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Background and purpose: Obesity is associated with an elevated risk of severe respiratory infections and inflammatory lung diseases. The objectives were to investigate (i) the production of adiponectin by human lung explants, (ii) the expression of the adiponectin receptors AdipoR1 and AdipoR2 by human lung macrophages (LMs), and (iii) the impact of recombinant human adiponectin and a small-molecule APN receptor agonist (AdipoRon) on LMs activation. Experimental approach: Human parenchyma explants and LMs were isolated from patients operated for carcinoma. The LMs were cultured with recombinant adiponectin or AdipoRon and stimulated with LPS (10 ng.mL-1), poly(I:C) (10 µg.mL-1) or interleukin(IL)-4 (10 ng.mL-1) for 24 h. Cytokines or adiponectin, released by explants or LMs, were measured using ELISAs. The mRNA levels of AdipoR1 and AdipoR2 were determined using real-time quantitative PCR. AdipoRs expression was also assessed with confocal microscopy. Key results: Adiponectin was released by lung explants at a level negatively correlated with the donor’s body mass index. AdipoR1 and AdipoR2 were both expressed in LMs. Adiponectin (3-30 µg.mL-1) and AdipoRon (25-50 μM) markedly inhibited the LPS- and poly(I:C)-induced release of Tumor Necrosis Factor-alpha, IL-6 and chemokines (CCL3, CCL4, CCL5, CXCL1, CXCL8, CXCL10) and the IL-4-induced release of chemokines (CCL13, CCL17, CCL22) in a concentration-dependent manner. Recombinant adiponectin produced in mammalian cells (lacking low molecular weight isoforms) had no effects on LMs. Conclusions and implications: The low-molecular-weight isoforms of adiponectin and AdipoRon have an anti-inflammatory activity in the lung environment. Targeting adiponectin receptors may constitute a new means of controlling airways inflammation.

Roflumilast is an oral, add-on option for treating patients with severe COPD and frequent exacerbations despite optimal therapy with inhaled drugs. The present study focused on whether this phosphodiesterase 4 inhibitor and its active metabolite roflumilast N-oxide affect the tone of human bronchial rings. We also investigated the interactions between roflumilast, roflumilast N-oxide and the long-acting β2 -agonist formoterol with regard to the relaxation of isolated human bronchial rings at basal tone or pre-contracted with histamine. Our results demonstrated for the first time that at a clinically relevant concentration (1 nm), roflumilast N-oxide and roflumilast induce a weak relaxation of the isolated human bronchus either at resting tone (22% and 16%, respectively) or even weaker on pre-contracted bronchus with histamine (7% and 5%, respectively). In addition, the combination of formoterol with roflumilast or roflumilast N-oxide is more potent than each component alone for relaxing pre-contracted isolated bronchi - the apparent pD2 of formoterol was significantly reduced for the threshold concentration of 1 nm of the phosphodiesterase 4 inhibitors by a factor of 2.4 for roflumilast N-oxide and 1.9 for roflumilast. The full inhibition of phosphodiesterase 4 activity is achieved at 100 nm but this high concentration only caused partial relaxations of the human bronchi. At a clinically relevant concentration, these oral phosphodiesterase 4 inhibitors are not effective direct bronchodilators but could enhance the efficacy of inhaled long-acting β2-agonists.

