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Introduction: Many patients with severe asthma continue to experience symptoms and exacerbations despite treatment with standard-of-care therapy. In the phase 3 NAVIGATOR study, tezepelumab significantly reduced exacerbations over 52 weeks compared with placebo in patients with severe, uncontrolled asthma. This analysis assessed the efficacy of tezepelumab in reducing asthma exacerbations in various clinically relevant subgroups of patients in NAVIGATOR., Methods: NAVIGATOR was a phase 3, multicentre, randomized, double-blind, placebo-controlled study. Participants (12-80 years old) with severe, uncontrolled asthma were randomized 1:1 to receive tezepelumab 210 mg or placebo subcutaneously every 4 weeks for 52 weeks. Pre-specified and post hoc analyses were performed to evaluate the annualized asthma exacerbation rate (AAER) over 52 weeks in clinically relevant subgroups of patients defined by baseline patient characteristics, medical history, exacerbation triggers, medication eligibility and medication use before and during the study., Results: Tezepelumab reduced the AAER over 52 weeks compared with placebo across a wide range of patient subgroups assessed. Reductions in exacerbations were similar across subgroups defined by baseline patient characteristics, ranging from 48% (95% confidence interval [CI]: 21, 65) to 60% (95% CI: 44, 71) in subgroups analysed by sex, smoking history and body mass index. Among the asthma-related comorbidity subgroups investigated, patients with aspirin or NSAID sensitivity had the greatest reductions in AAER with tezepelumab compared with placebo (83%; 95% CI: 66, 91). In patients eligible to receive dupilumab, tezepelumab reduced exacerbations compared with placebo by 64% (95% CI: 54, 71). Reductions in the AAER with tezepelumab compared with placebo were also observed irrespective of exacerbation trigger category and the number of asthma controller medications patients were receiving at baseline., Conclusion: These findings further support the benefits of tezepelumab in patients with severe, uncontrolled asthma and can help to inform healthcare providers' treatment decisions., Clinical Trial Registration: NAVIGATOR (NCT03347279)., (© 2024. The Author(s).)

Background: It remains unclear whether patients with asthma and/or chronic obstructive pulmonary disease (COPD) are at increased risk for severe coronavirus disease 2019 (COVID-19)., Objective: Compare in-hospital COVID-19 outcomes among patients with asthma, COPD, and no airway disease., Methods: A retrospective cohort study was conducted on 8,395 patients admitted with COVID-19 between March 2020 and April 2021. Airway disease diagnoses were defined using International Classification of Diseases, 10 th Revision codes. Mortality and sequential organ failure assessment (SOFA) scores were compared among groups. Logistic regression analysis was used to identify and adjust for confounding clinical features associated with mortality., Results: The median SOFA score in patients without airway disease was 0.32 and mortality was 11%. In comparison, asthma patients had lower SOFA scores (median 0.15; P < .01) and decreased mortality, even after adjusting for age, diabetes, and other confounders (odds ratio 0.65; P = .01). Patients with COPD had higher SOFA scores (median 0.86; P < .01) and increased adjusted odds of mortality (odds ratio 1.40; P < .01). Blood eosinophil count of 200 cells/μL or greater, a marker of type 2 inflammation, was associated with lower mortality across all groups. Importantly, patients with asthma showed improved outcomes even after adjusting for eosinophilia, indicating that noneosinophilic asthma was associated with protection as well., Conclusions: COVID-19 severity was increased in patients with COPD and decreased in those with asthma, eosinophilia, and noneosinophilic asthma, independent of clinical confounders. These findings suggest that COVID-19 severity may be influenced by intrinsic immunological factors in patients with airway diseases, such as type 2 inflammation., (Copyright © 2023 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

Background: Previous research has shown greater efficacy of dupilumab in patients with uncontrolled asthma and type 2 inflammation. We analyzed dupilumab's efficacy in patients from the TRAVERSE study with or without evidence of allergic asthma and type 2 inflammation per current GINA guidelines (≥150  eosinophils/μL or FeNO ≥20 ppb)., Methods: All patients aged ≥12 years who rolled over from the placebo-controlled QUEST study (NCT02414854) to TRAVERSE (NCT02134028) received add-on dupilumab 300 mg every 2 weeks for up to 96 weeks. We assessed annualized severe asthma exacerbation rates (AERs) and changes from parent-study baseline (PSBL) in pre-bronchodilator FEV 1 and 5-item asthma control questionnaire (ACQ-5) score in patients with moderate-to-severe type 2 asthma with and without evidence of allergic asthma at PSBL., Results: In TRAVERSE, dupilumab consistently reduced AER across all subgroups. By Week 96, dupilumab increased pre-bronchodilator FEV 1 from PSBL by 0.35-0.41 L in patients receiving placebo during QUEST (placebo/dupilumab) and 0.34-0.44 L in those receiving dupilumab during QUEST (dupilumab/dupilumab) with an allergic phenotype at baseline. In patients without evidence of allergic asthma, pre-bronchodilator FEV 1 improved by 0.38-0.41 L and 0.33-0.37 L, respectively. By Week 48, ACQ-5 scores decreased from PSBL by 1.63-1.69 (placebo/dupilumab) and 1.74-1.81 (dupilumab/dupilumab) points across subgroups with allergic asthma, and 1.75-1.83 (placebo/dupilumab) and 1.78-1.86 (dupilumab/dupilumab) in those without., Conclusions: Long-term treatment with dupilumab reduced exacerbation rates and improved lung function and asthma control in patients with asthma with type 2 inflammation as per current GINA guidance and irrespective of evidence of allergic asthma., (© 2023 Sanofi and The Authors. Allergy published by European Academy of Allergy and Clinical Immunology and John Wiley & Sons Ltd.)

