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Background: Clustering approaches using single omics platforms are increasingly used to characterise molecular phenotypes of eosinophilic and neutrophilic asthma. Effective integration of multi‐omics platforms should lead towards greater refinement of asthma endotypes across molecular dimensions and indicate key targets for intervention or biomarker development. Objectives: To determine whether multi‐omics integration of sputum leads to improved granularity of the molecular classification of severe asthma. Methods: We analyzed six ‐omics data blocks–microarray transcriptomics, gene set variation analysis of microarray transcriptomics, SomaSCAN proteomics assay, shotgun proteomics, 16S microbiome sequencing, and shotgun metagenomic sequencing–from induced sputum samples of 57 severe asthma patients, 15 mild‐moderate asthma patients, and 13 healthy volunteers in the U‐BIOPRED European cohort. We used Monti consensus clustering algorithm for aggregation of clustering results and Similarity Network Fusion to integrate the 6 multi‐omics datasets of the 72 asthmatics. Results: Five stable omics‐associated clusters were identified (OACs). OAC1 had the best lung function with the least number of severe asthmatics with sputum paucigranulocytic inflammation. OAC5 also had fewer severe asthma patients but the highest incidence of atopy and allergic rhinitis, with paucigranulocytic inflammation. OAC3 comprised only severe asthmatics with the highest sputum eosinophilia. OAC2 had the highest sputum neutrophilia followed by OAC4 with both clusters consisting of mostly severe asthma but with more ex/current smokers in OAC4. Compared to OAC4, there was higher incidence of nasal polyps, allergic rhinitis, and eczema in OAC2. OAC2 had microbial dysbiosis with abundant Moraxella catarrhalis and Haemophilus influenzae. OAC4 was associated with pathways linked to IL‐22 cytokine activation, with the prediction of therapeutic response to anti‐IL22 antibody therapy. Conclusion: Multi‐omics analysis of sputum in asthma has defined with greater granularity the asthma endotypes linked to neutrophilic and eosinophilic inflammation. Modelling diverse types of high‐dimensional interactions will contribute to a more comprehensive understanding of complex endotypes. Key Points: Unsupervised clustering on sputum multi‐omics of asthma subjects identified 3 out of 5 clusters with predominantly severe asthma.One severe asthma cluster was linked to type 2 inflammation and sputum eosinophilia while the other 2 clusters to sputum neutrophilia.One severe neutrophilic asthma cluster was linked to Moraxella catarrhalis and to a lesser extent Haemophilus influenzae while the second cluster to activation of IL‐22. [ABSTRACT FROM AUTHOR]

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 were 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. [ABSTRACT FROM AUTHOR]

Background: Severe neutrophilic asthma is resistant to treatment with glucocorticoids. The immunomodulatory protein macrophage migration inhibitory factor (MIF) promotes neutrophil recruitment to the lung and antagonises responses to glucocorticoids. We hypothesised that MIF promotes glucocorticoid resistance of neutrophilic inflammation in severe asthma.Methods: We examined whether sputum MIF protein correlated with clinical and molecular characteristics of severe neutrophilic asthma in the Unbiased Biomarkers for the Prediction of Respiratory Disease Outcomes (U-BIOPRED) cohort. We also investigated whether MIF regulates neutrophilic inflammation and glucocorticoid responsiveness in a murine model of severe asthma in vivo.Results: MIF protein levels positively correlated with the number of exacerbations in the previous year, sputum neutrophils and oral corticosteroid use across all U-BIOPRED subjects. Further analysis of MIF protein expression according to U-BIOPRED-defined transcriptomic-associated clusters (TACs) revealed increased MIF protein and a corresponding decrease in annexin-A1 protein in TAC2, which is most closely associated with airway neutrophilia and NLRP3 inflammasome activation. In a murine model of severe asthma, treatment with the MIF antagonist ISO-1 significantly inhibited neutrophilic inflammation and increased glucocorticoid responsiveness. Coimmunoprecipitation studies using lung tissue lysates demonstrated that MIF directly interacts with and cleaves annexin-A1, potentially reducing its biological activity.Conclusion: Our data suggest that MIF promotes glucocorticoid-resistance of neutrophilic inflammation by reducing the biological activity of annexin-A1, a potent glucocorticoid-regulated protein that inhibits neutrophil accumulation at sites of inflammation. This represents a previously unrecognised role for MIF in the regulation of inflammation and points to MIF as a potential therapeutic target for the management of severe neutrophilic asthma. [ABSTRACT FROM AUTHOR]

