Long-Term Cryopreservation of Nasal Polyp Tissue in a Biobank for the Isolation and Culture of Primary Epithelial Cells.

Epithelial cells may play an important role in the pathologic process of chronic rhinosinusitis with nasal polyps. Therefore, providing epithelial cells from a biobank could greatly contribute to further research. In the present work, the isolation of epithelial cells from long-term cryopreserved tissue is demonstrated. Polyp tissues were cryopreserved in a commercially available freezing medium with dimethyl sulfoxide and stored in liquid nitrogen. The outgrowth and proliferation of epithelial cells from cryopreserved tissue were evaluated and compared to that of fresh tissue. Flow cytometric analysis with anti-cytokeratin, anti-p63, and anti-Ki-67 was performed to identify epithelial cells and determine differentiation and proliferation. A functionality test was performed by determining type 2-relevant proteins, representatively thymic stromal lymphopoietin (TSLP) and periostin, using ELISA. Primary epithelial cells could be isolated from cryopreserved tissues. Cells from cryopreserved tissues showed comparable outgrowth and proliferation to that of fresh tissue. Isolated epithelial cells showed high cytokeratin, p63, and Ki-67 expression and secreted TSLP and periostin. In the present study, a method for long-term cryopreservation of polyp tissue was established, thereby enabling the isolation and cell culture of primary cell culture at a later time. Epithelial cell availability should be greatly improved by including this method in a biobank.

OR2AT4 and OR1A2 counterregulate molecular pathophysiological processes of steroid-resistant inflammatory lung diseases in human alveolar macrophages.

BACKGROUND: Therapeutic options for steroid-resistant non-type 2 inflammation in obstructive lung diseases are lacking. Alveolar macrophages are central in the progression of these diseases by releasing proinflammatory cytokines, making them promising targets for new therapeutic approaches. Extra nasal expressed olfactory receptors (ORs) mediate various cellular processes, but clinical data are lacking. This work investigates whether ORs in human primary alveolar macrophages could impact pathophysiological processes and could be considered as therapeutic targets. METHODS: Human primary alveolar macrophages were isolated from bronchoalveolar lavages of 50 patients with pulmonary diseases. The expression of ORs was validated using RT-PCR, immunocytochemical staining, and Western blot. Changes in intracellular calcium levels were analyzed in real-time by calcium imaging. A luminescent assay was used to measure the cAMP concentration after OR stimulation. Cytokine secretion was measured in cell supernatants 24 h after stimulation by ELISA. Phagocytic ability was measured by the uptake of fluorescent-labeled beads by flow cytometry. RESULTS: We demonstrated the expression of functional OR2AT4 and OR1A2 on mRNA and protein levels. Both ORs were primarily located in the plasma membrane. Stimulation with Sandalore, the ligand of OR2AT4, and Citronellal, the ligand of OR1A2, triggered a transient increase of intracellular calcium and cAMP. In the case of Sandalore, this calcium increase was based on a cAMP-dependent signaling pathway. Stimulation of alveolar macrophages with Sandalore and Citronellal reduced phagocytic capacity and release of proinflammatory cytokines. CONCLUSION: These are the first indications for utilizing olfactory receptors as therapeutic target molecules in treating steroid-resistant lung diseases with non-type 2 inflammation.

Biomarkers for Comorbidities Modulate the Activity of T-Cells in COPD.

In smoking-induced chronic obstructive pulmonary disease (COPD), various comorbidities are linked to systemic inflammation and infection-induced exacerbations. The underlying mechanisms are unclear but might provide therapeutic targets. T-cell activity is central in systemic inflammation and for infection-defense mechanisms and might be influenced by comorbidities. Hypothesis: Circulating biomarkers of comorbidities modulate the activity of T-cells of the T-helper type 1 (Th1) and/or T-cytotoxic type 1 (Tc1). T-cells in peripheral blood mononuclear cells (PBMCs) from non-smokers (NS), current smokers without COPD (S), and COPD subjects (total n = 34) were ex vivo activated towards Th1/Tc1 and were then stimulated with biomarkers for metabolic and/or cardiovascular comorbidities (Brain Natriuretic Peptide, BNP; chemokine (C-C motif) ligand 18, CCL18; C-X3-C motif chemokine ligand 1, CX3CL1; interleukin-18, IL-18) or for asthma- and/or cancer-related comorbidities (CCL22; epidermal growth factor, EGF; IL-17; periostin) each at 10 or 50 ng/mL. The Th1/Tc1 activation markers interferon-γ (IFNγ), tumor necrosis factor-α (TNFα), and granulocyte-macrophage colony-stimulating factor (GM-CSF) were analyzed in culture supernatants by Enzyme-Linked Immunosorbent Assay (ELISA). Ex-vivo activation induced IFNγ and TNFα without differences between the groups but GM-CSF more in S vs. NS. At 10 ng/mL, the different biomarkers increased or reduced the T-cell activation markers without a clear trend for one direction in the different categories of comorbidities or for the different T-cell activation markers. At 50 ng/mL, there was a clear shift towards suppressive effects, particularly for the asthma- and cancer-related biomarkers and in cells of S and COPD. Comorbidities might suppress T-cell immunity in COPD. This could explain the association of comorbidities with frequent exacerbations.

