Calu-3 Human Airway Epithelial Cells Research Articles

IL-13 protects epithelial cells from SARS-CoV-2 infection by inhibiting early ACE2-mediated events

Abstract Severe Acute Respiratory Syndrome coronavirus-2 (SARS-CoV-2) infection is mediated by binding of viral spike (S) protein to membrane ACE2 on host epithelial cells, preferentially in the airway. Asthmatics with type 2 (T2) inflammation are reportedly less susceptible to disease, but the underlying mechanisms are unclear. We recently showed that ACE2expression is decreased in airway epithelial cells from T2 asthmatics and is consistently suppressed in human primary epithelial cells cultured with IL-13 (PMID: 32422146). To dissect IL-13/SARS-CoV-2 interactions, we assessed the effects of IL-13 on discrete events in SARS-CoV-2 infection. S1 protein binding to green monkey kidney Vero E6, human lung Calu-3 epithelial cells and HEK293T cells stably transfected with human ACE2(hACE2-HEK293T) was measured by flow cytometry with His-tagged S1 protein. S protein-mediated viral entry into Vero E6 and hACE2-HEK293T cells was assessed using S protein-pseudotyped lentiviral particles carrying a GFP reporter. ACE2transcription in Vero E6, Calu-3 and primary human airway epithelial cells was measured by RT-qPCR. ACE2 expression on Vero E6, Calu-3 and hACE2-HTK293T cells was evaluated by flow cytometry. USA-WA1/2020 SARS-CoV-2 was used to infect Calu-3 cells. IL-13 strongly inhibited S protein binding, cell entry, and infection of epithelial cells by SARS-CoV-2. These effects resulted at least in part from IL-13-dependent inhibition of ACE2expression because IL-13 did not affect S protein binding to, and S protein-pseudotyped lentivirus entry into, hACE2-HEK293T cells that do not downregulate ACE2 in response to IL-13. We suggest that IL-13 protects epithelial cells from SARS-CoV-2 infection by inhibiting early ACE2-mediated events. Supported by grant from NIAID: U19 AI125357

Construction of Human Three-Dimensional Lung Model Using Layer-by-Layer Method.

The respiratory tract is one of the frontline barriers for biological defense. Lung epithelial intercellular adhesions provide protection from bacterial and viral infections and prevent invasion into deep tissues by pathogens. Dysfunction of lung epithelial intercellular adhesion caused by pathogens is associated with development of several diseases, such as acute respiratory distress syndrome, pneumonia, and asthma. To elucidate the pathological mechanism of respiratory infections, two-dimensional cell cultures and animal models are commonly used, although are not useful for evaluating host specificity or human biological response. With the rapid progression and worldwide spread of severe acute respiratory syndrome coronavirus-2, there is increasing interest in the development of a three-dimensional (3D) in vitro lung model for analyzing interactions between pathogens and hosts. However, some models possess unclear epithelial polarity or insufficient barrier functions and need the use of complex technologies, have high cost, and long cultivation terms. We previously reported about the fabrication of 3D cellular multilayers using a layer-by-layer (LbL) cell coating technique with extracellular matrix protein, fibronectin (FN), and gelatin (G). In the present study, such a LbL cell coating technique was utilized to construct a human 3D lung model in which a monolayer of the human lower airway epithelial adenocarcinoma cell line Calu-3 cells was placed on 3D-cellular multilayers composed of FN-G-coated human primary pulmonary fibroblast cells. The 3D lung model thus constructed demonstrated an epithelial-fibroblast layer that maintained uniform thickness until 7 days of incubation. Moreover, expressions of E-cadherin, ZO-1, and mucin in the epithelial layer were observed by immunohistochemical staining. Epithelial barrier integrity was evaluated using transepithelial electrical resistance values. The results indicate that the present constructed human 3D lung model is similar to human lung tissues and also features epithelial polarity and a barrier function, thus is considered useful for evaluating infection and pathological mechanisms related to pneumonia and several pathogens. Impact statement A novel in vitro model of lung tissue was established. Using a layer-by-layer cell coating technique, a three-dimensional cultured lung model was constructed. The present novel model was shown to have epithelial polarity and chemical barrier functions. This model may be useful for investigating interaction pathogens and human biology.