International audience; Lung macrophages (LM) are in the first line of defense against inhaled pathogens and can undergo phenotypic polarization to the proinflammatory M1 after stimulation with Toll-like receptor agonists. The objective of the present work was to characterize the metabolic alterations occurring during the experimental M1 LM polarization. Human LM were obtained from resected lungs and cultured for 24 hrs in medium alone or with 10 ng.mL-1 lipopolysaccharide. Cells and culture supernatants were subjected to extraction for metabolomic analysis with high-resolution LC-MS (HILIC and reverse phase -RP- chromatography in both negative and positive ionization modes) and GC-MS. The data were analyzed with R and the Worklow4Metabolomics and MetaboAnalyst online infrastructures. A total of 8,741 and 4,356 features were detected in the intracellular and extracellular content, respectively, after the filtering steps. Pathway analysis showed involvement of arachidonic acid metabolism, tryptophan metabolism and Krebs cycle in the response of LM to LPS, which was confirmed by the specific quantitation of selected compounds. This refined analysis highlighted a regulation of the kynurenin pathway as well as the serotonin biosynthesis pathway, and an involvement of aspartate-arginosuccinate shunt in the malate production. Macrophages M1 polarization is accompanied by changes in the cell metabolome, with the differential expression of metabolites involved in the promotion and regulation of inflammation and antimicrobial activity. The analysis of this macrophage immunometabolome may be of interest for the understanding of the pathophysiology of lung inflammatory disesases.

Fucoidans are sulfated polysaccharides from brown algae, known to have immunomodulatory activity. Their effects on the response of airway epithelial cells to Toll-like receptor 3 (TLR3) stimulation have not been characterized. Our objective was to evaluate the effects of a marine-sourced fucoidan solution (MFS) on the TLR3-induced expression and/or production of cytokines and prostaglandin by human primary bronchial epithelial cells as a model of the airway epithelium. The cells were incubated with MFS in the presence or absence of Poly(I:C) (a TLR3 agonist that mimics viral RNA). Cytokine expression and production were assessed using RT-qPCR and ELISA. The expression of cyclooxygenase-2 and the production of prostaglandin E2 were also measured. Relative to control, exposure to MFS was associated with lower Poly(I:C)-induced mRNA expression of various cytokines and chemokines, and lower COX-2 production. The MFS inhibited the production of some cytokines (IL-1α, IL-1β, TNFα, and IL-6), chemokines (CCL5, CCL22, CXCL1, CXCL5 and CXCL8) and prostaglandin E2 but did not alter the production of IL-12/25, CCL2 and CCL20. At clinically relevant concentrations, the MFS inhibited the TLR3-mediated production of inflammatory mediators by human primary bronchial epithelial cells - suggesting that locally applied MFS might help to reduce airway inflammation in viral infections.

Background: The Toll-like receptor (TLR) family is involved in the recognition of and response to microbial infections. These receptors are expressed in leukocytes. TLR stimulation induces the production of proinflammatory cytokines and chemokines. Given that human lung macrophages (LMs) constitute the first line of defense against inhaled pathogens, the objective of this study was to investigate the expression and function of TLR subtypes in this cell population. Methods: Human primary LMs were obtained from patients undergoing surgical resection. The RNA and protein expression levels of TLRs, chemokines, and cytokines were assessed after incubation with subtype-selective agonists. Results: In human LMs, the TLR expression level varied from one subtype to another. Stimulation with subtype-selective agonists induced an intense, concentration- and time-dependent increase in the production of chemokines and cytokines. TLR4 stimulation induced the strongest effect, whereas TLR9 stimulation induced a much weaker response. Conclusions: The stimulation of TLRs in human LMs induces intense cytokine and chemokine production, a characteristic of the proinflammatory M1 macrophage phenotype.

