Type I-like Cells Research Articles

Immunohistochemical analysis of glutamatergic and serotonergic signaling pathways in chemosensory cell clusters in the pharynx and larynx of rats

The present study examined cellular components and the localization of vesicular glutamate transporter (VGLUT) 1 and 2 and serotonin (5-HT) in chemosensory cell clusters in the rat pharynx and larynx. Triple immunolabeling for guanine nucleotide-binding protein G (t), subunit ⍺3 (GNAT3) and nucleoside triphosphate diphosphohydrolase 2 (NTPDase2) with synaptotagmin-1 (Syt1) revealed NTPDase2-immunoreactive type I-like cells in addition to GNAT3-immunoreactive type II-like and Syt1-immunoreactive type III-like cells in pharyngolaryngeal chemosensory cell clusters. Therefore, these clusters appear to comprise similar cell types to those in the lingual taste buds with slight morphological modifications. An immunofluorescence analysis of VGLUT1 or VGLUT2 and GNAT3 with P2X3 purinoceptors revealed that VGLUTs co-localized to P2X3‐immunoreactive spherical nerve terminals closely associated with GNAT3-immunoreactive type II-like cells. Moreover, triple immunolabeling for Syt1/synaptosomal-associated protein, 25 kDa (SNAP25) and P2X3 with VGLUT1 or VGLUT2 revealed punctate immunoreactive products for VGLUT1 and VGLUT2 within P2X3‐immunoreactive flat axon terminals wrapped around Syt1/SNAP25-immunoreactive type III-like cells. The afferent nerve fibers innervating cell clusters may contain glutamate and release it by exocytosis. On the other hand, immunoreactive products for 5-HT and dopa decarboxylase were detected in Syt1-immunoreactive cells, indicating the release of 5-HT by these cells. The present results suggest that chemosensory cell clusters in the pharynx and larynx may be modulated by intrinsic glutamate and 5-HT.

FOXO1 Couples KGF and PI-3K/AKT Signaling to NKX2.1-Regulated Differentiation of Alveolar Epithelial Cells.

NKX2.1 is a master regulator of lung morphogenesis and cell specification; however, interactions of NKX2.1 with various transcription factors to regulate cell-specific gene expression and cell fate in the distal lung remain incompletely understood. FOXO1 is a key regulator of stem/progenitor cell maintenance/differentiation in several tissues but its role in the regulation of lung alveolar epithelial progenitor homeostasis has not been evaluated. We identified a novel role for FOXO1 in alveolar epithelial cell (AEC) differentiation that results in the removal of NKX2.1 from surfactant gene promoters and the subsequent loss of surfactant expression in alveolar epithelial type I-like (AT1-like) cells. We found that the FOXO1 forkhead domain potentiates a loss of surfactant gene expression through an interaction with the NKX2.1 homeodomain, disrupting NKX2.1 binding to the SFTPC promoter. In addition, blocking PI-3K/AKT signaling reduces phosphorylated FOXO-1 (p-FOXO1), allowing accumulated nuclear FOXO1 to interact with NKX2.1 in differentiating AEC. Inhibiting AEC differentiation in vitro with keratinocyte growth factor (KGF) maintained an AT2 cell phenotype through increased PI3K/AKT-mediated FOXO1 phosphorylation, resulting in higher levels of surfactant expression. Together these results indicate that FOXO1 plays a central role in AEC differentiation by directly binding NKX2.1 and suggests an essential role for FOXO1 in mediating AEC homeostasis.

Functional expression of equilibrative and concentrative nucleoside transporters in alveolar epithelial cells.

