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Pieta C Wijsman,1,* Annika WM Goorsenberg,1,* Abilash Ravi,1,2,* Julia NS d’Hooghe,1 Barbara S Dierdorp,2 Tamara Dekker,2 Charlotte CLM van Schaik,1 Nick HT ten Hacken,3 Pallav L Shah,4– 6 Els JM Weersink,1 Elisabeth H Bel,1 Jouke T Annema,1 René Lutter,1,2 Peter I Bonta1 1Department of Pulmonary Medicine, Amsterdam UMC, University of Amsterdam, Amsterdam, the Netherlands; 2Department of Experimental Immunology, Amsterdam Infection and Immunity Institute, Amsterdam UMC, University of Amsterdam, Amsterdam, the Netherlands; 3Department of Pulmonology, University Medical Center Groningen, University of Groningen, Groningen, the Netherlands; 4Department of Pulmonology, Royal Brompton Hospital, London, UK; 5National Heart & Lung Institute, Imperial College, London, UK; 6Department of Pulmonology, Chelsea & Westminster Hospital, London, UK*These authors contributed equally to this workCorrespondence: Peter I Bonta, Email p.i.bonta@amsterdamumc.nlBackground: Bronchial thermoplasty (BT) is a bronchoscopic treatment for severe asthma, of which the working mechanism and responder profile are partly unknown. The aim of this study is to analyse whether BT alters airway inflammation by epithelial gene expression, inflammatory cell counts and cytokines, and whether this relates to treatment response.Methods: In this clinical trial, 28 severe asthma patients underwent bronchoscopy before and after treatment to obtain bronchial brushes and bronchoalveolar lavage fluid (BALF) from treated and untreated airways. RNA was extracted from bronchial brushes for transcriptome analysis, and BALF cells and cytokines were analysed. Asthma quality of life questionnaires were used to distinguish responders from non-responders. We compared results before and after treatment, between treated and untreated airways, and between responders and non-responders.Results: Gene expression of airway epithelium related to airway inflammation gene set was significantly downregulated in treated airways compared to untreated airways, although this did not differ for patients before and after treatment. No differences were observed in cell counts and cytokines, neither from the untreated compared to treated airways, nor before and after treatment. At baseline, compared to non-responders, the expression of genes related to glycolysis in bronchial epithelium was downregulated and both BALF and blood eosinophil counts were higher in responders.Conclusion: Local differences in gene sets pertaining to epithelial inflammatory status were identified between treated and untreated airways after treatment, not resulting in changes in differential cell counts and cytokine analyses in BALF. Secondly, baseline epithelial glycolysis genes and eosinophil counts in BALF and blood were different between responders and non-responders. The observations from this study demonstrate the potential impact of BT on epithelial gene expression related to airway inflammation while also identifying a possible responder profile.Keywords: airway inflammation, bronchial thermoplasty, bronchoalveolar lavage fluid cells, cytokines, epithelial transcriptome

COVID-19-related acute respiratory distress syndrome (ARDS) can lead to long-term pulmonary fibrotic lesions. Alveolar fibroproliferative response (FPR) is a key factor in the development of pulmonary fibrosis. N-terminal peptide of procollagen III (NT-PCP-III) is a validated biomarker for activated FPR in ARDS. This study aimed to assess the association between dynamic changes in alveolar FPR and long-term outcomes, as well as mortality in COVID-19 ARDS patients. We conducted a prospective cohort study of 154 COVID-19 ARDS patients. We collected bronchoalveolar lavage (BAL) and blood samples for measurement of 17 pulmonary fibrosis biomarkers, including NT-PCP-III. We assessed pulmonary function and chest computed tomography (CT) at 3 and 12 mo after hospital discharge. We performed joint modeling to assess the association between longitudinal changes in biomarker levels and mortality at day 90 after starting mechanical ventilation. 154 patients with 284 BAL samples were analyzed. Of all patients, 40% survived to day 90, of whom 54 completed the follow-up procedure. A longitudinal increase in NT-PCP-III was associated with increased mortality (HR 2.89, 95% CI: 2.55-3.28; P < 0.001). Forced vital capacity and diffusion for carbon monoxide were impaired at 3 mo but improved significantly at one year after hospital discharge ( P = 0.03 and P = 0.004, respectively). There was no strong evidence linking alveolar FPR during hospitalization and signs of pulmonary fibrosis in pulmonary function or chest CT images during 1-yr follow-up. In COVID-19 ARDS patients, alveolar FPR during hospitalization was associated with higher mortality but not with the presence of long-term fibrotic lung sequelae within survivors. NEW & NOTEWORTHY This is the first prospective study on the longitudinal alveolar fibroproliferative response in COVID-19 ARDS and its relationship with mortality and long-term follow-up. We used the largest cohort of COVID-19 ARDS patients who had consecutive bronchoalveolar lavages and measured 17 pulmonary fibroproliferative biomarkers. We found that a higher fibroproliferative response during admission was associated with increased mortality, but not correlated with long-term fibrotic lung sequelae in survivors.

