The TGF-beta-Pseudoreceptor BAMBI is strongly expressed in COPD lungs and regulated by nontypeable Haemophilus influenzae

<p>Abstract</p> <p>Background</p> <p>Nontypeable <it>Haemophilus influenzae </it>(NTHI) may play a role as an infectious trigger in the pathogenesis of chronic obstructive pulmonary disease (COPD). Few data are available regarding the influence of acute and persistent infection on tissue remodelling and repair factors such as transforming growth factor (TGF)-β.</p> <p>Methods</p> <p>NTHI infection in lung tissues obtained from COPD patients and controls was studied <it>in vivo </it>and using an <it>in vitro model</it>. Infection experiments were performed with two different clinical isolates. Detection of NTHI was done using <it>in situ </it>hybridization (ISH) in unstimulated and in <it>in vitro </it>infected lung tissue. For characterization of TGF-β signaling molecules a transcriptome array was performed. Expression of the TGF-pseudoreceptor BMP and Activin Membrane-bound Inhibitor (BAMBI) was analyzed using immunohistochemistry (IHC), ISH and PCR. CXC chemokine ligand (CXCL)-8, tumor necrosis factor (TNF)-α and TGF-β expression were evaluated in lung tissue and cell culture using ELISA.</p> <p>Results</p> <p>In 38% of COPD patients infection with NTHI was detected <it>in vivo </it>in contrast to 0% of controls (p < 0.05). Transcriptome arrays showed no significant changes of TGF-β receptors 1 and 2 and Smad-3 expression, whereas a strong expression of BAMBI with upregulation after <it>in vitro </it>infection of COPD lung tissue was demonstrated. BAMBI was expressed ubiquitously on alveolar macrophages (AM) and to a lesser degree on alveolar epithelial cells (AEC). Measurement of cytokine concentrations in lung tissue supernatants revealed a decreased expression of TGF-β (p < 0.05) in combination with a strong proinflammatory response (p < 0.01).</p> <p>Conclusions</p> <p>We show for the first time the expression of the TGF pseudoreceptor BAMBI in the human lung, which is upregulated in response to NTHI infection in COPD lung tissue <it>in vivo </it>and <it>in vitro</it>. The combination of NTHI-mediated induction of proinflammatory cytokines and inhibition of TGF-β expression may influence inflammation induced tissue remodeling.</p

Beta defensin-2 is reduced in central but not in distal airways of smoker COPD patients

Background: Altered pulmonary defenses in chronic obstructive pulmonary disease (COPD) may promote distal airways bacterial colonization. The expression/activation of Toll Like receptors (TLR) and beta 2 defensin (HBD2) release by epithelial cells crucially affect pulmonary defence mechanisms. Methods: The epithelial expression of TLR4 and of HBD2 was assessed in surgical specimens from current smokers COPD (s-COPD; n = 17), ex-smokers COPD (ex-s-COPD; n = 8), smokers without COPD (S; n = 12), and from non-smoker non-COPD subjects (C; n = 13). Results: In distal airways, s-COPD highly expressed TLR4 and HBD2. In central airways, S and s-COPD showed increased TLR4 expression. Lower HBD2 expression was observed in central airways of s-COPD when compared to S and to ex-s-COPD. s-COPD had a reduced HBD2 gene expression as demonstrated by real-time PCR on micro-dissected bronchial epithelial cells. Furthermore, HBD2 expression positively correlated with FEV1/FVC ratio and inversely correlated with the cigarette smoke exposure. In a bronchial epithelial cell line (16 HBE) IL-1β significantly induced the HBD2 mRNA expression and cigarette smoke extracts significantly counteracted this IL-1 mediated effect reducing both the activation of NFkB pathway and the interaction between NFkB and HBD2 promoter. Conclusions: This study provides new insights on the possible mechanisms involved in the alteration of innate immunity mechanisms in COPD. © 2012 Pace et al

Toll-like receptor 2 expression is decreased on alveolar macrophages in cigarette smokers and COPD patients

BACKROUND: Cigarette smoke exposure including biologically active lipopolysaccharide (LPS) in the particulate phase of cigarette smoke induces activation of alveolar macrophages (AM) and alveolar epithelial cells leading to production of inflammatory mediators. This represents a crucial mechanism in the pathogenesis of chronic obstructive pulmonary disease (COPD). Respiratory pathogens are a major cause of exacerbations leading to recurrent cycles of injury and repair. The interaction between pathogen-associated molecular patterns and the host is mediated by pattern recognition receptors (PRR's). In the present study we characterized the expression of Toll-like receptor (TLR)- 2, TLR4 and CD14 on human AM compared to autologous monocytes obtained from patients with COPD, healthy smokers and non-smokers. METHODS: The study population consisted of 14 COPD patients without evidence for acute exacerbation, 10 healthy smokers and 17 healthy non-smokers stratified according to age. The expression of TLR2, TLR4 and CD14 surface molecules on human AM compared to autologous monocytes was assessed ex vivo using FACS analysis. In situ hybridization was performed on bronchoalveolar lavage (BAL) cells by application of the new developed HOPE-fixative. RESULTS: The expression of TLR2, TLR4 and CD14 on AM from COPD patients, smokers and non-smokers was reduced as compared to autologous monocytes. Comparing AM we detected a reduced expression of TLR2 in COPD patients and smokers. In addition TLR2 mRNA and protein expression was increased after LPS stimulation on non-smokers AM in contrast to smokers and COPD patients. CONCLUSION: Our data suggest a smoke related change in the phenotype of AM's and the cellular response to microbial stimulation which may be associated with impairment of host defenses in the lower respiratory tract

