CCL11/eotaxin-1 is a potent eosinophilic CC chemokine expressed by primary human fibroblasts. The combination of TGF-β1 and IL-13 synergistically increases CCL11 expression, but the mechanisms behind the synergy are unclear. To address this, human airway fibroblast cultures from normal and asthmatic subjects were exposed to IL-13 alone or TGF-β1 plus IL-13. Transcriptional (nuclear run-on) and post-transcriptional (mRNA stability) assays confirmed that transcriptional regulation is critical for synergistic expression of CCL11. TGF-β1 plus IL-13 synergistically increased STAT-6 phosphorylation, nuclear translocation and binding to the CCL11 promoter as compared to IL-13 alone. STAT-6 siRNA significantly knocked down both STAT-6 mRNA expression and phosphorylation, and inhibited CCL11 mRNA and protein expression.
Regulation of the IL-4 receptor α (IL-4Rα) complex by TGF-β1 augmented IL-13 signaling by dampening IL-13 receptor α2 (IL-13Rα2) expression, overcoming IL-13's autoregulation of its pathway and enhancing the expression of CCL11. Our data suggest that TGF-β1 induced activation of the MEK-ERK pathway reduces IL-13Rα2 expression induced by IL-13. Thus, TGF-β1, a pleiotropic cytokine upregulated in asthmatic airways, can augment eosinophilic inflammation by interfering with IL-13's negative feedback autoregulatory loop under MEK/ERK dependent conditions.
human airway fibroblasts; CCL11/Eotaxin-1; IL-13; TGF-β1; actinomycin D; STAT-6; MEK-ERK; CHIP; siRNA; qRT-PCR; IL-13Rα
Eosinophilic inflammation is implicated in asthma. Eotaxin 1–3 regulate eosinophil trafficking into the airways along with other chemotactic factors. However, the epithelial and bronchoalveolar lavage (BAL) cell expression of these chemokines in relation to asthma severity and eosinophilic phenotypes has not been addressed.
To measure the expression of the three eotaxin isoforms in bronchoscopically obtained samples and compare them with clinically relevant parameters between normal subjects and patients with asthma.
Normal subjects and patients with asthma of varying severity recruited through the Severe Asthma Research Program underwent clinical assessment and bronchoscopy with airway brushing and BAL. Eotaxin 1–3 mRNA/protein were measured in epithelial and BAL cells and compared with asthma severity, control and eosinophilic inflammation.
Eotaxin-2 and eotaxin-3 mRNA and eotaxin-2 protein were increased in airway epithelial brushings from patients with asthma and were highest in cases of severe asthma (p values 0.0155, 0.0033 and 0.0006, respectively), with eotaxin-2 protein increased with age at onset. BAL cells normally expressed high levels of eotaxin-2 mRNA/protein but BAL fluid levels of eotaxin-2 were lowest in severe asthma. Epithelial eotaxin-2 and eotaxin-3 mRNA/protein was associated with sputum eosinophilia, lower forced expiratory volume in 1 s and more asthma exacerbations. Airway epithelial cell eotaxin-2 protein differed by asthma severity only in those with late onset disease, and tended to be highest in those with late onset eosinophilic asthma.
Epithelial eotaxin-2 and 3 are increased in asthma and severe asthma. Their expression may contribute to luminal migration of eosinophils, especially in later onset disease, asthma control and severity.
Airway inflammation in asthma involves innate immune responses. Toll-like receptors (TLRs) and thymic stromal lymphopoietin (TSLP) are thought to be involved in airway inflammation, but their expression in asthmatics’ both large and small airways has not been investigated.
To analyze the expression of TLR2, TLR3, TLR4 and TSLP in large and small airways of asthmatics and compare their expression in smoking and nonsmoking asthmatics; to investigate whether TLR expression is associated with eosinophilic or neutrophilic airway inflammation and with Mycoplasma pneumoniae and Chlamydophila pneumoniae infection.
Using immunohistochemistry and image analysis, we investigated TLR2, TLR3, TLR4 and TSLP expression in large and small airways of 24 victims of fatal asthma, FA, (13 nonsmokers, 11 smokers) and 9 deceased control subjects (DCtrl). TLRs were also measured in 18 mild asthmatics (MA) and 12 healthy controls (HCtrl). Mycoplasma pneumoniae and Chlamydophila pneumoniae in autopsy lung tissue was analyzed using real-time polymerase chain reaction. Airway eosinophils and neutrophils were measured in all subjects.
Fatal asthma patients had higher TLR2 in the epithelial and outer layers of large and small airways compared with DCtrls. Smoking asthmatics had lower TLR2 levels in the inner and outer layers of the small airways than nonsmoking asthmatics. TSLP was increased in the epithelial and outer layers of the large airways of FA. FA patients had greater TLR3 expression in the outer layer of large airways and greater TLR4 expression in the outer layer of small airways. Eosinophilic airway inflammation was associated with TLR expression in the epithelium of FA. No bacterial DNA was detected in FA or DCtrls. MA and HCtrls had only a small difference in TLR3 expression.
