BEAS-2B Airway Epithelial Cells Research Articles

The Cellular Accumulation of Vehicle Exhaust Particulates Changes the Acidic pH Environment of Lysosomes in BEAS-2B Airway Epithelial Cells.

Many people are exposed every day to vehicle exhaust particulates (VEPs), which are thought to be taken up by epithelial cells that are the first barrier in our biological defense. The study aim was to investigate how VEPs are processed in the lysosomal degradation system. BEAS-2B airway epithelial cells easily ingest VEPs and have been shown to accumulate in cells for several days, but no elevated cytotoxicity was observed over that time period. An analysis of 3D images confirmed the presence of VEPs in or near lysosomes, and an accumulation of VEPs resulted in an increase in the normal acidic pH in lysosomes and the extracellular release of the lysosomal enzyme β-hexosaminidase. Epithelial cells were thought to activate the lysosome-mediated secretion of extracellular vesicles to avoid damage caused by non-degradable foreign substances, such as VEPs, and as a side reaction, the acidic pH environment of the lysosomes could not be maintained.

Metabolomics of V2O5 nanoparticles and V2O5 nanofibers in human airway epithelial BEAS-2B cells

Vanadium is a toxic metal listed by the IARC as possibly carcinogenic to humans. Manufactured nanosize vanadium pentoxide (V2O5) materials are used in a wide range of industrial sectors and recently have been developed as nanomedicine for cancer therapeutics, yet limited information is available to evaluate relevant nanotoxicity. In this study we used high-resolution metabolomics to assess effects of two V2O5 nanomaterials, nanoparticles and nanofibers, at exposure levels (0.01, 0.1, and 1 ppm) that did not cause cell death (i.e., non-cytotoxic) in a human airway epithelial cell line, BEAS-2B. As prepared, V2O5 nanofiber exhibited a fibrous morphology, with a width approximately 63 ± 12 nm and length in average 420 ± 70 nm; whereas, V2O5 nanoparticles showed a typical particle morphology with a size 36 ± 2 nm. Both V2O5 nanoparticles and nanofibers had dose-response effects on aminosugar, amino acid, fatty acid, carnitine, niacin and nucleotide metabolism. Differential effects of the particles and fibers included dibasic acid, glycosphingolipid and glycerophospholipid pathway associations with V2O5 nanoparticles, and cholesterol and sialic acid metabolism associations with V2O5 nanofibers. Examination by transmission electron microscopy provided evidence for mitochondrial stress and increased lysosome fusion by both nanomaterials, and these data were supported by effects on mitochondrial membrane potential and lysosomal activity. The results showed that non-cytotoxic exposures to V2O5 nanomaterials impact major metabolic pathways previously associated with human lung diseases and suggest that toxico-metabolomics may be useful to evaluate health risks from V2O5 nanomaterials.

Apelin-13 facilitates mitochondria homeostasis via mitophagy to prevent against airway oxidative injury in asthma

Oxidative stress is a major mediator in the pathogenesis of allergens-induced asthma. Mitochondria damage and dysfunction is considered to be closely related with oxidative stress. Apelin-13 is a novel multifunctional protein with anti-inflammatory and anti-oxidative properties in neuroinflammation and ischemia-reperfusion injury. However, its role in mitochondria homeostasis under asthma-associated airway oxidative injury and the potential mechanisms have not been elucidated. A murine model of asthma was established by house dust mite (HDM) allergen sensitization and challenge. The mice were received Apelin-13 protein through intraperitoneal administration before HDM challenge. Airway inflammation, histopathological changes and oxidative stress were examined. The regulatory effects of Apelin-13 on mitochondria function were evaluated using airway epithelial BEAS-2B cells, including mitochondria membrane potential (MMP), mitophagy and the possible signaling pathway. The HDM-challenged mice group exhibited robust inflammation and apoptosis in airway epithelium compared to the control group. The airway impairment in asthmatic mice was partly lessened after Apelin-13 administration. Meanwhile, protein expressions of mitophagy-related markers PINK1, Parkin, Tomm20 and LC3 were significantly increased in the lungs of Apelin-13-treated asthmatic mice. In vitro, Apelin-13 treatment significantly improved MMP levels and reduced ROS production in BEAS-2B cells exposed to HDM, accompanied with the increase of cell viability. Furthermore, Apelin-13 was found to promote the activation of PINK1/Parkin signaling in BEAS-2B cells, thereby increasing mitophagy activity and facilitating mitochondria homeostasis. These results demonstrate that Apelin-13 acts as a regulator of mitochondria homeostasis by driving mitophagy to protect against HDM allergen-induced airway oxidative injury. Apelin-13 may serve as a promising protective agent for treating asthma.