Airway Delivery of Hydrogel-Encapsulated Niclosamide for the Treatment of Inflammatory Airway Disease.

Repurposing of the anthelminthic drug niclosamide was proposed as an effective treatment for inflammatory airway diseases such as asthma, cystic fibrosis, and chronic obstructive pulmonary disease. Niclosamide may also be effective for the treatment of viral respiratory infections, such as SARS-CoV-2, respiratory syncytial virus, and influenza. While systemic application of niclosamide may lead to unwanted side effects, local administration via aerosol may circumvent these problems, particularly when the drug is encapsulated into small polyethylene glycol (PEG) hydrospheres. In the present study, we examined whether PEG-encapsulated niclosamide inhibits the production of mucus and affects the pro-inflammatory mediator CLCA1 in mouse airways in vivo, while effects on mucociliary clearance were assessed in excised mouse tracheas. The potential of encapsulated niclosamide to inhibit TMEM16A whole-cell Cl− currents and intracellular Ca2+ signalling was assessed in airway epithelial cells in vitro. We achieved encapsulation of niclosamide in PEG-microspheres and PEG-nanospheres (Niclo-spheres). When applied to asthmatic mice via intratracheal instillation, Niclo-spheres strongly attenuated overproduction of mucus, inhibited secretion of the major proinflammatory mediator CLCA1, and improved mucociliary clearance in tracheas ex vivo. These effects were comparable for niclosamide encapsulated in PEG-nanospheres and PEG-microspheres. Niclo-spheres inhibited the Ca2+ activated Cl− channel TMEM16A and attenuated mucus production in CFBE and Calu-3 human airway epithelial cells. Both inhibitory effects were explained by a pronounced inhibition of intracellular Ca2+ signals. The data indicate that poorly dissolvable compounds such as niclosamide can be encapsulated in PEG-microspheres/nanospheres and deposited locally on the airway epithelium as encapsulated drugs, which may be advantageous over systemic application.

PCO-015 - Human airway epithelial Calu-3 cells as the potential platform to study the pathophysiology of SARS-CoV-2 isolated in Malaysia

PCO-015 - Human airway epithelial Calu-3 cells as the potential platform to study the pathophysiology of SARS-CoV-2 isolated in Malaysia

Treatment of human airway epithelial Calu-3cells with a peptide-nucleic acid (PNA) targeting the microRNA miR-101-3p is associated with increased expression of the cystic fibrosis Transmembrane Conductance Regulator () gene.

Since the identification of microRNAs (miRNAs) involved in the regulation of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene, miRNAs known to down-regulate the expression of the CFTR and associated proteins have been investigated as potential therapeutic targets. Here we show that miR-101-3p, targeting the 3'-UTR sequence of the CFTR mRNA, can be selectively inhibited by a peptide nucleic acid (PNA) carrying a full complementary sequence. With respect to clinical relevance of microRNA targeting, it is expected that reduction in concentration of miRNAs (the anti-miRNA approach) could be associated with increasing amounts of target mRNAs. Consistently to this hypothesis, we report that PNA-mediated inhibition of miR-101-3p was accompanied by CFTR up-regulation. Next Generation Sequencing (NGS) was performed in order to verify the effects of the anti-miR-101-3p PNA on the Calu-3 miRNome. Upon inhibition of miR-101-3p we observed a fold change (FC) expression <2 of the majority of miRNAs (403/479, 84.13%), whereas we identified a list of dysregulated miRNAs, suggesting that specific miRNA inhibition (in our case miR-101-3p) might be accompanied by alteration of expression of other miRNAs, some of them known to be involved in Cystic Fibrosis (CF), such as miR-155-5p and miR-125b-5p.

