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Background: Interleukin (IL)-33 is an upstream regulator of type 2 (T2) eosinophilic inflammation and has been proposed as a key driver of some asthma phenotypes., Objective: To derive gene signatures from in vitro studies of IL-33-stimulated cells and use these to determine IL-33-associated enrichment patterns in asthma., Methods: Signatures downstream of IL-33 stimulation were derived from our in vitro study of human mast cells and from public datasets of in vitro stimulated human basophils, type 2 innate lymphoid cells (ILC2), regulatory T cells (Treg) and endothelial cells. Gene Set Variation Analysis (GSVA) was used to probe U-BIOPRED and ADEPT sputum transcriptomics to determine enrichment scores (ES) for each signature according to asthma severity, sputum granulocyte status and previously defined molecular phenotypes., Results: IL-33-activated gene signatures were cell-specific with little gene overlap. Individual signatures, however, were associated with similar signalling pathways (TNF, NF-κB, IL-17 and JAK/STAT signalling) and immune cell differentiation pathways (Th17, Th1 and Th2 differentiation). ES for IL-33-activated gene signatures were significantly enriched in asthmatic sputum, particularly in patients with neutrophilic and mixed granulocytic phenotypes. IL-33 mRNA expression was not elevated in asthma whereas the expression of mRNA for IL1RL1, the IL-33 receptor, was up-regulated in the sputum of severe eosinophilic asthma. The mRNA expression for IL1RAP, the IL1RL1 co-receptor, was greatest in severe neutrophilic and mixed granulocytic asthma., Conclusions: IL-33-activated gene signatures are elevated in neutrophilic and mixed granulocytic asthma corresponding with IL1RAP co-receptor expression. This suggests incorporating T2-low asthma in anti-IL-33 trials., (© 2022 The Authors. Allergy published by European Academy of Allergy and Clinical Immunology and John Wiley & Sons Ltd.)

Inhaled glucocorticoids (GCs) are drugs widely used as treatment for asthma patients. They prevent the recruitment and activation of lung immune and inflammatory cells and, moreover, have profound effects on airway structural cells to reverse the effects of disease on airway inflammation. GCs bind to a specific receptor, the glucocorticoid receptor (GR), which is a member of the nuclear receptor superfamily and modulates pro- and anti-inflammatory gene transcription through a number of distinct and complementary mechanisms. Targets genes include many pro-inflammatory mediators such as chemokines, cytokines, growth factors and their receptors. Inhaled GCs are very effective for most asthma patients with little, if any, systemic side effects depending upon the dose. However, some patients show poor asthma control even after the administration of high doses of topical or even systemic GCs. Several mechanisms relating to inflammation have been considered to be responsible for the onset of the relative GC resistance observed in these patients. In these patients, the side-effect profile of GCs prevent continued use of high doses and new drugs are needed. Targeting the defective pathways associated with GC function in these patients may also reactivate GC responsiveness., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The lungs are exposed to reactive oxygen species oxygen (ROS) produced as a result of inhalation of oxygen, as well as smoke and other air pollutants. Cell metabolism and the NADPH oxidases (Nox) generate low levels of intracellular ROS that act as signal transduction mediators by inducing oxidative modifications of histones, enzymes and transcription factors. Redox signalling is also regulated by localised production and sensing of ROS in mitochondria, the endoplasmic reticulum (ER) and inside the nucleus. Intracellular ROS are maintained at low levels through the action of a battery of enzymatic and non-enzymatic antioxidants. Asthma is a heterogeneous airway inflammatory disease with different immune endotypes; these include atopic or non-atopic Th2 type immune response associated with eosinophilia, or a non-Th2 response associated with neutrophilia. Airway remodelling and hyperresponsiveness accompany the inflammatory response in asthma. Over-production of ROS resulting from infiltrating immune cells, particularly eosinophils and neutrophils, and a concomitant impairment of antioxidant responses lead to development of oxidative stress in asthma. Oxidative stress is augmented in severe asthma and during exacerbations, as well as by air pollution and obesity, and causes oxidative damage of tissues promoting airway inflammation and hyperresponsiveness. Furthermore, deregulated Nox activity, mitochondrial dysfunction, ER stress and/or oxidative DNA damage, resulting from exposure to irritants, inflammatory mediators or obesity, may lead to redox-dependent changes in cell signalling. ROS play a central role in airway epithelium-mediated sensing, development of innate and adaptive immune responses, and airway remodelling and hyperresponsiveness. Nonetheless, antioxidant compounds have proven clinically ineffective as therapeutic agents for asthma, partly due to issues with stability and in vivo metabolism of these compounds. The compartmentalised nature of ROS production and sensing, and the role of ROS in homeostatic responses and in the action of corticosteroids and β2-adrenergic receptor agonists, adds another layer of complexity to antioxidant therapy development. Nox inhibitors and mitochondrial-targeted antioxidants are in clinical development for a number of diseases but they have not yet been investigated in asthma. A better understanding of the complex role of ROS in the pathogenesis of asthma will highlight new opportunities for more targeted and effective redox