Methacholine-induced Airway Hyperresponsiveness Research Articles

Celastrol alleviates airway hyperresponsiveness and inflammation in obese asthma through mediation of alveolar macrophage polarization

Obese asthma is a unique asthma phenotype that decreases sensitivity to inhaled corticosteroids, and currently lacks efficient therapeutic medication. Celastrol, a powerful bioactive substance obtained naturally from the roots of Tripterygium wilfordii, has been reported to possess the potential effect of weight loss in obese individuals. However, its role in the treatment of obese asthma is not fully elucidated. In the present study, diet-induced obesity (DIO) mice were used with or without ovalbumin (OVA) sensitization, the therapeutic effects of celastrol on airway hyperresponsiveness (AHR) and airway inflammation were examined. We found celastrol significantly decreased methacholine-induced AHR in obese asthma, as well as reducing the infiltration of inflammatory cells and goblet cell hyperplasia in the airways. This effect was likely due to the inhibition of M1-type alveolar macrophages (AMs) polarization and the promotion of M2-type macrophage polarization. In vitro, celastrol yielded equivalent outcomes in Lipopolysaccharide (LPS)-treated RAW264.7 macrophage cells, featuring a reduction in the expression of M1 macrophage makers (iNOS, IL-1β, TNF-α) and heightened M2 macrophage makers (Arg-1, IL-10). Mechanistically, the PI3K/AKT signaling pathway has been implicated in these processes. In conclusion, we demonstrated that celastrol assisted in mitigating various parameters of obese asthma by regulating the balance of M1/M2 AMs polarization.

Design, Synthesis, and Biological Evaluation of Novel Spiro Imidazobenzodiazepines to Identify Improved Inhaled Bronchodilators.

Novel gamma-aminobutyric acid receptor (GABAAR) ligands structurally related to imidazobenzodiazepine MIDD0301 were synthesized using spiro-amino acid N-carboxyanhydrides (NCAs). These compounds demonstrated increased resistance to phase 2 metabolism and avoided the formation of a 6H isomer. Compound design was guided by molecular docking using the available crystal structure of the α1β3γ2 GABAAR and correlated with in vitro binding data. The carboxylic acid containing GABAAR ligands have high aqueous solubility, low permeability, and low cell toxicity. The inability of GABAAR ligands to cross the blood-brain barrier was confirmed in vivo by the absence of sensorimotor inhibition. Pharmacological activities at lung GABAARs were demonstrated by ex vivo relaxation of guinea pig airway smooth muscle and reduction of methacholine-induced airway hyperresponsiveness (AHR) in conscious mice. We identified bronchodilator 5c with an affinity of 9 nM for GABAARs that was metabolically stable in the presence of human and mouse microsomes.

Empagliflozin inhibits autophagy and mitigates airway inflammation and remodelling in mice with ovalbumin-induced allergic asthma

Empagliflozin, a selective inhibitor of Na+-glucose cotransporter-2, has been reported to exert anti-inflammatory and anti-fibrotic effects in addition to autophagy modulation. Addressing the role of autophagy in allergic asthma revealed controversial results. The potential effect of empagliflozin treatment on airway inflammation and remodelling as well as autophagy modulation in a murine model of allergic asthma was investigated. Over a 7-week period, male BALB/c mice were sensitized and challenged by intraperitoneal injection and inhalation of ovalbumin, respectively. Animals were treated with empagliflozin (10 mg/kg; orally) and/or rapamycin (an autophagy inducer; 4 mg/kg; intraperitoneally) before every challenge. Methacholine-induced airway hyperresponsiveness was evaluated one day after the last challenge. After euthanasia, serum, bronchoalveolar lavage fluid, and lung tissues were collected for biochemical, histopathological, and immunohistochemical assessment. Results revealed that empagliflozin decreased airway hyperresponsiveness, serum ovalbumin-specific immunoglobulin E, and bronchoalveolar lavage total and differential leukocytic counts. Levels of inflammatory and profibrotic cytokines (IL-4, IL-5, IL-13, IL-17, and transforming growth factor-β1) were all inhibited. Moreover, empagliflozin preserved pulmonary microscopic architecture and alleviated bronchiolar epithelial thickening, goblet cell hyperplasia, fibrosis and smooth muscle hypertrophy. These effects were associated with inhibition of ovalbumin-activated autophagic flux, as demonstrated by decreased LC3B expression and LC3BII/I ratio, as well as increased P62 expression. However, the therapeutic potential of empagliflozin was inhibited when rapamycin was co-administered. In conclusion, this study demonstrates that empagliflozin has immunomodulatory, anti-inflammatory, and anti-remodelling properties in ovalbumin-induced allergic asthma and suggests that autophagic flux inhibition may play a role in empagliflozin's anti-asthmatic effects.

