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The leukotriene receptors (nomenclature as agreed by the NC-IUPHAR subcommittee on Leukotriene Receptors [35, 38]) are activated by the endogenous ligands leukotrienes (LT), synthesized from lipoxygenase metabolism of arachidonic acid. The human BLT1 receptor is the high affinity LTB4 receptor whereas the BLT2 receptor in addition to being a low-affinity LTB4 receptor also binds several other lipoxygenase-products, such as 12S-HETE, 12S-HPETE, 15S-HETE, and the thromboxane synthase product 12-hydroxyheptadecatrienoic acid. The BLT receptors mediate chemotaxis and immunomodulation in several leukocyte populations and are in addition expressed on non-myeloid cells, such as vascular smooth muscle and endothelial cells. In addition to BLT receptors, LTB4 has been reported to bind to the peroxisome proliferator activated receptor (PPAR) α [201] and the vanilloid TRPV1 ligand-gated nonselective cation channel [223]. The crystal structure of the BLT1 receptor was initially determined in complex with selective antagonists [141, 231] and has recently been extended to the cryo-electron microscopy structure of LTB4-bound human BLT1 receptor at 2.91 Å resolution [389]. The receptors for the cysteinyl-leukotrienes (i.e. LTC4, LTD4 and LTE4) are termed CysLT1 and CysLT2 and exhibit distinct expression patterns in human tissues, mediating for example smooth muscle cell contraction, regulation of vascular permeability, and leukocyte activation. Quite recently, the the crystal structures of both receptors have been solved, the CysLT1 in complex with zafirlukast and pranlukast [203] and the CysLT2 in complex with three dual CysLT1/CysLT2 antagonists [122]. There is also evidence in the literature for additional CysLT receptor subtypes, derived from functional in vitro studies, radioligand binding and in mice lacking both CysLT1 and CysLT2 receptors [38]. Cysteinyl-leukotrienes have also been suggested to signal through the P2Y12 receptor [99, 251, 280], GPR17 [60] and GPR99 [173].

BackgroundEffectiveness studies with biological therapies for asthma lack standardised outcome measures. The COMSA (Core Outcome Measures sets for paediatric and adult Severe Asthma) Working Group sought to develop Core Outcome Measures (COM) sets to facilitate better synthesis of data and appraisal of biologics in paediatric and adult asthma clinical studies.MethodsCOMSA utilised a multi-stakeholder consensus process among patients with severe asthma, adult and paediatric clinicians, pharmaceutical representatives, and health regulators from across Europe. Evidence included a systematic review of development, validity and reliability of selected outcome measures plus a narrative review and a pan-European survey to better understand patients’ and carers’ views about outcome measures. It was discussed using a modified GRADE (Grading of Recommendations Assessment, Development and Evaluation) Evidence to Decision framework. Anonymous voting was conducted using predefined consensus criteria.ResultsBoth adult and paediatric COM sets include forced expiratory volume in 1 s (FEV1) as z-scores, annual frequency of severe exacerbations and maintenance oral corticosteroid use. Additionally, the paediatric COM set includes the Paediatric Asthma Quality of Life Questionnaire and Asthma Control Test or Childhood Asthma Control Test, while the adult COM set includes the Severe Asthma Questionnaire and Asthma Control Questionnaire-6 (symptoms and rescue medication use reported separately).ConclusionsThis patient-centred collaboration has produced two COM sets for paediatric and adult severe asthma. It is expected that they will inform the methodology of future clinical trials, enhance comparability of efficacy and effectiveness of biological therapies, and help assess their socioeconomic value. COMSA will inform definitions of non-response and response to biological therapy for severe asthma.

Background: SUCNR1 is a sensor of extracellular succinate, a Krebs cycle intermediate generated in excess during oxidative stress and has been linked to metabolic regulation and inflammation. While mast cells express SUCNR1, its role in mast cell reactivity and allergic conditions such as asthma remains to be elucidated. Methods: Cord blood-derived mast cells and human mast cell line LAD-2 challenged by SUCNR1 ligands were analyzed for the activation and mediator release. Effects on mast cell-dependent bronchoconstriction were assessed in guinea pig trachea and isolated human small bronchi challenged with antigen and anti-IgE, respectively. Results: SUCNR1 is abundantly expressed on human mast cells. Challenge with succinate, or the synthetic non-metabolite agonist cis-epoxysuccinate, renders mast cells hypersensitive to IgE-dependent activation, resulting in augmented degranulation and histamine release, de novo biosynthesis of eicosanoids and cytokine secretion. The succinate-potentiated mast cell reactivity was attenuated by SUCNR1 knockdown and selective SUCNR1 antagonists and could be tuned by pharmacologically targeting protein kinase C and extracellular signal-regulated kinase. Both succinate and cis-epoxysuccinate dose-dependently potentiated antigen-induced contraction in a mast cell-dependent guinea pig airway model, associated with increased generation of cysteinyl-leukotrienes and histamine in trachea. Similarly, cis-epoxysuccinate aggravated IgE-receptor-induced contraction of human bronchi, which was blocked by SUCNR1 antagonism. Conclusion: SUCNR1 amplifies IgE-receptor-induced mast cell activation and allergic bronchoconstriction, suggesting a role for this pathway in aggravation of allergic asthma, thus linking metabolic perturbations to mast cell-dependent inflammation.