Background: Allergen source‐derived proteases are a critical factor in the formation and development of asthma. The cysteine protease activity of house dust mite (HDM) disrupts the epithelial barrier function. The expression of cystatin SN (CST1) is elevated in asthma epithelium. CST1 inhibits the cysteine protease activity. We aimed to elucidate the role of epithelium‐derived CST1 in the development of asthma caused by HDM. Methods: CST1 protein levels in sputum supernatants and serum of patients with asthma and healthy volunteers were measured by ELISA. The ability of CST1 protein to suppress HDM‐induced bronchial epithelial barrier function was examined in vitro. The effects of exogenous CST1 protein on abrogating HDM‐induced epithelial barrier function and inflammation were examined in mice in vivo. Results: CST1 protein levels were higher in sputum supernatants (142.4 ± 8.95 vs 38.87 ± 6.85 ng/mL, P < 0.0001) and serum (1129 ± 73.82 vs 703.1 ± 57.02 pg/mL, P = 0.0035) in patients with asthma than in healthy subjects. The levels were significantly higher in patients with not well‐ and very poorly controlled asthma than those with well‐controlled asthma. Sputum and serum CST1 protein levels were negatively correlated with lung function in asthma. CST1 protein levels were significantly lower in the serum of HDM‐specific IgE (sIgE)‐positive asthmatics than in sIgE‐negative asthmatics. The HDM‐induced epithelial barrier function disruption was suppressed by recombinant human CST1 protein (rhCST1) in vitro and in vivo. Conclusion: Our data indicated that human CST1 protein suppresses asthma symptoms by protecting the asthmatic bronchial epithelial barrier through inhibiting allergenic protease activity. CST1 protein may serve as a potential biomarker for asthma control. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

To the Editor, COVID-19 can be a serious multisystem disease caused by the SARS-CoV-2 coronavirus, and the current pandemic has affected more than 80 million people and caused nearly two million deaths worldwide. Therefore, the aim of this study was to investigate associations of sputum endopeptidases gene expression with metagenomics composition and whether they could be used to stratify asthma patients according to risk of SARS-CoV-2 infection. In contrast, non-T2 asthma, particularly neutrophilic asthma, has been associated with higher ACE2 and endopeptidases (TMPRSS2 and furin) expression as compared with the T2-high phenotype4,5 that might imply a worse outcome with COVID-19 infection. [Extracted from the article]

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. [ABSTRACT FROM AUTHOR]

An amendment to this paper has been published and can be accessed via the original article. [ABSTRACT FROM AUTHOR]