TMPRSS2 and furin are both essential for proteolytic activation of SARS-CoV-2 in human airway cells.

The novel emerged SARS-CoV-2 has rapidly spread around the world causing acute infection of the respiratory tract (COVID-19) that can result in severe disease and lethality. For SARS-CoV-2 to enter cells, its surface glycoprotein spike (S) must be cleaved at two different sites by host cell proteases, which therefore represent potential drug targets. In the present study, we show that S can be cleaved by the proprotein convertase furin at the S1/S2 site and the transmembrane serine protease 2 (TMPRSS2) at the S2' site. We demonstrate that TMPRSS2 is essential for activation of SARS-CoV-2 S in Calu-3 human airway epithelial cells through antisense-mediated knockdown of TMPRSS2 expression. Furthermore, SARS-CoV-2 replication was also strongly inhibited by the synthetic furin inhibitor MI-1851 in human airway cells. In contrast, inhibition of endosomal cathepsins by E64d did not affect virus replication. Combining various TMPRSS2 inhibitors with furin inhibitor MI-1851 produced more potent antiviral activity against SARS-CoV-2 than an equimolar amount of any single serine protease inhibitor. Therefore, this approach has considerable therapeutic potential for treatment of COVID-19.

Comparative Transcriptomic Analysis of Rhinovirus and Influenza Virus Infection.

Rhinovirus (RV) and influenza virus are the most frequently detected respiratory viruses among adult patients with community acquired pneumonia. Previous clinical studies have identified major differences in the clinical presentations and inflammatory or immune response during these infections. A systematic transcriptomic analysis directly comparing influenza and RV is lacking. Here, we sought to compare the transcriptomic response to these viral infections. Human airway epithelial Calu-3 cells were infected with contemporary clinical isolates of RV, influenza A virus (IAV), or influenza B virus (IBV). Host gene expression was determined using RNA-seq. Differentially expressed genes (DEGs) with respect to mock-infected cells were identified using the overlapping gene-set of four different statistical models. Transcriptomic analysis showed that RV-infected cells have a more blunted host response with fewer DEGs than IAV or IBV-infected cells. IFNL1 and CXCL10 were among the most upregulated DEGs during RV, IAV, and IBV infection. Other DEGs that were highly expressed for all 3 viruses were mainly genes related to type I or type III interferons (RSAD2, IDO1) and chemokines (CXCL11). Notably, ICAM5, a known receptor for enterovirus D68, was highly expressed during RV infection only. Gene Set Enrichment Analysis (GSEA) confirmed that pathways associated with interferon response, innate immunity, or regulation of inflammatory response, were most perturbed for all three viruses. Network analysis showed that steroid-related pathways were enriched. Taken together, our data using contemporary virus strains suggests that genes related to interferon and chemokine predominated the host response associated with RV, IAV, and IBV infection. Several highly expressed genes, especially ICAM5 which is preferentially-induced during RV infection, deserve further investigation.

Luteolin reduces fluid hypersecretion by inhibiting TMEM16A in interleukin-4 treated Calu-3 airway epithelial cells.

