Nadia Khorasani

Relative corticosteroid insensitivity of alveolar macrophages in severe asthma compared with non-severe asthma

Background: About 5–10% of patients with asthma suffer from poorly controlled disease despite corticosteroid (CS) treatment, which may indicate the presence of CS insensitivity. A study was undertaken to determine whether relative CS insensitivity is present in alveolar macrophages from patients with severe asthma and its association with p38 mitogen-activated protein kinase (MAPK) activation and MAPK phosphatase-1 (MKP-1). Methods: Fibreoptic bronchoscopy and bronchoalveolar lavage (BAL) were performed in 20 patients with severe asthma and 19 with non-severe asthma and, for comparison, in 14 normal volunteers. Alveolar macrophages were exposed to lipopolysaccharide (LPS, 10 μg/ml) and dexamethasone (10−8 and 10−6 M). Supernatants were assayed for cytokines using an ELISA-based method. p38 MAPK activity and MKP-1 messenger RNA expression were assayed in cell extracts. Results: The inhibition of LPS-induced interleukin (IL)1β, IL6, IL8, monocyte chemotactic protein (MCP)-1 and macrophage inflammatory protein (MIP)-1α release by dexamethasone (10−6 M) was significantly less in macrophages from patients with severe asthma than in macrophages from patients with non-severe asthma. There was increased p38 MAPK activation in macrophages from patients with severe asthma. MKP-1 expression induced by dexamethasone and LPS, expressed as a ratio of LPS-induced expression, was reduced in severe asthma. Conclusion: Alveolar macrophages from patients with severe asthma demonstrate CS insensitivity associated with increased p38 MAPK activation that may result from impaired inducibility of MKP-1.

TGF-β regulates Nox4, MnSOD and catalase expression, and IL-6 release in airway smooth muscle cells

Reactive oxygen species (ROS) are generated as a result of normal cellular metabolism, mainly through the mitochondria and peroxisomes, but their release is enhanced by the activation of oxidant enzymes such as NADPH oxidases or downregulation of endogenous antioxidant enzymes such as manganese-superoxide dismutase (MnSOD) and catalase. Transforming growth factor-β (TGF-β), found to be overexpressed in airway smooth muscle (ASM) from asthmatic and chronic obstructive pulmonary disease patients, may be a pivotal regulator of abnormal ASM cell (ASMC) function in these diseases. An important effect of TGF-β on ASMC inflammatory responses is the induction of IL-6 release. TGF-β also triggers intracellular ROS release in ASMCs by upregulation of NADPH oxidase 4 (Nox4). However, the effect of TGF-β on the expression of key antioxidant enzymes and subsequently on oxidant/antioxidant balance is unknown. Moreover, the role of redox-dependent pathways in the mediation of the proinflammatory effects of TGF-β in ASMCs is unclear. In this study, we show that TGF-β induced the expression of Nox4 while at the same time inhibiting the expression of MnSOD and catalase. This change in oxidant/antioxidant enzymes was accompanied by elevated ROS levels and IL-6 release. Further studies revealed a role for Smad3 and phosphatidyl-inositol kinase-mediated pathways in the induction of oxidant/antioxidant imbalance and IL-6 release. The changes in oxidant/antioxidant enzymes and IL-6 release were reversed by the antioxidants N-acetyl-cysteine (NAC) and ebselen through inhibition of Smad3 phosphorylation, indicating redox-dependent activation of Smad3 by TGF-β. Moreover, these findings suggest a potential role for NAC in preventing TGF-β-mediated pro-oxidant and proinflammatory responses in ASMCs. Knockdown of Nox4 using small interfering RNA partially prevented the inhibition of MnSOD but had no effect on catalase and IL-6 expression. These findings provide novel insights into redox regulation of ASM function by TGF-β.