Introduction: Neutrophilic asthma has been suggested to be a clinically distinct phenotype characterised by more severe airflow obstruction and higher exacerbation risk. However, this has only been assessed in few and smaller studies, using different cut-offs to define neutrophilia, and with conflicting results. We used data from ATLANTIS, an observational longitudinal study including a large number of patients with asthma and healthy controls. The aim of the present study was to examine whether neutrophilic inflammation, either in sputum or blood, is more prevalent in asthma and whether it correlates with disease severity., Methods: ATLANTIS included 773 asthma patients, with blood collected from 767 (99%) and sputum from 228 patients (30%). Data were available from 244 healthy controls, all providing blood and 126 (52%) providing sputum. Asthma patients were characterised, including parameters of large and small airways disease at baseline and after 6 and 12 months of follow-up. Sputum and blood neutrophilia were defined as values exceeding the upper quartile in asthma patients., Results: The prevalence of sputum neutrophilia did not differ between asthma patients and healthy controls. Asthma patients with sputum neutrophilia did not display more severe symptoms, large or small airways disease or more frequent exacerbations. Blood neutrophilia was more common in asthma and was associated with higher body mass index, female sex, current smoking and systemic corticosteroid use. Patients with blood neutrophilia had a statistically significant, but small, increase in residual volume/total lung capacity. Blood neutrophilia was not associated with large or small airways disease or exacerbation risk., Conclusion: Sputum and blood neutrophilia do not define a distinct clinical phenotype in asthma., Competing Interests: Conflict of interest: P.J.M. Kuks reports support for the present study from the Dutch Ministry of Economic Affairs and Climate Policy by means of the public–private partnership programme. Conflict of interest: T.M. Kole reports support for the present study from the Dutch Ministry of Economic Affairs and Climate Policy by means of the public–private partnership programme. Conflict of interest: M. Kraft reports grants or contacts from the National Institutes of Health, American Lung Association, Synairgen, Janssen, AstraZeneca (AZ) and Sanofi (funds paid to the University of Arizona until June 2022 and currently to the Icahn School of Medicine at Mount Sinai, all pharmaceutical industry studies now completed); consulting fees from AZ, Sanofi, Chiesi, GSK, Kinaset and Genentech (funds paid to M. Kraft); payment or honoraria for lectures, presentations, speakers’ bureaus, manuscript writing or educational events from Chiesis (funds paid to M. Kraft); support from the European Respiratory Society (ERS) to attend the ERS Congress in 2023 (partial support paid to M. Kraft); that they are a cofounder and chief medical officer of RaeSedo Inc. (one patent issued and two files for the development of therapeutics for inflammatory lung disease); participation on the ALung data safety monitoring board (DSMB) (funds paid to M. Kraft); leadership or fiduciary roles on the National Heart, Lung and Blood Advisory Council (completed in 2022, funds paid to M. Kraft) and the Association of Professors of Medicine (no compensation); equity ownership in RaeSedo Inc. (company is developing therapeutics for asthma in the preclinical phase, no human trials or IND); and that they are a Section Editor of UpToDate (funds paid to M. Kraft), all in the past 36 months. Conflict of interest: S. Siddiqui reports consulting fees from CSL Behring, AZ, GSK, Areteia Therapeutics and Novartis; speaker fees from Chiesi for presenting ATLANTIS data; support from the ERS to attend ERS Science Council meetings; and being a member of the ATLANTIS scientific steering group, all in the past 36 months. Conflict of interest: L.M. Fabbri reports consulting fees from Chiesi, GSK, AZ, Novartis, Verona Pharma and ICON; payment or honoraria for lectures, presentations, speakers’ bureaus, manuscript writing or educational events from Chiesi and GSK; and participation on a DSMB or advisory board for Novartis and Chiesi, all in the past 36 months. Conflict of interest: K.F. Rabe reports payment or honoraria for lectures, presentations, speakers’ bureaus, manuscript writing or educational events from AZ, Boehringer Ingelheim, Chiesi, Novartis, Sanofi Regeneron, GSK, Berlin Chemie and Roche Pharma (payments made to K.F. Rabe); participation on a DSMB or advisory board for AZ, Boehringer Ingelheim and Sanofi Regeneron; and leadership or fiduciary roles in the German Center for Lung Research, German Chest Society and American Thoracic Society, all in the past 36 months. Conflict of interest: A. Papi reports that the ATLANTIS study was supported by Chiesi. They also report grants to their institution from Chiesi, AZ, GSK and Sanofi; consulting fees from Chiesi, AZ, GSK, Novartis, Sanofi, Iqvia, Avillion, Elpen Pharmaceuticals, Moderna and Roche (to A. Papi); payment or honoraria for lectures, presentations, speakers’ bureaus, manuscript writing or educational events from Chiesi, AZ, GSK, Menarini, Zambon, Mundipharma, Sanofi, Edmond Pharma, Iqvia, Avillion, Sanofi and Regeneron (to A. Papi); participation on advisory boards for Chiesi, AZ, GSK, Novartis, Sanofi, Iqvia, Avillion, Elpen Pharmaceuticals and Moderna (to A. Papi); and receipt of equipment, materials, drugs, medical writing, gifts or other services from Consorzio Futuro in Ricerca, all in the past 36 months. Conflict of interest: C. Brightling reports support for the ATLANTIS study from a grant from Chiesi and for the Leicester National Institute for Health and Care Research (NIHR) Biomedical Research Centre from the NIHR; and grants and consultancy fees from 4D Pharma, Areteia, AZ, Chiesi, Genentech, GSL, Mologic, Novartis, Regeneron Pharmaceuticals, Roche and Sanofi (paid to their institution), in the past 36 months. Conflict of interest: D. Singh reports consulting fees from Aerogen, AZ, Boehringer Ingelheim, Chiesi, Cipla, CSL Behring, EpiEndo, Genentech, GSK, Glenmark, Gossamer Bio, Kinaset Therapeutics, Menarini, Novartis, Orion, Pulmatrix, Sanofi, Synairgen, Teva, Theravance Biopharma and Verona Pharma, in the past 36 months. Conflict of interest: T. van der Molen reports that Chiesi funded the present study through their department; and Chiesi funded presentations, and GSK funded presentations and travel (to their company), in the past 36 months. Conflict of interest: J.W.W.H. Kocks reports grants or contracts from AZ, Boehringer Ingelheim, Chiesis, GSK and Valneva; consulting fees from AZ, Boehringer Ingelheim, Chiesi, GSK, Teva, MSD, COVIS Pharma and Janssen (payments made to their institution); and payment or honoraria for lectures, presentations, speakers’ bureaus, manuscript writing or educational events from Mundi Pharma and ALK-Bello (payments made to their institution); that they are an ERS group chair, President and a board member of the International Primary Care Respiratory Group, a member of the CAHAG scientific committee, and a board member of the Inhalation Institute Netherlands; and that they are Director of and hold stocks in the General Practitioners Research Institute, and hold <5% stocks in Lothar Medtec, all in the past 36 months. Conflict of interest: K.F. Chung reports a Medical Research Council (MRC) grant on precision medicine for severe asthma, an Engineering and Physical Sciences Research Council (EPSRC) on air pollution and asthma, a GSK grant on mepolizumab and eosinophils in asthma, a Merck grant on the effects of ATP on cough hypersensitivity, and a National Institute of Environmental Health Sciences grant on air pollution and lipid metabolites in asthma (all to their institution); speaking engagements for GSK, Novartis and AZ (payments to K.F. Chung); and advisory board meetings for GSK, AZ, Novartis, Roche, Merck, Trevi, Rickett-Beckinson, Nocion and Shionogi on asthma, COPD and chronic cough, and the Scientific Advisory Board of the Clean Breathing Institute supported by Haleon (all payments to K.F. Chung), all in the past 36 months. Conflict of interest: I.M. Adcock reports European Union Innovative Medicines Initiative funding for the U-BIOPRED project. They also report grants to their institution from GSK, MRC, EPSRC and Sanofi; consulting fees from GSK, Sanofi and Kinaset (all for advisory boards); and lecture fees from AZ and Sanofi, all in the past 36 months. Conflict of interest: P.K. Bhavsar is an associate editor of this journal. Conflict of interest: N.Z. Kermani has nothing to disclose. Conflict of interest: I.H. Heijink reports research grants from NWO, Longfonds and Health∼Holland outside the scope of the present study, in the past 36 months. Conflict of interest: S.D. Pouwels has nothing to disclose. Conflict of interest: H.A.M. Kerstjens reports support for the present study from Chiesi. They also report unrestricted research grants from Boehringer Ingelheim, GSK and Novartis; and participation on a DSMB or advisory board for GSK, AZ, Novartis and Teva (all payments to their institution), in the past 36 months. Conflict of interest: D-J. Slebos reports support for the ATLANTIS consortium from Chiesi. Conflict of interest: M. van den Berge reports research grants paid to their institution from GSK, Chiesi, AZ, Novartis, Genentech and Roche, in the past 36 months., (Copyright ©The authors 2025.)