Background. Because of altered airway microbiome in asthma, we analysed the bacterial species in sputum of patients with severe asthma. Methods. Whole genome sequencing was performed on induced sputum from non-smoking (SAn) and current or ex-smoker (SAs/ex) severe asthma patients, mild/moderate asthma (MMA) and healthy controls (HC). Data was analysed by asthma severity, inflammatory status and transcriptome-associated clusters (TACs). Results. α-diversity at the species level was lower in SAn and SAs/ex, with an increase in Haemophilus influenzae and Moraxella catarrhalis, and Haemophilus influenzae and Tropheryma whipplei, respectively, compared to HC. In neutrophilic asthma, there was greater abundance of Haemophilus influenzae and Moraxella catarrhalis and in eosinophilic asthma, Tropheryma whipplei was increased. There was a reduction in α-diversity in TAC1 and TAC2 that expressed high levels of Haemophilus influenzae and Tropheryma whipplei, and Haemophilus influenzae and Moraxella catarrhalis, respectively, compared to HC. Sputum neutrophils correlated positively with Moraxella catarrhalis and negatively with Prevotella, Neisseria and Veillonella species and Haemophilus parainfluenzae. Sputum eosinophils correlated positively with Tropheryma whipplei which correlated with pack-years of smoking. α- and β-diversities were stable at one year. Conclusions.Haemophilus influenzae and Moraxella catarrhalis were more abundant in severe neutrophilic asthma and TAC2 linked to inflammasome and neutrophil activation, while Haemophilus influenzae and Tropheryma whipplei were highest in SAs/ex and in TAC1 associated with highest expression of IL-13 Type 2 and ILC2 signatures with the abundance of Tropheryma whipplei correlating positively with sputum eosinophils. Whether these bacterial species drive the inflammatory response in asthma needs evaluation.

Background Mastocytosis encompasses a heterogeneous group of disorders characterized by accumulation of clonal mast cells (MCs) in the skin and/or internal organs. Patients typically present with a broad variety of recurrent mediator-related clinical symptoms, including severe anaphylaxis. However, not all patients with mastocytosis experience anaphylactic reactions. Objective We sought to identify disease-specific biomarkers in plasma that could be used to predict patients with mastocytosis with increased risk of anaphylaxis. Methods Nineteen patients (≥18 years) and 2 control groups (11 subjects with allergic asthma and 13 healthy volunteers without history of atopy) were recruited. In total, 248 plasma proteins were analyzed by Proximity Extension Assay using Olink Proseek Multiplex panels. Results We identified 4 novel proteins, in addition to tryptase, E-selectin, adrenomedullin, T-cell immunoglobulin, and mucin domain 1, and CUB domain–containing protein 1/CD138 to be significantly increased in patients with mastocytosis compared with both patients with asthma and healthy controls. Furthermore, we investigated whether we could discriminate between patients with mastocytosis with or without anaphylaxis. In addition to tryptase, we identified 3 novel proteins, that is, allergin-1, pregnancy-associated plasma protein-A, and galectin-3, with significantly different levels in patients with mastocytosis with anaphylaxis compared with those without anaphylaxis. Conclusions Newly identified proteomic biomarkers may be used to predict patients with mastocytosis with increased risk of anaphylaxis.

Background: Although estimates of suboptimal adherence to oral corticosteroids in asthma range from 30% to 50%, no ideal method for measurement exists; the impact of poor adherence in severe asthma is likely to be particularly high. Research Questions: What is the prevalence of suboptimal adherence detected by self-reporting and direct measures? Is suboptimal adherence associated with disease activity? Study Design and Methods: Data were included from individuals with severe asthma taking part in the U-BIOPRED (Unbiased Biomarkers for the Prediction of Respiratory Disease Outcomes) study and prescribed daily oral corticosteroids. Participants completed the Medication Adherence Report Scale, a five-item questionnaire used to grade adherence on a scale from 1 to 5, and provided a urine sample for analysis of prednisolone and metabolites by liquid chromatography-mass spectrometry. Results: Data from 166 participants were included in this study: mean (SD) age, 54.2 (± 11.9) years; FEV 1, 65.1% (± 20.5%) predicted; female, 58%; 37% completing the Medication Adherence Report Scale reported suboptimal adherence; and 43% with urinary corticosteroid data did not have detectable prednisolone or metabolites in their urine. Good adherence by both methods was detected in 49 of the 142 (35%) of participants in whom both methods were performed; adherence detection did not match between methods in 53%. Self-reported high adherers had better asthma control and quality of life, whereas directly measured high adherers had lower blood eosinophil levels. Interpretation: Low adherence is a common problem in severe asthma, whether measured directly or self-reported. We report poor agreement between the two methods, suggesting some disassociation between self-assessment of medication adherence and regular oral corticosteroid use, which suggests that each approach may provide complementary information in clinical practice.

