The D prostanoid receptor 2 (DP2; also known as chemoattractant receptor–homologous molecule expressed on TH2 cells) is implicated in the pathogenesis of asthma, but its expression within bronchial biopsy specimens is unknown.
We sought to investigate the bronchial submucosal DP2 expression in asthmatic patients and healthy control subjects and to explore its functional role in epithelial cells.
DP2 protein expression was assessed in bronchial biopsy specimens from asthmatic patients (n = 22) and healthy control subjects (n = 10) by using immunohistochemistry and in primary epithelial cells by using flow cytometry, immunofluorescence, and quantitative RT-PCR. The effects of the selective DP2 agonist 13, 14-dihydro-15-keto prostaglandin D2 on epithelial cell migration and differentiation were determined.
Numbers of submucosal DP2+ cells were increased in asthmatic patients compared with those in healthy control subjects (mean [SEM]: 78  vs 22 /mm2 submucosa, P < .001). The bronchial epithelium expressed DP2, but its expression was decreased in asthmatic patients compared with that seen in healthy control subjects (mean [SEM]: 21  vs 72 /10 mm2 epithelial area, P = .001), with similar differences observed in vitro by primary epithelial cells. Squamous metaplasia of the bronchial epithelium was increased in asthmatic patients and related to decreased DP2 expression (rs = 0.69, P < .001). 13, 14-Dihydro-15-keto prostaglandin D2 promoted epithelial cell migration and at air-liquid interface cultures increased the number of MUC5AC+ and involucrin-positive cells, which were blocked with the DP2-selective antagonist AZD6430.
DP2 is expressed by the bronchial epithelium, and its activation drives epithelial differentiation, suggesting that in addition to its well-characterized role in inflammatory cell migration, DP2 might contribute to airway remodeling in asthmatic patients.
Expression; asthma; immunohistochemistry; prostaglandin D2; biopsy; ALI, Air-liquid interface; COPD, Chronic obstructive pulmonary disease; CRTH2, Chemoattractant receptor–homologous molecule expressed on TH2 cells; DK-PGD2, 13, 14-Dihydro-15-keto prostaglandin D2; DP1, D prostanoid receptor 1; DP2, D prostanoid receptor 2; PGD2, Prostaglandin D2
Idiopathic pulmonary fibrosis is a common and invariably fatal disease with limited therapeutic options. Ca2+-activated KCa3.1 potassium channels play a key role in promoting TGFβ1 and bFGF-dependent profibrotic responses in human lung myofibroblasts (HLMFs). We hypothesised that KCa3.1 channel-dependent cell processes regulate HLMF αSMA expression via Smad2/3 signalling pathways.
In this study we have compared the phenotype of HLMFs derived from non-fibrotic healthy control lungs (NFC) with cells derived from IPF lungs. HLMFs grown in vitro were examined for αSMA expression by immunofluorescence (IF), RT-PCR and flow cytommetry. Basal Smad2/3 signalling was examined by RT-PCR, western blot and immunofluorescence. Two specific and distinct KCa3.1 blockers (TRAM-34 200 nM and ICA-17043 [Senicapoc] 100 nM) were used to determine their effects on HLMF differentiation and the Smad2/3 signalling pathways.
IPF-derived HLMFs demonstrated increased constitutive expression of both α-smooth muscle actin (αSMA) and actin stress fibres, indicative of greater myofibroblast differentiation. This was associated with increased constitutive Smad2/3 mRNA and protein expression, and increased Smad2/3 nuclear localisation. The increased Smad2/3 nuclear localisation was inhibited by removing extracellular Ca2+ or blocking KCa3.1 ion channels with selective KCa3.1 blockers (TRAM-34, ICA-17043). This was accompanied by de-differentiation of IPF-derived HLMFs towards a quiescent fibroblast phenotype as demonstrated by reduced αSMA expression and reduced actin stress fibre formation.