Introduction: Asthma is a complex disease with heterogeneous expression/severity. There is growing interest in defining asthma endotypes consistently associated with different responses to therapy, focusing on type 2 inflammation (Th2) as a key pathological mechanism. Current asthma endotypes are defined primarily by clinical/laboratory criteria. Each endotype is likely characterised by distinct molecular mechanisms that identify optimal therapies., Methods: We applied unsupervised (without a priori clinical criteria) principal component analysis on sputum airway cells RNA-sequencing transcriptomic data from 19 asthmatics from the Severe Asthma Research Program at baseline and 6-8 weeks follow-up after a 40 mg dose of intramuscular corticosteroids. We investigated principal components PC1, PC3 for association with 55 clinical variables., Results: PC3 was associated with baseline Th2 clinical features including blood (rank-sum p=0.0082) and airway (rank-sum p=0.0024) eosinophilia, FEV 1 change (Kendall tau-b R=-0.333 (-0.592 to -0.012)) and follow-up FEV 1 albuterol response (Kendall tau-b R=0.392 (0.079 to 0.634)). PC1 with blood basophlia (rank-sum p=0.0191). The top 5% genes contributing to PC1, PC3 were enriched for distinct immune system/inflammation ontologies suggesting distinct subject-specific clusters of transcriptomic response to corticosteroids. PC3 association with FEV 1 change was reproduced in silico in a comparable independent 14-subject (baseline, 8 weeks after daily inhaled corticosteroids (ICS)) airway epithelial cells microRNAome dataset., Conclusions: Transcriptomic PCs from this unsupervised methodology define molecular pharmacogenomic endotypes that may yield novel biology underlying different subject-specific responses to corticosteroid therapy in asthma, and optimal personalised asthma care. Top contributing genes to these PCs may suggest new therapeutic targets., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: The US population-level data on asthma morbidity and mortality are available primarily through state-level surveys. We hypothesize that considerable county-level heterogeneity may be obscured by state-level data, thus impeding focused initiatives to improve asthma outcomes., Objective: To assess heterogeneity in the prevalence of uncontrolled, severe, and severe uncontrolled asthma by evaluating state- and county-level morbidity reflected in large administrative claims data sets and identify relationships between pharmacotherapy-based morbidity and the Centers for Disease Control and Prevention's asthma mortality data., Methods: Asthma prevalence and morbidity were identified using medical and pharmacy claims from the IQVIA Longitudinal Access and Adjudication Data database (July 2015-June 2018). Heat maps ranked the prevalence of severe uncontrolled asthma by deciles in all 50 states and the District of Columbia, plus 2935 counties. Mortality in states (2016) and 3147 counties (1999-2018) was similarly mapped and ranked and contrasted with claims-based morbidity., Results: Among 4,506,527 individuals with asthma, 640,936 (14.2%) received age-specific therapy for severe asthma. Of those with severe asthma, 144,232 (22.5%) filled 2 or more annual courses of systemic steroids and were designated as having severe uncontrolled asthma. Most states with high mortality had relatively few patients with severe uncontrolled asthma. A marked correlation between mortality and morbidity and trends by urban vs rural and metropolitan status were found at the county level., Conclusion: Intrastate heterogeneity in the morbidity and mortality of severe uncontrolled asthma at the county level is not evident in state-level analyses. Increased local awareness of systemic corticosteroid use as an indicator of uncontrolled asthma should prompt regional educational and public health efforts to improve outcomes., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: The IL-33/ST2 pathway is linked with asthma susceptibility. Inhaled allergens, pollutants, and respiratory viruses, which trigger asthma exacerbations, induce release of IL-33, an epithelial-derived "alarmin." Astegolimab, a human IgG 2 mAb, selectively inhibits the IL-33 receptor, ST2. Approved biologic therapies for severe asthma mainly benefit patients with elevated blood eosinophils (type 2-high), but limited options are available for patients with low blood eosinophils (type 2-low). Inhibiting IL-33 signaling may target pathogenic pathways in a wider spectrum of asthmatics., Objectives: This study evaluated astegolimab efficacy and safety in patients with severe asthma., Methods: This double-blind, placebo-controlled, dose-ranging study (ZENYATTA [A Study to Assess the Efficacy and Safety of MSTT1041A in Participants With Uncontrolled Severe Asthma]) randomized 502 adults with severe asthma to subcutaneous placebo or 70-mg, 210-mg, or 490-mg doses of astegolimab every 4 weeks. The primary endpoint was the annualized asthma exacerbation rate (AER) at week 54. Enrollment caps ensured ∼30 patients who were eosinophil-high (≥300 cells/μL) and ∼95 patients who were eosinophil-low (<300 cells/μL) per arm., Results: Overall, adjusted AER reductions relative to placebo were 43% (P = .005), 22% (P = .18), and 37% (P = .01) for 490-mg, 210-mg, and 70-mg doses of astegolimab, respectively. Adjusted AER reductions for patients who were eosinophil-low were comparable to reductions in the overall population: 54% (P = .002), 14% (P = .48), and 35% (P = .05) for 490-mg, 210-mg, and 70-mg doses of astegolimab. Adverse events were similar in astegolimab- and placebo-treated groups., Conclusions: Astegolimab reduced AER in a broad population of patients, including those who were eosinophil-low, with inadequately controlled, severe asthma. Astegolimab was safe and well tolerated., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Comorbidities can complicate the management of severe asthma; therefore, the presence of comorbid conditions or traits often need to be considered when considering treatment options for patients with severe asthma. The aim of this analysis is to investigate the efficacy of mepolizumab in patients with severe eosinophilic asthma and comorbidities., Methods: This was a post hoc analysis (GSK ID:209140) of data from the Phase IIb/III studies DREAM, MENSA, SIRIUS, and MUSCA. Patients aged ≥ 12 years with severe eosinophilic asthma were randomized to: mepolizumab 750, 250, or 75 mg intravenously or placebo (DREAM); mepolizumab 75 mg intravenously or 100 mg subcutaneously or placebo (MENSA); or mepolizumab 100 mg subcutaneously or placebo (SIRIUS and MUSCA) every 4 weeks for 24 weeks in SIRIUS and MUSCA, 32 weeks in MENSA or 52 weeks in DREAM. In this analysis the primary endpoint was the annual rate of clinically significant exacerbations; secondary endpoints were Asthma Control Questionnaire-5 score, St George's Respiratory Questionnaire total score, and pre-bronchodilator forced expiratory volume in 1 s at study end. Subgroups were based on comorbidities at baseline., Results: Overall, 1878 patients received placebo (n = 689) or mepolizumab (n = 1189). Across all comorbidity subgroups mepolizumab reduced the rate of clinically significant exacerbations by 44-68% versus placebo, improved Asthma Control Questionnaire-5 score by 0.27-0.59 points, and improved St George's Respiratory Questionnaire total score by 5.0-11.6 points. Pre-bronchodilator forced expiratory volume in 1 s was improved by 27.1-286.9 mL in all but one comorbidity subgroup, the diabetes mellitus subgroup., Conclusions: Mepolizumab reduces exacerbations, and improves asthma control, health-related quality of life, and lung function in patients with severe eosinophilic asthma despite comorbid conditions, including upper respiratory conditions, psychopathologies, cardiovascular conditions, gastroesophageal reflux disease, diabetes mellitus, and obesity., Trial Registration: https://clinicaltrials.gov/ DREAM, MEA112997/NCT01000506; MENSA, MEA115588/NCT01691521; SIRIUS, MEA115575/NCT01842607; MUSCA, 200862/NCT02281318.

