IL-33 Expression Is Lower in Current Smokers at both Transcriptomic and Protein Levels.

Rationale: IL-33 is a proinflammatory cytokine thought to play a role in the pathogenesis of asthma and chronic obstructive pulmonary disease (COPD). A recent clinical trial using an anti-IL-33 antibody showed a reduction in exacerbation and improved lung function in ex-smokers but not current smokers with COPD. Objectives: This study aimed to understand the effects of smoking status on IL-33. Methods: We investigated the association of smoking status with the level of gene expression of IL-33 in the airways in eight independent transcriptomic studies of lung airways. Additionally, we performed Western blot analysis and immunohistochemistry for IL-33 in lung tissue to assess protein levels. Measurements and Main Results: Across the bulk RNA-sequencing datasets, IL-33 gene expression and its signaling pathway were significantly lower in current versus former or never-smokers and increased upon smoking cessation (P < 0.05). Single-cell sequencing showed that IL-33 is predominantly expressed in resting basal epithelial cells and decreases during the differentiation process triggered by smoke exposure. We also found a higher transitioning of this cellular subpopulation into a more differentiated cell type during chronic smoking, potentially driving the reduction of IL-33. Protein analysis demonstrated lower IL-33 levels in lung tissue from current versus former smokers with COPD and a lower proportion of IL-33-positive basal cells in current versus ex-smoking controls. Conclusions: We provide strong evidence that cigarette smoke leads to an overall reduction in IL-33 expression in transcriptomic and protein level, and this may be due to the decrease in resting basal cells. Together, these findings may explain the clinical observation that a recent antibody-based anti-IL-33 treatment is more effective in former than current smokers with COPD.

Label-Free Time-of-Flight Secondary Ion Mass Spectrometry Imaging of Sulfur-Producing Enzymes inside Microglia Cells following Exposure to Silver Nanowires.

There are no methods sensitive enough to detect enzymes within cells, without the use of analyte labeling. Here we show that it is possible to detect protein ion signals of three different H2S-synthesizing enzymes inside microglia after pretreatment with silver nanowires (AgNW) using time-of-flight secondary ion mass spectrometry (TOF-SIMS). Protein fragment ions, including the fragment of amino acid (C4H8N+ = 70 amu), fragments of the sulfur-producing cystathionine-containing enzymes, and the Ag+ ion signal could be detected without the use of any labels; the cells were mapped using the C4H8N+ amino acid fragment. Scanning electron microscopy imaging and energy-dispersive X-ray chemical analysis showed that the AgNWs were inside the same cells imaged by TOF-SIMS and transformed chemically into crystalline Ag2S within cells in which the sulfur-producing proteins were detected. The presence of these sulfur-producing cystathionine-containing enzymes within the cells was confirmed by Western blots and confocal microscopy images of fluorescently labeled antibodies against the sulfur-producing enzymes. Label-free TOF-SIMS is very promising for the label-free identification of H2S-contributing enzymes and their cellular localization in biological systems. The technique could in the future be used to identify which of these enzymes are most contributory.

Enhanced oxidative stress in smoking and ex-smoking severe asthma in the U-BIOPRED cohort.