Targeting of G-protein coupled receptor 40 alleviates airway hyperresponsiveness through RhoA/ROCK1 signaling pathway in obese asthmatic mice

Obesity increases the severity of airway hyperresponsiveness (AHR) in individuals with asthma, but the mechanism is not well elucidated. G-protein coupled receptor 40 (GPR40) has been found to induce airway smooth muscle contraction after activated by long-chain fatty acids (LC-FFAs), suggesting a close correlation between GPR40 and AHR in obese. In this study, C57BL/6 mice were fed a high-fat diet (HFD) to induce obesity with or without ovalbumin (OVA) sensitization, the regulatory effects of GPR40 on AHR, inflammatory cells infiltration, and the expression of Th1/Th2 cytokines were evaluated by using a small-molecule antagonist of GPR40, DC260126. We found that the free fatty acids (FFAs) level and GPR40 expression were greatly elevated in the pulmonary tissues of obese asthmatic mice. DC260126 greatly reduced methacholine-induced AHR, ameliorated pulmonary pathological changes and decreased inflammatory cell infiltration in the airways in obese asthma. In addition, DC260126 could down-regulate the levels of Th2 cytokines (IL-4, IL-5, and IL-13) and pro-inflammatory cytokines (IL-1β, TNF-α), but elevated Th1 cytokine (IFN-γ) expression. In vitro, DC260126 could remarkedly reduce oleic acid (OA)-induced cell proliferation and migration in HASM cells. Mechanistically, the effects that DC260126 alleviated obese asthma was correlated with the down-regulation of GTP-RhoA and Rho-associated coiled-coil-forming protein kinase 1 (ROCK1). Herein, we proved that targeting of GPR40 with its antagonist helped to mitigate multiple parameters of obese asthma effectively.

Genetic deletion of osteoprotegerin attenuates asthma development through suppression of inflammatory response in mice

To clarify the detailed molecular mechanisms underlying the development of asthma, we assessed the potential immune effects of prenatal osteoprotegerin (OPG) inhibition in the pathogenesis of asthma. The effects of OPG deficiency on the development of asthma were evaluated using an ovalbumin-induced asthma model in OPG knockout mice. Histological analysis demonstrated that OPG was mainly detected in airway epithelial cells in wild type mice. After ovalbumin sensitization and challenge, accumulation of inflammatory cells, gene expression of T helper 2-related cytokines and mucus hypersecretion in lung tissues were inhibited by OPG deficiency. Importantly, the serum level of IgE was not increased in OPG KO mice after ovalbumin sensitization and challenge. Based on these findings, OPG knockout mice were protected against methacholine-induced airway hyperresponsiveness. OPG expression is thought to be essential for induction of the allergic immune response in asthma.

Ammonia exposure by intratracheal instillation causes severe and deteriorating lung injury and vascular effects in mice

Objective Ammonia (NH3) is a corrosive alkaline gas that can cause life-threatening injuries by inhalation. The aim was to establish a disease model for NH3-induced injuries similar to acute lung injury (ALI) described in exposed humans and investigate the progression of lung damage, respiratory dysfunction and evaluate biomarkers for ALI and inflammation over time. Methods Female BALB/c mice were exposed to an NH3 dose of 91.0 mg/kg·bw using intratracheal instillation and the pathological changes were followed for up to 7 days. Results NH3 instillation resulted in the loss of body weight along with a significant increase in pro-inflammatory mediators in both bronchoalveolar lavage fluid (e.g. IL-1β, IL-6, KC, MMP-9, SP-D) and blood (e.g. IL-6, Fibrinogen, PAI-1, PF4/CXCL4, SP-D), neutrophilic lung inflammation, alveolar damage, increased peripheral airway resistance and methacholine-induced airway hyperresponsiveness compared to controls at 20 h. On day 7 after exposure, deteriorating pathological changes such as increased macrophage lung infiltration, heart weights, lung hemorrhages and coagulation abnormalities (elevated plasma levels of PAI-1, fibrinogen, endothelin and thrombomodulin) were observed but no increase in lung collagen. Some of the analyzed blood biomarkers (e.g. RAGE, IL-1β) were unaffected despite severe ALI and may not be significant for NH3-induced damages. Conclusions NH3 induces severe acute lung injuries that deteriorate over time and biomarkers in lungs and blood that are similar to those found in humans. Therefore, this model has potential use for developing diagnostic tools for NH3-induced ALI and for finding new therapeutic treatments, since no specific antidote has been identified yet.

