Lung Epithelial Barrier Research Articles

Treatment with natural AhR ligand leads to amelioration of LPS-induced acute lung injury by decreasing microRNA 29b-2-5p expression targeting increased IL-22 production

Abstract Acute lung injury and acute respiratory distress syndrome (ALI/ARDS) are severe pulmonary inflammatory diseases that cause destruction and remolding of the lung epithelial barrier, which may lead to restriction of pulmonary functionality and/or death. Indole-3-carbinol (I3C), a naturally occurring Ahr ligand has been shown to ameliorate multiple inflammatory disorders. Our studies are aimed at determining whether treatment with the I3C affects reparative reepithelialization and restores lung functioning through an immune-mediated mechanism. Towards this, 5mg/kg of LPS was intranasally injected in C57BL/6 mice to induce ALI. Three hours following LPS administration, the mice were treated with 40mg/kg I3C. After 48 hours, we examined lung functions using Buxco plethysmography and noticed LPS + I3C treated-mice have similar functionality as naïve mice in multiple parameters including ratio of time peak expiratory flow (Rpef), rejection index (Rinx), and tidal volume (TV) basal pulmonary functions whereas LPS+vehicle treated mice showed alterations. Histopathological analysis revealed fibrotic tissue in lung sections of LPS + Vehicle administered mice, but not in LPS + I3C treated mice. In addition, I3C increased the expression of Th22 (CD3+CD4+IL22+) cells, which are key mediators of epithelial cell layer protection, antimicrobial peptide production, and reepithelialization improvement. I3C increased IL-22 expression in bronchoalveolar lavage fluid, and decreased the expression in miR-29b-2-5p transcript that directly regulates IL-22 expression. In conclusion, our studies suggest that Ahr ligand I3C alleviates lung injury by maintaining lung epithelium integrity which may be mediated by IL-22 secreting T cells.

Assessing the Activity of Multidrug Resistance-Associated Protein 1 at the Lung Epithelial Barrier.

Multidrug resistance-associated protein 1 (adenosine triphosphate-binding cassette subfamily C member 1 [ABCC1]) is abundantly expressed at the lung epithelial barrier, where it may influence the pulmonary disposition of inhaled drugs and contribute to variability in therapeutic response. The aim of this study was to assess the impact of ABCC1 on the pulmonary disposition of 6-bromo-7-11C-methylpurine (11C-BMP), a prodrug radiotracer that is intracellularly conjugated with glutathione to form the ABCC1 substrate S-(6-(7-11C-methylpurinyl))glutathione (11C-MPG). Methods: Groups of Abcc1(-/-) rats, wild-type rats pretreated with the ABCC1 inhibitor MK571, and wild-type control rats underwent dynamic PET scans after administration of 11C-BMP intravenously or by intratracheal aerosolization. In vitro transport experiments were performed with unlabeled BMP on the human distal lung epithelial cell line NCI-H441. Results: The pulmonary kinetics of radioactivity significantly differed between wild-type and Abcc1(-/-) rats, but differences were more pronounced after intratracheal than after intravenous administration. After intravenous administration, lung exposure (area under the lung time-activity curve from 0 to 80 min after radiotracer administration [AUClung]) was 77% higher and the elimination slope of radioactivity washout from the lungs (kE,lung) was 70% lower in Abcc1(-/-) rats, whereas after intratracheal administration, AUClung was 352% higher and kE,lung was 86% lower in Abcc1(-/-) rats. Pretreatment with MK571 decreased kE,lung by 20% after intratracheal radiotracer administration. Intracellular accumulation of MPG in NCI-H441 cells was significantly higher and extracellular efflux was lower in the presence than in the absence of MK571. Conclusion: PET with pulmonary administered 11C-BMP can measure ABCC1 activity at the lung epithelial barrier and may be applicable in humans to assess the effects of disease, genetic polymorphisms, or concomitant drug intake on pulmonary ABCC1 activity.

Role of the Epithelial Glycocalyx in Maintaining the Alveolar‐Capillary Barrier During Acute Lung Injury