Nlrx1-Regulated Defense and Metabolic Responses to Aspergillus fumigatus Are Morphotype and Cell Type Specific.

The Nlr family member X1 (Nlrx1) is an immuno-metabolic hub involved in mediating effective responses to virus, bacteria, fungi, cancer, and auto-immune diseases. We have previously shown that Nlrx1 is a critical regulator of immune signaling and mortality in several models of pulmonary fungal infection using the clinically relevant fungus Aspergillus fumigatus. In the absence of Nlrx1, hosts produce an enhanced Th2 response primarily by CD103+ dendritic cell populations resulting in enhanced mortality via immunopathogenesis as well as enhanced fungal burden. Here, we present our subsequent efforts showcasing loss of Nlrx1 resulting in a decreased ability of host cells to process A. fumigatus conidia in a cell-type-specific manner by BEAS-2B airway epithelial cells, alveolar macrophages, bone marrow-derived macrophages, but not bone marrow-derived neutrophils. Furthermore, loss of Nlrx1 results in a diminished ability to generate superoxide and/or generic reactive oxygen species during specific responses to fungal PAMPs, conidia, and hyphae. Analysis of glycolysis and mitochondrial function suggests that Nlrx1 is needed to appropriately shut down glycolysis in response to A. fumigatus conidia and increase glycolysis in response to hyphae in BEAS-2B cells. Blocking glycolysis and pentose phosphate pathway (PPP) via 2-DG and NADPH production through glucose-6-phosphate dehydrogenase inhibitor resulted in significantly diminished conidial processing in wild-type BEAS-2B cells to the levels of Nlrx1-deficient BEAS-2B cells. Our findings suggest a need for airway epithelial cells to generate NADPH for reactive oxygen species production in response to conidia via PPP. In context to fungal pulmonary infections, our results show that Nlrx1 plays significant roles in host defense via PPP modulation of several aspects of metabolism, particularly glycolysis, to facilitate conidia processing in addition to its critical role in regulating immune signaling.

Syndecan-1 Amplifies Ovalbumin-Induced Airway Remodeling by Strengthening TGFβ1/Smad3 Action.

Syndecan-1 (SDC-1) is a transmembrane proteoglycan of heparin sulfate that can regulate various cell signal transduction pathways in the airway epithelial cells and fibroblasts. Airway epithelial cells and human bronchial fibroblasts are crucial in airway remodeling. However, the importance of SDC-1 in the remodeling of asthmatic airways has not been confirmed yet. The present study was the first to uncover SDC-1 overexpression in the airways of humans and mice with chronic asthma. This study also validated that an increase in SDC-1 expression was correlated with TGFβ1/Smad3-mediated airway remodeling in vivo and in vitro. A small interfering RNA targeting SDC-1 (SDC-1 siRNA) and homo-SDC-1 in pcDNA3.1 (pc-SDC-1) was designed to assess the effects of SDC-1 on TGFβ1/Smad3-mediated collagen I expression in Beas-2B (airway epithelial cells) and HLF-1 (fibroblasts) cells. Downregulation of the SDC-1 expression by SDC-1 siRNA remarkably attenuated TGFβ1-induced p-Smad3 levels and collagen I expression in Beas-2B and HLF-1 cells. In addition, SDC-1 overexpression with pc-SDC-1 enhanced TGFβ1-induced p-Smad3 level and collagen I expression in Beas-2B and HLF-1 cells. Furthermore, the levels of p-Smad3 and collagen I induced by TGFβ1 were slightly increased after the addition of the recombinant human SDC-1 protein to Beas-2B and HLF-1 cells. These findings in vitro were also confirmed in a mouse model. A short hairpin RNA targeting SDC-1 (SDC-1 shRNA) to interfere with SDC-1 expression considerably reduced the levels of p-Smad3 and remodeling protein (α-SMA, collagen I) in the airways induced by ovalbumin (OVA). Similarly, OVA-induced p-Smad3 and remodeling protein levels in airways increased after mice inhalation with the recombinant mouse SDC-1 protein. These results suggested that SDC-1 of airway epithelial cells and fibroblasts plays a key role in the development of airway remodeling in OVA-induced chronic asthma.

Progranulin Protects Against Airway Remodeling Through the Modulation of Autophagy via HMGB1 Suppression in House Dust Mite-Induced Chronic Asthma.

