The IL-4/13-induced production of M2 chemokines by human lung macrophages is enhanced by adenosine and PGE2.

BACKGROUND AND PURPOSE: Lung macrophages (LMs) are critically involved in respiratory diseases. The primary objective of the present study was to determine whether or not an adenosine analog (NECA) and prostaglandin E2 (PGE2) affected the interleukin (IL)-4- and IL-13-induced release of M2a chemokines (CCL13, CCL17, CCL18, and CCL22) by human LMs. EXPERIMENTAL APPROACH: Primary macrophages isolated from resected human lungs were incubated with NECA, PGE2, roflumilast, or vehicle and stimulated with IL-4 or IL-13 for 24 h. The levels of chemokines and PGE2 in the culture supernatants were measured using ELISAs and enzyme immunoassays. KEY RESULTS: Exposure to IL-4 (10 ng/mL) and IL-13 (50 ng/mL) was associated with greater M2a chemokine production but not PGE2 production. PGE2 (10 ng/mL) and NECA (10-6 M) induced the production of M2a chemokines to a lesser extent but significantly enhanced the IL-4/IL-13-induced production of these chemokines. At either a clinically relevant concentration (10-9 M) or at a concentration (10-7 M) that fully inhibited phosphodiesterase 4 (PDE4) activity, roflumilast did not increase the production of M2a chemokines and did not modulate their IL-13-induced production, regardless of the presence or absence of PGE2. CONCLUSIONS: NECA and PGE2 enhanced the IL-4/IL-13-induced production of M2a chemokines. The inhibition of PDE4 by roflumilast did not alter the production of these chemokines. These results contrast totally with the previously reported inhibitory effects of NECA, PGE2, and PDE4 inhibitors on the lipopolysaccharide-induced release of tumor necrosis factor alpha and M1 chemokines in human LMs.

Enhanced real-time mass spectrometry breath analysis for the diagnosis of COVID-19.

Background: Although rapid screening for and diagnosis of coronavirus disease 2019 (COVID-19) are still urgently needed, most current testing methods are long, costly or poorly specific. The objective of the present study was to determine whether or not artificial-intelligence-enhanced real-time mass spectrometry breath analysis is a reliable, safe, rapid means of screening ambulatory patients for COVID-19. Methods: In two prospective, open, interventional studies in a single university hospital, we used real-time, proton transfer reaction time-of-flight mass spectrometry to perform a metabolomic analysis of exhaled breath from adults requiring screening for COVID-19. Artificial intelligence and machine learning techniques were used to build mathematical models based on breath analysis data either alone or combined with patient metadata. Results: We obtained breath samples from 173 participants, of whom 67 had proven COVID-19. After using machine learning algorithms to process breath analysis data and further enhancing the model using patient metadata, our method was able to differentiate between COVID-19-positive and -negative participants with a sensitivity of 98%, a specificity of 74%, a negative predictive value of 98%, a positive predictive value of 72% and an area under the receiver operating characteristic curve of 0.961. The predictive performance was similar for asymptomatic, weakly symptomatic and symptomatic participants and was not biased by COVID-19 vaccination status. Conclusions: Real-time, noninvasive, artificial-intelligence-enhanced mass spectrometry breath analysis might be a reliable, safe, rapid, cost-effective, high-throughput method for COVID-19 screening.

Assessment of an e-nose performance for the detection of COVID-19 specific biomarkers.

Early, rapid and non-invasive diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is needed for the prevention and control of coronavirus disease 2019 (COVID-19). COVID-19 mainly affects the respiratory tract and lungs. Therefore, analysis of exhaled breath could be an alternative scalable method for reliable SARS-CoV-2 screening. In the current study, an experimental protocol using an electronic-nose ('e-nose') for attempting to identify a specific respiratory imprint in COVID-19 patients was optimized. Thus the analytical performances of the Cyranose®, a commercial e-nose device, were characterized under various controlled conditions. In addition, the effect of various experimental conditions on its sensor array response was assessed, including relative humidity, sampling time and flow rate, aiming to select the optimal parameters. A statistical data analysis was applied to e-nose sensor response using common statistical analysis algorithms in an attempt to demonstrate the possibility to detect the presence of low concentrations of spiked acetone and nonanal in the breath samples of a healthy volunteer. Cyranose®reveals a possible detection of low concentrations of these two compounds, in particular of 25 ppm nonanal, a possible marker of SARS-CoV-2 in the breath.

