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author:("folkers, Gert")
1.  Dietary galacto-oligosaccharides prevent airway eosinophilia and hyperresponsiveness in a murine house dust mite-induced asthma model 
Respiratory Research  2015;16(1):17.
Background
Allergic asthma is strongly associated with the exposure to house dust mite (HDM) and is characterized by eosinophilic pulmonary inflammation and airway hyperresponsiveness (AHR). Recently, there is an increased interest in using dietary oligosaccharides, also known as prebiotics, as a novel strategy to prevent the development of, or reduce, symptoms of allergy.
Aim
We investigated the preventive capacity of dietary galacto-oligosaccharides (GOS) compared to an intra-airway therapeutic treatment with budesonide on the development of HDM-induced allergic asthma in mice.
Methods
BALB/c mice were intranasally sensitized with 1 μg HDM on day 0 followed by daily intranasal challenge with PBS or 10 μg HDM on days 7 to 11. Two weeks prior to the first sensitization and throughout the experiment mice were fed a control diet or a diet containing 1% GOS. Reference mice were oropharyngeally instilled with budesonide (500 μg/kg) on days 7, 9, 11, and 13, while being fed the control diet. On day 14, AHR was measured by nebulizing increasing doses of methacholine into the airways. At the end of the experiment, bronchoalveolar lavage fluid (BALF) and lungs were collected.
Results
Sensitization and challenge with HDM resulted in AHR. In contrast to budesonide, dietary intervention with 1% GOS prevented the development of AHR. HDM sensitization and challenge resulted in a significant increase in BALF leukocytes numbers, which was suppressed by budesonide treatment and dietary intervention with 1% GOS. Moreover, HDM sensitization and challenge resulted in significantly enhanced concentrations of IL-6, CCL17, IL-33, CCL5 and IL-13 in lung tissue. Both dietary intervention with 1% GOS or budesonide treatment significantly decreased the HDM-induced increased concentrations of CCL5 and IL-13 in lung tissue, while budesonide also reduced the HDM-enhanced concentrations of IL-6 and CCL17 in lung tissue.
Conclusion
Not only did dietary intervention with 1% GOS during sensitization and challenge prevent the induction of airway eosinophilia and Th2-related cytokine and chemokine concentrations in the lung equally effective as budesonide treatment, it also prevented AHR development in HDM-allergic mice. GOS might be useful for the prevention and/or treatment of symptoms in asthmatic disease.
Electronic supplementary material
The online version of this article (doi:10.1186/s12931-015-0171-0) contains supplementary material, which is available to authorized users.
doi:10.1186/s12931-015-0171-0
PMCID: PMC4327967
Asthma; House dust mite; Galacto-oligosaccharide; Budesonide
2.  Measurement of airway function using invasive and non-invasive methods in mild and severe models for allergic airway inflammation in mice 
In this study a direct comparison was made between non-invasive and non-ventilated unrestrained whole body plethysmography (Penh) (conscious animals) and the invasive ventilated lung resistance (RL) method (anesthetized animals) in both mild and severe allergic airway inflammation models. Mild inflammation was induced by intraperitoneal sensitization and aerosols of ovalbumin. Severe inflammation was induced by intraperitoneal sensitization using trinitrophenyl-ovalbumin, followed by intranasal challenges with IgE-allergen complexes. A significant increase in airway responsiveness to methacholine was observed in the mild inflammation group when RL was measured. Significant changes in both RL and Penh were observed in the severe inflammation groups. There was a significant increase in the number of inflammatory cells in the Broncho-Alveolar Lavage Fluid (BALF) in both the mild and severe inflammation animals. The enforced ventilation of the animals during the RL measurement further increased the number of cells in the BALF. IL-2 and RANTES levels in the BALF were higher in the severe inflammation groups compared to the mild inflammation groups. Penh gave only reliable measurements during severe airway inflammation. Measuring RL gave consistent results in both mild and severe allergic airway inflammation models however, ventilation induced an additional cell influx into the airways.
doi:10.3389/fphar.2014.00190
PMCID: PMC4129625  PMID: 25161620
airway hyperresponsiveness; airway inflammation; lung resistance; Penh
3.  Elevated CXCL-8 expression in bronchoalveolar lavage correlates with disease severity in patients with acute respiratory distress syndrome resulting from tuberculosis 
Background
Tuberculosis (TB) is a rare but known cause of acute respiratory distress syndrome (ARDS). The role of inflammatory cytokines in the progression of ARDS in TB patients is unknown.
Objectives
In this study we investigated the possible link between the levels of inflammatory cytokines in bronchoalveolar lavage (BAL) in patients with TB or ARDS alone or in patients with TB-induced ARDS (ARDS + TB).