Purpose: Asthma is a clinical syndrome with various underlying pathomechanisms and clinical phenotypes. Genetic, ethnic, and geographic factors may influence the differences in clinical presentation, severity, and prognosis. We compared the characteristics of asthma based on the geographical background by analyzing representative cohorts from the United States, Europe, South America, and Asia using the Severe Asthma Research Program (SARP), Unbiased Biomarkers for the Prediction of Respiratory Disease Outcomes (U-BIOPRED), Program for Control of Asthma in Bahia (ProAR), and Cohort for Reality and Evolution of Adult Asthma in Korea (COREA), respectively., Methods: The clinical characteristics and medications for the SARP (n = 669), U-BIOPRED (n = 509), ProAR (n = 996), and COREA (n = 3,748) were analyzed. Subgroup analysis was performed for severe asthma., Results: The mean age was highest and lowest in the COREA and SARP, respectively. The asthma onset age was lowest in the ProAR. The mean body mass index was highest and lowest in the SARP and COREA, respectively. Baseline pulmonary function was lowest and highest in the U-BIOPRED and COREA, respectively. The number of patients with acute exacerbation in the previous year was highest in U-BIOPRED. The mean blood eosinophil count was highest in COREA. The total immunoglobulin E was highest in the ProAR. The frequency of atopy was highest in the SARP. The principal component analysis plot revealed differences among all cohorts., Conclusions: The cohorts from 4 different continents exhibited different clinical and physiological characteristics, probably resulting from the interplay between genetic susceptibility and geographical factors., Competing Interests: There are no financial or other issues that might lead to conflict of interest., (Copyright © 2024 The Korean Academy of Asthma, Allergy and Clinical Immunology • The Korean Academy of Pediatric Allergy and Respiratory Disease.)