Background and Purpose 15-Lipoxygenase (15-LOX) activity is associated with inflammation and immune regulation. The objectives of the present study were to investigate the expression of 15-LOX-1 and 15-LOX-2 and evaluate the enzymes' roles in the polarization of human lung macrophages (LMs) in response to LPS and Th2 cytokines (IL-4/-13). Experimental Approach LMs were isolated from patients undergoing surgery for carcinoma. The cells were cultured with a 15-LOX inhibitor (PD146176 or ML351), a COX inhibitor (indomethacin), a 5-LOX inhibitor (MK886) or vehicle and then stimulated with LPS (10 ng·mL−1), IL-4 (10 ng·mL−1) or IL-13 (50 ng·mL−1) for 24 h. Levels of ALOX15 (15-LOX-1) and ALOX15B (15-LOX-2) transcripts were determined by real-time quantitative PCR. Immunoassays were used to measure levels of LPS-induced cytokines (TNF-α, CCL2, CCL3, CCL4, CXCL1, CXCL8 and CXCL10) and Th2 cytokine-induced chemokines (CCL13, CCL18 and CCL22) in the culture supernatant. Key Results Stimulation of LMs with LPS was associated with increased expression of ALOX15B, whereas stimulation with IL-4/IL-13 induced the expression of ALOX15. PD146176 and ML351 (10 μM) reduced the release of the chemokines induced by LPS and Th2 cytokines. The effects of these 15-LOX inhibitors were maintained in the presence of indomethacin and MK886. Furthermore, indomethacin revealed the inhibitory effect of PD146176 on TNF-α release. Conclusions and Implications Inhibition of the 15-LOX pathways is involved in the down-regulation of the in vitro production of chemokines in LMs. Our results suggest that the 15-LOX pathways have a role in the pathogenesis of inflammatory lung disorders and may thus constitute a potential drug target.

International audience; Background: β2-adrenoceptor agonists have been shown to reduce the lipopolysaccharide (LPS)-induced cytokine release by human monocyte-derived macrophages (MDMs). We compare the expression of β2-adrenoceptors and the inhibitory effect of formoterol and salmeterol on the LPS-induced release of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6 and a range of chemokines (CCL2, 3, 4, and IL-8) by human lung macrophages (LMs) and MDMs. Methods: LMs were isolated from patients undergoing resection and MDMs were obtained from blood monocytes in the presence of GM-CSF. LMs and MDMs were incubated in the absence or presence of formoterol or salmeterol prior to stimulation with LPS. The effects of formoterol were also assessed in the presence of the phosphodiesterase inhibitor roflumilast. Results: LPS-induced cytokine production was higher in LMs than in MDMs. Salmeterol and formoterol exerted an inhibitory effect on the LPS-induced production of TNF-α, IL-6, CCL2, CCL3, and CCL4 in MDMs. In contrast, the β2-adrenoceptor agonists were devoid of any effect on LMs - even in the presence of roflumilast. The expression of β2-adrenergic receptors was detected on Western blots in MDMs but not in LMs. Conclusions: Concentrations of β2-adrenoceptor agonists that cause relaxation of the human bronchus can inhibit cytokine production by LPS-stimulated MDMs but not by LMs. © 2017 The Author(s).

Introduction Le ruxolitinib est un inhibiteur de Janus Kinase approuve pour le traitement de la myelofibrose et la reaction du greffon contre l’hote cutanee apres allogreffe de moelle. Nous avons precedemment rapporte des cas de proteinose alveolaire et d’infections pulmonaires a mycobacteries non tuberculeuses chez des patients sous ruxolitinib, ce qui suggere un deficit des fonctions macrophagiques alveolaires en lien avec le traitement. Nous avons voulu etudier in vitro l’effet du ruxolitinib sur la fonction des macrophages pulmonaires humains. Methodes Les macrophages pulmonaires (MP) ont ete isoles a partir de pieces operatoires de patients operes pour un carcinome bronchique. Ils ont ete traites par du ruxolitinib (0,5–10 μM), seul ou en presence de LPS (10 ng/mL), Poly I :C (10 ng/mL) ou GM-CSF (10 ng/mL). La production de cytokines a ete mesuree par ELISA. Pour evaluer la phagocytose, les MPs ont ete incubes avec des particules de carbone noir (diametre 14 nm) en presence de ruxolitinib (5 μM) et de LPS ; les modifications de morphologie et le pourcentage de cellules ayant phagocyte les particules ont ete releves. Resultats Le ruxolitinib n’a pas d’effet sur la production de cytokines par les MPs non stimules. Il reduit, de facon concentration dependante, la production induite par le LPS et le POLY (I :C) de TNFa, IL-6, CCL2, CXCL9, CXCL10, et IFNb mais pas celle de CXCL1 et de CXCL8. Ce resultat n’est pas modifie en cas de pre-traitement par du GM-CSF. Le GM-CSF amplifie la production d’IL-23 induite par le LPS ; cette production est inhibee par le ruxolitinib. Le ruxolitinib induit une morphologie fusiforme des MPs et reduit le nombre de cellules ayant phagocyte les particules de carbone, en basal et apres stimulation par du LPS. Conclusion Le ruxolitinib induit une diminution des reponses inflammatoires et de la phagocytose des macrophages pulmonaires humains. Ces modifications structurelles et fonctionnelles pourraient favoriser la survenue d’infections a mycobacteries et de proteinose alveolaire.