Taken together, these data suggest that Ca2+- and KCa3.1-dependent processes facilitate “constitutive” Smad2/3 signalling in IPF-derived fibroblasts, and thus promote fibroblast to myofibroblast differentiation. Importantly, inhibiting KCa3.1 channels reverses this process. Targeting KCa3.1 may therefore provide a novel and effective approach for the treatment of IPF and there is the potential for the rapid translation of KCa3.1-directed therapy to the clinic.
Idiopathic pulmonary fibrosis (IPF); Fibrosis; Lung; Myofibroblast; KCa3.1; Ion channel; Differentiation; Smad 2; Smad 3
Approximately 5% to 10% of patients with asthma have severe disease that is refractory or poorly responsive to inhaled corticosteroid therapy. These patients represent an important unmet clinical need because they experience considerable morbidity and mortality and consume a disproportionately large amount of health care resources. TNF-α is a proinflammatory cytokine that has been implicated in many aspects of the airway pathology in asthma. Evidence is emerging to suggest that it might play an important role in severe refractory disease. The development of novel TNF-α antagonists has allowed us to test the role of this cytokine in vivo. Preliminary studies have demonstrated an improvement in asthma quality of life, lung function, and airway hyperresponsiveness and a reduction in exacerbation frequency in patients treated with anti–TNF-α therapy. However, there is marked heterogeneity in response, suggesting that benefit is likely to be reserved to a small subgroup. Importantly, where efficacy is reported, this also needs to be considered in the context of concerns about the safety of anti–TNF-α therapies. Therefore the challenge for clinicians is to evaluate the risk/benefit ratio of these therapies in individual patients with asthma.
Asthma; refractory asthma; TNF-α; mast cells; airway smooth muscle
Identifying the factors responsible for relative glucocorticosteroid (GC) resistance present in patients with severe asthma and finding tools to reverse it are of paramount importance. In asthma there is in vivo evidence of GC-resistant pathways in airway smooth muscle (ASM) bundles which can be modelled in vitro by exposing cultured ASM cells to TNFα/IFNγ. This drives GC insensitivity via protein phosphatase-5 (PP5)-dependent impairment of GC receptor (GR) phosphorylation. Here, we investigated whether KCa3.1 ion channels modulate the activity of GC-resistant pathways using our ASM model of GC insensitivity. Immunohistochemical staining of endobronchial biopsies revealed that KCa3.1 channels are localized to the plasma membrane and nucleus of ASM in both healthy controls and asthmatic patients, irrespective of disease severity. Western blot assays and immunofluorescence staining confirmed the nuclear localisation of KCa3.1 channels in ASM cells. The functional importance of KCa3.1 channels in the regulation of GC-resistant chemokines induced by TNFα/IFNγ was assessed using complementary inhibitory strategies including KCa3.1 blockers (TRAM-34 and ICA-17043) or KCa3.1-specific shRNA delivered by adenoviruses. KCa3.1 channel blockade led to a significant reduction of fluticasone-resistant CX3CL1, CCL5 and CCL11 gene and protein expression. KCa3.1 channel blockade also restored fluticasone-induced GRα phosphorylation at ser211 and transactivation properties via the suppression of cytokine-induced PP5 expression. The effect of KCa3.1 blockade was evident in ASM cells from both healthy controls and asthmatic subjects. In summary KCa3.1 channels contribute to the regulation of GC-resistant inflammatory pathways in ASM cells: blocking KCa3.1 channels may enhance corticosteroid activity in severe asthma.