Equilibrative nucleoside transporters (ENTs) and concentrative nucleoside transporters (CNTs) mediate the cellular uptake of nucleosides and nucleobases across the plasma membrane and play important roles in the salvage pathways of nucleotide synthesis. However, information about nucleoside transport systems in the lung alveolar epithelial cells is limited. Therefore, in the present study, we examined the function and expression of nucleoside transporters using primary cultured alveolar type II cells and transdifferentiated type I-like cells. The uptake of uridine, a substrate for ENTs and CNTs, in type II and type I-like cells was time, temperature, and concentration dependent, and was inhibited by other nucleoside transporter substrates such as adenosine. Uridine uptake in both cells was insensitive to nanomolar concentrations of NBMPR, a potent ENT1 inhibitor, while it was inhibited by higher concentrations of NBMPR, suggesting that ENT2, but not ENT1, is involved in uridine uptake in these cells. Additionally, uridine uptake was higher in the presence of Na+ than in the absence of Na + and was partially inhibited by a CNT inhibitor phloridzin in these cells, suggesting that CNT is also involved in uridine uptake. In both cells, the mRNA expression of ENT1, ENT2, CNT2, and CNT3 was observed. Finally, the activity of uridine uptake was considerably higher in type II cells than in type I-like cells. In addition, the mRNA expression of ENT2, CNT2, and CNT3, but not ENT1, was lower in type I-like cells than in type II cells. These findings would help understand the functional roles of equilibrative and concentrative nucleoside transporters in alveolar epithelial cells.

The Transcription Factor Sox2 Is Required to Maintain the Cell Type-Specific Properties and Innervation of Type II Vestibular Hair Cells in Adult Mice.

The sense of balance relies on vestibular hair cells, which detect head motions. Mammals have two types of vestibular hair cell, I and II, with unique morphological, molecular, and physiological properties. Furthermore, each hair cell type signals to a unique form of afferent nerve terminal. Little is known about the mechanisms in mature animals that maintain the specific features of each hair cell type or its postsynaptic innervation. We found that deletion of the transcription factor Sox2 from Type II hair cells in adult mice of both sexes caused many cells in utricles to acquire features unique to Type I hair cells and to lose Type II-specific features. This cellular transdifferentiation, which included changes in nuclear size, chromatin condensation, soma and stereocilium morphology, and marker expression, resulted in a significantly higher proportion of Type I-like hair cells in all epithelial zones. Furthermore, Sox2 deletion from Type II hair cells triggered non-cell autonomous changes in vestibular afferent neurons; they retracted bouton terminals (normally present on only Type II cells) from transdifferentiating hair cells and replaced them with a calyx terminal (normally present on only Type I cells). These changes were accompanied by significant expansion of the utricle's central zone, called the striola. Our study presents the first example of a transcription factor required to maintain the type-specific hair cell phenotype in adult inner ears. Furthermore, we demonstrate that a single genetic change in Type II hair cells is sufficient to alter the morphology of their postsynaptic partners, the vestibular afferent neurons.SIGNIFICANCE STATEMENT The sense of balance relies on two types of sensory cells in the inner ear, Type I and Type II hair cells. These two cell types have unique properties. Furthermore, their postsynaptic partners, the vestibular afferent neurons, have differently shaped terminals on Type I versus Type II hair cells. We show that the transcription factor Sox2 is required to maintain the cell-specific features of Type II hair cells and their postsynaptic terminals in adult mice. This is the first evidence of a molecule that maintains the phenotypes of hair cells and, non-cell autonomously, their postsynaptic partners in mature animals.

EphB6 Regulates TFEB-Lysosomal Pathway and Survival of Disseminated Indolent Breast Cancer Cells.

