Obesity Enhances Non-Th2 Airway Inflammation in a Murine Model of Allergic Asthma.

Obese patients with asthma present with aggravated symptoms that are also harder to treat. Here, we used a mouse model of allergic asthma sensitised and challenged to house dust mite (HDM) extracts to determine whether high-fat-diet consumption would exacerbate the key features of allergic airway inflammation. C57BL/6 mice were intranasally sensitised and challenged with HDM extracts over a duration of 3 weeks. The impact of high-fat-diet (HFD) vs. normal diet (ND) chow was studied on HDM-induced lung inflammation and inflammatory cell infiltration as well as cytokine production. HFD-fed mice had greater inflammatory cell infiltration around airways and blood vessels, and an overall more severe degree of inflammation than in the ND-fed mice (semiquantitative blinded evaluation). Quantitative assessment of HDM-associated Th2 responses (numbers of lung CD4+ T cells, eosinophils, serum levels of allergen-specific IgE as well as the expression of Th2 cytokines (Il5 and Il13)) did not show significant changes between the HFD and ND groups. Interestingly, the HFD group exhibited a more pronounced neutrophilic infiltration within their lung tissues and an increase in non-Th2 cytokines (Il17, Tnfa, Tgf-b, Il-1b). These findings provide additional evidence that obesity triggered by a high-fat-diet regimen may exacerbate asthma by involving non-Th2 and neutrophilic pathways.

NADH Intraperitoneal Injection Prevents Lung Inflammation in a BALB/C Mice Model of Cigarette Smoke-Induced Chronic Obstructive Pulmonary Disease.

Cigarette smoke is one of the main factors in Chronic Obstructive Pulmonary Disease (COPD), a respiratory syndrome marked by persistent respiratory symptoms and increasing airway obstruction. Perturbed NAD+/NADH levels may play a role in various diseases, including lung disorders like COPD. In our study, we investigated the preventive effect of NADH supplementation in an experimental model of COPD induced by cigarette smoke extract (CSE). N = 64 mice randomly distributed in eight groups were injected with NADH (two doses of 100 mg/kg or 200 mg/kg) or dexamethasone (2 mg/kg) before being exposed to CSE for up to 9 weeks. Additionally, NADH supplementation preserved lung antioxidant defenses by preventing the functional loss of key enzymes such as superoxide dismutase (SOD), glutathione peroxidase (GPX), catalase, and the expression levels of glutathione (GSH) (n = 4, p < 0.001). It also reduced oxidative damage markers, such as malondialdehyde (MDA) and nitrites (n = 4, p < 0.001). A marked increase in tissue myeloperoxidase activity was assessed (MPO), confirming neutrophils implication in the inflammatory process. The latter was significantly ameliorated in the NADH-treated groups (p < 0.001). Finally, NADH prevented the CSE-induced secretion of cytokines such as Tumor Necrosis Factor alpha (TNF-α), IL-17, and IFN-y (n = 4, p < 0.001). Our study shows, for the first time, the clinical potential of NADH supplementation in preventing key features of COPD via its unique anti-inflammatory and antioxidant properties.

Aptamer decorated PDA@magnetic silica microparticles for bacteria purification.

One significant constraint in the advancement of biosensors is the signal-to-noise ratio, which is adversely affected by the presence of interfering factors such as blood in the sample matrix. In the present investigation, a specific aptamer binding was chosen for its affinity, while exhibiting no binding affinity towards non-target bacterial cells. This selective binding property was leveraged to facilitate the production of magnetic microparticles decorated with aptamers. A novel assay was developed to effectively isolate S. pneumoniae from PBS or directly from blood samples using an aptamer with an affinity constant of 72.8 nM. The capture experiments demonstrated efficiencies up to 87% and 66% are achievable for isolating spiked S. pneumoniae in 1 mL PBS and blood samples, respectively.

Stressed out - The role of oxidative stress in airway smooth muscle dysfunction in asthma and COPD.