Corticosteroid insensitivity; chemokines; GR phosphorylation; TNFα; transactivation; transrepression; KCa3.1; severe asthma; airway smooth muscle; transcription factors
Corticosteroid insensitivity (CSI) represents a profound challenge in managing patients with asthma. We recently demonstrated that short exposure of airway smooth muscle cells (ASMCs) to proasthmatic cytokines drastically reduced their responsiveness to glucocorticoids (GCs), an effect that was partially mediated via interferon regulatory factor-1, suggesting the involvement of additional mechanisms (Am J Respir Cell Mol Biol 2008;38:463–472). Although GC receptor (GR) can be phosphorylated at multiple serines in the N-terminal region, the major phosphorylation sites critical for GR transcriptional activity are serines 211 (Ser211) and 226 (Ser226). We tested the novel hypothesis that cytokine-induced CSI in ASMCs is due to an impaired GR phosphorylation. Cells were treated with TNF-α (10 ng/ml) and IFN-γ (500 UI/ml) for 6 hours and/or fluticasone (100 nm) added 2 hours before. GR was constitutively phosphorylated at Ser226 but not at Ser211 residues. Cytokines dramatically suppressed fluticasone-induced phosphorylation of GR on Ser211 but not on Ser226 residues while increasing the expression of Ser/Thr protein phosphatase (PP)5 but not that of PP1 or PP2A. Transfection studies using a reporter construct containing GC responsive elements showed that the specific small interfering RNA–induced mRNA knockdown of PP5, but not that of PP1 or PP2A, partially prevented the cytokine suppressive effects on GR-meditated transactivation activity. Similarly, cytokines failed to inhibit GC-induced GR-Ser211 phosphorylation when expression of PP5 was suppressed. We propose that the novel mechanism that proasthmatic cytokine-induced CSI in ASMCs is due, in part, to PP5-mediated impairment of GR-Ser211 phosphorylation.
serine/threonine protein phosphatase; airway smooth muscle; asthma; corticosteroid insensitivity; airway remodeling
Flaxseed (FS), a nutritional supplement consisting mainly of omega-3 fatty acids and lignan phenolics has potent anti-inflammatory, anti-fibrotic and antioxidant properties. The usefulness of flaxseed as an alternative and complimentary treatment option has been known since ancient times. We have shown that dietary FS supplementation ameliorates oxidative stress and inflammation in experimental models of acute and chronic lung injury in mice resulting from diverse toxicants. The development of lung tissue damage in response to direct or indirect oxidant stress is a complex process, associated with changes in expression levels of a number of genes. We therefore postulated that flaxseed might modulate gene expression of vital signaling pathways, thus interfering with the development of tissue injury.
We evaluated gene expression in lungs of flaxseed-fed (10%FS) mice under unchallenged, control conditions. We reasoned that array technology would provide a powerful tool for studying the mechanisms behind this response and aid the evaluation of dietary flaxseed intervention with a focus on toxicologically relevant molecular gene targets. Gene expression levels in lung tissues were analyzed using a large-scale array whereby 28,800 genes were evaluated.
3,713 genes (12.8 %) were significantly (p < 0.05) differentially expressed, of which 2,088 had a >1.5-fold change. Genes affected by FS include those in protective pathways such as Phase I and Phase II.
The array studies have provided information on how FS modulates gene expression in lung and how they might be related to protective mechanisms. In addition, our study has confirmed that flaxseed is a nutritional supplement with potentially useful therapeutic applications in complementary and alternative (CAM) medicine especially in relation to treatment of lung disease.
Antioxidant; CAM; Flaxseed; Genomic profiling; Lignans; Lung disease; Oxidative stress
In severe asthma, bronchodilator- and steroid-insensitive airflow obstruction develops through unknown mechanisms characterized by increased lung airway smooth muscle (ASM) mass and stiffness. We explored the role of a Regulator of G-protein Signaling protein (RGS4) in the ASM hyperplasia and reduced contractile capacity characteristic of advanced asthma. Using immunocytochemical staining, ASM expression of RGS4 was determined in endobronchial biopsies from healthy subjects and those from subjects with mild, moderate and severe asthma. Cell proliferation assays, agonist-induced calcium mobilization and bronchoconstriction were determined in cultured human ASM cells and in human precision cut lung slices. Using gain- and loss-of-function approaches, the precise role of RGS proteins was determined in stimulating human ASM proliferation and inhibiting bronchoconstriction. RGS4 expression was restricted to a subpopulation of ASM and was specifically upregulated by mitogens, which induced a hyperproliferative and hypocontractile ASM phenotype similar to that observed in recalcitrant asthma. RGS4 expression was markedly increased in bronchial smooth muscle of patients with severe asthma, and expression correlated significantly with reduced pulmonary function. Whereas RGS4 inhibited G protein-coupled receptor (GPCR)-mediated bronchoconstriction, unexpectedly RGS4 was required for PDGF-induced proliferation and sustained activation of PI3K, a mitogenic signaling molecule that regulates ASM proliferation. These studies indicate that increased RGS4 expression promotes a phenotypic switch of ASM, evoking irreversible airway obstruction in subjects with severe asthma.