Simple SummaryA large number of estrogen receptor-positive breast cancer patients show relapses at the metastatic site up to 20 years after the removal of the primary tumor. This phenomenon, called “metastatic dormancy”, is a particularly dangerous aspect of cancers, as it affects patients considered healed. A metastatic relapse after years since mastectomy implies that disseminated cells could survive in the metastatic organ for a long period of time. Our goal was to better understand the signals supporting the survival of the disseminated cancer cells with the aim of killing them before the relapse. We found a molecule, called EphB6, that supports the persistence of disseminated dormant cancer cells thanks to the activation of a cellular process, the lysosomal-flux, that is a central hub for nutrient sensing and recycling of the cell.Late relapse of disseminated cancer cells is a common feature of breast and prostate tumors. Several intrinsic and extrinsic factors have been shown to affect quiescence and reawakening of disseminated dormant cancer cells (DDCCs); however, the signals and processes sustaining the survival of DDCCs in a foreign environment are still poorly understood. We have recently shown that crosstalk with lung epithelial cells promotes survival of DDCCs of estrogen receptor-positive (ER+) breast tumors. By using a lung organotypic system and in vivo dissemination assays, here we show that the TFEB-lysosomal axis is activated in DDCCs and that it is modulated by the pro-survival ephrin receptor EphB6. TFEB lysosomal direct targets are enriched in DDCCs in vivo and correlate with relapse in ER+ breast cancer patients. Direct coculture of DDCCs with alveolar type I-like lung epithelial cells and dissemination in the lung drive lysosomal accumulation and EphB6 induction. EphB6 contributes to survival, TFEB transcriptional activity, and lysosome formation in DDCCs in vitro and in vivo. Furthermore, signaling from EphB6 promotes the proliferation of surrounding lung parenchymal cells in vivo. Our data provide evidence that EphB6 is a key factor in the crosstalk between disseminated dormant cancer cells and the lung parenchyma and that the TFEB-lysosomal pathway plays an important role in the persistence of DDCCs.

Heat-Not-Burn cigarette induces oxidative stress response in primary rat alveolar epithelial cells

There has been an increase in the usage of heat-not-burn (HNB) cigarette products. However, their effects on alveolar epithelial cells (AECs) remain unknown. AECs are the target cells of conventional cigarette smoking-related respiratory diseases such as chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis and lung cancer whose pathogenesis involves oxidative stress. In this study, primary rat AECs were isolated, cultured and stimulated by HNB cigarette smoke extract (CSE). Our data indicate that rat AECs exposed to HNB CSE induced oxidative stress response genes (e.g. Hmox-1, Gsta1, Gsta3 and Nqo1). We also compared the oxidative stress response between two different types of AECs, alveolar type I-like (ATI-like) cells and type II (ATII) cells, and between two different types of cigarette, HNB cigarettes and conventional cigarettes. The expressions of Gsta1, Gsta3 and Nqo1 were higher in ATII cells than ATI-like cells in response to HNB and conventional cigarettes, but there was no significant difference in their expression levels between HNB cigarette and conventional cigarette. Taken together, our results suggest that HNB cigarettes have the similar potential as conventional cigarette products to induce oxidative stress response in AECs.

Mechanistic profiling of the release kinetics of siRNA from lipidoid-polymer hybrid nanoparticles in vitro and in vivo after pulmonary administration

Understanding the release kinetics of siRNA from nanocarriers, their cellular uptake, their in vivo biodistribution and pharmacokinetics is a fundamental prerequisite for efficient optimisation of the design of nanocarriers for siRNA-based therapeutics. Thus, we investigated the influence of composition on the siRNA release from lipid-polymer hybrid nanoparticles (LPNs) consisting of cationic lipidoid 5 (L5) and poly(DL-lactic-co-glycolic acid) (PLGA) intended for pulmonary administration. An array of siRNA-loaded LPNs was prepared by systematic variation of: (i) the L5 content (10–20%, w/w), and (ii) the L5:siRNA ratio (10,1–30:1, w/w). For comparative purposes, L5-based lipoplexes, L5-based stable nucleic acid lipid nanoparticles (SNALPs). and dioleoyltrimethylammoniumpropane (DOTAP)-modified LPNs loaded with siRNA were also prepared. Release studies in buffer and lung surfactant-containing medium showed that siRNA release is dependent on the presence of both surfactant and heparin (a displacing agent) in the release medium, since these interact with the lipid shell structure thereby facilitating decomplexation of L5 and siRNA, as evident from the retarded siRNA release when the L5 content and the L5:siRNA ratio were increased. This confirms the hypothesis that siRNA loaded in LPNs is predominantly present as complexes with the cationic lipid and primarily is located near the particle surface. Cellular uptake and tolerability studies in the human macrophage cell line THP-1 and the type I-like human alveolar epithelial cell line hAELVi, which together represents a monolayer-based barrier model of lung epithelium, indicated that uptake of LPNs was much higher in THP-1 cells in agreement with their primary clearance role. In vivo biodistributions of formulations loaded with Alexa Fluor® 750-labelled siRNA after pulmonary administration in mice were compared by using quantitative fluorescence imaging tomography. The L5-modified LPNs, SNALPs and DOTAP-modified LPNs displayed significantly increased lung retention of siRNA as compared to L5-based lipoplexes, which had a biodistribution profile comparable to that of non-loaded siRNA, for which >50% of the siRNA dose permeated the air-blood barrier within 6 h and subsequently was excreted via the kidneys. Hence, the enhanced lung retention upon pulmonary administration of siRNA-loaded LPNs represents a promising characteristic that can be used to control the delivery of the siRNA cargo to lung tissue for local management of disease.