Background: Transfusion-related acute lung injury (TRALI) is a severe complication of blood transfusion that is thought of as a two-hit event: first the underlying patient condition (e.g., sepsis), and then the transfusion. Transfusion factors include human leukocyte antigen antibodies or biologic response modifiers (BRMs) accumulating during storage. Preclinical studies show an increased TRALI risk with longer stored platelets, clinical studies are conflicting. We aim to discover whether longer platelet concentrate (PC) storage time increases TRALI risk in a controlled human experiment., Study Design and Methods: In a randomized controlled trial, 18 healthy male volunteers received a first hit of experimental endotoxemia (2 ng/kg lipopolysaccharide), and a second hit of fresh (2-day old) or aged (7-day old) autologous PC, or physiological saline. After 6 h, changes in TRALI pathways were determined using spirometry, chest X-ray, and bronchoalveolar lavage (BAL)., Results: All subjects reacted adequately to lipopolysaccharide infusion and satisfied SIRS criteria (increased pulse [>90/min] and temperature [>38°C]). There were no differences between the saline, fresh, and aged PC groups in BAL-fluid protein (95 ± 33 μg/ml; 83 ± 21 μg/ml and 104 ± 29 μg/ml, respectively) and relative neutrophil count (1.5 ± 0.5%; 1.9 ± 0.8% and 1.3 ± 0.8%, respectively), nor in inflammatory BAL-fluid BRMs (Interleukin-6, CXCL8, TNFα , and myeloperoxidase), clinical respiratory parameters, and spirometry results. All chest X-rays were normal., Conclusions: In a human endotoxemia model of autologous platelet transfusion, with an adequate first hit and platelet storage lesion, transfusion of 7-day-old PC does not increase pulmonary inflammation compared with 2-day-old PC., (© 2022 The Authors. Transfusion published by Wiley Periodicals LLC on behalf of AABB.)

Background: Defective translocation of the translational repressor TIAR (T-cell internal antigen receptor) in bronchial epithelial cells (BECs) from asthma patients underlies epithelial hyperresponsiveness, reflected by an exaggerated production of a select panel of inflammatory cytokines such as CXCL-8, interleukin (IL)-6, granulocyte colony-stimulating factor, CXCL-10, upon exposure to tumour necrosis factor (TNF) and IL-17A. With this study we aimed to clarify whether epithelial hyperresponsiveness is a consistent finding, is changed upon in vivo exposure to rhinovirus (RV)-A16 and applies to the bronchoconstrictor endothelin-1., Methods: BECs were obtained from asthma patients (n=18) and healthy individuals (n=11), 1 day before and 6 days post-RV-A16 exposure. BECs were cultured and stimulated with TNF and IL-17A and inflammatory mediators were analysed. The bronchoalveolar lavage fluid (BALF) was obtained in parallel with BECs to correlate differential cell counts and inflammatory mediators with epithelial hyperresponsiveness., Results: Epithelial hyperresponsiveness was confirmed in sequential samples and even increased in BECs from asthma patients after RV-A16 exposure, but not in BECs from healthy individuals. Endothelin-1 tended to increase in BECs from asthma patients collected after RV-A16 exposure, but not in BECs from healthy individuals. In vitro CXCL-8 and endothelin-1 production correlated. In vivo relevance for in vitro CXCL-8 and endothelin-1 production was shown by correlations with forced expiratory volume in 1 s % predicted and CXCL-8 BALF levels., Conclusion: Epithelial hyperresponsiveness is an intrinsic defect in BECs from asthma patients, which increases upon viral exposure, but not in BECs from healthy individuals. This epithelial hyperresponsiveness also applies to the bronchoconstrictor endothelin-1, which could be involved in airway obstruction., Competing Interests: Conflict of interest: A. Ravi has nothing to disclose. Conflict of interest: S. Chowdhury has nothing to disclose. Conflict of interest: A. Dijkhuis has nothing to disclose. Conflict of interest: B.S. Dierdorp has nothing to disclose. Conflict of interest: T. Dekker has nothing to disclose. Conflict of interest: R. Kruize has nothing to disclose. Conflict of interest: Y.S. Sabogal Piñeros has nothing to disclose. Conflict of interest: C.J. Majoor has nothing to disclose. Conflict of interest: P.J. Sterk has nothing to disclose. Conflict of interest: R. Lutter reports support for the present manuscript from the Netherlands Asthma Foundation (now Netherlands Lung Foundation; grant numbers 3-2-07-012 and 3.2.10.069) and GlaxoSmithKline (CRT 114696)., (Copyright ©The authors 2022.)