Glucocorticoid (GC) insensitivity represents a profound challenge in managing patients with asthma. The mutual inhibition of transcriptional activity between GC receptor (GR) and other regulators is one of the mechanisms contributing to GC resistance in asthma. We recently reported that interferon regulatory factor (IRF)-1 is a novel transcription factor that promotes GC insensitivity in human airway smooth muscle (ASM) cells by interfering with GR signaling (Tliba et al., Am J Respir Cell Mol Biol 2008;38:463–472). Here, we sought to determine whether the inhibition of GR function by IRF-1 involves its interaction with the transcriptional co-regulator GR-interacting protein 1 (GRIP-1), a known GR transcriptional co-activator. We here found that siRNA-mediated GRIP-1 depletion attenuated IRF-1–dependent transcription of the luciferase reporter construct and the mRNA expression of an IRF-1–dependent gene, CD38. In parallel experiments, GRIP-1 silencing significantly reduced GR-mediated transactivation activities. Co-immunoprecipitation and GST pull-down assays showed that GRIP-1, through its repression domain, physically interacts with IRF-1 identifying GRIP-1 as a bona fide transcriptional co-activator for IRF-1. Interestingly, the previously reported inhibition of GR-mediated transactivation activities by either TNF-α and IFN-γ treatment or IRF-1 overexpression was fully reversed by increasing cellular levels of GRIP-1. Together, these data suggest that the cellular accumulation of IRF-1 may represent a potential molecular mechanism mediating altered cellular response to GC through the depletion of GRIP-1 from the GR transcriptional regulatory complexes.
glucocorticoid; cytokine; airway smooth muscle; IRF-1; GRIP-1
During pregnancy asthma may remain stable, improve or worsen. The factors underlying the deleterious effect of pregnancy on asthma remain unknown. Oxytocin is a neurohypophyseal protein that regulates a number of central and peripheral responses such as uterine contractions and milk ejection. Additional evidence suggests that oxytocin regulates inflammatory processes in other tissues given the ubiquitous expression of the oxytocin receptor. The purpose of this study was to define the role of oxytocin in modulating human airway smooth muscle (HASMCs) function in the presence and absence of IL-13 and TNFα, cytokines known to be important in asthma.
Expression of oxytocin receptor in cultured HASMCs was performed by real time PCR and flow cytomery assays. Responses to oxytocin was assessed by fluorimetry to detect calcium signals while isolated tracheal rings and precision cut lung slices (PCLS) were used to measure contractile responses. Finally, ELISA was used to compare oxytocin levels in the bronchoalveloar lavage (BAL) samples from healthy subjects and those with asthma.
PCR analysis demonstrates that OXTR is expressed in HASMCs under basal conditions and that both interleukin (IL)-13 and tumor necrosis factor (TNFα) stimulate a time-dependent increase in OXTR expression at 6 and 18 hr. Additionally, oxytocin increases cytosolic calcium levels in fura-2-loaded HASMCs that were enhanced in cells treated for 24 hr with IL-13. Interestingly, TNFα had little effect on oxytocin-induced calcium response despite increasing receptor expression. Using isolated murine tracheal rings and PCLS, oxytocin also promoted force generation and airway narrowing. Further, oxytocin levels are detectable in bronchoalveolar lavage (BAL) fluid derived from healthy subjects as well as from those with asthma.