Chlamydophila pneumoniae induces expression of Toll-like Receptor 4 and release of TNF-α and MIP-2 via an NF-κB pathway in rat type II pneumocytes

BACKGROUND: The role of alveolar type II cells in the regulation of innate and adaptive immunity is unclear. Toll-like receptors (TLRs) have been implicated in host defense. The purpose of the present study was to investigate whether Chlamydophila pneumoniae (I) alters the expression of TLR2 and/orTLR4 in type II cells in a (II) Rho-GTPase- and (III) NF-κB-dependent pathway, subsequently (IV) leading to the production of (IV) pro-inflammatory TNF-α and MIP-2. METHODS: Isolated rat type II pneumocytes were incubated with C. pneumoniae after pre-treatment with calcium chelator BAPTA-AM, inhibitors of NF-κB (parthenolide, SN50) or with a specific inhibitor of the Rho-GTPase (mevastatin). TLR2 and TLR4 mRNA expressions were analyzed by PCR. Activation of TLR4, Rac1, RhoA protein and NF-κB was determined by Western blotting and confocal laser scan microscopy (CLSM) and TNF-α and MIP-2 release by ELISA. RESULTS: Type II cells constitutively expressed TLR4 and TLR2 mRNA. A prominent induction of TLR4 but not TLR2 mRNA was detected after 2 hours of incubation with C. pneumoniae. The TLR4 protein expression reached a peak at 30 min, began to decrease within 1–2 hours and peaked again at 3 hours. Incubation of cells with heat-inactivated bacteria (56°C for 30 min) significantly reduced the TLR4 expression. Treated bacteria with polymyxin B (2 μg/ml) did not alter TLR4 expression. C. pneumoniae-induced NF-κB activity was blocked by TLR4 blocking antibodies. TLR4 mRNA and protein expression were inhibited in the presence of BAPTA-AM, SN50 or parthenolide. TNF-α and MIP-2 release was increased in type II cells in response to C. pneumoniae, whereas BAPTA-AM, SN50 or parthenolide decreased the C. pneumoniae-induced TNF-α and MIP-2 release. Mevastatin inhibited C. pneumoniae-mediated Rac1, RhoA and TLR4 expression. CONCLUSION: The TLR4 protein expression in rat type II cells is likely to be mediated by a heat-sensitive C. pneumoniae protein that induces a fast Ca(2+)-mediated NF-κB activity, necessary for maintenance of TLR4 expression and TNF-α and MIP-2 release through possibly Rac and Rho protein-dependent mechanism. These results indicate that type II pneumocytes play an important role in the innate pulmonary immune system and in inflammatory response mechanism of the alveolus

Cigarette smoke-exposed neutrophils die unconventionally but are rapidly phagocytosed by macrophages

Pulmonary accumulation of neutrophils is typical for active smokers who are also predisposed to multiple inflammatory and infectious lung diseases. We show that human neutrophil exposure to cigarette smoke extract (CSE) leads to an atypical cell death sharing features of apoptosis, autophagy and necrosis. Accumulation of tar-like substances in autophagosomes is also apparent. Before detection of established cell death markers, CSE-treated neutrophils are effectively recognized and non-phlogistically phagocytosed by monocyte-derived macrophages. Blockade of LOX-1 and scavenger receptor A, but not MARCO or CD36, as well as pre-incubation with oxLDL, inhibited phagocytosis, suggesting that oxLDL-like structures are major phagocytosis signals. Specific lipid (β-carotene and quercetin), but not aqueous, antioxidants increased the pro-phagocytic effects of CSE. In contrast to non-phlogistic phagocytosis, degranulation of secondary granules, as monitored by lactoferrin release, was apparent on CSE exposure, which is likely to promote pulmonary inflammation and tissue degradation. Furthermore, CSE-exposed neutrophils exhibited a compromised ability to ingest the respiratory pathogen, Staphylococcus aureus, which likely contributes to bacterial persistence in the lungs of smokers and is likely to promote further pulmonary recruitment of neutrophils. These data provide mechanistic insight into the lack of accumulation of apoptotic neutrophil populations in the lungs of smokers and their increased susceptibility to degradative pulmonary diseases and bacterial infections