Corticosteroid Inhibition of Growth-Related Oncogene Protein-α via Mitogen-Activated Kinase Phosphatase-1 in Airway Smooth Muscle Cells

Expression of the inflammatory chemokine, growth-related oncogene protein-α (GRO-α), from airway smooth muscle cells (ASMC) is regulated by pathways involving NF-κB and MAPK activation. We determined the effects of dexamethasone on GRO-α induced by IL-1β or TNF-α with respect to the role of MAPK pathways and of MAPK phosphatase-1 (MKP-1). Human ASMC were studied in primary culture at confluence. Dexamethasone (10−8–10−5 M) partially inhibited GRO-α expression and release induced by IL-1β and TNF-α; this was associated with an inhibition of JNK, but not of p38 or ERK phosphorylation. Together with IL-1β or TNF-α, dexamethasone rapidly induced mRNA and protein expression of MKP-1, which dephosphorylates MAPKs. Using MKP-1 small interfering RNA (siRNA) to block the expression of IL-1β- and dexamethasone-induced MKP-1 by 50%, JNK phosphorylation was doubled. The inhibitory effect of dexamethasone on GRO-α release was partially reversed in ASMC treated with MKP-1 siRNA compared with those treated with scrambled siRNA. In contrast, overexpression of MKP-1 led to a reduction in IL-1β-induced release of GRO-α, but the inhibitory effects of dexamethasone were preserved. Nuclear translocation of the glucocorticoid receptor was increased in ASMC exposed to dexamethasone and IL-1β. Using chromatin immunoprecipitation assay, glucocorticoid receptor binding to the MKP-1 promoter was increased by IL-1β and dexamethasone compared with either alone. Glucocorticoids and IL-1β or TNF-α modulate GRO-α release partly through the inhibition of JNK pathway, resulting from an up-regulation of MKP-1 expression.

Effect of p38 MAPK inhibition on corticosteroid suppression of cytokine release in severe asthma

Patients with severe asthma respond less well to corticosteroids than those with non-severe asthma. Increased p38 mitogen-activated protein kinase (MAPK) activation in alveolar macrophages (AMs) from severe asthma patients has been associated with a reduced inhibition of cytokine release by dexamethasone. We determined whether p38 MAPK inhibitors would modulate corticosteroid suppression of cytokine release from AMs and peripheral blood mononuclear cells (PBMCs). PBMCs were isolated from venous blood and AMs by bronchoalveolar lavage in severe and non-severe asthma patients. PBMCs and AMs were exposed to lipopolysaccharide (LPS) with and without the p38 MAPK inhibitor, SD282, or dexamethasone. We determined the concentration-dependent effects of another p38 MAPK inhibitor, GW-A, on dexamethasone-induced inhibition of interleukin (IL)-8 release from PBMCs. Cytokines were assayed using an ELISA-based method. SD282 (10−7 M), with dexamethasone (10−6 M), caused a greater inhibition of release of IL-1β, IL-6, macrophage inflammatory protein-1α and IL-10, than with dexamethasone alone in AMs from severe and non-severe asthma. At 10−9 and 10−10 M, GW-A, that had no direct effects, increased the inhibitory activity of dexamethasone (10−8 and 10−6 M) on LPS-induced IL-8 release in PBMCs from severe asthma. Corticosteroid insensitivity in severe asthma patients may be improved by inhibitors of p38 MAPK.

Corticosteroid insensitivity of chemokine expression in airway smooth muscle of patients with severe asthma.