RationaleDisruption of the alveolar‐capillary barrier is a signature feature of acute lung injury. Previous studies have shown the importance of the glycocalyx in maintaining the integrity and homeostasis of the lung endothelium during acute lung injury. However, the role of the glycocalyx on lung epithelial barriers during acute lung injury has not been fully explored. We hypothesize that the lung epithelial glycocalyx is important in maintaining lung epithelial tight junctions in the setting of acute lung injury.MethodsCultured mouse lung epithelial cells (MLE12) were treated with functional or heat‐inactivated heparinase I/III. Additionally, some MLE12 cells were treated with LPS after pretreatment with functional heparinase I/III. Glycocalyx shedding was measured by immunohistochemistry for heparan sulfate (HS). To assess paracellular permeability, electrical cell‐impedance sensing (ECIS) was used to measure the resistance across the epithelial monolayer. To determine whether loss of the glycocalyx affects epithelial tight junctions we measured gene and protein expression of ZO‐1, by PCR, western blot, and immunofluorescence.ResultsMLE12 cells in culture express a robust glycocalyx that is completely shed following treatment with active heparinase I/III at 24 hours. Active heparinase I/III did not cause significant change in monolayer resistance from baseline compared to inactive heparinase I/III by ECIS (Active −142±39.2 v Inactive −118.9±57.9 ohms, p=0.75). Active heparinase I/III did not decrease protein expression of ZO‐1 compared to inactive heparinase I/III (Active 1.55±0.33 v Inactive 1.8±0.25 fold change ZO‐1/Actin, p=0.61). MLE12 cells treated with activated heparinase I/III and LPS had a significantly greater decrease in resistance from baseline compared to inactive heparinase and LPS (Figure , Active + LPS −145±8.8 vs Inactive + LPS − 7.7±10.8 ohms, p=0.0022)ConclusionTreating MLE12 cells with functional heparinase I/III degrades the epithelial glycocalyx. Loss of the glycocalyx alone was not sufficient to cause permeability defects nor alter the expression of tight junction proteins. However, glycocalyx shedding did increase the susceptibility of MLE12 cells to LPS‐induced injury. In clinical situations, the shedding of the glycocalyx in ALI is often accompanied by compounding factors such as infection, inflammation, or mechanical stress. The current findings suggest that damage to the epithelial glycocalyx may play a key role in maintaining the alveolar epithelial barrier in acute lung injury.Support or Funding InformationSupported by a research grant from Department of DefenseMLE12 cells treated with active heparinase + LPS had a significantly greater decrease in resistance compared to baseline than cells treated with inactive heparinase + LPS p=0.0022.Figure 1

Zonulin antagonist protects against tight junction disruption by Pseudomonas aeruginosa in airway epithelial cells

Zonulin is known as a regulator of barrier function in both epithelial and endothelial cells in the intestine and kidney by acting through EGFR (epidermal growth factor receptor) and PAR2 (Proteinase activated receptors) to cause the dissociation of ZO‐1 from occludin and claudin‐1 from the cell junctions. Respiratory infection is characterized by increased lung permeability caused by the reduction in the abundance and/or distribution of proteins at the tight junction. Here the effect of the zonulin antagonist, larazotide acetate (LA) on airway epithelial cell barrier function after the addition of lipopolysaccharide (LPS) from Pseudomonas aeruginosa or undiluted P.aeruginosa filtrate isolated from an overnight culture was investigated. Two human airway epithelial cell models were used, H441 cells grown at air‐liquid interface and 16HBE14o‐cells grown as submerged monolayers. Cells were incubated with 10 μg/ml LPS and 12 mM larazotide acetate. Transepithelial electrical resistance (TEER) and permeability was measured after 1h and 6h. TEER was decreased with LPS which was prevented by LA, (p<0.05, n=3) LPS vs LPS/LA at 1 hour. Permeability was increased with LPS after 6h (p<0.05, n=3) LPS vs control. Cells were immunostained for ZO‐1 and E‐cadherin, LPS caused the disappearance of ZO‐1 staining at the apical surface which was conserved with LA. E‐cadherin localization did not change with either LPS or LPS/LA treatment. Total protein abundance assessed by western blot showed no changes. Exposure of 16HBE14o‐ cells to P.aeruginosa filtrate for 24 h lead to a 61% decrease in TEER compared to control (p<0.0001, n=3). P.aeruginosa filtrate/LA increased TEER by 39% (p<0.02, n=3). Tight junction localization of E‐cadherin and claudin‐1 were both disrupted with P.aeruginosa filtrate while incubation with LA attenuated this rearrangement. This study shows that larazotide acetate helped to counteract the damage to tight junctions caused by LPS and P.aeruginosa filtrate, this supports a role for zonulin as a regulator of lung epithelial barrier function.Support or Funding InformationFunded by Astra Zeneca

Macrophages exposed to HIV viral protein disrupt lung epithelial cell integrity and mitochondrial bioenergetics via exosomal microRNA shuttling