Background: Asthma has been associated with impaired interferon response. Multiple cell types have been implicated in such response impairment and may be responsible for asthma immunopathology. However, existing models to study the immune response in asthma are limited by bulk profiling of cells. Our objective was to Characterize a model of peripheral blood mononuclear cells (PBMCs) of patients with severe asthma (SA) and its response to the TLR3 agonist Poly I:C using two single-cell methods., Methods: Two complementary single-cell methods, DropSeq for single-cell RNA sequencing (scRNA-Seq) and mass cytometry (CyTOF), were used to profile PBMCs of SA patients and healthy controls (HC). Poly I:C-stimulated and unstimulated cells were analyzed in this study., Results: PBMCs (n = 9414) from five SA (n = 6099) and three HC (n = 3315) were profiled using scRNA-Seq. Six main cell subsets, namely CD4 + T cells, CD8 + T cells, natural killer (NK) cells, B cells, dendritic cells (DCs), and monocytes, were identified. CD4 + T cells were the main cell type in SA and demonstrated a pro-inflammatory profile characterized by increased JAK1 expression. Following Poly I:C stimulation, PBMCs from SA had a robust induction of interferon pathways compared with HC. CyTOF profiling of Poly I:C stimulated and unstimulated PBMCs (n = 160,000) from the same individuals (SA = 5; HC = 3) demonstrated higher CD8 + and CD8 + effector T cells in SA at baseline, followed by a decrease of CD8 + effector T cells after poly I:C stimulation., Conclusions: Single-cell profiling of an in vitro model using PBMCs in patients with SA identified activation of pro-inflammatory pathways at baseline and strong response to Poly I:C, as well as quantitative changes in CD8 + effector cells. Thus, transcriptomic and cell quantitative changes are associated with immune cell heterogeneity in this model to evaluate interferon responses in severe asthma.

Asthma is a chronic inflammatory disease of the airways that afflicts over 30 million individuals in the United States and over 300 million individuals worldwide. The inflammatory response in the airways is often characterized by the analysis of sputum, which contains multiple types of cells including neutrophils, macrophages, lymphocytes, and rare bronchial epithelial cells. Subtyping patients using microscopy of the sputum has identified both neutrophilic and eosinophilic infiltrates in airway inflammation. However, with the extensive heterogeneity among these cell types, a higher resolution understanding of the inflammatory cell types present in the sputum is needed to dissect the heterogeneity of disease. Improved recognition of the distinct phenotypes and sources of inflammation in asthmatic granulocytes may identify relevant pathways for clinical management or investigation of novel therapeutic mediators. Here, we employed mass cytometry or cytometry by time-of-flight to quantify frequency and define functional status of sputum derived airway cells in asthmatic patients and healthy controls. This in-depth single cell analysis method identified multiple distinct subtypes of airway immune cells, especially in neutrophils. Significance was discovered by statistical analysis as well as a data-driven unbiased clustering approach. Our multidimensional assessment method identifies differences in cellular function and supports identification of cellular status that may contribute to diverse clinical responses. This technical advance is relevant for studies of pathogenesis and may provide meaningful insights to advance our knowledge of asthmatic inflammation., (©2020 Society for Leukocyte Biology.)

Rationale: MicroRNAs are potent regulators of biologic systems that are critical to tissue homeostasis. Individual microRNAs have been identified in airway samples. However, a systems analysis of the microRNA-mRNA networks present in the sputum that contribute to airway inflammation in asthma has not been published. Objectives: Identify microRNA and mRNA networks in the sputum of patients with asthma. Methods: We conducted a genome-wide analysis of microRNA and mRNA in the sputum from patients with asthma and correlated expression with clinical phenotypes. Weighted gene correlation network analysis was implemented to identify microRNA networks (modules) that significantly correlate with clinical features of asthma and mRNA expression networks. MicroRNA expression in peripheral blood neutrophils and lymphocytes and in situ hybridization of the sputum were used to identify the cellular sources of microRNAs. MicroRNA expression obtained before and after ozone exposure was also used to identify changes associated with neutrophil counts in the airway. Measurements and Main Results: Six microRNA modules were associated with clinical features of asthma. A single module ( nely ) was associated with a history of hospitalizations, lung function impairment, and numbers of neutrophils and lymphocytes in the sputum. Of the 12 microRNAs in the nely module, hsa-miR-223-3p was the highest expressed microRNA in neutrophils and was associated with increased neutrophil counts in the sputum in response to ozone exposure. Multiple microRNAs in the nely module correlated with two mRNA modules enriched for TLR (Toll-like receptor) and T-helper cell type 17 (Th17) signaling and endoplasmic reticulum stress. hsa-miR-223-3p was a key regulator of the TLR and Th17 pathways in the sputum of subjects with asthma. Conclusions: This study of sputum microRNA and mRNA expression from patients with asthma demonstrates the existence of microRNA networks and genes that are associated with features of asthma severity. Among these, hsa-miR-223-3p, a neutrophil-derived microRNA, regulates TLR/Th17 signaling and endoplasmic reticulum stress.