Resume Les recepteurs responsables de la perception gustative permettent de distinguer les quatre gouts basiques : sale, sucre, amer et umami. Parmi eux, les recepteurs des gouts amers (TAS2R) sont des recepteurs couples a des proteines G, dont 25 sous-types ont ete identifies chez l’homme a ce jour. Meme si l’existence d’agonistes endogenes demeure incertaine, les recepteurs TAS2R ont la capacite de reconnaitre de nombreuses molecules naturelles ou de synthese, comme certaines molecules produites par des bacteries, ou encore la cafeine, la chloroquine, ou l’erythromycine. L’expression de ces recepteurs, que l’on pensait initialement circonscrite a la cavite buccale, a recemment ete decrite dans des tissus extra-oraux tels que le tractus gastro-intestinal ou les poumons. Les effets dans le tissu pulmonaire se situent essentiellement a trois niveaux : les recepteurs TAS2R exprimes sur les cils vibratiles des cellules epitheliales augmentent la frequence de vibration des cils ; la stimulation des recepteurs TAS2R exprimes dans les cellules musculaires lisses bronchiques entraine une relaxation des bronches, alors que les recepteurs TAS2R exprimes dans les cellules immunitaires du tissu pulmonaire, dont les macrophages, participent a la modulation de la production de cytokines pro-inflammatoires. En conclusion, au regard de ces differents mecanismes complementaires, les recepteurs TAS2R pourraient devenir une cible pharmacologique d’un interet certain pour le traitement des pathologies pulmonaires obstructives.

International audience; Untargeted metabolomics of human plasma with mass spectrometry is of particular interest in medical research to explore pathophysiology, find disease biomarkers or for the understanding of the response to pharmacotherapy. Since analytical performances may be impacted by the laboratory environment and the acquisition method settings, the objectives of this study were to assess the role of interfering compounds and to propose an acquisition method to maximize the metabolome coverage for human plasma metabolomic analysis. Human plasma samples were processed with liquid/liquid extraction then analysed with HILIC-high resolution mass spectrometry. A method with a single m/z range was compared to four methods with different split acquisition ranges and four sets of ionization source parameters were compared. The data were analysed with the R software and on the Worklow4Metabolomics online platform. The major interfering compounds were identified in blank samples where they accounted for up to 86% of the signal intensity. Splitting the acquisition range into 3 m/z ranges improved the number of detected features, the number of features with proposed annotation in the Human Metabolome Database, as well as signal intensity throughout the whole m/z range. The method performing best was the one using three m/z ranges of approximatively the same extent. Ionization source parameters also strongly affected the number of detected features. Splitting the acquisition range into 3 m/z ranges with optimized ionization source parameters allows a comprehensive analysis of the human plasma metabolome with perspectives for applications to pathophysiological studies.