PurposeAirway remodeling is an important feature of chronic asthma, and yet there are few effective therapeutic strategies. Progranulin (PGRN) has been shown to have lung protective functions, but the role of PGRN in asthmatic airway remodeling is unclear. We aim to explore the protective potential of PGRN on house dust mite (HDM)-induced airway remodeling and the underlying mechanisms.MethodsIn this study, a murine model of chronic asthma was established by HDM sensitization and challenge. Recombinant PGRN was intranasally administrated to mice during the phase of HDM challenge. TGF-β1-treated human airway epithelial BEAS-2B cells were utilized to explore the effect of PGRN on airway epithelia exposed to profibrotic conditions and molecular mechanisms.ResultsWe found that PGRN treatment attenuated HDM-induced airway remodeling, as evidenced by the suppression of collagen accumulation, mucus overproduction and airway smooth muscle synthesis in HDM-challenged asthmatic mice lungs. Meanwhile, PGRN also reversed the increased levels of autophagy markers and autophagosomes in airway epithelia under mimic asthmatic conditions, thereby controlling the fibrotic process in vivo and in vitro. Specifically, overexpressed HMGB1 and the subsequent RAGE/MAPKs signaling activation due to HDM exposure were abrogated in PGRN-treated asthmatic mice. Furthermore, knockdown of HMGB1 expression significantly restrained the fibrosis formation in TGF-β1-induced airway epithelia accompanied by the downregulation of autophagic activity. However, enhancement of extracellular HMGB1 levels blunted the inhibition of autophagic flux by PGRN in airway epithelia, thereby resulting in the augmentation of collagen synthesis and fibrosis.ConclusionTaken together, our data revealed that PGRN protected against asthmatic airway remodeling by negatively regulating autophagy via HMGB1 suppression, which might provide new insights into the therapeutic options for HDM-induced chronic asthma.

Role of nuclear factor‐κB (NF‐κB) in the regulation of pro‐inflammatory IκB‐kinase‐ε (IKKε) gene expression in human pulmonary epithelial cells

Rationale Clinically, mild-to-moderate asthma is well-controlled by inhaled glucocorticoids. However, severe asthma is poorly controlled by this key pharmacological intervention. Understanding which pro-inflammatory genes and signalling pathways avoid repression by glucocorticoids represents an important step towards improving current pharmacological interventions. IκB-kinase-ε (IKKε) was shown to escape full repression by glucocorticoids in airway epithelial cells, the primary site of action for glucocorticoid therapies in asthma. IKKε is also highly homologous to the canonical IκB kinases known to activate a family of pro-inflammatory transcription factors: nuclear factor-κB (NF-κB). However, the role and regulation of IKKε is largely uncharacterized. This study aimed to elucidate the regulation of IKKε gene expression by NF-κB in airway epithelial cells. Methods Human pulmonary epithelial A549 or bronchial airway epithelial BEAS-2B cells were used to model gene expression in airway epithelial cells. Cells were treated with cytokines interleukin-1β (IL1B, 1 ng/ml), tumour necrosis factor-α (TNF, 10 ng/ml), and/or glucocorticoid (dexamethasone, 1µM). Western blotting, qPCR, and RNA-sequencing were performed to characterize protein and mRNA expression of IKKε. Cells harbouring an NF-κB driven luciferase reporter construct were used to assess overall NF-κB activity. Chromatin immunoprecipitation (ChIP) sequencing data from BEAS-2B cells was used to explore binding of NF-κB subunit RelA/p65 to DNA using the UCSC genome browser. Results In A549 cells, IKKε protein was upregulated by both and IL1B and TNF with highest expression observed at 24h post stimulus. This peak expression was significantly, albeit partially, repressed by the glucocorticoid, dexamethasone. IKKε mRNA expression peaked at 6h, with partial repression by dexamethasone observed at 24h. ChIP-seq data from BEAS-2B cells treated with TNF showed p65 binding to the promoter region of IKKε. This suggests regulation of IKKε expression by NF-κB. In A549 cells, silencing of p65 decreased the ability of IL1B to induce NF-κB reporter activity and also reduced IL1B-induced expression of IKKε. Likewise, adenoviral-mediated overexpression of IκBαΔN, a dominant inhibitor of NF-κB, markedly reduced IKKε mRNA expression that was induced by IL1B. Conclusions These data suggest that induction of IKKε expression by IL1B or TNF is dependent on NF-κB activity in pulmonary epithelial cells. This supports the conclusion that early onset NF-κB activity leads to the expression of inflammatory genes, such as IKKε, to activate later onset signal transduction cascades to prolong inflammatory gene expression in response to a given stimuli. As IKKε induction was only partially repressed by glucocorticoid, the induction of downstream effector processes may also be largely resistant to the repressive effects of glucocorticoid. These findings represent an important step in characterizing the role of IKKε in inflammatory signalling cascades and paves the way for interventions in severe asthma therapy that may be more able to repress the activity of this pro-inflammatory pathway.