BACKGROUND Patients with severe asthma are less responsive to the beneficial effects of corticosteroid therapy. OBJECTIVE We investigated whether corticosteroid insensitivity was present in airway smooth muscle cells (ASMCs) of patients with severe asthma. METHODS ASMCs cultured from bronchial biopsy specimens of nonasthmatic control subjects (n = 12) and patients with nonsevere (n = 10) or severe (n = 10) asthma were compared for the effect of dexamethasone on suppression of TNF-α- and IFN-γ-induced CCL11 (eotaxin), CXCL8 (IL-8), and CX3CL1 (fractalkine) expression. The mechanisms of corticosteroid insensitivity are also determined. RESULTS CCL11 release was higher in ASMCs of patients with nonsevere but not severe asthma and nonasthmatic control subjects; CXCL8 and CX3CL1 release were similar in all groups. In patients with severe asthma, dexamethasone caused less suppression of CCL11 and CXCL8 release induced by TNF-α. Dexamethasone potentiated TNF-α- and IFN-γ-induced CX3CL1 release equally in all 3 groups. TNF-α-induced phosphorylated p38 mitogen-activated protein kinase levels were increased in ASMCs from patients with severe asthma compared with those from patients with nonsevere asthma and nonasthmatic subjects, whereas TNF-α-induced phosphorylated c-Jun N-terminal kinase and phosphorylated extracellular signal-related kinase levels were increased in all asthmatic groups. A p38 inhibitor increased the inhibitory effect of dexamethasone. CONCLUSIONS ASMCs of patients with severe asthma are corticosteroid insensitive; this might be secondary to heightened p38 mitogen-activated protein kinase levels.

Chromosome 17q21 SNP and severe asthma.

Asthma is a complex disease that is influenced by poorly understood genetic and environmental factors1. A genome-wide association study (GWAS) of 994 cases and 1243 controls from the UK and Germany found strong associations (P < 10−12) for SNPs at the 17q21 locus and childhood asthma using family and case-control panels2. The association between these SNPs and gene transcript levels in Epstein-Barr virus-transformed lymphoblastoid cell lines from the asthmatic children identified ORMDL3 as a candidate gene for asthma. One of the SNPs, rs7216389, located within a highly conserved region containing an element homologous to the proinflammatory transcription factor C/EBPb was associated to both asthma and ORMDL3 transcript levels with the highest degree of statistical significance in the initial study (uncorrected P = 9×10−11)2. The initial GWAS findings have subsequently replicated in a number of studies involving ethnically diverse populations and the variants were reported to contribute to early-onset asthma and interacting to early-life environmental tobacco smoke exposures3. A study published recently identified an association of rs7216389 variant with disease severity in early-onset asthma4. In the study, asthmatic cases were stratified according to asthma severity and they were classified into mild, moderate and severe asthmatics following national and international guidelines. Severe asthmatics were not recruited through severe asthma clinics; however, the study proposed rs7216389 is involved in early-onset severe asthma. Severe or “difficult/therapy-resistant” asthma refers to asthma that is poorly controlled in terms of persistent symptoms, episodic exacerbations and persistent and variable airway obstruction despite the use of high doses of inhaled corticosteroids, long-acting bronchodilators and short β2 agonists5. Studying individuals with an extreme phenotype can be very powerful when isolating the genetic determinants underlying a disease. Using this strategy we have consequently examined the role of rs7216389 in severe asthma. The case group consisted of 397 severe asthmatic adults identified through specialist severe asthma clinics at two UK centres, Royal Brompton Hospital, London and the Glenfield Hospital, Leicester. Asthma was defined using the international GINA (Global Initiative for Asthma: http://www.ginasthma.com) guidelines and the ATS criteria for refractory asthma5. For 226 subjects, the asthma age of onset was available. Childhood asthma-onset was present in 114 samples and adulthood asthma-onset in 112 subjects. The male to female ratio was 1:2, the mean age was 48.95 years (Standard Deviation 13.55) and mean IgE (kU/L) was 291.72 (Standard Deviation 456.09). We derived 1429 previously genotyped healthy UK adult controls from the 1958 British Birth Cohort study. The 1958 British birth cohort includes 17,638 males and females with sex ratio 1:1 enrolled in the Perinatal Mortality Survey at the time of their birth during 1 week in March 1958 across England, Wales and Scotland 6. A DNA collection was obtained during a follow-up in 2002 to 20047. Genome-wide genotyping data from the Illumina HumanHap550 Beadarray on 1430 subjects were deposited by the Wellcome Trust Sanger Institute8. Blood samples from cases were collected and DNA was extracted using whole blood DNA extraction protocols (Promega Wizard® Genomic DNA purification kit). TaqMan® SNP Genotyping Assays (Applied Biosystems 7300 Real-Time PCR System, 40 cycles of 10 min at 95 °C, 15 sec at 92 °C and 1 min at 60 °C) were used for the allelic discrimination (primer and probe sequences available upon request). Controls of known genotype were included. Deviation from Hardy Weinberg equilibrium (HWE) was calculated for the allele frequencies. Genotype and allele frequencies were compared between cases and controls by Fisher’s exact test and logistic regression. Associations between the genotypes and IgE were also examined by Kruskal-Wallis test. The genotyping success rate for rs7216389 was 97%. No significant deviation from HWE was detected (P > 0.05). The rs7216389 SNP was found to be significantly associated with severe adult asthma (OR 1.42, CI: 1.21-1.67, P = 1.8×10−5) (Table 1). In our study the frequency of the T allele in the asthmatic adults was 56%, which is lower than that reported by Moffatt et al. (62%)2. When the data was stratified according to the disease age of onset, a significant association between SNP rs7216389 and severe asthma was reported only in the childhood-onset asthmatics (OR 2.02, CI: 1.53-2.68, P=4.5×10−6) (Table 1). Interestingly, the frequency of the T allele in cases of childhood onset was 67%, similar to the figure reported by Moffatt et al.2 Adult-onset of the disease did not show any significant associations for this SNP (P=0.853) and minor allele frequency (T allele) was 49%, comparable to the control group (47%) (Table 1). No associations were found between genotypes and IgE levels. The results highlight the differences in the genetic components of childhood and adulthood asthma-onset. Table 1 Genotype frequencies and association test results for rs7216389 with severe asthma, childhood-onset and adult-onset asthma susceptibility. Our results confirm the role of the ORMDL3 genomic area as a locus conferring susceptibility to childhood asthma-onset of the most severe type of the disease. In combination with the recent published studies in ethnically diverse populations it highlights the importance of the ORMDL3 and other genes from the Chromosome 17q21 region in the development of this complex disease. Further studies are required to investigate the functional role of this polymorphism and its involvement in early-onset asthma that could contribute in elucidating the mechanisms underlying asthma and could be applied for therapeutic interventions.

