Cholinergic sensing of allergen exposure by airway epithelium promotes type 2 immunity in the lungs.

BACKGROUND: Nonneuronal cells, including epithelial cells, can produce acetylcholine (ACh). Muscarinic ACh receptor antagonists are used clinically to treat asthma and other medical conditions; however, knowledge regarding the roles of ACh in type 2 immunity is limited. OBJECTIVE: Our aim was to investigate the roles of epithelial ACh in allergic immune responses. METHODS: Human bronchial epithelial (HBE) cells were cultured with allergen extracts, and their ACh production and IL-33 secretion were studied in vitro. To investigate immune responses in vivo, naive BALB/c mice were treated intranasally with different muscarinic ACh receptor antagonists and then exposed intranasally to allergens. RESULTS: At steady state, HBE cells expressed cellular components necessary for ACh production, including choline acetyltransferase and organic cation transporters. Exposure to allergens caused HBE cells to rapidly release ACh into the extracellular medium. Pharmacologic or small-interfering RNA-based blocking of ACh production or autocrine action through the M3 muscarinic ACh receptors in HBE cells suppressed allergen-induced ATP release, calcium mobilization, and extracellular secretion of IL-33. When naive mice were exposed to allergens, ACh was quickly released into the airway lumen. A series of clinical M3 muscarinic ACh receptor antagonists inhibited allergen-induced IL-33 secretion and innate type 2 immune response in the mouse airways. In a preclinical murine model of asthma, an ACh receptor antagonist suppressed allergen-induced airway inflammation and airway hyperreactivity. CONCLUSIONS: ACh is released quickly by airway epithelial cells on allergen exposure, and it plays an important role in type 2 immunity. The epithelial ACh system can be considered a therapeutic target in allergic airway diseases.

ATP functions as a primary alarmin in allergen-induced type 2 immunity.

Environmental allergens that interact with the airway epithelium can activate cellular stress pathways that lead to the release of danger signals known as alarmins. The mechanisms of alarmin release are distinct from damage-associated molecular patterns (DAMPs), which typically escape from cells after loss of plasma membrane integrity. Oxidative stress represents a form of allergen-induced cellular stress that stimulates oxidant-sensing mechanisms coupled to pathways, which facilitate alarmin mobilization and efflux across the plasma membrane. In this review, we highlight examples of alarmin release and discuss their roles in the initiation of type 2 immunity and allergic airway inflammation. In addition, we discuss the concept of alarmin amplification, where "primary" alarmins, which are directly released in response to a specific cellular stress, stimulate additional signaling pathways that lead to secretion of "secondary" alarmins that include proinflammatory cytokines, such as IL-33, as well as genomic and mitochondrial DNA that coordinate or amplify type 2 immunity. Accordingly, allergen-evoked cellular stress can elicit a hierarchy of alarmin signaling responses from the airway epithelium that trigger local innate immune reactions, impact adaptive immunity, and exacerbate diseases including asthma and other chronic inflammatory conditions that affect airway function.

Allergen-induced DNA release by the airway epithelium amplifies type 2 immunity.

BACKGROUND: Alternaria alternata and house dust mite exposure evokes IL-33 secretion from the airway epithelium, which functions as an alarmin to stimulate type 2 immunity. Extracellular DNA (eDNA) is also an alarmin that intensifies inflammation in cystic fibrosis, chronic obstructive pulmonary disease, and asthma. OBJECTIVE: We investigated the mechanisms underlying allergen-evoked DNA mobilization and release from the airway epithelium and determined the role of eDNA in type 2 immunity. METHODS: Human bronchial epithelial (hBE) cells were used to characterize allergen-induced DNA mobilization and extracellular release using comet assays to measure DNA fragmentation, Qubit double-stranded DNA assays to measure DNA release, and DNA sequencing to determine eDNA composition. Mice were used to investigate the role of eDNA in type 2 immunity. RESULTS: Alternaria extract rapidly induces mitochondrial and nuclear DNA release from human bronchial epithelial cells, whereas house dust mite extract induces mitochondrial DNA release. Caspase-3 is responsible for nuclear DNA fragmentation and becomes activated after cleavage by furin. Analysis of secreted nuclear DNA showed disproportionally higher amounts of promotor and exon sequences and lower intron and intergenic regions compared to predictions of random DNA fragmentation. In mice, Alternaria-induced type 2 immune responses were blocked by pretreatment with a DNA scavenger. In caspase-3-deficient mice, Alternaria-induced DNA release was suppressed. Furthermore, intranasal administration of mouse genomic DNA with Alternaria amplified secretion of IL-5 and IL-13 into bronchoalveolar lavage fluid while DNA alone had no effect. CONCLUSION: These findings highlight a novel, allergen-induced mechanism of rapid DNA release that amplifies type 2 immunity in airways.