Exposure to welding fumes suppresses the activity of T-helper cells.

Welders have an increased susceptibility to airway infections with non-typeable Haemophilus influenzae (NTHi), which implicates immune defects and might promote pneumonia and chronic obstructive pulmonary disease (COPD). We hypothesized that welding-fume exposure suppresses Th1-lymphocyte activity. Non-effector CD4+ T-cells from blood of 45 welders (n = 23 gas metal arc welders, GMAW; n = 16 tungsten inert gas welders, TIG; n = 6 others) and 25 non-welders were ex vivo activated towards Th1 via polyclonal T-cell receptor stimulation and IL-12 (first activation step) and then stimulated with NTHi extract or lipopolysaccharide (LPS) (second activation step). IFNγ and IL-2 were measured by ELISA. In the first activation step, IFNγ was reduced in welders compared to non-welders and in the GMAW welders with higher concentrations of respirable particles compared to the lower exposed TIG welders. IFNγ was not influenced by tobacco smoking and correlated negatively with welding-fume exposure, respirable manganese, and iron. In the second activation step, NTHi and LPS induced additional IFNγ, which was reduced in current smokers compared to never smokers in welders as well as in non-welders. Analyzing both activation steps together, IFNγ production was lowest in smoking welders and highest in never smoking non-welders. IL-2 was not associated with any of these parameters. Welding-fume exposure might suppress Th1-based immune responses due to effects of particulate matter, which mainly consists of iron and manganese. For responses to NTHi this is strongest in smoking welders because welding fume suppresses T-cell activation towards Th1 and cigarette smoke suppresses the subsequent Th1-response to NTHi via LPS. Both effects are independent from IL-2-regulated T-cell proliferation. This might explain the increased susceptibility to infections and might promote COPD development.

Quantitative Secretome Analysis of Activated Jurkat Cells Using Click Chemistry-Based Enrichment of Secreted Glycoproteins.

Quantitative secretome analyses are a high-performance tool for the discovery of physiological and pathophysiological changes in cellular processes. However, serum supplements in cell culture media limit secretome analyses, but serum depletion often leads to cell starvation and consequently biased results. To overcome these limiting factors, we investigated a model of T cell activation (Jurkat cells) and performed an approach for the selective enrichment of secreted proteins from conditioned medium utilizing metabolic marking of newly synthesized glycoproteins. Marked glycoproteins were labeled via bioorthogonal click chemistry and isolated by affinity purification. We assessed two labeling compounds conjugated with either biotin or desthiobiotin and the respective secretome fractions. 356 proteins were quantified using the biotin probe and 463 using desthiobiotin. 59 proteins were found differentially abundant (adjusted p-value ≤0.05, absolute fold change ≥1.5) between inactive and activated T cells using the biotin method and 86 using the desthiobiotin approach, with 31 mutual proteins cross-verified by independent experiments. Moreover, we analyzed the cellular proteome of the same model to demonstrate the benefit of secretome analyses and provide comprehensive data sets of both. 336 proteins (61.3%) were quantified exclusively in the secretome. Data are available via ProteomeXchange with identifier PXD004280.

A systemic defect in Toll-like receptor 4 signaling increases lipopolysaccharide-induced suppression of IL-2-dependent T-cell proliferation in COPD.