Sputum induction is a non-invasive method to evaluate the airway environment, particularly for asthma. RNA sequencing (RNA-seq) of sputum samples can be challenging to interpret due to the complex and heterogeneous mixtures of human cells and exogenous (microbial) material. In this study, we develop a pipeline that integrates dimensionality reduction and statistical modeling to grapple with the heterogeneity. LDA(Latent Dirichlet allocation)-link connects microbes to genes using reduced-dimensionality LDA topics. We validate our method with single-cell RNA-seq and microscopy and then apply it to the sputum of asthmatic patients to find known and novel relationships between microbes and genes.

The chitinase-like protein YKL-40 mediates airway inflammation and serum levels are associated with asthma severity. However, asthma phenotypes associated with YKL-40 levels have not been precisely defined.We conducted an unsupervised cluster analysis of asthma patients treated at the Yale Center for Asthma and Airways Disease (n=156) to identify subgroups according to YKL-40 level. The resulting YKL-40 clusters were cross-validated in cohorts from the Severe Asthma Research Programme (n=167) and the New York University/Bellevue Asthma Repository (n=341). A sputum transcriptome analysis revealed molecular pathways associated with YKL-40 subgroups.Four YKL-40 clusters (C1-C4) were identified. C3 and C4 had high serum YKL-40 levels compared with C1 and C2. C3 was associated with earlier onset and longer duration of disease, severe airflow obstruction, and near-fatal asthma exacerbations. C4 had the highest serum YKL-40 levels, adult onset and less airflow obstruction, but frequent exacerbations. An airway transcriptome analysis in C3 and C4 showed activation of non-type 2 inflammatory pathways.Elevated serum YKL-40 levels were associated with two distinct clinical asthma phenotypes: one with irreversible airway obstruction and another with severe exacerbations. The YKL-40 clusters are potentially useful for identification of individuals with severe or exacerbation-prone asthma., Competing Interests: Conflict of interest: Disclosures can be found alongside this article at erj.ersjournals.com, (Copyright ©ERS 2017.)

Oxidative stress is important in the pathogenesis of allergic asthma. Extracellular superoxide dismutase (EC-SOD; SOD3) is the major antioxidant in lungs, but its role in allergic asthma is unknown. Here we report that asthmatics have increased SOD3 transcript levels in sputum and that a single nucleotide polymorphism (SNP) in SOD3 (R213G; rs1799895) changes lung distribution of EC-SOD, and decreases likelihood of asthma-related symptoms. Knockin mice analogous to the human R213G SNP had lower airway hyperresponsiveness, inflammation, and mucus hypersecretion with decreased interleukin-33 (IL-33) in bronchoalveolar lavage fluid and reduced type II innate lymphoid cells (ILC2s) in lungs. SOD mimetic (Mn (III) tetrakis (N-ethylpyridinium-2-yl) porphyrin) attenuated Alternaria-induced expression of IL-33 and IL-8 release in BEAS-2B cells. These results suggest that R213G SNP potentially benefits its carriers by resulting in high EC-SOD in airway-lining fluid, which ameliorates allergic airway inflammation by dampening the innate immune response, including IL-33/ST2-mediated changes in ILC2s.