The airway smooth muscle (ASM) surrounding the airways is dysfunctional in both asthma and chronic obstructive pulmonary disease (COPD), exhibiting; increased contraction, increased mass, increased inflammatory mediator release and decreased corticosteroid responsiveness. Due to this dysfunction, ASM is a key contributor to symptoms in patients that remain symptomatic despite optimal provision of currently available treatments. There is a significant body of research investigating the effects of oxidative stress/ROS on ASM behaviour, falling into the following categories; cigarette smoke and associated compounds, air pollutants, aero-allergens, asthma and COPD relevant mediators, and the anti-oxidant Nrf2/HO-1 signalling pathway. However, despite a number of recent reviews addressing the role of oxidative stress/ROS in asthma and COPD, the potential contribution of oxidative stress/ROS-related ASM dysfunction to asthma and COPD pathophysiology has not been comprehensively reviewed. We provide a thorough review of studies that have used primary airway, bronchial or tracheal smooth muscle cells to investigate the role of oxidative stress/ROS in ASM dysfunction and consider how they could contribute to the pathophysiology of asthma and COPD. We summarise the current state of play with regards to clinical trials/development of agents targeting oxidative stress and associated limitations, and the adverse effects of oxidative stress on the efficacy of current therapies, with reference to ASM related studies where appropriate. We also identify limitations in the current knowledge of the role of oxidative stress/ROS in ASM dysfunction and identify areas for future research.

TGFß1 induces resistance of human lung myofibroblasts to cell death via down-regulation of TRPA1 channels.

BACKGROUND AND PURPOSE: TGFß1-mediated myofibroblast activation contributes to pathological fibrosis in many diseases including idiopathic pulmonary fibrosis (IPF), where myofibroblast resistance to oxidant-mediated apoptosis is also evident. We therefore investigated the involvement of redox-sensitive TRPA1 ion channels on human lung myofibroblasts (HLMFs) cell death and TGFß1-mediated pro-fibrotic responses. EXPERIMENTAL APPROACH: The effects of TGFß1 stimulation on TRPA1 expression and cell viability was studied in HLMFs derived from IPF patients and non-fibrotic patients. We also examined a model of TGFß1-dependent fibrogenesis in human lung. We used qRT-PCR, immunofluorescent assays, overexpression with lentiviral vectors and electrophysiological methods. KEY RESULTS: TRPA1 mRNA, protein and ion currents were expressed in HLMFs derived from both non-fibrotic patient controls and IPF patients, and expression was reduced by TGFß1. TRPA1 mRNA was also down-regulated by TGFß1 in a model of lung fibrogenesis in human lung. TRPA1 over-expression or activation induced HLMF apoptosis, and activation of TRPA1 channel activation by H2 O2 induced necrosis. TRPA1 inhibition following TGFß1 down-regulation or pharmacological inhibition, protected HLMFs from both apoptosis and necrosis. Lentiviral vector mediated TRPA1 expression was also found to induce sensitivity to H2 O2 induced cell death in a TRPA1-negative HEK293T cell line. CONCLUSION AND IMPLICATIONS: TGFß1 induces resistance of HLMFs to TRPA1 agonist- and H2 O2 -mediated cell death via down-regulation of TRPA1 channels. Our data suggest that therapeutic strategies which prevent TGFß1-dependent down-regulation of TRPA1 may reduce myofibroblast survival in IPF and therefore improve clinical outcomes.

Potential Role of Mast Cells in Regulating Corticosteroid Insensitivity in Severe Asthma.

The mechanisms driving corticosteroid insensitivity in asthma are still unclear although evidence points toward a potential role of lung mast cells. Indeed, a number of in vitro studies using various cell types showed that different mediators produced by activated mast cells, including cytokines, have the capacity to interfere with the therapeutic action of corticosteroids. In patients with severe allergic refractory asthma, the anti-IgE monoclonal antibody (mAb), Omalizumab, has been shown to be associated with a marked reduction in inhaled and systemic use of corticosteroids, further suggesting a key role of mast cells in the poor response of patients to these drugs. The present chapter will discuss the possible underlying mechanisms by which mast cells could contribute to reducing corticosteroid sensitivity seen in patients with severe asthma.

Human Lung Mast Cells Impair Corticosteroid Responsiveness in Human Airway Smooth Muscle Cells.

The mechanisms underlying corticosteroid insensitivity in severe asthma have not been elucidated although some indirect clinical evidence points toward a role of mast cells. Here, we tested the hypothesis that mast cells can drive corticosteroid insensitivity in airway smooth muscle cells, a key player in asthma pathogenesis. Conditioned media from resting or FcεR1-activated human lung mast cells were incubated with serum-deprived ASM cells (1:4 dilution, 24 h) to determine their impact on the anti-inflammatory action of fluticasone on ASM cell chemokine expression induced by TNFα (10 ng/ml). Conditioned media from FcεR1-activated mast cells (but not that from non-activated mast cells or control media) significantly reduced the ability of 100 nM fluticasone to suppress ASM TNFα-dependent CCL5 and CXCL10 production at both mRNA and protein levels. In contrast, fluticasone inhibition of CXCL-8 production by TNFα was still preserved in the presence of activated mast cell conditioned media. Transcriptomic analysis validated by individual qPCR assays revealed that activated mast cell conditioned media dramatically reduced the number of anti-inflammatory genes induced by fluticasone in ASM cells. Our study demonstrates for the first time that conditioned media from FcεR1-activated mast cells blunt the anti-inflammatory action of corticosteroids in ASM cells by altering their transactivation properties. Because infiltration of mast cells within the ASM bundles is a defining feature of asthma, mast cell-derived mediators may contribute to the glucocorticoid insensitivity present in severe asthma.