Methods
90 patients were studied: 30 with TB alone, 30 with ARDS alone and 30 with ARDS + TB. BAL was collected by fiberoptic bronchoscopy and the concentrations of interleukin(IL)-6, CXCL8, TNF-α and IL-1β and the amounts of total protein were measured by ELISA and bicinchoninic acid assay (BCA) methods respectively. The correlation between disease severity measured by Murray scores, SOFA and APACHE II analysis and BAL mediators and cells was also determined.
Results
CXCL8 levels in BAL were significantly higher in the ARDS + TB group compared to TB and ARDS alone groups. Disease severity in the ARDS + TB group as determined by Murray score correlated with BAL CXCL8 and neutrophils but not with IL-6, IL-1β and TNF-α concentrations. In addition, CXCL8 levels and neutrophils were increased in non-miliary TB versus miliary TB. This difference in CXCL8 was lost in the presence of ARDS.
Conclusions
BAL CXCL8 levels were significantly higher in patients with ARDS induced by TB and could suggest an important role of CXCL8 in the pathogenesis of this form of ARDS. This further suggests that CXCL8 inhibitors or blockers may be useful to control the onset and/or development of these combined diseases.
doi:10.1186/1476-9255-11-21
PMCID: PMC4126912  PMID: 25110464
ARDS; TB; CXCL8 and neutrophils
4.  Targeting Prolyl Endopeptidase with Valproic Acid as a Potential Modulator of Neutrophilic Inflammation 
PLoS ONE  2014;9(5):e97594.
A novel neutrophil chemoattractant derived from collagen, proline-glycine-proline (PGP), has been recently characterized in chronic obstructive pulmonary disease (COPD). This peptide is derived via the proteolytic activity of matrix metalloproteases (MMP's)-8/9 and PE, enzymes produced by neutrophils and present in COPD serum and sputum. Valproic acid (VPA) is an inhibitor of PE and could possibly have an effect on the severity of chronic inflammation. Here the interaction site of VPA to PE and the resulting effect on the secondary structure of PE is investigated. Also, the potential inhibition of PGP-generation by VPA was examined in vitro and in vivo to improve our understanding of the biological role of VPA. UV- visible, fluorescence spectroscopy, CD and NMR were used to determine kinetic information and structural interactions between VPA and PE. In vitro, PGP generation was significantly inhibited by VPA. In vivo, VPA significantly reduced cigarette-smoke induced neutrophil influx. Investigating the molecular interaction between VPA and PE showed that VPA modified the secondary structure of PE, making substrate binding at the catalytic side of PE impossible. Revealing the molecular interaction VPA to PE may lead to a better understanding of the involvement of PE and PGP in inflammatory conditions. In addition, the model of VPA interaction with PE suggests that PE inhibitors have a great potential to serve as therapeutics in inflammatory disorders.
doi:10.1371/journal.pone.0097594
PMCID: PMC4023971  PMID: 24835793
5.  Bifidobacterium breve Attenuates Murine Dextran Sodium Sulfate-Induced Colitis and Increases Regulatory T Cell Responses 
PLoS ONE  2014;9(5):e95441.
While some probiotics have shown beneficial effects on preventing or treating colitis development, others have shown no effects. In this study, we have assessed the immunomodulating effects of two probiotic strains, Lactobacillus rhamnosus (L. rhamnosus) and Bifidobacterium breve (B. breve) on T cell polarization in vitro, using human peripheral blood mononuclear cells (PBMC), and in vivo, using murine dextran sodium sulfate (DSS) colitis model. With respect to the latter, the mRNA expression of T cell subset-associated transcription factors and cytokines in the colon was measured and the T helper type (Th) 17 and regulatory T cell (Treg) subsets were determined in the Peyer's patches. Both L. rhamnosus and B. breve incubations in vitro reduced Th17 and increased Th2 cell subsets in human PBMCs. In addition, B. breve incubation was also able to reduce Th1 and increase Treg cell subsets in contrast to L. rhamnosus. In vivo intervention with B. breve, but not L. rhamnosus, significantly attenuated the severity of DSS-induced colitis. In DSS-treated C57BL/6 mice, intervention with B. breve increased the expression of mRNA encoding for Th2- and Treg-associated cytokines in the distal colon. In addition, intervention with B. breve led to increases of Treg and decreases of Th17 cell subsets in Peyer's patches of DSS-treated mice. B. breve modulates T cell polarization towards Th2 and Treg cell-associated responses in vitro and in vivo. In vivo B. breve intervention ameliorates DSS-induced colitis symptoms and this protective effect may mediated by its effects on the T-cell composition.
doi:10.1371/journal.pone.0095441
PMCID: PMC4008378  PMID: 24787575
6.  Bifidobacterium breve and Lactobacillus rhamnosus treatment is as effective as budesonide at reducing inflammation in a murine model for chronic asthma 
Respiratory Research  2014;15(1):46.
