Distribution of type 2 biomarkers and association with severity, clinical characteristics and comorbidities in the BREATHE real-life asthma population.

Background: Type 2 (T2) high asthma is recognised as a heterogenous entity consisting of several endotypes; however, the prevalence and distribution of the T2 biomarkers in the general asthma population, across asthma severity, and across compartments is largely unknown. The objective of the present study was to describe expression and overlaps of airway and systemic T2 biomarkers in a clinically representative asthma population. Methods: Patients with asthma from the real-life BREATHE cohort referred to a specialist centre were included and grouped according to T2 biomarkers: blood and sputum eosinophilia (≥0.3×109 cells·L-1 and 3% respectively), total IgE (≥150 U·mL-1), and fractional exhaled nitric oxide (≥25 ppb). Results: Patients with mild-to-moderate asthma were younger (41 versus 49 years, p<0.001), had lower body mass index (25.9 versus 28.0 kg·m-2, p=0.002) and less atopy (47% versus 58%, p=0.05), higher forced expiratory volume in 1 s (3.2 versus 2.8 L, p<0.001) and forced vital capacity (4.3 versus 3.9 L, p<0.001) compared with patients with severe asthma, who had higher blood (0.22×109 versus 0.17×109 cells·L-1, p=0.01) and sputum (3.0% versus 1.5%, p=0.01) eosinophils. Co-expression of all T2 biomarkers was a particular characteristic of severe asthma (p<0.001). In patients with eosinophilia, sputum eosinophilia without blood eosinophilia was present in 45% of patients with mild-to-moderate asthma and 35% with severe asthma. Conclusion: Severe asthma is more commonly associated with activation of several T2 pathways, indicating that treatments targeting severe asthma may need to act more broadly on T2 inflammatory pathways. Implementation of airway inflammometry in clinical care is of paramount importance, as the best treatable trait is otherwise is overlooked in a large proportion of patients irrespective of disease severity.

Allergen Immunotherapy Enhances Airway Epithelial Antiviral Immunity in Patients with Allergic Asthma (VITAL Study): A Double-Blind Randomized Controlled Trial.

Rationale: Allergic asthma is linked to impaired bronchial epithelial secretion of IFNs, which may be causally linked to the increased risk of viral exacerbations. We have previously shown that allergen immunotherapy (AIT) effectively reduces asthma exacerbations and prevents respiratory infections requiring antibiotics; however, whether AIT alters antiviral immunity is still unknown. Objectives: To investigate the effect of house dust mite sublingual AIT (HDM-SLIT) on bronchial epithelial antiviral and inflammatory responses in patients with allergic asthma. Methods: In this double-blind, randomized controlled trial (VITAL [The Effect of Allergen Immunotherapy on Anti-viral Immunity in Patients with Allergic Asthma]), adult patients with HDM allergic asthma received HDM-SLIT 12-SQ or placebo for 24 weeks. Bronchoscopy was performed at baseline and at Week 24, which included sampling for human bronchial epithelial cells. Human bronchial epithelial cells were cultured at baseline and at Week 24 and stimulated with the viral mimic polyinosinic:polycytidylic acid (poly(I:C)). mRNA expression was quantified using qRT-PCR, and protein concentrations were measured using multiplex ELISA. Measurements and Main Results: Thirty-nine patients were randomized to HDM-SLIT (n = 20) or placebo (n = 19). HDM-SLIT resulted in increased polyinosinic:polycytidylic acid-induced expression of IFN-ß at both the gene (P = 0.009) and protein (P = 0.02) levels. IFN-λ gene expression was also increased (P = 0.03), whereas IL-33 tended to be decreased (P = 0.09). On the other hand, proinflammatory cytokines IL-6 (P = 0.009) and TNF-α (tumor necrosis factor-α) (P = 0.08) increased compared with baseline in the HDM-SLIT group. There were no significant changes in TSLP (thymic stromal lymphopoietin), IL-4, IL-13, and IL-10. Conclusions: HDM-SLIT improves bronchial epithelial antiviral resistance to viral infection. These results potentially explain the efficacy of HDM-SLIT in reducing exacerbations in allergic asthma. Clinical trial registered with www.clinicaltrials.gov (NCT04100902).

Impact of former smoking exposure on airway eosinophilic activation and autoimmunity in patients with severe asthma.