Non-steroidal anti-inflammatory drugs (NSAIDs) and other eicosanoid pathway modifiers are among the most ubiquitously used medications in the general population. Their broad anti-inflammatory, antipyretic and analgesic effects are applied against symptoms of respiratory infections, including SARS-CoV-2, as well as in other acute and chronic inflammatory diseases that often coexist with allergy and asthma. However, the current pandemic of COVID-19 also revealed the gaps in our understanding of their mechanism of action, selectivity and interactions not only during viral infections and inflammation, but also in asthma exacerbations, uncontrolled allergic inflammation, and NSAIDs-exacerbated respiratory disease (NERD). In this context, the consensus report summarises currently available knowledge, novel discoveries and controversies regarding the use of NSAIDs in COVID-19, and the role of NSAIDs in asthma and viral asthma exacerbations. We also describe here novel mechanisms of action of leukotriene receptor antagonists (LTRAs), outline how to predict responses to LTRA therapy and discuss a potential role of LTRA therapy in COVID-19 treatment. Moreover, we discuss interactions of novel T2 biologicals and other eicosanoid pathway modifiers on the horizon, such as prostaglandin D2 antagonists and cannabinoids, with eicosanoid pathways, in context of viral infections and exacerbations of asthma and allergic diseases. Finally, we identify and summarise the major knowledge gaps and unmet needs in current eicosanoid research.

Cysteinyl-maresins, also known as maresin-conjugates in tissue regeneration (MCTRs), are recently discovered lipid mediators proposed to reduce airway inflammation.To investigate the influence of MCTRs on IL-13-induced airway hyperresponsiveness in isolated human and mice airways.Before responsiveness to contractile agonists were assessed in myographs, human small bronchi were cultured for 2 days and mouse tracheas were cultured for 1-4 days. During the culture procedure airways were exposed to interleukin (IL)-13 in the presence or absence of MCTRs. Signalling mechanisms were explored using pharmacologic agonists and antagonists, and genetically modified mice.IL-13 treatment increased contractions to histamine, carbachol and leukotriene DMCTR3, but not LTD

Background There is a lack of early and precise biomarkers for personalized respiratory medicine. Breath contains an aerosol of droplet particles, which are formed from the epithelial lining fluid when the small airways close and re-open during inhalation succeeding a full expiration. These particles can be collected by impaction using the PExA method (Particles in Exhaled Air), and are derived from an area of high clinical interest previously difficult to access, making them a potential source of biomarkers reflecting pathological processes in the small airways. Research question Our aim was to investigate if PExA method is useful for discovery of biomarkers that reflect pathology of small airways. Methods and analysis Ten healthy controls and 20 subjects with asthma, of whom 10 with small airway involvement as indicated by a high lung clearance index (LCI ≥ 2.9 z-score), were examined in a cross-sectional design, using the PExA instrument. The samples were analysed with the SOMAscan proteomics platform (SomaLogic Inc.). Results Two hundred-seven proteins were detected in up to 80% of the samples. Nine proteins showed differential abundance in subjects with asthma and high LCI as compared to healthy controls. Two of these were less abundant (ALDOA4, C4), and seven more abundant (FIGF, SERPINA1, CD93, CCL18, F10, IgM, IL1RAP). sRAGE levels were lower in ex-smokers (n = 14) than in never smokers (n = 16). Gene Ontology (GO) annotation database analyses revealed that the PEx proteome is enriched in extracellular proteins associated with extracellular exosome-vesicles and innate immunity. Conclusion The applied analytical method was reproducible and allowed identification of pathologically interesting proteins in PEx samples from asthmatic subjects with high LCI. The results suggest that PEx based proteomics is a novel and promising approach to study respiratory diseases with small airway involvement.

Background Patients with severe asthma may have a greater risk of dying from COVID-19 disease. Angiotensin converting enzyme-2 (ACE2) and the enzyme proteases, transmembrane protease serine 2 (TMPRSS2) and FURIN, are needed for viral attachment and invasion into host cells. Methods We examined microarray mRNA expression of ACE2, TMPRSS2 and FURIN in sputum, bronchial brushing and bronchial biopsies of the European U-BIOPRED cohort. Clinical parameters and molecular phenotypes, including asthma severity, sputum inflammatory cells, lung functions, oral corticosteroid (OCS) use, and transcriptomic-associated clusters, were examined in relation to gene expression levels. Results ACE2 levels were significantly increased in sputum of severe asthma compared to mild-moderate asthma. In multivariate analyses, sputum ACE2 levels were positively associated with OCS use and male gender. Sputum FURIN levels were significantly related to neutrophils (%) and the presence of severe asthma. In bronchial brushing samples, TMPRSS2 levels were positively associated with male gender and body mass index, whereas FURIN levels with male gender and blood neutrophils. In bronchial biopsies, TMPRSS2 levels were positively related to blood neutrophils. The neutrophilic molecular phenotype characterised by high inflammasome activation expressed significantly higher FURIN levels in sputum than the eosinophilic Type 2-high or the pauci-granulocytic oxidative phosphorylation phenotypes. Conclusion Levels of ACE2 and FURIN may differ by clinical or molecular phenotypes of asthma. Sputum FURIN expression levels were strongly associated with neutrophilic inflammation and with inflammasome activation. This might indicate the potential for a greater morbidity and mortality outcome from SARS-CoV-2 infection in neutrophilic severe asthma.