The susceptibility to bacterial infections is increased in chronic obstructive pulmonary disease (COPD). This promotes exacerbations. IL-2 triggers CD4(+)/Th1-cell proliferation, which is important for infection defense. Bacterial endotoxin (LPS) activates MyD88/IRAK and TRIF/IKKε/TBK1 pathways via Toll-like receptor-4 (TLR4) in Th1 cells. Systemic defects in TLR pathways in CD4(+)/Th1 cells cause an impairment of IL-2-dependent immune responses to bacterial infections in COPD. Peripheral blood CD4(+) T cells of never smokers, smokers without COPD, and smokers with COPD (each n = 10) were ex vivo activated towards Th1 and stimulated with LPS. IL-2, MyD88, and TRIF expression, and cell proliferation was analyzed by ELISA, quantitative RT-PCR, and bromodeoxyuridine (BrdU) and trypan blue staining comparative among the cohorts. IL-2 release from activated T cells was increased in COPD vs. smokers and never smokers. LPS reduced IL-2 expression and T-cell proliferation. These effects were increased in COPD vs. never smokers and inversely correlated with FEV1 (%predicted). The MyD88/TRIF ratio was decreased in Th1 cells of COPD. The suppression of IL-2 by LPS was abolished by MyD88/IRAK blockade in never smokers but by TRIF/IKKε/TBK1 blockade in COPD. Moxifloxacin restored IL-2 expression and T-cell proliferation in the presence of LPS by blocking p38 MAPK. The increased IL-2 release from Th1 cells in COPD might contribute to airway inflammation in disease exacerbations. A switch from MyD88/IRAK to TRIF/IKKε/TBK1 signaling amplifies the suppression of IL-2-dependent proliferation of CD4(+) T cells by LPS in COPD. This molecular pathology is of systemic origin, might impair adaptive immune responses, and could explain the increased susceptibility to bacterial infections in COPD. Targeting TLR4-downstream signaling, for example, with moxifloxacin, might reduce exacerbation rates.

Endothelin receptor B protects granulocyte macrophage colony-stimulating factor mRNA from degradation.

Evidence is lacking on the differential effects of the two therapeutic concepts of endothelin receptor antagonists (ERAs): the blockade of only the endothelin receptor A (ETAR; selective antagonism) versus both ETAR and endothelin receptor B (ETBR; dual blockade). Ambrisentan, a selective ERA, and bosentan, a dual blocker, are both available for therapy. We hypothesized that there are differences in the potential of ERAs to ameliorate inflammatory processes in human airway smooth muscle cells (HASMCs) and aimed to unravel underlying mechanisms. We used HASMC culture, enzyme-linked immunosorbent assay, and quantitative reverse-transcription polymerase chain reaction. Tumor necrosis factor α (TNFα) induced transcription and expression of chemokine (C-X-C motif) ligand 2 (CXCL2), chemokine (C-X-C motif) ligand 3 (CXCL3), granulocyte macrophage colony-stimulating factor (GM-CSF), and matrix metalloproteinase 12 (MMP12) in HASMCs. In concentration-response experiments, bosentan led to a significantly greater reduction of GM-CSF and MMP12 protein release than ambrisentan, whereas there was no significant difference in their effect on GM-CSF and MMP12 mRNA. Both ERAs reduced CXCL3 protein and mRNA equally but had no effect on CXCL2. Blocking mitogen-activated protein kinases revealed that both ETAR and ETBR signal through p38 mitogen-activated protein kinase, but ETBR also signals through extracellular signal-regulated kinase (ERK) 1/2 to induce GM-CSF expression. In the presence of the transcription inhibitor actinomycin D, bosentan, but not ambrisentan, reduced GM-CSF but not MMP12 or CXCL3 mRNA. In conclusion, blockade of each endothelin receptor subtype reduces GM-CSF transcription, but blocking ETBR additionally protects GM-CSF mRNA from degradation via ERK-1/2. Accordingly, blocking both ETAR and ETBR leads to a stronger reduction of TNFα-induced GM-CSF protein expression. This mechanism might be specific to GM-CSF. Our data stress the anti-inflammatory potential of ERA and warrant further investigation of their utility in chronic inflammatory airway diseases.

Olfactory receptors impact pathophysiological processes of lung diseases in bronchial epithelial cells.