Ruxolitinib inhibits cytokine production by human lung macrophages without impairing phagocytic ability.

Background: The Janus kinase (JAK) 1/2 inhibitor ruxolitinib has been approved in an indication of myelofibrosis and is a candidate for the treatment of a number of inflammatory or autoimmune diseases. We assessed the effects of ruxolitinib on lipopolysaccharide (LPS)- and poly (I:C)-induced cytokine production by human lung macrophages (LMs) and on the LMs' phagocytic activity. Methods: Human LMs were isolated from patients operated on for lung carcinoma. The LMs were cultured with ruxolitinib (0.5 × 10-7 M to 10-5 M) or budesonide (10-11 to 10-8 M) and then stimulated with LPS (10 ng·ml-1) or poly (I:C) (10 µg·ml-1) for 24 h. Cytokines released by the LMs into the supernatants were measured using ELISAs. The phagocytosis of labelled bioparticles was assessed using flow cytometry. Results: Ruxolitinib inhibited both the LPS- and poly (I:C)-stimulated production of tumor necrosis factor alpha, interleukin (IL)-6, IL-10, chemokines CCL2, and CXCL10 in a concentration-dependent manner. Ruxolitinib also inhibited the poly (I:C)- induced (but not the LPS-induced) production of IL-1ß. Budesonide inhibited cytokine production more strongly than ruxolitinib but failed to mitigate the production of CXCL10. The LMs' phagocytic activity was not impaired by the highest tested concentration (10-5 M) of ruxolitinib. Conclusion: Clinically relevant concentrations of ruxolitinib inhibited the LPS- and poly (I:C)-stimulated production of cytokines by human LMs but did not impair their phagocytic activity. Overall, ruxolitinib's anti-inflammatory activities are less intense than (but somewhat different from) those of budesonide-particularly with regard to the production of the corticosteroid-resistant chemokine CXCL-10. Our results indicate that treatment with a JAK inhibitor might be a valuable anti-inflammatory strategy in chronic obstructive pulmonary disease, Th1-high asthma, and both viral and non-viral acute respiratory distress syndromes (including coronavirus disease 2019).

Biomedical detection dogs for the identification of SARS-CoV-2 infections from axillary sweat and breath samples*.

A Polymerase Chain Reaction (PCR) test of a nasal swab is still the 'gold standard' for detecting a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. However, PCR testing could be usefully complemented by non-invasive, fast, reliable, cheap methods for detecting infected individuals in busy areas (e.g. airports and railway stations) or remote areas. Detection of the volatile, semivolatile and non-volatile compound signature of SARS-CoV-2 infection by trained sniffer dogs might meet these requirements. Previous studies have shown that well-trained dogs can detect SARS-CoV-2 in sweat, saliva and urine samples. The objective of the present study was to assess the performance of dogs trained to detect the presence of SARS-CoV-2 in axillary-sweat-stained gauzes and on expired breath trapped in surgical masks. The samples were provided by individuals suffering from mild-to-severe coronavirus disease 2019 (COVID-19), asymptomatic individuals, and individuals vaccinated against COVID-19. Results: Seven trained dogs tested on 886 presentations of sweat samples from 241 subjects and detected SARS-CoV-2 with a diagnostic sensitivity (relative to the PCR test result) of 89.6% (95% confidence interval (CI): 86.4%-92.2%) and a specificity of 83.9% (95% CI: 80.3%-87.0%)-even when people with a low viral load were included in the analysis. When considering the 207 presentations of sweat samples from vaccinated individuals, the sensitivity and specificity were respectively 85.7% (95% CI: 68.5%-94.3%) and 86.0% (95% CI: 80.2%-90.3%). The likelihood of a false-positive result was greater in the two weeks immediately after COVID-19 vaccination. Four of the seven dogs also tested on 262 presentations of mask samples from 98 subjects; the diagnostic sensitivity was 83.1% (95% CI: 73.2%-89.9%) and the specificity was 88.6% (95% CI: 83.3%-92.4%). There was no difference (McNemar's testP= 0.999) in the dogs' abilities to detect the presence of SARS-CoV-2 in paired samples of sweat-stained gauzes vs surgical masks worn for only 10 min. Conclusion: Our findings confirm the promise of SARS-CoV-2 screening by detection dogs and broaden the method's scope to vaccinated individuals and easy-to-obtain face masks, and suggest that a 'dogs + confirmatory rapid antigen detection tests' screening strategy might be worth investigating.

