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Background: Patients with severe uncontrolled asthma represent a distinct endotype with persistent airway inflammation and remodeling that is refractory to corticosteroid treatment. CD4 + T H 2 cells play a central role in orchestrating asthma pathogenesis, and biologic therapies targeting their cytokine pathways have had promising outcomes. However, not all patients respond well to such treatment, and their effects are not always durable nor reverse airway remodeling. This observation raises the possibility that other CD4 + T cell subsets and their effector molecules may drive airway inflammation and remodeling., Methods: We performed single-cell transcriptome analysis of >50,000 airway CD4 + T cells isolated from bronchoalveolar lavage samples from 30 patients with mild and severe asthma., Findings: We observed striking heterogeneity in the nature of CD4 + T cells present in asthmatics' airways, with tissue-resident memory T (T RM ) cells making a dominant contribution. Notably, in severe asthmatics, a subset of CD4 + T RM cells (CD103-expressing) was significantly increased, comprising nearly 65% of all CD4 + T cells in the airways of male patients with severe asthma when compared to mild asthma (13%). This subset was enriched for transcripts linked to T cell receptor activation (HLA-DRB1, HLA-DPA1) and cytotoxicity (GZMB, GZMA) and, following stimulation, expressed high levels of transcripts encoding for pro-inflammatory non-T H 2 cytokines (CCL3, CCL4, CCL5, TNF, LIGHT) that could fuel persistent airway inflammation and remodeling., Conclusions: Our findings indicate the need to look beyond the traditional T2 model of severe asthma to better understand the heterogeneity of this disease., Funding: This research was funded by the NIH., Competing Interests: Declaration of interests S.H.A. received research funding unrelated to this work from Dyson Inc., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Background: Asthma is conventionally stratified as type 2 inflammation (T2)-high or T2-low disease. Identifying T2 status has therapeutic implications for patient management, but a real-world understanding of this T2 paradigm in difficult-to-treat and severe asthma remains limited., Objectives: To identify the prevalence of T2-high status in difficult-to-treat asthma patients using a multicomponent definition and compare clinical and pathophysiologic characteristics between patients classified as T2-high and T2-low., Methods: We evaluated 388 biologic-naive patients from the Wessex Asthma Cohort of difficult asthma (WATCH) study in the United Kingdom. Type 2-high asthma was defined as 20 parts per billion or greater FeNO , 150 cells/μL or greater peripheral blood eosinophils, the need for maintenance oral corticosteroids, and/or clinically allergy-driven asthma., Results: This multicomponent assessment identified T2-high asthma in 93% of patients (360 of 388). Body mass index, inhaled corticosteroid dose, asthma exacerbations, and common comorbidities did not differ by T2 status. Significantly worse airflow limitation was found in T2-high compared with T2-low patients (FEV 1 /FVC 65.9% vs 74.6%). Moreover, 75% of patients defined as having T2-low asthma had raised peripheral blood eosinophils within the preceding 10 years, which left only seven patients (1.8%) who had never had T2 signals. Incorporation of sputum eosinophilia 2% or greater into the multicomponent definition in a subset of 117 patients with induced sputum data similarly found that 96% (112 of 117) met criteria for T2-high asthma, 50% of whom (56 of 112) had sputum eosinophils 2% or greater., Conclusions: Almost all patients with difficult-to-treat asthma have T2-high disease; less than 2% of patients never display T2-defining criteria. This highlights a need to assess T2 status comprehensively in clinical practice before labeling a patient with difficult-to-treat asthma as T2-low., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Fungal sensitivity has been associated with severe asthma outcomes. However, the clinical implication of Aspergillus fumigatus sensitization in difficult-to-treat (or difficult) asthma is unclear., Objectives: To characterize the clinical implications of A fumigatus sensitization in a large difficult asthma cohort., Methods: Participants who underwent both skin prick and specific IgE testing to A fumigatus (n = 318) from the longitudinal real-life Wessex AsThma CoHort of difficult asthma, United Kingdom, were characterized by A fumigatus sensitization (either positive skin prick test result or specific IgE) and allergic bronchopulmonary aspergillosis status using clinical/pathophysiological disease measures., Results: A fumigatus sensitization was found in 23.9% (76 of 318) of patients with difficult asthma. Compared with A fumigatus nonsensitized subjects, those with sensitization were significantly more often male (50% vs 31%), older (58 years) with longer asthma duration (33 years), higher maintenance oral corticosteroid (39.7%) and asthma biologic use (27.6%), raised current/maximum log 10 total IgE+1 (2.43/2.72 IU/L), worse prebronchodilator airflow obstruction (FEV 1 62.2% predicted, FEV 1 /forced vital capacity 61.2%, forced expiratory flow between 25% and 75% exhalation 30.9% predicted), and frequent radiological bronchiectasis (40%), but had less psychophysiologic comorbidities. Allergic bronchopulmonary aspergillosis diagnosis was associated with higher treatment needs and stronger eosinophilic signals. Factors independently associated with A fumigatus sensitization in difficult asthma included maintenance oral corticosteroid use (odds ratio [OR], 3.34) and maximum log 10 total IgE+1 (OR, 4.30), whereas for allergic bronchopulmonary aspergillosis included maintenance oral corticosteroid use (OR, 6.98), maximum log 10 total IgE+1 (OR, 4.65), and radiological bronchiectasis (OR, 4.08)., Conclusions: A fumigatus sensitization in difficult asthma identifies a more severe form of airways disease associated with greater morbidity, treatment need, and airways dysfunction/damage, but fewer psychophysiologic comorbidities. Screening of A fumigatus status should be an early element in the comprehensive assessment of patients with difficult asthma., (Copyright © 2021 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