Background
Asthma is estimated to affect as many as 300 million people worldwide and its incidence and prevalence are rapidly increasing throughout the world, especially in children and within developing countries. Recently, there has been a growing interest in the use of potentially beneficial bacteria for allergic diseases. This study is aimed at exploring the therapeutic effects of long-term treatment with two different beneficial bacterial strains (Bifidobacterium breve M-16 V and Lactobacillus rhamnosus NutRes1) and a glucocorticoid (budesonide), as a reference treatment, on inflammatory response in a murine model for chronic allergic asthma.
Methods
To mimic the chronic disease in asthmatic patients, we used the murine ovalbumin-induced asthma model combined with prolonged allergen exposure. Airway function; pulmonary airway inflammation; airway remodelling, mRNA expression of pattern recognition receptors, Th-specific cytokines and transcription factors in lung tissue; mast cell degranulation; in vitro T cell activation; and expression of Foxp3 in blood Th cells were examined.
Results
Lactobacillus rhamnosus reduced lung resistance to a similar extent as budesonide treatment in chronically asthmatic mice. Pulmonary airway inflammation, mast cell degranulation, T cell activation and airway remodelling were suppressed by all treatments. Beneficial bacteria and budesonide differentially modulated the expression of toll-like receptors (TLRs), nod-like receptors (NLRs), cytokines and T cell transcription factors. Bifidobacterium breve induced regulatory T cell responses in the airways by increasing Il10 and Foxp3 transcription in lung tissue as well as systemic by augmenting the mean fluorescence intensity of Foxp3 in blood CD4+ T cells.
Conclusion
These findings show that Bifidobacterium breve M-16 V and Lactobacillus rhamnosus NutRes1 have strong anti-inflammatory properties that are comparable to budesonide and therefore may be beneficial in the treatment of chronic asthma.
doi:10.1186/1465-9921-15-46
PMCID: PMC4029990  PMID: 24735374
Allergic asthma; Beneficial bacteria; Glucocorticoids; Regulatory T cell
8.  N-acetylated Proline–Glycine–Proline induced G-protein dependent chemotaxis of neutrophils is independent of CXCL8 release 
European journal of pharmacology  2011;668(3):428-434.
Chronic inflammation in lung diseases contributes to lung tissue destruction leading to the formation of chemotactic collagen fragments such as N-acetylated Proline–Glycine–Proline (N-ac-PGP). In this study, we investigated in more detail the mechanism of action of N-ac-PGP in neutrophilic inflammation. N-ac-PGP was chemotactic for human neutrophils via pertussis toxin sensitive G protein-coupled receptors in vitro and directly activated this cell type, which led to cytosolic calcium mobilization and release of CXCL8. Furthermore, using a selective CXCR2 antagonist confirmed that N-ac-PGP-induced neutrophil chemotaxis is mediated through CXCR2 activation. To determine whether N-ac-PGP was solely responsible for the migration and activation of human neutrophils in vitro and not the released CXCL8 upon stimulation with N-ac-PGP, an antibody directed against CXCL8 was used. Performing chemotaxis and calcium influx assays in the presence of this antibody did not alter the effects of N-ac-PGP whereas effects of CXCL8 were attenuated. These experiments indicate that N-ac-PGP, in addition to the direct induction of chemotaxis, also directly activates neutrophils to release CXCL8. In vivo, this may lead in the long term to a self-maintaining situation enhanced by both N-ac-PGP and CXCL8, leading to a further increase in neutrophil infiltration and chronic inflammation.
doi:10.1016/j.ejphar.2011.03.022
PMCID: PMC3761398  PMID: 21458443
N-ac-PGP; Chemotaxis; Neutrophil; Chronic airway inflammation; CXCL8
9.  New Perspective on Dextran Sodium Sulfate Colitis: Antigen-Specific T Cell Development during Intestinal Inflammation 
PLoS ONE  2013;8(7):e69936.
CD4+ T cell responses against oral antigens can develop in inflammatory bowel disease (IBD) patients, which may modulate disease. Dextran sodium sulfate (DSS) colitis is commonly used to study IBD, however, it is not considered the best model in which to study T cell involvement in intestinal disease. Our aim was to determine if antigen-specific T cells could be induced during DSS colitis and if they could be detected after disease resolution. To induce antigen-specific T cells, the tracking antigen, ovalbumin (OVA), was administered orally during colitis initiation. Disease severity was monitored, and the antigen-reactivity of CD4+ T cells examined using CD69 expression. While OVA-directed, CD4+ Foxp3+ regulatory T cells could be detected in the spleens of both OVA-treated control and DSS mice, OVA-reactive, CD4+ Foxp3-T cells were only found in the OVA and DSS-treated mice. These results indicate that during DSS colitis T cells develop that are specific against oral antigens, and they are found systemically after colitis resolution. This gives added depth and utility to the DSS model as well as a way to track T cells that are primed against luminal antigens.
doi:10.1371/journal.pone.0069936
PMCID: PMC3723715  PMID: 23936123
10.  Differential Regulation of Inflammation and Immunity in Mild and Severe Experimental Asthma 
Mediators of Inflammation  2013;2013:808470.