INTRODUCTION: Severe eosinophilic asthma is characterised by frequent exacerbations and a relative insensitivity to steroids. Experimentally, smoking may induce eosinophilic airway inflammation, but the impact in patients with severe asthma is not clear. OBJECTIVE: To investigate the association between smoking exposure in patients with severe asthma, and eosinophilic inflammation and activation, as well as airway autoimmunity and steroid responsiveness. METHODS: Patients with severe asthma according to European Respiratory Society/American Thoracic Society criteria were assessed with sputum samples, analysed by cell differential count, and for the presence of free eosinophil granules (FEGs), autoantibodies against eosinophil peroxidase (EPX) and macrophage receptor with collagenous structure (MARCO). A subgroup of patients with eosinophilic airway inflammation was re-assessed after a 2-week course of prednisolone. RESULTS: 132 severe asthmatics were included in the study. 39 (29.5%) patients had ≥10 pack-years of smoking history: 36 (27.3%) were former smokers and three (2.3%) current smokers; and 93 (70.5%) had <10 pack-years exposure. Eosinophilic airway inflammation was more prevalent among patients with ≥10 pack-years (66.7%), compared to patients with <10 pack-years (38.7%, p=0.03), as was the level of FEGs (p=0.001) and both anti-EPX and anti-MARCO (p<0.05 and p<0.0001, respectively). Omitting current smokers did not affect these associations. Furthermore, prednisolone reduced, but did not normalise, sputum eosinophils in patients with a ≥10 pack-year smoking history. CONCLUSION: In patients with severe asthma, a former smoking history is associated with eosinophilic airway inflammation and activation and relative insensitivity to steroids, as well as airway autoimmunity.

Clinical characteristics of the BREATHE cohort - a real-life study on patients with asthma and COPD.

Background: The BREATHE study is a cross-sectional study of real-life patients with asthma and/or COPD in Denmark and Sweden aiming to increase the knowledge across severities and combinations of obstructive airway disease. Design: Patients with suspicion of asthma and/or COPD and healthy controls were invited to participate in the study and had a standard evaluation performed consisting of questionnaires, physical examination, FeNO and lung function, mannitol provocation test, allergy test, and collection of sputum and blood samples. A subgroup of patients and healthy controls had a bronchoscopy performed with a collection of airway samples. Results: The study population consisted of 1403 patients with obstructive airway disease (859 with asthma, 271 with COPD, 126 with concurrent asthma and COPD, 147 with other), and 89 healthy controls (smokers and non-smokers). Of patients with asthma, 54% had moderate-to-severe disease and 46% had mild disease. In patients with COPD, 82% had groups A and B, whereas 18% had groups C and D classified disease. Patients with asthma more frequently had childhood asthma, atopic dermatitis, and allergic rhinitis, compared to patients with COPD, asthma + COPD and Other, whereas FeNO levels were higher in patients with asthma and asthma + COPD compared to COPD and Other (18 ppb and 16 ppb vs 12.5 ppb and 14 ppb, p < 0.001). Patients with asthma, asthma + COPD and Other had higher sputum eosinophilia (1.5%, 1.5%, 1.2% vs 0.75%, respectively, p < 0.001) but lower sputum neutrophilia (39.3, 43.5%, 40.8% vs 66.8%, p < 0.001) compared to patients with COPD. Conclusions: The BREATHE study provides a unique database and biobank with clinical information and samples from 1403 real-life patients with asthma, COPD, and overlap representing different severities of the diseases. This research platform is highly relevant for disease phenotype- and biomarker studies aiming to describe a broad spectrum of obstructive airway diseases.

Interleukin-6 release is higher across arm than leg muscles during whole-body exercise.