Adiponectin Inhibits the Production of TNF-α, IL-6 and Chemokines by Human Lung Macrophages.

Background: Obesity is associated with an elevated risk of severe respiratory infections and inflammatory lung diseases. The objectives were to investigate 1) the production of adiponectin by human lung explants, 2) the expression of the adiponectin receptors AdipoR1 and AdipoR2 by human lung macrophages (LMs), and 3) the impact of recombinant human adiponectin and a small-molecule APN receptor agonist (AdipoRon) on LMs activation. Material and methods: Human parenchyma explants and LMs were isolated from patients operated for carcinoma. The LMs were cultured with recombinant adiponectin or AdipoRon and stimulated with lipopolysaccharide (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. 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-α, 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. Conclusion 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.

Metabolomics of exhaled breath in critically ill COVID-19 patients: A pilot study.

BACKGROUND: Early diagnosis of coronavirus disease 2019 (COVID-19) is of the utmost importance but remains challenging. The objective of the current study was to characterize exhaled breath from mechanically ventilated adults with COVID-19. METHODS: In this prospective observational study, we used real-time, online, proton transfer reaction time-of-flight mass spectrometry to perform a metabolomic analysis of expired air from adults undergoing invasive mechanical ventilation in the intensive care unit due to severe COVID-19 or non-COVID-19 acute respiratory distress syndrome (ARDS). FINDINGS: Between March 25th and June 25th, 2020, we included 40 patients with ARDS, of whom 28 had proven COVID-19. In a multivariate analysis, we identified a characteristic breathprint for COVID-19. We could differentiate between COVID-19 and non-COVID-19 ARDS with accuracy of 93% (sensitivity: 90%, specificity: 94%, area under the receiver operating characteristic curve: 0·94-0·98, after cross-validation). The four most prominent volatile compounds in COVID-19 patients were methylpent-2-enal, 2,4-octadiene 1-chloroheptane, and nonanal. INTERPRETATION: The real-time, non-invasive detection of methylpent-2-enal, 2,4-octadiene 1-chloroheptane, and nonanal in exhaled breath may identify ARDS patients with COVID-19. FUNDING: The study was funded by Agence Nationale de la Recherche (SoftwAiR, ANR-18-CE45-0017 and RHU4 RECORDS, Programme d'Investissements d'Avenir, ANR-18-RHUS-0004), Région Île de France (SESAME 2016), and Fondation Foch.

Clinical relevance of the relaxant effects of roflumilast on human bronchus: potentiation by a long-acting beta-2-agonist.

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 ß2 -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 ß2-agonists.

Clinical Relevance of the Anti-inflammatory Effects of Roflumilast on Human Bronchus: Potentiation by a Long-Acting Beta-2-Agonist.