Airway Exposure to Polyethyleneimine Nanoparticles Induces Type 2 Immunity by a Mechanism Involving Oxidative Stress and ATP Release.

Polyethyleneimine (PEI) induced immune responses were investigated in human bronchial epithelial (hBE) cells and mice. PEI rapidly induced ATP release from hBE cells and pretreatment with glutathione (GSH) blocked the response. PEI activated two conductive pathways, VDAC-1 and pannexin 1, which completely accounted for ATP efflux across the plasma membrane. Moreover, PEI increased intracellular Ca2+ concentration ([Ca2+]i), which was reduced by the pannexin 1 inhibitor, 10Panx (50 µM), the VDAC-1 inhibitor, DIDS (100 µM), and was nearly abolished by pretreatment with GSH (5 mM). The increase in [Ca2+]i involved Ca2+ uptake through two pathways, one blocked by oxidized ATP (oATP, 300 µM) and another that was blocked by the TRPV-1 antagonist A784168 (100 nM). PEI stimulation also increased IL-33 mRNA expression and protein secretion. In vivo experiments showed that acute (4.5 h) PEI exposure stimulated secretion of Th2 cytokines (IL-5 and IL-13) into bronchoalveolar lavage (BAL) fluid. Conjugation of PEI with ovalbumin also induced eosinophil recruitment and secretion of IL-5 and IL-13 into BAL fluid, which was inhibited in IL-33 receptor (ST2) deficient mice. In conclusion, PEI-induced oxidative stress stimulated type 2 immune responses by activating ATP-dependent Ca2+ uptake leading to IL-33 secretion, similar to allergens derived from Alternaria.

Fungal allergen-induced IL-33 secretion involves cholesterol-dependent, VDAC-1-mediated ATP release from the airway epithelium.

KEY POINTS: Alternaria aeroallergens induce the release of ATP from human bronchial epithelial (HBE) cells by activating a conductive pathway involving voltage-dependent anion channel-1 (VDAC-1) and by exocytosis of ATP localized within membrane vesicles. Inhibition of VDAC-1 blocked Alternaria-evoked Ca2+ uptake across the plasma membrane of HBE cells and interleukin (IL)-33 release into the extracellular media. Reducing cholesterol content with a cholesterol scavenger (ß-methylcyclodextrin) or statin compound (simvastatin) blocked ATP and IL-33 release by lowering the expression of VDAC-1 in the plasma membrane. Pretreatment with simvastatin for 24 h also inhibited the increase in tight junction macromolecule permeability that occurs following Alternaria exposure. These results establish a novel role for VDAC-1 as a mechanism underlying ATP release induced by fungal allergens and suggests a possible therapeutic use for cholesterol lowering compounds in reducing Alternaria-stimulated allergic inflammation. ABSTRACT: Human bronchial epithelial (HBE) cells exposed to allergens derived from the common saprophytic fungus, Alternaria alternata release ATP, which in turn stimulates P2X7 receptor-mediated Ca2+ uptake across the plasma membrane. The subsequent increase in intracellular calcium concentration induces proteolytic processing and secretion of interleukin (IL)-33, a critical cytokine involved in the initiation of allergic airway inflammation. A major objective of the present study was to identify the mechanism responsible for conductive ATP release. The results show that pretreatment of HBE cells with inhibitors of the voltage-dependent anion channel-1 (VDAC-1) or treatment with a VDAC-1 selective blocking antibody or silencing mRNA expression of the channel by RNA interference, inhibit Alternaria-evoked ATP release. Moreover, inhibition of VDAC-1 channel activity or reducing protein expression blocked the secretion of IL-33. Similarly, reducing the cholesterol content of HBE cells with simvastatin or the cholesterol scavenger ß-methylcyclodextrin also blocked ATP release and IL-33 secretion by decreasing the level of VDAC-1 expression in the plasma membrane. In addition, simvastatin inhibited the increase in tight junction macromolecule permeability that was previously observed after Alternaria exposure. These results demonstrate a novel function for VDAC-1 as the conductive mechanism responsible for Alternaria-induced ATP release, an essential early step in the processing, mobilization and secretion of IL-33 by the airway epithelium. Furthermore, the simvastatin-evoked reduction of VDAC-1 expression in the plasma membrane, suggests the possibility that cholesterol lowering compounds may be beneficial in alleviating allergic airway inflammation induced by fungal allergens.