This study aimed at exploring innate and adaptive immunity in allergic asthma by investigation of mRNA expression of pattern recognition receptors, T-cell-specific cytokines, and transcription factors. Mouse models for mild and severe asthma, with similar pathological characteristics observed in humans, were used to study the involved inflammatory markers as a first step in the development of phenotype-directed treatment approaches. In the mild model, mice were sensitized to ovalbumin-Imject Alum and challenged with ovalbumin. In the severe model, mice were sensitized to trinitrophenyl-conjugated ovalbumin and challenged with trinitrophenyl-ovalbumin/IgE immune complex. Pulmonary airway inflammation and mRNA expression of Toll-like receptors (TLRs), NOD-like receptors (NLRs), T cell cytokines, and transcription factors in lung tissue were examined. Different mRNA expression profiles of TLRs, NLRs, T cell cytokines, and transcription factors were observed. In the mild model, Il10 showed the largest increase in expression, whereas in the severe model, it was Infγ with the largest increase. Expression of Tbet was also significantly increased in the severe model. Inflammation and immunity are differentially regulated in mild and severe experimental asthma. This preclinical data may help in directing clinical research towards a better understanding and therapy in mild and severe asthmatic patients.
doi:10.1155/2013/808470
PMCID: PMC3679512  PMID: 23781124
11.  Probiotics in the Management of Lung Diseases 
Mediators of Inflammation  2013;2013:751068.
The physiology and pathology of the respiratory and gastrointestinal tracts are closely related. This similarity between the two organs may underlie why dysfunction in one organ may induce illness in the other. For example, smoking is a major risk factor for COPD and IBD and increases the risk of developing Crohn's disease. Probiotics have been defined as “live microorganisms which, when administered in adequate amounts, confer health benefits on the host.” In model systems probiotics regulate innate and inflammatory immune responses. Commonly used probiotics include lactic acid bacteria, particularly Lactobacillus, Bifidobacterium, and Saccharomyces, and these are often used as dietary supplements to provide a health benefit in gastrointestinal diseases including infections, inflammatory bowel disease, and colon cancer. In this respect, probiotics probably act as immunomodulatory agents and activators of host defence pathways which suggest that they could influence disease severity and incidence at sites distal to the gut. There is increasing evidence that orally delivered probiotics are able to regulate immune responses in the respiratory system. This review provides an overview of the possible role of probiotics and their mechanisms of action in the prevention and treatment of respiratory diseases.
doi:10.1155/2013/751068
PMCID: PMC3662166  PMID: 23737654
12.  Cigarette Smoke-Induced Collagen Destruction; Key to Chronic Neutrophilic Airway Inflammation? 
PLoS ONE  2013;8(1):e55612.
Background
Cigarette smoking induces inflammatory responses in all smokers and is the major risk factor for lung disease such as chronic obstructive pulmonary disease (COPD). In this progressive disease, chronic inflammation in the lung contributes to lung tissue destruction leading to the formation of chemotactic collagen fragments such as N-acetylated Proline-Glycine-Proline (N-ac-PGP). The generation of this tripeptide is mediated by a multistep pathway involving matrix metalloproteases (MMPs) 8 and 9 and prolyl endopeptidase (PE). Here we investigated whether cigarette smoke extract (CSE) stimulates human PMNs to breakdown whole matrix collagen leading to the generation of the chemotactic collagen fragment N-ac-PGP.
Methodology/Principal Findings
Incubating PMNs with CSE led to the release of chemo-attractant CXCL8 and proteases MMP8 and MMP9. PMNs constitutively expressed PE activity as well as PE protein. Incubating CSE-primed PMNs with collagen resulted in collagen breakdown and in N-ac-PGP generation. Incubation of PMNs with the tripeptide N-ac-PGP resulted in the release of CXCL8, MMP8 and MMP9. Moreover, we tested whether PMNs from COPD patients are different from PMNs from healthy donors. Here we show that the intracellular basal PE activity of PMNs from COPD patients increased 25-fold compared to PMNs from healthy donors. Immunohistological staining of human lung tissue for PE showed that besides neutrophils, macrophages and epithelial cells express PE.
