Search

Introduction: COL4A3 degradation is significantly increased in severe, exacerbating type 2 asthmatics and correlates with increased chymase positive mast cells (mouse model of asthma). However, whether chymase is sufficient/necessary for COL4A3 degradation and if this mode of action is responsive to therapeutic intervention, is unknown. We hypothesized, that mast cell chymase is necessary for COL4A3 degradation and that treatment with anti-IgE reduces COL4A3 degradation. Methods: The COL4A3 fibrolysis marker C4Ma3 (Nordic Bioscience, Denmark) was measured in female 6-8 week old Balb/C mice, which were sensitized (OVA i.p., day 1, 14, 21) and challenged with aerosol (OVA i.n., day 26, 27, 28) to induce acute allergic airway inflammation. Acute exacerbations were provoked by instillation of poly(cytidylic-inosinic, i.n. day 28) and Chymase was inhibited with Fulacimstat (i.t.). Allergic, uncontrolled asthmatics (n=19, Omalizumab treatment), were monitored over six months. Treatment response was set as Asthma Control Test (ACT) improvement of g3 points. Serum levels of C4Ma3 were determined at baseline, 3 and 6 months. Results: Chymase inhibition prevented OVA-challenge induced C4Ma3 increase. Recombinant chymase alone did not induce elevated C4Ma3 level. Ten out of 19 patients had ACT score increases of g3 points after treatment. Responders mean level of circulating C4Ma3 was 8.87 ng/mL (confidence interval (CI): 7.02-10.72ng/mL), which decreased to 6.57 ng/mL (CI: 4.95-8.1ng/mL, pl0.01) after six months treatment. Non-responders did not show any change in serum levels of after treatment. Conclusion: COL4A3 degradation depends on mast cell chymase in vivo and monitoring serum C4Ma3, may afford a novel avenue to stratify and monitor anti-IgE therapy in severe, allergic asthma.

Introduction: Remodeling of the airway wall is a hallmark feature of asthma. Under physiological conditions, a finely tuned balance maintains a functional state of the extracellular matrix. This balance is disrupted in asthma. COL4A3 is reduced 18-fold in lung tissue from asthmatics, however, the mechanism leading to the diminished levels of COL4A3 has remained elusive. Methods: The COL4A3 fibrolysis marker C4Ma3 (Nordic Bioscience, Denmark) was measured in the ALLIANCE cohort study participants (Fuchs et al. 2018). Pediatric controls/cases: 34/134; Adult control/cases 31/149. C4Ma3 data was correlated with clinical data, serum cytokine profiles (27-Bioplex, Biorad). Female 6-8 week old Balb/C mice were sensitized (OVA i.p., day 1, 14, 21) and challenged with aerosol (OVA i.n., day 26, 27, 28) to induce acute allergic airway inflammation. Acute exacerbations were provoked by instillation of poly(cytidylic-inosinic, i.n. day 28). Murine precision cut lung slices (PCLS) were used to elucidate the role of sensitisation for COL4A3 degradation. Results: Increased C4Ma3 correlated with asthma exacerbation (pl0.01) and elevated levels of Th2/Th9 cytokines in the ALLIANCE cohort. Serum levels of C4Ma3 were elevated in OVA challenged mice (pl0.0001) and correlated with lung tissue levels of mast cell chymase (pl0.01), but not with neutrophils or matrix-metallo-proteinase 9. C4MA3 was elevated in PCLS of allergic, HDM challenged mice (pl0.05) with no further increase after PolyIC. Conclusion: COL4A3 degradation (circulating C4Ma3), is elevated in severe, exacerbating type 2 asthmatics. C4Ma3 levels correlate with chymase positive mast cells in a mouse model of asthma exacerbation. These data highlight that the degradation of COL4A3 is a central part of the pathology of asthma.