Oxidative stress is believed to be a major driver of inflammation in smoking asthmatics. The U-BIOPRED project recruited a cohort of Severe Asthma smokers/ex-smokers (SAs/ex) and non-smokers (SAn) with extensive clinical and biomarker information enabling characterization of these subjects. We investigated oxidative stress in severe asthma subjects by analysing urinary 8-iso-PGF2α and the mRNA-expression of the main pro-oxidant (NOX2; NOSs) and anti-oxidant (SODs; CAT; GPX1) enzymes in the airways of SAs/ex and SAn. All the severe asthma U-BIOPRED subjects were further divided into current smokers with severe asthma (CSA), ex-smokers with severe asthma (ESA) and non-smokers with severe asthma (NSA) to deepen the effect of active smoking. Clinical data, urine and sputum were obtained from severe asthma subjects. A bronchoscopy to obtain bronchial biopsy and brushing was performed in a subset of subjects. The main clinical data were analysed for each subset of subjects (urine-8-iso-PGF2α; IS-transcriptomics; BB-transcriptomics; BBr-transcriptomics). Urinary 8-iso-PGF2α was quantified using mass spectrometry. Sputum, bronchial biopsy and bronchial brushing were processed for mRNA expression microarray analysis. Urinary 8-iso-PGF2α was increased in SAs/ex, median (IQR) = 31.7 (24.5-44.7) ng/mmol creatinine, compared to SAn, median (IQR) = 26.6 (19.6-36.6) ng/mmol creatinine (p< 0.001), and in CSA, median (IQR) = 34.25 (24.4-47.7), vs. ESA, median (IQR) = 29.4 (22.3-40.5), and NSA, median (IQR) = 26.5 (19.6-16.6) ng/mmol creatinine (p = 0.004). Sputum mRNA expression of NOX2 was increased in SAs/ex compared to SAn (probe sets 203922_PM_s_at fold-change = 1.05 p = 0.006; 203923_PM_s_at fold-change = 1.06, p = 0.003; 233538_PM_s_at fold-change = 1.06, p = 0.014). The mRNA expression of antioxidant enzymes were similar between the two severe asthma cohorts in all airway samples. NOS2 mRNA expression was decreased in bronchial brushing of SAs/ex compared to SAn (fold-change = -1.10; p = 0.029). NOS2 mRNA expression in bronchial brushing correlated with FeNO (Kendal's Tau = 0.535; p< 0.001). From clinical and inflammatory analysis, FeNO was lower in CSA than in ESA in all the analysed subject subsets (p< 0.01) indicating an effect of active smoking. Results about FeNO suggest its clinical limitation, as inflammation biomarker, in severe asthma active smokers. These data provide evidence of greater systemic oxidative stress in severe asthma smokers as reflected by a significant changes of NOX2 mRNA expression in the airways, together with elevated urinary 8-iso-PGF2α in the smokers/ex-smokers group. Trial registration ClinicalTrials.gov-Identifier: NCT01976767.

A Severe Asthma Disease Signature from Gene Expression Profiling of Peripheral Blood from U-BIOPRED Cohorts.

RATIONALE: Stratification of asthma at the molecular level, especially using accessible biospecimens, could greatly enable patient selection for targeted therapy. OBJECTIVES: To determine the value of blood analysis to identify transcriptional differences between clinically defined asthma and nonasthma groups, identify potential patient subgroups based on gene expression, and explore biological pathways associated with identified differences. METHODS: Transcriptomic profiles were generated by microarray analysis of blood from 610 patients with asthma and control participants in the U-BIOPRED (Unbiased Biomarkers in Prediction of Respiratory Disease Outcomes) study. Differentially expressed genes (DEGs) were identified by analysis of variance, including covariates for RNA quality, sex, and clinical site, and Ingenuity Pathway Analysis was applied. Patient subgroups based on DEGs were created by hierarchical clustering and topological data analysis. MEASUREMENTS AND MAIN RESULTS: A total of 1,693 genes were differentially expressed between patients with severe asthma and participants without asthma. The differences from participants without asthma in the nonsmoking severe asthma and mild/moderate asthma subgroups were significantly related (r = 0.76), with a larger effect size in the severe asthma group. The majority of, but not all, differences were explained by differences in circulating immune cell populations. Pathway analysis showed an increase in chemotaxis, migration, and myeloid cell trafficking in patients with severe asthma, decreased B-lymphocyte development and hematopoietic progenitor cells, and lymphoid organ hypoplasia. Cluster analysis of DEGs led to the creation of subgroups among the patients with severe asthma who differed in molecular responses to oral corticosteroids. CONCLUSIONS: Blood gene expression differences between clinically defined subgroups of patients with asthma and individuals without asthma, as well as subgroups of patients with severe asthma defined by transcript profiles, show the value of blood analysis in stratifying patients with asthma and identifying molecular pathways for further study. Clinical trial registered with www.clinicaltrials.gov (NCT01982162).

Corticosteroid modulation of immunoglobulin expression and B-cell function in COPD.