Oxidative stress, autophagy and airway ion transport.

Mucociliary clearance is critically important in protecting the airways from infection and from the harmful effects of smoke and various inspired substances known to induce oxidative stress and persistent inflammation. An essential feature of the clearance mechanism involves regulation of the periciliary liquid layer on the surface of the airway epithelium, which is necessary for normal ciliary beating and maintenance of mucus hydration. The underlying ion transport processes associated with airway surface hydration include epithelial Na+ channel-dependent Na+ absorption occurring in parallel with CFTR and Ca2+-activated Cl- channel-dependent anion secretion, which are coordinately regulated to control the depth of the periciliary liquid layer. Oxidative stress is known to cause both acute and chronic effects on airway ion transport function, and an increasing number of studies in the past few years have identified an important role for autophagy as part of the physiological response to the damaging effects of oxidation. In this review, recent studies addressing the influence of oxidative stress and autophagy on airway ion transport pathways, along with results showing the potential of autophagy modulators in restoring the function of ion channels involved in transepithelial electrolyte transport necessary for effective mucociliary clearance, are presented.

Knob protein enhances epithelial barrier integrity and attenuates airway inflammation.

BACKGROUND: Altered epithelial physical and functional barrier properties along with TH1/TH2 immune dysregulation are features of allergic asthma. Regulation of junction proteins to improve barrier function of airway epithelial cells has the potential for alleviation of allergic airway inflammation. OBJECTIVE: We sought to determine the immunomodulatory effect of knob protein of the adenoviral capsid on allergic asthma and to investigate its mechanism of action on airway epithelial junction proteins and barrier function. METHODS: Airway inflammation, including junction protein expression, was evaluated in allergen-challenged mice with and without treatment with knob. Human bronchial epithelial cells were exposed to knob, and its effects on expression of junction proteins and barrier integrity were determined. RESULTS: Administration of knob to allergen-challenged mice suppressed airway inflammation (eosinophilia, airway hyperresponsiveness, and IL-5 levels) and prevented allergen-induced loss of airway epithelial occludin and E-cadherin expression. Additionally, knob decreased expression of TH2-promoting inflammatory mediators, specifically IL-33, by murine lung epithelial cells. At a cellular level, treatment of human bronchial epithelial cells with knob activated c-Jun N-terminal kinase, increased expression of occludin and E-cadherin, and enhanced epithelial barrier integrity. CONCLUSION: Increased expression of junction proteins mediated by knob leading to enhanced epithelial barrier function might mitigate the allergen-induced airway inflammatory response, including asthma.

P2Y receptor regulation of K2P channels that facilitate K+ secretion by human mammary epithelial cells.