Conclusions and Clinical Relevance
Increased expression of TLR 2, 3 and 4 and TSLP in fatal asthma may contribute to the acute inflammation surrounding asthma deaths.
lung; innate immunity; immunohistochemistry
IL4RA; genetics; pharmacogenetics; interleukin 4; interleukin 13; interleukin 4 receptor; allergen; asthma therapy
Patients with severe or difficult-to-treat asthma are an understudied population but account for considerable asthma morbidity, mortality, and costs. The Epidemiology and Natural History of Asthma: Outcomes and Treatment Regimens (TENOR) study was a large, 3-year, multicenter, observational cohort study of 4756 patients (n = 3489 adults ≥18 years of age, n = 497 adolescents 13-17 years of age, and n = 770 children 6-12 years of age) with severe or difficult-to-treat asthma. TENOR's primary objective was to characterize the natural history of disease in this cohort. Data assessed semiannually and annually included demographics, medical history, comorbidities, asthma control, asthma-related health care use, medication use, lung function, IgE levels, self-reported asthma triggers, and asthma-related quality of life. We highlight the key findings and clinical implications from more than 25 peer-reviewed TENOR publications. Regardless of age, patients with severe or difficult-to-treat asthma demonstrated high rates of health care use and substantial asthma burden despite receiving multiple long-term controller medications. Recent exacerbation history was the strongest predictor of future asthma exacerbations. Uncontrolled asthma, as defined by the 2007 National Heart, Lung, and Blood Institute guidelines’ impairment domain, was highly prevalent and predictive of future asthma exacerbations; this assessment can be used to identify high-risk patients. IgE and allergen sensitization played a role in the majority of severe or difficult-to-treat asthmatic patients.
TENOR; severe or difficult-to-treat asthma; asthma control; asthma exacerbations; burden; medication; quality of life; allergy; IgE
Acute exacerbations of chronic obstructive pulmonary disease (AECOPD) are a significant cause of mortality of COPD patients, and pose a huge burden on healthcare. One of the major causes of AECOPD is airway bacterial (e.g. nontypeable Haemophilus influenzae [NTHi]) infection. However, the mechanisms underlying bacterial infections during AECOPD remain poorly understood. As neutrophilic inflammation including increased release of human neutrophil elastase (HNE) is a salient feature of AECOPD, we hypothesized that HNE impairs airway epithelial defense against NTHi by degrading airway epithelial host defense proteins such as short palate, lung, and nasal epithelium clone 1 (SPLUNC1).
Recombinant human SPLUNC1 protein was incubated with HNE to confirm SPLUNC1 degradation by HNE. To determine if HNE-mediated impairment of host defense against NTHi was SPLUNC1-dependent, SPLUNC1 protein was added to HNE-treated primary normal human airway epithelial cells. The in vivo function of SPLUNC1 in NTHi defense was investigated by infecting SPLUNC1 knockout and wild-type mice intranasally with NTHi. We found that: (1) HNE directly increased NTHi load in human airway epithelial cells; (2) HNE degraded human SPLUNC1 protein; (3) Recombinant SPLUNC1 protein reduced NTHi levels in HNE-treated human airway epithelial cells; (4) NTHi levels in lungs of SPLUNC1 knockout mice were increased compared to wild-type mice; and (5) SPLUNC1 was reduced in lungs of COPD patients.
Our findings suggest that SPLUNC1 degradation by neutrophil elastase may increase airway susceptibility to bacterial infections. SPLUNC1 therapy likely attenuates bacterial infections during AECOPD.
Exhaled nitric oxide (FeNO) associates with asthma and eosinophilic inflammation. However, relationships between nitric oxide synthases, arginase, FeNO, asthma severity and inflammation remain poorly understood.
To determine the relationships of iNOS expression/activation and arginase 2 expression with asthma severity, FeNO, nitrotyrosine (NT) and eosinophilic inflammation.
Bronchial brushings and sputum were obtained from 25 Normal Controls, 8 Mild/no inhaled corticosteroids (ICS), 16 Mild-Moderate/with ICS and 35 Severe Asthmatics. FeNO was measured the same day by ATS/ERS standards. iNOS, Arginase2 mRNA/protein and NT protein were measured in lysates from bronchial brushings by quantitative real time PCR and Western Blot. Induced sputum differentials were obtained.
Severe asthma was associated with the highest levels of iNOS protein and mRNA, although the index of iNOS mRNA to arginase2 mRNA most strongly differentiated severe from milder asthma. When evaluating NO-related enzyme functionality, iNOS mRNA/protein expression both strongly predicted FeNO (r=0.61, p<0.0001 for both). Only iNOS protein predicted NT levels (r=0.48, p=0.003) with the strongest relationship in severe asthma (r=0.61, p=0.009). iNOS protein, FeNO and NT all correlated with sputum eosinophils, but the relationships were again strongest in severe asthma. Controlling for arginase 2 mRNA/protein did not impact any functional outcome.