Rhinorrhea in allergic rhinitis (AR) is characterized by the secretion of electrolytes in the nasal discharge. The secretion of Cl– and HCO3– is mainly regulated by cystic fibrosis transmembrane conductance regulator (CFTR) or via the calcium-activated Cl– channel anoctamin-1 (ANO1) in nasal gland serous cells. Interleukin-4 (IL-4), which is crucial in the development of allergic inflammation, increases the expression and activity of ANO1 by stimulating histamine receptors. In this study, we investigated ANO1 as a potential therapeutic target for rhinorrhea in AR using an ANO1 inhibitor derived from a natural herb. Ethanolic extracts (30%) of Spirodela polyrhiza (SPEtOH) and its five major flavonoids constituents were prepared. To elucidate whether the activity of human ANO1 (hANO1) was modulated by SPEtOH and its chemical constituents, a patch clamp experiment was performed in hANO1-HEK293T cells. Luteolin, one of the major chemical constituents in SPEtOH, significantly inhibited hANO1 activity in hANO1-HEK293T cells. Further, SPEtOH and luteolin specifically inhibited the calcium-activated chloride current, but not CFTR current in human airway epithelial Calu-3 cells. Calu-3 cells were cultured to confluency on transwell inserts in the presence of IL-4 to measure the electrolyte transport by Ussing chamber. Luteolin also significantly inhibited the ATP-induced increase in electrolyte transport, which was increased in IL-4 sensitized Calu-3 cells. Our findings indicate that SPEtOH and luteolin may be suitable candidates for the prevention and treatment of allergic rhinitis. SPEtOH- and luteolin-mediated ANO1 regulation provides a basis for the development of novel approaches for the treatment of allergic rhinitis-induced rhinorrhea.

In Vitro and In Vivo Assessment of PEGylated PEI for Anti-IL-8/CxCL-1 siRNA Delivery to the Lungs

Inhalation offers a means of rapid, local delivery of siRNA to treat a range of autoimmune or inflammatory respiratory conditions. This work investigated the potential of a linear 10 kDa Poly(ethylene glycol) (PEG)-modified 25 kDa branched polyethyleneimine (PEI) (PEI-LPEG) to effectively deliver siRNA to airway epithelial cells. Following optimization with anti- glyceraldehyde 3-phosphate dehydrogenase (GAPDH) siRNA, PEI and PEI-LPEG anti-IL8 siRNA nanoparticles were assessed for efficacy using polarised Calu-3 human airway epithelial cells and a twin stage impinger (TSI) in vitro lung model. Studies were then advanced to an in vivo lipopolysaccharide (LPS)-stimulated rodent model of inflammation. In parallel, the suitability of the siRNA-loaded nanoparticles for nebulization using a vibrating mesh nebuliser was assessed. The siRNA nanoparticles were nebulised using an Aerogen® Pro vibrating mesh nebuliser and characterised for aerosol output, droplet size and fine particle fraction. Only PEI anti-IL8 siRNA nanoparticles were capable of significant levels of IL-8 knockdown in vitro in non-nebulised samples. However, on nebulization through a TSI, only PEI-PEG siRNA nanoparticles demonstrated significant decreases in gene and protein expression in polarised Calu-3 cells. In vivo, both anti-CXCL-1 (rat IL-8 homologue) nanoparticles demonstrated a decreased CXCL-1 gene expression in lung tissue, but this was non-significant. However, PEI anti-CXCL-1 siRNA-treated rats were found to have significantly less infiltrating macrophages in their bronchoalveolar lavage (BAL) fluid. Overall, the in vivo gene and protein inhibition findings indicated a result more reminiscent of the in vitro bolus delivery rather than the in vitro nebulization data. This work demonstrates the potential of nebulised PEI-PEG siRNA nanoparticles in modulating pulmonary inflammation and highlights the need to move towards more relevant in vitro and in vivo models for respiratory drug development.

Characterization of biofilm formation and induction of apoptotic DNA fragmentation by nontypeable Haemophilus influenzae on polarized human airway epithelial cells

Nontypeable Haemophilus influenzae (NTHi) is a common airway commensal and opportunistic pathogen that persists within biofilm communities in vivo. Biofilm studies so far are mainly based on assays on plastic surfaces. The aim of this work was to investigate the capacity of clinical NTHi strains to form biofilm structures on polarized Calu-3 human airway epithelial cells and primary normal human bronchial epithelial cells and to characterize the biofilm architecture. Formation of adherent NTHi biofilms post colonization of host cells at multiple time-points was evaluated using confocal laser scanning microscopy and electron microscopy. NTHi biofilms were analyzed in terms of biofilm height and presence of extracellular matrix components, and their apoptotic effects on epithelial cells were measured by TUNEL assay. Strain Fi176 was observed to form robust biofilms on airway epithelia over time, while disrupting the integrity of Calu-3 monolayer by 72 h of co-culture. NTHi biofilms were observed to induce apoptotic DNA fragmentation in host cells at 24 h post infection. Biofilm formation on cell monolayers by Fi176ΔpilA strain was markedly reduced compared to WT strain. Biofilm inhibition and disruption assays by crystal violet staining indicated that DNA and proteins are part of NTHi biofilms in vitro. Our findings highlight critical stages of NTHi pathogenesis following host colonization and provide useful biofilm models for future antimicrobial drug discovery investigations.