Exercising muscle releases interleukin-6 (IL-6), but the mechanisms controlling this process are poorly understood. This study was performed to test the hypothesis that the IL-6 release differs in arm and leg muscle during whole-body exercise, owing to differences in muscle metabolism. Sixteen subjects (10 men and six women, with body mass index 24 ± 1 kg m(-2) and peak oxygen uptake 3.4 ± 0.6 l min(-1)) performed a 90 min combined arm and leg cycle exercise at 60% of maximal oxygen uptake. The subjects arrived at the laboratory having fasted overnight, and catheters were placed in the femoral artery and vein and in the subclavian vein. During exercise, arterial and venous limb blood was sampled and arm and leg blood flow were measured by thermodilution. Lean limb mass was measured by dual-energy X-ray absorbtiometry scanning. Before and after exercise, biopsies were obtained from vastus lateralis and deltoideus. During exercise, IL-6 release was similar between men and women and higher (P < 0.05) from arms than legs (1.01 ± 0.42 and 0.33 ± 0.12 ng min(-1) (kg lean limb mass)(-1), respectively). Blood flow (425 ± 36 and 554 ± 35 ml min(-1) (kg lean limb mass)(-1)) and fatty acid uptake (26 ± 7 and 47 ± 7 µmol min(-1) (kg lean limb mass)(-1)) were lower, glucose uptake similar (51 ± 12 and 41 ± 8 mmol min(-1) (kg lean limb mass)(-1)) and lactate release higher (82 ± 32 and -2 ± 12 µmol min(-1) (kg lean limb mass)(-1)) in arms than legs, respectively, during exercise (P < 0.05). No correlations were present between IL-6 release and exogenous substrate uptakes. Muscle glycogen was similar in arms and legs before exercise (388 ± 22 and 428 ± 25 mmol (kg dry weight)(-1)), but after exercise it was only significantly lower in the leg (219 ± 29 mmol (kg dry weight)(-1)). The novel finding of a markedly higher IL-6 release from the exercising arm compared with the leg during whole-body exercise was not directly correlated to release or uptake of exogenous substrate, nor to muscle glycogen utilization.

Dysregulation of glycogen synthase COOH- and NH2-terminal phosphorylation by insulin in obesity and type 2 diabetes mellitus.

CONTEXT: Insulin-stimulated glucose disposal is impaired in obesity and type 2 diabetes mellitus (T2DM) and is tightly linked to impaired skeletal muscle glucose uptake and storage. Impaired activation of glycogen synthase (GS) by insulin is a well-established defect in both obesity and T2DM, but the underlying mechanisms remain unclear. DESIGN AND PARTICIPANTS: Insulin action was investigated in a matched cohort of lean healthy, obese nondiabetic, and obese type 2 diabetic subjects by the euglycemic-hyperinsulinemic clamp technique combined with muscle biopsies. Activity, site-specific phosphorylation, and upstream signaling of GS were evaluated in skeletal muscle. RESULTS: GS activity correlated inversely with phosphorylation of GS site 2+2a and 3a. Insulin significantly decreased 2+2a phosphorylation in lean subjects only and induced a larger dephosphorylation at site 3 in lean compared with obese subjects. The exaggerated insulin resistance in T2DM compared with obese subjects was not reflected by differences in site 3 phosphorylation but was accompanied by a significantly higher site 1b phosphorylation during insulin stimulation. Hyperphosphorylation of another Ca(2+)/calmodulin-dependent kinase-II target, phospholamban-Thr17, was also evident in T2DM. Dephosphorylation of GS by phosphatase treatment fully restored GS activity in all groups. CONCLUSIONS: Dysregulation of GS phosphorylation plays a major role in impaired insulin regulation of GS in obesity and T2DM. In obesity, independent of T2DM, this is associated with impaired regulation of site 2+2a and likely site 3, whereas the exaggerated insulin resistance to activate GS in T2DM is linked to hyperphosphorylation of at least site 1b. Thus, T2DM per se seems unrelated to defects in the glycogen synthase kinase-3 regulation of GS.

Lack of AMPKalpha2 enhances pyruvate dehydrogenase activity during exercise.