Sex differences in the induction of angiotensin converting enzyme 2 (ACE-2) in mouse lungs after e-cigarette vapor exposure and its relevance to COVID-19

The COVID-19 pandemic has affected over 114 million people and has resulted in >2.5 million deaths so far. Some people have greater susceptibility which influences both SARS-CoV-2 infectivity and COVID-19...

Mast cell-derived serotonin enhances methacholine-induced airway hyperresponsiveness in house dust mite-induced experimental asthma.

Airway hyperresponsiveness (AHR) is a feature of asthma in which airways are hyperreactive to stimuli causing extensive airway narrowing. Methacholine provocations assess AHR in asthma patients mainly by direct stimulation of smooth muscle cells. Using in vivo mouse models, mast cells have been implicated in AHR, but the mechanism behind has remained unknown. Cpa3Cre/+ mice, which lack mast cells, were used to assess the role of mast cells in house dust mite (HDM)-induced experimental asthma. Effects of methacholine in presence or absence of ketanserin were assessed on lung function and in lung mast cells in vitro. Airway inflammation, mast cell accumulation and activation, smooth muscle proliferation, and HDM-induced bronchoconstriction were evaluated. Repeated intranasal HDM sensitization induced allergic airway inflammation associated with accumulation and activation of lung mast cells. Lack of mast cells, absence of activating Fc-receptors, or antagonizing serotonin (5-HT)2A receptors abolished HDM-induced trachea contractions. HDM-sensitized mice lacking mast cells had diminished lung-associated 5-HT levels, reduced AHR and methacholine-induced airway contraction, while blocking 5-HT2A receptors in wild types eliminated AHR, implying that mast cells contribute to AHR by releasing 5-HT. Primary mouse and human lung mast cells express muscarinic M3 receptors. Mouse lung mast cells store 5-HT intracellularly, and methacholine induces release of 5-HT from lung-derived mouse mast cells and Ca2+ flux in human LAD-2 mast cells. Methacholine activates mast cells to release 5-HT, which by acting on 5-HT2A receptors enhances bronchoconstriction and AHR. Thus, M3-directed asthma treatments like tiotropium may also act by targeting mast cells.

Subacute TGFβ Exposure Drives Airway Hyperresponsiveness in Cystic Fibrosis Mice through the PI3K Pathway.

Cystic fibrosis (CF) is a lethal genetic disease characterized by progressive lung damage and airway obstruction. The majority of patients demonstrate airway hyperresponsiveness (AHR), which is associated with more rapid lung function decline. Recent studies in the neonatal CF pig demonstrated airway smooth muscle (ASM) dysfunction. These findings, combined with observed CF transmembrane conductance regulator (CFTR) expression in ASM, suggest that a fundamental defect in ASM function contributes to lung function decline in CF. One established driver of AHR and ASM dysfunction is transforming growth factor (TGF) β1, a genetic modifier of CF lung disease. Prior studies demonstrated that TGFβ exposure in CF mice drives features of CF lung disease, including goblet cell hyperplasia and abnormal lung mechanics. CF mice displayed aberrant responses to pulmonary TGFβ, with elevated PI3K signaling and greater increases in lung resistance compared with controls. Here, we show that TGFβ drives abnormalities in CF ASM structure and function through PI3K signaling that is enhanced in CFTR-deficient lungs. CF and non-CF mice were exposed intratracheally to an adenoviral vector containing the TGFβ1 cDNA, empty vector, or PBS only. We assessed methacholine-induced AHR, bronchodilator response, and ASM area in control and CF mice. Notably, CF mice demonstrated enhanced AHR and bronchodilator response with greater ASM area increases compared with non-CF mice. Furthermore, therapeutic inhibition of PI3K signaling mitigated the TGFβ-induced AHR and goblet cell hyperplasia in CF mice. These results highlight a latent AHR phenotype in CFTR deficiency that is enhanced through TGFβ-induced PI3K signaling.

