrier dysfunction of airway epithelium contributes to the development of allergies, airway hyper-responsiveness and immunological respiratory diseases. Short-chain fatty acids (SCFA) enhance and restore the barrier function of the intestinal epithelium. This study investigated whether acetate, propionate and butyrate enhance the integrity of bronchial epithelial cells. Differentiating human bronchial epithelial cells (16HBE) grown on transwells were exposed to butyrate, propionate and acetate while trans-epithelial electrical resistance was monitored over time. Restorative effects of SCFA were investigated by subsequent incubation of cells with IL-4, IL-13 or house dust mite extract and SCFA. SCFA effects on IL-4-induced cytokine production and the expression of zonula occludens-1 (ZO-1) and Mitogen-activated protein kinases (MAPK) signalling pathways were investigated by ELISA and Western blot assays. Propionate and butyrate enhanced the barrier function of differentiating 16HBE cells and induced complete recovery of the barrier function after exposure to the above-mentioned stimuli. Butyrate decreased IL-4-induced IL-6 production. IL-4 decreased ZO-1 protein expression and induced phosphorylation of extracellular signal-regulated protein kinases 1/2 (ERK1/2) and c-Jun N-terminal kinases (JNK) in 16HBE cells, both of which could be restored by SCFA. SCFA showed prophylactic and restorative effects on airway epithelial barrier function, which might be induced by increased ZO-1 expression.

Inducible expression of heat shock protein 20 protects airway epithelial cells against oxidative injury involving the Nrf2-NQO-1 pathway

BackgroundHeat shock protein (HSP) 20 is a molecular chaperone that exerts multiple protective functions in various kinds of tissues. However, the expression of HSP20 and its specific functions in airway epithelial cells (AECs) remain elusive.ResultsIn current study, we first confirmed the inducible expression of HSP20 in mouse AECs and in a human bronchial epithelial cell line BEAS-2B cells, under different oxidant stressors. Then by establishing a HSP20-abundant mouse model with repeated low-level-ozone exposures and stimulating this model with a single high-level ozone exposure, we found that the HSP20 abundance along with its enhanced phosphorylation potentially contributed to the alleviation of oxidative injuries, evidenced by the decreases in the bodyweight reduction, the BAL neutrophil accumulation, the AECs shedding, and the BAL concentrations of albumin and E-cadherin. The biological function of HSP20 and its molecular mechanisms were further investigated in BEAS-2B cells that were transfected with HSP20-, unphosphorylatable HSP20(Ala) or empty vector plasmids prior to the stimulation of H2O2, of which its oxidant capacity has been proved to be similar with those of ozone in an air–liquid culture system. We found that the H2O2-induced intracellular ROS level and the early cell apoptosis were attenuated in the HSP20- but not HSP20(Ala)- transfected cells. The intracellular expression of NQO-1 (mRNA and protein) and the intranuclear content of Nrf2 were significantly increased in the HSP20- transfected cells but not in the HSP20(Ala)- and empty vector-transfected cells after the stimulation of H2O2.ConclusionsThe inducible expression of HSP20 in AECs by oxidative stress exerts protective roles against oxidative damages, which may involve the activation of the Nrf2-NQO-1 pathway.

Pathogenesis of chronic rhinosinusitis with nasal polyps: role of IL-6 in airway epithelial cell dysfunction

BackgroundChronic rhinosinusitis with nasal polyps (CRSwNP) is characterized by an alteration in airway epithelial cell functions including barrier function, wound repair mechanisms, mucociliary clearance. The mechanisms leading to epithelial cell dysfunction in nasal polyps (NPs) remain poorly understood. Our hypothesis was that among the inflammatory cytokines involved in NPs, IL-6 could alter epithelial repair mechanisms and mucociliary clearance. The aim of this study was to evaluate the in vitro effects of IL-6 on epithelial repair mechanisms in a wound repair model and on ciliary beating in primary cultures of Human Nasal Epithelial Cells (HNEC).MethodsPrimary cultures of HNEC taken from 38 patients during surgical procedures for CRSwNP were used in an in vitro model of wound healing. Effects of increasing concentrations of IL-6 (1 ng/mL, 10 ng/mL, and 100 ng/mL) and other ILs (IL-5, IL-9, IL-10) on wound closure kinetics were compared to cultures without IL-modulation. After wound closure, the differentiation process was characterized under basal conditions and after IL supplementation using cytokeratin-14, MUC5AC, and βIV tubulin as immunomarkers of basal, mucus, and ciliated cells, respectively. The ciliated edges of primary cultures were analyzed on IL-6 modulation by digital high-speed video-microscopy to measure: ciliary beating frequency (CBF), ciliary length, relative ciliary density, metachronal wavelength and the ciliary beating efficiency index.ResultsOur results showed that: (i) IL-6 accelerated airway wound repair in vitro, with a dose–response effect whereas no effect was observed after other ILs-stimulation. After 24 h, 79% of wounded wells with IL6-100 were fully repaired, vs 46% in the IL6-10 group, 28% in the IL6-1 group and 15% in the control group; (ii) specific migration analyses of closed wound at late repair stage (Day 12) showed IL-6 had the highest migration compared with other ILs (iii) The study of the IL-6 effect on ciliary function showed that CBF and metachronal wave increased but without significant modifications of ciliary density, length of cilia and efficiency index.ConclusionThe up-regulated epithelial cell proliferation observed in polyps could be induced by IL-6 in the case of prior epithelial damage. IL-6 could be a major cytokine in NP physiopathology.