Modulation of ozone-induced airway hyperresponsiveness and inflammation by interleukin-13

The present study aimed to determine whether the T-helper cell type 2-derived cytokines, interleukin (IL)-4 and -13, can modulate the lung response to ozone exposure. IL-13-/-, IL-4/13-/- and IL-13-overexpressing transgenic (Tg) mice were exposed to ozone (3 ppm; 3 h) or air. Wild-type (Wt) Balb/c mice and transgenic-negative littermates (IL-13Wt) were used as controls for gene-deficient and IL-13Tg mice, respectively. IL-4/13-/- and IL-13-/- mice developed a lesser degree of ozone-induced airway hyperresponsiveness (AHR) while IL-13Tg mice developed a greater degree of AHR compared with ozone-exposed wild-type or IL-13Wt mice, respectively. Ozone caused a time-dependent increase of bronchoalveolar lavage (BAL) neutrophils and macrophages in wild-type mice, maximal at 20–24 h, which was attenuated in the IL-13-/- and IL-4/13-/- mice. In IL-13Tg mice, there was a greater increase in BAL neutrophils after ozone exposure compared with IL-13Wt mice. Using quantitative real-time PCR, ozone-induced mRNA expression for IL-6 and keratinocyte chemokine was further enhanced in IL-13-/- and IL-4/13-/- mice, and was inhibited in IL-13Tg mice. Macrophage inflammatory protein (MIP)-3α/CCL20 expression was enhanced after ozone exposure in wild-type mice, inhibited in IL-13-/- and IL-4/13-/- mice, while in IL-13Tg mice it was enhanced. A similar pattern of expression was observed with lipopolysaccharide-induced cytokine (LIX/CXCL5/ENA-78) expression. In conclusion, interleukin-13 augments ozone-induced airway hyperresponsiveness and neutrophilic inflammation, possibly through modulation of certain cytokines induced by ozone exposure.

