Annette T. Hastie

Sphingosine 1-phosphate modulates human airway smooth muscle cell functions that promote inflammation and airway remodeling in asthma

Asthma is characterized by airway inflammation, remodeling, and hyperresponsiveness to contractile stimuli that promote airway constriction and wheezing. Here we present evidence that sphingosine 1‐phosphate (SPP) is a potentially important inflammatory mediator implicated in the pathogenesis of airway inflammation and asthma. SPP levels were elevated in the airways of asthmatic (but not control) subjects following segmental antigen challenge, and this increase was correlated with a concomitant increase in airway inflammation. Because human airway smooth muscle (ASM) cells expressed EDG receptors for SPP (EDG‐1, ‐3, ‐5, and ‐6), we examined whether SPP may play a role in airway inflammation and remodeling, by affecting ASM cell growth, contraction, and cytokine secretion. SPP is mitogenic and augments EGF‐ and thrombin‐induced DNA proliferation by increasing G1/S progression. SPP increased phosphoinositide turnover and intracellular calcium mobilization, the acute signaling events that affect ASM contraction. By modulating adenylate cyclase activity and cAMP accumulation, SPP had potent effects on cytokine secretion. Although SPP inhibited TNF‐α–induced RANTES release, it induced substantial IL‐6 secretion alone and augmented production of IL‐6 induced by TNF‐α. These studies are the first to associate SPP with airway inflammation and to identify SPP as an effective regulator of ASM growth, contraction and synthetic functions.

Analyses of Asthma Severity Phenotypes and Inflammatory Proteins in Subjects Stratified by Sputum Granulocytes

BACKGROUND Patients with severe asthma have increased granulocytes in their sputum compared with patients with mild to moderate asthma. OBJECTIVE We hypothesized that inflammatory granulocytes in sputum may identify specific asthma severity phenotypes and are associated with different patterns of inflammatory proteins in sputum supernatants. METHODS This hypothesis was tested in 242 patients with asthma enrolled in the Severe Asthma Research Program who provided sputum samples for cell count, differential cell determinations, cell lysates for Western blot, and supernatant analyses by inflammatory protein microarrays and ELISAs. ANOVA and multiple linear regression models tested mediator associations. RESULTS Stratified by sputum granulocytes, <2% or > or = 2% eosinophils and <40% or > or = 40% neutrophils, subjects with both increased eosinophils and neutrophils had the lowest lung function and increased symptoms and health care use. Subjects with elevated eosinophils with or without increased neutrophils had significantly increased fraction exhaled nitric oxide (FeNO) and serum eosinophils and greater frequency of daily beta-agonist use. Microarray data stratified by granulocytes revealed 25 to 28 inflammatory proteins increased >2-fold in sputa with > or = 40% neutrophils. Microarray analyses stratified by severity of asthma identified 6 to 9 proteins increased >2-fold in sputa in subjects with severe asthma compared with nonsevere asthma. ELISA data stratified by sputum granulocytes showed significant increases in brain-derived neurotrophic factor, IL-1beta, and macrophage inflammatory protein 3alpha/CCL20 for those with > or = 40% neutrophils; these mediators demonstrated positive associations with neutrophil counts. CONCLUSION Combined increased sputum eosinophils and neutrophils identified patients with asthma with the lowest lung function, worse asthma control, and increased symptoms and health care requirements. Inflammatory protein analyses of sputum supernatants found novel mediators increased in patients with asthma, predominantly associated with increased sputum neutrophils.

Bronchoalveolar lavage fluid concentrations of transforming growth factor (TGF)‐β1, TGF‐β2, interleukin (IL)‐4 and IL‐13 after segmental allergen challenge and their effects on α‐smooth muscle actin and collagen III synthesis by primary human lung fibroblasts

Rationale Asthmatic airway remodelling is characterized by myofibroblast hyperplasia and subbasement membrane collagen deposition. We hypothesized that cytokines and growth factors implicated in asthmatic airway remodelling are increased in bronchoalveolar lavage (BAL) fluid of asthmatics after segmental allergen challenge (SAC), and that these growth factors and cytokines increase α‐smooth muscle actin (α‐SMA) and collagen III synthesis by human lung fibroblasts (HLFs).