Conclusions and Clinical Relevance
These data suggest that while iNOS expression from epithelial brushings is highest in severe asthma, factors controlling arginase2 mRNA expression significantly improve differentiation of severity. In contrast, functionality of the NO pathway as measured by FeNO, NT and eosinophilic inflammation, is strongly associated with iNOS expression alone, particularly in severe asthma.
exhaled nitric oxide; airway epithelial cells; iNOS; arginase 2; nitrotyrosine
The effect of sleep quality on asthma control independent from common comorbidities like gastroesophageal reflux disease (GERD) and obstructive sleep apnea (OSA) is unknown. This study examined the association between sleep quality and asthma control and quality of life after accounting for OSA and GERD in non-severe (NSA) and severe (SA) asthma.
Cross-sectional data from 60 normal controls, 143 with NSA, and 79 with SA participating in the Severe Asthma Research Program was examined. Those who reported using positive airway pressure therapy or were at high risk for OSA were excluded.
Both SA and NSA had poorer sleep quality than controls, with SA reporting the worst sleep quality. All asthmatics with GERD and 92% of those without GERD had poor sleep quality (p =.02). The majority (88%–100%) of NSA and SA participants who did not report nighttime asthma disturbances still reported having poor sleep quality. In both NSA and SA, poor sleep quality was associated with worse asthma control and quality of life after controlling for GERD and other covariates.
These results suggest that poor sleep quality is associated with poor asthma control and quality of life among asthmatics and cannot be explained by comorbid GERD and nighttime asthma disturbances.
Asthma control; Gastroesphogeal reflux disease; Sleep
Despite the high prevalence of respiratory symptoms and obstructive lung disease in HIV-infected persons, the prevalence of bronchodilator reversibility (BDR) and asthma has not been systematically studied during the era of combination antiretroviral therapy (ART).
To determine the prevalence of asthma diagnosis and related pulmonary function abnormalities in an HIV-infected cohort and to identify potential mechanisms.
A cross-sectional analysis of 223 HIV-infected individuals with data on respiratory symptoms and diagnoses, pulmonary function, sputum cell counts, and asthma-related cytokines and chemokines in serum/sputum.
Doctor-diagnosed asthma was present in 46 (20.6%) and BDR (≥200ml and ≥12% increase in FEV1 or FVC) in 20 participants (9.0%). Pulmonary symptoms and function were worse in those with doctor-diagnosed asthma. Doctor-diagnosed asthma was independently associated with female sex (p=0.04), body mass index >29.6kg/m2 (vs.<29.6kg/m2) (p=0.03), history of bacterial or Pneumocystis pneumonia (p=0.01), and with not currently taking ART (p=0.04), and in univariate analysis with parental history of asthma (n=180; p=0.004). High sputum eosinophil percentages (>2.3% based on the highest decile) were more likely in those with doctor-diagnosed asthma (p=0.02) or BDR (p=0.02). Doctor-diagnosed asthma tended to be more common with high sputum IL-4 (p=0.02) and RANTES (p=0.02), while BDR was associated with high plasma macrophage inflammatory protein (MIP)-1α (p=0.002), and sputum MIP-1β levels (p=0.001).
Asthma diagnosis and BDR are prevalent in an HIV-infected outpatient cohort, and associations with family history, obesity, allergic inflammation, prior infection, the absence of ART, and elevated HIV-stimulated cytokines suggest possible mechanisms of HIV-associated asthma.
HIV; asthma; airway obstruction; allergy
Primary human distal lung/parenchymal fibroblasts (DLFs) exhibit a different phenotype from airway fibroblasts (AFs), including the expression of high levels of α–smooth muscle actin (α-SMA). The scope of the differences between these anatomically differentiated fibroblasts, or the mechanisms driving them, has remained unknown. To determine whether the different characteristics of regional fibroblasts are predicted by distinct genomic differences in AFs versus DLFs, matched human fibroblast pairs were isolated from proximal and distal lung tissue and evaluated. Microarray analysis was performed on 12 matched fibroblast pairs (four normal and eight asthmatic samples) and validated by quantitative real-time PCR. The potential functional implications of these differences were analyzed using computational approaches. Four hundred seventy-four transcripts were up-regulated in AFs, and 611 were up-regulated in DLFs via microarray analysis. No differences in normal and asthmatic fibroblasts were evident, and the data were combined for subsequent analyses. Gene ontology and network analyses suggested distinct patterns of pathway activation between AFs and DLFs. The up-regulation of extracellular matrix–associated molecules in AFs was observed, whereas genes associated with actin binding and cytoskeletal organization were up-regulated in DLFs. The up-regulation of activated/total SMAD3 and c-Jun N-terminal kinase in DLFs may partly explain these myofibroblast-like characteristics in DLFs. Thus, marked genomic differences exist between these two populations of regional lung fibroblasts. These striking differences may help identify potential mechanisms by which AFs and DLFs differ in their responses to injury, regeneration, and remodeling in the lung.
human lung fibroblasts; α–smooth muscle actin; microarray; SMAD; JNK; MAPK8
Secreted phospholipase A2s (sPLA2) may be important mediators of asthma, but the specific sPLA2s involved in asthma are not known.