Background: Mepolizumab, an anti-interleukin-5 monoclonal antibody approved as add-on therapy to standard of care for patients with severe eosinophilic asthma, has been shown in previous studies to reduce exacerbations and dependency on oral corticosteroids compared with placebo. We aimed to further assess mepolizumab in patients with severe eosinophilic asthma by examining its effect on health-related quality of life (HRQOL)., Methods: We did a randomised, double-blind, placebo-controlled, parallel-group, multicentre, phase 3b trial (MUSCA) in 146 hospitals or research centres in 19 countries worldwide. Eligible participants were patients aged 12 years or older with severe eosinophilic asthma and a history of at least two exacerbations requiring treatment in the previous 12 months before screening despite regular use of high-dose inhaled corticosteroids plus other controller medicines. Exclusion criteria included current smokers or former smokers with a history of at least ten pack-years. We randomly assigned participants (1:1) by country to receive a subcutaneous injection of either mepolizumab 100 mg or placebo, plus standard of care, every 4 weeks for 24 weeks (the final dose was given at week 20). We did the randomisation using an interactive voice response system and a centralised, computer-generated, permuted-block design of block size six. The two treatments were identical in appearance and administered in a masked manner; patients, investigators, other site staff and the entire study team including those assessing outcomes data were also masked to group assignment. The primary endpoint was the mean change from baseline in the St George's Respiratory Questionnaire (SGRQ) total score at week 24 in the modified intention-to-treat (modified ITT) population (analysed according to their randomly assigned treatment). Safety was assessed in all patients who received at least one dose of trial medication (analysed according to the actual treatment received). This trial is registered with ClinicalTrials.gov, number NCT02281318., Findings: We recruited patients between Dec 11, 2014, and Nov 20, 2015, and the study was undertaken between Dec 11, 2014, and June 10, 2016. The modified ITT population comprised 274 patients assigned to mepolizumab 100 mg and 277 assigned to placebo. Mepolizumab versus placebo showed significant improvements at week 24 from baseline in SGRQ total score (least squares mean [SE] change from baseline -15·6 (1·0) vs -7·9 (1·0), a treatment difference of -7·7 (95% CI -10·5 to -4·9; p<0·0001). No deaths occurred during the study. 192 (70%) of 273 patients who received mepolizumab and 207 (74%) of 278 who received placebo reported at least one on-treatment adverse event, the most common of which were headache (in 45 [16%] given mepolizumab vs 59 [21%] given placebo) and nasopharyngitis (in 31 [11%] given mepolizumab vs 46 [17%] given placebo). 15 (5%) and 22 (8%) patients had an on-treatment serious adverse event in the mepolizumab and placebo groups, respectively; the most common was asthma in both groups (in three [1%] given mepolizumab vs nine [3%] given placebo)., Interpretation: Mepolizumab was associated with significant improvements in HRQOL in patients with severe eosinophilic asthma, and had a safety profile similar to that of placebo. These results add to and support the use of mepolizumab as a favourable add-on treatment option to standard of care in patients with severe eosinophilic asthma., Funding: GlaxoSmithKline., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Airway diseases affect over 7% of the U.S. population and millions of patients worldwide. Asthmatic patients have wide variation in clinical severity with different clinical and physiologic manifestations of disease that may be driven by distinct biologic mechanisms. Further, the immunologic underpinnings of this complex trait disease are heterogeneous and treatment success depends on defining subgroups of asthmatics. Because of the limited availability and number of cells from the lung, the active site, in-depth investigation has been challenging. Recent advances in technology support transcriptional analysis of cells from induced sputum. Flow cytometry studies have described cells present in the sputum but a detailed analysis of these subsets is lacking. Mass cytometry or CyTOF (Cytometry by Time-Of-Flight) offers tremendous opportunities for multiparameter single cell analysis. Experiments can now allow detection of up to ∼40 markers to facilitate unprecedented multidimensional cellular analyses. Here we demonstrate the use of CyTOF on primary airway samples obtained from well-characterized patients with asthma and cystic fibrosis. Using this technology, we quantify cellular frequency and functional status of defined cell subsets. Our studies provide a blueprint to define the heterogeneity among subjects and underscore the power of this single cell method to characterize airway immune status. © 2016 International Clinical Cytometry Society., (© 2016 International Clinical Cytometry Society.)

Background: Single nucleotide polymorphisms (SNPs) in the chitinase 3-like 1 (CHI3L1) promoter, the gene encoding YKL-40, are associated with circulating YKL-40 levels and asthma prevalence. However, the effects of gene polymorphisms on asthma severity and airway expression of YKL-40 have not been examined., Objective: We sought to determine the effect of genetic variation in CHI3L1 on asthma severity and YKL-40 expression in subjects from the Yale Center for Asthma and Airways Disease and the Severe Asthma Research Program., Methods: SNPs spanning the CHI3L1 gene were genotyped in 259 Yale Center for Asthma and Airways Disease and 919 Severe Asthma Research Program subjects. Association and haplotype analyses were conducted to identify effects on airflow obstruction, YKL-40 levels, and asthma severity., Results: Fifteen SNPs in CHI3L1 were associated with FEV1, serum YKL-40 levels, or both. rs12141494 (intron 6) was the only SNP in subjects of European ancestry in both cohorts that was associated with serum YKL-40 levels and postbronchodilator FEV1. Conditional analysis demonstrated that the effect on lung function was independent of the promoter SNP rs4950928, and haplotype analysis demonstrated that G alleles at rs12141494 and rs4950928 are associated with lower YKL-40 expression and higher FEV1 percent predicted values. In asthmatic subjects the risk allele A at rs12141494 was associated with severe asthma and higher YKL-40 expression in the airway (P ≤ .05)., Conclusion: In contrast to the promoter SNP rs4950928, the intronic SNP rs12141494 in CHI3L1 is associated with asthma severity, lung function, and YKL-40 expression in the blood and airway. These data suggest that SNP rs12141494 modulates YKL-40 expression in the airway and contributes to airway remodeling and asthma severity., (Copyright © 2014 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

Rationale: The airway transcriptome includes genes that contribute to the pathophysiologic heterogeneity seen in individuals with asthma., Objectives: We analyzed sputum gene expression for transcriptomic endotypes of asthma (TEA), gene signatures that discriminate phenotypes of disease., Methods: Gene expression in the sputum and blood of patients with asthma was measured using Affymetrix microarrays. Unsupervised clustering analysis based on pathways from the Kyoto Encyclopedia of Genes and Genomes was used to identify TEA clusters. Logistic regression analysis of matched blood samples defined an expression profile in the circulation to determine the TEA cluster assignment in a cohort of children with asthma to replicate clinical phenotypes., Measurements and Main Results: Three TEA clusters were identified. TEA cluster 1 had the most subjects with a history of intubation (P = 0.05), a lower prebronchodilator FEV1 (P = 0.006), a higher bronchodilator response (P = 0.03), and higher exhaled nitric oxide levels (P = 0.04) compared with the other TEA clusters. TEA cluster 2, the smallest cluster, had the most subjects that were hospitalized for asthma (P = 0.04). TEA cluster 3, the largest cluster, had normal lung function, low exhaled nitric oxide levels, and lower inhaled steroid requirements. Evaluation of TEA clusters in children confirmed that TEA clusters 1 and 2 are associated with a history of intubation (P = 5.58 × 10(-6)) and hospitalization (P = 0.01), respectively., Conclusions: There are common patterns of gene expression in the sputum and blood of children and adults that are associated with near-fatal, severe, and milder asthma.