Tyrosine Kinase Inhibitor Gold Nanoconjugates for the Treatment of Non-Small Cell Lung Cancer.

Gold nanoparticles (AuNPs) have emerged as promising drug delivery candidates that can be leveraged for cancer therapy. Lung cancer (LC) is a heterogeneous disease that imposes a significant burden on society, with an unmet need for new therapies. Chemotherapeutic drugs such as afatinib (Afb), which is clinically approved for the treatment of epidermal growth factor receptor positive LC, is hydrophobic and has low bioavailability leading to spread around the body, causing severe side effects. Herein, we present a novel afatinib-AuNP formulation termed Afb-AuNPs, with the aim of improving drug efficacy and biocompatibility. This was achieved by synthesis of an alkyne-bearing Afb derivative and reaction with azide-functionalized lipoic acid using copper-catalyzed click chemistry, then conjugation to AuNPs via alkylthiol-gold bond formation. The Afb-AuNPs were found to possess up to 3.7-fold increased potency when administered to LC cells in vitro and were capable of significantly inhibiting cancer cell proliferation, as assessed by MTT assay and electric cell-substrate impedance sensing, respectively. Furthermore, when exposed to Afb-AuNPs, human alveolar epithelial type I-like cells, a model of the healthy lung epithelium, maintained viability and were found to release less proinflammatory cytokines when compared to free drug, demonstrating the biocompatibility of our formulation. This study provides a new platform for the development of nontraditional AuNP conjugates which can be applied to other molecules of therapeutic or diagnostic utility, with potential to be combined with photothermal therapy in other cancers.

Oligopeptide Transport in Rat Lung Alveolar Epithelial Cells is Mediated by Pept2.

Studies were conducted in primary cultured rat alveolar epithelial cell monolayers to characterize peptide transporter expression and function. Freshly isolated rat lung alveolar epithelial cells were purified and cultured on permeable support with and without keratinocyte growth factor (KGF). Messenger RNA and protein expression of Pept1 and Pept2 in alveolar epithelial type I- and type II-like cell monolayers (±KGF, resp.) were examined by RT-PCR and Western blotting. 3H-Glycyl-sarcosine (3H-gly-sar) transmonolayer flux and intracellular accumulation were evaluated in both cell types. RT-PCR showed expression of Pept2, but not Pept1, mRNA in both cell types. Western blot analysis revealed presence of Pept2 protein in type II-like cells, and less in type I-like cells. Bi-directional transmonolayer 3H-gly-sar flux lacked asymmetry in transport in both types of cells. Uptake of 3H-gly-sar from apical fluid of type II-like cells was 7-fold greater than that from basolateral fluid, while no significant differences were observed from apical vs. basolateral fluid of type I-like cells. This study confirms the absence of Pept1 from rat lung alveolar epithelium in vitro. Functional Pept2 expression in type II-like cell monolayers suggests its involvement in oligopeptide lung disposition, and offers rationale for therapeutic development of di/tripeptides, peptidomimetics employing pulmonary drug delivery.

Integrated Stress Response Mediates Epithelial Injury in Mechanical Ventilation.