BACKGROUND: Therapeutic options for steroid-resistant non-type 2 inflammation in obstructive lung diseases are limited. Bronchial epithelial cells are key in the pathogenesis by releasing the central proinflammatory cytokine interleukine-8 (IL-8). Olfactory receptors (ORs) are expressed in various cell types. This study examined the drug target potential of ORs by investigating their impact on associated pathophysiological processes in lung epithelial cells. METHODS: Experiments were performed in the A549 cell line and in primary human bronchial epithelial cells. OR expression was investigated using RT-PCR, Western blot, and immunocytochemical staining. OR-mediated effects were analyzed by measuring 1) intracellular calcium concentration via calcium imaging, 2) cAMP concentration by luminescence-based assays, 3) wound healing by scratch assays, 4) proliferation by MTS-based assays, 5) cellular vitality by Annexin V/PI-based FACS staining, and 6) the secretion of IL-8 in culture supernatants by ELISA. RESULTS: By screening 100 potential OR agonists, we identified two, Brahmanol and Cinnamaldehyde, that increased intracellular calcium concentrations. The mRNA and proteins of the corresponding receptors OR2AT4 and OR2J3 were detected. Stimulation of OR2J3 with Cinnamaldehyde reduced 1) IL-8 in the absence and presence of bacterial and viral pathogen-associated molecular patterns (PAMPs), 2) proliferation, and 3) wound healing but increased cAMP. In contrast, stimulation of OR2AT4 by Brahmanol increased wound healing but did not affect cAMP and proliferation. Both ORs did not influence cell vitality. CONCLUSION: ORs might be promising drug target candidates for lung diseases with non-type 2 inflammation. Their stimulation might reduce inflammation or prevent tissue remodeling by promoting wound healing.

Inflammatory responses of airway smooth muscle cells and effects of endothelin receptor antagonism.

Endothelin receptor antagonists (ETRAs), authorized for pulmonary hypertension, have failed to prove their utility in chronic lung diseases with corticosteroid-resistant airway inflammation when applied at late disease stages with emphysema/fibrosis. Earlier administration might prove effective by targeting the interaction between airway inflammation and tissue remodeling. We hypothesized that human airway smooth muscle cells (HASMCs) participate in linking inflammation with remodeling and that associated genes become differentially suppressed by ambrisentan (A-receptor selective ETRA) and bosentan (nonselective/dual ETRA). Inflammatory responses of ex vivo-cultivated HASMCs to TNF-α were investigated by whole-genome microarray analyses. qRT-PCR and ELISA were used to test inflammatory and remodeling genes for sensitivity to bosentan and ambrisentan and to investigate differential sensitivities mechanistically. ETRA and corticosteroid effects were compared in HASMCs from patients with chronic obstructive pulmonary disease. TNF-α induced the expression of 18 cytokines/chemokines and five tissue remodeling genes involved in severe, corticosteroid-insensitive asthma, chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis, and/or pulmonary hypertension. Thirteen cytokines/chemokines, MMP13, and WISP1 were suppressed by ETRAs. Eight genes had differential sensitivity to bosentan and ambrisentan depending on the endothelin-B receptor impact on transcriptional regulation and mRNA stabilization. Chemokine (C-C motif) ligands 2 and 5, granulocyte macrophage colony-stimulating factor, and MMP13 had increased sensitivity to bosentan or bosentan/dexamethasone combination versus dexamethasone alone. Suppression of cytokine and remodeling gene expression by ETRAs was confirmed in TNF-α-activated human bronchial epithelial cells. HASMCs and human bronchial epithelial cells participate in the interaction of inflammation and tissue remodeling. This interaction is targeted differentially by selective and nonselective ETRAs, which could be used in therapies of chronic lung diseases with corticosteroid-resistant airway inflammation at early disease stages to attenuate inflammation-induced airway remodeling.

Endothelin receptor antagonists attenuate the inflammatory response of human pulmonary vascular smooth muscle cells to bacterial endotoxin.