Severe asthma: lessons learned from the National Heart, Lung, and Blood Institute Severe Asthma Research Program.

The National Heart, Lung, and Blood Institute Severe Asthma Research Program (SARP) has characterized over the past 10 years 1,644 patients with asthma, including 583 individuals with severe asthma. SARP collaboration has led to a rapid recruitment of subjects and efficient sharing of samples among participating sites to conduct independent mechanistic investigations of severe asthma. Enrolled SARP subjects underwent detailed clinical, physiologic, genomic, and radiological evaluations. In addition, SARP investigators developed safe procedures for bronchoscopy in participants with asthma, including those with severe disease. SARP studies revealed that severe asthma is a heterogeneous disease with varying molecular, biochemical, and cellular inflammatory features and unique structure-function abnormalities. Priorities for future studies include recruitment of a larger number of subjects with severe asthma, including children, to allow further characterization of anatomic, physiologic, biochemical, and genetic factors related to severe disease in a longitudinal assessment to identify factors that modulate the natural history of severe asthma and provide mechanistic rationale for management strategies.

Differential Expression of TRAIL and TRAIL Receptors in Allergic Asthmatics Following Segmental Antigen Challenge: Evidence for a Role of TRAIL in Eosinophil Survival

Asthma is a chronic lung disease exhibiting airway obstruction, hyperresponsiveness, and inflammation, characterized by the infiltration of eosinophils into the airways and the underlying tissue. Prolonged eosinophilic inflammation depends on the balance between the cell’s inherent tendency to undergo apoptosis and the local eosinophil-viability enhancing activity. TRAIL, a member of the TNF family, induces apoptosis in most transformed cells; however, its role in health and disease remains unknown. To test the hypothesis that Ag-induced inflammation is associated with TRAIL/TRAIL-R interactions, we used a segmental Ag challenge (SAC) model in ragweed-allergic asthmatics and nonasthmatic patients and analyzed bronchoalveolar lavage (BAL) material for 2 wk. In asthmatic patients, the level of TRAIL in BAL fluid dramatically increased 24 h after SAC, which significantly correlated with BAL eosinophil counts. Immunohistochemical analysis of bronchial biopsies from asthmatic patients demonstrated that TRAIL staining was increased in epithelial, airway smooth muscle, and vascular smooth muscle cells and throughout the interstitial tissue after SAC. This was confirmed by quantitative immunocytochemical image analysis of BAL eosinophils and alveolar macrophages, which demonstrated that expression levels of TRAIL and DcR2 increased, whereas expression levels of the TRAIL-Rs DR4 and DR5 decreased in asthmatic subjects after SAC. We also determined that TRAIL prolongs eosinophil survival ex vivo. These data provide the first in vivo evidence that TRAIL expression is increased in asthmatics following Ag provocation and suggest that modulation of TRAIL and TRAIL-R interactions may play a crucial role in promoting eosinophil survival in asthma.

Genome-wide association study identifies TH1 pathway genes associated with lung function in asthmatic patients