Application of green tea catechins, polysaccharides, and flavonol prevent fine dust induced bronchial damage by modulating inflammation and airway cilia

Airborne fine dust particles (FDPs) have been identified as major toxins in air pollution that threaten human respiratory health. While searching for an anti-FDP reagent, we found that green tea extract (GTE) and fractions rich in flavonol glycosides (FLGs) and crude tea polysaccharides (CTPs) had protective effects against FDP-stimulated cellular damage in the BEAS-2B airway epithelial cell line. The GTE, FLGs, and CTPs significantly increased viability and lowered oxidative stress levels in FDP-treated cells. Combined treatment with GTE, FLGs, and CTPs also exerted synergistic protective effects on cells and attenuated FDP-induced elevations in inflammatory gene expression. Moreover, the green tea components increased the proportion of ciliated cells and upregulated ciliogenesis in the airway in FDP-stimulated BEAS-2B cells. Our findings provide insights into how natural phytochemicals protect the airway and suggest that green tea could be used to reduce FDP-induced airway damage as an ingredient in pharmaceutical, nutraceutical, and also cosmeceutical products.

STAT1 participates in the induction of substance P expression in airway epithelial cells by respiratory syncytial virus

Purpose The regulation effect and mechanism of respiratory syncytial virus (RSV) infection on the expression of tachykinin substance P (SP) in airway epithelial cells was investigated. Methods The regulation of SP expression by RSV was investigated in the BEAS-2B airway epithelial cell line. RT-qPCR, immunofluorescence, and ELISA assay were used to examine the expression of the SP encoding gene TAC1, the intracellular SP protein expression, and the extracellular SP secretion. Results The mRNA expression of TAC1 and the intracellular SP protein level in BEAS-2B cells were significantly enhanced by RSV infection with multiplicity of infection (MOI) values of both 1 and 0.1 at 48 hours post infection. Heat-inactivated and UV-inactivated RSV, but not live RSV, significantly induced SP secretion in both control BEAS-2B cells and CX3CR1 receptor knockout cells without affecting the TAC1 gene expression or cell viability. RSV G protein (2–10 μg/ml) and fractalkine (10–50 ng/ml), both CX3CR1 receptor ligands, did not affect SP secretion in BEAS-2B cells. Inhibition of STAT1 phosphorylation by fludarabine (1 μM) markedly reduced the RSV-induced TAC1 gene expression and antagonized the inhibition of RSV replication by interferon-α in BEAS-2B cells. Conclusions STAT1 participates in RSV infection-induced SP expression in airway epithelial cells.

NLRX1 is a key regulator of immune signaling during invasive pulmonary aspergillosis.

Aspergillus fumigatus is an opportunistic fungal pathogen of immunocompromised patient populations. Mortality is thought to be context-specific and occurs via both enhanced fungal growth and immunopathogenesis. NLRX1 is a negative regulator of immune signaling and metabolic pathways implicated in host responses to microbes, cancers, and autoimmune diseases. Our study indicates loss of Nlrx1 results in enhanced fungal burden, pulmonary inflammation, immune cell recruitment, and mortality across immuno-suppressed and immuno-competent models of IPA using two clinically derived isolates (AF293, CEA10). We observed that the heightened mortality is due to enhanced recruitment of CD103+ dendritic cells (DCs) that produce elevated amounts of IL-4 resulting in a detrimental Th2-mediated immune response. Adoptive transfer of Nlrx1-/- CD103+ DCs in neutropenic NRG mice results in enhanced mortality that can be ablated using IL-4 neutralizing antibodies. In vitro analysis of CD103+ DCs indicates loss of Nlrx1 results in enhanced IL-4 production via elevated activation of the JNK/JunB pathways. Interestingly, loss of Nlrx1 also results in enhanced recruitment of monocytes and neutrophils. Chimeras of irradiated Nlrx1-/- mice reconstituted with wild type bone marrow have enhanced neutrophil recruitment and survival during models of IPA. This enhanced immune cell recruitment in the absence of Nlrx1 is mediated by excessive production of CXCL8/IL-8 family of chemokines and IL-6 via early and enhanced activation of P38 in response to A. fumigatus conidia as shown in BEAS-2B airway epithelial cells. In summary, our results point strongly towards the cell-specific and contextual function of Nlrx1 during invasive pulmonary aspergillosis and may lead to novel therapeutics to reduce Th2 responses by CD103+ DCs or heightened recruitment of neutrophils.