BackgroundAsthma is a heterogeneous syndrome substantiating the urgent requirement for endotype-specific biomarkers. Dysbalance of fibrosis and fibrolysis in asthmatic lung tissue leads to reduced levels of the inflammation-protective collagen 4 (COL4A3).ObjectiveTo delineate the degradation of COL4A3 in allergic airway inflammation and evaluate the resultant product as a biomarker for anti-IgE therapy response.MethodsThe serological COL4A3 degradation marker C4Ma3 (Nordic Bioscience, Denmark) and serum cytokines were measured in the ALLIANCE cohort (paediatric cases/controls: n=134/n=35; adult cases/controls: n=149/n=31). Exacerbation of allergic airway disease in mice was induced by sensitising to ovalbumin (OVA), challenge with OVA aerosol and instillation of poly(cytidylic-inosinic). Fulacimstat (chymase inhibitor; Bayer) was used to determine the role of mast cell chymase in COL4A3 degradation. Patients with cystic fibrosis (n=14) and cystic fibrosis with allergic bronchopulmonary aspergillosis (ABPA; n=9) as well as patients with severe allergic uncontrolled asthma (n=19) were tested for COL4A3 degradation. Omalizumab (anti-IgE) treatment was assessed using the Asthma Control Test.ResultsSerum levels of C4Ma3 were increased in asthma in adults and children alike and linked to a more severe, exacerbating allergic asthma phenotype. In an experimental asthma mouse model, C4Ma3 was dependent on mast cell chymase. Serum C4Ma3 was significantly elevated in cystic fibrosis plus ABPA and at baseline predicted the success of the anti-IgE therapy in allergic, uncontrolled asthmatics (diagnostic OR 31.5).ConclusionC4Ma3 levels depend on lung mast cell chymase and are increased in a severe, exacerbating allergic asthma phenotype. C4Ma3 may serve as a novel biomarker to predict anti-IgE therapy response.

Exposure to respiratory pathogens is a leading cause of exacerbations of airway diseases such as asthma and chronic obstructive pulmonary disease (COPD). Pellino-1 is an E3 ubiquitin ligase known to regulate virally-induced inflammation. We wished to determine the role of Pellino-1 in the host response to respiratory viruses in health and disease. Pellino-1 expression was examined in bronchial sections from patients with GOLD stage two COPD and healthy controls. Primary bronchial epithelial cells (PBECs) in which Pellino-1 expression had been knocked down were extracellularly challenged with the TLR3 agonist poly(I:C). C57BL/6 Peli1−/− mice and wild type littermates were subjected to intranasal infection with clinically-relevant respiratory viruses: rhinovirus (RV1B) and influenza A. We found that Pellino-1 is expressed in the airways of normal subjects and those with COPD, and that Pellino-1 regulates TLR3 signaling and responses to airways viruses. In particular we observed that knockout of Pellino-1 in the murine lung resulted in increased production of proinflammatory cytokines IL-6 and TNFα upon viral infection, accompanied by enhanced recruitment of immune cells to the airways, without any change in viral replication. Pellino-1 therefore regulates inflammatory airway responses without altering replication of respiratory viruses.

COPD is characterized by chronic lung inflammation and irreversible lung tissue damage. Inhaled noxious gases, including cigarette smoke, are the major risk factor for COPD. Inhaled smoke first encounters the epithelial lining of the lungs, causing oxidative stress and mitochondrial dysfunction. We investigated whether a mitochondrial defect may contribute to increased lung epithelial pro-inflammatory responses, impaired epithelial repair and reduced corticosteroid sensitivity as observed in COPD. We used wild-type alveolar epithelial cells A549 and mitochondrial DNA-depleted A549 cells (A549 Rho-0) and studied pro-inflammatory responses using (multiplex) ELISA as well as epithelial barrier function and repair (real-time impedance measurements), in the presence and absence of the inhaled corticosteroid budesonide. We observed that A549 Rho-0 cells secrete higher levels of pro-inflammatory cytokines than wild-type A549 cells and display impaired repair upon wounding. Budesonide strongly suppressed the production of neutrophil attractant CXCL8, and promoted epithelial integrity in A549 wild-type cells, while A549 Rho-0 cells displayed reduced corticosteroid sensitivity compared to wild-type cells. The reduced corticosteroid responsiveness may be mediated by glycolytic reprogramming, specifically glycolysis-associated PI3K signaling, as PI3K inhibitor LY294002 restored the sensitivity of CXCL8 secretion to corticosteroids in A549 Rho-0 cells. In conclusion, mitochondrial defects may lead to increased lung epithelial pro-inflammatory responses, reduced epithelial repair and reduced corticosteroid responsiveness in lung epithelium, thus potentially contributing to the pathogenesis of COPD.

Human lungs enable efficient gas exchange and form an interface with the environment, which depends on mucosal immunity for protection against infectious agents. Tightly controlled interactions between structural and immune cells are required to maintain lung homeostasis. Here, we use single-cell transcriptomics to chart the cellular landscape of upper and lower airways and lung parenchyma in healthy lungs, and lower airways in asthmatic lungs. We report location-dependent airway epithelial cell states and a novel subset of tissue-resident memory T cells. In the lower airways of patients with asthma, mucous cell hyperplasia is shown to stem from a novel mucous ciliated cell state, as well as goblet cell hyperplasia. We report the presence of pathogenic effector type 2 helper T cells (TH2) in asthmatic lungs and find evidence for type 2 cytokines in maintaining the altered epithelial cell states. Unbiased analysis of cell-cell interactions identifies a shift from airway structural cell communication in healthy lungs to a TH2-dominated interactome in asthmatic lungs.