GTS-21, an α7nAChR agonist, suppressed the production of key inflammatory mediators by PBMCs that are elevated in COPD patients and associated with impaired lung function.

Chronic obstructive pulmonary disease (COPD) is a lung inflammatory disease characterized by progressive airflow limitation, chronic respiratory symptoms and frequent exacerbations. There is an unmet need to identify novel therapeutic alternatives beside bronchodilators that prevent disease progression. Levels of both Nitric Oxide (NO) and IL-6 were significantly increased in the plasma of patients in the exacerbation phase (ECOPD, n = 13) when compared to patients in the stable phase (SCOPD, n = 38). Levels of both NO and IL-6 were also found to inversely correlate with impaired lung function (%FEV1 predicted). In addition, there was a strong positive correlation between levels of IL-6 and NO found in the plasma of patients and those spontaneously produced by their peripheral blood mononuclear cells (PBMCs), identifying these cells as a major source of these key inflammatory mediators in COPD. GTS-21, an agonist for the alpha 7 nicotinic receptors (α7nAChR), was found to exert immune-modulatory actions in PBMCs of COPD patients by suppressing the production of IL-6 and NO. This study provides the first evidence supporting the therapeutic potential of α7nAChR agonists in COPD due to their ability to suppress the production of key inflammatory markers associated with disease severity.

Important lessons learned from studies on the pharmacology of glucocorticoids in human airway smooth muscle cells: Too much of a good thing may be a problem.

Glucocorticoids (GCs) are the treatment of choice for chronic inflammatory diseases such as asthma. Despite proven effective anti-inflammatory and immunosuppressive effects, long-term and/or systemic use of GCs can potentially induce adverse effects. Strikingly, some recent experimental evidence suggests that GCs may even exacerbate some disease outcomes. In asthma, airway smooth muscle (ASM) cells are among the targets of GC therapy and have emerged as key contributors not only to bronchoconstriction, but also to airway inflammation and remodeling, as implied by experimental and clinical evidence. We here will review the beneficial effects of GCs on ASM cells, emphasizing the differential nature of GC effects on pro-inflammatory genes and on other features associated with asthma pathogenesis. We will also summarize evidence describing how GCs can potentially promote pro-inflammatory and remodeling features in asthma with a specific focus on ASM cells. Finally, some of the possible solutions to overcome these unanticipated effects of GCs will be discussed.

Tensin1 expression and function in chronic obstructive pulmonary disease.

Chronic obstructive pulmonary disease (COPD) constitutes a major cause of morbidity and mortality. Genome wide association studies have shown significant associations between airflow obstruction or COPD with a non-synonymous SNP in the TNS1 gene, which encodes tensin1. However, the expression, cellular distribution and function of tensin1 in human airway tissue and cells are unknown. We therefore examined these characteristics in tissue and cells from controls and people with COPD or asthma. Airway tissue was immunostained for tensin1. Tensin1 expression in cultured human airway smooth muscle cells (HASMCs) was evaluated using qRT-PCR, western blotting and immunofluorescent staining. siRNAs were used to downregulate tensin1 expression. Tensin1 expression was increased in the airway smooth muscle and lamina propria in COPD tissue, but not asthma, when compared to controls. Tensin1 was expressed in HASMCs and upregulated by TGFß1. TGFß1 and fibronectin increased the localisation of tensin1 to fibrillar adhesions. Tensin1 and α-smooth muscle actin (αSMA) were strongly co-localised, and tensin1 depletion in HASMCs attenuated both αSMA expression and contraction of collagen gels. In summary, tensin1 expression is increased in COPD airways, and may promote airway obstruction by enhancing the expression of contractile proteins and their localisation to stress fibres in HASMCs.

Airway smooth muscle cells are insensitive to the anti-proliferative effects of corticosteroids: The novel role of insulin growth factor binding Protein-1 in asthma.