Rationale: Obesity, especially truncal obesity, is a risk factor for asthma incidence, prevalence, and severity. Chitinase 3-like-1 (Chi3l1) is an evolutionarily conserved moiety that plays a critical role in antipathogen and Th2 responses. However, the mechanisms that underlie the association between asthma and obesity and the role(s) of Chi3l1 in fat accumulation have not been defined., Objectives: To determine whether Chi3l1 is regulated by a high-fat diet (HFD) and simultaneously plays an important role(s) in the pathogenesis of asthma and obesity., Methods: We evaluated the regulation of Chi3l1 by an HFD and Th2 inflammation. We also used genetically modified mice to define the roles of Chi3l1 in white adipose tissue (WAT) accumulation and Th2 inflammation and blockers of sirtuin 1 (Sirt1) to define its roles in these responses. Finally, the human relevance of these findings was assessed with a case-control study involving obese and lean control subjects and those with asthma., Measurements and Main Results: These studies demonstrate that an HFD and aeroallergen challenge augment the expression of WAT and pulmonary Chi3l1. Chi3l1 also played a critical role in WAT accumulation and lung Th2 inflammation. In addition, Chi3l1 inhibited Sirt1 expression, and the deficient visceral fat and Th2 responses in Chi3l1 null mice were reversed by Sirt1 inhibition. Finally, serum and sputum Chi3l1 were positively associated with truncal adiposity, and serum Chi3l1 was associated with persistent asthma and low lung function in obese subjects with asthma., Conclusions: Chi3l1 is induced by an HFD and Th2 inflammation, and simultaneously contributes to the genesis of obesity and asthma.

Rationale: Cytokine receptors can be markers defining different T-cell subsets and considered as therapeutic targets. The association of IL-6 and IL-6 receptor α (IL-6Rα) with asthma was reported, suggesting their involvement in asthma., Objectives: To determine whether and how IL-6Rα defines a distinct effector memory (EM) CD8+ T-cell population in health and disease., Methods: EM CD8+ T cells expressing IL-6Rα (IL-6Rα(high)) were identified in human peripheral blood and analyzed for function, gene, and transcription factor expression. The relationship of these cells with asthma was determined using blood and sputum., Measurements and Main Results: A unique population of IL-6Rα(high) EM CD8+ T cells was found in peripheral blood. These cells that potently proliferated, survived, and produced high levels of the Th2-type cytokines IL-5 and IL-13 had increased levels of GATA3 and decreased levels of T-bet and Blimp-1 in comparison with other EM CD8+ T cells. In fact, GATA3 was required for IL-6Rα expression. Patients with asthma had an increased frequency of IL-6Rα(high) EM CD8+ T cells in peripheral blood compared with healthy control subjects. Also, IL-6Rα(high) EM CD8+ T cells exclusively produced IL-5 and IL-13 in response to asthma-associated respiratory syncytial virus and bacterial superantigens., Conclusions: Human IL-6Rα(high) EM CD8+ T cells is a unique cell subset that may serve as a reservoir for effector CD8+ T cells, particularly the ones producing Th2-type cytokines, and expand in asthma.

Background: Observational studies suggest that asthma control improves after adenotonsillectomy, but longitudinal studies that correlate the effect of the procedure on the levels of biomarkers associated with airway inflammation are limited., Methods: We conducted a longitudinal, observational study on pediatric patients, both with and without asthma, undergoing adenotonsillectomy. Asthma control test (ACT) scores and chitinase activity in the circulation were measured at time of surgery and at 6-mo follow-up., Results: Sixty-six children with asthma and 64 control subjects were enrolled. Mean ACT scores improved by three points (P < 0.001) after 6 mo. 85% of children with poorly controlled asthma demonstrated an increase in ACT score of at least three points or a decrease in emergency department/urgent care visits, oral corticosteroid courses, or rescue short acting bronchodilator usage. Chitinase activity decreased significantly in asthmatics who improved (P < 0.01). Higher chitinase activity levels at baseline were associated with improved asthma control following surgery (P < 0.01)., Conclusion: In children with high preoperative circulating chitinase activity levels, asthma control and healthcare utilization were significantly improved after adenotonsillecotmy. Chitinase activity decreased after surgery in children with improved control. This suggests that adenotonsillectomy modulates chitinase activity, affecting airway inflammation and improving airway disease.