BACKGROUND: Persistent airflow limitation (PAL) occurs in a subset of patients with asthma. Previous studies on PAL in asthma have included relatively small populations, mostly restricted to severe asthma, or have no included longitudinal data. The aim of this post-hoc analysis was to investigate the determinants, clinical implications, and outcome of PAL in patients with asthma who were included in the ATLANTIS study.METHODS: In this post-hoc analysis of the ATLANTIS study, we assessed the prevalence, clinical characteristics, and implications of PAL across the full range of asthma severity. The study population included patients aged 18-65 years who had been diagnosed with asthma at least 6 months before inclusion. We defined PAL as a post-bronchodilator FEV1/forced vital capacity (FVC) of less than the lower limit of normal at recruitment. Asthma severity was defined according to the Global Initiative for Asthma. We used Mann-Whitney U test, t test, or χ2 test to analyse differences in baseline characteristics between patients with and without PAL. Logistic regression was used for multivariable analysis of the associations between PAL and baseline data. Cox regression was used to analyse risk of exacerbation in relation to PAL, and a linear mixed-effects model was used to analyse change in FEV1 over time in patients with versus patients without PAL. Results were validated in the U-BIOPRED cohort.FINDINGS: Between June 30, 2014 and March 3, 2017, 773 patients were enrolled in the ATLANTIS study of whom 760 (98%) had post-bronchodilator FEV1/FVC data available. Of the included patients with available data, mean age was 44 years (SD 13), 441 (58%) of 760 were women, 578 (76%) were never-smokers, and 248 (33%) had PAL. PAL was not only present in patients with severe asthma, but also in 21 (16%) of 133 patients with GINA step 1 and 24 (29%) of 83 patients with GINA step 2. PAL was independently associated with older age at baseline (46 years in PAL group vs 43 years in non-PAL group), longer duration of asthma (24 years vs 12 years), male sex (51% vs 38%), higher blood eosinophil counts (median 0·27 × 109 cells per L vs 0·20 × 109 cells per L), more small airway dysfunction, and more exacerbations during 1 year of follow-up. Associations between PAL, age, and eosinophilic inflammation were validated in the U-BIOPRED cohort, whereas associations with sex, duration of asthma, and risk of exacerbations were not validated.INTERPRETATION: PAL is not only present in severe disease, but also in a considerable proportion of patients with milder disease. In patients with mild asthma, PAL is associated with eosinophilic inflammation and a higher risk of exacerbations. Our findings are important because they suggest that increasing treatment intensity should be considered in patients with milder asthma and PAL.FUNDING: Chiesi Farmaceutici and Dutch Ministry of Economic Affairs and Climate Policy (by means of the public-private partnership programme).

BACKGROUND: Electronic noses (eNoses) are emerging point-of-care tools that may help in the subphenotyping of chronic respiratory diseases such as asthma.OBJECTIVE: We aimed to investigate whether eNoses can classify atopy in pediatric and adult patients with asthma.METHODS: Participants with asthma and/or wheezing from 4 independent cohorts were included; BreathCloud participants (n = 429), Unbiased Biomarkers in Prediction of Respiratory Disease Outcomes adults (n = 96), Unbiased Biomarkers in Prediction of Respiratory Disease Outcomes pediatric participants (n = 100), and Pharmacogenetics of Asthma Medication in Children: Medication with Anti-Inflammatory Effects 2 participants (n = 30). Atopy was defined as a positive skin prick test result (≥3 mm) and/or a positive specific IgE level (≥0.35 kU/L) for common allergens. Exhaled breath profiles were measured by using either an integrated eNose platform or the SpiroNose. Data were divided into 2 training and 2 validation sets according to the technology used. Supervised data analysis involved the use of 3 different machine learning algorithms to classify patients with atopic versus nonatopic asthma with reporting of areas under the receiver operating characteristic curves as a measure of model performance. In addition, an unsupervised approach was performed by using a bayesian network to reveal data-driven relationships between eNose volatile organic compound profiles and asthma characteristics.RESULTS: Breath profiles of 655 participants (n = 601 adults and school-aged children with asthma and 54 preschool children with wheezing [68.2% of whom were atopic]) were included in this study. Machine learning models utilizing volatile organic compound profiles discriminated between atopic and nonatopic participants with areas under the receiver operating characteristic curves of at least 0.84 and 0.72 in the training and validation sets, respectively. The unsupervised approach revealed that breath profiles classifying atopy are not confounded by other patient characteristics.CONCLUSION: eNoses accurately detect atopy in individuals with asthma and wheezing in cohorts with different age groups and could be used in asthma phenotyping.