Previously, we observed increased serum levels of damage-associated molecular patterns (DAMPs) during COPD exacerbations. Here, gene expression of DAMP receptors was measured in peripheral blood neutrophils of COPD patients during stable disease and severe acute exacerbation. The expression of toll-like receptor (TLR)2, TLR4 and NLR family, pyrin domain-containing 3 (NLRP3) was significantly increased, while serum levels of the soluble form of the decoy receptor for advanced glycation end-product (sRAGE) were decreased during exacerbation. Together, these data indicate that increased DAMP signalling contributes to activation of neutrophils during COPD exacerbations.

Wingless/integrase-1 (WNT) signalling is associated with lung inflammation and repair, but its role in chronic obstructive pulmonary disease (COPD) pathogenesis is unclear. We investigated whether cigarette smoke-induced dysregulation of WNT-5B contributes to airway remodelling in COPD.We analysed WNT-5B protein expression in the lung tissue of COPD patients and (non)smoking controls, and investigated the effects of cigarette smoke exposure on WNT-5B expression in COPD and control-derived primary bronchial epithelial cells (PBECs). Additionally, we studied downstream effects of WNT-5B on remodelling related genes fibronectin, matrix metalloproteinase (MMP)-2, MMP-9 and SnaiI in BEAS-2B and air–liquid interface (ALI)-cultured PBECs.We observed that airway epithelial WNT-5B expression is significantly higher in lung tissue from COPD patients than controls. Cigarette smoke extract significantly increased mRNA expression of WNT-5B in COPD, but not control-derived PBECs. Exogenously added WNT-5B augmented the expression of remodelling related genes in BEAS-2B cells, which was mediated by transforming growth factor (TGF)-β/Smad3 signalling. In addition, WNT-5B upregulated the expression of these genes in ALI-cultured PBECs, particularly PBECs from COPD patients.Together, our results provide evidence that exaggerated WNT-5B expression upon cigarette smoke exposure in the bronchial epithelium of COPD patients leads to TGF-β/Smad3-dependent expression of genes related to airway remodelling.

The aim was to investigate whether microRNA (miRNA) expression is modulated by inhaled corticosteroid (ICS) treatmentWe performed genome-wide miRNA analysis on bronchial biopsies of 69 moderate/severe chronic obstructive pulmonary disease (COPD) patients at baseline and after 6- and 30-month treatment with the ICS fluticasone propionate or placebo. The effect of ICS on miRNA expression was validated in differentiated primary bronchial epithelial cultures, and functional studies were conducted in BEAS-2B cells. MiRNAs affected by ICS and their predicted targets were compared to an independent miRNA dataset of bronchial brushings from COPD patients and healthy controls.Treatment with ICS for both 6 and 30 months significantly altered the expression of four miRNAs, including miR-320d, which was increased during ICS treatment compared with placebo. The ICS-induced increase of miR-320d was confirmed in primary airway epithelial cells. MiR-320d negatively correlated targets were enriched for pro-inflammatory genes and were increased in the bronchial brushes of patients with lower lung function in the independent dataset. Overexpression of miR-320d in BEAS-2B cells dampened cigarette smoke extract-induced pro-inflammatory activity via inhibition of nuclear factor-κB.Collectively, we identified miR-320d as a novel mediator of ICS, regulating the pro-inflammatory response of the airway epithelium.

Introduction and objectives Exposure to respiratory pathogens is a leading cause of exacerbations of airway diseases such as asthma and chronic obstructive pulmonary disease (COPD). We have shown that Pellino-1, an E3 ubiquitin ligase, is involved in viral-induced TLR3 signalling; thus we sought to describe the role of Pellino-1 in the host response to viral stimulation of the airway. Methods Pellino-1 expression was examined in bronchial sections from patients with GOLD stage 2 COPD (n=7) and healthy controls (n=8). Primary bronchial epithelial cells (PBECs, n=5) in which Pellino-1 expression had been knocked down using siRNA, were extracellularly challenged with the TLR3 agonist poly(I:C). C57BL/6 Peli1-/- mice and wild type littermates were subjected to intranasal infection with the clinically-relevant respiratory viruses rhinovirus (RV1B) and influenza A, and responses monitored up to 8 days later. Results We show that Pellino-1 is expressed in the airways of normal subjects and those with COPD. In the absence of Pellino-1, PBECs showed significant reduction in proinflammatory cytokines, including CXCL8 and IL-6, upon TLR3 activation. Surprisingly however, knockout of Peli1 in the murine lung resulted in increased production of the proinflammatory cytokines IL-6 and TNFα with viral infection. This was accompanied by an enhanced recruitment of immune cells to the airways in these animals, including a population of innate B cells, without loss of viral replication. Conclusions We conclude that Pellino-1 functions as a positive regulator for antiviral responses in isolated bronchial epithelial cells and in systemic responses to TLR3 activation, but it has the opposite effect in the lung. Here, Pellino-1 has a distinct function in the down-regulation of antiviral inflammation; a role dissociated from viral replication. Our data therefore suggest that Pellino-1 may offer therapeutic potential to limit viral inflammation in COPD and asthma.