MUC5AC overexpression by organic extract of SRM 1648 urban particulate matter via ERK, JNK and p38 MAPK activation in Calu-3 human airway epithelial cells

MUC5AC overexpression by organic extract of SRM 1648 urban particulate matter via ERK, JNK and p38 MAPK activation in Calu-3 human airway epithelial cells

Multifactorial inhibition of lactobacilli against the respiratory tract pathogen Moraxella catarrhalis

Probiotics, mainly lactic acid bacteria (LAB), are widely focused on gastrointestinal applications. However, recent microbiome studies indicate that LAB can be endogenous members of other human body sites such as the upper respiratory tract (URT). Interestingly, DNA-based microbiome research suggests an inverse correlation between the presence of LAB and the occurrence of potential pathogens, such as Moraxella catarrhalis, an important URT pathogen linked to otitis media, sinusitis and chronic obstructive pulmonary disease. However, a direct interaction between these microbes has not been explored in detail. This study investigated the direct antipathogenic effects of Lactobacillus species, including several well-documented probiotic strains, on M. catarrhalis using agar-based assays, time course analysis, biofilm assays and minimal inhibitory concentration (MIC) testing. These assays were performed using spent culture supernatans (SCS) at two pHs (4.3 and 7) and D- and/or L-lactic acid at three pHs (2, 4 and 7). In addition, cell line assays for adhesion competition and immunomodulation were used to substantiate the inhibitory effect of lactobacilli against M. catarrhalis. A proportion of Lactobacillus strains, including the model probiotic Lactobacillus rhamnosus GG, showed a strong and direct activity against M. catarrhalis. Screening of the activity of the SCS after different treatments demonstrated that lactic acid has an important antimicrobial activity against this pathogen - at least in vitro - with mean MIC values for D- and L-lactic acid varying between 0.5 and 27 g/l depending on the pH. Furthermore, L. rhamnosus GG also decreased the adhesion of M. catarrhalis to human airway epithelial Calu-3 cells with more than 50%, and the expression of mucin MUC5AC, pro-inflammatory cytokines interleukin (IL)-8, IL-1β, and tumor necrosis factor-α at least 1.2 fold. This study suggests that several lactobacilli and their key metabolite lactic acid are possible candidates for probiotic therapeutic interventions against URT infections.

Identifying contact-mediated, localized toxic effects of MWCNT aggregates on epithelial monolayers: a single-cell monitoring toxicity assay

Aggregates of multiwalled carbon nanotubes (MWCNT) impair the barrier properties of human airway cell monolayers. To resolve the mechanism of the barrier alteration, monolayers of Calu-3 human airway epithelial cells were exposed to aggregated MWCNT. At the cell-population level, trans-epithelial electrical resistance (TEER) was used as an indicator of barrier competence, caspase activity was assessed with standard biochemical assays, and cell viability was investigated by biochemical techniques and high-throughput screening (HTS) technique based on automated epifluorescence microscopy. At cell level, the response to MWCNT was investigated with confocal microscopy, by evaluating cell death (calcein/propidium iodide (PI)), proliferation (Ki-67), and apoptosis (caspase activity). At the cell-population level, exposure to aggregated MWCNT caused a decrease in TEER, which was not associated with a decrease in cell viability or onset of apoptosis even after an 8-d exposure. In contrast, confocal imaging demonstrated contact with MWCNT aggregates triggered cell death after 24 h of exposure. In the presence of a natural surfactant, both TEER decrease and contact-mediated toxicity were mitigated. With confocal imaging, increased proliferation and apoptosis were detected in Calu-3 cells next to the aggregates. Contact-mediated cytotoxicity was recorded in two additional cell lines (BEAS-2B and A549) derived from human airways. Similar results were confirmed by adopting two additional MWCNT preparations with different physico-chemical features. This indicates MWCNT caused localized damage to airway epithelial monolayers in vitro and altered the apoptotic and proliferative rate of epithelial cells in close proximity to the aggregates. These findings provide evidence on the pathway by which MWCNT aggregates impair airway barrier function, and support the use of imaging techniques as a possible regulatory-decision supporting tool to identify effects of aggregated nanomaterials not readily detected at cell population level.