Hesperetin-5,7,3'-O-Trimethylether Dually Inhibits Phosphodiesterase 3/4 and Methacholine-Induced Airway Hyperresponsiveness in Sensitized and Challenged Mice.

IntroductionHesperetin-5,7,3ʹ-O-trimethylether (HTME), a synthetic liposoluble hesperetin, has been reported to be a dual phosphodiesterase (PDE)3/4 inhibitor. We investigated its inhibitory effects on methacholine (MCh)-induced airway hyperresponsiveness (AHR) and its potential for treating atypical asthma and COPD.MethodsFlexiVent system was used to determine AHR in ovalbumin (OVA) sensitized and challenged mice. Determination of cytokines was performed by using mouse T helper (Th)1/Th2 cytokine CBA kits, and of total immunoglobulin (Ig)E and OVA-specific IgE using ELISA kits. The number of inflammatory cells was counted using a hemocytometer. Xylazine/ketamine-induced anesthesia was to assess nausea, vomiting, and gastric hypersecretion in these mice.ResultsHTME dually and competitively inhibited PDE3/4 activities in the Lineweaver–Burk analysis. HTME (30 and 100 μmol/kg) dose-dependently and significantly decreased the airway resistance (RL) and increased lung dynamic compliance (Cdyn) values induced by MCh. It significantly suppressed numbers of total inflammatory cells and neutrophils, and levels of cytokines in bronchoalveolar lavage fluid (BALF). HTME dose-dependently and significantly inhibited total and OVA-specific IgE levels in the BALF and serum. However, HTME did not influence xylazine/ketamine-induced anesthesia.ConclusionHTME exerted anti-inflammatory and bronchodilator effects and may be useful in treating chronic obstructive pulmonary disease and allergic atypical asthma with no gastrointestinal side effects.

Effects of the Toll-like receptor 7 (TLR7) agonist, AZD8848, on allergen-induced responses in patients with mild asthma: a double-blind, randomised, parallel-group study

BackgroundAlthough allergic asthma is a complex area with many interacting factors involved, the ‘hygiene hypothesis’ proposes that a lack of exposure to infection during childhood may polarise the immune system towards allergen-reactive Th2-type responses in genetically susceptible individuals. Toll-like receptors (TLRs) play a key role within the innate immune system and TLR7 agonists have previously been shown to up-regulate Th1 responses and down-regulate Th2 responses to allergens in murine models of allergic or chronic asthma. This study aimed to examine the efficacy and safety of the novel TRL7 agonist AZD8848, which has been developed as an antedrug.MethodsIn this double-blind, randomised, parallel-group study, AZD8848 60 μg or placebo was administered intranasally once-weekly for 8 weeks in patients with mild-to-moderate allergic asthma (NCT00999466). Efficacy assessments were performed at 1 and 4 weeks after the last dose. The primary outcome was the late asthmatic response (LAR) fall in forced expiratory volume in 1 s (FEV1) after allergen challenge at 1-week post-treatment.ResultsAZD8848 significantly reduced average LAR fall in FEV1 by 27% vs. placebo at 1 week after treatment (p = 0.035). This effect was sustained at 4 weeks post-treatment; however, it did not reach clinical significance. AZD8848 reduced post-allergen challenge methacholine-induced airway hyper-responsiveness (AHR) vs. placebo at 1 week post-dosing (treatment ratio: 2.20, p = 0.024), with no effect at 4 weeks. There was no significant difference between the two groups in plasma cytokine, sputum Th2 cytokine or eosinophil responses post-allergen challenge at 1 week after treatment. The incidence of adverse events was similar in the two groups. AZD8848 was generally well tolerated.Conclusions and clinical relevanceIn patients with allergic asthma, TLR7 agonists could potentially reduce allergen responsiveness by stimulating Type 1 interferon responses to down-regulate the dominant Th2 responses.Trial registrationclinicaltrials.gov identifier NCT00999466.