BACKGROUND Recent meta-analyses of genome-wide association studies in general populations of European descent have identified 28 loci for lung function. OBJECTIVE We sought to identify novel lung function loci specifically for asthma and to confirm lung function loci identified in general populations. METHODS Genome-wide association studies of lung function (percent predicted FEV1 [ppFEV1], percent predicted forced vital capacity, and FEV1/forced vital capacity ratio) were performed in 4 white populations of European descent (n = 1544), followed by meta-analyses. RESULTS Seven of 28 previously identified lung function loci (HHIP, FAM13A, THSD4, GSTCD, NOTCH4-AGER, RARB, and ZNF323) identified in general populations were confirmed at single nucleotide polymorphism (SNP) levels (P < .05). Four of 32 loci (IL12A, IL12RB1, STAT4, and IRF2) associated with ppFEV1 (P < 10(-4)) belong to the TH1 or IL-12 cytokine family pathway. By using a linear additive model, these 4 TH1 pathway SNPs cumulatively explained 2.9% to 7.8% of the variance in ppFEV1 values in 4 populations (P = 3 × 10(-11)). Genetic scores of these 4 SNPs were associated with ppFEV1 values (P = 2 × 10(-7)) and the American Thoracic Society severe asthma classification (P = .005) in the Severe Asthma Research Program population. TH2 pathway genes (IL13, TSLP, IL33, and IL1RL1) conferring asthma susceptibility were not associated with lung function. CONCLUSION Genes involved in airway structure/remodeling are associated with lung function in both general populations and asthmatic subjects. TH1 pathway genes involved in anti-virus/bacterial infection and inflammation modify lung function in asthmatic subjects. Genes associated with lung function that might affect asthma severity are distinct from those genes associated with asthma susceptibility.

Effect of rare variants in ADRB2 on risk of severe exacerbations and symptom control during longacting β agonist treatment in a multiethnic asthma population: a genetic study

BACKGROUND Severe adverse life-threatening events associated with longacting β agonist (LABA) use have caused the US Food and Drug Administration (FDA) to review the safety of these drugs, resulting in a boxed warning and a mandatory safety study in 46 800 patients with asthma. Identification of an at-risk, susceptible subpopulation on the basis of predictive biomarkers is crucial for understanding LABA safety. The β2-adrenergic receptor gene (ADRB2) contains a common, non-synonymous single nucleotide polymorphism, Gly16Arg, that is unlikely to account for the rare, life-threatening events seen with LABA use. We hypothesise that rare ADRB2 variants modulate therapeutic responses to LABA therapy and contribute to rare, severe adverse events. METHODS In this genetic study, ADRB2 was sequenced in 197 African American, 191 non-Hispanic white, and 73 Puerto Rican patients. Sequencing identified six rare variants, which were genotyped in 1165 patients with asthma. The primary hypothesis was that severe asthma exacerbations requiring hospital admission were associated with rare ADRB2 variants in patients receiving LABA therapy. This outcome was assessed overall and by ethnic group. Replication was done in 659 non-Hispanic white patients with asthma. FINDINGS Patients receiving LABA with a rare ADRB2 variant had increased asthma-related hospital admissions (15 [44%] of 34 patients with rare variant vs 121 [22%] of 553 patients with common ADRB2 alleles admitted to hospital in past 12 months; meta-analysis for all ethnic groups, p=0·0003). Specifically, increases in hospital admission rates were recorded in LABA-treated non-Hispanic white patients with the rare Ile 164 allele compared with non-Hispanic white patients with the common allele (odds ratio [OR] 4·48, 95% CI 1·40-13·96, p=0·01) and African American patients with a 25 bp promoter polynucleotide insertion, -376ins, compared with African American patients with the common allele (OR 13·43, 95% CI 2·02-265·42, p=0·006). The subset of non-Hispanic white and African American patients receiving LABAs with these rare variants had increased exacerbations requiring urgent outpatient health-care visits (non-Hispanic white patients with or without the rare Ile 164 allele, 2·6 [SD 3·5] vs 1·1 [2·1] visits, p<0·0001; and African American patients with or without the rare insertion, 3·7 [4·6] vs 2·4 [3·4] visits, p=0·01), and more frequently were treated with chronic systemic corticosteroids (OR 4·25, 95% CI 1·38-14·41, p=0·01, and 12·83, 1·96-251·93, p=0·006). Non-Hispanic white patients from the primary and replication cohorts with the rare Ile 164 allele were more than twice as likely as Thr 164 homozygotes to have uncontrolled, persistent symptoms during LABA treatment (p=0·008-0·04). INTERPRETATION The rare ADRB2 variants Ile164 and -376ins are associated with adverse events during LABA therapy and should be evaluated in large clinical trials including the current FDA-mandated safety study. FUNDING US National Institutes of Health.