Severe asthma accounts for almost half the cost associated with asthma. Severe asthma is driven by heterogeneous molecular mechanisms. Conventional clinical trial design often lacks the power and efficiency to target subgroups with specific pathobiological mechanisms. Furthermore, the validation and approval of new asthma therapies is a lengthy process. A large proportion of that time is taken by clinical trials to validate asthma interventions. The National Institutes of Health Precision Medicine in Severe and/or Exacerbation Prone Asthma (PrecISE) program was established with the goal of designing and executing a trial that uses adaptive design techniques to rapidly evaluate novel interventions in biomarker-defined subgroups of severe asthma, while seeking to refine these biomarker subgroups, and to identify early markers of response to therapy. The novel trial design is an adaptive platform trial conducted under a single master protocol that incorporates precision medicine components. Furthermore, it includes innovative applications of futility analysis, cross-over design with use of shared placebo groups, and early futility analysis to permit more rapid identification of effective interventions. The development and rationale behind the study design are described. The interventions chosen for the initial investigation and the criteria used to identify these interventions are enumerated. The biomarker-based adaptive design and analytic scheme are detailed as well as special considerations involved in the final trial design., (Copyright © 2021 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

CD4 + T helper (T H ) cells and regulatory T (T reg ) cells that respond to common allergens play an important role in driving and dampening airway inflammation in patients with asthma. Until recently, direct, unbiased molecular analysis of allergen-reactive T H and T reg cells has not been possible. To better understand the diversity of these T cell subsets in allergy and asthma, we analyzed the single-cell transcriptome of ~50,000 house dust mite (HDM) allergen-reactive T H cells and T reg cells from asthmatics with HDM allergy and from three control groups: asthmatics without HDM allergy and nonasthmatics with and without HDM allergy. Our analyses show that HDM allergen-reactive T H and T reg cells are highly heterogeneous and certain subsets are quantitatively and qualitatively different in individuals with HDM-reactive asthma. The number of interleukin-9 (IL-9)-expressing HDM-reactive T H cells is greater in asthmatics with HDM allergy compared with nonasthmatics with HDM allergy, and this IL-9-expressing T H subset displays enhanced pathogenic properties. More HDM-reactive T H and T reg cells expressing the interferon response signature (T H IFNR and T reg IFNR) are present in asthmatics without HDM allergy compared with those with HDM allergy. In cells from these subsets (T H IFNR and T reg IFNR), expression of TNFSF10 was enriched; its product, tumor necrosis factor-related apoptosis-inducing ligand, dampens activation of T H cells. These findings suggest that the T H IFNR and T reg IFNR subsets may dampen allergic responses, which may help explain why only some people develop T H 2 responses to nearly ubiquitous allergens., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Single-cell next-generation sequencing assays are powerful tools to understand the nature of the immune cells that drive disease pathogenesis. In this brief review we explain the value of performing assays at single-cell resolution to better understand the pathogenesis of allergy, asthma, and other lung diseases. We explain the challenges in performing single-cell studies of airways and lung samples from patients with lung diseases. A major limitation comes from the amount of diseased tissue that can be used for research purposes. Finally, we discuss which sequencing strategies can be used to successfully investigate airway and lung diseases at single-cell resolution., (Copyright © 2019 American Academy of Allergy, Asthma & Immunology. All rights reserved.)