Background: Asthma is a heterogeneous disease with differences in onset, severity, and inflammation. Bronchial epithelial cells (BECs) contribute to asthma pathophysiology., Objective: We determined whether transcriptomes of BECs reflect heterogeneity in inflammation and severity in asthma, and whether this was affected in BECs from patients with severe asthma after their regeneration by bronchial thermoplasty., Methods: RNA sequencing was performed on BECs obtained by bronchoscopy from healthy controls (n = 16), patients with mild asthma (n = 17), patients with moderate asthma (n = 5), and patients with severe asthma (n = 17), as well as on BECs from treated and untreated airways of the latter (also 6 months after bronchial thermoplasty) (n = 23). Lipidome and metabolome analyses were performed on cultured BECs from healthy controls (n = 7); patients with severe asthma (n = 9); and, for comparison, patients with chronic obstructive pulmonary disease (n = 7)., Results: Transcriptome analysis of BECs from patients showed a reduced expression of oxidative phosphorylation (OXPHOS) genes, most profoundly in patients with severe asthma but less profoundly and more heterogeneously in patients with mild asthma. Genes related to fatty acid metabolism were significantly upregulated in asthma. Lipidomics revealed enhanced levels of lipid species (phosphatidylcholines, lysophosphatidylcholines. and bis(monoacylglycerol)phosphate), whereas levels of OXPHOS metabolites were reduced in BECs from patients with severe asthma. BECs from patients with mild asthma characterized by hyperresponsive production of mediators implicated in neutrophilic inflammation had decreased expression of OXPHOS genes compared with that in BECs from patients with mild asthma with normoresponsive production. BECs obtained after thermoplasty had significantly increased expression of OXPHOS genes and decreased expression of fatty acid metabolism genes compared with BECs obtained from untreated airways., Conclusion: BECs in patients with asthma are metabolically different from those in healthy individuals. These differences are linked with inflammation and asthma severity, and they can be reversed by bronchial thermoplasty., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Asthma patients suffer from periodic acute worsening of symptoms (i.e. loss of asthma control or exacerbations), triggered by a variety of exogenous stimuli. With the growing awareness that air pollutants impact respiratory diseases, we investigated whether particulate matter (PM) derived from various livestock farms (BioPM) differentially affected innate and oxidative stress responses in asthma and health., Methods: Peripheral blood mononuclear cells (PBMCs), collected from patients sequentially before and during loss of asthma control and from healthy individuals, were exposed to BioPM collected from chicken, goat and pig farms (1 and 5 μg/ml), with or without pre-treatment with antioxidants. Cytokine release and oxidative stress were assessed., Results: PBMCs produced IFNγ, IL-1β, IL-10 and TNFα upon stimulation with BioPM, with that from pig farms inducing the highest cytokine levels. Overall, cytokine production was irrespective of the presence or state of disease. However, PBMCs from stable asthma patients upon exposure to the three BioPM showed more extreme TNFα responses than those from healthy subjects. Furthermore, PBMCs obtained during loss of asthma control that were exposed to BioPM from pig farms showed enhanced IFNγ release as well as decreased oxidative stress levels upon pre-treatment with N-acetylcysteine (NAC) compared to stable disease. NAC, but not superoxide dismutase and catalase, also counteracted BioPM-induced cytokine release, indicating the importance of intracellular reactive oxygen species in the production of cytokines., Conclusions: BioPM triggered enhanced pro-inflammatory responses by PBMCs from both healthy subjects and asthma patients, with those from patients during loss of asthma control showing increased susceptibility to BioPM from pig farms in particular.