EMMPRIN (CD147) regulation of MMP-9 in bronchial epithelial cells in COPD.

Background and objective:  Extracellular matrix metalloproteinase inducer (EMMPRIN or CD147) induces the production of matrix metalloproteinases (MMP) such as MMP‐9, which plays an important role in COPD. We determined its cellular origin and role in MMP‐9 production in COPD.

Reversal of corticosteroid insensitivity by p38 MAPK inhibition in peripheral blood mononuclear cells from COPD

Background Corticosteroids (CS) have limited efficacy in the treatment of chronic obstructive pulmonary disease (COPD). p38 mitogen-activated protein kinase (MAPK) activation is increased in lung macrophages of COPD. We investigated whether p38 MAPK inhibition can modulate CS insensitivity of peripheral blood mononuclear cells (PBMCs) from patients with COPD. Methods PBMCs from patients with COPD (n=8) or healthy smokers (n=8) were exposed to lipopolysaccharide (LPS) with a selective p38 MAPK inhibitor (GW856553; 10−10–10−6 M), with dexamethasone (10−10–10−6 M), or with both. Phosphorylated glucocorticoid receptor (GR) was measured by Western blot. Results Baseline (P<0.01) and LPS-induced (P<0.05) CXCL8 release was greater in PBMCs from COPD compared to healthy smokers. Inhibition of LPS-induced CXCL8 release by dexamethasone (10−6 M) was reduced, and baseline and LPS-induced p38 MAPK activation increased in PBMCs of COPD. GW856553 (10−9 and 10−10 M) synergistically increased the inhibitory effect of dexamethasone (10−8 and 10−6 M) on LPS-induced CXCL8 release in COPD. Similar results were obtained for IL-6 release. GW856553 inhibited dexamethasone- and LPS-activated phosphorylation of serine 211 on GR. CS insensitivity in COPD PBMCs is reversed by inhibition of p38 MAPK activity, partly by preventing phosphorylation of GR at serine 211. Conclusion p38 MAPK inhibition may be beneficial in COPD by restoring CS sensitivity.

Oxidants Induce a Corticosteroid-Insensitive Phosphorylation of Histone 3 at Serine 10 in Monocytes

Oxidative stress enhances inflammation and reduces the effectiveness of corticosteroids, but the inflammatory signalling pathways induced by oxidants remain ill-defined. Phosphorylation of histone 3 at serine 10 (H3-Pser10) marks out a subset of inflammatory genes for transcription, several of which are induced in oxidant-associated inflammation. However, the influence of oxidants or of corticosteroids on this modification remains unknown. We assessed the regulation of H3-Pser10 by oxidants and lipopolysaccharide (LPS) in human blood monocytes and lung macrophages and the effectiveness of its abolition in controlling inflammatory gene expression in cells from asthmatic subjects compared to corticosteroids alone. Both oxidants and LPS promoted the induction of H3-Pser10 which was unaffected by corticosteroids. The induction of H3-Pser10 was mediated through p38α mitogen-activated protein kinase (MAPK) and IκB kinase 2 (IKK-2) signalling. Consequently, inhibitors of p38α MAPK or IKK-2 used in combination with dexamethasone were more effective at controlling inflammatory gene expression from monocytes and lung macrophages from asthmatic patients than the corticosteroid alone. Therefore, reduction of H3-Pser10 by inhibition of p38α MAPK or of IKK-2 may provide greater anti-inflammatory control than corticosteroids alone in oxidant-associated inflammation such as severe asthma.