Asthma and autoimmune disease susceptibility has been strongly linked to genetic variants in the 17q21 haploblock that alter the expression of ORMDL3; however, the molecular mechanisms by which these variants perturb gene expression and the cell types in which this effect is most prominent are unclear. We found several 17q21 variants overlapped enhancers present mainly in primary immune cell types. CD4 + T cells showed the greatest increase (threefold) in ORMDL3 expression in individuals carrying the asthma-risk alleles, where ORMDL3 negatively regulated interleukin-2 production. The asthma-risk variants rs4065275 and rs12936231 switched CTCF-binding sites in the 17q21 locus, and 4C-Seq assays showed that several distal cis-regulatory elements upstream of the disrupted ZPBP2 CTCF-binding site interacted with the ORMDL3 promoter region in CD4 + T cells exclusively from subjects carrying asthma-risk alleles. Overall, our results suggested that T cells are one of the most prominent cell types affected by 17q21 variants.

Allergic asthma and rhinitis are two common chronic allergic diseases that affect the lungs and nose, respectively. Both diseases share clinical and pathological features characteristic of excessive allergen-induced type 2 inflammation, orchestrated by memory CD4(+) T cells that produce type 2 cytokines (Th2 cells). However, a large majority of subjects with allergic rhinitis do not develop asthma, suggesting divergence in disease mechanisms. Because Th2 cells play a pathogenic role in both these diseases and are also present in healthy nonallergic subjects, we performed global transcriptional profiling to determine whether there are qualitative differences in Th2 cells from subjects with allergic asthma, rhinitis, and healthy controls. Th2 cells from asthmatic subjects expressed higher levels of several genes that promote their survival as well as alter their metabolic pathways to favor persistence at sites of allergic inflammation. In addition, genes that enhanced Th2 polarization and Th2 cytokine production were also upregulated in asthma. Several genes that oppose T cell activation were downregulated in asthma, suggesting enhanced activation potential of Th2 cells from asthmatic subjects. Many novel genes with poorly defined functions were also differentially expressed in asthma. Thus, our transcriptomic analysis of circulating Th2 cells has identified several molecules that are likely to confer pathogenic features to Th2 cells that are either unique or common to both asthma and rhinitis., (Copyright © 2016 by The American Association of Immunologists, Inc.)

A characteristic feature of asthma is the aberrant accumulation, differentiation or function of memory CD4(+) T cells that produce type 2 cytokines (TH2 cells). By mapping genome-wide histone modification profiles for subsets of T cells isolated from peripheral blood of healthy and asthmatic individuals, we identified enhancers with known and potential roles in the normal differentiation of human TH1 cells and TH2 cells. We discovered disease-specific enhancers in T cells that differ between healthy and asthmatic individuals. Enhancers that gained the histone H3 Lys4 dimethyl (H3K4me2) mark during TH2 cell development showed the highest enrichment for asthma-associated single nucleotide polymorphisms (SNPs), which supported a pathogenic role for TH2 cells in asthma. In silico analysis of cell-specific enhancers revealed transcription factors, microRNAs and genes potentially linked to human TH2 cell differentiation. Our results establish the feasibility and utility of enhancer profiling in well-defined populations of specialized cell types involved in disease pathogenesis.