IL-33 and its soluble receptor and cell-associated receptor (ST2L) are all increased in clinical and experimental asthma. The present study addressed the hypothesis that ST2L impairs the therapeutic effects of CpG in a fungal model of asthma. C57BL/6 mice were sensitized to Aspergillus fumigatus and challenged via i.t. instillation with live A. fumigatus conidia. Mice were treated with IgG alone, anti-ST2L monoclonal antibody (mAb) alone, CpG alone, IgG plus CpG, or anti-ST2L mAb plus CpG every other day from day 14 to day 28 and investigated on day 28 after conidia. Lung ST2L and toll-like receptor 9 protein expression levels concomitantly increased in a time-dependent manner during fungal asthma. Therapeutic blockade of ST2L with an mAb attenuated key pathological features of this model. At subtherapeutic doses, neither anti-ST2L mAb nor CpG alone affected fungal asthma severity. However, airway hyperresponsiveness, mucus cell metaplasia, peribronchial fibrosis, and fungus retention were markedly reduced in asthmatic mice treated with the combination of both. Whole lung CXCL9 levels were significantly elevated in the combination group but not in the controls. Furthermore, in asthmatic mice treated with the combination therapy, dendritic cells generated significantly greater IL-12p70 with CpG in vitro compared with control dendritic cells. The combination of anti-ST2L mAb with CpG significantly attenuated experimental asthma, suggesting that targeting ST2L might enhance the therapeutic efficacy of CpG during allergic inflammation., (Copyright © 2011 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Background: Asthma exacerbations remain a major unmet clinical need. The difficulty in obtaining airway tissue and bronchoalveolar lavage samples during exacerbations has greatly hampered study of naturally occurring exacerbations. This study was conducted to determine if mRNA profiling of peripheral blood mononuclear cells (PBMCs) could provide information on the systemic molecular pathways involved during asthma exacerbations., Methodology/principal Findings: Over the course of one year, gene expression levels during stable asthma, exacerbation, and two weeks after an exacerbation were compared using oligonucleotide arrays. For each of 118 subjects who experienced at least one asthma exacerbation, the gene expression patterns in a sample of peripheral blood mononuclear cells collected during an exacerbation episode were compared to patterns observed in multiple samples from the same subject collected during quiescent asthma. Analysis of covariance identified genes whose levels of expression changed during exacerbations and returned to quiescent levels by two weeks. Heterogeneity among visits in expression profiles was examined using K-means clustering. Three distinct exacerbation-associated gene expression signatures were identified. One signature indicated that, even among patients without symptoms of respiratory infection, genes of innate immunity were activated. Antigen-independent T cell activation mediated by IL15 was also indicated by this signature. A second signature revealed strong evidence of lymphocyte activation through antigen receptors and subsequent downstream events of adaptive immunity. The number of genes identified in the third signature was too few to draw conclusions on the mechanisms driving those exacerbations., Conclusions/significance: This study has shown that analysis of PBMCs reveals systemic changes accompanying asthma exacerbation and has laid the foundation for future comparative studies using PBMCs.

Fungi can exacerbate symptoms in patients with asthma. To our knowledge, genetic risk factors for fungal-associated asthma have not been described. We present here the cases of 6 children who carried the diagnosis of severe asthma with fungal sensitization, 3 of whom were treated with and responded clinically to itraconazole therapy. All 6 patients were heterozygous for a 24-base pair duplication in the CHIT1 gene, which has been associated with decreased levels of circulating chitotriosidase and susceptibility to fungal infection.

Purpose of Review: The present review provides an overview of the chitinase and chitinase-like proteins, chitotriosidase (CHIT1), YKL-40, and acid mammalian chitinase, and summarizes the genetic studies of asthma and immune-mediated diseases with polymorphisms in the genes encoding these proteins, CHIT1, CHI3L1, and CHIA, respectively., Recent Findings: Polymorphisms in the CHIT1, CHIA, and CHI3L1 genes influence chitotriosidase enzyme activity, acid mammalian chitinase activity, and YKL-40 levels, respectively. Regulatory SNPs in CHI3L1 were also associated with asthma, atopy, and immune-mediated diseases, and nonsynonymous SNPs in CHIA were associated with asthma. No CHIT1 polymorphisms, including a common nonfunctional 24-bp duplication allele, have been associated with asthma., Summary: These genes represent novel asthma susceptibility genes. Variations in CHI3L1 and CHIA have been associated with asthma risk. Polymorphisms in CHIT1 have not yet been associated with asthma, but few studies have been reported. Given that chitotriosidase is the major chitinase in the airways and a common nonfunctional allele is present in many populations, additional studies of this gene are warranted. Lastly, studies of all three genes need to be conducted in populations of diverse ancestries.

Purpose of Review: Allergic asthma is a frequent lung disease in Western civilizations and is characterized by airway inflammation and tissue remodeling. Without early diagnosis and specific treatment, asthma results in a loss of lung function, impaired quality of life and the risk to die from uncontrolled asthma attacks. Thus, there is a need for specific biomarkers to detect asthma as soon as possible and to initiate the correct clinical treatment., Recent Findings: Recent studies have highlighted the potential role of the chemokine (C-C motif) ligand 17 and the chitinase-like protein YKL-40 as novel biomarkers in asthma. Patient studies suggest that these proteins could be useful to identify asthmatics, to characterize disease severity or both in patients with asthma. Functional studies indicate that these molecules are more than correlated epiphenomena and instead contribute in significant ways to asthma pathogenesis., Summary: Assessments of chemokine (C-C motif) ligand 17 and YKL-40 may allow physicians to more accurately diagnose and predict the course of asthma and thereby allow therapy to be appropriately tailored for a given patient.

Background: The chitinase-like protein YKL-40 is involved in inflammation and tissue remodeling. We recently showed that serum YKL-40 levels were elevated in patients with asthma and were correlated with severity, thickening of the subepithelial basement membrane, and pulmonary function. We hypothesized that single-nucleotide polymorphisms (SNPs) that affect YKL-40 levels also influence asthma status and lung function., Methods: We carried out a genomewide association study of serum YKL-40 levels in a founder population of European descent, the Hutterites, and then tested for an association between an implicated SNP and asthma and lung function. One associated variant was genotyped in a birth cohort at high risk for asthma, in which YKL-40 levels were measured from birth through 5 years of age, and in two populations of unrelated case patients of European descent with asthma and controls., Results: A promoter SNP (-131C-->G) in CHI3L1, the chitinase 3-like 1 gene encoding YKL-40, was associated with elevated serum YKL-40 levels (P=1.1 x 10(-13)), asthma (P=0.047), bronchial hyperresponsiveness (P=0.002), and measures of pulmonary function (P=0.046 to 0.002) in the Hutterites. The same SNP could be used to predict the presence of asthma in the two case-control populations (combined P=1.2 x 10(-5)) and serum YKL-40 levels at birth (in cord-blood specimens) through 5 years of age in the birth cohort (P=8.9 x 10(-3) to 2.5 x 10(-4))., Conclusions: CHI3L1 is a susceptibility gene for asthma, bronchial hyperresponsiveness, and reduced lung function, and elevated circulating YKL-40 levels are a biomarker for asthma and decline in lung function., (Copyright 2008 Massachusetts Medical Society.)