Increased intracellular Cl− concentration by activating FAK promotes airway epithelial BEAS-2B cells proliferation and wound healing

An increase in intracellular Cl− concentration ([Cl−]i) may be a general response of airway epithelial cells to various stimuli and may participate in some basic cellular functions. However, whether the basic functional activities of cells, such as proliferation and wound healing, are related to Cl− activities remains unclear. This study aimed to investigate the effects and potential mechanisms of [Cl−]i on the proliferation and wound healing ability of airway epithelial BEAS-2B cells. BEAS-2B cells were treated with four Cl− channel inhibitors (T16Ainh-A01, CFTRinh-172, CaCCinh-A01, and IAA-94), and the Cl− fluorescence probe N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide was used. Results showed that all Cl− channel inhibitors could increase [Cl−]i in BEAS-2B cells. The increased [Cl−]i induced by Cl− channel inhibitors or clamping [Cl−]i at high levels enhanced the phosphorylation of focal adhesion kinase (FAK) and subsequently promoted the proliferation and wound healing ability of BEAS-2B cells. By contrast, the FAK inhibitor PF573228 abrogated these effects induced by the increased [Cl−]i. FAK also activated the PI3K/AKT signaling pathway. In conclusion, increased [Cl−]i promotes the proliferation and wound healing ability of BEAS-2B cells by activating FAK to activate the PI3K/AKT signaling pathway. Intracellular Cl− may act as a signaling molecule to regulate the proliferation and wound healing ability of airway epithelial cells.

Airway Epithelial Cells Generate Pro-inflammatory Tenascin-C and Small Extracellular Vesicles in Response to TLR3 Stimuli and Rhinovirus Infection.

Viral infections are a common cause of asthma exacerbations, with human rhinoviruses (RV) the most common trigger. RV signals through a number of different receptors, including toll-like receptor (TLR)3. Tenascin-C (TN-C) is an immunomodulatory extracellular matrix protein present in high quantities in the airway of people with asthma, and expression is also upregulated in nasal lavage fluid in response to RV infection. Respiratory viral infection has been demonstrated to induce the release of small extracellular vesicles (sEV) such as exosomes, whilst exosomal cargo can also be modified in the bronchoalveolar lavage fluid of people with asthma. These sEVs may potentiate airway inflammation and regulate the immune response to infection. This study characterizes the relationship between RV infection of bronchial epithelial cells and the release of TN-C, and the release of sEVs following stimulation with the TLR3 agonist and synthetic viral mimic, poly(I:C), as well as the function of the released protein/vesicles. The BEAS-2B airway epithelial cell line and primary human bronchial epithelial cells (PBECs) from asthmatic and non-asthmatic donors were infected with RV or treated with poly(I:C). TN-C expression, release and localization to sEVs was quantified. TN-C expression was also assessed following intra-nasal challenge of C57BL/6 mice with poly(I:C). BEAS-2B cells and macrophages were subsequently challenged with TN-C, or with sEVs generated from BEAS-2B cells pre-treated with siRNA targeted to TN-C or control. The results revealed that poly(I:C) stimulation induced TN-C release in vivo, whilst both poly(I:C) stimulation and RV infection promoted release in vitro, with elevated TN-C release from PBECs obtained from people with asthma. Poly(I:C) also induced the release of TN-C-rich sEVs from BEAS-2B cells. TN-C, and sEVs from poly(I:C) challenged cells, induced cytokine synthesis in macrophages and BEAS-2B cells, whilst sEVs from control cells did not. Moreover, sEVs with ~75% reduced TN-C content did not alter the capacity of sEVs to induce inflammation. This study identifies two novel components of the inflammatory pathway that regulates the immune response following RV infection and TLR3 stimulation, highlighting TN-C release and pro-inflammatory sEVs in the airway as relevant to the biology of virally induced exacerbations of asthma.

Impact of Phosphodiesterase 4 Inhibition on the Operational Efficacy, Response Maxima, and Kinetics of Indacaterol-Induced Gene Expression Changes in BEAS-2B Airway Epithelial Cells: A Global Transcriptomic Analysis

The effects of phosphodiesterase (PDE) 4 inhibitors on gene expression changes in BEAS-2B human airway epithelial cells are reported and discussed in relation to the mechanism(s) of action of roflumilast in chronic obstructive pulmonary disease (COPD). Microarray-based gene expression profiling failed to identify mRNA transcripts that were differentially regulated by the PDE4 inhibitor 6-[3-(dimethylcarbamoyl)benzenesulphonyl]-4-[(3-methoxyphenyl)amino]-8-methylquinoline-3-carboxamide (GSK 256066) after 1, 2, 6, or 18 hours of exposure. However, real-time polymerase chain reaction analysis revealed that GSK 256066 was a weak stimulus, and the negative microarray results reflected low statistical power due to small sample sizes. Furthermore, GSK 256066, roflumilast, and its biologically active metabolite roflumilast N-oxide generally potentiated gene expression changes produced by the long-acting β 2-adrenoceptor agonists (LABAs) salmeterol, indacaterol, and formoterol. Many of these genes encode proteins with antiviral, anti-inflammatory, and antibacterial activities that could contribute to the clinical efficacy of roflumilast in COPD. RNA-sequencing experiments established that the sensitivity of genes to salmeterol varied by ∼7.5-fold. Consequently, the degree to which a PDE4 inhibitor potentiated the effect of a given concentration of LABA was gene-dependent. Operational model fitting of concentration-response curve data from cells subjected to fractional, β 2-adrenoceptor inactivation determined that PDE4 inhibition increased the potency and doubled the efficacy of LABAs. Thus, adding roflumilast to standard triple therapy, as COPD guidelines recommend, may have clinical relevance, especially in target tissues where LABAs behave as partial agonists. Collectively, these results suggest that the genomic impact of roflumilast, including its ability to augment LABA-induced gene expression changes, may contribute to its therapeutic activity in COPD.