Conclusions
This study indicates that neutrophils activated by cigarette smoke extract can breakdown collagen into N-ac-PGP and that this collagen fragment itself can activate neutrophils, which may lead in vivo to a self-propagating cycle of neutrophil infiltration, chronic inflammation and lung emphysema. MMP-, PE- or PGP-inhibitors can serve as an attractive therapeutic target and may open new avenues towards effective treatment of COPD.
doi:10.1371/journal.pone.0055612
PMCID: PMC3561332  PMID: 23383243
13.  Proline-Glycine-Proline as a Potential Biomarker in Chronic Obstructive Pulmonary Disease and Cystic Fibrosis 
Tanaffos  2012;11(2):12-15.
The present review discusses the role of tri-peptide Proline –Glycine –Proline (PGP) as a potential player, biomarker and therapeutic target in this process.
PMCID: PMC4153190  PMID: 25191408
Chemotactic factors; Cystic fibrosis; Chronic obstructive pulmonary disease; Extracellular matrix; Neutrophil activation praline; Interleukin-8 A and B; Serine ednopeptidases
14.  Role of P2X7 Receptors in Release of IL-1β: A Possible Mediator of Pulmonary Inflammation 
Tanaffos  2012;11(2):6-11.
Extracellular ATP is a signaling molecule which plays an important role in alerting the immune system in case of any tissue damage. Recent studies show that binding of ATP to the ionotropic P2X7 receptor of inflammatory cells (macrophages and monocytes) will induce caspase 1 activation. Stimulation of caspase 1 activity results in maturation and release of IL-1β in the inflammasome in Chronic Obstructive Pulmonary Disease (COPD) patients. COPD is an inflammatory disease characterized by emphysema and/or chronic bronchitis and is mostly associated with cigarette smoking. It is one of the leading causes of death in humans and there is currently no medication to stop the progression of disease. A deeper understanding of the mechanism by which the P2X7 receptor triggers IL-1β maturation and release, may open new opportunities for the treatment of inflammatory diseases such as COPD.
PMCID: PMC4153200
Pulmonary inflammation; Interleukin-1β; P2X7 receptor
15.  Cigarette smoke induces β2-integrin-dependent neutrophil migration across human endothelium 
Respiratory Research  2011;12(1):75.
Background
Cigarette smoking induces peripheral inflammatory responses in all smokers and is the major risk factor for neutrophilic lung disease such as chronic obstructive pulmonary disease. The aim of this study was to investigate the effect of cigarette smoke on neutrophil migration and on β2-integrin activation and function in neutrophilic transmigration through endothelium.
Methods and results
Utilizing freshly isolated human PMNs, the effect of cigarette smoke on migration and β2-integrin activation and function in neutrophilic transmigration was studied. In this report, we demonstrated that cigarette smoke extract (CSE) dose dependently induced migration of neutrophils in vitro. Moreover, CSE promoted neutrophil adherence to fibrinogen. Using functional blocking antibodies against CD11b and CD18, it was demonstrated that Mac-1 (CD11b/CD18) is responsible for the cigarette smoke-induced firm adhesion of neutrophils to fibrinogen. Furthermore, neutrophils transmigrated through endothelium by cigarette smoke due to the activation of β2-integrins, since pre-incubation of neutrophils with functional blocking antibodies against CD11b and CD18 attenuated this transmigration.
Conclusion
This is the first study to describe that cigarette smoke extract induces a direct migratory effect on neutrophils and that CSE is an activator of β2-integrins on the cell surface. Blocking this activation of β2-integrins might be an important target in cigarette smoke induced neutrophilic diseases.
doi:10.1186/1465-9921-12-75
PMCID: PMC3128861  PMID: 21651795
16.  Inflammatory changes in the airways of mice caused by cigarette smoke exposure are only partially reversed after smoking cessation 
Respiratory Research  2010;11(1):99.
Background
Tobacco smoking irritates and damages the respiratory tract and contributes to a higher risk of developing lung emphysema. At present, smoking cessation is the only effective treatment for reducing the progression of lung emphysema, however, there is hardly anything known about the effects of smoking cessation on cytokine and chemokine levels in the airways. To the best of our knowledge, this is the first reported in vivo study in which cytokine profiles were determined after cessation of cigarette smoke exposure.
Methods
The severity of airway remodeling and inflammation was studied by analyzing alveolar enlargement, heart hypertrophy, inflammatory cells in the bronchoalveolar lavage fluid (BALF) and lung tissue and by determining the cytokine and chemokine profiles in the BALF of A/J mice exposed to cigarette smoke for 20 weeks and 8 weeks after smoking cessation.