Immunomodulatory function of the cystic fibrosis modifier gene BPIFA1

BackgroundCystic fibrosis (CF) is characterized by a progressive decline in lung function due to airway obstruction, infection, and inflammation. CF patients are particularly susceptible to respiratory infection by a variety of pathogens, and the inflammatory response in CF is dysregulated and prolonged. BPI fold containing family A, member 1 (BPIFA1) and BPIFB1 are proteins expressed in the upper airways that may have innate immune activity. We previously identified polymorphisms in the BPIFA1/BPIFB1 region associated with CF lung disease severity.MethodsWe evaluated whether the BPIFA1/BPIFB1 associations with lung disease severity replicated in individuals with CF participating in the International CF Gene Modifier Consortium (n = 6,365). Furthermore, we investigated mechanisms by which the BPIFA1 and BPIFB1 proteins may modify lung disease in CF.ResultsThe association of the G allele of rs1078761 with reduced lung function was replicated in an independent cohort of CF patients (p = 0.001, n = 2,921) and in a meta-analysis of the full consortium (p = 2.39x10-5, n = 6,365). Furthermore, we found that rs1078761G which is associated with reduced lung function was also associated with reduced BPIFA1, but not BPIFB1, protein levels in saliva from CF patients. Functional assays indicated that BPIFA1 and BPIFB1 do not have an anti-bacterial role against P. aeruginosa but may have an immunomodulatory function in CF airway epithelial cells. Gene expression profiling using RNAseq identified Rho GTPase signaling pathways to be altered in CF airway epithelial cells in response to treatment with recombinant BPIFA1 and BPIFB1 proteins.ConclusionsBPIFA1 and BPIFB1 have immunomodulatory activity and genetic variation associated with low levels of these proteins may increase CF lung disease severity.

Contribution of cyclic AMP and β‐arrestin‐2‐dependent mechanisms to β2‐adrenoceptor‐mediated gene expression changes in human airway epithelial cells