Background Animal models are extensively used to study underlying mechanisms in asthma. Guinea pigs share anatomical, pharmacological and physiological features with human airways and may enable the development of a pre-clinical in vivo model that closely resembles asthma. Objectives To develop an asthma model in guinea pigs using the allergen house dust mite (HDM). Methods Guinea pigs were intranasally sensitized to HDM which was followed by HDM challenges once weekly for five weeks. Antigen-induced bronchoconstriction (AIB) was evaluated as alterations in Rn (Newtonian resistance), G (tissue damping) and H (tissue elastance) at the first challenge with forced oscillation technique (FOT), and changes in respiratory pattern upon each HDM challenge were assessed as enhanced pause (Penh) using whole-body plethysmography. Airway responsiveness to methacholine was measured one day after the last challenge by FOT. Inflammatory cells and cytokines were quantified in bronchoalveolar lavage fluid, and HDM-specific immunoglobulins were measured in serum by ELISA. Airway pathology was evaluated by conventional histology. Results The first HDM challenge after the sensitization generated a marked increase in Rn and G, which was abolished by pharmacological inhibition of histamine, leukotrienes and prostanoids. Repeated weekly challenges of HDM caused increase of Penh and a marked increase in airway hyperresponsiveness for all three lung parameters (Rn , G and H) and eosinophilia. Levels of IgE, IgG1 , IgG2 and IL-13 were elevated in HDM-treated guinea pigs. HDM exposure induced infiltration of inflammatory cells into the airways with a pronounced increase of mast cells. Subepithelial collagen deposition, airway wall thickness and goblet cell hyperplasia were induced by repeated HDM challenge. Conclusion and clinical relevance Repeated intranasal HDM administration induces mast cell activation and hyperplasia together with an asthma-like pathophysiology in guinea pigs. This model may be suitable for mechanistic investigations of asthma, including evaluation of the role of mast cells.

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 FceR1-stimulated and IFN-g–stimulated signatures were enriched in SA. A FceR1-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-kB (nuclear factor-kB), and IL-1b/TNF-a (tumor necrosis factor-a) pathway activation. The IFN-g–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.

IntroductionAsthma is a heterogeneous disease with poorly defined phenotypes. Patients with severe asthma often receive multiple treatments including oral corticosteroids (OCS). Treatment may modify the observed metabotype, rendering it challenging to investigate underlying disease mechanisms. Here, we aimed to identify dysregulated metabolic processes in relation to asthma severity and medication.MethodsBaseline urine was collected prospectively from healthy participants (n=100), patients with mild-to-moderate asthma (n=87) and patients with severe asthma (n=418) in the cross-sectional U-BIOPRED cohort; 12–18-month longitudinal samples were collected from patients with severe asthma (n=305). Metabolomics data were acquired using high-resolution mass spectrometry and analysed using univariate and multivariate methods.ResultsA total of 90 metabolites were identified, with 40 significantly altered (p−20), OCS-treated asthmatic patients differed significantly from non-treated patients (p=9.5×10−4), and longitudinal metabotypes demonstrated temporal stability. Carnitine levels evidenced the strongest OCS-independent decrease in severe asthma. Reduced carnitine levels were associated with mitochondrial dysfunction via decreases in pathway enrichment scores of fatty acid metabolism and reduced expression of the carnitine transporter SLC22A5 in sputum and bronchial brushings.ConclusionsThis is the first large-scale study to delineate disease- and OCS-associated metabolic differences in asthma. The widespread associations with different therapies upon the observed metabotypes demonstrate the need to evaluate potential modulating effects on a treatment- and metabolite-specific basis. Altered carnitine metabolism is a potentially actionable therapeutic target that is independent of OCS treatment, highlighting the role of mitochondrial dysfunction in severe asthma.

BackgroundValid outcome measures are imperative to evaluate treatment response, yet the suitability of existing end-points for severe asthma is unclear. This review aimed to identify outcome measures for severe asthma and appraise the quality of their measurement properties.MethodsA literature search was performed to identify “candidate” outcome measures published between 2018 and 2020. A modified Delphi exercise was conducted to select “key” outcome measures within healthcare professional, patient, pharmaceutical and regulatory stakeholder groups. Initial validation studies for “key” measures were rated against modified quality criteria from COnsensus-based Standards for the selection of health Measurement INstruments (COSMIN). The evidence was discussed at multi-stakeholder meetings to ratify “priority” outcome measures. Subsequently, four bibliographic databases were searched from inception to 20 July 2020 to identify development and validation studies for these end-points. Two reviewers screened records, extracted data, assessed their methodological quality and graded the evidence according to COSMIN.Results96 outcome measures were identified as “candidates”, 55 as “key” and 24 as “priority” for severe asthma, including clinical, healthcare utilisation, quality of life, asthma control and composite. 32 studies reported measurement properties of 17 “priority” end-points from the latter three domains. Only the Severe Asthma Questionnaire and Childhood Asthma Control Test were developed with input from severe asthma patients. The certainty of evidence was “low” to “very low” for most “priority” end-points across all measurement properties and none fulfilled all quality standards.ConclusionsOnly two outcome measures had robust developmental data for severe asthma. This review informed development of core outcome measures sets for severe asthma.