Background: The evolutionarily conserved 18-glycosyl-hydrolase family contains true chitinases and chitinase-like proteins that lack enzymatic activity. Acidic mammalian chitinase has recently been associated with animal models of asthma. The related chitinase-like protein, YKL-40 (also called human cartilage glycoprotein 39 [HCgp-39] and chitinase 3-like 1), can be readily measured in the serum. However, its relationship to asthma has not been evaluated., Methods: We quantified serum YKL-40 levels in three cohorts of patients with asthma--one recruited from the patient population at Yale University, one from the University of Paris, and one from the University of Wisconsin--as well as in controls from the surrounding communities. In the Paris cohort, immunohistochemical analysis and morphometric quantitation were used to evaluate the locus of expression of YKL-40 in the lung. The clinical characteristics of the patients with high serum or lung YKL-40 levels were also evaluated., Results: Serum YKL-40 levels were significantly elevated in patients with asthma as compared with controls. In the Paris cohort, lung YKL-40 levels were elevated and were correlated with circulating YKL-40 levels (r=0.55, P<0.001) and with airway remodeling (measured as the thickness of the subepithelial basement membrane) (r=0.51, P=0.003). In all three cohorts, serum YKL-40 levels correlated positively with the severity of asthma and inversely with the forced expiratory volume in 1 second. Patients with elevated levels of YKL-40 had significantly more frequent rescue-inhaler use, greater oral corticosteroid use, and a greater rate of hospitalization than patients with lower levels., Conclusions: YKL-40 is found in increased quantities in the serum and lungs in a subgroup of patients with asthma, in whom expression of chitinase in both compartments correlates with the severity of asthma. The recovery of YKL-40 from these patients indicates either a causative or a sentinel role for this molecule in asthma., (Copyright 2007 Massachusetts Medical Society.)

When asthma is diagnosed, eosinophilic inflammation and airway remodeling are established in the bronchial airways and can no longer be separated as cause and effect because both processes contribute to persistence and progression of disease, despite anti-inflammatory therapy. Th2 cells are continually active in the airways, even when disease is quiescent. IL-13 is the key effector cytokine in asthma and stimulates airway fibrosis through the action of matrix metalloproteinases on TGF-beta and promotes epithelial damage, mucus production, and eosinophilia. The production of IL-13 and other Th2 cytokines by non-T cells augments the inflammatory response. Inflammation is amplified by local responses of the epithelium, smooth muscle, and fibroblasts through the production of chemokines, cytokines, and proteases. Injured cells produce adenosine that enhances IL-13 production. We review human and animal data detailing the cellular and molecular interactions in established allergic asthma that promote persistent disease, amplify inflammation, and, in turn, cause disease progression.

Globally, a small proportion (5–12%) of asthma patients are estimated to have severe disease. However, severe asthma accounts for disproportionately high healthcare resource utilization. The Global Initiative for Asthma (GINA) management committee recommends treating patients with asthma with inhaled corticosteroids plus long-acting β2-agonists and, when needed, adding a long-acting muscarinic receptor antagonist or biologic agent. Five biologics, targeting different effectors in the type 2 inflammatory pathway, are approved for asthma treatment. However, biologics have not been compared against each other or add-on inhaled therapies in head-to-head clinical trials. As a result, their positioning versus that of current and anticipated small-molecule strategies is largely unknown. Furthermore, with the emergence of biomarkers for predicting response to biologics, a more personalized treatment approach—currently lacking with inhaled therapies—may be possible. To gain perspective, we reviewed recent advances in asthma pathophysiology, phenotypes, and biomarkers; the place of biologics in the management and personalized treatment of severe asthma; and the future of biologics and small-molecule drugs. We propose an algorithm for the stepwise treatment of severe asthma based on recommendations in the GINA strategy document that accounts for the broad range of phenotypes targeted by inhaled therapies and the specificity of biologics. In the future, both biologics and small molecules will continue to play key roles in the individualized treatment of severe asthma. However, as targeted therapies, their application will continue to be focused on patients with certain phenotypes who meet the specific criteria for use as identified in pivotal clinical trials. [ABSTRACT FROM AUTHOR]

In most living cells, the second-messenger roles for adenosine 39,59-cyclic monophosphate (cAMP) are short-lived, confined to the intracellular space, and tightly controlled by the binary switch-like actions of Gas (stimulatory G protein)-activated adenylyl cyclase (cAMP production) and cAMP-specific PDE (cAMP breakdown). Here, by using human airway smooth muscle (HASM) cells in culture as a model, we report that activation of the cell-surface b2AR (b2-adrenoceptor), a Gs-coupled GPCR (G protein-coupled receptor), evokes cAMP egress to the extracellular space. Increased extracellular cAMP levels ([cAMP]e) are long-lived in culture and are induced by receptor-dependent and receptor-independent mechanisms in such a way as to define a universal response class of increased intracellular cAMP levels ([cAMP]i). We find that HASM cells express multiple ATP-binding cassette (ABC) membrane transporters, with ABCC1 (ABC subfamily member C 1) being the most highly enriched transcript mapped to MRPs (multidrug resistance-associated proteins). We show that pharmacological inhibition or downregulation of ABCC1 with siRNA markedly reduces b2AR-evoked cAMP release from HASM cells. Furthermore, inhibition of ABCC1 activity or expression decreases basal tone and increases b-agonist-induced HASM cellular relaxation. These findings identify a previously unrecognized role for ABCC1 in the homeostatic regulation of [cAMP]i in HASM that may be conserved traits of the Gs-GPCRs (Gs-coupled family of GPCRs). Hence, the general features of this activation mechanism may uncover new disease-modifying targets in the treatment of airflow obstruction in asthma. Surprisingly, we find that serum cAMP levels are elevated in a small cohort of patients with asthma as compared with control subjects, which warrants further investigation. [ABSTRACT FROM AUTHOR]