Background: Loss of asthma control and asthma exacerbations are associated with increased sputum eosinophil counts. However, whether eosinophils, or the also present neutrophils, actively contribute to the accompanying inflammation has not been extensively investigated., Methods: Twenty-three patients with mild to moderate asthma were included in a standardized prospective inhaled corticosteroid (ICS) withdrawal study; 22 of the patients experienced loss of asthma control. The study assessed various immune, inflammatory, and oxidative stress parameters, as well as markers of eosinophil and neutrophil activity, in exhaled breath condensate, plasma, and sputum collected at three phases (baseline, during loss of control, and following recovery)., Results: Loss of asthma control was characterized by increased sputum eosinophils, whereas no differences were detected between the three phases for most inflammatory and oxidative stress responses. There were also no differences detected for markers of activated eosinophils (eosinophil cationic protein and bromotyrosine) and neutrophils (myeloperoxidase and chlorotyrosine). However, free eosinophilic granules and citrullinated histone H 3 , suggestive of eosinophil cytolysis and potentially eosinophil extracellular trap formation, were enhanced. Baseline blood eosinophils and changes in asymmetric dimethylarginine (an inhibitor of nitric oxide synthase) in plasma were found to correlate with the decrease in FEV 1 percent predicted upon ICS withdrawal (both, r s  = 0.46; P = .03)., Conclusions: The clinical effect in mild to moderate asthma upon interruption of ICS therapy is not related to the classic inflammatory activation of eosinophils and neutrophils. It may, however, reflect another pathway underlying the onset of loss of disease control and asthma exacerbations., Trial Registry: The Netherlands Trial Register; No.: NTR3316; URL: trialregister.nl/trial/3172., (Copyright © 2019 American College of Chest Physicians. Published by Elsevier Inc. All rights reserved.)

Background: Activated eosinophils cause major pathology in stable and exacerbating asthma; however, they can also display protective properties like an extracellular antiviral activity. Initial murine studies led us to further explore a potential intracellular antiviral activity by eosinophils., Methods: To follow eosinophil-virus interaction, respiratory syncytial virus (RSV) and influenza virus were labeled with a fluorescent lipophilic dye (DiD). Interactions with eosinophils were visualized by confocal microscopy, electron microscopy, and flow cytometry. Eosinophil activation was assessed by both flow cytometry and ELISA. In a separate study, eosinophils were depleted in asthma patients using anti-IL-5 (mepolizumab), followed by a challenge with rhinovirus-16 (RV16)., Results: DiD-RSV and DiD-influenza rapidly adhered to human eosinophils and were internalized and inactivated (95% in ≤ 2 hours) as reflected by a reduced replication in epithelial cells. The capacity of eosinophils to capture virus was reduced up to 75% with increasing severity of asthma. Eosinophils were activated by virus in vitro and in vivo. In vivo this correlated with virus-induced loss of asthma control., Conclusions: This previously unrecognized and in asthma attenuated antiviral property provides a new perspective to eosinophils in asthma. This is indicative of an imbalance between protective and cytotoxic properties by eosinophils that may underlie asthma exacerbations., (© 2019 The Authors. Allergy Published by John Wiley & Sons Ltd.)

Background: Influenza virus triggers severe asthma exacerbations for which no adequate treatment is available. It is known that IL-33 levels correlate with exacerbation severity, but its role in the immunopathogenesis of exacerbations has remained elusive., Objective: We hypothesized that IL-33 is necessary to drive asthma exacerbations. We intervened with the IL-33 cascade and sought to dissect its role, also in synergy with thymic stromal lymphopoietin (TSLP), in airway inflammation, antiviral activity, and lung function. We aimed to unveil the major source of IL-33 in the airways and IL-33-dependent mechanisms that underlie severe asthma exacerbations., Methods: Patients with mild asthma were experimentally infected with rhinovirus. Mice were chronically exposed to house dust mite extract and then infected with influenza to resemble key features of exacerbations in human subjects. Interventions included the anti-IL-33 receptor ST2, anti-TSLP, or both., Results: We identified bronchial ciliated cells and type II alveolar cells as a major local source of IL-33 during virus-driven exacerbation in human subjects and mice, respectively. By blocking ST2, we demonstrated that IL-33 and not TSLP was necessary to drive exacerbations. IL-33 enhanced airway hyperresponsiveness and airway inflammation by suppressing innate and adaptive antiviral responses and by instructing epithelial cells and dendritic cells of house dust mite-sensitized mice to dampen IFN-β expression and prevent the T H 1-promoting dendritic cell phenotype. IL-33 also boosted luminal NETosis and halted cytolytic antiviral activities but did not affect the T H 2 response., Conclusion: Interventions targeting the IL-33/ST2 axis could prove an effective acute short-term therapy for virus-induced asthma exacerbations., (Copyright © 2018 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