Short-term effect of once-daily fluticasone furoate on methacholine-induced bronchoconstriction in mild asthmatics.

Inhaled corticosteroids (ICS) decrease airway inflammation and airway hyperresponsiveness (AHR). Previous studies have generally investigated the effect of ICS on methacholine-induced AHR following weeks or months of medium to high dose treatment. The short-term effects of once-daily fluticasone furoate (FF) 100 mcg on methacholine-induced AHR and airway inflammation were examined over the course of one week. Eleven mild asthmatics completed this randomized, double-blind crossover study. Once-daily FF (100 mcg) and identical appearing placebo Ellipta® inhalers were given for 7 days with a 2-week washout. Methacholine challenges were performed before and 24 h after the first, third and seventh doses. Fractional exhaled nitric oxide (FeNO) was measured initially and at 7 days. FF significantly (p = 0.0009-0.0078) increased methacholine PD20 (provocative dose causing 20% fall in forced expiratory volume in 1 s) at all times. Doubling dose shifts (95% CI) were 1.23 (0.60-1.86), 1.17 (0.68-1.67) and 1.44 (0.93-1.94) after the first, third and seventh dose respectively. FeNO (geometric mean, 95% CI) decreased significantly (p = 0.0049) following FF treatment from 37.9 ppb (23.7-60.5) initially to 22.9 ppb (14.8-35.5) at 7 days. Placebo did not affect methacholine PD20 or FeNO. Single-dose FF 100 mcg decreased methacholine AHR at 24 h without significant further improvement with continued daily use over 7 days. The inhibition in AHR after one week of daily dosing coincided with a significant decrease in FeNO at 7 days. Contrary to past assumptions, the ICS FF appears to rapidly reduce AHR to methacholine.

APD588, a novel, selective S1P- receptor modulator, inhibits inflammation and airway hyperresponsiveness in the ovalbumin-induced model of allergic asthma

Abstract Background FTY720 is a non-selective sphingosine-1-phosphate (S1P) receptor modulator that has shown efficacy in mouse asthma models. APD588 is an investigational stage S1P receptor modulator with increased selectivity and potential for an improved safety profile. We hypothesized that the optimized S1P receptor profile of APD588 would confer beneficial effects in ovalbumin (OVA)-induced asthma. Aim To evaluate efficacy of prophylactic APD588 in the OVA-induced allergic asthma model Methods Receptor potency and selectivity profile of APD588 was determined (b-arrestin, GTPgS) in cell lines expressing recombinant human S1P1–5. Efficacy in allergic asthma was assessed in BALB/c mice which were sensitized with intraperitoneal OVA on days 0, 7, and 17, then exposed to intratracheal (IT) vehicle or OVA on days 21–23. From day 20, vehicle, APD588 or FTY720 were dosed IT, QD. On day 24, methacholine-induced airway hyperresponsiveness (AHR), immune cell infiltration and BAL cytokines were measured. Results APD588 was a potent modulator of S1P1, with less potency at S1P4, 5, and no detectable activity at S1P2,3. IT administration of APD588 produced a dose-dependent inhibition of AHR, significantly inhibited airway eosinophil and lymphocyte infiltration, and BAL cytokines IL-4 and IL-5. FTY720 also reduced AHR and neutrophil and lymphocyte BAL cell counts, but had no effect on eosinophil counts or cytokines. Conclusions APD588 was an effective IT therapy for OVA-induced asthma. Lung function improvements correlated with reduced Th2 cytokines and lymphocyte and eosinophil infiltration in the BAL. Based on these results, APD588 may have therapeutic potential in asthma, and broadly indicate potential in other atopic Th2 diseases.