Results
The alveolar enlargement and right ventricle heart hypertrophy found in smoke-exposed mice remained unchanged after smoking cessation. Although the neutrophilic inflammation in the BALF of cigarette smoke-exposed animals was reduced after smoking cessation, a sustained inflammation in the lung tissue was observed. The elevated cytokine (IL-1α and TNF-α) and chemokine (CCL2 and CCL3) levels in the BALF of smoke-exposed mice returned to basal levels after smoking cessation, while the increased IL-12 levels did not return to its basal level. The cigarette smoke-enhanced VEGF levels did not significantly change after smoking cessation. Moreover, IL-10 levels were reduced in the BALF of smoke-exposed mice and these levels were still significantly decreased after smoking cessation compared to the control animals.
Conclusion
The inflammatory changes in the airways caused by cigarette smoke exposure were only partially reversed after smoking cessation. Although smoking cessation should be the first step in reducing the progression of lung emphysema, additional medication could be provided to tackle the sustained airway inflammation.
doi:10.1186/1465-9921-11-99
PMCID: PMC2918562  PMID: 20649997
17.  Cigarette smoke attenuates the production of cytokines by human plasmacytoid dendritic cells and enhances the release of IL-8 in response to TLR-9 stimulation 
Respiratory Research  2009;10(1):47.
Myeloid and plasmacytoid dendritic cells (mDCs, pDC) are crucial to the immune system, detecting microorganisms and linking the innate and adaptive immunity. pDC are present in small quantities in tissues that are in contact with the external environment; mainly the skin, the inner lining of the nose, lungs, stomach and intestines. They produce large amounts of IFN-α after stimulation and are pivotal for the induction of antiviral responses. Chronic obstructive pulmonary disease (COPD) patients are known to be more susceptible to viral infections. We have demonstrated that exposure of mDC to cigarette smoke extract (CSE) leads to the release of chemokines, however, not much is known about the role of pDC in COPD. In this study, we addressed several key questions with respect to the mechanism of action of CSE on human pDC in an in vitro model. Human pDCs were isolated from normal healthy volunteers and subjected to fresh CSE and the levels of IL-8, TNF-α, IP-10, IL-6, IL-1, IL-12 and IL-10 and IFN-α were studied by both ELISA and real time PCR methods. We observed that CSE augmented the production of IL-8 and suppressed the release of TNF-α, IL-6 and IFN-α. Moreover, CSE suppressed PI3K/Akt signalling in pDC. In conclusion, our data indicate that CSE has both the potential to diminish anti-viral immunity by downregulating the release of IFN-α and other pro-inflammatory cytokines while, at the same time, augmenting the pathogenesis of COPD via an IL-8 induced recruitment of neutrophils.
doi:10.1186/1465-9921-10-47
PMCID: PMC2701931  PMID: 19515231
18.  Cigarette smoke regulates the expression of TLR4 and IL-8 production by human macrophages 
Background
Toll-like receptors (TLRs) are present on monocytes and alveolar macrophages that form the first line of defense against inhaled particles. The importance of those cells in the pathophysiology of chronic obstructive pulmonary disease (COPD) has well been documented. Cigarette smoke contains high concentration of oxidants which can stimulate immune cells to produce reactive oxygen species, cytokines and chemokines.
Methods
In this study, we evaluated the effects of cigarette smoke medium (CSM) on TLR4 expression and interleukin (IL)-8 production by human macrophages investigating the involvement of ROS.
Results and Discussion
TLR4 surface expression was downregulated on short term exposure (1 h) of CSM. The downregulation could be explained by internalization of the TLR4 and the upregulation by an increase in TLR4 mRNA. IL-8 mRNA and protein were also increased by CSM. CSM stimulation increased intracellular ROS-production and decreased glutathione (GSH) levels. The modulation of TLR4 mRNA and surface receptors expression, IRAK activation, IκB-α degradation, IL-8 mRNA and protein, GSH depletion and ROS production were all prevented by antioxidants such as N-acetyl-L-cysteine (NAC).
Conclusion
TLR4 may be involved in the pathogenesis of lung emphysema and oxidative stress and seems to be a crucial contributor in lung inflammation.
doi:10.1186/1476-9255-6-12
PMCID: PMC2683828  PMID: 19409098
19.  Effect of Cigarette Smoke Extract on Dendritic Cells and Their Impact on T-Cell Proliferation 
PLoS ONE  2009;4(3):e4946.