The 3TR (Taxonomy, Treatments, Targets and Remission) consortium is the largest IMI (Innovative Medicine Initiative) project ever started in the field of immune diseases (https://3tr-imi.eu/). 3TR is unique in bringing several medical specialties, encompassing respiratory medicine, rheumatology, neurology and gastroenterology, together, to study disease mechanisms across seven disease entities: asthma, COPD, systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, ulcerative colitis and Crohn's disease. [...]

Patients with severe asthma may have a greater risk of dying from COVID-19 disease. Angiotensin converting enzyme-2 (ACE2) and the enzyme proteases, transmembrane protease serine 2 (TMPRSS2) and FURIN, are needed for viral attachment and invasion into host cells.We examined microarray mRNA expression of ACE2, TMPRSS2 and FURIN in sputum, bronchial brushing and bronchial biopsies of the European U-BIOPRED cohort. Clinical parameters and molecular phenotypes, including asthma severity, sputum inflammatory cells, lung functions, oral corticosteroid (OCS) use, and transcriptomic-associated clusters, were examined in relation to gene expression levels.ACE2 levels were significantly increased in sputum of severe asthma compared to mild-moderate asthma. In multivariate analyses, sputum ACE2 levels were positively associated with OCS use and male gender. Sputum FURIN levels were significantly related to neutrophils (%) and the presence of severe asthma. In bronchial brushing samples, TMPRSS2 levels were positively associated with male gender and body mass index, whereas FURIN levels with male gender and blood neutrophils. In bronchial biopsies, TMPRSS2 levels were positively related to blood neutrophils. The neutrophilic molecular phenotype characterised by high inflammasome activation expressed significantly higher FURIN levels in sputum than the eosinophilic Type 2-high or the pauci-granulocytic oxidative phosphorylation phenotypes.Levels of ACE2 and FURIN may differ by clinical or molecular phenotypes of asthma. Sputum FURIN expression levels were strongly associated with neutrophilic inflammation and with inflammasome activation. This might indicate the potential for a greater morbidity and mortality outcome from SARS-CoV-2 infection in neutrophilic severe asthma.

Severe asthma is a heterogeneous disease with different phenotypes based on clinical, functional or inflammatory parameters. In particular, the eosinophilic phenotype is associated with type 2 inflammation and increased levels of interleukin (IL)‐4, IL‐5 and IL‐13). Monoclonal antibodies that target the eosinophilic inflammatory pathways (IL‐5R and IL‐5), namely mepolizumab, reslizumab, and benralizumab, are effective and safe for severe eosinophilic asthma. Eosinophils threshold represents the most indicative biomarker for response to treatment with all three monoclonal antibodies. Improvement in asthma symptoms scores, lung function, the number of exacerbations, history of late‐onset asthma, chronic rhinosinusitis with nasal polyposis, low oral corticosteroids use and low body mass index represent predictive clinical markers of response. Novel Omics studies are emerging with proteomics data and exhaled breath analyses. These may prove useful as biomarkers of response and non‐response biologics. Moreover, future biomarker studies need to be undertaken in paediatric patients affected by severe asthma. The choice of appropriate biologic therapy for severe asthma remains challenging. The importance of finding biomarkers that can predict response continuous an open issue that needs to be further explored. This review describes the clinical effects of targeting the IL‐5 pathway in severe asthma in adult and paediatric patients, focusing on predictors of response and non‐response.