We investigated changes in gene expression that occur in chronic obstructive pulmonary disease (COPD) after corticosteroid treatment and sought to identify the mechanisms that regulate these changes. Biopsy samples were taken from patients with COPD (Global Initiative for Chronic Obstructive Lung Disease stage I to II) before and after treatment with fluticasone propionate (FP)/salmeterol (SM) (50/500, 4 wk). Gene expression was measured by microarray and was confirmed by real-time reverse transcription-quantitative PCR (RT-qPCR). The effect of FP on IgG expression and B-cell proliferation in the presence of oxidative stress was also studied. FP/SM significantly increased the expression of 180 genes while repressing 343 genes. The top 5 down-regulated genes were associated with immunoglobulin production, whereas the immunomodulatory FK506 binding protein (FK506BP) was up-regulated. Genes including IL6, IL8, and TBET-encoding TBX21 were unaffected. FP reduced IgG protein and mRNA expression and proliferation of human B cells through the dephosphorylation of ERK-1/2 via increased DUSP1 (dual-specificity protein phosphatase 1) expression. Consistent with in vivo data, oxidative stress did not prevent FP-induced suppression of IgG expression in human B cells in vitro Changes in expression were validated by RT-qPCR and by gene set enrichment analysis in distinct COPD cohorts. FP may reduce the adaptive immune response in COPD and may be more effective in patients with an increased B-cell/antibody response indicated by high autoantibody titers.-Lee, J., Machin, M., Russell, K. E., Pavlidis, S., Zhu, J., Barnes, P. J., Chung, K. F., Adcock, I. M., Durham, A. L. Corticosteroid modulation of immunoglobulin expression and B-cell function in COPD.

Oxidative stress-induced antibodies to carbonyl-modified protein correlate with severity of chronic obstructive pulmonary disease.

RATIONALE: There is increasing evidence for the presence of autoantibodies in chronic obstructive pulmonary disease (COPD). Chronic oxidative stress is an essential component in COPD pathogenesis and can lead to increased levels of highly reactive carbonyls in the lung, which could result in the formation of highly immunogenic carbonyl adducts on "self" proteins. OBJECTIVES: To determine the presence of autoantibodies to carbonyl-modified protein in patients with COPD and in a murine model of chronic ozone exposure. To assess the extent of activated immune responses toward carbonyl-modified proteins. METHODS: Blood and peripheral lung were taken from patients with COPD, age-matched smokers, and nonsmokers with normal lung function, as well as patients with severe persistent asthma. Mice were exposed to ambient air or ozone for 6 weeks. Antibody titers were measured by ELISA, activated compliment deposition by immunohistochemistry, and cellular activation by ELISA and fluorescence-activated cell sorter. MEASUREMENTS AND MAIN RESULTS: Antibody titer against carbonyl-modified self-protein was significantly increased in patients with Global Initiative for Chronic Obstructive Lung Disease stage III COPD compared with control subjects. Antibody levels inversely correlated with disease severity and showed a prevalence toward an IgG1 isotype. Deposition of activated complement in the vessels of COPD lung as well as autoantibodies against endothelial cells were also observed. Ozone-exposed mice similarly exhibited increased antibody titers to carbonyl-modified protein, as well as activated antigen-presenting cells in lung tissue and splenocytes sensitized to activation by carbonyl-modified protein. CONCLUSIONS: Carbonyl-modified proteins, arising as a result of oxidative stress, promote antibody production, providing a link by which oxidative stress could drive an autoimmune response in COPD.

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. METHODS: Bronchial biopsies, alveolar macrophages (AM) and blood monocytes (BM) from patients with COPD, healthy smokers and non-smokers, and bronchial epithelial cells (EC) from surgically resected airways from patients with COPD were stimulated with LPS or CRP in the presence and absence of an anti-EMMPRIN blocking antibody. EMMPRIN in BAL, plasma, conditioned media and cell lysates was quantified and immunohistochemical localization of EMMPRIN was determined in bronchial biopsies. MMP-9 activity and mRNA was also determined. RESULTS: EMMPRIN levels in BAL fluid were higher in patients with COPD compared with non-smokers and smokers. There was greater EMMPRIN expression in EC from patients with COPD compared with smokers and non-smokers. EC secreted and expressed more EMMPRIN protein than BM and AM. Blocking EMMPRIN decreased MMP-9 activity in supernatant of EC, but not in those from AM and BM, and decreased MMP-9 mRNA expression in EC. CONCLUSIONS: The increased EMMPRIN expression in COPD is reflected by an increased release from bronchial EC, which are one of the main source of EMMPRIN. EMMPRIN regulates MMP-9 expression in COPD.

Pharmacological studies of the mechanism and function of interleukin-1beta-induced miRNA-146a expression in primary human airway smooth muscle.