Introduction Les recepteurs a l’amertume (TAS2Rs) sont une famille de recepteurs couples aux proteines G, comprenant 25 membres chez l’homme, dont l’expression a ete recemment documentee dans les leucocytes, dans les cellules epitheliales et les cellules musculaires lisses des voies aeriennes. Leur stimulation entraine une relaxation bronchique une inhibition de la production de cytokines par les leucocytes. Cette etude vise a evaluer l’expression et la fonction des recepteurs a l’amertume dans les macrophages pulmonaires humains. Methodes Des macrophages pulmonaires ont ete isoles chez des patients operes pour un carcinome pulmonaire, puis soumis a une agression par des agonistes TAS2R (1 μM a 1 mM), en presence de lipopolysaccharide (LPS). Les agonistes ont ete selectionnes sur la base de leur selectivite connue envers les differents sous-types de recepteurs. L’expression de seize TAS2R a ete evaluee avec RT-qPCR et la production de cytokines (TNF-a, CCL3, CXCL8 et IL-10) a ete mesuree par ELISA. Resultats L’expression des seize TAS2R a ete trouvee dans les macrophages pulmonaires humains : TAS2R3, 4, 5, 7, 8, 9, 10, 14, 19, 20, 31, 38, 39, 43, 45 et 46. Parmi les agonistes non-selectifs des TAS2Rs, la quinine et le denatonium inhibaient la liberation induite par le LPS de TNF-α, CCL3 et CXCL8, mais le diphenidol etait inactif. Les agonistes partiellement selectifs de TAS2Rs (dapsone, colchicine, strychnine et chloroquine) et les agonistes selectifs de TAS2R (erythromycine [TAS2R10], phenanthroline [TAS2R5], ofloxacine [TAS2R9] et carisoprodol [TAS2R14]) inhibaient egalement la liberation de cytokines induite par le LPS. En revanche, deux autres agonistes selectifs (le cromoglycate de sodium [TAS2R20] et la saccharine [TAS2R31 et 43]) etaient inactifs. L’inhibition de la production d’IL-10 par les agonistes de TAS2R suggere que la production de cette cytokine anti-inflammatoire n’etait pas impliquee dans l’inhibition de la production de cytokines induite par le LPS. Conclusion La stimulation des TAS2R entraine une inhibition de la production de cytokines par les macrophages pulmonaires humains. Malgre l’absence d’antagonistes selectifs des TAS2R, l’analyse croisee des resultats suggere l’implication de TAS2R3, 4, 5, 9, 10, 30, 39, 40 et, possiblement, celle de TAS2R14. Dans l’ensemble, les TAS2R peuvent constituer une nouvelle cible pharmacologique pour le traitement des maladies pulmonaires obstructives inflammatoires.

Introduction The epithelial-derived cytokine, thymic stromal lymphopoietin (TSLP), has been identified as an important cytokine in allergic inflammation. Various studies demonstrated elevate levels and expression of TSLP in airways of asthmatic. Environmental factors such as toll-like receptor ligands, viruses, microbes, allergen sources, diesel exhaust and cigarette smoke trigger TSLP production by epithelial cells. It has been described that TSLP amplifies the differentiation of peritoneal macrophages and bone marrow derived macrophages in M2-type in mice. Our aims were to investigate the role of TSLP in the M1/M2 polarization of human lung macrophages (LM). Material and methods Lung macrophages were isolated from tissues obtained after surgical resection for cancer. Macrophages were treated with LPS (10 ng/mL) or IL-4 (10 ng/mL) in presence or not of TSLP (10–30 ng/mL) for 24 h or 48 h. Production of cytokines of the M1-type (TNF-α, CCL-2, CXCL-8 and CXCL-1) and the M2-type (CCL-13, CCL-17, CCL-18 and CCL-22) was determined by ELISA and cytokine expression by RT-qPCR. Results Exposure to TSLP did not alter the production of TNF-α, CXCL-8 and CXCL-1 either in unstimulated LM or in LPS-stimulated LM. However, CCL-2 production was increased by TSLP at 20 ng/mL in unstimulated LM. In addition, TSLP (10 ng/mL but not 30 ng/mL) increased M2-type cytokine production (CCL-13 and CCL-17, but did not CCL-18 and CCL-22). TSLP also increased IL4-induced CCL-17 and CCL-22. Expression of CCL-17 and CCL-13 was also increased by TSLP in unstimulated LM, and expression of CCL-17 and CCL-22 was increased in IL-4-stimulantes LM. Conclusion Our results show that TSLP can activate unstimulated LM and modulate the IL-4-induced polarization of LM in the M2 subtype.