To evaluate sPLA2 group IIA, V, and X proteins (sPLA2-IIA, sPLA2-V and sPLA2-X) in bronchoalveolar lavage (BAL) fluid, BAL cells and airway epithelial cells of subjects with and without asthma, and examine the relationship between the levels of specific sPLA2 enzymes and airway inflammation, asthma severity, and lung function.
The expression of sPLA2-IIA, sPLA2-V and sPLA2-X in BAL cells and epithelial brushings was assessed by qPCR. The levels of these sPLA2 proteins and sPLA2 activity with and without group II and group X-specific inhibitors were measured in BAL fluid from 18 controls and 39 asthmatics.
The airway epithelium expressed sPLA2-X at higher levels than either sPLA2-IIA or sPLA2-V, whereas BAL cells expressed sPLA2-IIA and sPLA2-X at similar levels. The majority of sPLA2 activity in BAL fluid was attributed to either sPLA2-IIA or sPLA2-X. After 10-fold concentration of BAL fluid, the levels of sPLA2-X normalized to total protein were increased in asthma and were associated with lung function, the concentration of induced sputum neutrophils, and prostaglandin E2. The levels of sPLA2-IIA were elevated in asthma when normalized to total protein, but where not related to lung function, markers of airway inflammation or eicosanoid formation.
Conclusions and Clinical Relevance
These data indicate that sPLA2-IIA and sPLA2-X are the major sPLA2s in human airways, and suggest a link between the levels of sPLA2-X in the airways and several features of asthma.
Asthma; Eicosanoid; Epithelial Cell; Leukotriene; Phospholipase
Rationale: Severe asthma (SA) remains poorly understood. Mast cells (MC) are implicated in asthma pathogenesis, but it remains unknown how their phenotype, location, and activation relate to asthma severity.
Objectives: To compare MC-related markers measured in bronchoscopically obtained samples with clinically relevant parameters between normal subjects and subjects with asthma to clarify their pathobiologic importance.
Methods: Endobronchial biopsies, epithelial brushings, and bronchoalveolar lavage were obtained from subjects with asthma and normal subjects from the Severe Asthma Research Program (N = 199). Tryptase, chymase, and carboxypeptidase A (CPA)3 were used to identify total MC (MCTot) and the MCTC subset (MCs positive for both tryptase and chymase) using immunostaining and quantitative real-time polymerase chain reaction. Lavage was analyzed for tryptase and prostaglandin D2 (PGD2) by ELISA.
Measurements and Main Results: Submucosal MCTot (tryptase-positive by immunostaining) numbers were highest in “mild asthma/no inhaled corticosteroid (ICS) therapy” subjects and decreased with greater asthma severity (P = 0.002). In contrast, MCTC (chymase-positive by immunostaining) were the predominant (MCTC/MCTot > 50%) MC phenotype in SA (overall P = 0.005). Epithelial MCTot were also highest in mild asthma/no ICS, but were not lower in SA. Instead, they persisted and were predominantly MCTC. Epithelial CPA3 and tryptase mRNA supported the immunostaining data (overall P = 0.008 and P = 0.02, respectively). Lavage PGD2 was higher in SA than in other steroid-treated groups (overall P = 0.02), whereas tryptase did not differentiate the groups. In statistical models, PGD2 and MCTC/MCTot predicted SA.
Conclusions: Severe asthma is associated with a predominance of MCTC in the airway submucosa and epithelium. Activation of those MCTC may contribute to the increases in PGD2 levels. The data suggest an altered and active MC population contributes to SA pathology.
prostaglandin D2; chymase; carboxypeptidase A
Asthma in children is a heterogeneous disorder with many phenotypes. Although unsupervised cluster analysis is a useful tool for identifying phenotypes, it has not been applied to school-age children with persistent asthma across a wide range of severities.
This study determined how children with severe asthma are distributed across a cluster analysis and how well these clusters conform to current definitions of asthma severity.
Cluster analysis was applied to 12 continuous and composite variables from 161 children at 5 centers enrolled in the Severe Asthma Research Program (SARP).
Four clusters of asthma were identified. Children in Cluster 1 (n = 48) had relatively normal lung function and less atopy, while children in Cluster 2 (n = 52) had slightly lower lung function, more atopy, and increased symptoms and medication usage. Cluster 3 (n = 32) had greater co-morbidity, increased bronchial responsiveness and lower lung function. Cluster 4 (n = 29) had the lowest lung function and the greatest symptoms and medication usage. Predictors of cluster assignment were asthma duration, the number of asthma controller medications, and baseline lung function. Children with severe asthma were present in all clusters, and no cluster corresponded to definitions of asthma severity provided in asthma treatment guidelines.