Expression of Toll-like Receptor 9 in nose, peripheral blood and bone marrow during symptomatic allergic rhinitis

BACKGROUND: Allergic rhinitis is an inflammatory disease of the upper airway mucosa that also affects leukocytes in bone marrow and peripheral blood. Toll-like receptor 9 (TLR9) is a receptor for unmethylated CpG dinucleotides found in bacterial and viral DNA. The present study was designed to examine the expression of TLR9 in the nasal mucosa and in leukocytes derived from different cellular compartments during symptomatic allergic rhinitis. METHODS: The study was based on 32 patients with seasonal allergic rhinitis and 18 healthy subjects, serving as controls. Nasal biopsies were obtained before and after allergen challenge. Bone marrow, peripheral blood and nasal lavage fluid were sampled outside and during pollen season. The expression of TLR9 in tissues and cells was analyzed using immunohistochemistry and flow cytometry, respectively. RESULTS: TLR9 was found in several cell types in the nasal mucosa and in different leukocyte subpopulations derived from bone marrow, peripheral blood and nasal lavage fluid. The leukocyte expression was generally higher in bone marrow than in peripheral blood, and not affected by symptomatic allergic rhinitis. CONCLUSION: The widespread expression of TLR9 in the nasal mucosa along with its rich representation in leukocytes in different compartments, demonstrate the possibility for cells involved in allergic airway inflammation to directly interact with bacterial and viral DNA

Human lung cancer cells express functionally active Toll-like receptor 9

BACKGROUND: CpG-oligonucleotides (CpG-ODN), which induce signaling through Toll-like receptor 9 (TLR9), are currently under investigation as adjuvants in therapy against infections and cancer. CpG-ODN function as Th-1 adjuvants and are able to activate dendritic cells. In humans TLR9 has been described to be strongly expressed in B-lymphocytes, monocytes, plasmacytoid dendritic cells and at low levels in human respiratory cells. We determined whether a direct interaction of bacterial DNA with the tumor cells themselves is possible and investigated the expression and function of TLR9 in human malignant solid tumors and cell lines. TLR9 expression by malignant tumor cells, would affect treatment approaches using CpG-ODN on the one hand, and, on the other hand, provide additional novel information about the role of tumor cells in tumor-immunology. METHODS: The expression of TLR9 in HOPE-fixed non-small lung cancer, non-malignant tissue and tumor cell lines was assessed using immunohistochemistry, confocal microscopy, in situ hybridization, RT-PCR and DNA-sequencing. Apoptosis and chemokine expression was detected by FACS analysis and the Bio-Plex system. RESULTS: We found high TLR9 signal intensities in the cytoplasm of tumor cells in the majority of lung cancer specimens as well as in all tested tumor cell lines. In contrast to this non-malignant lung tissues showed only sporadically weak expression. Stimulation of HeLa and A549 cells with CpG-ODN induced secretion of monocyte chemoattractant protein-1 and reduction of spontaneous and tumor necrosis factor-alpha induced apoptosis. CONCLUSIONS: Here we show that TLR9 is expressed in a selection of human lung cancer tissues and various tumor cell lines. The expression of functionally active TLR9 in human malignant tumors might affect treatment approaches using CpG-ODN and shows that malignant cells can be regarded as active players in tumor-immunology

Expression of Toll-like receptor 2 is up-regulated in monocytes from patients with chronic obstructive pulmonary disease

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is characterised by pulmonary and systemic inflammation which flare-up during episodes of acute exacerbation (AECOPD). Given the role of Toll-like receptors (TLRs) in the induction of inflammatory responses we investigated the involvement of TLRs in COPD pathogenesis. METHODS: The expression of TLR-2, TLR-4 and CD14 in monocytes was analyzed by flow cytometry. To study the functional responses of these receptors, monocytes were stimulated with peptidoglycan or lipopolysaccharide and the amounts of TNFα and IL-6 secreted were determined by ELISA. RESULTS: We found that the expression of TLR-2 was up-regulated in peripheral blood monocytes from COPD patients, either clinically stable or during AECOPD, as compared to never smokers or smokers with normal lung function. Upon stimulation with TLR-2 ligand monocytes from COPD patients secreted increased amounts of cytokines than similarly stimulated monocytes from never smokers and smokers. In contrast, the expressions of TLR-4 and CD14 were not significantly different between groups, and the response to lipopolysaccharide (a TLR-4 ligand) stimulation was not significantly different either. At discharge from hospital TLR-2 expression was down-regulated in peripheral blood monocytes from AECOPD patients. This could be due to the treatment with systemic steroids because, in vitro, steroids down-regulated TLR-2 expression in a dose-dependent manner. Finally, we demonstrated that IL-6, whose plasma levels are elevated in patients, up-regulated in vitro TLR-2 expression in monocytes from never smokers. CONCLUSION: Our results reveal abnormalities in TLRs expression in COPD patients and highlight its potential relationship with systemic inflammation in these patients