Human Bronchial Airway Epithelial Cells Research Articles

Comparison of a novel potentiator of CFTR channel activity to ivacaftor in ameliorating mucostasis caused by cigarette smoke in primary human bronchial airway epithelial cells

BackgroundCystic Fibrosis causing mutations in the gene CFTR, reduce the activity of the CFTR channel protein, and leads to mucus aggregation, airway obstruction and poor lung function. A role for CFTR in the pathogenesis of other muco-obstructive airway diseases such as Chronic Obstructive Pulmonary Disease (COPD) has been well established. The CFTR modulatory compound, Ivacaftor (VX-770), potentiates channel activity of CFTR and certain CF-causing mutations and has been shown to ameliorate mucus obstruction and improve lung function in people harbouring these CF-causing mutations. A pilot trial of Ivacaftor supported its potential efficacy for the treatment of mucus obstruction in COPD. These findings prompted the search for CFTR potentiators that are more effective in ameliorating cigarette-smoke (CS) induced mucostasis.MethodsSmall molecule potentiators, previously identified in CFTR binding studies, were tested for activity in augmenting CFTR channel activity using patch clamp electrophysiology in HEK-293 cells, a fluorescence-based assay of membrane potential in Calu-3 cells and in Ussing chamber studies of primary bronchial epithelial cultures. Addition of cigarette smoke extract (CSE) to the solutions bathing the apical surface of Calu-3 cells and primary bronchial airway cultures was used to model COPD. Confocal studies of the velocity of fluorescent microsphere movement on the apical surface of CSE exposed airway epithelial cultures, were used to assess the effect of potentiators on CFTR-mediated mucociliary movement.ResultsWe showed that SK-POT1, like VX-770, was effective in augmenting the cyclic AMP-dependent channel activity of CFTR. SK-POT-1 enhanced CFTR channel activity in airway epithelial cells previously exposed to CSE and ameliorated mucostasis on the surface of primary airway cultures.ConclusionTogether, this evidence supports the further development of SK-POT1 as an intervention in the treatment of COPD.

FBXL19 Targeted STK11 Degradation Enhances Cigarette Smoke-Induced Airway Epithelial Cell Cytotoxicity

Objective: To investigate the effects of cigarette smoke (CS) on Serine/Threonine Kinase 11 (STK11) and to determine STK11’s role in CS-induced airway epithelial cell cytotoxicity.Methods: STK11 expression levels in the lung tissues of smokers with or without COPD and mice exposed to CS or room air (RA) were determined by immunoblotting and RT-PCR. BEAS-2Bs–human bronchial airway epithelial cells were exposed to CS extract (CSE), and the changes in STK11 expression levels were determined by immunoblotting and RT-PCR. BEAS-2B cells were transfected with STK11-specific siRNA or STK11 expression plasmid, and the effects of CSE on airway epithelial cell cytotoxicity were measured. To determine the specific STK11 degradation-proteolytic pathway, BEAS-2Bs were treated with cycloheximide alone or combined with MG132 or leupeptin. Finally, to identify the F-box protein mediating the STK11 degradation, a screening assay was performed using transfection with a panel of FBXL E3 ligase subunits.Results: STK11 protein levels were significantly decreased in the lung tissues of smokers with COPD relative to smokers without COPD. STK11 protein levels were also significantly decreased in mouse lung tissues exposed to CS compared to RA. Exposure to CSE shortened the STK11 mRNA and protein half-life to 4 h in BEAS-2B cells. STK11 protein overexpression attenuated the CSE-induced cytotoxicity; in contrast, its knockdown augmented CSE-induced cytotoxicity. FBXL19 mediates CSE-induced STK11 protein degradation via the ubiquitin-proteasome pathway in cultured BEAS-2B cells. FBXL19 overexpression led to accelerated STK11 ubiquitination and degradation in a dose-dependent manner.Conclusions: Our results suggest that CSE enhances the degradation of STK11 protein in airway epithelial cells via the FBXL19-mediated ubiquitin-proteasomal pathway, leading to augmented cell death. HIGHLIGHTS Lung tissues of COPD-smokers exhibited a decreased STK11 RNA and protein expression. STK11 overexpression attenuates CS-induced airway epithelial cell cytotoxicity. STK11 depletion augments CS-induced airway epithelial cell cytotoxicity. CS diminishes STK11 via FBXL19-mediated ubiquitin-proteasome degradation.