Long‐acting β2‐adrenoceptor agonists (LABAs) are mainstay drugs used routinely in asthma management. Our recent work revealed that β2‐adrenoceptor (β2‐AR) agonists promote a significant number of gene expression changes in BEAS‐2B airway epithelial cells that could contribute to their therapeutic activity and adverse effects (AEs) in asthma. In this study, we investigated the extent to which canonical (G‐protein/cAMP/protein kinase A [PKA]) and non‐canonical (β‐arrestin/extracellular signal‐regulated kinase [ERK]) signalling regulate gene expression in human primary airway epithelial cells (HpAECs) using formoterol and salmeterol as an example of a high and low efficacy LABA, respectively.Previous studies have shown that β‐arrestin‐2 is responsible for β2‐AR internalization and ERK1/2 phosphorylation. To investigate these functions in airway epithelial cells, we transfected BEAS‐2B cells with HA‐tagged β2‐AR and monitored internalization by confocal microscopy. When stimulated with formoterol or salmeterol, HA‐tagged β2‐AR redistributed from the plasma membrane to endosomes by a mechanism that was inhibited in cells deficient in β‐arrestin‐2. In BEAS‐2B cells and HpAECs, salmeterol (100nM) and formoterol (1nM) rapidly dephosphorylated basal ERK1/2 expression in a time‐dependent manner. This dephosphorylation was abolished in cells pre‐treated with a selective, β2‐AR antagonist, ICI‐118551 (1μM), and by overexpressing a PKA inhibitor transgene via adenoviral transduction with no evidence of ERK phosphorylation. Moreover, salmeterol‐ and formoterol‐induced ERK dephosphorylation was unaffected in BEAS‐2B cells lacking in β‐arrestin‐2.To evaluate the genomic effect of β2‐AR agonists, HpAECs from five normal subjects were treated with vehicle, formoterol (1nM), forskolin (FSK,10μM), tumor necrosis factor‐α (TNFα, 10ng/ml) and formoterol/TNFα or FSK/TNFα in combination for 1, 2, 6 and 18h. Total RNA was extracted, quantified, and sequenced on a NextSeq 500 instrument (Illumina). The sequenced reads were quantified by Kallisto and differential gene expression analysis performed using the R package, Sleuth. At a FDR of 5%, formoterol promoted a significant number of gene expression changes at both early and late time points; functional annotation clustering determined that many of these genes have both beneficial and AE potential. Formoterol also significantly enhanced or repressed many gene expression changes induced by TNFα at early and late time points. The gene expression profile induced by formoterol in HpAECs was replicated by FSK in the absence and presence of TNFα highlighting the cAMP dependency of this genomic effect.These data indicate that cAMP/PKA signalling plays a dominant role in regulating LABA‐induced gene expression changes in human airway epithelial cells. We submit that changes in gene expression through canonical signalling represents a previously unappreciated mechanism that contributes to the therapeutic activity and AEs of β2‐adrenoceptor agonists in asthma.Support or Funding InformationCanadian Institutes for Health Research (CIHR) ‐ The Lung Association of Alberta & NWTThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Simvastatin Inhibits Allergen‐induced IL‐33 Secretion in Human Bronchial Epithelial Cells by Blocking ATP Release and Ca2+ Uptake

Previous studies have demonstrated that simvastatin possesses immunomodulatory effects in airways, but the precise mechanisms and therapeutic potential are poorly understood. The objective of the present study was to investigate the inhibitory effects of simvastatin and b‐methyl‐cyclodextrin (β‐MCD) on the innate immune response of the airway epithelium induced by Alternaria alternata (100 μg/ml) and house dust mite (200 μg/ml, HDM) allergens. Experiments were performed with cultured human bronchial epithelial cells transfected with the human IL‐33 gene to produce a robust and reproducible cell model for the study of IL‐33 secretion. Cells were pretreated with varying concentrations of simvastatin ranging from 0.1 to 10 μM for 24 hours prior to allergen exposure. IL‐33 release was quantified by ELISA (Quantikine®) following the procedure provided by the manufacturer. Simvastatin produced a concentration‐dependent inhibition of IL‐33 release that was nearly abolished at 10 μM (IC50; 1.0 μM). In contrast, simvastatin was ineffective at blocking the effects of HDM on IL‐33 release (IC50; >10 μM). Similarly, pretreatment with β‐MCD (5 mM) for 30 minutes also inhibited the Alternaria response, suggesting that depletion of cholesterol from the plasma membrane was responsible for the effects of simvastatin and β‐MCD. Subsequent experiments explored the effects of simvastatin and β‐MCD on Alternaria‐evoked Ca2+ uptake, which is necessary for activating IL‐33 release. Simvastatin (10 μM, 24 hrs) and β‐MCD (5 mM; 30 minutes) both abolished the increase in [Ca2+]i induced by Alternaria. We next examined the possibility that abolition of Ca2+ uptake resulted from inhibition of ATP release, since ATP activates P2X7 receptors that mediate Ca2+ influx in response to Alternaria exposure. ATP release was measured in real time using a luciferin/luciferase‐based photon counting assay. Both simvastatin and β‐MCD blocked ATP release. Moreover, addition of 100 μM cholesterol to the extracellular media rescued the effects of simvastatin. These results demonstrated that inhibition of IL‐33 secretion by simvastatin and β‐MCD following Alternaria exposure involved suppression of ATP release that was dependent on membrane cholesterol content. Thus, membrane cholesterol likely plays a pivotal role in innate immune function of airway epithelial cells.Support or Funding InformationThis study was supported by NIH R01 AI128729 to HK and SMO.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A Nanosensor Toolbox for Rapid, Label-Free Measurement of Airway Surface Liquid and Epithelial Cell Function.