Antiretroviral therapy extends survival but does not eliminate HIV from its cellular reservoirs. Between immune and stromal cells in the tissue microenvironment, a dynamic intercellular communication might influence host viral immune responses via intercellular transfer of extracellular vehicles (EVs) (microvesicles, exosome, or apoptotic bodies). It is increasingly recognized that HIV-infected macrophage-secreted nucleotide-rich exosomes might play a critical role in mediating communication between macrophages and other structural cells; however, molecular mechanisms underlying cell–cell crosstalk remain unknown. Here we show that HIV-1-infected macrophages and HIV-1 proteins Tat or gp120-treated macrophages express high levels of microRNAs, including miR-23a and miR-27a. Identical miRNAs expression patterns were detected in macrophage-secreted exosomes isolated from bronchoalveolar lavage fluid of HIV transgenic rats. Tat-treated macrophage-derived exosomal miR-23a attenuated posttranscriptional modulation of key tight junction protein zonula occludens (ZO-1) 3′-UTR in epithelial cells. In parallel, exosomal miR-27a released from Tat-treated macrophages altered the mitochondrial bioenergetics of recipient lung epithelial cells by targeting peroxisome proliferator-activated receptor gamma (PPARγ), while simultaneously stimulating glycolysis. Together, exosomal miRNAs shuttle from macrophages to epithelial cells and thereby explain in part HIV-mediated lung epithelial barrier dysfunction. These studies suggest that targeting miRNAs may be of therapeutic value to enhance lung health in HIV.

Activation of mas restores hyperoxia-induced loss of lung epithelial barrier function through inhibition of apoptosis

Neonatal therapy with a high concentration of oxygen (hyperoxia) is a known cause of bronchopulmonary dysplasia (BPD). BPD is characterized by increased pulmonary permeability and diffuse infiltration of various inflammatory cells. Disruption of the epithelial barrier may lead to altered pulmonary permeability and airways fluid accumulation. Mas receptor is a component of the renin angiotensin system and is the receptor for the protective endogenous peptide angiotensin 1-7. The activation of the Mas receptor was previously shown to have protective pulmonary responses. However, the effect of Mas receptor activation on epithelial barrier integrity has not been tested. To determine the effects of hyperoxia with or without Mas receptor activation on epithelial cell barrier integrity. Human epithelial cell line A549 was cultured on transwell polycarbonate porous membrane to confluence and treated with 95% oxygen (hyperoxia) for 72 hours with or without the Mas receptor agonist (AVE0991), or the apoptotic inhibitors Z-VAD-FMK or aurintricarboxylic acid. The cells were then challenged with Rhodamine labeled bovine serum albumin (Rh-BSA) on one side of the membrane. Fluorescent quantitation of Rh-BSA (albumin flux) was performed on the media in the other side of the membrane 3 hours later and was compared with 21% oxygen (Normoxia) control group. A549 cells were also cultured with or without AVE0991 in hyperoxia or normoxia and used for nuclear fragmentation apoptosis assay using propidium iodide staining. Hyperoxia induced an increase in albumin flux that was significantly prevented by AVE0991 treatment and by the apoptosis inhibitors. AVE0991 also significantly decreased the hyperoxia-induced nuclear fragmentation. These results suggest that hyperoxia causes a disruption in the epithelial barrier integrity, and that this disruption is inhibited by the Mas receptor agonist AVE0991 through inhibition of epithelial apoptosis. These results reveal a novel potential drug for BPD and pulmonary edema treatment.

Protective effect of resveratrol on the integrity of alveolar and intestinal epithelial barrier in SEB-induced acute lung injury

Abstract Acute lung injury (ALI) is a state of inflammation that breaks down the lung endothelial and epithelial cell barriers. In the current study, we investigated the role of resveratrol (RES) in regulating the expression and functions of tight junction proteins (TJP) in epithelial cell responses following exposure to this superantigen. To this end, C3H mice were given resveratrol orally twice prior to intranasal challenge with lethal SEB doses. 16S rRNA results showed that there were microbes transported in the blood in addition to the lung and colonic tissues. For this purpose, we used a reporter E. coli-GFP labeled bacterium to monitor and examine the viability of this bacterium in case of leak from lung into blood and other body compartments during the peak of ALI. The flow cytometry results showed significant reduction in copy numbers of this reporter in the blood in RES-treated mice in comparison to vehicle-treated mice. However, bacteriological examination for viability by culture of the blood showed all were negative for all experimental groups. Mouse transcriptome array showed increase in TJP gene expression in colonic and alveolar epithelial cells of RES-treated mice. Some of these genes were validated with qPCR. We found that there was an increase in the expression of some TJP genes following RES treatment. Commensals and probiotics are also known to decrease intestinal barrier dysfunction caused by inflammatory cytokines. Therefore, mass spectrometry (MS) analysis showed increase in the metabolome in colons of RES-treated group that help in strengthening of intestinal barrier. In conclusion, RES treatment increased directly or indirectly the rigidity of cell boundaries resulting in amelioration of ALI-dependent complications.