Cigarette smoke is one of the major risk factors for development of chronic obstructive pulmonary disease (COPD), characterized by chronic inflammation. Bacterial infections can increase inflammatory responses and cause exacerbations in COPD. Extracellular Hsp70 (eHsp70) can act pro-inflammatory via Toll-like receptors (TLR) 2 or 4, and was found elevated in sera of COPD patients. We hypothesized that eHsp70 modulates the inflammatory response provoked by bacterial compounds and cigarette smoke. Therefore, we aimed to investigate the effect of eHsp70 present together with lipopolysaccharide (LPS), lipoteichoic acid (LTA) or cigarette smoke extract (CSE) on interleukin-8 (IL-8) secretion. We treated primary tracheo-bronchial epithelial cells (PBEC) isolated from healthy controls and COPD patients either with recombinant human (rh) Hsp70 (0.1, 1 and 3 µg/ml), 0.1 µg/ml LPS, 1 µg/ml LTA and CSE (2.5 and 15%) alone or with their combinations. IL-8 levels in cell-free supernatants were determined by ELISA. RhHsp70, LTA and CSE alone increased IL-8 secretion in PBEC both from healthy volunteers and COPD patients, while LPS had no significant effect. Instead of an additive effect, combined treatment with rhHsp70 reduced IL-8 levels compared to the presence of LPS, LTA or CSE alone in COPD-derived PBEC, but not those from healthy controls. In conclusion, rhHsp70 reduces the pro-inflammatory effects of bacterial components and CSE in PBEC cells from COPD patients. Their combined presence could lead to attenuation of pro-inflammatory epithelial responses in COPD patients, and Hsp70 could have a protective instead of detrimental effect in COPD.

Nasal gene expression profiling is a promising method to characterize COPD non-invasively. We aimed to identify a nasal gene expression profile to distinguish COPD patients from healthy controls. We investigated whether this COPD-associated gene expression profile in nasal epithelium is comparable with the profile observed in bronchial epithelium. Genome wide gene expression analysis was performed on nasal epithelial brushes of 31 severe COPD patients and 22 controls, all current smokers, using Affymetrix Human Gene 1.0 ST Arrays. We repeated the gene expression analysis on bronchial epithelial brushes in 2 independent cohorts of mild-to-moderate COPD patients and controls. In nasal epithelium, 135 genes were significantly differentially expressed between severe COPD patients and controls, 21 being up- and 114 downregulated in COPD (false discovery rate

Introduction: Nasal gene expression profiling is a promising method to characterize COPD non-invasively. First, we aimed to identify a nasal gene expression profile that distinguishes COPD patients from healthy controls. Next, we investigated whether this COPD-associated gene expression profile in nasal epithelium is comparable with the profile in the lower airways, i.e. the bronchial epithelium. Methods: Genome wide gene expression analysis was performed on nasal epithelial brushes of 76 COPD patients and 39 healthy controls, using Affymetrix Human Gene 1.0 ST Arrays. To compare findings in nasal and bronchial epithelium, we repeated the gene expression analysis on bronchial epithelial brushes in 2 independent cohorts of COPD patients and controls. Results: We found 2673 genes to be significantly differentially expressed in nasal epithelium between COPD patients and controls, 1158 being up- and 1515 downregulated in COPD (false discovery rate Conclusion: We identified a nasal gene expression profile that differentiates individuals with and without COPD. Of interest, part of the nasal gene expression changes in COPD is comparable to differentially expressed genes in the bronchus. These findings indicate that nasal gene expression has the potential to be developed as a non-invasive biomarker in COPD.