Rationale: Eosinophils drive pathophysiology in stable and exacerbating eosinophilic asthma, and therefore treatment is focused on the reduction of eosinophil numbers. Mepolizumab, a humanized monoclonal antibody that neutralizes IL-5 and efficiently attenuates eosinophils, proved clinically effective in severe eosinophilic asthma but not in mild asthma., Objectives: To study the effect of mepolizumab on virus-induced immune responses in mild asthma., Methods: Patients with mild asthma, steroid-naive and randomized for eosinophil numbers, received 750 mg mepolizumab intravenously in a placebo-controlled double-blind trial, 2 weeks after which patients were challenged with rhinovirus (RV) 16. FEV 1 , FVC, fractional exhaled nitric oxide, symptom scores (asthma control score), viral load (PCR), eosinophil numbers, humoral (luminex, ELISA), and cellular (flow cytometry) immune parameters in blood, BAL fluid, and sputum, before and after mepolizumab and RV16, were assessed., Measurements and Main Results: Mepolizumab attenuated baseline blood eosinophils and their activation, attenuated trendwise sputum eosinophils, and enhanced circulating natural killer cells. Mepolizumab did not affect FEV 1 , FVC, and fractional exhaled nitric oxide, neither at baseline nor after RV16. On RV16 challenge mepolizumab did not prevent eosinophil activation but did enhance local B lymphocytes and macrophages and reduce neutrophils and their activation. Mepolizumab also enhanced secretory IgA and reduced tryptase in BAL fluid. Finally, mepolizumab affected particularly RV16-induced macrophage inflammatory protein-3a, vascular endothelial growth factor-A, and IL-1RA production in BAL fluid., Conclusions: Mepolizumab failed to prevent activation of remaining eosinophils and changed RV16-induced immune responses in mild asthma. Although these latter effects likely are caused by attenuated eosinophil numbers, we cannot exclude a role for basophils. Clinical trial registered with www.clinicaltrials.gov (NCT 01520051).

Background: Acute worsening of asthma symptoms (exacerbation) is predominantly triggered by respiratory viruses, with influenza causing the most severe exacerbations. The lack of an adequate animal model hampers mechanistic insight and the development of new therapeutics., Aim: We developed and characterized a robust, consistent, and reproducible mouse model of severe exacerbation of chronic allergic asthma., Methods: Chronic allergic airway inflammation was induced following a house dust mite (HDM) sensitization protocol. HDM-sensitized mice and controls were infected with influenza virus A/X31 H3N2 and either or not treated with inhaled fluticasone propionate (FP), systemic corticosteroids (Pred), or anti-IL-5. Mice were killed at different time points after infection: Cellular accumulation and cytokines levels in the airways, PenH as a measure of airway hyper-responsiveness (AHR), and lung histology and viral replication were assessed., Results: Infection with low-dose A/X31 H3N2 led to prolonged deterioration of lung function, aggravated mucus production, peri-vascular, peri-bronchial, and allergic inflammation that was unresponsive to inhaled corticosteroids, but responsive to systemic corticosteroids. The exacerbation was preceded at 14 h after virus exposure by a marked innate, but no Th2 and Th1 response subsequently followed by enhanced numbers of eosinophils, neutrophils, dendritic, and T cells into the lung lumen, parenchyma, and draining lymph nodes in HDM-sensitized mice. Anti-IL-5 treatment attenuated eosinophils and prevented the X31-induced exacerbation., Conclusions: Together, these findings indicate that an early innate response that involves eosinophils underlies the exacerbation. This model recapitulates all major features of severe asthma exacerbations and can serve to discern driving mechanisms and promote the development of novel therapeutics., (© 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Objective: Angiotensin-converting enzyme and its effector peptide angiotensin II have been implicated in the pathogenesis of acute respiratory distress syndrome. Recently, angiotensin-converting enzyme 2 was identified as the counter-regulatory enzyme of angiotensin-converting enzyme that converts angiotensin II into angiotensin-(1-7). The aim of this study was to determine pulmonary angiotensin-converting enzyme and angiotensin-converting enzyme 2 activity in patients with acute respiratory distress syndrome., Design: Prospective observational pilot study., Setting: A PICU of a university hospital., Patients: Fourteen patients admitted, requiring mechanical ventilation for respiratory syncytial virus lower respiratory tract infection., Interventions: None., Measurements and Main Results: Two groups of patients were distinguished at admission: a group fulfilling the criteria for acute respiratory distress syndrome and a non-acute respiratory distress syndrome group. Angiotensin-converting enzyme and angiotensin-converting enzyme 2 activity were measured in bronchoalveolar lavage fluid. Patients with acute respiratory distress syndrome had increased angiotensin-converting enzyme activity and decreased angiotensin-converting enzyme 2 activity (p < 0.001) compared with the control group., Conclusion: It is shown for the first time that in acute respiratory distress syndrome, enhanced angiotensin-converting enzyme activity is paralleled by a reduced angiotensin-converting enzyme 2 activity, similar to that found in an experimental rat model of acute respiratory distress syndrome. The reduced angiotensin-converting enzyme 2 activity may be counteracted by restoring angiotensin-(1-7) level, thereby offering a novel treatment modality for this syndrome.