The objective of this study was to determine the molecular identity of ion channels involved in K+ secretion by the mammary epithelium and to examine their regulation by purinoceptor agonists. Apical membrane voltage-clamp experiments were performed on human mammary epithelial cells where the basolateral membrane was exposed to the pore-forming antibiotic amphotericin B dissolved in a solution with intracellular-like ionic composition. Addition of the Na+ channel inhibitor benzamil reduced the basal current, consistent with inhibition of Na+ uptake across the apical membrane, whereas the KCa3.1 channel blocker TRAM-34 produced an increase in current resulting from inhibition of basal K+ efflux. Treatment with two-pore potassium (K2P) channel blockers quinidine, bupivacaine and a selective TASK1/TASK3 inhibitor (PK-THPP) all produced concentration-dependent inhibition of apical K+ efflux. qRT-PCR experiments detected mRNA expression for nine K2P channel subtypes. Western blot analysis of biotinylated apical membranes and confocal immunocytochemistry revealed that at least five K2P subtypes (TWIK1, TREK1, TREK2, TASK1, and TASK3) are expressed in the apical membrane. Apical UTP also increased the current, but pretreatment with the PKC inhibitor GF109203X blocked the response. Similarly, direct activation of PKC with phorbol 12-myristate 13-acetate produced a similar increase in current as observed with UTP. These results support the conclusion that the basal level of K+ secretion involves constitutive activity of apical KCa3.1 channels and multiple K2P channel subtypes. Apical UTP evoked a transient increase in KCa3.1 channel activity, but over time caused persistent inhibition of K2P channel function leading to an overall decrease in K+ secretion.

Large-conductance Ca2+ -activated K+ channel activation by apical P2Y receptor agonists requires hydrocortisone in differentiated airway epithelium.

KEY POINTS: Hydrocortisone (HC) is required for activation of large-conductance Ca2+ -activated K+ current (BK) by purinergic receptor agonists. HC reduces insertion of the stress-regulated exon (STREX) in the KCNMA1 gene, permitting protein kinase C (PKC)-dependent channel activation. Overlapping and unique purinergic signalling regions exist at the apical border of differentiated surface cells. BK channels localize in the cilia of surface cells. ABSTRACT: In the present study we investigated the role of hydrocortisone (HC) on uridine-5'-triphosphate (UTP)-stimulated ion transport in differentiated, pseudostratified epithelia derived from normal human bronchial basal cells. The presence of a UTP-stimulated, paxilline-sensitive large-conductance Ca2+ -activated K+ (BK) current was demonstrated in control epithelia but was not stimulated in epithelia differentiated in the absence of HC (HC0). Addition of the BK channel opener NS11021 directly activated channels in control epithelia; however, under HC0 conditions, activation only occurred when UTP was added after NS11021. The PKC inhibitors GF109203x and Gö6983 blocked BK activation by UTP in control epithelia, suggesting that PKC-mediated phosphorylation plays a permissive role in purinoceptor-stimulated BK activation. Moreover, HC0 epithelia expressed significantly more KCNMA1 containing the stress-regulated exon (STREX), a splice-variant of the α-subunit that displays altered channel regulation by phosphorylation, compared to control epithelia. Furthermore, BK channels as well as purinergic receptors were shown to localize in unique and overlapping domains at the apical membrane of ciliated surface cells. These results establish a previously unrecognized role for glucocorticoids in regulation of BK channels in airway epithelial cells.

Airway epithelial anion secretion and barrier function following exposure to fungal aeroallergens: role of oxidative stress.