Th17-mediated neutrophilic airway inflammation has been implicated in decreased response to glucocorticoids in asthma. We aimed to investigate the effect of glucocorticoids on the airway epithelial release of the neutrophilic and Th17-cell chemoattractant CCL20.We studied CCL20 and CXCL8 sputum levels in asthmatic subjects using inhaled glucocorticoids or not, and the effect of budesonide on CCL20 and CXCL8 production in primary bronchial epithelial cells. The mechanism behind the effect of budesonide-induced CCL20 production was studied in 16HBE14o- cells using inhibitors for the glucocorticoid receptor, intracellular pathways and metalloproteases.We observed higher levels of CCL20, but not CXCL8, in the sputum of asthmatics who used inhaled glucocorticoids. CCL20 levels correlated with inhaled glucocorticoid dose and sputum neutrophils. Budesonide increased tumour necrosis factor (TNE)-alpha-induced CCL20 by primary bronchial epithelium, while CXCL8 was suppressed. In 16HBE14o- cells, similar effects were observed at the CCL20 protein and mRNA levels, indicating transcriptional regulation. Although TNF-alpha-induced CCL20 release was dependent on the ERK, p38 and STAT3 pathways, the increase by budesonide was not. Inhibition of glucocorticoid receptor or ADAM17 abrogated the budesonide-induced increase in CCL20 levels.We show that glucocorticoids enhance CCL20 production by bronchial epithelium, which may constitute a novel mechanism in Th17-mediated glucocorticoid-insensitive inflammation in asthma.

Chronic obstructive pulmonary disease (COPD) is characterized by abnormal repair in the lung resulting in airway obstruction associated with emphysema and peripheral airway fibrosis. Because the presence and degree of airways disease and emphysema varies between COPD patients, this may explain the heterogeneity in the response to treatment. It is currently unknown whether and to what extent inhaled steroids can affect the abnormal repair process in the airways and lung parenchyma in COPD. We investigated the effects of fluticasone on transforming growth factor (TGF)-β- and cigarette smoke-induced changes in mothers against decapentaplegic homolog (Smad) signaling and extracellular matrix (ECM) production in airway and parenchymal lung fibroblasts from patients with severe COPD. We showed that TGF-β-induced ECM production by pulmonary fibroblasts, but not activation of the Smad pathway, was sensitive to the effects of fluticasone. Fluticasone induced decorin production by airway fibroblasts and partly reversed the negative effects of TGF-β treatment. Fluticasone inhibited biglycan production in both airway and parenchymal fibroblasts and procollagen 1 production only in parenchymal fibroblasts, thereby restoring the basal difference in procollagen 1 production between airway and parenchymal fibroblasts. Our findings suggest that the effects of steroids on the airway compartment may be beneficial for patients with severe COPD, i.e., restoration of decorin loss around the airways, whereas the effects of steroids on the parenchyma may be detrimental, since the tissue repair response, i.e., biglycan and procollagen production, is inhibited. More research is needed to further disentangle these differential effects of steroid treatment on the different lung compartments and its impact on tissue repair and remodeling in COPD.

BACKGROUND: House dust mite (HDM) affects the immunological and physical barrier function of airway epithelium, leading to allergic sensitization, airway remodeling, and eosinophilic inflammation in mouse models, although the mechanisms are still largely unknown.OBJECTIVE: Given the implications for adenosine triphosphate (ATP)-dependent Ca(2+) signaling in allergic sensitization in mice, we sought to determine the role of intracellular Ca(2+) concentration ([Ca(2+)](i)) in HDM-induced barrier dysfunction and pro-inflammatory activity of bronchial epithelium.METHODS: We investigated the effect of HDM on accumulation of [Ca(2+)](i) levels, barrier function, and CCL20 release in human bronchial epithelial 16HBE cells and primary bronchial epithelial cells (PBECs) from healthy subjects and asthma patients. Involvement of ATP-dependent activation of purinergic receptors and downstream Ca(2+) influx was studied, using the ATP hydrolyzing agent apyrase, the purinergic receptor agonist PPADS, the calcium chelator BAPTA-AM, and calpain inhibitors.RESULTS: Asthma PBECs were more susceptible to HDM-induced barrier dysfunction, CCL20 secretion, and Ca(2+) influx than healthy PBECs. Furthermore, we show that the HDM-induced increase in CCL20 in PBECs and 16HBE cells and the HDM-induced barrier dysfunction in 16HBE cells are dependent on [Ca(2+)](i) accumulation. Additionally, we demonstrate that [Ca(2+)](i) accumulation is initiated partly through the activation of purinergic receptors, which contributes to HDM-induced epithelial barrier dysfunction by disruption of cell-cell contacts, but not CCL20 secretion.CONCLUSION: Our data show for the first time that Ca(2+) signaling plays a crucial role in barrier dysfunction and the pro-inflammatory response of bronchial epithelium upon HDM exposure and may thus have important implications for the development of allergic asthma.