Protective effects of VGX-1027 in PM2.5-induced airway inflammation and bronchial hyperresponsiveness

Fine particulate matter (PM2.5) can penetrate into alveolar spaces and induce airway inflammation. Recent evidence suggests that the activation of Toll-like receptor 4 (TLR4) signaling may participate in PM2.5-induced acute lung injury. We investigated the effect of VGX-1027, a TLR4 blocker, on PM2.5-induced airway inflammation and bronchial hyperresponsiveness (BHR) in a murine model in vivo and on inflammatory mechanisms in vitro in human airway epithelial cells. Mice were injected intraperitoneally with vehicle (PBS) or VGX-1027 (25 mg/kg) one hour before intranasal instillation of vehicle (PBS) or PM2.5 (7.8 mg/kg) for two consecutive days and inflammatory events and BHR studied 24 h later. Human airway epithelial Beas-2b cells were pretreated with vehicle or VGX-1027 (50 μM) in vitro one hour before incubation with vehicle or PM2.5 (150 ng/ml) for 24 h and effects on inflammatory mediators and mechanisms studied. VGX-1027 pretreatment attenuated PM2.5-induced BHR and elevated total and neutrophils, macrophages, lymphocytes and eosinophils numbers in bronchoalveolar lavage (BAL) fluid in vivo. PM2.5-induced BAL fluid inflammatory mediator levels including TNF-α, chemokine (C-X-C motif) ligand1, IL-1β, IL-6 and IL-18 were reduced by VGX-1027. PM2.5-induced increases in TNF-α, IL-1β, IL-6 and IL-18 mRNA levels in Beas-2b cells were also reduced by VGX-1027. Mechanistically, VGX-1027 inhibited PM2.5-induced activation of the TLR4-NF-κB-p38 MAPK and NLRP3-caspase-1 pathways as well as the dysregulation of mitochondrial fusion/fission proteins in vivo and in vitro. VGX-1027 may be a potential prophylactic treatment for PM2.5-induced acute lung injury that has airway inflammation, BHR and mitochondrial damage.

Long-Acting β2-Adrenoceptor Agonists Enhance Glucocorticoid Receptor (GR)–Mediated Transcription by Gene-Specific Mechanisms Rather Than Generic Effects via GR

In asthma, the clinical efficacy of inhaled corticosteroids (ICSs) is enhanced by long-acting β2-adrenoceptor agonists (LABAs). ICSs, or more accurately, glucocorticoids, promote therapeutically relevant changes in gene expression, and, in primary human bronchial epithelial cells (pHBECs) and airway smooth muscle cells, this genomic effect can be enhanced by a LABA. Modeling this interaction in human bronchial airway epithelial BEAS-2B cells transfected with a 2× glucocorticoid response element (2×GRE)-driven luciferase reporter showed glucocorticoid-induced transcription to be enhanced 2- to 3-fold by LABA. This glucocorticoid receptor (GR; NR3C1)-dependent effect occurred rapidly, was insensitive to protein synthesis inhibition, and was maximal when glucocorticoid and LABA were added concurrently. The ability of LABA to enhance GR-mediated transcription was not associated with changes in GR expression, serine (Ser203, Ser211, Ser226) phosphorylation, ligand affinity, or nuclear translocation. Chromatin immunoprecipitation demonstrated that glucocorticoid-induced recruitment of GR to the integrated 2×GRE reporter and multiple gene loci, whose mRNAs were unaffected or enhanced by LABA, was also unchanged by LABA. Transcriptomic analysis revealed glucocorticoid-induced mRNAs were variably enhanced, unaffected, or repressed by LABA. Thus, events leading to GR binding at target genes are not the primary explanation for how LABAs modulate GR-mediated transcription. As many glucocorticoid-induced genes are independently induced by LABA, gene-specific control by GR- and LABA-activated transcription factors may explain these observations. Because LABAs promote similar effects in pHBECs, therapeutic relevance is likely. These data illustrate the need to understand gene function(s), and the mechanisms leading to gene-specific induction, if existing ICS/LABA combination therapies are to be improved.