Anti-inflammatory and anti-fibrotic treatment in a rodent model of acute lung injury induced by sulfur dioxide

Context: Inhalation of sulfur dioxide (SO2) affects the lungs and exposure to high concentrations can be lethal. The early pulmonary response after inhaled SO2 involves tissue injury, acute neutrophilic lung inflammation and airway hyperresponsiveness (AHR). In rats, long-term pulmonary fibrosis is evident 14 days post-exposure as indicated by analysis of collagen deposition in lung tissue. Early treatment with a single dose of dexamethasone (DEX,10 mg/kg) significantly attenuates the acute inflammatory response in airways. However, this single DEX-treatment is not sufficient for complete protection against SO2-induced injuries.Methods: Female Sprague–Dawley rats exposed to SO2 (2200 ppm, nose-only exposure, 10 min) were given treatments (1, 5 and 23 h after SO2-exposure) with the anti-fibrotic and anti-inflammatory substance Pirfenidone (PFD, 200 mg/kg) or DEX (10 mg/kg) to evaluate whether the inflammatory response, AHR and lung fibrosis could be counteracted.Results: Both treatment approaches significantly reduced the total leukocyte response in bronchoalveolar lavage fluid and suppressed pulmonary edema. In contrast to DEX-treatment, PFD-treatment reduced the methacholine-induced AHR to almost control levels and partially suppressed the acute mucosal damage whereas multiple DEX-treatment was the only treatment that reduced collagen formation in lung tissue.Conclusions: To enable an accurate extrapolation of animal derived data to humans, a detailed understanding of the underlying mechanisms of the injury, and potential treatment options, is needed. The findings of the present study suggest that treatments with the capability to reduce both AHR, the inflammatory response, and fibrosis are needed to achieve a comprehensive mitigation of the acute lung injury caused by SO2.

Ginsenoside Rg1 Exhibits Anti-asthmatic Activity in an Aspergillus Protease-Induced Asthma Model in Mice

Asthma is one of the most common chronic airway diseases and is characterized by symptoms, such as, wheezing and coughing. Its prevalence in Korea is gradually increasing among children and adults, especially among those older than 60 years. In this study, we investigated the effects of ginsenoside Rg1 (Rg1), one of the major constituents of Panax ginseng Meyer on ovalbumin (OVA) plus Aspergillus protease ( A. protease) allergen-induced asthmatic mice. Mice were orally administered Rg1 for 10 days from 8 days after OVA + A. protease sensitization and intranasally administered booster doses of OVA + A. protease for 4 days from 14 days after sensitization. The effects of Rg1 administration on airway hyperresponsiveness (AHR), immune cell distributions in bronchoalveolar lavage fluid (BALF), and serum level of immunoglobulin E (IgE) were investigated at 18 days after sensitization. Histopathological changes in hematoxylin and eosin (H&E)-stained lung sections were also examined. Treatment of OVA + A. protease-sensitized/challenged asthmatic mice with Rg1 significantly decreased total eosinophil counts in BALF as compared with those of phosphate-buffered saline (PBS)-treated sensitized/challenged controls, and significantly decreased methacholine-induced AHR. Furthermore, IgE serum levels were significantly lower in Rg1 administered OVA + A. protease-sensitized asthmatic mice than in PBS-treated sensitized/challenged controls. The study shows Rg1 protects against A. protease allergen-induced asthma in mice.

Prevention of Asthma Exacerbation in a Mouse Model by Simultaneous Inhibition of NF-κB and STAT6 Activation Using a Chimeric Decoy Strategy.

Transactivation of inflammatory and immune mediators in asthma is tightly regulated by nuclear factor κB (NF-κB) and signal transducer and activator of transcription 6 (STAT6). Therefore, we investigated the efficacy of simultaneous inhibition of NF-κB and STAT6 using a chimeric decoy strategy to prevent asthma exacerbation. The effects of decoy oligodeoxynucleotides were evaluated using an ovalbumin-induced mouse asthma model. Ovalbumin-sensitized mice received intratracheal administration of decoy oligodeoxynucleotides 3 days before ovalbumin challenge. Fluorescent-dye-labeled decoy oligodeoxynucleotides could be detected in lymphocytes and macrophages in the lung, and activation of NF-κB and STAT6 was inhibited by chimeric decoy oligodeoxynucleotide transfer. Consequently, treatment with chimeric or NF-κB decoy oligodeoxynucleotides protected against methacholine-induced airway hyperresponsiveness, whereas the effect of chimeric decoy oligodeoxynucleotides was significantly greater than that of NF-κB decoy oligodeoxynucleotides. Treatment with chimeric decoy oligodeoxynucleotides suppressed airway inflammation through inhibition of overexpression of interleukin-4 (IL-4), IL-5, and IL-13 and inflammatory infiltrates. Histamine levels in the lung were reduced via suppression of mast cell accumulation. A significant reduction in mucin secretion was observed due to suppression of MUC5AC gene expression. Interestingly, the inhibitory effects on IL-5, IL-13, and histamine secretion were achieved by transfer of chimeric decoy oligodeoxynucleotides only. This novel therapeutic approach could be useful to treat patients with various types of asthma.