Ciliated lung epithelial cells and the airway surface liquid (ASL) comprise one of the body's most important protective systems. This system is finely tuned, and perturbations to ASL rheology, ASL depth, ASL pH, the transepithelial potential, and the cilia beat frequency are all associated with disease pathology. Further, these apparently distinct properties interact with each other in a complex manner. For example, changes in ASL rheology can result from altered mucin secretion, changes in ASL pH, or changes in ASL depth. Thus, one of the great challenges in trying to understand airway pathology is that the properties of the ASL/epithelial cell system need to be assessed near-simultaneously and without perturbing the sample. Here, we show that nanosensor probes mounted on a scanning ion conductance microscope make this possible for the first time, without any need for labeling. We also demonstrate that ASL from senescence-retarded human bronchial epithelial cells retains its native properties. Our results demonstrate that by using a nanosensor approach, it is possible to pursue faster, more accurate, more coherent, and more informative studies of ASL and airway epithelia in health and disease.

Nix/BNIP3L-dependent mitophagy accounts for airway epithelial cell injury induced by cigarette smoke.

Cigarette smoke-induced airway epithelial cell mitophagy is an important mechanism in the pathogenesis of chronic obstructive pulmonary disease (COPD). Mitochondrial protein Nix (also known as BNIP3L) is a selective autophagy receptor and participates in several human diseases. However, little is known about the role of Nix in airway epithelial cell injury during the development of COPD. The aim of the present study is to investigate the effects of Nix on mitophagy and mitochondrial function in airway epithelial cells exposed to cigarette smoke extract (CSE). Our present study has found that CSE could increase Nix protein expression and induce mitophagy in airway epithelial cells. And Nix siRNA significantly inhibited mitophagy and attenuated mitochondrial dysfunction and cell injury when airway epithelial cells were stimulated with 7.5% CSE. In contrast, Nix overexpression enhanced mitophagy and aggravated mitochondrial dysfunction and cell injury when airway epithelial cells were incubated with 7.5% CSE. These data suggest that Nix-dependent mitophagy promotes airway epithelial cell and mitochondria injury induced by cigarette smoke, and may be involved in the pathogenesis of COPD and other cigarette smoke-associated diseases.

Airway Epithelial Cell Function and Respiratory Host Defense in Chronic Obstructive Pulmonary Disease.

Airway Epithelial Cell Function and Respiratory Host Defense in Chronic Obstructive Pulmonary Disease.

Role of GPR40/120 omega‐3 fatty acid receptors on barrier function of airway epithelial cells

The airway epithelium serves as a physical barrier, whose integrity is maintained by tight junctions. Disruption of epithelial barrier function leads to increased permeability of harmful particles and pathogens, causing the development of various airway diseases. G protein‐coupled receptor 40 (GPR40) and G protein‐coupled receptor 120 (GPR120) have been discovered as receptors of omega‐3 fatty acids, and activation of these receptors induces AMP‐activated protein kinase (AMPK) activation, which is known to promote tight junction assembly. This study aimed to investigate the effect of omega‐3 fatty acid receptors, namely GPR40 and GPR120, on tight junction integrity of airway epithelial cells (Calu‐3 cells). Cell junctional assembly was measured by calcium switch assays and transepithelial electrical resistance (TER) analyses. We found that GW9508, an agonist of GPR40/120, promoted tight junction assembly of airway epithelial cells. This effect was abolished by specific antagonists of GPR40 and GPR120. In addition, the enhancement of airway barrier function by GPR40/120 agonist was mediated via a phospholipase C‐ calcium/calmodulin‐dependent protein kinase kinase (CaMKK)‐AMPK pathway. Taken together, these results indicate that activation of GPR40/120 has a beneficial effect in promoting airway epithelial barrier integrity and may be of therapeutic potential in airway diseases resulting from airway epithelial disruption including chronic obstructive pulmonary disease.Support or Funding InformationThis work was supported by Thailand Research Fund and Mahidol University (Grant BRG5980008), Faculty of Science, Mahidol University, and Faculty of Medicine Ramathibodi Hospital, Mahidol University (Tonkla Ramathibodi Project).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Exposure to Silver Nanospheres Leads to Altered Respiratory Mechanics and Delayed Immune Response in an in Vivo Murine Model.