Taken together, we show that cytokines modulate the expression of functional oxytocin receptors in HASMCs suggesting a potential role for inflammation-induced changes in oxytocin receptor signaling in the regulation of airway hyper-responsiveness in asthma.
Chronic psychosocial stress exacerbates asthma but the underlying mechanisms remain poorly understood. We hypothesized that psychosocial stress aggravates allergic airway inflammation by altering innate immune cell function. The effects of stress on airway inflammation, lung function and glucocorticoid responsiveness were studied in a novel in vivo murine model of combined social disruption stress and allergic sensitization. The effects of corticosterone were assessed on cytokine profile and glucocorticoid receptor activation in LPS-stimulated spleen cell cultures in vitro.
Airway inflammation resolved 48 hours after a single allergen provocation in sensitized control mice but not in animals that were repeatedly exposed to stress prior to allergen challenge. The enhanced eosinophilic airway inflammation 48 hours after allergen challenge in these mice was associated with increased levels of IL-5, GM-CSF, IgG1, TARC, TNF-α and IL-6 in the airways and a diminished inhibition of these mediators by corticosterone in LPS-stimulated splenocyte cultures in vitro. Stress-induced reduction of the corticosteroid effects paralleled increased p65 expression and a decreased DNA binding capability of the glucocorticoid receptor in vitro. Further, glucocorticoid receptor mRNA and protein expression in the lungs of mice exposed to both stress and allergen was markedly reduced in comparison with that in either condition alone or in naïve mice. Thus, exposure to repeated social stress prior to allergen inhalation enhances and prolongs airway inflammation and alters corticosterone responsiveness. We speculate that these effects were mediated at least in part by impaired glucocorticoid receptor expression and function.
Rodent; Allergy; Lung; Inflammation; Th1/Th2 Cells
We have previously shown that long-term treatment of airway smooth muscle (ASM) cells with a combination of TNF-α and IFN-γ impaired steroid anti-inflammatory action through the up-regulation of glucocorticoid receptor beta isoform (GRβ) (Mol Pharmacol 2006;69:588–596). We here found that steroid actions could also be suppressed by short-term exposure of ASM cells to TNF-α and IFN-γ (6 h) as shown by the abrogated glucocorticoid responsive element (GRE)-dependent gene transcription; surprisingly, neither GRα nuclear translocation nor GRβ expression was affected by cytokine mixture. The earlier induction of CD38, a molecule recently involved in asthma, seen with TNF-α and IFN-γ combination but not with cytokine alone, was also completely insensitive to steroid pretreatment. Chromatin-immunoprecipitation (IP) and siRNA strategies revealed not only increased binding of interferon regulatory factor 1 (IRF-1) transcription factor to CD38 promoter, but also its implication in regulating CD38 gene transcription. Interestingly, the capacity of fluticasone to completely inhibit TNF-α–induced IRF-1 expression, IRF-1 DNA binding, and transactivation activities was completely lost in cells exposed to TNF-α and IFN-γ in combination. This early steroid dysfunction seen with cytokine combination could be reproduced by enhancing IRF-1 cellular levels using constitutively active IRF-1, which dose-dependently inhibited GRE-dependent gene transcription. Consistently, reducing IRF-1 cellular levels using siRNA approach significantly restored steroid transactivation activities. Collectively, our findings demonstrate for the first time that IRF-1 is a novel alternative GRβ-independent mechanism mediating steroid dysfunction induced by pro-asthmatic cytokines, in part via the suppression of GRα activities.
transcription factor; glucocorticoid; inflammation; asthma; mesenchymal cells
The present article will describe the potential role of airway smooth muscle (ASM) in mediating both deleterious/beneficial effects of interferons (IFNs) in asthma. First described as beneficial in treating the main features of asthma, the interplay between IFNs and ASM could explain their deleterious actions recently described in a number of different studies. Through multiple mechanisms, including the suppression of steroid action, the synergistic pro-inflammatory actions when combined with other cytokines, and the modulation of calcium metabolism, IFNs are now seen as critical mediators in the pathogenesis of asthma.