Severe asthma in children is highly heterogeneous. Unique phenotypic clusters previously identified in adults can also be identified in children, but with important differences. Larger validation and longitudinal studies are needed to determine the baseline and predictive validity of these phenotypic clusters in the larger clinical setting.
Allergic sensitization; Asthma; Severe asthma; Asthma guidelines; Children; Cluster analysis; Lung function; Phenotype
Rationale: Before the introduction of combination antiretroviral (ARV) therapy, patients infected with HIV had an increased prevalence of respiratory symptoms and lung function abnormalities. The prevalence and exact phenotype of pulmonary abnormalities in the current era are unknown. In addition, these abnormalities may be underdiagnosed.
Objectives: Our objective was to determine the current burden of respiratory symptoms, pulmonary function abnormalities, and associated risk factors in individuals infected with HIV.
Methods: Cross-sectional analysis of 167 participants infected with HIV who underwent pulmonary function testing.
Measurements and Main Results: Respiratory symptoms were present in 47.3% of participants and associated with intravenous drug use (odds ratio [OR] 3.64; 95% confidence interval [CI], 1.32–10.046; P = 0.01). Only 15% had previous pulmonary testing. Pulmonary function abnormalities were common with 64.1% of participants having diffusion impairment and 21% having irreversible airway obstruction. Diffusion impairment was independently associated with ever smoking (OR 2.46; 95% CI, 1.16–5.21; P = 0.02) and Pneumocystis pneumonia prophylaxis (OR 2.94; 95% CI, 1.10–7.86; P = 0.01), whereas irreversible airway obstruction was independently associated with pack-years smoked (OR 1.03 per pack-year; 95% CI, 1.01–1.05; P < 0.01), intravenous drug use (OR 2.87; 95% CI, 1.15–7.09; P = 0.02), and the use of ARV therapy (OR 6.22; 95% CI, 1.19–32.43; P = 0.03).
Conclusions: Respiratory symptoms and pulmonary function abnormalities remain common in individuals infected with HIV. Smoking and intravenous drug use are still important risk factors for pulmonary abnormalities, but ARV may be a novel risk factor for irreversible airway obstruction. Obstructive lung disease is likely underdiagnosed in this population.
HIV; respiratory function tests; smoking; antiretroviral therapy, highly active; AIDS
In humans, environmental exposure to a high dose of lipopolysaccharide (LPS) protects from allergic asthma the immunological underpinnings of which are not well understood. In mice, exposure to a high LPS dose blunted house dust mite-induced airway eosinophilia and Th2 cytokine production. While adoptively transferred Th2 cells induced allergic airway inflammation in control mice, they were unable to do so in LPS-exposed mice. LPS promoted the development of a CD11b+Gr1intF4/80+ lung-resident cell resembling myeloid-derived suppressor cells in a TLR4- and MyD88-dependent fashion that suppressed lung dendritic cell (DC)-mediated reactivation of primed Th2 cells. LPS effects switched from suppressive to stimulatory in MyD88-/- mice. Suppression of Th2 effector function was reversed by anti-IL-10 or inhibition of Arginase 1. Lineageneg bone marrow progenitor cells could be induced by LPS to develop into CD11b+Gr1intF4/80+ cells both in vivo and in vitro which when adoptively transferred suppressed allergen-induced airway inflammation in recipient mice. These data suggest that CD11b+Gr1intF4/80+ cells contribute to the protective effects of LPS in allergic asthma by tempering Th2 effector function in the tissue.
LPS; lung; myeloid cells; asthma; suppression
Rationale: The Severe Asthma Research Program cohort includes subjects with persistent asthma who have undergone detailed phenotypic characterization. Previous univariate methods compared features of mild, moderate, and severe asthma.
Objectives: To identify novel asthma phenotypes using an unsupervised hierarchical cluster analysis.
Methods: Reduction of the initial 628 variables to 34 core variables was achieved by elimination of redundant data and transformation of categorical variables into ranked ordinal composite variables. Cluster analysis was performed on 726 subjects.
Measurements and Main Results: Five groups were identified. Subjects in Cluster 1 (n = 110) have early onset atopic asthma with normal lung function treated with two or fewer controller medications (82%) and minimal health care utilization. Cluster 2 (n = 321) consists of subjects with early-onset atopic asthma and preserved lung function but increased medication requirements (29% on three or more medications) and health care utilization. Cluster 3 (n = 59) is a unique group of mostly older obese women with late-onset nonatopic asthma, moderate reductions in FEV1, and frequent oral corticosteroid use to manage exacerbations. Subjects in Clusters 4 (n = 120) and 5 (n = 116) have severe airflow obstruction with bronchodilator responsiveness but differ in to their ability to attain normal lung function, age of asthma onset, atopic status, and use of oral corticosteroids.