Background: Roflumilast is an option for treating patients with severe COPD and frequent exacerbations despite optimal therapy with inhaled drugs. The present study focused on whether the phosphodiesterase (PDE) 4 inhibitor roflumilast and its active metabolite roflumilast N-oxide affect the release of tumor necrosis factor (TNF)-α and chemokines by lipopolysaccharide (LPS)-stimulated human bronchial explants. We also investigated the interactions between roflumilast, roflumilast N-oxide and the ß2-agonist formoterol with regard to cytokine release by the bronchial preparations. Methods: Bronchial explants from resected lungs were incubated with roflumilast, roflumilast N-oxide and/or formoterol and then stimulated with LPS. An ELISA was used to measure levels of TNF-α and chemokines in the culture supernatants. Results: At a clinically relevant concentration (1 nM), roflumilast N-oxide and roflumilast consistently reduced the release of TNF-α, CCL2, CCL3, CCL4, CCL5 and CXCL9 (but not CXCL1, CXCL5, CXCL8 and IL-6) from human bronchial explants. Formoterol alone decreased the release of TNF-α, CCL2, and CCL3. The combination of formoterol with roflumilast (1 nM) was more potent than roflumilast alone for inhibiting the LPS-induced release of TNF-α, CCL2, CCL3, CCL4, and CXCL9 by the bronchial explants. Conclusions: At a clinically relevant concentration, roflumilast N-oxide and its parent compound, roflumilast, reduced the LPS-induced production of TNF-α and chemokines involved in monocyte and T-cell recruitment but did not alter the release of chemokines involved in neutrophil recruitment. The combination of formoterol with roflumilast enhanced the individual drugs' anti-inflammatory effects.

Chloroquine Inhibits the Release of Inflammatory Cytokines by Human Lung Explants.

On human lung parenchymal explants, chloroquine concentration clinically achievable in the lung (100 µM) inhibited the lipopolysaccharide-induced release of TNF-ɑ (by 76%), IL-6 (by 68%), CCL2 (by 72%), and CCL3 (by 67%). Besides its antiviral activity, chloroquine might also mitigate the cytokine storm associated with severe pneumonia caused by coronaviruses.

Metabolic reprograming of LPS-stimulated human lung macrophages involves tryptophan metabolism and the aspartate-arginosuccinate shunt.

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.

Contrasting Effects of Adipokines on the Cytokine Production by Primary Human Bronchial Epithelial Cells: Inhibitory Effects of Adiponectin.

BACKGROUND: Obesity is associated with an elevated risk of respiratory infections and inflammatory lung diseases. The objective was to investigate (i) the effects of adipokines (adiponectin (APN), leptin, chemerin, and visfatin) on the production of cytokines by unstimulated and poly(I:C)- and TNF-α-activated human primary bronchial epithelial cells (hBECs), (ii) the cells' expression of the APN receptors (AdipoR1 and AdipoR2), and (iii) the cells' production of APN. METHODS: The hBECs were isolated from patients undergoing surgery for lung carcinoma. The cells were then cultured with human recombinant adipokines in the absence or presence of TNF-α or poly(I:C) for 24 h. Supernatant levels of cytokines (IL-6, CCL2, CCL5, CCL20, CXCL1, CXCL8) and APN were measured using ELISAs. The mRNA levels of AdipoR1 and AdipoR2 in hBECs were determined using a real-time quantitative PCR. RESULTS: Of the four adipokines tested, only APN significantly influenced the basal production and the TNF-α poly(I:C)-induced production of cytokines by hBECs. APN (3-30 µg.ml-1) was associated with greater basal production of IL-6, CCL20, and CXCL8, lower basal production of CCL2 and CXCL1 and no difference in CCL5 production. APN inhibited the poly(I:C)-induced production of these five cytokines and the TNF-α-induced production of CCL2 and CXCL1. AdipoR1 and AdipoR2 were both expressed in hBECs. In contrast to human bronchial explants, isolated hBECs did not produce APN. CONCLUSIONS: The APN concentrations are abnormally low in obese individuals, and this fall may contribute to the susceptibility to viral lung infections and the severity of these infections in obese individuals.

The Role of Toll-Like Receptors in the Production of Cytokines by Human Lung Macrophages.

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.

Bitter Taste Receptors (TAS2Rs) in Human Lung Macrophages: Receptor Expression and Inhibitory Effects of TAS2R Agonists.