Ventilator-induced lung injury (VILI) is a severe complication of mechanical ventilation that can lead to acute respiratory distress syndrome. VILI is characterized by damage to the epithelial barrier with subsequent pulmonary edema and profound hypoxia. Available lung-protective ventilator strategies offer only a modest benefit in preventing VILI because they cannot impede alveolar overdistension and concomitant epithelial barrier dysfunction in the inflamed lung regions. There are currently no effective biochemical therapies to mitigate injury to the alveolar epithelium. We hypothesize that alveolar stretch activates the integrated stress response (ISR) pathway and that the chemical inhibition of this pathway mitigates alveolar barrier disruption during stretch and mechanical ventilation. Using our established rat primary type I-like alveolar epithelial cell monolayer stretch model and in vivo rat mechanical ventilation that mimics the alveolar overdistension seen in acute respiratory distress syndrome, we studied epithelial responses to mechanical stress. Our studies revealed that the ISR signaling pathway is a key modulator of epithelial permeability. We show that prolonged epithelial stretch and injurious mechanical ventilation activate the ISR, leading to increased alveolar permeability, cell death, and proinflammatory signaling. Chemical inhibition of protein kinase RNA-like endoplasmic reticulum kinase, an upstream regulator of the pathway, resulted in decreased injury signaling and improved barrier function after prolonged cyclic stretch and injurious mechanical ventilation. Our results provide new evidence that therapeutic targeting of the ISR can mitigate VILI.

Effect of cigarette smoke extract on P-glycoprotein function in primary cultured and newly developed alveolar epithelial cells

The effect of cigarette smoke extract (CSE) on P-glycoprotein (P-gp) function in the distal lung is unclear. In this study, we first examined the expression and function of P-gp and the effect of CSE in rat primary cultured alveolar epithelial cells. The expression of P-gp protein was observed in type I-like cells, but not in type II cells. In type I-like cells, rhodamine 123 (Rho123) accumulation was enhanced by various P-gp inhibitors such as verapamil and cyclosporine A. In addition, the expression of P-gp mRNAs, mdr1a and mdr1b, as well as P-gp activity increased along with the transdifferentiation. When type I-like cells were co-incubated with CSE, P-gp activity was suppressed. Next, we attempted to clarify the effect of CSE on P-gp function in human-derived cultured alveolar epithelial cells. For this purpose, we isolated an A549 clone (A549/P-gp) expressing P-gp, because P-gp expression in native A549 cells was negligible. In A549/P-gp cells, P-gp was functionally expressed, and the inhibitory effect of CSE on P-gp was observed. These results suggested that smoking would directly suppress P-gp activity, and that A549/P-gp cell line should be a useful model to further study the effect of xenobiotics on P-gp function in the alveolar epithelial cells.

Pulmonary surfactant mitigates silver nanoparticle toxicity in human alveolar type-I-like epithelial cells

Accompanying increased commercial applications and production of silver nanomaterials is an increased probability of human exposure, with inhalation a key route. Nanomaterials that deposit in the pulmonary alveolar region following inhalation will interact firstly with pulmonary surfactant before they interact with the alveolar epithelium. It is therefore critical to understand the effects of human pulmonary surfactant when evaluating the inhalation toxicity of silver nanoparticles. In this study, we evaluated the toxicity of AgNPs on human alveolar type-I-like epithelial (TT1) cells in the absence and presence of Curosurf® (a natural pulmonary surfactant substitute), hypothesising that the pulmonary surfactant would act to modify toxicity. We demonstrated that 20nm citrate-capped AgNPs induce toxicity in human alveolar type I-like epithelial cells and, in agreement with our hypothesis, that pulmonary surfactant acts to mitigate this toxicity, possibly through reducing AgNP dissolution into cytotoxic Ag+ ions. For example, IL-6 and IL-8 release by TT1 cells significantly increased 10.7- and 35-fold, respectively (P<0.01), 24h after treatment with 25μg/ml AgNPs. In contrast, following pre-incubation of AgNPs with Curosurf®, this effect was almost completely abolished. We further determined that the mechanism of this toxicity is likely associated with Ag+ ion release and lysosomal disruption, but not with increased reactive oxygen species generation. This study provides a critical understanding of the toxicity of AgNPs in target human alveolar type-I-like epithelial cells and the role of pulmonary surfactant in mitigating this toxicity. The observations reported have important implications for the manufacture and application of AgNPs, in particular for applications involving use of aerosolised AgNPs.