Extracellular Nucleotides and Histamine Suppress TLR3- and RIG-I-Mediated Release of Antiviral IFNs from Human Airway Epithelial Cells.

Respiratory viruses stimulate the release of antiviral IFNs from the airway epithelium. Previous studies have shown that asthmatic patients show diminished release of type I and type III IFNs from bronchial epithelia. However, the mechanism of this suppression is not understood. In this study, we report that extracellular nucleotides and histamine, which are elevated in asthmatic airways, strongly inhibit release of type I and type III IFNs from human bronchial airway epithelial cells (AECs). Specifically, ATP, UTP, and histamine all inhibited the release of type I and type III IFNs from AECs induced by activation of TLR3, retinoic acid-inducible gene I (RIG-I), or cyclic GMP-AMP synthase-STING. This inhibition was at least partly mediated by Gq signaling through purinergic P2Y2 and H1 receptors, but it did not involve store-operated calcium entry. Pharmacological blockade of protein kinase C partially reversed inhibition of IFN production. Conversely, direct activation of protein kinase C with phorbol esters strongly inhibited TLR3- and RIG-I-mediated IFN production. Inhibition of type I and type III IFNs by ATP, UTP, histamine, and the proteinase-activated receptor 2 (PAR2) receptor agonist SLIGKV also occurred in differentiated AECs grown at an air-liquid interface, indicating that the suppression is conserved following mucociliary differentiation. Importantly, histamine and, more strikingly, ATP inhibited type I IFN release from human airway cells infected with live influenza A virus or rhinovirus 1B. These results reveal an important role for extracellular nucleotides and histamine in attenuating the induction of type I and III IFNs from AECs and help explain the molecular basis of the suppression of IFN responses in asthmatic patients.

Printer center nanoparticles alter the DNA repair capacity of human bronchial airway epithelial cells

Nano-enabled, toner-based printing equipment emit nanoparticles during operation. The bioactivity of these nanoparticles as documented in a plethora of published toxicological studies raises concerns about their potential health effects. These include pro-inflammatory effects that can lead to adverse epigenetic alterations and cardiovascular disorders in rats. At the same time, their potential to alter DNA repair pathways at realistic doses remains unclear. In this study, size-fractionated, airborne particles from a printer center in Singapore were sampled and characterized. The PM0.1 size fraction (particles with an aerodynamic diameter less than 100 nm) of printer center particles (PCP) were then administered to human lung adenocarcinoma (Calu-3) or lymphoblastoid (TK6) cells. We evaluated plasma membrane integrity, mitochondrial activity, and intracellular reactive oxygen species (ROS) generation. Moreover, we quantified DNA damage and alterations in the cells' capacity to repair 6 distinct types of DNA lesions. Results show that PCP altered the ability of Calu-3 cells to repair 8oxoG:C lesions and perform nucleotide excision repair, in the absence of acute cytotoxicity or DNA damage. Alterations in DNA repair capacity have been correlated with the risk of various diseases, including cancer, therefore further genotoxicity studies are needed to assess the potential risks of PCP exposure, at both occupational settings and at the end-consumer level.

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

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

Intracellular Colocalization of Influenza Viral RNA and Rab11A Is Dependent upon Microtubule Filaments.