Aeroallergens produced by Alternaria alternata can elicit life-threatening exacerbations of asthma in patients sensitized to this fungus. In this study, the effect of Alternaria on ion transport mechanisms underlying mucociliary clearance and airway epithelial barrier function was investigated in human airway epithelial cells. Apical exposure to Alternaria induced an increase in anion secretion that was inhibited by blockers of CFTR and Ca2+-activated Cl- channels. Stimulation of anion secretion was dependent on Ca2+ uptake from the apical solution. Alternaria exposure also produced an increase in reactive oxygen species (ROS) that was blocked by pretreatment with the oxidant scavenger glutathione (GSH). GSH and the NADPH oxidase inhibitor/complex 1 electron transport inhibitor diphenylene iodonium chloride (DPI) blocked ATP release and the increase in intracellular [Ca2+] evoked by AlternariaAlternaria also decreased transepithelial resistance, and a portion of this effect was dependent on the increase in ROS. However, the Alternaria-induced increase in unidirectional dextran (molecular mass = 4,000 Da) flux across the epithelium could not be accounted for by increased oxidative stress. These results support the conclusion that oxidative stress induced by Alternaria was responsible for regulating Ca2+-dependent anion secretion and tight junction electrical resistance that would be expected to affect mucociliary clearance.

The neuropeptide TLQP-21 opposes obesity via C3aR1-mediated enhancement of adrenergic-induced lipolysis.

OBJECTIVES: Obesity is characterized by excessive fat mass and is associated with serious diseases such as type 2 diabetes. Targeting excess fat mass by sustained lipolysis has been a major challenge for anti-obesity therapies due to unwanted side effects. TLQP-21, a neuropeptide encoded by the pro-peptide VGF (non-acronymic), that binds the complement 3a receptor 1 (C3aR1) on the adipocyte membrane, is emerging as a novel modulator of adipocyte functions and a potential target for obesity-associated diseases. The molecular mechanism is still largely uncharacterized. METHODS: We used a combination of pharmacological and genetic gain and loss of function approaches. 3T3-L1 and mature murine adipocytes were used for in vitro experiments. Chronic in vivo experiments were conducted on diet-induced obese wild type, ß1, ß2, ß3-adrenergic receptor (AR) deficient and C3aR1 knockout mice. Acute in vivo lipolysis experiments were conducted on Sprague Dawley rats. RESULTS: We demonstrated that TLQP-21 does not possess lipolytic properties per se. Rather, it enhances ß-AR activation-induced lipolysis by a mechanism requiring Ca2+ mobilization and ERK activation of Hormone Sensitive Lipase (HSL). TLQP-21 acutely potentiated isoproterenol-induced lipolysis in vivo. Finally, chronic peripheral TLQP-21 treatment decreases body weight and fat mass in diet induced obese mice by a mechanism involving ß-adrenergic and C3a receptor activation without associated adverse metabolic effects. CONCLUSIONS: In conclusion, our data identify an alternative pathway modulating lipolysis that could be targeted to diminish fat mass in obesity without the side effects typically observed when using potent pro-lipolytic molecules.

Desensitization of idiopathic pulmonary fibrosis fibroblasts to Alternaria alternata extract-mediated necrotic cell death.

Alternaria alternata is an allergenic fungus and known to cause an upper respiratory tract infection and asthma in humans with compromised immunity. Although A. alternata's effect on airway epithelial cells has previously been examined, the potential role of A. alternata on lung fibroblast viability is not understood. Since lung fibroblasts derived from patients with idiopathic pulmonary fibrosis (IPF) display a distinct phenotype that is resistant to stress and cell death inducing conditions, the investigation of the role of Alternaria on pathological IPF fibroblasts provides a better understanding of the fibrotic process induced by an allergenic fungus. Therefore, we examined cell viability of control and IPF fibroblasts (n = 8 each) in response to A. alternata extract. Control fibroblast cell death was increased while IPF fibroblasts were resistant when exposed to 50-100 µg/mL of A. alternata extract. However, there was no significant difference in kinetics or magnitude of Ca2+ responses from control lung and IPF fibroblasts. In contrast, unlike control fibroblasts, intracellular reactive oxygen species (ROS) levels remained low when IPF cells were treated with A. alternata extracts as a function of time. Caspase 3/7 and TUNEL assay revealed that enhanced cell death caused by A. alternata extract was likely due to necrosis, and 7-AAD assay and the use of sodium pyruvate for ATP generation further supported our findings that IPF fibroblasts become resistant to A. alternata extract-induced necrotic cell death. Our results suggest that exposure to A. alternata potentially worsens the fibrotic process by promoting normal lung fibroblast cell death in patients with IPF.