Bacterial infections induce exacerbations in chronic lung diseases, e.g., chronic obstructive pulmonary disease (COPD), by enhancing airway inflammation. Exacerbations are frequently associated with right heart decompensation and accelerate disease progression. Endothelin receptor antagonists (ERAs) might have therapeutic potential as pulmonary vasodilators and anti-inflammatory agents, but utility in exacerbations of chronic lung diseases is unknown. We hypothesized that cytokine releases induced by lipopolysaccharide (LPS), the major bacterial trigger of inflammation, are reduced by ERAs in pulmonary vascular smooth muscle cells (PVSMCs). Ex vivo cultivated human PVSMCs were preincubated with the endothelin-A-receptor selective inhibitor ambrisentan, with the endothelin-B-receptor selective inhibitor BQ788 [sodium (2R)-2-{[(2S)-2-({[(2R,6S)-2,6-dimethyl-1-piperidinyl]carbonyl}amino)-4,4-dimethylpentanoyl][1-(methoxycarbonyl)-d-tryptophyl]amino}hexanoate], or with the dual blocker bosentan before stimulation with smooth LPS (S-LPS), rough LPS (Re-LPS), or a mixture of long and short forms (M-LPS). Expression of cytokines and LPS receptors (TLR4, CD14) were analyzed via enzyme-linked immunosorbent assay (ELISA) and/or quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). All LPS forms induced interleukin (IL)-6-, IL-8-, and granulocyte macrophage-colony stimulating factor (GM-CSF) release. Bosentan and BQ788 inhibited M-LPS-induced release of all cytokines and soluble CD14 (sCD14) but not TLR4 expression. Ambrisentan blocked M-LPS-induced IL-6 release but not IL-8, GM-CSF, or LPS receptors. IL-8 release induced by S-LPS, which requires CD14 to activate TLR4, was blocked by bosentan and BQ788. IL-8 release induced by Re-LPS, which does not require CD14 to activate TLR4, was insensitive to both bosentan and BQ788. In conclusion, PVSMCs contribute to inflammation in bacteria-induced exacerbations of chronic lung diseases. Inhibition of the endothelin-B receptor suppresses cytokine release induced by long/smooth LPS attributable to sCD14 downregulation. ERAs, particularly when targeting the endothelin-B receptor, might have therapeutic utility in exacerbations of chronic lung diseases.

Simple, low-cost, and well-performing method, the outgrowth technique, for the isolation of cells from nasal polyps.

BACKGROUND: Epithelial cells are an important part of the pathomechanism in chronic rhinosinusitis with nasal polyps. It is therefore essential to establish a robust method for the isolation and culture of epithelial cells from nasal polyps to enable further research. In this study, the feasibility of the outgrowth technique for the isolation of the epithelial cells from the nasal polyps was evaluated. RESULTS: Using the outgrowth technique, epithelial cells could be isolated from all tissue samples. Isolated epithelial cells showed a proliferation rate of approximately 7- to 23-fold every 6 days up to the 3rd passage. Over 97% of isolated cells were shown to be cytokeratin- and p63-positive, and over 86% of them were Ki-67-positive in flow cytometry. Interleukin-33 and periostin were detectable in the supernatant. CONCLUSIONS: We introduce a simple, low-cost, and well-performing method for isolating epithelial cells from nasal polyps with the outgrowth technique.

The T-helper cell type 1 immune response to gram-negative bacterial infections is impaired in COPD.

RATIONALE: The increased susceptibility to bacterial infections in chronic obstructive pulmonary disease (COPD) is critical for exacerbations. Toll-like receptor-4 (TLR4) detects bacteria via LPS and induces IFN-γ-based immune responses. The direct responsiveness of Th1 lymphocytes to LPS is disputed because they lack surface expression of the TLR4 coreceptor CD14. OBJECTIVES: We hypothesized that the Th1-mediated adaptive immune response to bacterial infections is impaired in COPD. METHODS: LPS-induced TLR4 expression and IFN-γ release in and from ex vivo-generated Th1 cells was compared among nonsmokers (n = 14), smokers without COPD (n = 13), and smokers with COPD (n = 25) via quantitative reverse transcription polymerase chain reaction, Western blot, and ELISA. TLR4 transfection experiments were performed to functionally link receptor to IFN-γ dysregulation in COPD. MEASUREMENTS AND MAIN RESULTS: Short-chain LPS from Salmonella species and nontypeable Haemophilus influenzae and nontypeable Haemophilus influenzae whole-cell extract all induced TLR4 expression via TLR4/MyD88/IRAK/mitogen-activated protein-kinase signaling and IFN-γ release via TLR4/TRIF/IKKε/TBK1 signaling in Th1 cells of nonsmokers. These effects were all impaired in smokers with and without COPD. The LPS responses were partially dependent on soluble CD14 and correlated positively to lung-function parameters but negatively to cigarette smoking (pack-years). Endogenous MyD88/IRAK signaling antagonists were up-regulated in Th1 cells of smokers and COPD, and TLR4 overexpression in Th1 cells of COPD restored LPS-dependent IFN-γ release. CONCLUSIONS: Th1 cells directly respond to short-chain LPS. Cigarette smoking suppresses Th1-mediated immune responses to gram-negative bacterial infections by interfering with MyD88/IRAK signaling thereby reducing LPS-induced TLR4 expression. This can explain the increased susceptibility to bacterial infections in COPD. Targeting TLR signaling might be useful to reduce exacerbation rates.