Chronic obstructive pulmonary disease (COPD) is characterized by chronic airway inflammation. Cigarette smoke has been considered a major player in the pathogenesis of COPD. The inflamed airways of COPD patients contain several inflammatory cells including neutrophils, macrophages,T lymphocytes, and dendritic cells (DCs). The relative contributions of these various inflammatory cells to airway injury and remodeling are not well documented. In particular, the potential role of DCs as mediators of inflammation in the smoker's airways and COPD patients is poorly understood. In the current study we analyzed the effects of cigarette smoke extract on mouse bone marrow derived DC and the production of chemokines and cytokines were studied. In addition, we assessed CSE-induced changes in cDC function in the mixed lymphocyte reaction (MLR) examining CD4+ and CD8+ T cell proliferation. Cigarette smoke extract induces the release of the chemokines CCL3 and CXCL2 (but not cytokines), via the generation of reactive oxygen species (ROS). In a mixed-leukocyte reaction assay, cigarette smoke-primed DCs potentiate CD8+T cell proliferation via CCL3. In contrast, proliferation of CD4+T cells is suppressed via an unknown mechanism. The cigarette smoke-induced release of CCL3 and CXCL2 by DCs may contribute to the influx of CD8+T cells and neutrophils into the airways, respectively.
doi:10.1371/journal.pone.0004946
PMCID: PMC2655711  PMID: 19293939
20.  Salmeterol with fluticasone enhances the suppression of IL-8 release and increases the translocation of glucocorticoid receptor by human neutrophils stimulated with cigarette smoke 
The combination of inhaled corticosteroids and long-acting β2-adrenoceptor agonists is increasingly used in chronic obstructive pulmonary disease (COPD). Recently, we have demonstrated that combination of salmeterol and fluticasone propionate (FP) additionally suppress the production of IL-8 by human monocyte. In this study, the molecular mechanism behind the effectiveness of this combination therapy is investigated in human neutrophils. Human neutrophils were preincubated with salmeterol or FP or the combination. The amount of interleukin-8 (IL-8), elastase and matrix metalloproteinases (MMP)-2 and -9 releases, and reactive oxygen species (ROS) generation and expression of MAP kinase phosphatase (MKP-1) and glucocorticoid receptor (GR) were determined. Cigarette smoke medium (CSM) induces an increased expression of CXC receptors and the production of ROS that may explain the strong production of IL-8 by neutrophils. The expression of CXC receptors, the production of ROS, and the release of elastase and MMP-2 and -9 were not influenced by salmeterol, FP, or the combination. Interestingly, the combination therapy had an additive suppressive effect on the CSM-induced production of IL-8. The latter could be explained by an increased mRNA expression of MKP-1, the GR and an increased translocation of the GR to the nucleus. This leads eventually to suppression of both the NF-κB and MAPK pathways and, hence, to less IL-8 production by the neutrophil. These data are in support for the use of a combination therapy in COPD patients.
doi:10.1007/s00109-008-0360-0
PMCID: PMC2517086  PMID: 18600309
Glucocorticoids; Neutrophil; Chemokine
21.  Chlamydophila pneumoniae induces a sustained airway hyperresponsiveness and inflammation in mice 
Respiratory Research  2007;8(1):83.
Background
It has been reported that Chlamydophila (C.) pneumoniae is involved in the initiation and promotion of asthma and chronic obstructive pulmonary diseases (COPD). Surprisingly, the effect of C. pneumoniae on airway function has never been investigated.
Methods
In this study, mice were inoculated intranasally with C. pneumoniae (strain AR39) on day 0 and experiments were performed on day 2, 7, 14 and 21.
Results
We found that from day 7, C. pneumoniae infection causes both a sustained airway hyperresponsiveness and an inflammation. Interferon-γ (IFN-γ) and macrophage inflammatory chemokine-2 (MIP-2) levels in bronchoalveolar lavage (BAL)-fluid were increased on all experimental days with exception of day 7 where MIP-2 concentrations dropped to control levels. In contrast, tumor necrosis factor-α (TNF-α) levels were only increased on day 7. From day 7 to 21 epithelial damage and secretory cell hypertrophy was observed. It is suggested that, the inflammatory cells/mediators, the epithelial damage and secretory cell hypertrophy contribute to initiation of airway hyperresponsiveness.
Conclusion
Our study demonstrates for the first time that C. pneumoniae infection can modify bronchial responsiveness. This has clinical implications, since additional changes in airway responsiveness and inflammation-status induced by this bacterium may worsen and/or provoke breathlessness in asthma and COPD.
doi:10.1186/1465-9921-8-83
PMCID: PMC2194694  PMID: 18021431
22.  Toll-like receptor-4 mediates cigarette smoke-induced cytokine production by human macrophages 
Respiratory Research  2006;7(1):66.
Background
The major risk factor for the development of COPD is cigarette smoking. Smoking causes activation of resident cells and the recruitment of inflammatory cells into the lungs, which leads to release of pro-inflammatory cytokines, chemotactic factors, oxygen radicals and proteases. In the present study evidence is found for a new cellular mechanism that refers to a link between smoking and inflammation in lungs.
Methods
Employing human monocyte-derived macrophages, different techniques including FACS analysis, Cytometric Bead Array Assay and ELISA were achieved to evaluate the effects of CS on pro-inflammatory cytokine secretion including IL-8. Then, Toll-like receptor neutralization was performed to study the involvement of Toll-like receptor-4 in IL-8 production. Finally, signaling pathways in macrophages after exposure to CS medium were investigated performing ELISA and Western analysis.