Background We hypothesised that increased oxidative stress, as present in the airways of asthma and chronic obstructive pulmonary disease (COPD) patients, induces epithelial damage and reduces epithelial responsiveness to suppressive effects of corticosteroids on proinflammatory cytokine production and barrier function.Methods We induced oxidative stress by H2O2 and/or cigarette smoke extract (CSE) in human bronchial epithelial 16HBE cells and primary bronchial epithelial cells (PBEC) derived by brushings from asthma patients, COPD patients, and smoking and non-smoking control individuals. We investigated effects of budesonide on barrier function (electrical resistance) and TNF-alpha-induced proinflammatory cytokine production (IL-8/CXCL8, granulocyte macrophage-colony stimulating factor (GM-CSF)).Results We observed that H2O2 and CSE reduce epithelial resistance. Budesonide significantly counteracted this effect, likely by protection against epidermal growth factor receptor-dependent cell-cell contact disruption. Furthermore, budesonide suppressed proinflammatory cytokine production. H2O2 pretreatment reduced this effect of budesonide on cytokine production in both 16HBE cells and PBECs. Importantly, PBECs from asthma and COPD patients were less sensitive to budesonide with respect to cytokine production and barrier function than PBECs from control subjects.Conclusions Together, our data indicate that budesonide suppresses epithelial proinflammatory responses and barrier dysfunction and that oxidative stress reduces these effects in airway epithelium from asthma and COPD patients. Therefore, restoration of corticosteroid responsiveness in asthma and COPD may act to improve the airway epithelial barrier.

Rationale: Clinical data from large cohorts of COPD patients suggest an increased risk of pneumonia in patients treated with fluticasone propionate (FP) vs no use of inhaled corticosteroids (Thorax 2013;68:1029) and vs budesonide (BUD) (BMJ 2013;346:f3306).Mechanisms underlying this difference are unknown. We hypothesized that BUD and FP differentially affect the susceptibility to bacterial infection upon concomitant viral exposure. Methods: We assessed the effect of 2-hour pre-treatment with equipotent concentrations of BUD (16 nM) and FP (10 nM) on adhesion and/or internalization of S. pneumoniae and barrier function (trans-epithelial resistance; TER) in the human bronchial epithelial 16HBE cells upon concomitant exposure to viral mimetic poly-(I:C). Data were normalized to the poly-(I:C) response. Results: Exposure to poly-(I:C) increased the adhesion and/or internalization of S. pneumoniae administered to the apical side of the cells 2-fold (p S. pneumoniae caused a 7-fold (p Conclusions: BUD, but not FP, counteracted S. pneumoniae adhesion and/or internalization and barrier dysfunction upon concomitant exposure to viral mimetic, suggesting that BUD is more effective than FP in the protection of the epithelial barrier against secondary bacterial infection. This may contribute to the difference in the risk of pneumonia between FP and BUD in COPD. Supported by AstraZeneca.

Background: COPD patients have a higher risk of pneumonia when treated with fluticasone propionate (FP) than with placebo, and a lower risk with budesonide (BUD). We hypothesized that BUD and FP differentially affect the mucosal barrier in response to viral infection and/or cigarette smoke.Methods: We assessed protective effects of equivalent concentrations of BUD and FP on cytokine production and barrier function (electrical resistance) in human bronchial epithelial 16HBE cells and primary bronchial epithelial cells (PBECs) upon exposure to viral mimetic poly-(I:C) and/or cigarette smoke extract (CSE) or epidermal growth factor (EGF).Results: BUD and FP were equally effective in suppressing poly-(I:C)-and/or CSE-induced IL-8 secretion in 16HBE and PBECs. Poly-(I:C) substantially decreased electrical resistance in 16HBE cells and both BUD and FP fully counteracted this effect. However, FP hardly affected 16HBE barrier dysfunction induced by CSE with/without poly-(I:C), whereas BUD (16 nM) provided full protection, an effect likely mediated by affecting EGFR-downstream target GSK-3 beta. Similarly, BUD, but not FP, significantly improved CSE-induced barrier dysfunction in PBECs. Finally, BUD, but not FP, exerted a modest but significant protective effect against Streptococcus Pneumoniae-induced barrier dysfunction, and BUD, but not FP, prevented cellular adhesion and/or internalization of these bacteria induced by poly-(I:C) in 16HBE.Conclusions: Collectively, both BUD and FP efficiently control epithelial pro-inflammatory responses and barrier function upon mimicry of viral infection. Of potential clinical relevance, BUD more effectively counteracted CSE-induced barrier dysfunction, reinforcing the epithelial barrier and potentially limiting access of pathogens upon smoking in vivo.