Chronic damage and repair of the bronchial epithelium are features of asthma. We have previously reported that ex vivo stimulation of normal bronchial epithelial cells with epidermal growth factor (EGF), a key factor of epithelial repair, enhances the mechanisms of neutrophil accumulation, thereby promoting neutrophil defences during acute injury but potentially enhancing inflammation in chronic airway diseases. We have now sought to (i) determine whether this EGF-dependent pro-neutrophil activity is increased in asthma, where EGF and its epithelial receptor are over-expressed, and (ii) elucidate some of the mechanisms underlying this asthmatic epithelial-neutrophil interaction. Primary bronchial epithelial cells (PBEC) from healthy subjects, mild asthmatics and moderate-to-severe asthmatics (Mod/Sev) were stimulated with EGF, a model that mimics a repairing epithelium. Conditioned culture media (EGF-CM) were assessed for neutrophil chemotactic and anti-apoptotic activities and inflammatory mediator production. EGF induced the epithelium to produce soluble mediators with neutrophil chemotactic (p<0.001) and pro-survival (p = 0.021) activities which were related to the clinical severity of asthma (trend p = 0.010 and p = 0.009, respectively). This was associated with enhanced IL-6, IL-8, GM-CSF and TNF-α release, and cytokine-neutralising experiments using EGF-CM from Mod/Sev asthmatics demonstrated a role for GM-CSF in neutrophil survival (p<0.001). Pre-treatment of neutrophils with specific inhibitors of the myeloid-restricted class I phosphatidylinositol-3-OH kinase (PI(3)K) isoforms showed that the EGF-CM from Mod/Sev asthmatics depended on the γ (p<0.021) but not δ isoforms, while neutrophil survival required multiple class I PI(3)Ks. The EGF-induced chemotactic, but not pro-survival activity, involved RhoA signaling in neutrophils (p = 0.012). EGF whose activity is upregulated in asthma induces ex vivo the epithelium from asthmatic patients to produce pro-neutrophil activities; these are related to asthma severity and, in moderate-to-severe asthmatics, involves class IB PI(3)Kγ signaling, providing a potential therapeutic target for neutrophilic forms of asthma.

Genome-wide association studies (GWASs) identify single nucleotide polymorphisms (SNPs) that are enriched in individuals suffering from a given disease. Most disease-associated SNPs fall into non-coding regions, so that it is not straightforward to infer phenotype or function; moreover, many SNPs are in tight genetic linkage, so that a SNP identified as associated with a particular disease may not itself be causal, but rather signify the presence of a linked SNP that is functionally relevant to disease pathogenesis. Here, we present an analysis method that takes advantage of the recent rapid accumulation of epigenomics data to address these problems for some SNPs. Using asthma as a prototypic example; we show that non-coding disease-associated SNPs are enriched in genomic regions that function as regulators of transcription, such as enhancers and promoters. Identifying enhancers based on the presence of the histone modification marks such as H3K4me1 in different cell types, we show that the location of enhancers is highly cell-type specific. We use these findings to predict which SNPs are likely to be directly contributing to disease based on their presence in regulatory regions, and in which cell types their effect is expected to be detectable. Moreover, we can also predict which cell types contribute to a disease based on overlap of the disease-associated SNPs with the locations of enhancers present in a given cell type. Finally, we suggest that it will be possible to re-analyze GWAS studies with much higher power by limiting the SNPs considered to those in coding or regulatory regions of cell types relevant to a given disease.

T lymphocytes of the Th2 type are central orchestrators of airway inflammation in asthma. The mechanisms that regulate their accumulation in the asthmatic airways remains poorly understood. We tested the hypothesis that CCR4, preferentially expressed on T lymphocytes of the Th2 type, plays a critical role in this process. We enumerated by flow cytometry the CCR4-expressing T cells from blood, induced sputum, and biopsy samples of patients with asthma and control subjects. We showed a positive correlation between the numbers of peripheral blood CCR4+ T cells and asthma severity, provided evidence of preferential accumulation of CCR4+ T cells in asthmatic airways, and demonstrated that CCR4+ but not CCR4- cells from patients with asthma produce Th2 cytokines. Explanted airway mucosal biopsy specimens, acquired by bronchoscopy from subjects with asthma, were challenged with allergen and the explant supernatants assayed for T cell chemotactic activity. Allergen-induced ex vivo production of the CCR4 ligand, CCL17 was raised in explants from patients with asthma when compared with healthy controls. Using chemotaxis assays, we showed that the T cell chemotactic activity generated by bronchial explants can be blocked with a selective CCR4 antagonist or by depleting CCR4+ cells from responder cells. These results provide evidence that CCR4 might play a role in allergen-driven Th2 cell accumulation in asthmatic airways. Targeting this chemokine receptor in patients with asthma might reduce Th2 cell-driven airway inflammation; therefore, CCR4 antagonists could be an effective new therapy for asthma. This study also provides wider proof of concept for using tissue explants to study immunomodulatory drugs for asthma.