The regulation of Dkk1 expression during embryonic development.

During embryogenesis, the Dkk1 mediated Wnt inhibition controls the spatiotemporal dynamics of cell fate determination, cell differentiation and cell death. Furthermore, the Dkk1 dose is critical for the normal Wnt homeostasis, as alteration of the Dkk1 activity is associated with various diseases. We investigated the regulation of Dkk1 expression during embryonic development. We identified nine conserved non-coding elements (CNEs), located 3' to the Dkk1 locus. Analyses of the regulatory potential revealed that four of these CNEs in combination drive reporter expression very similar to Dkk1 expression in several organs of transgenic embryos. We extended the knowledge of Dkk1 expression during hypophysis, external genitalia and kidney development, suggesting so far to unexplored functions of Dkk1 during the development of these organs. Characterization of the regulatory potential of four individual CNEs revealed that each of these promotes Dkk1 expression in brain and kidney. In combination, two enhancers are responsible for expression in the pituitary and the genital tubercle. Furthermore, individual CNEs mediates craniofacial, optic cup and limb specific Dkk1 regulation. Our study substantially improves the knowledge of Dkk1 regulation during embryonic development and thus might be of high relevance for therapeutic approaches.

Resveratrol impairs the release of steroid-resistant inflammatory cytokines from human airway smooth muscle cells in chronic obstructive pulmonary disease.

Chronic obstructive pulmonary disease (COPD) therapy is complicated by corticosteroid resistance of the interleukin 8 (IL-8)-dependent and granulocyte macrophage-colony stimulating factor (GM-CSF)-dependent chronic airway inflammation, for whose establishment human airway smooth muscle cells (HASMCs) might be crucial. It is unclear whether the release of inflammatory mediators from HASMCs is modulated by cigarette smoking and is refractory to corticosteroids in COPD. Resveratrol, an antiaging drug with protective effects against lung cancer, might be an alternative to corticosteroids in COPD therapy. Vascular endothelial growth factor (VEGF) might offer protection from developing emphysema. We tested the following hypotheses for HASMCs: 1) smoking with or without airway obstruction modulates IL-8, GM-CSF, and VEGF release; and 2) corticosteroids, but not resveratrol, fail to inhibit cytokine release in COPD. Cytokine release from HASMCs exposed to tumor necrosis factor α (TNFα), dexamethasone, and/or resveratrol was measured via enzyme-linked immunosorbent assay and compared between nonsmokers (NS), smokers without COPD (S), and smokers with COPD (all n = 10). In response to TNFα, IL-8 release was increased, but GM-CSF and VEGF release was decreased in S and COPD compared with NS. Dexamethasone and resveratrol inhibited concentration-dependently TNFα-induced IL-8, GM-CSF, and VEGF release. For IL-8 and GM-CSF efficiency of dexamethasone was NS > S > COPD. That of resveratrol was NS = S = COPD for IL-8 and NS = S < COPD for GM-CSF. For VEGF the efficiency of dexamethasone was NS = S = COPD, and that of resveratrol was NS = S > COPD. All resveratrol effects were partially based on p38 mitogen-activated protein kinase blockade. In conclusion, smoking modulates cytokine release from HASMCs. Corticosteroid refractoriness of HASMCs in COPD is cytokine-dependent. Resveratrol might be superior to corticosteroids in COPD therapy, because it more efficiently reduces the release of inflammatory mediators and has limited effects on VEGF in COPD.

LABAs and p38MAPK Inhibitors Reverse the Corticosteroid-Insensitivity of IL-8 in Airway Smooth Muscle Cells of COPD.