BACKGROUND: Despite the widespread induction of miR-146a during the innate immune response little is known regarding its biogenesis, function and mechanism. We have therefore examined the role of miR-146a during the interleukin (IL)-1beta-stimulated IL-6 and IL-8 release and proliferation in primary human airway smooth muscle (HASM) cells. METHODS: HASM cells were isolated from human lung re-section, cultured to a maximum of 3 - 6 passages and then exposed to IL-1beta. miR-146a expression were determined by qRT-PCR, IL-6 and IL-8 release by ELISA and proliferation using bromodeoxyuridine incorporation. The role of NF-kappaB and the MAP kinase pathways was assessed using pharmacological inhibitors of IKK2 (TPCA-1), JNK (SP600125), p38 MAP kinase (SB203580) and MEK-1/2 (PD98059). miR-146a function was determined following transfection of HASM with inhibitors and mimics using Amaxa electroporation. RESULTS: IL-1beta induced a time-dependent and prolonged 100-fold induction in miR-146a expression, which correlated with release of IL-6 and IL-8. Exposure to IL-1beta had no effect upon HASM proliferation. Pharmacological studies showed that expression of primary miR-146a was regulated at the transcriptional levels by NF-kappaB whilst post-transcriptional processing to mature miR-146a was regulated by MEK-1/2 and JNK-1/2. Functional studies indicated that IL-1beta-induced miR-146a expression does not negatively regulate IL-6 and IL-8 release or basal proliferation. However, inhibition of IL-1beta-induced IL-6 and IL-8 release was observed at the super-maximal intracellular miR-146a levels obtained by transfection with miR-146a mimics and indicates that studies using miRNA mimics can produce false positive results. Mechanistic studies showed that in the presence of super-maximal levels, the action of miR-146a mimics was mediated at a step following IL-6 and IL-8 mRNA transcription and not through down-regulation of IL-1 receptor associated kinase 1 (IRAK-1) and TNF receptor-associated factor 6 (TRAF6) protein expression, two predicted miR-146a targets involved in IL-1beta signalling. CONCLUSIONS: We have shown that IL-1beta-induced miR-146a expression in HASM and that this was regulated at the transcriptional level by NF-kappaB and at the post-transcriptional level by the MEK-1/2 and JNK-1/2. Unlike previous reports, studies using miRNA inhibitors showed that miR-146a expression did not regulate IL-6 and IL-8 release or proliferation and suggest miR-146a function and mechanism is cell-type dependent.

MUC5AC expression is increased in bronchial submucosal glands of stable COPD patients.

AIMS: Mucus expectoration is a common feature of chronic obstructive pulmonary disease (COPD). MUC5AC and MUC5B, the major mucins, are released predominantly from submucosal glands in the central airways. The aim was to investigate gland size and MUC5AC and MUC5B expression in bronchial rings from smokers with COPD compared with control groups. METHODS AND RESULTS: Bronchial rings from 10 non-smoking subjects, 20 smokers with normal lung function and 20 smokers with COPD were studied. Periodic acid-Schiff (PAS) and Alcian blue histochemistry and MUC5AC and MUC5B immunohistochemistry followed by quantification of the immunoreactive area was performed. The area occupied by MUC5AC+ cells in bronchial submucosal glands was increased in COPD [20% (5.5-31.7%) gland area] compared with smokers with normal lung function [9.5% (2.5-17.5%); P < 0.05] and non-smoking subjects [2% (0.4-6.2%); P < 0.05]. The area occupied by MUC5AC+ cells in the bronchial surface epithelium was also increased in smokers (with/without COPD) [73.5% (25-92%) epithelial area] compared with non-smoking subjects [15% (2.7-32%); P < 0.01]. Gland size, PAS, Alcian blue staining and MUC5B expression were not significantly different among groups. MUC5AC expression correlated with the degree of airflow obstruction. MUC5AC and MUC5B expression correlated with pack-years. CONCLUSIONS: COPD is associated with increased MUC5AC expression in submucosal glands, indicating that MUC5AC may be involved in the pathophysiology of the disease.

Cigarette smoke induces IL-8, but inhibits eotaxin and RANTES release from airway smooth muscle.