Role of IGSF3 in Lung Epithelial Barrier Disruption Induced by Cigarette Smoke Exposure

RationaleCigarette smoke (CS) exposure disrupts lung epithelial and endothelial cell barrier function which is linked to lung injury and inflammation. We identified a tetraspanin‐interacting protein, IGSF3, with roles in cell proliferation, adhesion, and migration via unclear mechanisms. We have shown that lung IGSF3 is decreased by cigarette smoke (CS) exposure and that loss of IGSF3 can disrupt sphingolipid metabolites. Of these, sphingosine‐1 phosphate (S1P) protects endothelial barrier function but may disrupt epithelial barrier via distinct receptors. We investigated if IGSF3 and S1P are required for maintaining the lung epithelial barrier in response to CS.MethodsHuman bronchial lung epithelial cells (Beas2b) were stably transfected with lentivirus control or IGSF3 shRNA (shIGSF3). Trans‐epithelial resistance (TER) was measured using electric cell‐substrate impedance sensing. CS extract was prepared using research cigarettes (3R4F, University of KY). Phosphatidylinositol lipid strips (Echelon) were used to study IGSF3‐lipid binding.ResultsCompared to control shRNA, knockdown of IGSF3 did not alter epithelial barrier function as measured by TER. Exposure to CS (7.5%), as expected, decreased epithelial cell monolayers TER in control cells by 35%. CS‐induced effects on TER were attenuated by treatment with N‐acetylcysteine or with (R)‐FTY720‐phosphonate (1R), a sphingosine‐1 phosphate receptor 1 (S1PR1) agonist, and were worsened by sphingosine kinase‐1 inhibition using SKI2. The CS‐induced decrease in TER was significantly worsened (by 55%, p<0.01 vs. control cells exposed to CS) in shIGSF3 ‐ transfected cells which responded in turn to the barrier‐enhancing effect of N‐acetylcysteine treatment (by 30%, p<0.05). A putative Pleckstrin‐homology (PH) domain was identified in the C‐terminal cytoplasmic domain of IGSF3 which predicted binding to PI(3,4)P2, which was then experimentally confirmed. Further, site directed mutagenesis within PH domain disrupted binding of IGSF3 with tetraspanin CD81.ConclusionsIGSF3 is required for partial maintenance of the lung epithelial barrier function in response to CS injury. Loss of IGSF3 may worsen CS injury of the airways, contributing to inflammation, an effect that could be alleviated by antioxidants and S1P1‐mediated signaling. The mechanisms of IGSF3 barrier effects may include binding to and tethering PI(3,4)P2 and CD81 at the cell membrane via its PH domain.Support or Funding InformationFunding: Wollowick Chair of Pulmonary Research (IP), Department of Medicine Research Microgrant (KSS) from National Jewish Health.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Establishment of a pulmonary epithelial barrier on biodegradable poly-L-lactic-acid membranes.

Development of biocompatible and functional scaffolds for tissue engineering is a major challenge, especially for development of polarised epithelia that are critical structures in tissue homeostasis. Different in vitro models of the lung epithelial barrier have been characterized using non-degradable polyethylene terephthalate membranes which limits their uses for tissue engineering. Although poly-L-lactic acid (PLLA) membranes are biodegradable, those prepared via conventional Diffusion Induced Phase Separation (DIPS) lack open-porous geometry and show limited permeability compromising their use for epithelial barrier studies. Here we used PLLA membranes prepared via a modification of the standard DIPS protocol to control the membrane surface morphology and permeability. These were bonded to cell culture inserts for use in barrier function studies. Pulmonary epithelial cells (H441) readily attached to the PLLA membranes and formed a confluent cell layer within two days. This was accompanied by a significant increase in trans-epithelial electrical resistance and correlated with the formation of tight junctions and vectorial cytokine secretion in response to TNFα. Our data suggest that a structurally polarized and functional epithelial barrier can be established on PLLA membranes produced via a non-standard DIPS protocol. Therefore, PLLA membranes have potential utility in lung tissue engineering applications requiring bio-absorbable membranes.