Transport Mechanism of Nicotine in Primary Cultured Alveolar Epithelial Cells

Nicotine is absorbed from the lungs into the systemic circulation during cigarette smoking. However, there is little information concerning the transport mechanism of nicotine in alveolar epithelial cells. In this study, we characterized the uptake of nicotine in rat primary cultured type II (TII) and transdifferentiated type I-like (TIL) epithelial cells. In both TIL and TII cells, [3H]nicotine uptake was time and temperature-dependent, and showed saturation kinetics. [3H]Nicotine uptake in these cells was not affected by Na+, but was sensitive to extracellular and intracellular pH, suggesting the involvement of a nicotine/proton antiport system. The uptake of [3H]nicotine in these cells was potently inhibited by organic cations such as clonidine, diphenhydramine, and pyrilamine, but was not affected by substrates and/or inhibitors of known organic cation transporters such as carnitine, 1-methyl-4-phenylpyridinium, and tetraethylammonium. In addition, the uptake of [3H]nicotine in TIL cells was stimulated by preloading the cells with unlabeled nicotine, pyrilamine, and diphenhydramine, but not with tetraethylammonium. These results suggest that a novel proton-coupled antiporter is involved in the uptake of nicotine in alveolar epithelial cells and its absorption from the lungs into the systemic circulation.

Human alveolar epithelial cells expressing tight junctions to model the air-blood barrier

This paper describes a new human alveolar epithelial cell line (hAELVi - human Alveolar Epithelial Lentivirus immortalized) with type I-like characteristics and functional tight junctions, suitable to model the air-blood barrier of the peripheral lung. Primary human alveolar epithelial cells were immortalized by a novel regimen, grown as monolayers on permeable filter supports and characterized morphologically, biochemically and biophysically. hAELVi cells maintain the capacity to form tight intercellular junctions, with high trans-epithelial electrical resistance (> 1000 Ω*cm²). The cells could be kept in culture over several days, up to passage 75, under liquid-liquid as well as air-liquid conditions. Ultrastructural analysis and real time PCR revealed type I-like cell properties, such as the presence of caveolae, expression of caveolin-1, and absence of surfactant protein C. Accounting for the barrier properties, inter-digitations sealed with tight junctions and desmosomes were also observed. Low permeability of the hydrophilic marker sodium fluorescein confirmed the suitability of hAELVi cells for in vitro transport studies across the alveolar epithelium. These results suggest that hAELVi cells reflect the essential features of the air-blood barrier, as needed for an alternative to animal testing to study absorption and toxicity of inhaled drugs, chemicals and nanomaterials.

Organic cation transporter function in different in vitro models of human lung epithelium

Organic cation transporters (OCT) encoded by members of the solute carrier (SLC) 22 family of genes are involved in the disposition of physiological substrates and xenobiotics, including drugs used in the treatment of chronic obstructive lung diseases and asthma. The aim of this work was to identify continuously growing epithelial cell lines that closely mimic the organic cation transport of freshly isolated human alveolar type I-like epithelial cells (ATI) in primary culture, and which consequently, can be utilised as in vitro models for the study of organic cation transport at the air–blood barrier. OCT activity was investigated by measuring [14C]-tetraethylammonium (TEA) uptake into monolayers of Calu-3, NCI-H441 and A549 lung epithelial cell lines in comparison to ATI-like cell monolayers in primary culture. Levels of time-dependent TEA uptake were highest in A549 and ATI-like cells. In A549 cells, TEA uptake had a saturable and a non-saturable component with Km=528.5±373.1μM, Vmax=0.3±0.1nmol/min/mg protein and Kd=0.02μl/min/mg protein. TEA uptake into Calu-3 and NCI-H441 cells did not reach saturation within the concentration range studied. RNAi experiments in A549 cells confirmed that TEA uptake was mainly facilitated by OCT1 and OCT2. Co-incubation studies using pharmacological OCT modulators suggested that organic cation uptake pathways share several similarities between ATI-like primary cells and the NCI-H441 cell line, whereas more pronounced differences exist between primary cells and the A549 and Calu-3 cell lines.