BACKGROUND: Bitter-taste receptors (TAS2Rs) are involved in airway relaxation but are also expressed in human blood leukocytes. We studied TAS2R expression and the effects of TAS2R agonists on the lipopolysaccharide (LPS)-induced cytokine release in human lung macrophages (LMs). METHODS: Lung macrophages were isolated from patients undergoing surgery for carcinoma. We used RT-qPCR to measure transcripts of 16 TAS2Rs (TAS2Rs 3/4/5/7/8/9/10/14/19/20/31/38/39/43/45 and 46) in unstimulated and LPS-stimulated (10 ng.mL-1) LMs. The macrophages were also incubated with TAS2R agonists for 24 h. Supernatant levels of the cytokines TNF-α, CCL3, CXCL8 and IL-10 were measured using ELISAs. RESULTS: The transcripts of all 16 TAS2Rs were detected in macrophages. The addition of LPS led to an increase in the expression of most TAS2Rs, which was significant for TAS2R7 and 38. Although the promiscuous TAS2R agonists, quinine and denatonium, inhibited the LPS-induced release of TNF-α, CCL3 and CXCL8, diphenidol was inactive. Partially selective agonists (dapsone, colchicine, strychnine, and chloroquine) and selective agonists [erythromycin (TAS2R10), phenanthroline (TAS2R5), ofloxacin (TAS2R9), and carisoprodol (TAS2R14)] also suppressed the LPS-induced cytokine release. In contrast, two other agonists [sodium cromoglycate (TAS2R20) and saccharin (TAS2R31 and 43)] were inactive. TAS2R agonists suppressed IL-10 production - suggesting that this anti-inflammatory cytokine is not involved in the inhibition of cytokine production. CONCLUSION: Human LMs expressed TAS2Rs. Experiments with TAS2R agonists' suggested the involvement of TAS2Rs 3, 4, 5, 9, 10, 14, 30, 39 and 40 in the inhibition of cytokine production. TAS2Rs may constitute new drug targets in inflammatory obstructive lung disease.

A split-range acquisition method for the non-targeted metabolomic profiling of human plasma with hydrophilic interaction chromatography - high-resolution mass spectrometry.

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.

CCR10+ ILC2s with ILC1-like properties exhibit a protective function in severe allergic asthma.

BACKGROUND: We previously showed that patients with severe allergic asthma have high numbers of circulating ILC2s expressing CCR10. METHOD: Herein, CCR10+ ILC2s were further analyzed in the blood of healthy individuals or patients with allergic and non-allergic asthma. Characteristics of human CCR10+ and CCR10- ILC2s were assessed by flow cytometry as well as single-cell multiplex RT-qPCR. The role of CCR10+ ILC2s in asthma pathophysiology was studied in allergen-treated mice. RESULTS: When compared to healthy controls, CCR10+ ILC2s are enriched in the blood of both allergic and non-allergic severe asthmatic patients, and these cells are recruited to the lungs. Plasma concentrations of the CCR10 ligand CCL27 are significantly increased in severe asthmatics when compared to non-asthmatic patients. CCR10+ ILC2s secrete little TH 2 cytokines, but exhibit ILC1-like properties, including a capacity to produce IFN-γ. Also, single-cell analysis reveals that the CCR10+ ILC2 subset is enriched in cells expressing amphiregulin. CCR10+ ILC2 depletion, as well as blocking of IFN-γ activity, exacerbates airway hyperreactivity in allergen-challenged mice, providing evidence for a protective role of these cells in allergic inflammation. CONCLUSIONS: Frequencies of circulating CCR10+ ILC2s and CCL27 plasma concentrations represent candidate markers of asthma severity. The characterization of CCR10+ ILC2s in human samples and in mouse asthma models suggests that these cells downregulate allergic inflammation through IFN-γ production.

Comparison of the in vitro pharmacological profiles of long-acting muscarinic antagonists in human bronchus.

BACKGROUND AND PURPOSE: Long-acting muscarinic antagonists (LAMAs) have been recommended for the treatment of chronic obstructive pulmonary disease and (more recently) asthma. However, the in vitro pharmacological profiles of the four LAMAs currently marketed (tiotropium, umeclidinium, aclidinium and glycopyrronium) have not yet been compared (relative to ipratropium) by using the same experimental approach. EXPERIMENTAL APPROACH: With a total of 560 human bronchial rings, we investigated the antagonists' potency, onset and duration of action for inhibition of the contractile response evoked by electrical field stimulation. We also evaluated the antagonists' potency for inhibiting cumulative concentration-contraction curves for acetylcholine and carbachol. KEY RESULTS: The onset and duration of action were concentration-dependent. At submaximal, equipotent concentrations, the antagonists' onsets of action were within the same order of magnitude. However, the durations of action differed markedly. After washout, ipratropium's inhibitory activity decreased rapidly (within 30-90 min) but those of tiotropium and umeclidinium remained stable (at above 70%) for at least 9 h. Aclidinium and glycopyrronium displayed less stable inhibitory effects, with a progressive loss of inhibition at submaximal concentrations. In contrast to ipratropium, all the LAMAs behaved as insurmountable antagonists by decreasing the maximum responses to both acetylcholine and carbachol. CONCLUSIONS AND IMPLICATIONS: The observed differences in the LAMAs' in vitro pharmacological profiles in the human bronchus provide a compelling pharmacological rationale for the differences in the drugs' respective recommended daily doses and frequencies of administration.