The Neutrophilic Response to Pseudomonas Damages the Airway Barrier, Promoting Infection by Klebsiella pneumoniae

Pseudomonas aeruginosa and Klebsiella pneumoniae are two common gram-negative pathogens that are associated with bacterial pneumonia and can often be isolated from the same patient. We used a mixed-pathogen pneumonia infection model in which mice were infected with sublethal concentrations of P. aeruginosa and K. pneumoniae, resulting in significant lethality, outgrowth of both bacteria in the lung, and systemic dissemination of K. pneumoniae. Inflammation, induced by P. aeruginosa activation of Toll-like receptor 5, results in prolonged neutrophil recruitment to the lung and increased levels of neutrophil elastase in the airway, resulting in lung damage and epithelial barrier dysfunction. Live P. aeruginosa was not required to potentiate K. pneumoniae infection, and flagellin alone was sufficient to induce lethality when delivered along with Klebsiella. Prophylaxis with an anti-Toll-like receptor 5 antibody or Sivelestat, a neutrophil elastase inhibitor, reduced neutrophil influx, inflammation, and mortality. Furthermore, pathogen-specific monoclonal antibodies targeting P. aeruginosa or K. pneumoniae prevented the outgrowth of both bacteria and reduced host inflammation and lethality. These findings suggest that coinfection with P. aeruginosa may enable the outgrowth and dissemination of K. pneumoniae, and that a pathogen- or host-specific prophylactic approach targeting P. aeruginosa may prevent or limit the severity of such infections by reducing neutrophil-induced lung damage.

Carbon nanodots: Opportunities and limitations to study their biodistribution at the human lung epithelial tissue barrier.

Inhalation of combustion-derived ultrafine particles (≤0.1 μm) has been found to be associated with pulmonary and cardiovascular diseases. However, correlation of the physicochemical properties of carbon-based particles such as surface charge and agglomeration state with adverse health effects has not yet been established, mainly due to limitations related to the detection of carbon particles in biological environments. The authors have therefore applied model particles as mimics of simplified particles derived from incomplete combustion, namely, carbon nanodots (CNDs) with different surface modifications and fluorescent properties. Their possible adverse cellular effects and their biodistribution pattern were assessed in a three-dimensional (3D) lung epithelial tissue model. Three different CNDs, namely, nitrogen, sulfur codoped CNDs ( N,S-CNDs) and nitrogen doped CNDs ( N-CNDs-1 and N-CNDs-2), were prepared by microwave-assisted hydrothermal carbonization using different precursors or different microwave systems. These CNDs were found to possess different chemical and photophysical properties. The surfaces of nanodots N-CNDs-1 and N-CNDs-2 were positively charged or neutral, respectively, arguably due to the presence of amine and amide groups, while the surfaces of N,S-CNDs were negatively charged, as they bear carboxylic groups in addition to amine and amide groups. Photophysical measurements showed that these three types of CNDs displayed strong photon absorption in the UV range. Both N-CNDs-1 and N,S-CNDs showed weak fluorescence emission, whereas N-CNDs-2 showed intense emission. A 3D human lung model composed of alveolar epithelial cells (A549 cell line) and two primary immune cells, i.e., macrophages and dendritic cells, was exposed to CNDs via a pseudo-air-liquid interface at a concentration of 100 μg/ml. Exposure to these particles for 24 h induced no harmful effect on the cells as assessed by cytotoxicity, cell layer integrity, cell morphology, oxidative stress, and proinflammatory cytokines release. The distribution of the CNDs in the lung model was estimated by measuring the fluorescence intensity in three different fractions, e.g., apical, intracellular, and basal, after 1, 4, and 24 h of incubation, whereby reliable results were only obtained for N-CNDs-2. It was shown that N-CNDs-2 translocate rapidly, i.e., >40% in the basal fraction within 1 h and almost 100% after 4 h, while ca. 80% of the N-CNDs-1 and N,S-CNDs were still located on the apical surface of the lung cells after 1 h. This could be attributed to the agglomeration behavior of N-CNDs-1 or N,S-CNDs. The surface properties of the N-CNDs bearing amino and amide groups likely induce greater uptake as N-CNDs could be detected intracellularly. This was less evident for N,S-CNDs, which bear carboxylic acid groups on their surface. In conclusion, CNDs have been designed as model systems for carbon-based particles; however, their small size and agglomeration behavior made their quantification by fluorescence measurement challenging. Nevertheless, it was demonstrated that the surface properties and agglomeration affected the biodistribution of the particles at the lung epithelial barrier in vitro.

Epithelial Heparan Sulfate Contributes to Alveolar Barrier Function and Is Shed during Lung Injury.