Exercise-induced bronchoconstriction in children with asthma: An observational cohort study.

The diagnosis of exercise-induced bronchoconstriction (EIB) was established by changes in lung function after exercise challenge. The prevalence of EIB and factors related to EIB were not fully described in children with asthma. The aim of this study was to investigate the prevalence and predictors of EIB in children with asthma. A total of 149 children with physician-diagnosed asthma above 5 years of age underwent standardized treadmill exercise challenge for EIB and methacholine challenge for airway hyper-responsiveness from October 2015 to December 2016. EIB presented in 52.5% of children with asthma. Compared with children without EIB, there were more patients with atopic dermatitis in children with EIB (p=0.038). Allergic to Dermatohagoides pteronyssinus and Dermatophagoides farinae were also found more in children with EIB (p=0.045 and 0.048 respectively). Maximal decrease in forced expiratory volume in 1s (FEV1) were highest in patients who were most sensitive to methacholine provocation (provocation concentration causing 20% fall in FEV1 [PC20]≤1mg/mL). Patients, who were more sensitive to methacholine challenge (with lower PC20 levels), develop EIB with more decline in FEV1 after exercise challenge (p=0.038). Among patients with EIB, airflow limitation development in patient with methacholine-induced airway hyper-responsiveness was more abrupt and severe compared with patients without airway hyper-responsiveness (p=0.045 and 0.033 respectively). EIB presented in 52.5% of children with asthma. The more severe methacholine-induced hyper-responsiveness, the higher prevalence of EIB as well as the severity.

The effect of glycopyrronium and indacaterol, as monotherapy and in combination, on the methacholine dose-response curve of mild asthmatics: a randomized three-way crossover study

BackgroundMethacholine dose-response curves illustrate pharmacologic bronchoprotection against methacholine-induced airway hyperresponsiveness and can be used to quantitate changes in airway sensitivity (position), reactivity (slope), and maximal responsiveness following drug administration. Our objective was to determine the influence of single-dose glycopyrronium (long-acting muscarinic antagonist) and indacaterol (ultra-long acting β2 agonist), as monotherapy and in combination, on the methacholine dose-response curve of mild asthmatics and to compare these findings with a non-asthmatic control curve.MethodsThis was a randomized, double blind, double dummy, three-way crossover study. For asthmatic participants (n = 14), each treatment arm included a baseline methacholine challenge, drug administration, and repeat methacholine challenges at 1, 24, and 48 h. Non-asthmatic control participants (n = 15) underwent a single methacholine challenge and did not receive any study treatment. Methacholine dose-response curves were graphed as the percent fall in forced expiratory volume in 1 s (FEV1) for each methacholine concentration administered. Best-fit curves were then generated. Differences in airway reactivity were calculated through linear regression. Changes in airway sensitivity were assessed as the shift in the provocative concentration of methacholine causing a 20% fall in FEV1.ResultsCompared to baseline, all treatments significantly reduced airway sensitivity to methacholine at 1 h post-dose (indacaterol ~1.5 doubling concentrations; glycopyrronium ~5 doubling concentrations; combination ~5 doubling concentrations). Bronchoprotection at 24 and 48 h remained significant with glycopyrronium and combination therapy only. Airway reactivity was not influenced by indacaterol whereas glycopyrronium significantly reduced airway reactivity at all time-points (p = 0.003-0.027). The combination significantly decreased slope at 1 (p = 0.021) and 24 (p = 0.039) hours only. The non-asthmatic control and 1-h glycopyrronium curves are nearly identical. Only the non-asthmatic control and 1-h post-combination therapy curves appeared to generate a true response plateau (three data points within 5%), which occurred at a 14% fall in FEV1.ConclusionsMethacholine dose-response curves differentiate the bronchoprotective mechanisms triggered by different classes of asthma medications. Assessment of bronchoprotection using methacholine dose-response curves may be useful during clinical development of respiratory medications when performing superiority, equivalence, or non-inferiority trials.Trial registrationclinicaltrials.gov (NCT02953041). Retrospectively registered on October 24th 2016.