Conclusions: Five distinct clinical phenotypes of asthma have been identified using unsupervised hierarchical cluster analysis. All clusters contain subjects who meet the American Thoracic Society definition of severe asthma, which supports clinical heterogeneity in asthma and the need for new approaches for the classification of disease severity in asthma.
asthma phenotype; definition; cluster analysis; severe asthma
An important problem in realizing personalized medicine is the development of methods for identifying disease subtypes using quantitative proteomics. Recently we found that bronchoalveolar lavage (BAL) cytokine patterns contain information about dynamic lung responsiveness. In this study, we examined physiological data from 1048 subjects enrolled in the US Severe Asthma Research Program (SARP) to identify four largely separable, quantitative intermediate phenotypes. Upper extremes in the study population were identified for eosinophil- or neutrophil- predominant inflammation, bronchodilation in response to albuterol treatment, or methacholine sensitivity. We evaluated four different statistical (“machine”) learning methods to predict each intermediate phenotypes using BAL cytokine measurements on a 76 subject subset. Comparison of these models using area under the ROC curve and overall classification accuracy indicated that logistic regression and multivariate adaptive regression splines produced the most accurate methods to predict intermediate asthma phenotypes. These robust classification methods will aid future translational studies in asthma targeted at specific intermediate phenotypes.
Asthma; logistic regression; multivariate regression splines; quantitative phenotypes; personalized medicine
Rationale: The hallmarks of allergic asthma are airway inflammation, obstruction, and remodeling. Airway remodeling may lead to irreversible airflow obstruction with increased morbidity and mortality. Despite advances in the treatment of asthma, the mechanisms underlying airway remodeling are still poorly understood. We reported that insulin-like growth factor (IGF) binding proteins (IGFBPs) contribute to extracellular matrix deposition in idiopathic pulmonary fibrosis; however, their contribution to airway remodeling in asthma has not been established.
Objectives: We hypothesized that IGFBP-3 is overexpressed in asthma and contributes to airway remodeling.
Methods: We evaluated levels of IGFBP-3 in tissues and bronchoalveolar lavage fluid from patients with asthma at baseline and 48 hours after allergen challenge, in reparative epithelium in an in vitro wounding assay, and in conditioned media from cytokine- and growth factor–stimulated primary epithelial cells.
Measurements and Main Results: IGFBP-3 levels and distribution were evaluated by Western blot, ELISA, and immunofluorescence. IGFBP-3 is increased in vivo in the airway epithelium of patients with asthma compared with normal control subjects. The concentration of IGFBP-3 is increased in the bronchoalveolar lavage fluid of patients with asthma after allergen challenge, its levels are increased in reparative epithelium in an in vitro wounding assay and in the conditioned medium of primary airway epithelial cell cultures stimulated with IGF-I.
Conclusions: Our results suggest that one mechanism of allergic airway remodeling is through the secretion of the profibrotic IGFBP-3 from IGF-I–stimulated airway epithelial cells during allergic inflammation.
asthma; bronchoalveolar lavage; primary epithelial cells; insulin-like growth factor binding protein; fibrosis
New therapeutic approaches are needed for severe asthmatics who are refractory to standard therapy with high doses of inhaled corticosteroids plus long-acting β2-agonists. Current treatment guidelines for severe asthmatics from the National Asthma Education and Prevention Program recommend the addition of oral corticosteroids, which are associated with significant morbidity, and for those with allergic asthma, anti-IgE. Genetic and translational studies, as well as clinical trials, suggest that in a sub-group of patients the pathobiology of severe asthma is mediated by immune pathways driven by Th2-type CD4+ T cells which produce a characteristic repertoire of interleukins, including IL-4, IL-5 and IL-13. Therefore, biological modifiers of Th2-type interleukins, such as monoclonal antibodies, soluble receptors and receptor antagonists, represent a rational strategy for developing new treatment approaches, but will need to be targeted to selected individuals in whom the appropriate Th2 immune pathway is “active.” The benefits of immune modifier therapies targeting Th2-type cytokines, however, will need to be weighed against the toxicities associated with inhibition of key biological pathways, as well as the expense of future medications. Therefore, future clinical trials will need to clearly establish the efficacy and safety of biological modifiers of Th2 immune pathways before these approaches can enter routine clinical practice for the treatment of severe asthma.
Rationale: 15-Lipoxygenase-1 (15LO1) and MUC5AC are highly expressed in asthmatic epithelial cells. IL-13 is known to induce 15LO1 and MUC5AC in human airway epithelial cells in vitro. Whether 15LO1 and/or its product 15-HETE modulate MUC5AC expression is unknown.
Objectives: To determine the expression of 15LO1 in freshly harvested epithelial cells from subjects with asthma and normal control subjects and to determine whether IL-13–induced 15LO1 expression and activation regulate MUC5AC expression in human bronchial epithelial cells in vitro.