Plasma interleukin-6 concentrations, metabolic dysfunction, and asthma severity: a cross-sectional analysis of two cohorts

BACKGROUND Severe asthma is a complex heterogeneous disease associated with older age and obesity. The presence of eosinophilic (type 2) inflammation in some but not all patients with severe asthma predicts responsiveness to current treatments, but new treatment approaches will require a better understanding of non-type 2 mechanisms of severe asthma. We considered the possibility that systemic inflammation, which arises in subgroups of obese and older patients, increases the severity of asthma. Interleukin-6 (IL-6) is a biomarker of systemic inflammation and metabolic dysfunction, and we aimed to explore the association between IL-6 concentrations, metabolic dysfunction, and asthma severity. METHODS In this cross-sectional analysis, patients were recruited from two cohorts: mainly non-severe asthmatics from the University of California San Francisco (UCSF) and mainly severe asthmatics from the Severe Asthma Research Program (SARP). We generated a reference range for plasma IL-6 in a cohort of healthy control patients. We compared the clinical characteristics of asthmatics with plasma IL-6 concentrations above (IL-6 high) and below (IL-6 low) the upper 95% centile value for plasma IL-6 concentration in the healthy cohort. We also compared how pulmonary function, frequency of asthma exacerbations, and frequency of severe asthma differed between IL-6 low and IL-6 high asthma populations in the two asthma cohorts. FINDINGS Between Jan 1, 2005, and Dec 31, 2014, we recruited 249 patients from UCSF and between Nov 1, 2012, and Oct 1, 2014, we recruited 387 patients from SARP. The upper 95th centile value for plasma IL-6 concentration in the healthy cohort (n=93) was 3·1 pg/mL, and 14% (36/249) of UCSF cohort and 26% (102/387) of the SARP cohort had plasma IL-6 concentrations above this upper limit. The IL-6 high patients in both asthma cohorts had a significantly higher average BMI (p<0·0001) and a higher prevalence of hypertension (p<0·0001) and diabetes (p=0·04) than the IL-6 low patients. IL-6 high patients also had significantly worse lung function and more frequent asthma exacerbations than IL-6 low patients (all p values <0·0001). Although 80% (111/138) of IL-6 high asthmatic patients were obese, 62% (178/289) of obese asthmatic patients were IL-6 low. Among obese patients, the forced expiratory volume in 1 s (FEV1) was significantly lower in IL-6 high than in IL-6 low patients (mean percent predicted FEV1=70·8% [SD 19·5] vs 78·3% [19·7]; p=0·002), and the percentage of patients reporting an asthma exacerbation in the past 1-2 years was higher in IL-6 high than in IL-6 low patients (66% [73/111] vs 48% [85/178]; p=0·003). Among non-obese asthmatics, FEV1 values and the frequency of asthma exacerbations within the past 1-2 years were also significantly worse in IL-6 high than in IL-6 low patients (mean FEV1 66·4% [SD 23·1] vs 83·2% [20·4] predicted; p<0·0001; 59% [16/27] vs 34% [108/320]; p=0·01). INTERPRETATION Systemic IL-6 inflammation and clinical features of metabolic dysfunction, which occur most commonly in a subset of obese asthma patients but also in a small subset of non-obese patients, are associated with more severe asthma. These data provide strong rationale to undertake clinical trials of IL-6 inhibitors or treatments that reduce metabolic dysfunction in a subset of patients with severe asthma. Plasma IL-6 is a biomarker that could guide patient stratification in these trials. FUNDING NIH and the Parker B Francis Foundation.