Cystic Fibrosis Transmembrane Conductance Regulator Is Expressed in Mucin Granules from Calu-3 and Primary Human Airway Epithelial Cells

Cystic fibrosis (CF) is caused by mutations in the tightly regulated anion channel cystic fibrosis transmembrane conductance regulator (CFTR), yet much of the pathology in this disease results from mucus obstruction of the small airways and other organs. Mucus stasis has been attributed to the abnormal luminal environment of CF airways, which results from dehydration of the mucus gel or low bicarbonate concentration. We show here that CFTR and MUC5AC are present in single mucin-containing granules isolated from a human airway epithelial cell line and from highly differentiated airway primary cell cultures. CFTR was not detected in MUC5AC granules from CFTR knockdown cells or CF primary cells. The results suggest a direct link between CFTR and the mucus defect.

Establishing a liquid-covered culture of polarized human airway epithelial Calu-3 cells to study host cell response to respiratory pathogens in vitro.

The apical and basolateral surfaces of airway epithelial cells demonstrate directional responses to pathogen exposure in vivo. Thus, ideal in vitro models for examining cellular responses to respiratory pathogens polarize, forming apical and basolateral surfaces. One such model is differentiated normal human bronchial epithelial cells (NHBE). However, this system requires lung tissue samples, expertise isolating and culturing epithelial cells from tissue, and time to generate an air-liquid interface culture. Calu-3 cells, derived from a human bronchial adenocarcinoma, are an alternative model for examining the response of proximal airway epithelial cells to respiratory insult, pharmacological compounds, and bacterial and viral pathogens, including influenza virus, rhinovirus and severe acute respiratory syndrome-associated coronavirus. Recently, we demonstrated that Calu-3 cells are susceptible to respiratory syncytial virus (RSV) infection in a manner consistent with NHBE. Here, we detail the establishment of a polarized, liquid-covered culture (LCC) of Calu-3 cells, focusing on the technical details of growing and culturing Calu-3 cells, maintaining cells that have been cultured into LCC, and we present the method for performing respiratory virus infection of polarized Calu-3 cells. To consistently obtain polarized Calu-3 LCC, Calu-3 cells must be carefully subcultured before culturing in Transwell inserts. Calu-3 monolayer cultures should remain below 90% confluence, should be subcultured fewer than 10 times from frozen stock, and should regularly be supplied with fresh medium. Once cultured in Transwells, Calu-3 LCC must be handled with care. Irregular media changes and mechanical or physical disruption of the cell layers or plates negatively impact polarization for several hours or days. Polarization is monitored by evaluating trans-epithelial electrical resistance (TEER) and is verified by evaluating the passive equilibration of sodium fluorescein between the apical and basolateral compartments . Once TEER plateaus at or above 1,000 Ω×cm(2), Calu-3 LCC are ready to use to examine cellular responses to respiratory pathogens.