Background: The number of type 2 helper CD4+ T cells is increased in the airways of persons with asthma. Whether the majority of these cells are class II major-histocompatibility-complex-restricted cells or are among the recently identified CD1d-restricted invariant natural killer T cells is a matter of controversy. We studied the frequency of invariant natural killer T cells in the airways of subjects with mild or moderately severe asthma to investigate the possibility of an association between the number of invariant natural killer T cells in the airway and disease severity. We also studied whether an increased number of these cells is a feature of chronic obstructive pulmonary disease (COPD)., Methods: We enumerated invariant natural killer T cells by flow cytometry with the use of CD1d tetramers loaded with alpha-galactosylceramide and antibodies specific to the invariant natural killer T-cell receptor in samples of bronchoalveolar-lavage fluid, induced sputum, and bronchial-biopsy specimens obtained from subjects with mild or moderately severe asthma, subjects with COPD, and healthy control subjects. Real-time polymerase-chain-reaction analysis was performed on bronchoalveolar-lavage cells for evidence of gene expression of the invariant natural killer T-cell receptor., Results: Fewer than 2% of the T cells obtained from all subjects on airway biopsy, bronchoalveolar lavage, and sputum induction were invariant natural killer T cells, with no significant differences among the three groups of subjects. No expression of messenger RNA for the invariant natural killer T-cell-receptor domains Valpha24 and Vbeta11 was detected in bronchoalveolar-lavage cells from subjects with asthma., Conclusions: Invariant natural killer T cells are found in low numbers in the airways of subjects with asthma, subjects with COPD, and controls., (Copyright 2007 Massachusetts Medical Society.)

Despite recent advances in asthma management with anti-IL-5 therapies, many patients have eosinophilic asthma that remains poorly controlled. IL-3 shares a common β subunit receptor with both IL-5 and GM-CSF but, through α-subunit-specific properties, uniquely influences eosinophil biology and may serve as a potential therapeutic target. We aimed to globally characterize the transcriptomic profiles of GM-CSF, IL-3, and IL-5 stimulation on human circulating eosinophils and identify differences in gene expression using advanced statistical modeling. Human eosinophils were isolated from the peripheral blood of healthy volunteers and stimulated with either GM-CSF, IL-3, or IL-5 for 48 h. RNA was then extracted and bulk sequencing performed. DESeq analysis identified differentially expressed genes and weighted gene coexpression network analysis independently defined modules of genes that are highly coexpressed. GM-CSF, IL-3, and IL-5 commonly upregulated 252 genes and downregulated 553 genes, producing a proinflammatory and survival phenotype that was predominantly mediated through TWEAK signaling. IL-3 stimulation yielded the most numbers of differentially expressed genes that were also highly coexpressed (n = 119). These genes were enriched in pathways involving JAK/STAT signaling. GM-CSF and IL-5 stimulation demonstrated redundancy in eosinophil gene expression. In conclusion, IL-3 produces a distinct eosinophil gene expression program among the β-chain receptor cytokines. IL-3-upregulated genes may provide a foundation for research into therapeutics for patients with eosinophilic asthma who do not respond to anti-IL-5 therapies.

Profiling miRNA expression in cells that directly contribute to human disease pathogenesis is likely to aid the discovery of novel drug targets and biomarkers. However, tissue heterogeneity and the limited amount of human diseased tissue available for research purposes present fundamental difficulties that often constrain the scope and potential of such studies. We established a flow cytometry-based method for isolating pure populations of pathogenic T cells from bronchial biopsy samples of asthma patients, and optimized a high-throughput nano-scale qRT-PCR method capable of accurately measuring 96 miRNAs in as little as 100 cells. Comparison of circulating and airway T cells from healthy and asthmatic subjects revealed asthma-associated and tissue-specific miRNA expression patterns. These results establish the feasibility and utility of investigating miRNA expression in small populations of cells involved in asthma pathogenesis, and set a precedent for application of our nano-scale approach in other human diseases. The microarray data from this study (Figure 7) has been submitted to the NCBI Gene Expression Omnibus (GEO; http://ncbi.nlm.nih.gov/geo) under accession no. GSE31030.

The article focuses on a study that examines the association of corticosteroid refractories and neutrophil-dominant inflammatory response, including tumour necrosis factor and interleukin (IL)-6, on the severity of asthma. It notes that eighteen severe asthmatic patients were included in the study. Findings confirmed strong negative correlations between forced expiratory volume in 1s (FEV1) and sputum IL-6 and IL-8 levels and a weak correlation with asthma control.

The article focuses on a study that examines the association of corticosteroid refractories and neutrophil-dominant inflammatory response, including tumour necrosis factor and interleukin (IL)-6, on the severity of asthma. It notes that eighteen severe asthmatic patients were included in the study. Findings confirmed strong negative correlations between forced expiratory volume in 1s (FEV1) and sputum IL-6 and IL-8 levels and a weak correlation with asthma control.