Results
We demonstrate that especially human monocytes are sensitive to produce IL-8 upon cigarette smoke stimulation compared to lymphocytes or neutrophils. Moreover, monocyte-derived macrophages produce high amounts of the cytokine. The IL-8 production is dependent on Toll-like receptor 4 stimulation and LPS is not involved. Further research resolved the cellular mechanism by which cigarette smoke induces cytokine production in monocyte-derived macrophages. Cigarette smoke causes subsequently a concentration-dependent phosphorylation of IRAK and degradation of TRAF6. Moreover, IκBα was phosphorylated which suggests involvement of NF-κB. In addition, NFκB -inhibitor blocked cigarette smoke-induced IL-8 production.
Conclusion
These findings link cigarette smoke to inflammation and lead to new insights/therapeutic strategies in the pathogenesis of lung emphysema.
doi:10.1186/1465-9921-7-66
PMCID: PMC1481582  PMID: 16620395
23.  Overexpression of endothelial nitric oxide synthase suppresses features of allergic asthma in mice 
Respiratory Research  2006;7(1):58.
Background
Asthma is associated with airway hyperresponsiveness and enhanced T-cell number/activity on one hand and increased levels of exhaled nitric oxide (NO) with expression of inducible NO synthase (iNOS) on the other hand. These findings are in paradox, as NO also relaxes airway smooth muscle and has immunosuppressive properties. The exact role of the endothelial NOS (eNOS) isoform in asthma is still unknown. We hypothezised that a delicate regulation in the production of NO and its bioactive forms by eNOS might be the key to the pathogenesis of asthma.
Methods
The contribution of eNOS on the development of asthmatic features was examined. We used transgenic mice that overexpress eNOS and measured characteristic features of allergic asthma after sensitisation and challenge of these mice with the allergen ovalbumin.
Results
eNOS overexpression resulted in both increased eNOS activity and NO production in the lungs. Isolated thoracic lymph nodes cells from eNOS overexpressing mice that have been sensitized and challenged with ovalbumin produced significantly less of the cytokines IFN-γ, IL-5 and IL-10. No difference in serum IgE levels could be found. Further, there was a 50% reduction in the number of lymphocytes and eosinophils in the lung lavage fluid of these animals. Finally, airway hyperresponsiveness to methacholine was abolished in eNOS overexpressing mice.
Conclusion
These findings demonstrate that eNOS overexpression attenuates both airway inflammation and airway hyperresponsiveness in a model of allergic asthma. We suggest that a delicate balance in the production of bioactive forms of NO derived from eNOS might be essential in the pathophysiology of asthma.
doi:10.1186/1465-9921-7-58
PMCID: PMC1456969  PMID: 16597326
24.  Collagen degradation and neutrophilic infiltration: a vicious circle in inflammatory bowel disease 
Gut  2013;63(4):578-587.
Objective
Proline–glycine–proline (PGP) has been shown to have chemotactic effects on neutrophils via CXCR2 in several lung diseases. PGP is derived from collagen by the combined action of matrix metalloproteinase (MMP) 8 and/or MMP9 and prolyl endopeptidase (PE). We investigated the role of PGP in inflammatory bowel disease (IBD).
Design
In intestinal tissue from patients with IBD and mice with dextran sodium sulfate (DSS)-induced colitis, MMP8, MMP9 and PE were evaluated by ELISA, immunoblot and immunohistochemistry. Peripheral blood polymorphonuclear cell (PMN) supernatants were also analysed accordingly and incubated with collagen to assess PGP generation ex vivo. PGP levels were measured by mass spectrometry, and PGP neutralisation was achieved with a PGP antagonist and PGP antibodies.
Results
In the intestine of patients with IBD, MMP8 and MMP9 levels were elevated, while PE was expressed at similar levels to control tissue. PGP levels were increased in intestinal tissue of patients with IBD. Similar results were obtained in intestine from DSS-treated mice. PMN supernatants from patients with IBD were far more capable of generating PGP from collagen ex vivo than healthy controls. Furthermore, PGP neutralisation during DSS-induced colitis led to a significant reduction in neutrophil infiltration in the intestine.
Conclusions
The proteolytic cascade that generates PGP from collagen, as well as the tripeptide itself, is present in the intestine of patients with IBD and mice with DSS-induced colitis. PGP neutralisation in DSS-treated mice showed the importance of PGP-guided neutrophilic infiltration in the intestine and indicates a vicious circle in neutrophilic inflammation in IBD.
doi:10.1136/gutjnl-2012-303252
PMCID: PMC3963538  PMID: 23525573

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