The lung epithelial glycocalyx is a carbohydrate-enriched layer lining the pulmonary epithelial surface. Although epithelial glycocalyx visualization has been reported, its composition and function remain unknown. Using immunofluorescence and mass spectrometry, we identified heparan sulfate (HS) and chondroitin sulfate within the lung epithelial glycocalyx. In vivo selective enzymatic degradation of epithelial HS, but not chondroitin sulfate, increased lung permeability. Using mass spectrometry and gel electrophoresis approaches to determine the fate of epithelial HS during lung injury, we detected shedding of 20 saccharide-long or greater HS into BAL fluid in intratracheal LPS-treated mice. Furthermore, airspace HS in clinical samples from patients with acute respiratory distress syndrome correlated with indices of alveolar permeability, reflecting the clinical relevance of these findings. The length of HS shed during intratracheal LPS-induced injury (≥20 saccharides) suggests cleavage of the proteoglycan anchoring HS to the epithelial surface, rather than cleavage of HS itself. We used pharmacologic and transgenic animal approaches to determine that matrix metalloproteinases partially mediate HS shedding during intratracheal LPS-induced lung injury. Although there was a trend toward decreased alveolar permeability after treatment with the matrix metalloproteinase inhibitor, doxycycline, this did not reach statistical significance. These studies suggest that epithelial HS contributes to the lung epithelial barrier and its degradation is sufficient to increase lung permeability. The partial reduction of HS shedding achieved with doxycycline is not sufficient to rescue epithelial barrier function during intratracheal LPS-induced lung injury; however, whether complete attenuation of HS shedding is sufficient to rescue epithelial barrier function remains unknown.

Transgenic up-regulation of Claudin-6 decreases fine diesel particulate matter (DPM)-induced pulmonary inflammation.

Claudin-6 (Cldn6) is a tetraspanin transmembrane protein that contributes to tight junctional complexes and has been implicated in the maintenance of lung epithelial barriers. In the present study, we tested the hypothesis that genetic up-regulation of Cldn-6 influences inflammation in mice exposed to short-term environmental diesel particulate matter (DPM). Mice were subjected to ten exposures of nebulized DPM (PM2.5) over a period of 20days via a nose-only inhalation system (Scireq, Montreal, Canada). Using real-time RT-PCR, we discovered that the Cldn6 gene was up-regulated in control mice exposed to DPM and in lung-specific transgenic mice that up-regulate Cldn-6 (Cldn-6 TG). Interestingly, DPM did not further enhance Cldn-6 expression in Cldn-6 TG mice. DPM caused increased cell diapedesis into bronchoalveolar lavage fluid (BALF) from control mice; however, Cldn-6 TG mice had less total cells and PMNs in BALF following DPM exposure. Because Cldn-6 TG mice had diminished cell diapedesis, other inflammatory intermediates were screened to characterize the impact of increased Cldn-6 on inflammatory signaling. Cytokines that mediate inflammatory responses including TNF-α and IL-1β were differentially regulated in Cldn6 TG mice and controls following DPM exposure. These results demonstrate that epithelial barriers organized by Cldn-6 mediate, at least in part, diesel-induced inflammation. Further work may show that Cldn-6 is a key target in understanding pulmonary epithelial gateways exacerbated by environmental pollution.

Modifications in MUC2 and OCLN Expression in Human Alveolar Adenocarcinoma Epithelial Cells Following E‐Cigarette Chemical Exposure

Electronic cigarettes (e‐cigarettes) have entered the market promoting a healthy and clean alternative to conventional tobacco cigarettes. Aggressive marketing has led to an increase in usage of e‐cigarettes among adults and adolescents. Despite the promotion of e‐cigarettes as an alternative for tobacco users, the chemical composition of these two products is very similar. Two of the predominant chemicals found in e‐cigarettes are formalin and methylbenzaldehyde, of which, formalin is also found in tobacco cigarettes. We hypothesize that exposure of the alveolar adenocarcinoma (A549) cells to these common e‐cigarette chemicals will alter the lung epithelial barrier as measured by occludin (OCLN) and mucin‐2 (MUC2) expression.Mucin‐2 is a protein that secretes a protective mucus membrane and occludin is a tight junction protein, both contributing to a functional and protective lung epithelial barrier. Overexpression of MUC2 can lead to hypersecretion of mucus, while under expression of OCLN can cause an increase in epithelial permeability, representing a disruption of this barrier. A549 cells will be treated with physiological concentrations of formalin and methylbenzaldehyde to mimic e‐cigarette exposure. We will show that exposure to formalin and methylbenzaldehyde alters MUC2 and OCLN, indicating a disruption of the lung epithelial barrier. These results represent the negative health effects associated with e‐cigarette use, which contradicts the current advertisements marketing e‐cigarettes as a safe product.Support or Funding InformationNational Science Foundation Grant# 1458212This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Biodistribution of single and aggregated gold nanoparticles exposed to the human lung epithelial tissue barrier at the air-liquid interface