OCTN1‐Mediated Ergothioneine Uptake Protects Lung Epithelial Cells From Tobacco Smoke‐Induced Damage

Smoking is a major contributing factor for chronic obstructive pulmonary disease (COPD). Tobacco smoke exposure causes oxidative stress which results in alveolar wall destruction, mucus hyper secretion, inflammation and defective tissue repair. Ergothioneine (ET), an amino acid transported by OCTN1, has been shown to exhibit antioxidant and cytoprotective capacities in vitro. Hence, the objective of this study is to understand the role of OCTN1 and the effect of ET on human alveolar cells when subjected to tobacco smoke‐induced oxidative stress.Confocal microscopy and Western blot was used to determine the presence of OCTN1 in A549 cells and freshly isolated human alveolar epithelial type II (ATII) and type I‐like (ATI‐like) cells in primary culture. A549 cells were cultured in medium containing 1 mM of ET and its intracellular accumulation was measured by LC‐MS/MS over a 3‐week period. A549 cells were also was exposed to various concentrations of cigarette smoke extract (CSE). Prior to the experiment, cells were either cultured for 1 week in the presence or absence of 1 mM ET. An MTT assay was used to assess cell viability for the difference exposures to CSE.Immunoblot and CLSM showed OCTN1 expression in A549, ATII and ATI‐like cells. The uptake of ET by A549 epithelial cells was saturated after approximately 7 days of culture. Western blot data supported the CLSM results and also indicated that OCTN1 levels were increased in the presence of ET. The MTT assay showed that cells pre‐treated with ET were less affected by the cytotoxic effects of CSE.These preliminary data indicate that OCTN1 might play a role in the pathogenesis of tobacco smoke‐induced COPD by regulating ergothioneine transport.

Expression and function of the epithelial sodium channel δ-subunit in human respiratory epithelial cells in vitro.

Using human airway epithelial cell lines (i.e. NCI-H441 and Calu-3) as well as human alveolar epithelial type I-like (ATI) cells in primary culture, we studied the contribution of the epithelial sodium channel δ-subunit (δ-ENaC) to transepithelial sodium transport in human lung in vitro. Endogenous δ-ENaC protein was present in all three cell types tested; however, protein abundance was low, and no expression was detected in the apical cell membrane of these cells. Similarly, known modulators of δ-ENaC activity, such as capsazepine and icilin (activators) and Evans blue (inhibitor), did not show effects on short-circuit current (I SC), suggesting that δ-ENaC is not involved in the modulation of transcellular sodium absorption in NCI-H441 cell monolayers. Over-expression of δ-ENaC in NCI-H441 cells resulted in detectable protein expression in the apical cell membrane, as well as capsazepine and icilin-stimulated increases in I SC that were effectively blocked by Evans blue and that were consistent with δ-ENaC activation and inhibition, respectively. Consequently, these observations suggest that δ-ENaC expression is low in NCI-H441, Calu-3, and ATI cells and does not contribute to transepithelial sodium absorption.

Expression and function of PEPT2 during transdifferentiation of alveolar epithelial cells