Serum pregnancy-associated plasma protein A (PAPPA) as a predictor of eosinophilic Type-2 high asthmaTo the Editor,Pregnancy-associated plasma protein A (PAPPA), a metalloproteinase that cleaves insulin-like growth factor (IGF)-binding proteins (IGFBPs) to increase IGF availability, is expressed systemically in pregnant women but also in other tissues (1). Higher serum PAPPA levels are reported in patients with newly-diagnosed asthma (1) and allergic rhinitis compared to healthy controls and are decreased following omalizumab treatment (2). We determined whether PAPPA could represent a novel biomarker for Type-2 (T2) asthma by exploring the relationship between asthma severity and phenotypes of severe asthma and PAPPA gene and protein expression (3).We recruited 288 severe non-smoking asthma (NSA), 102 smokers and ex-smokers with severe asthma (SSA), 86 mild/moderate non-smoking asthmatics (MMA) and 95 healthy non-smoking controls (HC) from the U-BIOPRED cohort (NCT01976767) (4) (Table S1 ). Transcriptomic and proteomic profiling of blood and sputum samples and specific serum periostin ELISA were performed (3). Gene set variation analysis (GSVA) was used to calculate the enrichment score (ES) of 34 genes that were upregulated following in vitro stimulation of primary human bronchial epithelial cells with IL-13 (T2_IL-13_IVS) (3). Eosinophilic inflammation was defined by sputum eosinophilia >1.49% (3). Local Ethics Committees of the recruiting centres approved the study and all participants gave written informed consent.Sputum cell PAPPA mRNA was elevated in NSA compared to SSA, MMA and HC subjects particularly in granulocytic asthmatics and in the transcriptomic-associated cluster (TAC)1; an eosinophilic cluster (5) (Figure 1A-C ). This was more pronounced with sputum PAPPA protein analysis according to asthma severity, in eosinophilic and mixed granulocytic asthmatics and in T2-high asthmatics identified by the T2_IL-13_IVS signature (Figure 1D-F ).PAPPA mRNA expression in blood cells was similar across asthma severities, blood granulocytes and molecular phenotypes (Supplementary Figure 1A-C ). However, serum PAPPA protein levels supported the discrimination seen in sputum with significant elevation seen in SA compared to HC, in eosinophilic and mixed granulocytic asthma and in T2-high asthma (SupplementaryFigure 1D-F ).Sputum eosinophil percentages were significantly correlated with sputum (r=0.88, p=10-6) and serum (r=0.41, p=10-6) PAPPA protein levels. Overall, sputum PAPPA protein gave a greater distinction between asthma severity, granulocyte composition and T2-high asthma than with serum although fewer samples were available.These results were validated in sputum from the Airways Disease Endotyping for Personalized Therapeutics (ADEPT) study (6) (Supplementary Figure S2 ). Elevated PAPPA protein in the serum and sputum of severe asthmatics and in eosinophilic compared to non- eosinophilic subjects was seen (SupplementaryFigure S2A-D ). In addition, sputum PAPPA mRNA levels were also elevated in eosinophilic versus non-eosinophilic asthma in the ADEPT cohort (Supplementary Figure S2E ).The ES score of the T2_IL-13_IVS gene signature in bronchial brushings was significantly, but weakly, correlated with blood eosinophil counts (r=0.329, p=10-6), serum PAPPA (r=0.356, p=10-6), but not with serum periostin levels (r=0.07, p-value=0.48). In contrast, the T2 IL-13 IVS ES score was strongly correlated with sputum PAPPA levels (r=0.72, p=10-3). Sputum PAPPA protein levels also significantly correlated with markers of remodelling such as MMP10 (r=0.646, p

The formylpeptide receptors (nomenclature agreed by the NC-IUPHAR Subcommittee on the formylpeptide receptor family [196]) respond to exogenous ligands such as the bacterial product fMet-Leu-Phe (fMLP) and endogenous ligands such as lipoxin A4 (LXA4), 15-epi-lipoxin A4, annexin I , cathepsin G, amyloid β42, serum amyloid A and spinorphin, derived from β-haemoglobin. FPR1 also serves as a plague receptor for selective destruction of human immune cells by Y. pestis [135]. The FPR1/2 agonists 'compound 17b' and 'compound 43' have shown cardiac protective functions [149, 64].

Inflammation, structural, and functional abnormalities within the airways are key features of asthma. Although these processes are well documented, their expression varies across the heterogeneous spectrum of asthma. Type 2 inflammatory responses are characterized by increased levels of eosinophils, FeNO, and type 2 cytokines in blood and/or airways. Presently, type 2 asthma is the best-defined endotype, typically found in patients with allergic asthma, but surprisingly also in nonallergic patients with (severe) asthma. The etiology of asthma with non-type 2 inflammation is less clear. During the past decade, targeted therapies, including biologicals and small molecules, have been increasingly integrated into treatment strategies of severe asthma. These treatments block specific inflammatory pathways or single mediators. Single or composite biomarkers help to identify patients who will benefit from these treatments. So far, only a few inflammatory biomarkers have been validated for clinical application. The European Academy of Allergy & Clinical Immunology Task Force on Biomarkers in Asthma was initiated to review different biomarker sampling methods and to investigate clinical applicability of new and existing inflammatory biomarkers (point-of-care) to support diagnosis, targeted treatment, and monitoring of severe asthma. Subsequently, we discuss existing and novel targeted therapies for asthma as well as applicable biomarkers.

The eicosanoids are a family of lipid mediators of pain and inflammation involved in multiple pathologies, including asthma, hypertension, cancer, atherosclerosis, and neurodegenerative diseases. These signaling mediators act locally, but are rapidly metabolized and transported to the systemic circulation as a mixture of primary and secondary metabolites. Accordingly, urine has become a useful readily accessible biofluid for monitoring the endogenous synthesis of these molecules. Herein, we present the validation of a rapid, repeatable, and precise method for the extraction and quantification of 32 eicosanoid urinary metabolites by LC-MS/MS. For 12 out of 17 deconjugated glucuronide eicosanoids, there was no improvement in recovered signal. These metabolites cover the major synthetic pathways, including prostaglandins, leukotrienes, and isoprostanes. The method linearity was >0.99 for all metabolites analyzed, the limit of detection ranged from 0.05–5 ng/ml, and the average extraction recoveries were >90%. All analytes were stable for at least three freeze/thaw cycles. The method was formatted for large-scale analysis of clinical cohorts, and the long-term repeatability was demonstrated over 15 months of acquisition, evidencing high precision (CV