ABSTRACTInfluenza A virus (IAV) consists of eight viral RNA (vRNA) segments that are replicated in the host cell nucleus and transported to the plasma membrane for packaging into progeny virions. We have previously proposed a model where subcomplexes of vRNA are exported from the nucleus and assembled en route to the plasma membrane. However, the role of host cytoskeletal proteins in the cytoplasmic assembly of IAV vRNA segments remains unknown. Previous studies have suggested that IAV vRNA segments are transported via Rab11A-containing recycling endosomes (RE) and use both microtubules (MT) and actin. Rab11A RE transport primarily along MT; therefore, investigation of the role of MT in vRNA assembly is warranted. We explored the role of MT in vRNA assembly and replication by using multiple IAV strains in various cell types, including primary human airway epithelial cells. We observed that Rab11A localization was altered in the presence of MT-depolymerizing drugs, but growth of IAV in all of the cell types tested was unchanged. Fluorescent in situ hybridization was performed to determine the role of MT in the assembly of multiple vRNA segments. Unexpectedly, we found that vRNA-vRNA association in cytoplasmic foci was independent of MT. Given the disparity of localization between Rab11A and vRNA segments in the absence of intact MT filaments, we analyzed the three-dimensional spatial relationship between Rab11A and vRNA in the cytoplasm of infected cells. We found that Rab11A and vRNA colocalization is dependent upon dynamic MT filaments. Taken together, our data suggest that cytoplasmic transport of influenza vRNA may include a Rab11A RE-independent mechanism.IMPORTANCE IAV infections cause a large public health burden through seasonal epidemics and sporadic pandemics. Pandemic IAVs emerge through reassortment of vRNA in animal or human hosts. Elucidation of the mechanism of intracellular dynamics of IAV assembly is necessary to understand reassortment. Our results describing the role of MT in vRNA transport and assembly expand upon previous studies characterizing vRNA assembly. This study is the first to assess the role of MT in influenza virus replication in human bronchial airway epithelial cells. In addition, we present novel data on the role of MT in facilitating the association between distinct vRNA segments. Interestingly, our results suggest that progressive assembly of vRNA segments may be cell type dependent and that vRNA may be transported through the cytoplasm without Rab11A RE in the absence of intact MT. These results enhance our understanding of vRNA assembly and the role of cytoskeletal proteins in that process.

Role of Smad3 and p38 Signalling in Cigarette Smoke-induced CFTR and BK dysfunction in Primary Human Bronchial Airway Epithelial Cells

Mucociliary clearance (MCC) is a major airway host defence system that is impaired in patients with smoking-associated chronic bronchitis. This dysfunction is partially related to a decrease of airway surface liquid (ASL) volume that is in part regulated by apically expressed cystic fibrosis transmembrane conductance regulator (CFTR) and large-conductance, Ca2+-activated, and voltage dependent K+ (BK) channels. Here, data from human bronchial epithelial cells (HBEC) confirm that cigarette smoke not only downregulates CFTR activity but also inhibits BK channel function, thereby causing ASL depletion. Inhibition of signalling pathways involved in cigarette smoke-induced channel dysfunction reveals that CFTR activity is downregulated via Smad3 signalling whereas BK activity is decreased via the p38 cascade. In addition, pre-treatment with pirfenidone, a drug presently used to inhibit TGF-β signalling in idiopathic pulmonary fibrosis, ameliorated BK dysfunction and ASL volume loss. Taken together, our results highlight the importance of not only CFTR but also BK channel function in maintaining ASL homeostasis and emphasize the possibility that pirfenidone could be employed as a novel therapeutic regimen to help improve MCC in smoking-related chronic bronchitis.

Novel function of tetraspanin‐interacting protein IGSF3 in the regulation of glycosphingolipid metabolism

RationaleWe have previously identified in a patient with severe emphysema with stable chromosomal translocation a disruption in the gene encoding for Immunoglobulin Superfamily, Member 3 (Igsf3) accompanied by low levels of this membrane‐anchored protein. We set out to identify the function of Igsf3, which is predicted to interact with other membrane proteins, tetraspanins. cDNA microarray analyses identified that loss of IGSF3 significantly altered gene expression in the glycosphingolipid (GSL) pathway. Since GLS interaction with tetraspanins at the plasma membrane (dubbed as glycosynapse microdomain) inhibits integrin‐mediated cell functions, we hypothesized that Igsf3 is required for the glycosynapse control of cell motility.MethodsTetraspanin‐enriched microdomains (TEMs) were isolated using density gradient‐ultracentrifugation from human bronchial airway epithelial cells (Beas2b) and from primary immortalized human lung lymphocytes isolated from the consenting subject. Beas2b were transduced or transfected with adenoviral‐ and mammalian‐ IGSF3 plasmid vectors; or with siRNA and lentiviral‐shRNA. GSL were measured by LC‐MS/MS and cell migration and proliferation were assessed by standard techniques.ResultsCo‐immunoprecipitation studies identified that within TEMs, Igsf3 colocalized with the tetraspanins CD9, CD81, CD82, and CD151, as well as with integrinb1 (Itgb1). As expected, when compared to other cellular fractions, TEMs were enriched in cholesterol, ceramides, and glycosylceramides (GC). IGSF3 knockdown significantly increased C16‐GC and C22‐GC (by 5.2‐ and 6.2‐ fold, respectively; p<0.0001), whereas IGSF3 overexpression tended to decrease glycosylceramide levels. Loss of Igsf3 by either siRNA or shRNA significantly delayed wound repair in a cell monolayer scratch assay (p<0.0001) and inhibited cell migration (p<0.03), but did not affect cell proliferation.ConclusionsIgsf3 controls glucosylceramide abundance in TEMs and is required for cell migration. While the mechanism by which Igsf3 regulates the GSL metabolism remains to be investigated, our results suggest that Igsf3 may be an important component of the glycosynapse with a functional role in cellular motility and injury repair.Support or Funding InformationWollowick Chair in Respiratory Research; Medresearch AEF_Schweitzer