therapies., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Rationale: Mast cells (MCs) play a role in inflammation and both innate and adaptive immunity, but their involvement in severe asthma (SA) remains undefined. Objectives: We investigated the phenotypic characteristics of the U-BIOPRED (Unbiased Biomarkers for the Prediction of Respiratory Diseases Outcomes) asthma cohort by applying published MC activation signatures to the sputum cell transcriptome. Methods: Eighty-four participants with SA, 20 with mild/moderate asthma (MMA), and 16 healthy participants without asthma were studied. We calculated enrichment scores (ESs) for nine MC activation signatures by asthma severity, sputum granulocyte status, and three previously defined sputum molecular phenotypes or transcriptome-associated clusters (TACs) 1, 2, and 3 using gene set variation analysis. Measurements and Main Results: MC signatures except unstimulated, repeated FcεR1-stimulated and IFN-γ-stimulated signatures were enriched in SA. A FcεR1-IgE-stimulated and a single-cell signature from asthmatic bronchial biopsies were highly enriched in eosinophilic asthma and in the TAC1 molecular phenotype. Subjects with a high ES for these signatures had elevated sputum amounts of similar genes and pathways. IL-33- and LPS-stimulated MC signatures had greater ES in neutrophilic and mixed granulocytic asthma and in the TAC2 molecular phenotype. These subjects exhibited neutrophil, NF-κB (nuclear factor-κB), and IL-1β/TNF-α (tumor necrosis factor-α) pathway activation. The IFN-γ-stimulated signature had the greatest ES in TAC2 and TAC3 that was associated with responses to viral infection. Similar results were obtained in an independent ADEPT (Airway Disease Endotyping for Personalized Therapeutics) asthma cohort. Conclusions: Gene signatures of MC activation allow the detection of SA phenotypes and indicate that MCs can be induced to take on distinct transcriptional phenotypes associated with specific clinical phenotypes. IL-33-stimulated MC signature was associated with severe neutrophilic asthma, whereas IgE-activated MC was associated with an eosinophilic phenotype.

Accumulating evidence highlights links between iron regulation and respiratory disease. Here, we assessed the relationship between iron levels and regulatory responses in clinical and experimental asthma.We show that cell-free iron levels are reduced in the bronchoalveolar lavage (BAL) supernatant of severe or mild-moderate asthma patients and correlate with lower forced expiratory volume in 1 s (FEV 1 ). Conversely, iron-loaded cell numbers were increased in BAL in these patients and with lower FEV 1 /forced vital capacity (FVC) ratio. The airway tissue expression of the iron sequestration molecules divalent metal transporter 1 ( DMT1 ) and transferrin receptor 1 ( TFR1 ) are increased in asthma, with TFR1 expression correlating with reduced lung function and increased Type-2 (T2) inflammatory responses in the airways. Furthermore, pulmonary iron levels are increased in a house dust mite (HDM)-induced model of experimental asthma in association with augmented Tfr1 expression in airway tissue, similar to human disease. We show that macrophages are the predominant source of increased Tfr1 and Tfr1 + macrophages have increased Il13 expression. We also show that increased iron levels induce increased pro-inflammatory cytokine and/or extracellular matrix (ECM) responses in human airway smooth muscle (ASM) cells and fibroblasts ex vivo and induce key features of asthma in vivo , including airway hyper-responsiveness (AHR) and fibrosis, and T2 inflammatory responses.Together these complementary clinical and experimental data highlight the importance of altered pulmonary iron levels and regulation in asthma, and the need for a greater focus on the role and potential therapeutic targeting of iron in the pathogenesis and severity of disease., Competing Interests: Conflict of interest: M.K. Ali has nothing to disclose. Conflict of interest: R.Y. Kim has nothing to disclose. Conflict of interest: A.C. Brown has nothing to disclose. Conflict of interest: J.R. Mayall has nothing to disclose. Conflict of interest: R. Karim has nothing to disclose. Conflict of interest: J.W. Pinkerton has nothing to disclose. Conflict of interest: G. Liu has nothing to disclose. Conflict of interest: K.L. Martin has nothing to disclose. Conflict of interest: M.R. Starkey has nothing to disclose. Conflict of interest: A.L. Pillar has nothing to disclose. Conflict of interest: C. Donovan has nothing to disclose. Conflict of interest: P.S. Pathinayake has nothing to disclose. Conflict of interest: O.R. Carroll has nothing to disclose. Conflict of interest: D. Trinder has nothing to disclose. Conflict of interest: H.L. Tay has nothing to disclose. Conflict of interest: Y.E. Badi has nothing to disclose. Conflict of interest: N.Z. Kermani has nothing to disclose. Conflict of interest: Y-K. Guo has nothing to disclose. Conflict of interest: R. Aryal has nothing to disclose. Conflict of interest: S. Mumby has nothing to disclose. Conflict of interest: S. Pavlidis has nothing to disclose. Conflict of interest: I.M. Adcock reports grants from EU-IMI, during the conduct of the study. Conflict of interest: J. Weaver has nothing to disclose. Conflict of interest: D. Xenaki has nothing to disclose. Conflict of interest: B.G. Oliver has nothing to disclose. Conflict of interest: E.G. Holliday has nothing to disclose. Conflict of interest: P.S. Foster has nothing to disclose. Conflict of interest: P.A. Wark has nothing to disclose. Conflict of interest: D.M. Johnstone has nothing to disclose. Conflict of interest: E.A. Milward has nothing to disclose. Conflict of interest: P.M. Hansbro has nothing to disclose. Conflict of interest: J.C. Horvat has nothing to disclose., (Copyright ©ERS 2020.)