AimsThe purpose of this study was to clarify the expression and function of peptide transporter 2 (PEPT2) in primary cultured alveolar type II epithelial cells and in transdifferentiated type I-like cells. Main methodsReal-time PCR analysis, uptake study of [3H]Gly-Sar, and immunostaining were performed in alveolar epithelial cells. Key findingsThe expression of PEPT2 mRNA in type II cells isolated from rat lungs was highest at day 0, and decreased rapidly during culture of the cells. In accordance with this change, PEPT2 activity estimated as cefadroxil-sensitive [3H]Gly-Sar uptake also decreased along with transdifferentiation. The expression of PEPT2 protein in type II cells was confirmed by immunostaining and Western blot analysis. The uptake of [3H]Gly-Sar in type II cells was time- and pH-dependent. In contrast, minimal time-dependence and no pH-dependence of [3H]Gly-Sar uptake were observed in type I-like cells. The maximal [3H]Gly-Sar uptake was observed at pH6.0, and the uptake decreased at higher pHs in type II cells. The uptake of [3H]Gly-Sar in type II cells was inhibited by cefadroxil in a concentration-dependent manner, the IC50 value being 4.3μM. On the other hand, no significant inhibition by cefadroxil was observed in type I-like cells. In addition, [3H]Gly-Sar uptake in type II cells was saturable, the Km value being 72.0μM. SignificancePEPT2 is functionally expressed in alveolar type II epithelial cells, but the expression decreases along with transdifferentiation, and PEPT2 would be almost completely lost in type I cells.

Human Coronavirus HKU1 Infection of Primary Human Type II Alveolar Epithelial Cells: Cytopathic Effects and Innate Immune Response

Because they are the natural target for respiratory pathogens, primary human respiratory epithelial cells provide the ideal in vitro system for isolation and study of human respiratory viruses, which display a high degree of cell, tissue, and host specificity. Human coronavirus HKU1, first discovered in 2005, has a worldwide prevalence and is associated with both upper and lower respiratory tract disease in both children and adults. Research on HCoV-HKU1 has been difficult because of its inability to be cultured on continuous cell lines and only recently it was isolated from clinical specimens using primary human, ciliated airway epithelial cells. Here we demonstrate that HCoV-HKU1 can infect and be serially propagated in primary human alveolar type II cells at the air-liquid interface. We were not able to infect alveolar type I-like cells or alveolar macrophages. Type II alveolar cells infected with HCoV-HKU1 demonstrated formation of large syncytium. At 72 hours post inoculation, HCoV-HKU1 infection of type II cells induced increased levels of mRNAs encoding IL29,CXCL10, CCL5, and IL-6 with no significant increases in the levels of IFNβ. These studies demonstrate that type II cells are a target cell for HCoV-HKU1 infection in the lower respiratory tract, that type II alveolar cells are immune-competent in response to infection exhibiting a type III interferon and proinflammatory chemokine response, and that cell to cell spread may be a major factor for spread of infection. Furthermore, these studies demonstrate that human alveolar cells can be used to isolate and study novel human respiratory viruses that cause lower respiratory tract disease.

Cyclic Stretch–Induced Oxidative Stress Increases Pulmonary Alveolar Epithelial Permeability

Mechanical ventilation with high tidal volumes has been associated with pulmonary alveolar flooding. Understanding the mechanisms underlying cyclic stretch-induced increases in alveolar epithelial permeability may be important in designing preventive measures for acute lung injury. In this work, we assessed whether cyclic stretch leads to the generation of reactive oxygen species in type I-like alveolar epithelial cells, which increase monolayer permeability via activation of NF-κB and extracellular signal-regulated kinase (ERK). We cyclically stretched type I-like rat primary alveolar epithelial cells at magnitudes of 12, 25, and 37% change in surface area (ΔSA) for 10 to 120 minutes. High levels of reactive oxygen species and of superoxide and NO specifically were detected in cells stretched at 37% ΔSA for 10 to 120 minutes. Exogenous superoxide and NO stimulation increased epithelial permeability in unstretched cells, which was preventable by the NF-κB inhibitor MG132. The cyclic stretch-induced increase in permeability was decreased by the superoxide scavenger tiron and by MG132. Furthermore, tiron had a dramatic protective effect on in vivo lung permeability under mechanical ventilation conditions. Cyclic stretch increased the activation of the NF-κB signaling pathway, which was significantly decreased with the ERK inhibitor U0126. Altogether, our in vitro and in vivo data demonstrate the sensitivity of permeability to stretch- and ventilation-induced superoxide production, suggesting that using antioxidants may be helpful in the prevention and treatment of ventilator-induced lung injury.