FKBP38 Peptidylprolyl Isomerase Promotes the Folding of Cystic Fibrosis Transmembrane Conductance Regulator in the Endoplasmic Reticulum

FK506-binding protein 38 (FKBP38), a membrane-anchored, tetratricopeptide repeat (TPR)-containing immunophilin, associates with nascent plasma membrane ion channels in the endoplasmic reticulum (ER). It promotes the maturation of the human ether-à-go-go-related gene (HERG) potassium channel and maintains the steady state level of the cystic fibrosis transmembrane conductance regulator (CFTR), but the underlying mechanisms remain unclear. Using a combination of steady state and pulse-chase analyses, we show that FKBP38 knockdown increases protein synthesis but inhibits the post-translational folding of CFTR, leading to reduced steady state levels of CFTR in the ER, decreased processing, and impaired cell surface functional expression in Calu-3 human airway epithelial cells. The membrane anchorage of FKBP38 is necessary for the inhibition of protein synthesis but not for CFTR post-translational folding. In contrast, the peptidylprolyl cis/trans isomerase active site is utilized to promote CFTR post-translational folding but is not important for regulation of protein synthesis. Uncoupling FKBP38 from Hsp90 by substituting a conserved lysine in the TPR domain modestly enhances CFTR maturation and further reduces its synthesis. Removing the N-terminal glutamate-rich domain (ERD) slightly enhances CFTR synthesis but reduces its maturation, suggesting that the ERD contributes to FKBP38 biological activities. Our data support a dual role for FKBP38 in regulating CFTR synthesis and post-translational folding. In contrast to earlier prediction but consistent with in vitro enzymological studies, FKBP38 peptidylprolyl cis/trans isomerase plays an important role in membrane protein biogenesis on the cytoplasmic side of the ER membrane, whose activity is negatively regulated by Hsp90 through the TPR domain.

Quantification of intracellular and extracellular prostanoids stimulated by A23187 by liquid chromatography/electrospray ionization tandem mass spectrometry

Prostanoids are bioactive substances that contribute to various biological and pathological processes. To evaluate both extracellular and intracellular levels of prostanoids at the same time, we developed methods for quantification of extracellular and intracellular levels of prostanoids, including prostaglandin E 2 (PGE 2), PGD 2, PGF 2α, 6-keto PGF 1α, and TXB 2, in cultured cells using liquid chromatography/tandem mass spectrometry (LC/MS/MS), and we validated the LC/MS/MS methods. A solid-phase extraction cartridge was used for extraction of prostanoids. The prostanoids were separated by a C 18 column with an isocratic flow of acetonitrile/water/acetic acid (40:60:0.1, v/v/v). Calibration curves of extracellular measurement for the prostanoids were linear in the range from 0.1 to 100 ng/mL ( r 2 > 0.999), and those of intracellular measurement were linear in the range from 0.05 to 50 ng ( r 2 > 0.999). Validation assessment showed that both methods of extracellular and intracellular measurements were highly reliable with good accuracy and precision. We also applied the methods to human airway epithelial Calu-3 cells and human lung adenocarcinoma epithelial A549 cells.

Capsaicinoids Regulate Airway Anion Transporters through Rho Kinase– and Cyclic AMP–Dependent Mechanisms