Differentiation of human bronchial epithelial cells: role of hydrocortisone in development of ion transport pathways involved in mucociliary clearance.

Glucocorticoids strongly influence the mucosal-defense functions performed by the bronchial epithelium, and inhaled corticosteroids are critical in the treatment of patients with inflammatory airway diseases such as asthma, chronic obstructive pulmonary disease, and cystic fibrosis. A common pathology associated with these diseases is reduced mucociliary clearance, a defense mechanism involving the coordinated transport of salt, water, and mucus by the bronchial epithelium, ultimately leading to retention of pathogens and particles in the airways and to further disease progression. In the present study we investigated the role of hydrocortisone (HC) in differentiation and development of the ion transport phenotype of normal human bronchial epithelial cells under air-liquid interface conditions. Normal human bronchial epithelial cells differentiated in the absence of HC (HC0) showed significantly less benzamil-sensitive short-circuit current than controls, as well as a reduced response after stimulation with the selective ß2-adrenergic receptor agonist salbutamol. Apical membrane localization of epithelial Na(+) channel α-subunits was similarly reduced in HC0 cells compared with controls, supporting a role of HC in the trafficking and density of Na(+) channels in the plasma membrane. Additionally, glucocorticoid exposure during differentiation regulated the transcription of cystic fibrosis transmembrane conductance regulator and ß2-adrenergic receptor mRNAs and appeared to be necessary for the expression of cystic fibrosis transmembrane conductance regulator-dependent anion secretion in response to ß2-agonists. HC had no significant effect on surface cell differentiation but did modulate the expression of mucin mRNAs. These findings indicate that glucocorticoids support mucosal defense by regulating critical transport pathways essential for effective mucociliary clearance.

Carvedilol binding to ß2-adrenergic receptors inhibits CFTR-dependent anion secretion in airway epithelial cells.

Carvedilol functions as a nonselective ß-adrenergic receptor (AR)/α1-AR antagonist that is used for treatment of hypertension and heart failure. Carvedilol has been shown to function as an inverse agonist, inhibiting G protein activation while stimulating ß-arrestin-dependent signaling and inducing receptor desensitization. In the present study, short-circuit current (Isc) measurements using human airway epithelial cells revealed that, unlike ß-AR agonists, which increase Isc, carvedilol decreases basal and 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate-stimulated current. The decrease in Isc resulted from inhibition of the cystic fibrosis transmembrane conductance regulator (CFTR). The carvedilol effect was abolished by pretreatment with the ß2-AR antagonist ICI-118551, but not the ß1-AR antagonist atenolol or the α1-AR antagonist prazosin, indicating that its inhibitory effect on Isc was mediated through interactions with apical ß2-ARs. However, the carvedilol effect was blocked by pretreatment with the microtubule-disrupting compound nocodazole. Furthermore, immunocytochemistry experiments and measurements of apical CFTR expression by Western blot analysis of biotinylated membranes revealed a decrease in the level of CFTR protein in monolayers treated with carvedilol but no significant change in monolayers treated with epinephrine. These results demonstrate that carvedilol binding to apical ß2-ARs inhibited CFTR current and transepithelial anion secretion by a mechanism involving a decrease in channel expression in the apical membrane.

Agonist binding to ß-adrenergic receptors on human airway epithelial cells inhibits migration and wound repair.