Human lung and monocyte-derived macrophages differ with regard to the effects of ß2-adrenoceptor agonists on cytokine release.

BACKGROUND: ß2-adrenoceptor agonists have been shown to reduce the lipopolysaccharide (LPS)-induced cytokine release by human monocyte-derived macrophages (MDMs). We compare the expression of ß2-adrenoceptors and the inhibitory effect of formoterol and salmeterol on the LPS-induced release of tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, 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 ß2-adrenoceptor agonists were devoid of any effect on LMs - even in the presence of roflumilast. The expression of ß2-adrenergic receptors was detected on Western blots in MDMs but not in LMs. CONCLUSIONS: Concentrations of ß2-adrenoceptor agonists that cause relaxation of the human bronchus can inhibit cytokine production by LPS-stimulated MDMs but not by LMs.

In smokers, Sonic hedgehog modulates pulmonary endothelial function through vascular endothelial growth factor.

BACKGROUND: Tobacco-induced pulmonary vascular disease is partly driven by endothelial dysfunction. The Sonic hedgehog (SHH) pathway is involved in vascular physiology. We sought to establish whether the SHH pathway has a role in pulmonary endothelial dysfunction in smokers. METHODS: The ex vivo endothelium-dependent relaxation of pulmonary artery rings in response to acetylcholine (Ach) was compared in 34 current or ex-smokers and 8 never-smokers. The results were expressed as a percentage of the contraction with phenylephrine. We tested the effects of SHH inhibitors (GANT61 and cyclopamine), an SHH activator (SAG) and recombinant VEGF on the Ach-induced relaxation. The level of VEGF protein in the pulmonary artery ring was measured in an ELISA. SHH pathway gene expression was quantified in reverse transcriptase-quantitative polymerase chain reactions. RESULTS: Ach-induced relaxation was much less intense in smokers than in never-smokers (respectively 24 ± 6% and 50 ± 7% with 10-4M Ach; p = 0.028). All SHH pathway genes were expressed in pulmonary artery rings from smokers. SHH inhibition by GANT61 reduced Ach-induced relaxation and VEGF gene expression in the pulmonary artery ring. Recombinant VEGF restored the ring's endothelial function. VEGF gene and protein expression levels in the pulmonary artery rings were positively correlated with the degree of Ach-induced relaxation and negatively correlated with the number of pack-years. CONCLUSION: SHH pathway genes and proteins are expressed in pulmonary artery rings from smokers, where they modulate endothelial function through VEGF.

Metabolomics in the Diagnosis and Pharmacotherapy of Lung Diseases.

Metabolomics is of the increasing interests in medical research and the study of respiratory diseases. This novel type of small molecule analysis can be performed not only on conventional biological samples such as plasma or urine but also on sputum, bronchoalveolar fluid, exhaled breath condensate or exhaled breath itself (which is particularly relevant for research on respiratory diseases). On one hand, powerful analytical methodologies (including mass spectrometry and nuclear magnetic resonance spectroscopy) are labour-intensive but enable the exhaustive identification of metabolites. On the other hand, electronic noses can be used for exhaled breath analysis. Although the latter devices do not contain metabolite-specific sensors, they produce a condition- or treatment-specific "breathprint" based on pattern recognition. Breath analysis with electronic noses is noninvasive, and can be performed at the bedside in real time. Here, we review the literature on metabolomics studies in respiratory medicine, with a focus on the evaluation of responses to pharmacotherapy.