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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

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 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.

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).

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.