Airway remodeling in asthma manifests, in part, as enhanced airway smooth muscle (ASM) mass, due to myocyte proliferation. While the anti-proliferative effects of glucocorticoid (GC) were investigated in normal ASM cells (NASMC), little is known about such effects in ASM cells derived from asthma subjects (AASMC). We posit that GC differentially modulates mitogen-induced proliferation of AASMC and NASMC. Cells were cultured, starved, then treated with Epidermal growth factor (EGF) (10 ng/ml) and Platelet-derived growth factor (PDGF) (10 ng/ml) for 24 h and/or fluticasone propionate (FP) (100 nM) added 2 h before. Cell counts and flow cytometry analyses showed that FP failed to decrease the cell number of and DNA synthesis in AASMC irrespective of mitogens used. We also examine the ability of Insulin Growth Factor Binding Protein-1 (IGFBP-1), a steroid-inducible gene that deters cell growth in other cell types, to inhibit proliferation of AASMC where FP failed. We found that FP increased IGFBP1 mRNA and protein levels. Interestingly, the addition of IGFBP1 (1 µg/ml) to FP completely inhibited the proliferation of AASMC irrespective to the mitogens used. Further investigation of different signaling molecules involved in ASM growth and GC receptor functions (Protein kinase B (PKB/AKT), Mitogen-activated protein kinases (MAPKs), Focal Adhesion Kinase (FAK)) showed that IGFBP-1 selectively decreased mitogen-induced p38 phosphorylation in AASMC. Collectively, our results show the insensitivity of AASMC to the anti-proliferative effects of GC, and demonstrate the ability of IGFBP1 to modulate AASMC growth representing, hence, a promising strategy to control ASM growth in subjects with GC insensitive asthma.

Non-genomic Effects of Glucocorticoids: An Updated View.

Glucocorticoid (GC) anti-inflammatory effects generally require a prolonged onset of action and involve genomic processes. Because of the rapidity of some of the GC effects, however, the concept that non-genomic actions may contribute to GC mechanisms of action has arisen. While the mechanisms have not been completely elucidated, the non-genomic effects may play a role in the management of inflammatory diseases. For instance, we recently reported that GCs 'rapidly' enhanced the effects of bronchodilators, agents used in the treatment of allergic asthma. In this review article, we discuss (i) the non-genomic effects of GCs on pathways relevant to the pathogenesis of inflammatory diseases and (ii) the putative role of the membrane GC receptor. Since GC side effects are often considered to be generated through its genomic actions, understanding GC non-genomic effects will help design GCs with a better therapeutic index.

A model of human lung fibrogenesis for the assessment of anti-fibrotic strategies in idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a progressive interstitial lung disease with limited therapeutic options. KCa3.1 ion channels play a critical role in TGFß1-dependent pro-fibrotic responses in human lung myofibroblasts. We aimed to develop a human lung parenchymal model of fibrogenesis and test the efficacy of the selective KCa3.1 blocker senicapoc. 2 mm3 pieces of human lung parenchyma were cultured for 7 days in DMEM ± TGFß1 (10 ng/ml) and pro-fibrotic pathways examined by RT-PCR, immunohistochemistry and collagen secretion. Following 7 days of culture with TGFß1, 41 IPF- and fibrosis-associated genes were significantly upregulated. Immunohistochemical staining demonstrated increased expression of ECM proteins and fibroblast-specific protein after TGFß1-stimulation. Collagen secretion was significantly increased following TGFß1-stimulation. These pro-fibrotic responses were attenuated by senicapoc, but not by dexamethasone. This 7 day ex vivo model of human lung fibrogenesis recapitulates pro-fibrotic events evident in IPF and is sensitive to KCa3.1 channel inhibition. By maintaining the complex cell-cell and cell-matrix interactions of human tissue, and removing cross-species heterogeneity, this model may better predict drug efficacy in clinical trials and accelerate drug development in IPF. KCa3.1 channels are a promising target for the treatment of IPF.

ß2-Adrenoceptor Function in Asthma.