signaling cross talk; interferons; asthma; cytokines; TNF-α
We recently identified autocrine interferon (IFN)β as a novel mechanism mediating tumor necrosis factor (TNF)α-induced expression of inflammatory genes in airway smooth muscle (ASM) cells, including CD38, known to regulate calcium signaling. Here, we investigated the putative involvement of IFNβ in regulating TNFα-induced airway hyper-responsiveness (AHR), a defining feature of asthma. Using our pharmacodynamic model to assess ex vivo AHR isolated murine tracheal rings, we found that TNFα-induced enhanced contractile responses to carbachol and bradykinin was abrogated by neutralizing anti-IFNβ antibody or in tracheal rings deficient in CD38. In cultured human ASM cells, where CD38 has been involved in TNFα-induced enhanced calcium signals to carbachol and bradykinin, we found that neutralizing anti-IFNβ prevented TNFα enhancing action only on carbachol responses but not to that induced by bradykinin. In a well-characterized model of allergic asthma (mice sensitized and challenged with Aspergillus fumigatus (Af)), we found heightened expression of both IFNβ and CD38 in the airways. Furthermore, allergen-associated AHR to methacholine, assessed by lung resistance and dynamic compliance, was completely suppressed in CD38-deficient mice, despite the preservation of airway inflammation. These data provide the first evidence that ASM-derived IFNβ and CD38 may play a significant role in the development of TNFα-associated AHR.
Allergic asthma; Cytokine; Airway smooth muscle; Inflammation; Calcium signaling; Hypercontractility
Growing evidence shows that interleukin 13 (IL-13) may play an essential role in the development of airway inflammation and bronchial hyper-responsiveness (BHR), two defining features of asthma. Although the underlying mechanisms remain unknown, a number of reports have shown that IL-13 may exert its deleterious effects in asthma by directly acting on airway resident cells, including epithelial cells and airway smooth muscle cells. In this report, we hypothesize that IL-13 may participate in the pathogenesis of asthma by activating a set of "pro-asthmatic" genes in airway smooth muscle (ASM) cells.
Microarray technology was used to study the modulation of gene expression of airway smooth muscle by IL-13 and IL-13R130Q. TaqMan™ Real Time PCR and flow cytometry was used to validate the gene array data.
IL-13 and the IL-13 polymorphism IL-13R130Q (Arg130Gln), recently associated with allergic asthma, seem to modulate the same set of genes, which encode many potentially interesting proteins including vascular cellular adhesion molecule (VCAM)-1, IL-13Rα2, Tenascin C and Histamine Receptor H1, that may be relevant for the pathogenesis of asthma.
The data supports the hypothesis that gene modulation by IL-13 in ASM may be essential for the events leading to the development of allergic asthma.
The cellular and molecular mechanisms that are involved in airway hyper-responsiveness are unclear. Current studies suggest that tumor necrosis factor (TNF)-α, a cytokine that is produced in considerable quantities in asthmatic airways, may potentially be involved in the development of bronchial hyper-responsiveness by directly altering the contractile properties of the airway smooth muscle (ASM). The underlying mechanisms are not known, but growing evidence now suggests that most of the biologic effects of TNF-α on ASM are mediated by the p55 receptor or tumor necrosis factor receptor (TNFR)1. In addition, activation of TNFR1 coupled to the tumor necrosis factor receptor-associated factor (TRAF)2-nuclear factor-κB (NF-κB) pathway alters calcium homeostasis in ASM, which appears to be a new potential mechanism underlying ASM hyper-responsiveness.
airway hyper-responsiveness; airway remodeling; airway smooth muscle; tumor necrosis factor-α; tumor necrosis factor receptor 1; thapsigargin