The human lung T cell compartment contains many CD8⁺ T cells specific for respiratory viruses, suggesting that the lung is protected from recurring respiratory infections by a resident T cell pool. The entry site for respiratory viruses is the epithelium, in which a subset of lung CD8⁺ T cells expressing CD103 (αE integrin) resides. Here, we determined the specificity and function of CD103⁺CD8⁺ T cells in protecting human lung against viral infection. Mononuclear cells were isolated from human blood and lung resection samples. Variable numbers of CD103⁺CD8⁺ T cells were retrieved from the lung tissue. Interestingly, expression of CD103 was seen only in lung CD8⁺ T cells specific for influenza but not in those specific for EBV or CMV. CD103⁺ and influenza-reactive cells preferentially expressed NKG2A, an inhibitor of CD8⁺ T cell cytotoxic function. In contrast to CD103⁻CD8⁺ T cells, most CD103⁺CD8⁺ cells did not contain perforin or granzyme B. However, they could quickly upregulate these cytotoxic mediators when exposed to a type I IFN milieu or via contact with their specific antigen. This mechanism may provide a rapid and efficient response to influenza infection, without inducing cytotoxic damage to the delicate epithelial barrier.

Background: Eosinophilic airway inflammation has successfully been used to tailor anti-inflammatory therapy in chronic obstructive pulmonary disease (COPD). Airway hyperresponsiveness (AHR) by indirect challenges is associated with airway inflammation. We hypothesized that AHR to inhaled mannitol captures eosinophilia in induced sputum in COPD., Methods: Twenty-eight patients (age 58 ± 7.8 yr, packyears 40 ± 15.5, post-bronchodilator FEV1 77 ± 14.0%predicted, no inhaled steroids ≥4 wks) with mild-moderate COPD (GOLD I-II) completed two randomized visits with hypertonic saline-induced sputum and mannitol challenge (including sputum collection). AHR to mannitol was expressed as response-dose-ratio (RDR) and related to cell counts, ECP, MPO and IL-8 levels in sputum., Results: There was a positive correlation between RDR to mannitol and eosinophil numbers (r = 0.47, p = 0.03) and level of IL-8 (r = 0.46, p = 0.04) in hypertonic saline-induced sputum. Furthermore, significant correlations were found between RDR and eosinophil numbers (r = 0.71, p = 0.001), level of ECP (r = 0.72, p = 0.001), IL-8 (r = 0.57, p = 0.015) and MPO (r = 0.64, p = 0.007) in sputum collected after mannitol challenge. ROC-curves showed 60% sensitivity and 100% specificity of RDR for >2.5% eosinophils in mannitol-induced sputum., Conclusions: In mild-moderate COPD mannitol hyperresponsiveness is associated with biomarkers of airway inflammation. The high specificity of mannitol challenge suggests that the test is particularly suitable to exclude eosinophilic airways inflammation, which may facilitate individualized treatment in COPD., Trial Registration: Netherlands Trial Register (NTR): NTR1283.