Analysis of the Indacaterol-Regulated Transcriptome in Human Airway Epithelial Cells Implicates Gene Expression Changes in the Adverse and Therapeutic Effects of β2-Adrenoceptor Agonists.

The contribution of gene expression changes to the adverse and therapeutic effects of β2-adrenoceptor agonists in asthma was investigated using human airway epithelial cells as a therapeutically relevant target. Operational model-fitting established that the long-acting β2-adrenoceptor agonists (LABA) indacaterol, salmeterol, formoterol, and picumeterol were full agonists on BEAS-2B cells transfected with a cAMP-response element reporter but differed in efficacy (indacaterol ≥ formoterol > salmeterol ≥ picumeterol). The transcriptomic signature of indacaterol in BEAS-2B cells identified 180, 368, 252, and 10 genes that were differentially expressed (>1.5- to <0.67-fold) after 1-, 2-, 6-, and 18-hour of exposure, respectively. Many upregulated genes (e.g., AREG, BDNF, CCL20, CXCL2, EDN1, IL6, IL15, IL20) encode proteins with proinflammatory activity and are annotated by several, enriched gene ontology (GO) terms, including cellular response to interleukin-1, cytokine activity, and positive regulation of neutrophil chemotaxis The general enriched GO term extracellular space was also associated with indacaterol-induced genes, and many of those, including CRISPLD2, DMBT1, GAS1, and SOCS3, have putative anti-inflammatory, antibacterial, and/or antiviral activity. Numerous indacaterol-regulated genes were also induced or repressed in BEAS-2B cells and human primary bronchial epithelial cells by the low efficacy LABA salmeterol, indicating that this genomic effect was neither unique to indacaterol nor restricted to the BEAS-2B airway epithelial cell line. Collectively, these data suggest that the consequences of inhaling a β2-adrenoceptor agonist may be complex and involve widespread changes in gene expression. We propose that this genomic effect represents a generally unappreciated mechanism that may contribute to the adverse and therapeutic actions of β2-adrenoceptor agonists in asthma.

MicroRNA-150 protects against cigarette smoke-induced lung inflammation and airway epithelial cell apoptosis through repressing p53: MicroRNA-150 in CS-induced lung inflammation.

Cigarette smoke (CS) exposure is an important risk factor for chronic obstructive pulmonary disease (COPD). MicroRNA-150 (miR-150) is involved in several inflammatory diseases. However, little is known about the role of miR-150 in the pathogenesis of COPD. In this study, we established a CS-related mouse model of COPD and evaluated the impact of miR-150 on CS-induced lung inflammation. We further investigated the effects of miR-150 overexpression on pro-inflammatory cytokine production and apoptosis in airway epithelial cells exposed to CS extract (CSE). It was found that miR-150 was significantly ( p < 0.05) downregulated in the lungs of CS-exposed mice, compared to control mice under normal air. The CSE-exposed BEAS-2B airway epithelial cells displayed a four- to six-fold reduction in miR-150 levels, compared to control cells ( p < 0.05). Delivery of miR-150 mimic attenuated CS-induced lung inflammation and accumulation of neutrophils, lymphocytes, and macrophages in bronchoalveolar lavage fluid. Moreover, miR-150 overexpression prevented the induction of interleukin-6, tumor necrosis factor alpha, and interleukin-8 expression and nuclear factor kappa B (NF-κB) transcriptional activity in BEAS-2B cells by CSE. Additionally, miR-150 protected BEAS-2B cells from CSE-induced apoptosis, which was associated with reduced p53 expression. Co-expression of p53 restored apoptotic response to CSE in miR-150-overexpressing BEAS-2B cells. Collectively, miR-150 suppresses CS-induced lung inflammation and airway epithelial cell apoptosis, which is causally linked to repression of p53 expression and NF-κB activity. Restoration of miR-150 expression may represent a potential therapeutic strategy for CS-related COPD.

Astragalin Inhibits Allergic Inflammation and Airway Thickening in Ovalbumin-Challenged Mice.