Here we examine the organ level toxicology of both carbon black (CB) and silver nanoparticles (AgNP). We aim to determine metal-specific effects to respiratory function, inflammation and potential interactions with lung lining fluid (LLF). C57Bl6/J male mice were intratracheally instilled with saline (control), low (0.05 μg/g) or high (0.5 μg/g) doses of either AgNP or CB 15 nm nanospheres. Lung histology, cytology, surfactant composition and function, inflammatory gene expression, and pulmonary function were measured at 1, 3, and 7 days post-exposure. Acutely, high dose CB resulted in an inflammatory response, increased neutrophilia and cytokine production, without alteration in surfactant composition or respiratory mechanics. Low dose CB had no effect. Neither low nor high dose AgNPs resulted in an acute inflammatory response, but there was an increase in work of breathing. Three days post-exposure with CB, a persistent neutrophilia was noted. High dose AgNP resulted in an elevated number of macrophages and invasion of lymphocytes. Additionally, AgNP treated mice displayed increased expression of IL1B, IL6, CCL2, and IL10. However, there were no significant changes in respiratory mechanics. At day 7, inflammation had resolved in AgNP-treated mice, but tissue stiffness and resistance were significantly decreased, which was accompanied by an increase in surfactant protein D (SP-D) content. These data demonstrate that the presence of metal alters the response of the lung to nanoparticle exposure. AgNP-surfactant interactions may alter respiratory function and result in a delayed immune response, potentially due to modified airway epithelial cell function.

Knob protein enhances epithelial barrier integrity and attenuates airway inflammation

Altered epithelial physical and functional barrier properties along with TH1/TH2 immune dysregulation are features of allergic asthma. Regulation of junction proteins to improve barrier function of airway epithelial cells has the potential for alleviation of allergic airway inflammation. We sought to determine the immunomodulatory effect of knob protein of the adenoviral capsid on allergic asthma and to investigate its mechanism of action on airway epithelial junction proteins and barrier function. Airway inflammation, including junction protein expression, was evaluated in allergen-challenged mice with and without treatment with knob. Human bronchial epithelial cells were exposed to knob, and its effects on expression of junction proteins and barrier integrity were determined. Administration of knob to allergen-challenged mice suppressed airway inflammation (eosinophilia, airway hyperresponsiveness, and IL-5 levels) and prevented allergen-induced loss of airway epithelial occludin and E-cadherin expression. Additionally, knob decreased expression of TH2-promoting inflammatory mediators, specifically IL-33, by murine lung epithelial cells. At a cellular level, treatment of human bronchial epithelial cells with knob activated c-Jun N-terminal kinase, increased expression of occludin and E-cadherin, and enhanced epithelial barrier integrity. Increased expression of junction proteins mediated by knob leading to enhanced epithelial barrier function might mitigate the allergen-induced airway inflammatory response, including asthma.

Effects of human rhinovirus on epithelial barrier integrity and function in children with asthma.

Bronchial epithelial tight junctions (TJ) have been extensively assessed in healthy airway epithelium. However, no studies have yet assessed the effect of human rhinovirus (HRV) infection on the expression and resultant barrier function in epithelial tight junctions (TJ) in childhood asthma. To investigate the impact of HRV infection on airway epithelial TJ expression and barrier function in airway epithelial cells (AECs) of children with and without asthma. Furthermore, to test the hypothesis that barrier integrity and function is compromised to a greater extent by HRV in AECs from asthmatic children. Primary AECs were obtained from children with and without asthma, differentiated into air-liquid interface (ALI) cultures and infected with rhinovirus. Expression of claudin-1, occludin and zonula occluden-1 (ZO-1) was assessed via qPCR, immunocytochemistry (ICC), in-cell western (ICW) and confocal microscopy. Barrier function was assessed by transepithelial electrical resistance (TER; RT ) and permeability to fluorescent dextran. Basal TJ gene expression of claudin-1 and occludin was significantly upregulated in asthmatic children compared to non-asthmatics; however, no difference was seen with ZO-1. Interestingly, claudin-1, occludin and ZO-1 protein expression was significantly reduced in AEC of asthmatic children compared to non-asthmatic controls suggesting possible post-transcriptional inherent differences. HRV infection resulted in a transient dissociation of TJ and airway barrier integrity in non-asthmatic children. Although similar dissociation of TJ was observed in asthmatic children, a significant and sustained reduction in TJ expression concurrent with both a significant decrease in TER and an increase in permeability in asthmatic children was observed. This study demonstrates novel intrinsic differences in TJ gene and protein expression between AEC of children with and without asthma. Furthermore, it correlates directly the relationship between HRV infection and the resultant dissociation of epithelial TJ that causes a continued altered barrier function in children with asthma.