Inflammatory and Comorbid Features of Patients with Severe Asthma and Frequent Exacerbations

Rationale: Reducing asthma exacerbation frequency is an important criterion for approval of asthma therapies, but the clinical features of exacerbation‐prone asthma (EPA) remain incompletely defined. Objectives: To describe the clinical, physiologic, inflammatory, and comorbidity factors associated with EPA. Methods: Baseline data from the NHLBI Severe Asthma Research Program (SARP)‐3 were analyzed. An exacerbation was defined as a burst of systemic corticosteroids lasting 3 days or more. Patients were classified by their number of exacerbations in the past year: none, few (one to two), or exacerbation prone (≥3). Replication of a multivariable model was performed with data from the SARP‐1 + 2 cohort. Measurements and Main Results: Of 709 subjects in the SARP‐3 cohort, 294 (41%) had no exacerbations and 173 (24%) were exacerbation prone in the prior year. Several factors normally associated with severity (asthma duration, age, sex, race, and socioeconomic status) did not associate with exacerbation frequency in SARP‐3; bronchodilator responsiveness also discriminated exacerbation proneness from asthma severity. In the SARP‐3 multivariable model, blood eosinophils, body mass index, and bronchodilator responsiveness were positively associated with exacerbation frequency (rate ratios [95% confidence interval], 1.6 [1.2‐2.1] for every log unit of eosinophils, 1.3 [1.1‐1.4] for every 10 body mass index units, and 1.2 [1.1‐1.4] for every 10% increase in bronchodilatory responsiveness). Chronic sinusitis and gastroesophageal reflux were also associated with exacerbation frequency (1.7 [1.4‐2.1] and 1.6 [1.3‐2.0]), even after adjustment for multiple factors. These effects were replicated in the SARP‐1 + 2 multivariable model. Conclusions: EPA may be a distinct susceptibility phenotype with implications for the targeting of exacerbation prevention strategies. Clinical trial registered with www.clinicaltrials.gov (NCT 01760915).

The IL6R variation Asp358Ala is a potential modifier of lung function in subjects with asthma

BACKGROUND The IL6R single nucleotide polymorphism (SNP) rs4129267 has recently been identified as an asthma susceptibility locus in subjects of European ancestry but has not been characterized with respect to asthma severity. The SNP rs4129267 is in linkage disequilibrium (r(2) = 1) with the IL6R coding SNP rs2228145 (Asp(358)Ala). This IL6R coding change increases IL-6 receptor (IL-6R) shedding and promotes IL-6 transsignaling. OBJECTIVES We sought to evaluate the IL6R SNP rs2228145 with respect to asthma severity phenotypes. METHODS The IL6R SNP rs2228145 was evaluated in subjects of European ancestry with asthma from the Severe Asthma Research Program (SARP). Lung function associations were replicated in the Collaborative Study on the Genetics of Asthma (CSGA) cohort. Serum soluble IL-6R levels were measured in subjects from SARP. Immunohistochemistry was used to qualitatively evaluate IL-6R protein expression in bronchoalveolar lavage cells and endobronchial biopsies. RESULTS The minor C allele of IL6R SNP rs2228145 was associated with a lower percent predicted FEV(1) in the SARP cohort (P= .005), the CSGA cohort (P= .008), and in a combined cohort analysis (P= .003). Additional associations with percent predicted forced vital capacity (FVC), FEV(1)/FVC ratio, and PC(20) were observed. The rs2228145 C allele (Ala(358)) was more frequent in severe asthma phenotypic clusters. Elevated serum soluble IL-6R levels were associated with lower percent predicted FEV(1) (P= .02) and lower percent predicted FVC (P= .008) (n= 146). IL-6R protein expression was observed in bronchoalveolar lavage macrophages, airway epithelium, vascular endothelium, and airway smooth muscle. CONCLUSIONS The IL6R coding SNP rs2228145 (Asp(358)Ala) is a potential modifier of lung function in subjects with asthma and might identify subjects at risk for more severe asthma. IL-6 transsignaling might have a pathogenic role in the lung.