Background: Immunohistochemical analysis of granule-associated proteases has revealed that human lung mast cells constitute a heterogeneous population of cells, with distinct subpopulations identified. However, a systematic and comprehensive analysis of cell-surface markers to study human lung mast cell heterogeneity has yet to be performed. Methods: Human lung mast cells were obtained from lung lobectomies, and the expression of 332 cell-surface markers was analyzed using flow cytometry and the LEGENDScreen kit. Markers that exhibited high variance were selected for additional analyses to reveal whether they were correlated and whether discrete mast cell subpopulations were discernable. Results: We identified the expression of 102 surface markers on human lung mast cells. Several markers showed high continuous variation in expression within the mast cell population. Six of these markers were correlated: SUSD2, CD49a, CD326, CD34, CD66 and HLA-DR. The expression of these markers was also correlated with the size and granularity of mast cells. However, no marker produced an expression profile consistent with a bi- or multimodal distribution. Conclusions: LEGENDScreen analysis identified more than 100 cell-surface markers on mast cells, including 23 that, to the best of our knowledge, have not been previously described on human mast cells. Several of the newly described markers are known to be involved in sensing the microenvironment, and their identification can shed new light on mast cell functions. The exhaustive expression profiling of the 332 surface markers failed to detect distinct mast cell subpopulations. Instead, we demonstrate the continuous nature of human lung mast cell heterogeneity.

The mechanism by which cyclooxygenase (COX) inhibition increases antigen-induced responses in airways remains unknown. Male albino guinea pigs were sensitized to ovalbumin (OVA). Intact rings of the trachea were isolated and mounted in organ baths for either force measurements or lipid mediator release analysis by UPLC-MS/MS or EIA following relevant pharmacological interventions. First, challenge with OVA increased the release of all primary prostanoids (prostaglandin (PG) D

Background The major mast cell prostanoid PGD(2) is targeted for therapy of asthma and other diseases, because the biological actions include bronchoconstriction, vasodilation and regulation of immune cells mediated by three different receptors. It is not known if the alternative to selectively inhibit the biosynthesis of PGD(2) affects release of other prostanoids in human mast cells. Objectives To determine the biochemical consequences of inhibition of the hematopoietic prostaglandin D synthase (hPGDS) PGD(2) in human mast cells. Methods Four human mast cell models, LAD2, cord blood derived mast cells (CBMC), peripheral blood derived mast cells (PBMC) and human lung mast cells (HLMC), were activated by anti-IgE or ionophore A23187. Prostanoids were measured by UPLC-MS/MS. Results All mast cells almost exclusively released PGD(2) when activated by anti-IgE or A23187. The biosynthesis was in all four cell types entirely initiated by COX-1. When pharmacologic inhibition of hPGDS abolished formation of PGD(2), PGE(2) was detected and release of TXA(2) increased. Conversely, when the thromboxane synthase was inhibited, levels of PGD(2) increased. Adding exogenous PGH(2) confirmed predominant conversion to PGD(2) under control conditions, and increased levels of TXB2 and PGE(2) when hPGDS was inhibited. However, PGE(2) was formed by non-enzymatic degradation. Conclusions Inhibition of hPGDS effectively blocks mast cell dependent PGD(2) formation. The inhibition was associated with redirected use of the intermediate PGH(2) and shunting into biosynthesis of TXA(2). However, the levels of TXA(2) did not reach those of PGD(2) in naive cells. It remains to determine if this diversion occurs in vivo and has clinical relevance.

Background: Asthma is a heterogeneous disease characterized by distinct phenotypes with associated microbial dysbiosis. Objectives: Our aim was to identify severe asthma phenotypes based on sputum microbiome profiles and assess their stability after 12 to 18 months. A further aim was to evaluate clusters’ robustness after inclusion of an independent cohort of patients with mild-to-moderate asthma. Methods: In this longitudinal multicenter cohort study, sputum samples were collected for microbiome profiling from a subset of the Unbiased Biomarkers in Prediction of Respiratory Disease Outcomes adult patient cohort at baseline and after 12 to 18 months of follow-up. Unsupervised hierarchical clustering was performed by using the Bray-Curtis β-diversity measure of microbial profiles. For internal validation, partitioning around medoids, consensus cluster distribution, bootstrapping, and topological data analysis were applied. Follow-up samples were studied to evaluate within-patient clustering stability in patients with severe asthma. Cluster robustness was evaluated by using an independent cohort of patients with mild-to-moderate asthma. Results: Data were available for 100 subjects with severe asthma (median age 55 years; 42% males). Two microbiome-driven clusters were identified; they were characterized by differences in asthma onset, smoking status, residential locations, percentage of blood and/or sputum neutrophils and macrophages, lung spirometry results, and concurrent asthma medications (all P values < .05). The cluster 2 patients displayed a commensal-deficient bacterial profile that was associated with worse asthma outcomes than those of the cluster 1 patients. Longitudinal clusters revealed high relative stability after 12 to 18 months in those with severe asthma. Further inclusion of an independent cohort of 24 patients with mild-to-moderate asthma was consistent with the clustering assignments. Conclusion: Unbiased microbiome-driven clustering revealed 2 distinct robust phenotypes of severe asthma that exhibited relative overtime stability. This suggests that the sputum microbiome may serve as a biomarker for better characterizing asthma phenotypes.