ß2-adrenoceptor agonists, often used in combination with corticosteroids, have been extensively used for the treatment of asthma. However, concerns have been raised regarding their adverse effects and safety including poor asthma control, life-threatening exacerbations, exacerbations that often require hospitalization, and asthma-related deaths. The question as to whether these adverse effects relate to the loss of their bronchoprotective action remains an interesting possibility. In the chapter, we will review the experimental evidence that describes the different potential factors and associated mechanisms that can blunt the therapeutic action of ß2-adrenoceptor agonists in asthma. We show here evidence that various key inflammatory cytokines, growth factors, some respiratory viruses, certain allergens, unknown factors present in serum from atopic asthmatics have the capacity to impair ß2-adrenoceptor function in airway smooth muscle, the main target of these drugs. More importantly, we present our latest research describing the role played by mast cells in impairing ß2-adrenoceptor function. Although no definitive conclusion could be made regarding the implication of one single mechanism, receptor uncoupling, or receptor desensitization due to phosphorylation represents the main inhibitory pathways associated with a loss of ß2-adrenoceptor function in airway smooth muscle. Targeting the pathways leading to ß2-adrenoceptor dysfunction will likely provide novel therapies to improve the efficacy of ß2-agonists in asthma.

Mapping physiological G protein-coupled receptor signaling pathways reveals a role for receptor phosphorylation in airway contraction.

G protein-coupled receptors (GPCRs) are known to initiate a plethora of signaling pathways in vitro. However, it is unclear which of these pathways are engaged to mediate physiological responses. Here, we examine the distinct roles of Gq/11-dependent signaling and receptor phosphorylation-dependent signaling in bronchial airway contraction and lung function regulated through the M3-muscarinic acetylcholine receptor (M3-mAChR). By using a genetically engineered mouse expressing a G protein-biased M3-mAChR mutant, we reveal the first evidence, to our knowledge, of a role for M3-mAChR phosphorylation in bronchial smooth muscle contraction in health and in a disease state with relevance to human asthma. Furthermore, this mouse model can be used to distinguish the physiological responses that are regulated by M3-mAChR phosphorylation (which include control of lung function) from those responses that are downstream of G protein signaling. In this way, we present an approach by which to predict the physiological/therapeutic outcome of M3-mAChR-biased ligands with important implications for drug discovery.

Bidirectional Counterregulation of Human Lung Mast Cell and Airway Smooth Muscle ß2 Adrenoceptors.

Human lung mast cells (HLMCs) play a central role in asthma pathogenesis through their relocation to the airway smooth muscle (ASM) bundles. ß2 adrenoceptor (ß2-AR)-agonists are used to relieve bronchoconstriction in asthma, but may reduce asthma control, particularly when used as monotherapy. We hypothesized that HLMC and human ASM cell (HASMC) responsiveness to ß2-AR agonists would be attenuated when HLMCs are in contact with HASMCs. Cells were cultured in the presence of the short-acting ß2-agonist albuterol, and the long-acting ß2-agonists formoterol and olodaterol. Constitutive and FcεRI-dependent HLMC histamine release, HASMC contraction, and ß2-AR phosphorylation at Tyr(350) were assessed. Constitutive HLMC histamine release was increased in HLMC-HASMC coculture and this was enhanced by ß2-AR agonists. Inhibition of FcεRI-dependent HLMC mediator release by ß2-agonists was greatly reduced in HLMC-HASMC coculture. These effects were reversed by neutralization of stem cell factor (SCF) or cell adhesion molecule 1 (CADM1). ß2-AR agonists did not prevent HASMC contraction when HLMCs were present, but this was reversed by fluticasone. ß2-AR phosphorylation at Tyr(350) occurred within 5 min in both HLMCs and HASMCs when the cells were cocultured, and was inhibited by neutralizing SCF or CADM1. HLMC interactions with HASMCs via CADM1 and Kit inhibit the potentially beneficial effects of ß2-AR agonists on these cells via phosphorylation of the ß2-AR. These results may explain the potentially adverse effects of ß2-ARs agonists when used for asthma therapy. Targeting SCF and CADM1 may enhance ß2-AR efficacy, particularly in corticosteroid-resistant patients.

Fetal human airway smooth muscle cell production of leukocyte chemoattractants is differentially regulated by fluticasone.