To investigate the effects of capsaicinoids on airway anion transporters, we recorded and analyzed transepithelial currents in human airway epithelial Calu-3 cells. Application of capsaicin (100 μM) attenuated vectorial anion transport, estimated as short-circuit currents (I(SC)), before and after stimulation by forskolin (10 μM) with concomitant reduction of cytosolic cyclic AMP (cAMP) levels. The capsaicin-induced inhibition of I(SC) was also observed in the response to 8-bromo-cAMP (1 mM, a cell-permeable cAMP analog) and 3-isobutyl-1-methylxanthine (1 mM, an inhibitor of phosphodiesterases). The capsaicin-induced inhibition of I(SC) was attributed to suppression of bumetanide (an inhibitor of the basolateral Na(+)-K(+)-2 Cl(-) cotransporter 1)- and 4,4'-dinitrostilbene-2,2'-disulfonic acid (an inhibitor of basolateral HCO(3)(-)-dependent anion transporters)-sensitive components, which reflect anion uptake via basolateral cAMP-dependent anion transporters. In contrast, capsaicin potentiated apical Cl(-) conductance, which reflects conductivity through the cystic fibrosis transmembrane conductance regulator, a cAMP-regulated Cl(-) channel. All these paradoxical effects of capsaicin were mimicked by capsazepine. Forskolin application also increased phosphorylated myosin phosphatase target subunit 1, and the phosphorylation was prevented by capsaicin and capsazepine, suggesting that these capsaicinoids assume aspects of Rho kinase inhibitors. We also found that the increments in apical Cl(-) conductance were caused by conventional Rho kinase inhibitors, Y-27632 (20 μM) and HA-1077 (20 μM), with selective inhibition of basolateral Na(+)-K(+)-2 Cl(-) cotransporter 1. Collectively, capsaicinoids inhibit cAMP-mediated anion transport through down-regulation of basolateral anion uptake, paradoxically accompanied by up-regulation of apical cystic fibrosis transmembrane conductance regulator-mediated anion conductance. The latter is mediated by inhibition of Rho-kinase, which is believed to interact with actin cytoskeleton.

Lymphocytes Accelerate Epithelial Tight Junction Assembly: Role of AMP-Activated Protein Kinase (AMPK)

The tight junctions (TJs), characteristically located at the apicolateral borders of adjacent epithelial cells, are required for the proper formation of epithelial cell polarity as well as for sustaining the mucosal barrier to the external environment. The observation that lymphocytes are recruited by epithelial cells to the sites of infection [1] suggests that they may play a role in the modulation of epithelial barrier function and thus contribute to host defense. To test the ability of lymphocytes to modulate tight junction assembly in epithelial cells, we set up a lymphocyte-epithelial cell co-culture system, in which Madin-Darby canine kidney (MDCK) cells, a well-established model cell line for studying epithelial TJ assembly [2], were co-cultured with mouse lymphocytes to mimic an infection state. In a typical calcium switch experiment, the TJ assembly in co-culture was found to be accelerated compared to that in MDCK cells alone. This accelaration was found to be mediated by AMP-activated protein kinase (AMPK). AMPK activation was independent of changes in cellular ATP levels but it was found to be activated by the pro-inflammatory cytokine TNF-α. Forced suppression of AMPK, either with a chemical inhibitor or by knockdown, abrogated the accelerating effect of lymphocytes on TJ formation. Similar results were also observed in a co-culture with lymphocytes and Calu-3 human airway epithelial cells, suggesting that the activation of AMPK may be a general mechanism underlying lymphocyte-accelerated TJ assembly in different epithelia. These results suggest that signals from lymphocytes, such as cytokines, facilitate TJ assembly in epithelial cells via the activation of AMPK.

Mechanosensitive gating of CFTR

Cystic fibrosis transmembrane conductance regulator (CFTR) is an anion and intracellular ligand-gated channel associated with cystic fibrosis, a lethal genetic disorder common among Caucasians. Here we show that CFTR is robustly activated by membrane stretch induced by negative pressures as small as 5 mmHg at the single-channel, cellular and tissue levels. Stretch increased the product of the number of channels present and probability of being open (NPo), and also increased the unitary conductance of CFTR in cell-attached membrane patches. CFTR stretch-mediated activation appears to be an intrinsic property independent of cytosolic factors and kinase signalling. CFTR stretch-mediated activation resulted in chloride transport in Calu-3 human airway epithelial cells and mouse intestinal tissues. Our study has revealed an unexpected function of CFTR in mechanosensing, in addition to its roles as a ligand-gated anion channel and a regulator of other membrane transporters, demonstrating for the first time a mechanosensitive anion channel with a clearly defined molecular identity. Given that CFTR is often found in mechanically dynamic environments, its mechanosensitivity has important physiological implications in epithelial ion transport and cell volume regulation in vivo.