Airway inflammation in chronic obstructive pulmonary disease (COPD) is partially insensitive/resistant to inhaled corticosteroids (ICS). ICS plus bronchodilator therapy has been discussed for COPD phenotypes with frequent exacerbations and participation of corticosteroid-sensitive type 2/eosinophilic inflammation. Neutralization of non-type 2/IL-8-associated airway inflammation by reversion of its corticosteroid-resistance might be a future strategy for other phenotypes. Human airway smooth muscle cells (HASMCs) produce corticosteroid-insensitive IL-8 in response to TNFα or LPS in stable disease stages or bacteria-induced exacerbations, respectively. p38-mitogen-activated-protein-kinases (p38MAPKs) are alternative therapeutic targets. Hypothesis: long-acting-ß2-agonists (LABAs) reverse the corticosteroid-insensitivity of IL-8 by p38MAPK inhibition in HASMCs. Cultivated HASMCs from COPD subjects were pre-incubated with formoterol, salmeterol, fluticasone-propionate, BIRB796 (p38MAPKα, -γ, -δ inhibitor), and/or SB203580 (p38MAPKα and -ß inhibitor) before stimulation with TNFα or LPS. IL-8 and MAPK-activities were measured by ELISA. Formoterol, salmeterol, and fluticasone did not or hardly reduced TNFα- or LPS-induced IL-8. BIRB796 and SB203580 reduced TNFα-induced IL-8. SB203580 reduced LPS-induced IL-8. Fluticasone/formoterol, fluticasone/salmeterol, and fluticasone/BIRB796, but not fluticasone/SB203580 combinations, reduced TNFα-induced IL-8 stronger than single treatments. All combinations including fluticasone/SB203580 reduced LPS-induced IL-8 stronger than single treatments. TNFα induced p38MAPKα and -γ activity. LPS induced p38MAPKα activity. Formoterol reduced TNFα-induced p38MAPKγ and LPS-induced p38MAPKα activity. LABAs reverse the corticosteroid-insensitivity of IL-8 in airway smooth muscles via p38MAPKγ in stable disease and via p38MAPKα in exacerbations. Our pre-clinical data indicate a utility for also adding ICS in non-type 2 inflammatory COPD phenotypes to bronchodilator therapy. Depending on phenotype and disease stage, isoform-specific p38MAPK blockers might also reverse corticosteroid-resistance in COPD.

The monocyte-dependent immune response to bacteria is suppressed in smoking-induced COPD.

COPD patients have an increased susceptibility to bacterial airway infections that can induce exacerbations. In response to infections, circulating monocytes become recruited to the infected tissue and secrete cytokines. We hypothesized that this cytokine response is reduced in COPD. Cultured peripheral blood monocytes of never smokers (NS) and smokers without (S) and with COPD (3 study populations, n = 36-37) were stimulated with extracts of Haemophilus influenzae, Staphylococcus aureus, or Streptococcus pneumoniae or with four different pathogen-associated molecular patterns (PAMPs). Four cytokines and 9 PAMP-related signaling molecules were measured and compared between the groups. Granulocyte-macrophage-colony-stimulating-factor responses to all stimulants were reduced in S and COPD compared to NS. Tumor-necrosis-factor-α responses to all bacterial extracts, peptidoglycan, and lipopolysaccharide were reduced in S and/or COPD. Interleukin-10 responses to S. aureus and lipoteichoic acid were increased in COPD. Correlations to pack-years and lung function were found. The peptidoglycan-receptor NOD2 and the mRNA of the lipopolysaccharide-receptor TLR4 were reduced in S and COPD. Cytokine responses of monocytes to bacteria are suppressed by smoking and in COPD possibly due to NOD2 and TLR4 reduction and/or interleukin-10 increase. This might help to explain the increased susceptibility to bacterial infections. These systemic molecular pathologies might be targets for therapeutic strategies to prevent infection-induced exacerbations. KEY MESSAGES: COPD subjects have an increased susceptibility to bacterial infections. This implies defects in the immune response to bacteria and is critical for disease progression. The cytokine response of monocytes to bacteria is reduced in COPD. This might be due to a reduced NOD2 and TLR4 and an increased IL-10 expression. This can explain the increased susceptibility to infections and help to identify drug targets.

Thalidomide induces limb deformities by perturbing the Bmp/Dkk1/Wnt signaling pathway.