BackgroundThe lung represents the primary entry route for airborne particles into the human body. Most studies addressed possible adverse effects using single (nano)particles, but aerosolic nanoparticles (NPs) tend to aggregate and form structures of several hundreds nm in diameter, changing the physico-chemical properties and interaction with cells. Our aim was to investigate how aggregation might affect the biodistribution; cellular uptake and translocation over time of aerosolized NPs at the air-blood barrier interface using a multicellular lung system.ResultsModel gold nanoparticles (AuNPs) were engineered and well characterized to compare single NPs with aggregated NPs with hydrodynamic diameter of 32 and 106 nm, respectively. Exposures were performed by aerosolization of the particles onto the air-liquid interface of a three dimensional (3D) lung model. Particle deposition, cellular uptake and translocation kinetics of single and aggregated AuNPs were determined for various concentrations, (30, 60, 150 and 300 ng/cm2) and time points (4, 24 and 48 h) using transmission electron microscopy and inductively coupled plasma optical emission spectroscopy. No apparent harmful effect for single and aggregated AuNPs was observed by lactate dehydrogenase assay, nor pro-inflammation response by tumor necrosis factor α assessment. The cell layer integrity was also not impaired. The bio-distribution revealed that majority of the AuNPs, single or aggregated, were inside the cells, and only a minor fraction, less than 5%, was found on the basolateral side. No significant difference was observed in the translocation rate. However, aggregated AuNPs showed a significantly faster cellular uptake than single AuNPs at the first time point, i.e. 4 h.ConclusionsOur studies revealed that aggregated AuNPs showed significantly faster cellular uptake than single AuNPs at the first time point, i.e. 4 h, but the uptake rate was similar at later time points. In addition, aggregation did not affect translocation rate across the lung barrier model since similar translocation rates were observed for single as well as aggregated AuNPs.

Alteration in Claudin-4 Contributes to Airway Inflammation and Responsiveness in Asthma.

PurposeClaudin-4 has been reported to function as a paracellular sodium barrier and is one of the 3 major claudins expressed in lung alveolar epithelial cells. However, the possible role of claudin-4 in bronchial asthma has not yet been fully studied. In this study, we aimed to elucidate the role of claudin-4 in the pathogenesis of bronchial asthma.MethodsWe determined claudin-4 levels in blood from asthmatic patients. Moreover, using mice sensitized and challenged with OVA, as well as sensitized and challenged with saline, we investigated whether claudin-4 is involved in the pathogenesis of bronchial asthma. Der p1 induced the inflammatory cytokines in NHBE cells.ResultsWe found that claudin-4 in blood from asthmatic patients was increased compared with that from healthy control subjects. Plasma claudin-4 levels were significantly higher in exacerbated patients than in control patients with bronchial asthma. The plasma claudin-4 level was correlated with eosinophils, total IgE, FEV1% pred, and FEV1/FVC. Moreover, lung tissues from the OVA-OVA mice showed significant increases in transcripts and proteins of claudin-4 as well as in TJ breaks and the densities of claudin-4 staining. When claudin-4 was knocked down by transfecting its siRNA, inflammatory cytokine expressions, which were induced by Der p1 treatment, were significantly increased.ConclusionsThese findings thus raise the possibility that regulation of lung epithelial barrier proteins may constitute a therapeutic approach for asthma.

Serum surfactant protein D as a marker for bronchopulmonary dysplasia

Background: Lung epithelial cells express surfactant protein D (SP-D), a calcium-dependent lectin that plays an important role in antibody-independent pulmonary host defense. Previous studies have shown that it is found in the peripheral circulation in patients with pulmonary disease, likely because of translocation into the blood when lung epithelial barriers are disrupted by inflammation or acute injury. In adults, serum SP-D levels are biomarkers for the progression and severity of chronic lung disease. In neonates, elevated SP-D levels in cord blood and on day 1 have been associated with prenatal risk factors and with an increased risk of respiratory distress syndrome and infections. It is not known whether serum SP-D during the first week of life is a marker for bronchopulmonary dysplasia (BPD), a form of chronic lung disease of prematurity that is associated with lung parenchymal maldevelopment and injury.Objective: The goal of this study is to determine whether serum SP-D on days 3 and 7 of life are associated with the development of BPD in preterm infants.Design/methods: Serum samples were obtained on postnatal days 3 and 7 from 106 preterm infants (500–2000 g birth weight, 23–32-week gestation). SP-D was quantified by Western blot. BPD was determined at 36 weeks PMA using NICHD criteria.Results: The mean birth weight was 1145 ± 347 g and gestational age 29.2 ± 7.4 weeks. BPD was diagnosed in 7 and “BPD or death” in 16 infants. Days 3 and 7 values tracked significantly (r = 0.648), and did not correlate with birth weight or gestational age. Contrary to expectations, serum SP-D was not associated with BPD. Significant gender differences were noted, with SP-D dropping from day 3 to day 7 in males, while increasing in females (p < .05).Conclusion: Elevated serum SP-D does not appear to be a useful marker for BPD. Decreasing serum SP-D levels in males, as compared to females, during the first week of life are likely related to gender differences in lung maturation, consistent with the higher incidence of BPD in males.