Fluticasone propionate and Salmeterol combination induces SOCS-3 expression in airway epithelial cells

Fluticasone propionate (FP) and Salmeterol (SAL) are commonly used in combination therapy for patients with Chronic obstructive pulmonary disease (COPD). Clinical studies show that FP/SAL used in combination therapy was found to inhibit airway inflammation in COPD patients. However, the mechanisms associated with FP/SAL induced anti-inflammatory effects were not clear. We have evaluated the effect of FP/SAL and tobacco smoke (TS) on SOCS-3 and interleukine-6 expression in bronchial airway epithelial cells (BAEpCs). Human BAEpCs were exposed to TS and subsequently treated with FP or SAL alone or in combinations in the presence and absence of mitogen activated protein kinase (MAPK) inhibitors for either Erk1/ Erk2, or p38 or PI3 kinase. In BAEpCs, TS induced IL-6 expression via ERK1/ERK2 MAPK pathway and FP/SAL inhibited TS mediated IL-6 expression. TS down regulated the SOCS-3 expression via activation of Erk1/ Erk2, and p38 MAPK signaling. When TS exposed BAEpCs were treated with FP/SAL SOCS-3 expression was restored. FP/SAL combinations induced significantly higher expression of SOCS-3 in BAEpCs when compared to individual drug. Pretreatment with Ly294002 a PI3 MAPK inhibitor significantly attenuated FP/SAL induced SOCS-3 expression in BAEpCs. Furthermore, FP/SAL blunted TS induced phosphorylation of Erk1/ Erk2 and p38 MAPK in BAEpCs. Our study suggests that TS inhibits SOCS-3, combination of FP/SAL has a profound synergistic effect on SOCS-3 induction in BAEpCs and it is dependent on PI3 kinase signaling pathway. SOCS-3 may represent a potential biomarker for understanding the efficacy and a novel anti-inflammatory mechanism of FP/SAL combination therapy in the treatment of COPD.

Airway Epithelial Inflammation-induced Endoplasmic Reticulum Ca2+ Store Expansion Is Mediated by X-box Binding Protein-1

Inflamed cystic fibrosis (CF) human bronchial epithelia (HBE), or normal HBE exposed to supernatant from mucopurulent material (SMM) from CF airways, exhibit endoplasmic reticulum (ER)/Ca(2+) store expansion and amplified Ca(2+)-mediated inflammation. HBE inflammation triggers an unfolded protein response (UPR) coupled to mRNA splicing of X-box binding protein-1 (XBP-1). Because spliced XBP-1 (XBP-1s) promotes ER expansion in other cellular models, we hypothesized that XBP-1s is responsible for the ER/Ca(2+) store expansion in inflamed HBE. XBP-1s was increased in freshly isolated infected/inflamed CF in comparison with normal HBE. The link between airway epithelial inflammation, XBP-1s, and ER/Ca(2+) store expansion was then addressed in murine airways challenged with phosphate-buffered saline or Pseudomonas aeruginosa. P. aeruginosa-challenged mice exhibited airway epithelial ER/Ca(2+) store expansion, which correlated with airway inflammation. P. aeruginosa-induced airway inflammation triggered XBP-1s in ER stress-activated indicator (ERAI) mice. To evaluate the functional role of XBP-1s in airway inflammation linked to ER/Ca(2+) store expansion, control, XBP-1s, or dominant negative XBP-1 (DN-XBP-1) stably expressing 16HBE14o(-) cell lines were used. Studies with cells transfected with an unfolded protein response element (UPRE) luciferase reporter plasmid confirmed that the UPRE was activated or inhibited by expression of XBP-1s or DN-XBP-1, respectively. Expression of XBP-1s induced ER/Ca(2+) store expansion and potentiated bradykinin-increased interleukin (IL)-8 secretion, whereas expression of DN-XBP-1 inhibited bradykinin-dependent IL-8 secretion. In addition, expression of DN-XBP-1 blunted SMM-induced ER/Ca(2+) store expansion and SMM-induced IL-8 secretion. These findings suggest that, in inflamed HBE, XBP-1s is responsible for the ER/Ca(2+) store expansion that confers amplification of Ca(2+)-dependent inflammatory responses.