5'-AMP-activated protein kinase (AMPK) was recently suggested to regulate pyruvate dehydrogenase (PDH) activity and thus pyruvate entry into the mitochondrion. We aimed to provide evidence for a direct link between AMPK and PDH in resting and metabolically challenged (exercised) skeletal muscle. Compared with rest, treadmill running increased AMPKalpha1 activity in alpha(2)KO mice (90%, P < 0.01) and increased AMPKalpha2 activity in wild-type (WT) mice (110%, P < 0.05), leading to increased AMPKalpha Thr(172) (WT: 40%, alpha(2)KO: 100%, P < 0.01) and ACCbeta Ser(227) phosphorylation (WT: 70%, alpha(2)KO: 210%, P < 0.01). Compared with rest, exercise significantly induced PDH-E(1)alpha site 1 (WT: 20%, alpha(2)KO: 62%, P < 0.01) and site 2 (only alpha(2)KO: 83%, P < 0.01) dephosphorylation and PDH(a) [ approximately 200% in both genotypes (P < 0.01)]. Compared with WT, PDH dephosphorylation and activation was markedly enhanced in the alpha(2)KO mice both at rest and during exercise. The increased PDH(a) activity during exercise was associated with elevated glycolytic flux, and muscles from the alpha(2)KO mice displayed marked lactate accumulation and deranged energy homeostasis. Whereas mitochondrial DNA content was normal, the expression of several mitochondrial proteins was significantly decreased in muscle of alpha(2)KO mice. In isolated resting EDL muscles, activation of AMPK signaling by AICAR did not change PDH-E(1)alpha phosphorylation in either genotype. PDH is activated in mouse skeletal muscle in response to exercise and is independent of AMPKalpha2 expression. During exercise, alpha(2)KO muscles display deranged energy homeostasis despite enhanced glycolytic flux and PDH(a) activity. This may be linked to decreased mitochondrial oxidative capacity.

AMPK-mediated AS160 phosphorylation in skeletal muscle is dependent on AMPK catalytic and regulatory subunits.

AMP-activated protein kinase (AMPK) is a heterotrimeric protein that regulates glucose transport mediated by cellular stress or pharmacological agonists such as 5-aminoimidazole-4-carboxamide 1 beta-d-ribonucleoside (AICAR). AS160, a Rab GTPase-activating protein, provides a mechanism linking AMPK signaling to glucose uptake. We show that AICAR increases AMPK, acetyl-CoA carboxylase, and AS160 phosphorylation by insulin-independent mechanisms in isolated skeletal muscle. Recombinant AMPK heterotrimeric complexes (alpha1beta1gamma1 and alpha2beta2gamma1) phosphorylate AS160 in a cell-free assay. In mice deficient in AMPK signaling (alpha2 AMPK knockout [KO], alpha2 AMPK kinase dead [KD], and gamma3 AMPK KO), AICAR effects on AS160 phosphorylation were severely blunted, highlighting that complexes containing alpha2 and gamma3 are necessary for AICAR-stimulated AS160 phosphorylation in intact skeletal muscle. Contraction-mediated AS160 phosphorylation was also impaired in alpha2 AMPK KO and KD but not gamma3 AMPK KO mice. Our results implicate AS160 as a downstream target of AMPK.

Oral glucose ingestion attenuates exercise-induced activation of 5'-AMP-activated protein kinase in human skeletal muscle.

5'-AMP-activated protein kinase (AMPK) has been suggested to be a 'metabolic master switch' regulating various aspects of muscle glucose and fat metabolism. In isolated rat skeletal muscle, glucose suppresses the activity of AMPK and in human muscle glycogen loading decreases exercise-induced AMPK activation. We hypothesized that oral glucose ingestion during exercise would attenuate muscle AMPK activation. Nine male subjects performed two bouts of one-legged knee-extensor exercise at 60% of maximal workload. The subjects were randomly assigned to either consume a glucose containing drink or a placebo drink during the two trials. Muscle biopsies were taken from the vastus lateralis before and after 2 h of exercise. Plasma glucose was higher (6.0 +/- 0.2 vs. 4.9 +/- 0.1 mmol L-1, P < 0.001), whereas glycerol (44.8 +/- 7.8 vs. 165.7 +/- 22.3 micromol L-1), and free fatty acid (169.3 +/- 9.5 vs. 1161 +/- 144.9 micromol L-1) concentrations were lower during the glucose compared to the placebo trial (both P < 0.001). Calculated fat oxidation was lower during the glucose trial (0.17 +/- 0.02 vs. 0.25 +/- 0.03 g min-1, P < 0.001). Activation of alpha2-AMPK was attenuated in the glucose trial compared to the placebo trial (0.24 +/- 0.07 vs. 0.46 +/- 0.14 pmol mg-1 min-1, P = 0.03), whereas the alpha1-AMPK activity was not different between trials or affected by exercise. AMPK and the downstream target of AMPK, acetyl-CoA carboxylase-beta, were phosphorylated as a response to exercise, but neither was significantly different between the two trials. We conclude that oral glucose ingestion attenuates the exercise-induced activation of alpha2-AMPK, bringing further support for a fuel-sensing role of AMPK in skeletal muscle.