Thalidomide, a sedative originally used to treat morning sickness and now used to treat leprosy and multiple myeloma, is also a teratogen that induces birth defects in humans such as limb truncations and microphthalmia. However, the teratogenic mechanism of action of this drug remains obscure. Thalidomide induces limb and eye defects in the chicken embryo at an EC50 of 50 microg/kg egg wt and apoptosis in primary human embryonic fibroblasts (HEFs) at an EC50 of 8.9 microM. Using these model systems, we demonstrate by semiquantitative reverse transcriptase-polymerase chain reaction and whole-mount in situ hybridization that thalidomide-induced oxidative stress enhances signaling through bone morphogenetic proteins (Bmps). This leads to up-regulation of the Bmp target gene and Wnt antagonist Dickkopf1 (Dkk1) with subsequent inhibition of canonical Wnt/beta-catenin signaling and increased cell death as shown by trypan blue and terminal deoxynucleotidyl transferase-mediated nick end labeling staining. Thalidomide-induced cell death was dramatically reduced in HEFs and in embryonic limb buds by the use of inhibitors against Bmps, Dkk1, and Gsk3beta, a beta-catenin antagonist acting downstream of Dkk1 in the Wnt pathway. Most interestingly, blocking of Dkk1 or Gsk3beta dramatically counteracts thalidomide-induced limb truncations and microphthalmia. From this, we conclude that perturbing of Bmp/Dkk1/Wnt signaling is central to the teratogenic effects of thalidomide.

Herbimycin A suppresses mitotic activity and egg production of female Schistosoma mansoni.

The eggs of the endoparasite Schistosoma are the causative agent of schistosomiasis, an important disease of humans, which is endemic in (sub-) tropical regions. The absence of a vaccine with sufficient protective qualities and increasing resistance to approved and established drugs like praziquantel, justify the exploration of novel ways to fight schistosomes. Our strategy is based on interference with the sexual maturation of the female. Prerequisites for gonad development in adult females are a continuous pairing contact with the male and significantly increased mitotic activity. In this study we show that the male governs sexual maturation of the female, as the separation of couples causes a clear reduction of female mitotic activity and, consequently, egg production. We demonstrate that treatment of schistosomes with Herbimycin A, an inhibitor of protein tyrosine kinases (PTKs), mimics the separation of couples as the drug blocks mitotic activity and egg production of paired females. However, the synthesis of the eggshell precursor protein p14 is elevated. Furthermore, we show for the first time in invertebrates that Herbimycin A decreases tyrosine phosphorylation and PTK stability in schistosomes. Summarised, our data provide evidence that PTKs have key functions in regulating gonad development, eggshell gene expression and, consequently, egg production. Therefore, we suggest envisaging schistosome PTKs as novel targets for strategies to combat schistosomiasis.

TNFα-induced airway smooth muscle cell proliferation depends on endothelin receptor signaling, GM-CSF and IL-6.

UNLABELLED: Pathological proliferation of human airway smooth muscle cells (HASMCs) causes hyperplasia in chronic lung diseases. Signaling pathways that link airway inflammation to HASMC proliferation might provide therapeutic targets for the prevention of airway remodeling and chronic lung diseases. Endothelin-1 (ET-1) signals via endothelin-A- and B-receptors (ETAR, ETBR) to perpetuate HASMC-associated and TNFα-dependent inflammatory processes. HYPOTHESIS: endothelin receptor antagonists (ERAs) suppress HASMC proliferation induced by inflammatory cytokines. HASMCs were stimulated ex vivo with cytokines in the presence or absence of ERAs (ETAR-specific/selective: BQ123, ambrisentan; ETBR-specific: BQ788; non-selective: bosentan, macitentan, ACT-132577) or cytokine-blocking antibodies. Cell counts, DNA-synthesis (BrdU-incorporation assay), cytokine production (ELISA) and ETBR expression (whole-genome microarray data, western blot) were analyzed. ET-1-induced HASMC proliferation and DNA-synthesis were reduced by protein kinase inhibitors and ETAR-specific/selective ERAs but not by BQ788. TNFα-induced HASMC proliferation and DNA-synthesis were reduced by all ERAs. TNFα induced ET-1 and ETBR expression. TNFα- and ET-1-induced GM-CSF releases were both reduced by BQ123 and BQ788. TNFα- and ET-1-induced IL-6 releases were both reduced by BQ123 but not by BQ788. Combined but not single blockade of GM-CSF-receptor-α-chain and IL-6 reduced TNFα- and ET-1-induced HASMC proliferation and DNA-synthesis. Combined but not single treatment with GM-CSF and IL-6 induced HASMC proliferation and DNA-synthesis in the presence of ET-1. In conclusion, TNFα induces HASMC proliferation via ET-1/GM-CSF/IL-6. ETBR requires up-regulation by TNFα to mediate ET-1 effects on HASMC proliferation. This signaling cascade links airway inflammation to HASMC-associated remodeling processes and is sensitive to ERAs. Therefore, ERAs could prevent inflammation-induced airway smooth muscle hyperplasia.