Human airway epithelial cells express ß-adrenergic receptors (ß-ARs), which regulate mucociliary clearance by stimulating transepithelial anion transport and ciliary beat frequency. Previous studies using airway epithelial cells showed that stimulation with isoproterenol increased cell migration and wound repair by a cAMP-dependent mechanism. In the present study, impedance-sensing arrays were used to measure cell migration and epithelial restitution following wounding of confluent normal human bronchial epithelial (NHBE) and Calu-3 cells by electroporation. Stimulation with epinephrine or the ß2-AR-selective agonist salbutamol significantly delayed wound closure and reduced the mean surface area of lamellipodia protruding into the wound. Treatment with the ß-AR bias agonist carvedilol or isoetharine also produced a delay in epithelial restitution similar in magnitude to epinephrine and salbutamol. Measurements of extracellular signal-regulated kinase phosphorylation following salbutamol or carvedilol stimulation showed no significant change in the level of phosphorylation compared with untreated control cells. However, inhibition of protein phosphatase 2A activity completely blocked the delay in wound closure produced by ß-AR agonists. In Calu-3 cells, where CFTR expression was inhibited by RNAi, salbutamol did not inhibit wound repair, suggesting that ß-AR agonist stimulation and loss of CFTR function share a common pathway leading to inhibition of epithelial repair. Confocal images of the basal membrane of Calu-3 cells labeled with anti-ß1-integrin (clone HUTS-4) antibody showed that treatment with epinephrine or carvedilol reduced the level of activated integrin in the membrane. These findings suggest that treatment with ß-AR agonists delays airway epithelial repair by a G protein- and cAMP-independent mechanism involving protein phosphatase 2A and a reduction in ß1-integrin activation in the basal membrane.

Differential Induction of Type I and Type III Interferons by Swine and Human Origin H1N1 Influenza A Viruses in Porcine Airway Epithelial Cells.

Interferons (IFNs) have been shown to inhibit influenza A virus (IAV) replication and play an essential role in controlling viral infection. Here we studied the kinetics and magnitude of induction of type I and type III IFN transcripts by primary porcine airway epithelial cells (pAECs) in response to swine and human origin IAV. We observed that swine influenza viruses (SIV) replicate more efficiently than the human pandemic influenza A/California/2009 (pH1N1 CA/09) in pAECs. Interestingly, we also found significant difference in kinetics of IFN-ß, IFN-λ1 and IFN-λ3 gene expression by these viruses. While there was delay of up to 12 hours post infection (h p.i.) in induction of IFN genes in pAECs infected with swine IAV A/Sw/Illinois/2008 (H1N1 IL/08), human pH1N1 CA/09 rapidly induced IFN-ß, IFN-λ1 and IFN-λ3 gene expression as early as 4 h p.i. However, the magnitude of IFN-ß and IFN-λ3 induction at 24 h p.i. was not significantly different between the viral strains tested. Additionally, we found that swine H1N1 IL/08 was less sensitive to dsRNA induced antiviral response compared to human pH1N1 CA/09. Our data suggest that the human and swine IAVs differ in their ability to induce and respond to type I and type III interferons in swine cells. Swine origin IAV may have adapted to the pig host by subverting innate antiviral responses to viral infection.

Nuclear localization and functional characteristics of voltage-gated potassium channel Kv1.3.

It is widely known that ion channels are expressed in the plasma membrane. However, a few studies have suggested that several ion channels including voltage-gated K(+) (Kv) channels also exist in intracellular organelles where they are involved in the biochemical events associated with cell signaling. In the present study, Western blot analysis using fractionated protein clearly indicates that Kv1.3 channels are expressed in the nuclei of MCF7, A549, and SNU-484 cancer cells and human brain tissues. In addition, Kv1.3 is located in the plasma membrane and the nucleus of Jurkat T cells. Nuclear membrane hyperpolarization after treatment with margatoxin (MgTX), a specific blocker of Kv1.3 channels, provides evidence for functional channels at the nuclear membrane of A549 cells. MgTX-induced hyperpolarization is abolished in the nuclei of Kv1.3 silenced cells, and the effects of MgTX are dependent on the magnitude of the K(+) gradient across the nuclear membrane. Selective Kv1.3 blockers induce the phosphorylation of cAMP response element-binding protein (CREB) and c-Fos activation. Moreover, Kv1.3 is shown to form a complex with the upstream binding factor 1 in the nucleus. Chromatin immunoprecipitation assay reveals that Sp1 transcription factor is directly bound to the promoter region of the Kv1.3 gene, and the Sp1 regulates Kv1.3 expression in the nucleus of A549 cells. These results demonstrate that Kv1.3 channels are primarily localized in the nucleus of several types of cancer cells and human brain tissues where they are capable of regulating nuclear membrane potential and activation of transcription factors, such as phosphorylated CREB and c-Fos.