I12 Repositioning andrographolide for airway inflammation: Mapping its mechanistic networks

Inhaled corticosteroid is the first-line controller for asthma and COPD. However, about 10% of the asthmatics (severe/refractory asthma) and most of the COPD patients are resistant to the beneficial effects of corticosteroids. There is a pressing unmet medical need to develop novel therapeutic agents to restore corticosteroid efficacy in affected patients. Andrographolide, a bioactive molecule isolated from the plant Andrographis paniculata, has demonstrated anti-inflammatory and antioxidant properties in both asthma and COPD experimental models. In a severe asthma mouse model using combined IFN- γ /LPS exposure, production of IL-27 and methacholine-induced airway hyperresponsiveness (AHR) were found to be corticosteroid-resistant. Andrographolide significantly restored the suppressive effect of dexamethasone on LPS/IFN- γ -induced IL-27 levels in bronchoalveolar lavage (BAL) fluid and AHR in mice. LPS/IFN- γ markedly reduced the nuclear level of histone deacetylase-2 (HDAC2), an essential epigenetic enzyme that mediates corticosteroid anti-inflammatory actions. Andrographolide significantly restored nuclear HDAC2 levels and diminished total HAT/HDAC activity ratio in mouse lungs exposed to LPS/IFN- γ , possibly via suppression of PI3K/Akt/HDAC2 phosphorylation and up-regulation of the antioxidant transcription factor Nrf2 level. In a cigarette smoke (CS)-induced COPD model, andrographolide markedly restored dexamethasone actions in inhibiting CS-induced lung neutrophilia. In addition, andrographolide facilitated dexamethasone actions to suppress BAL fluid IL-6, IL-1b, KC and IL-17 levels. In lung lysates, andrographolide markedly restored total nuclear HDAC activity. The complete steroid re-sensitization mechanism of andrographolide remains to be unravelled. Nevertheless, our existing data strongly implicate a potentially novel steroid re-sensitizing activity of andrographolide in both severe asthma and COPD models. Acknowledgements This work was supported by a NUHS Bridging Grant R-184-000-267-733 and a CREATE Grant R-184-000-269-592 by the National Research Foundation (NRF) of Singapore to W.S.F.W.

Ribosomal protein S3 gene silencing protects against experimental allergic asthma.

Ribosomal protein S3 (RPS3) is a 40S ribosomal protein of the S3P family essential for implementing protein translation. RPS3 has recently been found to interact with the p65 subunit of the NF-κB complex and promote p65 DNA-binding activity. Persistent activation of the NF-κB pathway is evident in allergic asthma. We hypothesized that gene silencing of lung RPS3 can ameliorate allergic airway inflammation. The gene silencing efficacy of RPS3 siRNA was screened in three different mouse cell lines by real-time PCR and immunoblotting. Protective effects of intratracheal RPS3 siRNA in a house dust mite (HDM) mouse asthma model were determined by measuring cell counts in lung lavage fluid and lung sections, lung cytokine profiles and airway hyperresponsiveness (AHR). RPS3 siRNA markedly knocked down RPS3 levels in all mouse cell lines tested, and in mouse lung tissues, blocked TNF-α- or HDM-induced release of mediators by the cultured cells and reduced eosinophil counts in lung lavage fluid from the HDM mouse asthma model. RPS3 siRNA lessened HDM-induced airway mucus hypersecretion, cytokine production and serum IgE elevation. Moreover, RPS3 knockdown significantly suppressed methacholine-induced AHR in experimental asthma. RPS3 siRNA disrupted TNF-α-induced NF-κB activation in a NF-κB reporter gene assay in vitro and prevented the nuclear accumulation of p65 subunit and p65 transcriptional activation in HDM-challenged lungs and cells. RPS3 gene silencing ameliorates experimental asthma, probably by disrupting NF-κB activity. RPS3 could be a novel therapeutic target for allergic airway inflammation.