Induction of cell cycle arrest and apoptosis by BCG infection in cultured human bronchial airway epithelial cells

Bronchial airway epithelial cells (BAEpC) are among the first cells to encounter M. tuberculosis following airborne infection. However, the response of BAEpC to M. tuberculosis infection has been little studied. This study investigates the response of a human BAEpC cell line (BEAS-2B) to infection with Mycobacterium bovis Bacille Calmette Guerin (BCG). Cultured human BEAS-2B cells were experimentally infected with BCG. Uninfected BEAS-2B cultures were included as controls. Following infection, BEAS-2B cells were evaluated by various methods at various time points up to 3 days. Cell proliferation was evaluated by cellular bioreduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Distribution of cells along the cell cycle was evaluated by FACS analysis of cellular DNA. Apoptotic cells were identified by cell death ELISA and the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling method. Eighty-four apoptosis-relevant genes were screened by PCR gene microarray. Translation of Fas, Fas ligand (Fas-L), and Fas-associated death domain (FADD) were evaluated quantitatively by real-time PCR. Expression of Fas and FADD proteins was evaluated by immunofluorescence and Western blot. Activity of caspase-3 and caspase-8 was evaluated by colorimetric assay of their enzymatic activity. BCG infection of BEAS-2B cells inhibits proliferation, induces cell cycle arrest at the G(0)/G(1) phase, causes apoptosis, modulates transcription of multiple apoptosis-relevant genes, promotes translation of Fas, Fas-L, and FADD, upregulates expression of Fas and FADD proteins, and increases activity of caspase-3 and caspase-8. Infection with BCG does not cause any significant change in the secretion of TGF-beta. The roles of Fas and FADD as mediators of BCG-induced apoptosis in BEAS-2B cells were tested by partial blockade of Fas and FADD expression with silencing RNA. Partial blockade of Fas or FADD expression results in a decreased apoptotic response to BCG infection. In conclusion, BCG induces cell cycle arrest and apoptosis in BEAS-2B cells. BCG induced apoptosis of BEAS-2B cells via the Fas death receptor pathway.

Inducible expression of the proallergic cytokine thymic stromal lymphopoietin in airway epithelial cells is controlled by NFκB

The epithelial-derived cytokine thymic stromal lymphopoietin (TSLP) is important for the initiation of allergic airway inflammation through a dendritic cell-mediated T helper 2 response. To identify the factors that control TSLP expression, we examined the ability of inflammatory mediators to regulate TSLP production in human airway epithelial cells. We found that both IL-1beta and TNF-alpha were capable of inducing rapid TSLP production in primary human bronchial airway epithelial cells. We further characterized the human TSLP gene promoter, using two human epithelial cell lines, 16HBEo(-) and A549, and showed that IL-1beta- and TNF-alpha-mediated human TSLP promoter activation in these cells was mediated by an upstream NFkappaB site. Mutation of this NFkappaB site abolished activation, as did overexpression of a dominant-negative version of IkappaB kinase (IKK)beta (a kinase acting on IkappaB, the inhibitor of NFkappaB). Interestingly, human TSLP mRNA levels were also increased after exposure to Toll-like receptor (TLR) 2, TLR8, and TLR9 ligands, further supporting an important role for NFkappaB in TSLP gene regulation. Similarly, analysis of the mouse TSLP gene promoter revealed the presence of a similarly situated NFkappaB site that was also critical for IL-1beta-inducible expression of mouse TSLP. Taken together, these results demonstrate that the inflammatory mediators IL-1beta and TNF-alpha regulate human TSLP gene expression in an NFkappaB-dependent manner.