Biokinetics of Aerosolized Liposomal Ciclosporin A in Human Lung Cells In Vitro Using an Air-Liquid Cell Interface Exposure System.

Inhalation of aerosolized drugs is a promising route for noninvasive targeted drug delivery to the lung. Nanocarrier systems such as liposomes have been explored for inhalation therapy opening new avenues, including stabilization of nonsoluble drugs (e.g., Ciclosporin A [CsA]) and controlled release. The biokinetic behavior of the immunosuppressive drug CsA encapsulated in liposomes (L-CsA) at the lung epithelial barrier was studied in vitro. Human lung epithelial cells (alveolar A549 and bronchial 16HBE14o- epithelial cells) were exposed to aerosolized L-CsA at the air-liquid interface (ALI) using a dose-controlled air-liquid interface cell exposure (ALICE) system and the temporal profile of the L-CsA dose in the apical, basal, and cell compartment was monitored up to 24 hours. Aerosolization of different volumes of L-CsA solution with the ALICE resulted in dose-controlled, spatially uniform, and reproducible L-CsA delivery. Cell viability at 24 hours postexposure was not impaired and immunofluorescence staining revealed the typical epithelial cell morphology in control as well as in L-CsA-exposed cells. The (pro-)inflammatory interleukin-8 levels were not elevated under any condition. The biokinetic analysis revealed that both cell types formed a tight, but imperfect, barrier for L-CsA resulting in initially high transbarrier L-CsA transport rates, which ceased after about 4 hours. Although substantial transbarrier L-CsA transport was observed for both cell types, respectively, a 150-fold higher L-CsA concentration was established in the apical and cell compared to the basal compartment. Most importantly, for pulmonary drug targeting, a high cellular L-CsA dose level (20%-25% of the delivered dose) was obtained rapidly (<1 hour) and maintained for at least 24 hours. The ALICE system combined with lung epithelial cells cultured at the ALI offers a reliable and relevant in vitro platform technology to study the effects of inhalable substances such as L-CsA under biomimetic conditions.

Industrial PM2.5 cause pulmonary adverse effect through RhoA/ROCK pathway

According to the Chinese Ministry of Health, industrial pollution-induced health impacts have been the leading cause of death in China. While industrial fine particulate matter (PM2.5) is associated with adverse health effects, the major action mechanisms of different compositions of PM2.5 are currently unclear. In this study, we treated normal human lung epithelial BEAS-2B cells with industrial organic and water-soluble PM2.5 extracts under daily alveolar deposition dose to elucidate the molecular mechanisms underlying adverse pulmonary effects induced by PM2.5, including oxidative damage, inflammatory response, lung epithelial barrier dysfunction, and the recruitment of macrophages. We found that water-soluble PM2.5 extracts caused more severe cytotoxic effects on BEAS-2B cells compared with that of organic extracts. Both organic and water-soluble PM2.5 extracts induced activation of the RhoA/ROCK pathway. Inflammatory response, epithelial barrier dysfunction, and the activation of NF-кB caused by both PM2.5 extracts were attenuated by ROCK inhibitor Y-27632. This indicated that both PM2.5 extracts could cause damage to epithelial cells through RhoA/ROCK-dependent NF-кB activation. Furthermore, the upregulation of macrophage adhesion induced by both PM2.5 extracts was also attenuated by Y-27632 in a co-culture model of macrophages and the epithelial cells. Therefore, our results support that industrial PM2.5 extracts-induced activation of the RhoA/ROCK-dependent NF-кB pathway induces pulmonary adverse effect. Thus, pharmacological inhibition of ROCK activation might have therapeutic potential in preventing lung disease associated with PM2.5.

A novel technique to determine the cell type specific response within an in vitro co-culture model via multi-colour flow cytometry

Determination of the cell type specific response is essential towards understanding the cellular mechanisms associated with disease states as well as assessing cell-based targeting of effective therapeutic agents. Recently, there have been increased calls for advanced in vitro multi-cellular models that provide reliable and valuable tools correlative to in vivo. In this pursuit the ability to assess the cell type specific response is imperative. Herein, we report a novel approach towards resolving each specific cell type of a multi-cellular model representing the human lung epithelial tissue barrier via multi-colour flow cytometry (FACS). We proved via ≤ five-colour FACS that the manipulation of this in vitro model allowed each cell type to be resolved with no impact upon cell viability. Subsequently, four-colour FACS verified the ability to determine the biochemical effect (e.g. oxidative stress) of each specific cell type. This technique will be vital in gaining information upon cellular mechanics when using next-level, multi-cellular in vitro strategies.