BACKGROUND: Cigarette smoke is the leading risk factor for the development of chronic obstructive pulmonary disease (COPD) an inflammatory condition characterised by neutrophilic inflammation and release of proinflammatory mediators such as interleukin-8 (IL-8). Human airway smooth muscle cells (HASMC) are a source of proinflammatory cytokines and chemokines. We investigated whether cigarette smoke could directly induce the release of chemokines from HASMC. METHODS: HASMC in primary culture were exposed to cigarette smoke extract (CSE) with or without TNFalpha. Chemokines were measured by enzyme-linked immunosorbent assay (ELISA) and gene expression by real time polymerase chain reaction (PCR). Data were analysed using one-way analysis of variance (ANOVA) followed by Bonferroni's t test RESULTS: CSE (5, 10 and 15%) induced IL-8 release and expression without effect on eotaxin or RANTES release. At 20%, there was less IL-8 release. TNFalpha enhanced CSE-induced IL-8 release and expression. However, CSE (5-30%) inhibited TNFalpha-induced eotaxin and RANTES production. The effects of CSE on IL-8 release were inhibited by glutathione (GSH) and associated with the induction of the oxidant sensing protein, heme oxygenase-1. CONCLUSION: Cigarette smoke may directly cause the release of IL-8 from HASMC, an effect enhanced by TNF-alpha which is overexpressed in COPD. Inhibition of eotaxin and RANTES by cigarette smoke is consistent with the predominant neutrophilic but not eosinophilic inflammation found in COPD.

Inflammatory mediators in chronic obstructive pulmonary disease.

Chronic obstructive pulmonary disease (COPD) is characterised by chronic obstruction of expiratory flow affecting peripheral airways, associated with chronic bronchitis (mucus hypersecretion with goblet cell and submucosal gland hyperplasia) and emphysema (destruction of airway parenchyma), together with fibrosis and tissue damage, and inflammation of the small airways. Inflammatory mediators include lipid mediators, chemokines, cytokines, growth factors, reactive oxygen species and proteinases. Increased levels of interleukin (IL)-6, IL-1beta, tumour necrosis factor-alpha (TNF-alpha) and IL-8 have been measured in sputum, with further increases during exacerbations, and the bronchiolar epithelium over-expresses MCP-1 and IL-8. IL-8 and LTB4 can account for neutrophil chemotactic activity of sputum. The expression of chemokines such as RANTES and eotaxin may underlie the airway eosinophilia observed in some COPD patients. Reactive oxygen species can increase gene expression of many inflammatory mediators, such as IL-1 and TNFalpha from macrophages, alveolar and bronchial epithelial cells. TNFalpha and IL-1beta stimulate macrophages to produced matrix metalloproteinase-9 (MMP-9), and bronchial epithelial cells to produce extracellular matrix glycoproteins such as tenascin. Increased expression of transforming growth factor-beta (TGFbeta) and of epidermal growth factor (EGF) occurs in the epithelium and submucosal cells of patients with chronic bronchitis. TGFbeta and EGF activate proliferation of fibroblasts, while activation of the EGF receptor leads to mucin gene expression.

p38 Mitogen-activated protein kinase-induced glucocorticoid receptor phosphorylation reduces its activity: role in steroid-insensitive asthma.

BACKGROUND: Although glucocorticoids are the most effective treatment for chronic inflammatory diseases, such as asthma, some patients show a poor response. IL-2 combined with IL-4 can alter glucocorticoid receptor (GR) ligand-binding affinity and modulate glucocorticoid function. OBJECTIVE: We sought to confirm the altered ligand-binding affinity in a distinct group of steroid-dependent asthmatic subjects and examine the mechanism by which IL-2 and IL-4 modify the ligand-binding affinity of the GR. METHODS: We examined PBMCs from healthy subjects, subjects with mild asthma, and steroid-dependent subjects with severe asthma using dexamethasone-binding assays and Western blot analysis of GR and phosphorylated activated transcription factor 2 expression. GR phosphorylation was measured after orthophosphate labeling and immunoprecipitation and cytokine production by means of ELISA. RESULTS: GR ligand-binding affinity was reduced in the nucleus but not in the cytoplasm of steroid-dependent asthmatic subjects compared with that seen in healthy subjects (dissociation constant, 39.8 +/- 4.6 vs. 6.79 +/- 0.8 nmol/L). This difference in ligand-binding affinity could be mimicked by IL-2 and IL-4 cotreatment and was blocked by the p38 mitogen-activated kinase (MAPK) inhibitor SB203580. Activation of p38 MAPK by IL-2 and IL-4, as shown by means of phosphorylation of activated transcription factor 2, resulted in GR phosphorylation and reduced dexamethasone repression of LPS-stimulated GM-CSF release. p38 MAPK phosphorylation of CD2(+) T cells occurred on serine residues. The ability of dexamethasone to modulate IL-10 release was also inhibited by IL-2 and IL-4 cotreatment. These effects were also inhibited by SB203580. CONCLUSION: These data show that p38 MAPK inhibitors may have potential in reversing glucocorticoid insensitivity and reestablishing the beneficial effects of glucocorticoids in patients with severe asthma.