Methods: Human airway epithelial cells from subjects with asthma and normal subjects were evaluated ex vivo for 15LO1 and MUC5AC expression. The impact of 15LO1 on MUC5AC expression in vitro was analyzed by inhibiting 15LO1 through pharmacologic (PD146176) and siRNA approaches in human bronchial epithelial cells cultured under air–liquid interface. We analyzed 15 hydroxyeicosatetraenoic acid (15-HETE) by liquid chromatography/UV/mass spectrometry. MUC5AC and 15LO1 were analyzed by real-time RT-PCR, immunofluoresence, and Western blot.
Measurements and Main Results: Epithelial 15LO1 expression increased with asthma severity (P < 0.0001). 15LO1 significantly correlated with MUC5AC ex vivo and in vitro. IL-13 increased 15LO1 expression and stimulated formation of two molecular species of 15-HETE esterified to phosphotidylethanolamine (15-HETE-PE). Inhibition of 15LO1 suppressed 15-HETE-PE and decreased MUC5AC expression in the presence of IL-13 stimulation. The addition of exogenous 15-HETE partially restored MUC5AC expression.
Conclusions: Epithelial 15LO1 expression increases with increasing asthma severity. IL-13 induction of 15-HETE-PE enhances MUC5AC expression in human airway epithelial cells. High levels of 15LO1 activity could contribute to the increases of MUC5AC observed in asthma.
15-lipoxygenase-1; MUC5AC; asthma pathogenesis; inflammation; lipid mediator
Immunoglobulin E (IgE)-mediated responses contribute to allergy and asthma. Little is understood regarding the relationship of tissue IgE to systemic IgE, inflammation or clinical outcomes.
To evaluate local IgE expression and cellular inflammation in proximal and distal lung of normal subjects and subjects with asthma of varying severity and relate those tissue parameters to systemic IgE levels, atopy, lung function and history of severe exacerbations of asthma.
Tissue from over 90 subjects with eosinophilic (SAeo+) and non-eosinophilic (SAeo−) severe asthma, mild asthma and normal subjects were immunostained for IgE, signal-amplifying isoform of IgE receptor (FcεRIβ) and markers of mast cells, eosinophils and lymphocytes. Tissue expression of IgE, FcεRIβ, cellular inflammation, serum IgE and atopy were compared. Regression models were used to determine the relationship of local and systemic IgE to lung function and severe exacerbations of asthma.
Mast cell-bound IgE was present along airways, but absent in lung parenchyma. While the groups were similar in systemic/serum IgE and atopy, local/tissue IgE was highest in SAeo+ and correlated with eosinophils and lymphocytes (rs=0.52; p<0.0001 and rs=0.23; p=0.03, respectively). Higher local IgE was associated with better lung function, but also with more severe exacerbations of asthma.
Local IgE appears to be primarily a component of responses within the mucosal immune compartment and is related to cellular inflammation, lung function and clinical outcomes in asthma.
Local/airway IgE-related processes rather than systemic markers of atopy may be relevant in determining clinical outcomes in asthma.
The study reports mucosal distribution of mast cell-bound IgE in human lung and suggests that local IgE and related responses rather than systemic/serum IgE and atopy are more relevant in determining clinical outcomes in asthma.
Immunoglobulin E; mucosal immune response; asthma exacerbation; mast cells; eosinophils; lymphocytes
Background: Severe asthma has been associated with severe exacerbations, lower lung function and greater tissue inflammation. Previous studies have suggested that mutations in interleukin-4 receptor α (IL4Rα) are associated with lower lung function, higher IgE, and a gain in receptor function. However, an effect on exacerbations and tissue inflammation has not been shown.
Hypothesis: Allelic substitutions in IL4Rα are associated with asthma exacerbations, lower lung function, and tissue inflammation, in particular to mast cells and IgE.
Methods: Two well-characterized cohorts of subjects with severe asthma were analyzed for five single nucleotide polymorphisms (SNPs) in IL4Rα. These polymorphisms were compared with the history of severe asthma exacerbations and lung function. In the primary (National Jewish) cohort, these polymorphisms were also compared with endobronchial tissue inflammatory cells and local IgE.
Results: In both cohorts, the presence of the minor alleles at E375A and Q551R, which were more common in African Americans, was associated with a history of severe exacerbations and lower lung function. In the National Jewish cohort, the C allele at E375A was associated with higher tissue mast cells and higher levels of IgE bound to mast cells. The significance for most of these associations remained when whites (the larger racial subgroup) were analyzed separately.
Conclusions: SNPs in IL4Rα, which are more common in African Americans, are associated with severe asthma exacerbations, lower lung function, and increased mast cell–related tissue inflammation. Further studies of the impact of these mutations in African Americans and on receptor function are indicated.
asthma; genetics; IL4Rα; exacerbations; mast cells; IgE
Rationale: Excessive deposition of extracellular matrix occurs in proximal airways of individuals with asthma, but fibrosis in distal lung has not been observed. Whether differing fibrotic capacities of fibroblasts from these two regions contribute to this variability is unknown.
Objectives: We compared morphologic and functional characteristics of fibroblasts isolated from proximal airways and distal lung parenchyma to determine phenotypic differences.