Inflammation is a central feature of asthma and chronic obstructive pulmonary disease (COPD). Despite recent advances in the knowledge of the pathogenesis of asthma and COPD, much more research on the molecular mechanisms of asthma and COPD are needed to aid the logical development of new therapies for these common and important diseases, particularly in COPD where no new effective treatments currently exist. In the future the role of the activation/repression of different transcription factors and the genetic regulation of their expression in asthma and COPD may be an increasingly important aspect of research, as this may be one of the critical mechanisms regulating the expression of different clinical phenotypes and their responsiveness to therapy, particularly to anti-inflammatory drugs., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The most effective anti-inflammatory drugs used to treat patients with airways disease are topical glucocorticosteroids (GCs). These act on virtually all cells within the airway to suppress airway inflammation or prevent the recruitment of inflammatory cells into the airway. They also have profound effects on airway structural cells to reverse the effects of disease on their function. Glucorticosteroids act via specific receptors-the glucocorticosteroid receptor (GR)-which are a member of the nuclear receptor family. As such, many of the important actions of GCs are to modulate gene transcription through a number of distinct and complementary mechanisms. Targets genes include most inflammatory mediators such as chemokines, cytokines, growth factors and their receptors. GCs delivered by the inhaled route are very effective for most patients and have few systemic side effects. However, in some patients, even high doses of topical or even systemic GCs fail to control their disease. A number of mechanisms relating to inflammation have been reported to be responsible for the failure of these patients to respond correctly to GCs and these provide insight into GC actions within the airways. In these patients, the side-effect profile of GCs prevent continued use of high doses and new drugs are needed for these patients. Targeting the defective pathways associated with GC function in these patients may also reactivate GC responsiveness.

Aims: Pro-oxidant iron provides a potential measure of iron-catalysed oxidative stress in biological fluids. This study aimed, to investigate if the Bleomycin technique for measurement of pro-oxidant iron in biological fluids could be utilised for determinations in exhaled breath condensate (EBC). Secondly, to measure levels of pro-oxidant iron in EBC from asthmatics after exposure to polluting city environments., Methods: Retrospective analysis of samples of EBC and bronchoalveolar lavage fluid (BALF). Pro-oxidant iron levels were determined by the Bleomycin method. Transferrin levels were determined by radial diffusion immunoassay and lactoferrin by ELISA., Subjects: Patients undergoing surgery necessitating cardiopulmonary bypass, normal healthy controls, "healthy" smokers, and asthmatics (mild and moderate)., Results: Pro-oxidant iron was significantly decreased (p<0.05) post cardiac surgery in both EBC and BALF. In smokers levels of pro-oxidant iron in EBC were significantly (p<0.05) increased verses healthy controls. In asthmatics with more severe disease, there were significant increases in EBC pro-oxidant iron content post exposure to city environments (p<0.001), with levels most elevated after exposure to the most polluted setting., Conclusion: Similar patterns in the levels of pro-oxidant iron detectable in EBC and paired BALF from patients undergoing cardiopulmonary bypass (pre and post surgery) suggest a potential for EBC determinations. Significantly elevated levels in EBC from smokers relative to control subjects provide further support for this technique. In asthma disease severity and environmental exposure influenced levels of pro-oxidant iron measured in EBC indicating a potential for enhanced iron-catalysed oxidative stress., (Copyright © 2011 Elsevier Ltd. All rights reserved.)