Introduction: MicroRNAs (miRNA) are small noncoding RNA transcripts that repress gene-expression. Data from the GLUCOLD study showed that miR-320d is down regulated in COPD airway epithelium compared to healthy controls and again upregulated by inhaled corticosteroid treatment in bronchial biopsies from COPD patients. We aimed to identify the role of miR-320d in airway epithelium and further investigate gene targets using the combined gene and miRNA expression data available in the GLUCOLD study. Methods: Affy- miRNA 2.0 and Hugene ST1.0 arrays were performed on bronchial biopsies at baseline and after 6 and 30-month treatment with fluticasone with or without added salmeterol (F/FS) compared to placebo in 69 patients with COPD. MiR-320d was overexpressed in bronchial epithelial BEAS2B cells using miRNA mimics and were then stimulated with 10% cigarette smoke extract (CSE) for 24 hours and pro-inflammatory chemokine IL8 levels were measured using ELISA. Results: Pathway analysis using GeneNetworks identified miR-320d to inversely correlate with genes associated with the innate immune system (p Conclusion: Our data suggest that miR-320d is an anti-inflammatory miRNA, which may influence neutrophil infiltration by repressing IL8 release from airway epithelial cells, providing a novel pathway in the regulation of inflammation in the lung.

Although inhaled corticosteroids reduce the number of exacerbations in COPD, it has been established that fluticasone propionate(FP) increases the risk of pneumonia, a finding not reported or in lower frequency with budesonide (BUD). The mechanisms underlying this difference are unknown. We hypothesized that FP is more efficacious than BUD in the suppression of immune defense genes upon viral infection. Methods: Primary bronchial epithelial cells from healthy donors (n=11) were cultured for 4 weeks at air-liquid interface to induce mucociliary differentiation. Cells were pre-treated for 2 h with equivalent concentrations of FP (10 nM) or BUD (16 nM) and exposed to rhinovirus (RV) for 24 h. The mRNA expression of antimicrobial defense genes RIG-I, INF-β, S100A8 and lactoferrin, and epithelial barrier molecule E-cadherin, was assessed by qPCR. Results: Exposure to RV significantly increased the expression of RIG-I, IFN-β and S100A8, but not of lactoferrin or E-cadherin. In RV-exposed epithelium, FP and BUD significantly and similarly reduced RIG-I expression, but had no effect on IFN-β, S100A8 or lactoferrin. Interestingly, E-cadherin expression was only reduced by FP (p=0.03), and E-cadherin, S100A8 and lactoferrin expression was significantly higher upon RV+BUD than RV+FP treatment (p

Rationale: Periostin plays a role in epithelial-to-mesenchymal transition (EMT). In addition, periostin is emerging as a biomarker for type 2-mediated eosinophilic airway inflammation in asthma. The role of periostin in modulating the phenotype of bronchial epithelial cells (BECs), leading to changes as observed in asthma, is unclear. We hypothesized that the type 2 cytokine IL-13 induces airway epithelial expression of periostin, which in turn contributes to epithelial changes and airway remodeling in asthma. Methods: We studied the effect of IL-13 on epithelial morphology and on the mRNA expression of the epithelial marker E-cadherin and the EMT markers periostin and fibronectin in the human bronchial epithelial cell line BEAS-2B and in human primary epithelial cells obtained from trachea-bronchial tissue of healthy individuals. TGF-β was used as positive control and IL-5 as negative control. Results: Upon 48 hours of incubation, both TGF-β and IL-13, but not IL-5, induced a spindle-like shape in BEAS-2B cells, characteristic of EMT. This was accompanied by the up-regulation of periostin and fibronectin mRNA expression, and down-regulation of E-cadherin expression in these cells. Similar changes were observed in air-liquid interface (ALI)-cultured primary human BECs. Conclusions: Our data show that IL-13 induces periostin expression along with EMT-like changes in BEC and may thus contribute to epithelial remodeling as observed in asthma.

Background: House dust mite (HDM) acts on the airway epithelium to induce airway inflammation in asthma. We previously showed that the ability of HDM to induce allergic sensitization in mice is related to airway epithelial CCL20 secretion.Objective: As a disintegrin and metalloprotease (ADAM)s have been implicated in chemokine shedding, we sought to determine their involvement in HDM-induced release of chemokines, including CCL20, by airway epithelial cells.Methods: We studied the effects of pharmacological ADAM inhibitors as well as ADAM10 and ADAM17 siRNA downregulation on chemokine release using (multiplex) ELISA in supernatants from HDM-exposed human bronchial epithelial 16HBE cells and primary normal human bronchial epithelial cells (NHBE) at 4-24 h.Results: House dust) mite markedly increased CCL20 levels in both 16HBE and NHBE cells (16-24 h). In 16HBE cells, the HDM-induced increase was observed as early as 4 h upon exposure and the use of specific inhibitors indicated the involvement of ADAM10/17-mediated shedding. siRNA knockdown of ADAM 10, but not of ADAM 17, significantly reduced the HDM-induced release of CCL20 in both 16HBE and NHBE cells. A similar effect was observed for HDM-induced CCL2, CCL5, and CXCL8 release in NHBE cells. The HDM-induced increase in CCL20 levels was not affected by protein synthesis inhibitor cycloheximide nor protein transport inhibitor monensin, indicating that HDM induces surface shedding of chemokines.Conclusion: Our data show for the first time that ADAM10 activity contributes to HDM-induced shedding of chemokines, including CCL20. The ADAM10/CCL20 axis may be a target for novel therapeutic strategies in asthma.