Methods: Concurrent proximal airway and distal lung biopsies were obtained by bronchoscopy from subjects with asthma to isolate airway and distal lung fibroblasts, respectively. The following characteristics were compared: morphology, proliferation, α-smooth muscle actin expression, and synthesis of procollagen type I and eotaxin-1.
Results: Airway fibroblasts (AFs) are morphologically distinct from distal lung fibroblasts (DLFs): they are larger (2.3-fold greater surface area vs. matched DLFs; p = 0.02), stellate in appearance, and with more cytoplasmic projections compared with the spindle-shaped DLFs. AFs synthesized more procollagen type I than did DLFs at baseline (twofold higher; p = 0.003) and after transforming growth factor-β stimulation (1.4-fold higher; p = 0.02). Similarly, AFs produced more eotaxin-1 than did DLFs at baseline (2.5-fold higher; p = 0.004) and after interleukin-13 stimulation (13-fold higher; p = 0.0001). In contrast, DLFs proliferate more than AFs with serum stimulation (about sixfold greater; p = 0.03). Unstimulated DLFs also expressed more α-smooth muscle actin than did corresponding AFs (p = 0.006).
Conclusions: These studies suggest that at least two phenotypes of fibroblast exist in the lung. These phenotypic differences may partially explain the variable responses to injury and repair between proximal airways and distal lung/parenchyma in asthma and other respiratory diseases.
asthma; fibroblast; interleukin 13; remodeling; transforming growth factor β
Rationale: Increased oxidative stress and decreased superoxide dismutase (SOD) activity in the asthmatic airway are correlated to airflow limitation and hyperreactivity. We hypothesized that asthmatic individuals with higher levels of oxidative stress may have greater loss of SOD activity, which would be reflected systemically in loss of circulating SOD activity and clinically by development of severe asthma and/or worsening airflow limitation. Methods: To investigate this, serum SOD activity and proteins, the glutathione peroxidase/glutathione antioxidant system, and oxidatively modified amino acids were measured in subjects with asthma and healthy control subjects. Results: SOD activity, but not Mn-SOD or Cu,Zn-SOD protein, was lower in asthmatic serum as compared with control, and activity loss was significantly related to airflow limitation. Further, serum SOD activity demonstrated an inverse correlation with circulating levels of 3-bromotyrosine, a posttranslational modification of proteins produced by the eosinophil peroxidase system of eosinophils. Exposure of purified Cu,Zn-SOD to physiologically relevant levels of eosinophil peroxidase-generated reactive brominating species, reactive nitrogen species, or tyrosyl radicals in vitro confirmed that eosinophil-derived oxidative pathways promote enzyme inactivation. Conclusion: These findings are consistent with greater oxidant stress in asthma leading to greater inactivation of SOD, which likely amplifies inflammation and progressive airflow obstruction.
asthma; superoxide dismutase; glutathione; pulmonary functions; peroxidase
Genome-wide association studies (GWASs) of asthma have consistently implicated the ORM1-like 3 and gasdermin B (ORMDL3-GSDMB), IL33, IL-1 receptor–like 1 and IL-18 receptor 1 (IL1RL1-IL18R1), RAD50-IL13, thymic stromal lymphopoietin and WD repeat domain 36 region (TSLP-WDR36), and HLA-DR/DQ regions.
A GWAS of asthma was performed in a non-Hispanic white population.
A GWAS was performed in 813 Severe Asthma Research Program/Collaborative Studies on the Genetics of Asthma/Chicago Asthma Genetics Study cases and 1564 control subjects. The GWAS results were compared with those of the published GWASs of autoimmune diseases.
Multiple single nucleotide polymorphisms in the TNFAIP3 interacting protein 1 (TNIP1) gene, which interacts with TNFAIP3 and inhibits the TNF-α–induced nuclear factor κB inflammation pathway, were associated with asthma: rs1422673 (P = 3.44 × 10−7) and rs10036748 (P = 1.41 × 10−6, r2 = 0.67). rs1422673 was also associated with asthma in the published GABRIEL (P = .018) and EVE (P = 1.31 × 10−5) studies. The minor allele T of rs20541 in IL13 is the risk allele for asthma but the protective allele for psoriasis. The minor allele T of rs2395185 in HLA-DRA is the risk allele for asthma but the protective allele for ulcerative colitis. The minor allele A of rs2872507 in GSDMB is the protective allele for asthma but the risk allele for rheumatoid arthritis, Crohn disease, and ulcerative colitis. The T allele of rs10036748 in the TNIP1 gene is the minor protective allele for asthma but the minor or major risk allele for systemic lupus erythematosus and systemic sclerosis in non-Hispanic white or Chinese subjects, respectively.
Our study suggests that single nucleotide polymorphisms associated with both asthma and autoimmune diseases might have opposite effects on immunopathogenesis. (J Allergy Clin Immunol 2012;130:861-8.)
Asthma; genetics; genome-wide association study; TNFAIP3 interacting protein 1