BACKGROUND: Adult human airway smooth muscle (ASM) produce cytokines involved in recruitment and survival of leukocytes within airway walls. Cytokine generation by adult ASM is glucocorticoid-sensitive. Whether developing lung ASM produces cytokines in a glucocorticoid-sensitive fashion is unknown. METHODS: Cultured fetal human ASM cells stimulated with TNF-α (0-20 ng/ml) were incubated with TNF-α receptor-blocking antibodies, fluticasone (1 and 100 nm), or vehicle. Supernatants and cells were assayed for the production of CCL5, CXCL10, and CXCL8 mRNA and protein and glucocorticoid receptor phosphorylation. RESULTS: CCL5, CXCL10, and CXCL8 mRNA and protein production by fetal ASM cell was significantly and dose-dependently following TNF-α treatment. Cytokine mRNA and protein production were effectively blocked by TNF-α R1 and R2 receptor neutralizing antibodies but variably inhibited by fluticasone. TNF-α-induced TNF-R1 and R2 receptor mRNA expression was only partially attenuated by fluticasone. Glucocorticoid receptor phosphorylation at serine (Ser) 211 but not at Ser 226 was enhanced by fluticasone. CONCLUSION: Production of CCL5, CXCL10, and CXCL8 by fetal ASM appears to involve pathways that are both qualitatively and mechanistically distinct to those described for adult ASM. The findings imply developing ASM has potential to recruit leukocyte into airways and, therefore, of relevance to childhood airway diseases.

Lipoxin A4 Attenuates Constitutive and TGF-ß1-Dependent Profibrotic Activity in Human Lung Myofibroblasts.

Idiopathic pulmonary fibrosis (IPF) is a common, progressive, and invariably lethal interstitial lung disease with no effective therapy. The key cell driving the development of fibrosis is the myofibroblast. Lipoxin A4 (LXA4) is an anti-inflammatory lipid, important in the resolution of inflammation, and it has potential antifibrotic activity. However, the effects of LXA4 on primary human lung myofibroblasts (HLMFs) have not previously been investigated. Therefore, the aim of this study was to examine the effects of LXA4 on TGF-ß1-dependent responses in IPF- and nonfibrotic control (NFC)-derived HLMFs. HLMFs were isolated from IPF and NFC patients and grown in vitro. The effects of LXA4 on HLMF proliferation, collagen secretion, α-smooth muscle actin (αSMA) expression, and Smad2/3 activation were examined constitutively and following TGF-ß1 stimulation. The LXA4 receptor (ALXR) was expressed in both NFC- and IPF-derived HLMFs. LXA4 (10(-10) and 10(-8) mol) reduced constitutive αSMA expression, actin stress fiber formation, contraction, and nuclear Smad2/3, indicating regression from a myofibroblast to fibroblast phenotype. LXA4 also significantly inhibited FBS-dependent proliferation and TGF-ß1-dependent collagen secretion, αSMA expression, and Smad2/3 nuclear translocation in IPF-derived HLMFs. LXA4 did not inhibit Smad2/3 phosphorylation. In summary, LXA4 attenuated profibrotic HLMF activity and promoted HLMF regression to a quiescent fibroblast phenotype. LXA4 or its stable analogs delivered by aerosol may offer a novel approach to the treatment of IPF.

IFN-γ-induced JAK/STAT, but not NF-κB, signaling pathway is insensitive to glucocorticoid in airway epithelial cells.

Although the majority of patients with asthma are well controlled by inhaled glucocorticoids (GCs), patients with severe asthma are poorly responsive to GCs. This latter group is responsible for a disproportionate share of health care costs associated with asthma. Recent studies in immune cells have incriminated interferon-γ (IFN-γ) as a possible trigger of GC insensitivity in severe asthma; however, little is known about the role of IFN-γ in modulating GC effects in other clinically relevant nonimmune cells, such as airway epithelial cells. We hypothesized that IFN-γ-induced JAK/STAT-associated signaling pathways in airway epithelial cells are insensitive to GCs and that strategies aimed at inhibiting JAK/STAT pathways can restore steroid responsiveness. Using Western blot analysis we found that all steps of the IFN-γ-induced JAK/STAT signaling pathway were indeed GC insensitive. Transfection of cells with reporter plasmid showed IFN-γ-induced STAT1-dependent gene transcription to be also GC insensitive. Interestingly, real-time PCR analysis showed that IFN-γ-inducible genes (IIGs) were differentially affected by GC, with CXCL10 being GC sensitive and CXCL11 and IFIT2 being GC insensitive. Further investigation showed that the differential sensitivity of IIGs to GC was due to their variable dependency to JAK/STAT vs. NF-κB signaling pathways with GC-sensitive IIGs being more NF-κB dependent and GC-insensitive IIGs being more JAK/STAT dependent. Importantly, transfection of cells with siRNA-STAT1 was able to restore steroid responsiveness of GC-insensitive IIGs. Taken together, our results show the insensitivity of IFN-γ-induced JAK/STAT signaling pathways to GC effects in epithelial cells and also suggest that targeting STAT1 could restore GC responsiveness in patients with severe asthma.