Reduced GM1 ganglioside in CFTR-deficient human airway cells results in decreased ß1-integrin signaling and delayed wound repair.

Loss of cystic fibrosis transmembrane conductance regulator (CFTR) function reduces chloride secretion and increases sodium uptake, but it is not clear why CFTR mutation also results in progressive lung inflammation and infection. We previously demonstrated that CFTR-silenced airway cells migrate more slowly during wound repair than CFTR-expressing controls. In addition, CFTR-deficient cells and mouse models have been reported to have altered sphingolipid levels. Here, we investigated the hypothesis that reduced migration in CFTR-deficient airway epithelial cells results from altered sphingolipid composition. We used cell lines derived from a human airway epithelial cell line (Calu-3) stably transfected with CFTR short hairpin RNA (CFTR-silenced) or nontargeting short hairpin RNA (controls). Cell migration was measured by electric cell substrate impedance sensing (ECIS). Lipid analyses, addition of exogenous glycosphingolipids, and immunoblotting were performed. We found that levels of the glycosphingolipid, GM1 ganglioside, were ~60% lower in CFTR-silenced cells than in controls. CFTR-silenced cells exhibited reduced levels of activated ß1-integrin, phosphorylated tyrosine 576 of focal adhesion kinase (pFAK), and phosphorylation of Crk-associated substrate (pCAS). Addition of GM1 (but not GM3) ganglioside to CFTR-silenced cells restored activated ß1-integrin, pFAK, and pCAS to near control levels and partially restored (~40%) cell migration. Our results suggest that decreased GM1 in CFTR-silenced cells depresses ß1-integrin signaling, which contributes to the delayed wound repair observed in these cells. These findings have implications for the pathology of cystic fibrosis, where altered sphingolipid levels in airway epithelial cells could result in a diminished capacity for wound repair after injury.

ATP release and Ca2+ signalling by human bronchial epithelial cells following Alternaria aeroallergen exposure.

Exposure of human bronchial epithelial (HBE) cells from normal and asthmatic subjects to extracts from Alternaria alternata evoked a rapid and sustained release of ATP with greater efficacy observed in epithelial cells from asthmatic patients. Previously, Alternaria allergens were shown to produce a sustained increase in intracellular Ca2+ concentration ([Ca2+]i) that was dependent on the coordinated activation of specific purinergic receptor (P2Y2 and P2X7) subtypes. In the present study, pretreatment with a cell-permeable Ca2+-chelating compound (BAPTA-AM) significantly inhibited ATP release, indicating dependency on [Ca2+]i. Alternaria-evoked ATP release exhibited a greater peak response and a slightly lower EC50 value in cells obtained from asthmatic donors compared to normal control cells. Furthermore, the maximum increase in [Ca2+]i resulting from Alternaria treatment was greater in cells from asthmatic patients compared to normal subjects. The vesicle transport inhibitor brefeldin A and BAPTA-AM significantly blocked Alternaria-stimulated incorporation of fluorescent lipid (FM1-43)-labelled vesicles into the plasma membrane and ATP release. In addition, inhibiting uptake of ATP into exocytotic vesicles with bafilomycin also reduced ATP release comparable to the effects of brefeldin A and BAPTA-AM. These results indicate that an important mechanism for Alternaria-induced ATP release is Ca2+ dependent and involves exocytosis of ATP. Serine and cysteine protease inhibitors also reduced Alternaria-induced ATP release; however, the sustained increase in [Ca2+]i typically observed following Alternaria exposure appeared to be independent of protease-activated receptor (PAR2) stimulation.