Background Chronic Obstructive Pulmonary Disease (COPD) is characterized by defective extracellular matrix (ECM) turnover as a result of prolonged cigarette smoking. Fibroblasts have a central role in ECM turnover. The TGFβ induced Smad pathway provides intracellular signals to regulate ECM production. We address the following hypothesis: fibroblasts have abnormal expression of genes in the Smad pathway in COPD, resulting in abnormal proteoglycan modulation, the ground substance of ECM. Methods We compared gene expression of the Smad pathway at different time points after stimulation with TGFβ, TNF or cigarette smoke extract (CSE) in pulmonary fibroblasts of GOLD stage II and IV COPD patients, and controls. Results Without stimulation, all genes were similarly expressed in control and COPD fibroblasts. TGFβ stimulation: downregulation of Smad3 and upregulation of Smad7 occurred in COPD and control fibroblasts, indicating a negative feedback loop upon TGFβ stimulation. CSE hardly influenced gene expression of the TGFβ-Smad pathway in control fibroblasts, whereas it reduced Smad3 and enhanced Smad7 gene expression in COPD fibroblasts. Furthermore, decorin gene expression decreased by all stimulations in COPD but not in control fibroblasts. Conclusion Fibroblasts of COPD patients and controls differ in their regulation of the Smad pathway, the contrast being most pronounced under CSE exposure. This aberrant responsiveness of COPD fibroblasts to CSE might result in an impaired tissue repair capability and is likely important with regard to the question why only a subset of smokers demonstrates an excess ECM destruction under influence of cigarette smoking.

Background: Regulatory T-cells (Treg) are natural Suppressors of autoimmunity. Previous studies indicate that immunosuppressive drugs, especially calcineurin-inhibitors, may interfere with Treg homeostasis. Inflammatory bowel disease (IBD) can relapse or develop de novo after liver transplantation. IBD is associated with a relative deficiency of Treg. The aim of this study was to determine the effect of long-term immunosuppression on the presence of Treg in the noninflamed colonic mucosa of liver transplant recipients.Methods: Colonic biopsies of normal mucosa of 36 liver transplant recipients on different types of immunosuppression and 11 controls were studied. Treg marker Foxp3 and Treg products transforming growth factor-beta (TGF-beta) and interleukin-10 (IL-10) were studied by quantitative polymerase chain reaction (Q-PCR) and immunohistochemistry. TGF-beta-induced Smad-protein 3 and 7 were studied by Q-PCR.Results: No significant differences between controls and patients were observed in IL-10, TGF-beta, and Smad expression. Mucosal Foxp3 mRNA levels and Foxp3+CD3+ cells were significantly 4 reduced in transplant recipients using prednisone/azathioprine/tacrolimus compared with controls but no direct relationship between Foxp3 expression and I specific drug was detected.Conclusions: These results challenge the hypothesis that calcineurin-induced reduction of Treg or TGF-beta expression predisposes nontransplanted tissue to inflammation, but indicate that combined immunosuppression hampers Treg development in the intestine.

Chronic obstructive pulmonary disease (COPD) is characterised by destruction of extracellular matrix (ECM) in parenchymal areas, whereas the bronchial walls can show fibrosis. In addition, an extensive inflammatory process is observed. CD8+ T-cells, located throughout the lung, and epithelial cells in centrally located airways, produce cytokines involved in the inflammatory process. These cytokines may influence the present fibroblasts, the key effectors in the defective ECM repair and maintenance in COPD.The current authors explored the effects of the cytokine microenvironment on cell-cell interaction gene expression in pulmonary fibroblasts of controls (n=6), and Global Initiative for Chronic Obstructive Lung Disease stage II (n=7) and stage IV (n=7) COPD patients. The current authors simulated the in vivo microenvironment using supernatants of CD3/CD28 stimulated CD8+ T-cells isolated from peripheral blood of COPD patients, supernatant of a bronchial-epithelial cell line, or a combination of both.The present data show that fibroblasts of chronic obstructive pulmonary disease patients display an altered response to the cytokine microenvironment, depending on both the disease stage and the central or peripheral location in the lung. Especially adhesion-related genes are upregulated in fibroblasts of chronic obstructive pulmonary disease patients, which can indicate a more pronounced role of fibroblasts in the inflammatory process in chronic obstructive pulmonary disease, possibly resulting in reduced function as effectors of extracellular matrix repair.