BACKGROUND: Regular use of β2-agonists may enhance non-specific airway responsiveness. The wingless/integrated (Wnt) signaling pathways are responsible for several cellular processes, including airway inflammation and remodeling while cAMP-PKA cascade can activate the Wnt signaling. We aimed to investigate whether the Wnt signaling pathways are involved in the bronchial hyperresponsiveness induced by prolonged exposure to β2-adrenoceptor agonists in human isolated airways. METHODS: Bronchi were surgically removed from 44 thoracic surgery patients. After preparation, bronchial rings and primary cultures of bronchial epithelial cells were incubated with fenoterol (0.1 µM, 15 hours, 37 °C), a β2-agonist with high intrinsic efficacy. The effects of inhibitors/blockers of Wnt signaling on the fenoterol-induced airway sensitization were examined and the impact of fenoterol exposure on the mRNA expression of genes interacting with Wnt signaling or cAMP-PKA cascade was assessed in complete bronchi and in cultured epithelial cells. RESULTS: Compared to paired controls, fenoterol-sensitization was abolished by inhibition/blockage of the Wnt/β-catenin signaling, especially the cell-surface LRP5/6 co-receptors or Fzd receptors (1 µM SFRP1 or 1 µM DKK1) and the nuclear recruitment of TCF/LEF transcriptions factors (0.3 µM FH535). Wnt proteins secretion did not seem to be involved in the fenoterol-induced sensitization since the mRNA expression of Wnt remained low after fenoterol exposure and the inactivator of Wnt secretion (1 µM IWP2) had no effect on the fenoterol-sensitization. Fenoterol exposure did not change the mRNA expression of genes regulating Wnt signaling or cAMP-PKA cascade. CONCLUSIONS: Collectively, our pharmacological investigations indicate that fenoterol-sensitization is modulated by the inhibition/blockage of canonical Wnt/β-catenin pathway, suggesting a phenomenon of biased agonism in connection with the β2-adrenoceptor stimulation. Future experiments based on the results of the present study will be needed to determine the impact of prolonged fenoterol exposure on the extra- and intracellular Wnt signaling pathways at the protein expression level.

Background Bitter-taste receptors (TAS2Rs) have recently been involved in the relaxation of mouse and guinea pig airways, and increased expression of TAS2Rs was shown in blood leucocytes from asthmatic children. We sought to identify and characterize the TAS2Rs expressed in isolated human bronchi and the subtypes involved in relaxation. Methods Human bronchi were isolated from resected lungs and TAS2R transcripts were assessed with RT-qPCR. Relaxation to TAS2R agonists was tested in organ bath in the presence or absence of pharmacological modulators of the signalling pathways involved in bronchial relaxation. Results We detected the expression of TAS2R transcripts in human bronchi. The non-selective agonists chloroquine, quinine, caffeine, strychnine and diphenidol produced a bronchial relaxation as effective and potent as theophylline but much less potent than formoterol and isoproterenol. Denatonium, saccharin and colchicine did not produce relaxation. Receptor expression analysis together with the use of selective agonists suggest a predominant role for TAS2R5, 10 and 14 in bitter taste agonist-induced relaxation. The mechanism of relaxation was independent of the signalling pathways modulated by conventional bronchodilators and may be partly explained by the inhibition of phosphatidylinositol-3-kinases. Conclusions The TAS2Rs may constitute a new therapeutic target in chronic obstructive lung diseases such as asthma.