Lung inflammation and oxidative stress are the major contributors to the development of obstructive pulmonary diseases. Macrophages are involved in pulmonary inflammation and alveolar damage in emphysema. Astragalin is an anti-inflammatory flavonoid present in persimmon leaves and green tea seeds. This study elucidated that astragalin inhibited inflammatory cell infiltration induced by 20 μM H2O2 and blocked airway thickening and alveolar emphysema induced by 20 μg of ovalbumin (OVA) in mice. OVA induced mouse pulmonary MCP-1, and H2O2 enhanced the expression of MCP-1/ICAM-1/αv integrin in bronchial airway epithelial BEAS-2B cells. Such induction was inhibited by supplying 10-20 mg/kg of astragalin to OVA-challenged mice and 1-20 μM astragalin to oxidant-stimulated cells. Oral administration of 20 mg/kg of astragalin reduced the induction of F4/80/CD68/CD11b in airways of mice challenged with OVA. Additionally, emphysema tissue damage was observed in OVA-exposed alveoli. Mast cell recruitment in the airway subepithelium was blocked by supplementing astragalin to OVA-challenged mice. Orally treating 20 mg/kg of astragalin reduced α-SMA induction in inflammation-occurring airways and appeared to reverse airway thickening and constriction induced by an OVA episode. These results revealed that astragalin may improve airway thickening and alveolar destruction with blockade of allergic inflammation in airways. Therefore, astragalin may be a therapeutic agent antagonizing asthma and obstructive pulmonary diseases.

MUC4 impairs the anti-inflammatory effects of corticosteroids in patients with chronic rhinosinusitis with nasal polyps

Current evidence suggests that membrane-tethered mucins could mediate corticosteroid efficacy, interacting with glucocorticoid receptor (GR) in patients with chronic rhinosinusitis with nasal polyps (CRSwNP). Mucin 4 (MUC4)-tethered mucin is expressed in nasal polyp (NP) epithelial cells and upregulated under inflammatory conditions. Moreover, MUC4β has the capacity to interact with other intracellular proteins. We hypothesized that MUC4 modulates corticosteroid efficacy of patients with CRSwNP. We sought to analyze the role of MUC4 in corticosteroid effectiveness in different cohorts of patients with CRSwNP and elucidate the possible mechanisms involved. Eighty-one patients with CRSwNP took oral corticosteroids for 15days. Corticosteroid resistance was evaluated by using nasal endoscopy. Expression of MUC4 and MUC4β was evaluated by means of real-time PCR, Western blotting, and immunohistochemistry. BEAS-2B knockdown with RNA interference for MUC4 (small interfering RNA [siRNA]-MUC4) was used to analyze the role of MUC4 in the anti-inflammatory effects of dexamethasone. Twenty-two patients had NPs resistant to oral corticosteroids. MUC4 expression was upregulated in these patients. In siRNA-MUC4 BEAS-2B airway epithelial cells dexamethasone produced higher anti-inflammatory effects, increased inhibition of phospho-extracellular signal-regulated kinase 1/2, increased mitogen-activated protein kinase phosphatase 1 expression, and increased glucocorticoid response element activation. Immunoprecipitation and immunofluorescence experiments revealed that MUC4β forms a complex with GRα in the nuclei of NP epithelial cells from corticosteroid-resistant patients. MUC4β participates in the corticosteroid resistance process, inhibiting normal GRα nuclear function. The high expression of MUC4 in patients with CRSwNP might participate in corticosteroid resistance.

Kaempferol Inhibits Endoplasmic Reticulum Stress-Associated Mucus Hypersecretion in Airway Epithelial Cells And Ovalbumin-Sensitized Mice.

Mucus hypersecretion is an important pathological feature of chronic airway diseases, such as asthma and pulmonary diseases. MUC5AC is a major component of the mucus matrix forming family of mucins in the airways. The initiation of endoplasmic reticulum (ER)-mediated stress responses contributes to the pathogenesis of airway diseases. The present study investigated that ER stress was responsible for airway mucus production and this effect was blocked by the flavonoid kaempferol. Oral administration of ≥10 mg/kg kaempferol suppressed mucus secretion and goblet cell hyperplasia observed in the bronchial airway and lung of BALB/c mice sensitized with ovalbumin (OVA). TGF-β and tunicamycin promoted MUC5AC induction after 72 h in human bronchial airway epithelial BEAS-2B cells, which was dampened by 20 μM kaempferol. Kaempferol inhibited tunicamycin-induced ER stress of airway epithelial cells through disturbing the activation of the ER transmembrane sensor ATF6 and IRE1α. Additionally, this compound demoted the induction of ER chaperones such as GRP78 and HSP70 and the splicing of XBP-1 mRNA by tunicamycin. The in vivo study further revealed that kaempferol attenuated the induction of XBP-1 and IRE1α in epithelial tissues of OVA-challenged mice. TGF-β and tunicamycin induced TRAF2 with JNK activation and such induction was deterred by kaempferol. The inhibition of JNK activation encumbered the XBP-1 mRNA splicing and MUC5AC induction by tunicamycin and TGF-β. These results demonstrate that kaempferol alleviated asthmatic mucus hypersecretion through blocking bronchial epithelial ER stress via the inhibition of IRE1α-TRAF2-JNK activation. Therefore, kaempferol may be a potential therapeutic agent targeting mucus hypersecretion-associated pulmonary diseases.